EP4683508A1

EP4683508A1 - Elicitation method for stimulating an immune system response in plants

Info

Publication number: EP4683508A1
Application number: EP24711177.6A
Authority: EP
Inventors: Maria BLASCO BIGATÁ
Original assignee: Mbp 4 Life SL
Current assignee: Mbp 4 Life SL
Priority date: 2023-03-22
Filing date: 2024-03-10
Publication date: 2026-01-28
Also published as: WO2024194047A1; CO2025012780A2; CL2025002812A1; IL323467A; MX2025011131A

Abstract

The invention refers to the use of monolaurin as a biostimulant for eliciting a defensive response in plants against biotic and abiotic stress by way of genetic stimulation of the immune system.

Description

ELICITATION METHOD FOR STIMULATING AN IMMUNE SYSTEM RESPONSE IN PLANTS.

TECHNICAL FIELD

The invention relates to the field of biostimulation in plants and crops. It specifically relates to the use of 1 -monoglyceride of lauric (C12) fatty acid (monolaurin), or a composition containing it, for eliciting an immune system response in plants and crops.

BACKGROUND ART

Plants and crops are exposed to various biotic and abiotic stresses in their environment. Abiotic stress is the negative impact of non-living factors on the living organisms in a specific environment. The main abiotic stresses that affect plants and crops include drought, salinity, heat, cold, chilling, freezing, nutrient stress, high light intensity, ozone and anaerobic stresses. On the other hand, biotic stress occurs as a result of damage done by other living organisms, such as bacteria, viruses, fungi, parasites, beneficial and harmful insects, herbivorous animals and competing plants.

Abiotic stresses are often prevented by optimizing plant growth conditions by means of using fertilizers, nutrients and plant growth regulators. Meanwhile, synthetic pesticides and phytochemical compounds are almost essential for the control of plant diseases and crop production to avoid biotic stress. Nevertheless, the excessive use of these synthetic chemical products (fertilizers, pesticides and similar) is a global concern because of their harmful effect on human health and the environment. In addition, the overuse of chemical products is the main cause of the development of resistance in pathogens. In this context, the promotion of eco-friendly alternatives appears necessary to reduce the use of chemicals in agriculture and hence the attenuation of their environment effect.

Plants have developed the capacity to activate defences in response to both types of stresses leading to different degrees of resistance which may be effective at the site of infection or systemically. The plant chemistry response against biotic and abiotic stresses is encoded in genes. Complex signalling networks are activated according to the type of invading organism, external attack or problematic situation. Defence-related signalling responses involve phosphorylation events, ionic fluxes and accumulation of phytohormones leading to transcriptional activation of gene coding for the synthesis of antimicrobial compounds such as phytoalexins or pathogenesis related (PR) proteins. These PR proteins are lytic enzymes that can destroy the integrity of the pathogen cell wall and inhibit growth Conversely, the suppression or avoidance of plant immunity is critical for pathogens to successfully infect their hosts and it is well-known that many pathogens (such as Candidatus Liberibacter, Pseudomonas syringae, Xanthomonas campestris, Cladosporium fulvum, Phytophthora infestans, Globodera rostochiensis, Pseudocercospora fuligena, Magnaporthe oryzae, Zymoseptoria tritici, Colletotrichum pathogens or Blumeria graminis) exhibit several mechanisms to disable, minimize, and/or delay the response of the host plant’s immune system. As an example, Candidatus Liberibacter does not cause a considerable induction of immune responses until 5 to 9 weeks after inoculation.

From this perspective, plant immune system stimulators, or biostimulants, or elicitors, are compounds capable of activating the plant immune system in order to defeat and/or limit an infection by pathogens, deal with herbivorous animals and/or mitigate extreme environmental conditions. These elicitors neither have a direct toxic effect on pathogens and pests nor provide nutrients directly to the plants, but they can induce plant defence signalling pathways against biotic and abiotic stresses, which makes them an option for replacing traditional agrochemicals in a sustainable crop production system. Since some pathogens can disable, minimize and/or delay the response of the host plant’s immune system, the biostimulation thereof achieves an enhanced immune response in plants in the very first moments of infection, maximizing the chances of defeating it.

Different biostimulants use different mechanisms. In terms of biochemical composition, biostimulants include a wide variety of inorganic and organic compounds, plant extracts, essential oils, algal extracts, bacteria, fungus, humic acids, peptides and polysaccharides; in terms of outcomes, biostimulants improve nutrient absorption, regulate plant growth and/or activate the plant’s defences.

On the other hand, the antibacterial and anti-fungal activity of monolaurin (glycerol alfa monolaurate-GML) is well known. Monolaurin is a lipophilic monoglyceride which means that, biologically, the target site of its attack is the cytoplasmic membrane of cells. Although the mechanism for the antibacterial activity of monolaurin has not been fully recognized, it has been found to causes structural damage to the cell membrane used as a permeable barrier and inhibit the transport of amino acids to the cell. Another hypothesis is based on the penetration of monolaurin in a non-dissociated form into bacterial cells and its dissociation inside the cells, which leads to acidification of the cell contents. Generally speaking, gram-negative bacteria are more likely to be resistant to the effects of the monoglyceride presumably due to the presence of an outer membrane. In terms of antifungal activity, in vitro studies suggest that monolaurin generally requires a higher concentration in media, although some fungi like Alternaria Alternata or Fusarium ssp. appear to be more sensitive. The bactericidal and fungicidal properties of monolaurin have been described in many fields like food preservation, animal feed, human bacterial diseases and plant infections and pests, as described in documents US6103768A and WO2021/064075. In terms of plant biostimulation, document EP1096853A1 describes aqueous preparations of fatty alcohols and/or partial esters of fatty acids with lower polyfunctional alcohols mixed with ecologically compatible surfactant compounds as a mixture of valuable substances with plant strengthening and/or plant-sanitizing action against attack by phytopathogenic fungi and/or pests from the soil. Document EP1570735B1 describes a plant growthpromoting composition comprising a glycerol derivative as a plant growth-promoting agent, a surfactant and a chelating agent. Document WO1996/019111 describes isopropyl and 2- ethyl-hexyl esters of lauric acid for increasing the activity of agrochemical active substances.

SUMMARY OF INVENTION

The inventors have found that monolaurin can stimulate the plant immune system against biotic and abiotic stress.

Unless stated otherwise, all terms used in this application shall be understood as having their ordinary meaning as known in the state of the art. Other more specific terms used in this application are as set out below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

For the purposes of this invention, any ranges given include both the lower and the upper endpoints of the range. Ranges given, such as temperatures, times, weights, and similar, should be considered approximate, unless specifically stated otherwise.

For the purpose of the invention, monolaurin is also known by the non-proprietary names glycerol monolaurate, glyceryl laurate and 1-lauroyl-glycerol, which are considered to be interchangeable. Monolaurin is the alfa monoester formed from glycerol and lauric acid that has the IUPAC name 2,3-dihydroxypropyl dodecanoate with CAS number 142-18-7 and NSC 698570.

As used herein, the term "plant" refers to both monocotyledonous plants and dicotyledonous plants, and includes familiar organisms such as but not limited to trees, herbs, shrubs, and grasses.

The term “immune system”, as used in this application, refers to all molecules, biomolecules, interactions, biochemical reactions, cellular processes, genetic processes and expressions, proteins and phytohormones, molecular signalling pathways and chemical and physical events involved in phytochemistry that give plants the capacity to prevent and/or withstand, environmental adverse circumstances and/or a biological attack by pathogens, pests and herbivores.

The plant molecular responses to abiotic and biotic stresses involve interactions and crosstalk with many other molecular pathways. Studies have shown that there is interplay (or overlap) between immune responses against abiotic and biotic factors, which is explained by the convergence between signalling and downstream biochemical events induced by both layers of defences. These pathways are regulated by proteins and phytohormones that affect gene expression: the changes in phytohormones and proteins influence plant responses to biotic and abiotic stress because they modulate defence signalling pathways that culminate in the production of secondary metabolites and activate defence priming. Thus, as demonstrated in the experimental section, monolaurin, as an external compound able to elicit the overexpression of these genes, has a biostimulating effect on the plant immune system.

One of the earliest signals in many abiotic stresses involves reactive oxygen species (ROS) and reactive nitrogen species (RNS), which modify enzyme activity and gene regulation. ROS signalling in response to abiotic stresses and its interactions with hormones has been thoroughly reviewed. ROS and RNS form a coordinated network that regulates many plant responses to the environment. The two most important phytohormones that regulate plant responses to abiotic stress are abscisic acid (ABA) and ethylene. ABA is a central regulator of many plant responses to environmental stresses, particularly osmotic and salinity stresses. Activation of ABA signalling cascades results in enhanced plant tolerance to dehydration stress. Ethylene is also involved in many abiotic stress responses, including drought, ozone, flooding (hypoxia and anoxia), heat, chilling, wounding and IIV-B light.

Plants also respond to biotic stresses caused by biological agents, like pathogens or insects. These plant defences can be classified broadly as constitutive (permanent) or induced (temporary). Constitutive defences are always present in the plant and do not depend on the attack of biological agents, they are constantly activated but not always needed, which entails high costs for the plants. On the other hand, induced defences are only activated in the presence of an attacker. Within this second possibility, induced resistance is a physiological state of enhanced defensive capacity of the plant, triggered by biological or chemical inducers, which protects plant tissues not exposed to the initial attack against future attack by pathogens and herbivorous insects. Induced resistance can be triggered in plants by the infection of pathogens, in response to insect herbivory, or upon root colonization by certain rhizosphere mutualistic microbes. Two of the most studied forms of induced resistance are Systemic Acquired Resistance (SAR), triggered by plant pathogens, and Induced Systemic Resistance (ISR), triggered by root-colonizing mutualistic microbes. SAR and ISR are mainly differentiated based on the elicitor and the regulatory pathways involved, although the signalling pathways that regulate SAR and ISR share some components.

In systemic tissues, SAR is characterized by increased levels of the hormone salicylic acid (SA) which activates the expression of a large set of Pathogenesis-Related (PR) genes involved in defence responses. In contrast to SAR, ISR is generally mediated by an SA- independent pathway where jasmonic acid (JA) and ethylene are the key players, and typically functions without PR gene activation.

Gene overexpression encodes the proteins involved in the plant immune system against either pathogen attacks (biotic stress), but also in certain conditions of heat, drought, salinity (abiotic stress). Thereby, genetic biostimulation is promoted when, after the monolaurin application, the plant receives the elicitation signal and triggers certain biological reactions: firstly, the stress factor is identified generally in membrane receptors, then this signal is transduced in the cell by intramolecular calcium and kinase proteins. Afterwards, the secondary metabolites are synthesized as transduction signals (secondary messengers). These secondary metabolites include phytohormones like ethylene, abscisic acid, jasmonic acid or salicylic acid, among others. These messengers activate the gene expression that encodes the defence proteins.

Therefore, this invention describes the stimulation, by way of monolaurin application, of key genes involved in these processes. These genes are as follows: Basic p-1 , 3-endoglucanse, Harpin-induced protein-like (Harp), Chitinase endochitinase family 19 PR3 (CHI3), pathogenesis-related protein-1 (PR1A), Peroxidase, Polyphenol oxidase (PPO), Chitinase induced by ethylene PR3 (ChiEt), PR2 (GluB), Blue copper protein (BCP), Osmotin-like protein, PR5 (0sm2) and Subtilisin-like protease, PR7 (Sub1). The known contribution of these genes to the plant immune system is described below:

-Harp (Harpin-induced protein-like) is involved in plant defence responses against biotic and some abiotic agents. Harpins are glycine-rich and heat-stable proteins that are secreted by gram-negative plant-pathogenic bacteria, such as Phytophthora infestans. Harpin proteins induce multiple responses in plants, such as Systemic Acquired Resistance (SAR), hypersensitive response, enhancement of growth, resistance to some pests and tolerance to drought by way of their involvement in ABA signalling and reactive oxygen species (ROS). Overexpression of Harpin-encoding genes enhances plant resistance to diseases and some abiotic stresses.

-CHI genes (endochitinases) are involved in plant defence responses against abiotic agents (salt solutions, ozone, UV light) and biotic factors (fungi, bacteria, viruses, viroids, fungal cell wall components, and oligosaccharides). These genes promote endochitinase enzymatic proteins capable of hydrolyzing chitin polymers. Specifically, the overexpression of CHI3 genes is involved in ethylene and SA pathways, so they enhance plant resistance to fungal, bacterial and pest diseases and some abiotic factors such as salinity and heavy metal stresses.

-PR1A (Pathogenesis-related protein-1) genes are involved in plant defence responses against biotic factors (fungi, bacteria, virus) as a marker for a salicylic acid mediated response and Systemic Acquired Resistance (SAR). PR1 genes constitute between 1% and 2% of all proteins in plant leaves. The overexpression of PR1A genes enhances plant resistance to fungal, bacterial and pest diseases.

-PPO (polyphenol oxidase) genes are involved in plant defence responses against wounds (by herbivore animals and/or insects) and pathogens. These genes promote polyphenol oxidase enzymatic proteins that use molecular oxygen to oxidize orthodiphenols to orthoquinones. These commonly cause browning reactions following tissue damage, and this is important in plant defence. They are involved in jasmonic acid (JA) pathways in the plant defence response throughout Induced Systemic Resistance (ISR). The overexpression of PPO genes enhances plant resistance to wounds.

-ChiEt (ethylene-induced chitinases) genes are involved in responses to a variety of stresses, such as drought, flooding, pathogen attack and high salinity. Ethylene, a key phytohormone involved in plant-pathogen interaction, plays a positive role in plant resistance against fungal pathogens, but ethylene also mediates adaptive responses to a variety of abiotic stresses, such as drought, flooding and high salinity. The overexpression of ChiEt genes enhances plant resistance to biotic and abiotic stress.

-GluB (Beta Endoqlucanase) genes are involved in responses to pathogen infections. Xyloglucan-specific endo-p-glucanases are enzymes that can attack xyloglucan and cellulose. They can greatly damage the plant cell wall as xyloglucan binds with cellulose microfibrils contributing to the structural integrity of the cell walls. Those enzymes are produced by pathogenic fungi in plant colonization in order to break the extracellular space of plant tissues. Plants produce xyloglucan-specific endo-glucanase inhibitor proteins that bind to the enzyme and inhibit its activity. Thus the overexpression of GluB genes enhances the production of these inhibitors and, therefore, the plant’s resistance to pathogen colonization.

-BCP (Blue copper-binding proteins) genes prevent copper toxicity for plants at high concentrations. Plants have developed a series of mechanisms to prevent the consequences of a copper excess or deficit. This genes family encodes proteins involved in oxidation/reduction processes carried out in response to high salinity and heavy metal stresses. Overexpression of BCP genes significantly increases the plant cell growth rate under abiotic factors, such as Cu(2+), Zn(2+) and high-salinity stresses.

-0sm2 (Osmotin-like protein) genes are involved in responses to both biotic and abiotic stress. Osmotin-like proteins belong to the PR-5 group and they were originally isolated from tobacco cells under osmotic stress. The overexpression of 0sm2 genes induces abiotic stress tolerance by reducing the production of reactive oxygen species (ROS) and also provides protection from fungal infections by increasing plasma membrane permeability and dissipating the membrane potential of infecting fungi. It also has antibacterial activity against many food-borne pathogens. -Sub1 (Subtilisin-like proteases) genes are involved in responses to both biotic and abiotic stress. Subtilisin-like proteases (subtilases) are serine proteases that fulfil highly specific functions in plant development and signalling cascades. In plant-pathogen interactions, the expression of subtilases was induced following pathogen attack and salicylic acid (SA) application. Furthermore, subtilases have been reported to be involved in drought and salt resistance mechanisms. The overexpression of Sub1 genes induces abiotic stress tolerance and also provides protection from pathogen infections.

As mentioned above, the use of an effective amount of monolaurin stimulates the plant immune system against biotic and abiotic stress. In an embodiment, the use of monolaurin is for stimulating the plant immune system against abiotic stresses such as drought, salinity, heat, cold, chilling, freezing, nutrient, high light intensity, ozone, heavy metal stresses and combinations thereof. In an embodiment, the use of monolaurin is for stimulating the plant immune system against biotic factors such as pests, bacteria, viruses, fungi, parasites, beneficial and harmful insects and combinations thereof.

The term “effective amount” refers to the amount that is sufficient to achieve the alleged technical effect, which means the amount of monolaurin that allows the stimulation of the plant immune system against biotic and abiotic stress. Furthermore, the term “biostimulant effective amount” as used herein refers to the amount of monolaurin as an active ingredient that is suitable for being administered or applied in plants by any method (including foliar, radicular or intravascular) that allows the stimulation of the immune system of plants against biotic and abiotic stress.

In an embodiment of the invention, the biostimulant effective amount of monolaurin, for stimulating the plant immune system against biotic and abiotic stress is from 0,001 to 5.000 mg per plant. In an embodiment of the invention, the biostimulant effective amount of monolaurin, for stimulating the immune system of plants against biotic and abiotic stress, is from 0,01 to 2.000 mg per plant. In an embodiment of the invention, the biostimulant effective amount of monolaurin, for stimulating the immune system of plants against biotic and abiotic stress, is from 0,1 to 1000 mg per plant.

In an embodiment of the invention, monolaurin forms part of an “agrochemical composition” comprising the biostimulant effective amount of monolaurin, and one or more “acceptable agrochemical excipients and/or carriers”. For the purpose of this invention, the term “agrochemical composition” refers to the chemical composition suitable for use in agriculture for any purpose. Furthermore, the term “acceptable agrochemical excipients and/or carriers” refers to the excipient or carrier suitable for use in the agriculture technology for preparing compositions with agrochemical use. It means that either the composition or the excipients or carriers are suitable for use in contact with plants without undue toxicity, incompatibility, instability, undesirable response, among others. The appropriate excipients and/or carriers, and their amounts, can readily be determined by those skilled in the art according to the type of formulation being prepared. Examples of appropriate excipients or carriers include, but are not limited to, solvents, cosolvents, diluents, surfactants, emulsifying agents, wetting agents, moisturizing agents, thickeners, stabilizers, rheologic modifiers, adhesive agents, pigments, and dyes, among others. In an embodiment, monolaurin forms part of an “agrochemical composition”, as defined herein above and below comprising from 0,01 to 99,5% by weight of monolaurin. In another embodiment, monolaurin forms part of an “agrochemical composition” as defined herein above and below comprising from 15 to 75% by weight of monolaurin. In another embodiment, monolaurin forms part of an “agrochemical composition” as defined herein above and below comprising from 20 to 50% by weight of monolaurin. In another embodiment, monolaurin forms part of an “agrochemical composition” as defined herein above and below comprising from 35 to 45% by weight of monolaurin.

In an embodiment of the invention, monolaurin forms part of an “agrochemical composition”, as defined herein above, which is in the form of a liquid composition, solid composition and semisolid composition such as gels and creams. In an embodiment of the invention, the use of monolaurin for stimulating the plant immune system against biotic and abiotic stress comprises a foliar application, a stem application, a stem injection, a root application, a root injection, a soil application or a combination thereof. The appropriate form of the agrochemical composition, its excipients and/or carriers, and their amounts, can readily be determined by those skilled in the art according to the type of application. In an embodiment of the invention, monolaurin forms part of an “agrochemical composition”, which is a topical foliar composition in the form of a liquid or a gel. In an embodiment of the invention, monolaurin forms part of an “agrochemical composition”, which is a liquid stem or root injectable and/or microinjectable composition. The term “liquid” composition covers solutions, suspensions, emulsions, liposomes, micelles, colloids, particles (microparticles and nanoparticles). The term “solid” composition covers powder (soluble or dispersible powder), granules (including dispersible or soluble granules) and/or capsules.

In an embodiment of the invention, monolaurin forms part of an “agrochemical composition” selected from ready-to-use composition or concentrated composition. The ready-to-use composition covers compositions that are in such a form that they are appropriate for direct use in agriculture. However, the concentrated compositions require a subsequent step for dilution until the biostimulant effective amount of monolaurin is achieved.

In an embodiment, monolaurin forms part of an “agrochemical composition” adsorbed and/or absorbed in a solid support. In an embodiment, monolaurin forms part of an “agrochemical composition” adsorbed and/or absorbed in a solid support which is selected from the group consisting of a mineral-based support, a polymeric based support and/or an organic-based support. In an embodiment, monolaurin forms part of an “agrochemical composition” absorbed in a solid support, wherein the solid support is a mineral-based support selected from the group consisting of silica, bentonite, sepiolite, active carbon, diatomaceous earth, and a combination thereof. In an embodiment, monolaurin forms part of an “agrochemical composition” absorbed in a solid support, wherein the solid support is a polymeric-based support selected from the group consisting of starch polymers, cellulose polymers, lignin polymers, chitosan and nanochitosan polymers, polyacrylamide, polyvinyl alcohols, polycaprolactone, polylactic acid, polyacrylate, polyhydroxyalkanoate, polyvinylacetate. In an embodiment, monolaurin forms part of an “agrochemical composition” absorbed in a solid support, wherein the solid support is an organic-based support selected from the group consisting of compost, manure, lignin, cellulose, chitosan, humic acids, fulvic acids, treatment plants sludge, sawdust, straw and organic slurries and wastes from biomass industries.

In an embodiment of the invention, monolaurin forms part of an “agrochemical composition”, as defined herein above, as a unique active ingredient for stimulating the plant immune system against biotic and abiotic stress.

In an embodiment of the invention, monolaurin forms part of an “agrochemical composition”, as defined herein above, further comprising one or more additional active ingredients. In an embodiment of the invention, monolaurin forms part of an “agrochemical composition”, as defined herein above, further comprising one or more additional active ingredients selected from the group consisting of pesticides, antifungal agents, antibacterial agents, essential oils, biostimulants, amino acid-based compounds and mixtures thereof.

In an embodiment of the invention, monolaurin forms part of an “agrochemical composition”, as defined herein above, further comprising one or more active biostimulant ingredients selected from the group consisting of humic and fulvic acids, protein hydrolysates and other N-containing compounds, sea weed extracts and botanicals, chitosan and other biopolymers, inorganic compounds, beneficial fungi, beneficial bacteria and mixtures thereof.

In an embodiment, the use monolaurin is for stimulating the plant immune system, against biotic and abiotic stress, and the application comprises one or more additional applications. In an embodiment, the use of monolaurin is for stimulating the plant immune system, against biotic and abiotic stress, and the application comprises a first application and at least one additional application at a daily, weekly, monthly, or annual frequency after the first application.

The agriculture compositions comprising a biostimulant effective amount of monolaurin used in this invention can be prepared according to methods well known in the state of the art. The appropriate method and conditions can readily be determined by those skilled in the art according to the type of formulation and method of application.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the severity of Xcv infection in pepper plants treated with monolaurin.

Figure 2 illustrates the impact of drought conditions in biomass of tomato plants treated with monolaurin (foliar application of 1%, 0,5% and 0,1% solutions; soil application of 40 mg/L, 20 mg/L and 5 mg/L solutions).

Figure 3 shows a comparison of tomato plant biomass under drought conditions with different monolaurin treatments (foliar application of 1%, 0,5% and 0,1 % solutions; soil application of 40 mg/L, 20 mg/L and 5 mg/L solutions).

DESCRIPTION OF EMBODIMENTS

The following examples are provided by way of illustration, and they are not intended to be limiting of this invention. Furthermore, this invention covers all possible combinations of particular and preferred embodiments described herein.

1. Immune system stimulation in tomato plants.

1.1. Plants.

Tomato seeds (Rio Grande variety) were placed in rock wool inert substrate (GrodanOPIugs), germinated and grown (25 ± 2 °C and 16 h of daylight, 18 ± 2 °C and 8 h of darkness, and relative humidity of 50%). Three weeks after seeding (two cotyledon phenological stage), the plants were transferred to pieces of rock wool measuring 7.5x7.5x6.5 cm (GrodanODelta), that were previously dipped in nutritive solution [Epsomita (MgSO4 49 %) and Hakaphos® (NPK 15:15:10)]. The tomato plants were acclimatized in a greenhouse until they reached 15 cm high (about 7 weeks from seeding). Fungicide and bactericide treatments were avoided in order to exclude interferences with the experiment. Plants were kept in the described conditions and watered with nutritive solution every two days throughout the entire experiment.

1.2. Method and sampling.

Monolaurin (99% purity, including 1 % of free glycerol, diglyceride, and triglyceride) was dissolved in 96% ethanol with a concentration of 2% by weight. The experiment consisted of two treatments, one active application with monolaurin (2% by weight) in ethanol and one control with no activity (CNT-OH), consisting of distilled water and ethanol.

The active solution was prepared by dissolving 1 g of monolaurin in 5 ml of pure ethanol. Once the monolaurin was dissolved, 45 ml of distilled water was added. No active control was prepared by mixing 5 ml of pure ethanol in 45 ml of distilled water in order to exclude the potential interference of ethanol in the experiment results. Both treatments were applied using foliar spraying until droplet fall. The experiment design consisted of three repetitions with three plants each (nine plants per treatment).

Leaf samples were collected 24 hours after the application of the treatments and immediately immersed in liquid nitrogen to avoid the overexpression of defence genes as a consequence of the wound caused by cutting off the leaves to be analyzed. Total mRNA was obtained using PureLink® Plant RNA Reagent kit extraction and treated with DNAsa enzyme to eliminate possible genomic DNA contaminants. RNA was quantified using a Nanodrop N-200 UV-Vis spectrophotometer (Thermo Scientific) and its integrity was verified by analyzing an aliquot of RNA sample on denaturing agarose gel, stained with ethidium bromide. The total RNA was stored at -80 °C. This RNA was used as a template for cDNA (complementary DNA) synthesis by reverse transcription (RT-PCR) and used to quantify the expression levels of selected defence genes by qPCR, using Sybr®Green reagent, and compared to the untreated control (CNT-OH). RT-qPCR quantifies the amplification product by fluorescence. The amount of fluorescence is proportional to the amount of product generated, this fluorescence is monitored during each PCR cycle and the analysis generates the Ct value (cycle threshold), which means the cycle in which the first significant detectable increase of fluorescence occurs. The Ct values obtained for each treatment and each repetition were used to estimate the ratio of the expression level of the target defence genes with regard to the endogenous reference gene (actin gene, which is constitutively expressed and has constant expression levels in all cells and invariant in the different study conditions). Relative gene expression levels were quantified, using the Ct values for each gene studied, by AACt method. In addition, the data was analyzed with a second method, the Pfaffl method, which is similar to AACt, but corrected for the efficiencies of the different genes, using Rest 2009 analysis software (Pfaffl, M. W., 2001 , Nucleic Acids Res. 29(9): e45).

The nine described marker genes (plus one reference gene) were chosen as defence markers of different pathways related to defence induction in tomato plants. The gene encoding actin protein was used as a reference in the Pfaffl method.

The criterion of the present invention considers as "gene overexpression" an expression value twice the value expressed in the control plants. However, other studies consider as "overexpressed gene" those with a value 1.2 times higher. 1.3. Results.

The application of the product monolaurin (99% purity, 2% in weight) on tomato plants by foliar spraying until droplet fall showed an increase in relative gene expression in the nine studied genes, 24 h after treatment. These results were validated using the two methodologies described above (AACt Method and Pfaffl method), as described in Table 1.

Table 1. Gene overexpression in tomato plants after monolaurin application using two methodologies (AACt Method and Pfaffl method).

1.4. Conclusions.

Treatment of tomato plants by foliar spraying with a single application of the product monolaurin (2%) produced significant overexpression of genes encoding polyphenol oxidase (PPO), endoglucanase (GluB), harpin (Harp), endochitinase (CHI3 and ChiEt), Blue Copper Protein (BCP), Osmotin (0sm2) and pathogenesis-related proteins (PR1A, Sub1). These results confirm an astonishing overexpression of key genes in the plant immune system by way of monolaurin application. The expression of the mentioned genes in plants triggers a cascade of cross-talking signalling paths, through different biomolecules (proteins, hormones, etc), with the capability of activating several defensive strategies against adverse environmental conditions and/or a biological attack by pathogens, pests and herbivores. Therefore, the use of monolaurin stimulates the plant immune system to elicit a response against biotic and abiotic stress.

2. Immune system stimulation in bell biotic stress.

The efficacy of monolaurin biostimulation was evaluated in bell pepper plants (Capsicum annuum - sweet Italian pepper, Batlle variety) against the biotic stress caused by the infection of phytopathogenic bacteria Xanthomonas campestris pv. Vesicatoria 206 (Xcv). 2.1. In vitro bactericidal activity assay.

In vitro monolaurin antibacterial activity against Xcv was evaluated in order to exclude potential bactericidal activity of monolaurin. The determination of the bactericidal activity was performed by means of the so-called “killing assay” with the suspension of the bacteria in liquid medium.

The commercially available 1 g of monolaurin capsule was dissolved in ethanol following the instructions of the supplier for sample concentrations of 250 pg/ml. For comparative purposes, a commercial bactericide agent, ZZ Cuprocol, was also tested. ZZ Curpocol was provided as a suspension (Sygenta) comprising copper oxychloride at 70% w/v and was tested at a concentration of 0.75 ml/L or 1.5 ml/L. In addition, several negative control samples (which do not promote any bactericide effect) were also included. Comparative samples are disclosed herein below:

-Comparative control 1 wherein monolaurin was replaced with distillate water, -Comparative control-OH 2 wherein monolaurin was replaced with ethanol.

The method comprised mixing 10OpI of the appropriate concentration of the sample to be tested with 1 OOpI of the bacterial suspension at a stock concentration of 2x10⁸ CFU/ml, obtaining a final volume of 200pl in each well of the microplate (bacterial suspension at a final concentration of approximately 10⁸ CFU/ml). The multi-well plates were incubated at 28°C under constant agitation (150 rpm). After 30 and 120 minutes (min) of exposure of the bacterial strain to the different concentrations, samples were taken, and their survival (CFU/ml) was analyzed by counting viable cells in agar. In this particular embodiment, 250 pg/ml of monolaurin and 1 .5 ml/l of ZZ Cuprocol were tested.

The colony-forming units were counted after 24 hours of incubation forXcv, and the survival (CFU/ml) was compared with control samples. Three samples for each concentration and time were carried out. The results are shown in table 2.

Table 2. Antibacterial activity of monolaurin against Xcv by means of the so-called “killing assay” method. Therefore, the monolaurin does not display any bactericidal effect in Xcv at bactericidal concentrations of 250 pg/ml.

2.2. In vivo biostimulant activity assay in bell peppers.

2.2.1. Method and sampling.

The plant material used for the experiment were bell pepper seeds of the Dulce Italiano variety (Batlle). These seeds were sown in alveoli for germination and, once they presented the first two true leaves, were transferred to 750 ml containers. The plants were fertilized once a week with a solution of 200 ppm NPK (20:10:20). The use of fungicides and bactericides was avoided to exclude interference in the development of the experiment. Plants were maintained in pots using standard growing conditions, in a controlled environment in terms of temperature, relative humidity and light (25 ± 2°C and 16 hours of light, 20 ± 2°C and 8 hours of darkness). The experiment was started two to three weeks after transfer (4 true leaves were present) and once the plants were acclimatized to greenhouse conditions.

Two treatments were carried out: monolaurin at a concentration of 2% and an untreated control (CNT). The monolaurin and CNT solutions were prepared as in point 1.2. For the experimental design, nine plants were treated with each product, consisting of three replicates with three plants per replicate.

A curative strategy was followed for the product efficacy evaluation in the control of Xcv infection in bell pepper plants. This strategy consisted of inoculating the plants with the pathogen and, one hour after inoculation, monolaurin solution (2%) was applied. Inoculation of the pathogen and application of the product was done by foliar spraying.

Disease levels were determined 7 and 12 days after pathogen inoculation (dapi) by assigning an index of infection intensity (severity index) with values from 0 to 4, depending on the percentage of leaf surface affected: 0, no symptoms; 1 , 1-25% affected surface; 2, 26-50% affected surface; 3, 51-75% affected surface; and 4, 76-100% affected surface. Between 4 and 5 leaves were evaluated per plant.

2.2.2. Results.

Table 3 and Figure 1 shows the results for the efficacy of the assay. At 7 and 12 dapi, plants curatively treated with monolaurin showed a significant reduction in infection severity compared to the untreated control, with a reduction in the severity index of 58% and 37%, respectively. Table 3. Severity of Xcv infection in pepper plants treated with monolaurin.

Unlike the CNT control, plants treated with monolaurin (2%) showed an increase in the severity index at 7 and 12 dapi. Since this situation occurred in treated plants, it is possible to conclude that Xcv infection was not suppressed nor controlled, thus neither bactericide nor bacteriostatic effects can be claimed. Moreover, it is well known that pathogen Xcv translocates about 30 type-3 effector proteins into pepper plants (Capsicum annuum) to suppress plant immune responses, resulting in a delayed immune response. Hence, the low level of infection in treated plants has been elicited by early plant immune retaliation. Therefore, the use of monolaurin stimulates the plant immune system to elicit a defensive response against Xcv infection, and this response achieves a reduction in the severity index at 7 and 12 dapi.

2.3. Conclusions.

These results confirm the stimulation of the immune system in Bell Pepper plants to elicit a defensive response against Xcv bacterial infection by means of monolaurin application. Once bactericide and/or bacteriostatic effects were ruled out, the defensive strategies of treated plants, triggered by monolaurin application, display more efficient results against infection than natural strategies of Systemic Acquired Resistance in non-treated plants. Therefore, genetic overexpression, caused by monolaurin biostimulation, develops an enhanced defensive capacity against pathogen infections.

3. Stimulation of immune system defences against abiotic stress in tomato plants.

The efficacy of monolaurin biostimulation was evaluated in tomato plants (Solanum lycopersicum L, Tres Cantos Zaragozano variety) against the abiotic stress caused by drought. The experiment was based in a biostimulants screening method for crop seedlings under water deficit stress developed by Jimenez-Arias et col, where the final biomass weight of the seedlings (dry weight) is the variable used to compare and study the effect of biostimulants (Jimenez-Arias, D.; Morales-Sierra, S.; Borges, A. A.; Herrera, A. J.; Luis, J.C. New Biostimulants Screening Method for Crop Seedlings under Water Deficit Stress. Agronomy 2022, 72, 728). 3.1. Plants.

Eight standard 54-cell tomato seedling trays were bought at a local commercial crop nursery. Tomato seeds were sown in the trays, using an automatic sowing machine to ensure germination and growth uniformity, until the two true-leaf stage (two weeks). Then, seedling trays were transferred to a greenhouse with controlled conditions (temperature 20-27°C, photoperiod 16-8 h, humidity 60-75%) and plants were acclimatized in this greenhouse during one week, watered with 50 ml of half-strength Hoagland solution per day (Hoagland's No. 2 Basal Salt Mixture, Sigma-Aldrich), divided in two dosages of 25 ml. Fungicide and bactericide treatments were avoided in order to exclude interferences with the assay.

3.2. Method and sampling processing.

The experiment consisted in eight treatments:

1) active foliar application with monolaurin (1 % in weight) in ethanol,

2) active foliar application with monolaurin (0,5% in weight) in ethanol,

3) active foliar application with monolaurin (0,1 % in weight) in ethanol,

4) active soil application with monolaurin (40 mg/L) in temperate water,

5) active soil application with monolaurin (20 mg/L) in temperate water,

6) active soil application with monolaurin (5 mg/L) in temperate water,

7) one plant control with optimal watered plants (CNT-W), and

8) one plant control with drought stressed plants (CNT-D).

In foliar application, monolaurin and CNT-OH solutions were prepared as point 1.2. and diluted with mixture of water/ethanol in same proportion. All treatments were applied by foliar spraying till droplet fall.

The experiment was performed for seven days after the acclimatization period in greenhouse. The CNT-W plants were watered with 50 ml of half-strength Hoagland solution per day (two dosages of 25 ml), CNT-D and the rest of treatments were watered with 25 ml of half-strength Hoagland solution per day (two dosages of 12,5 ml). In soil application, the amount of monolaurin (40, 20 and 5 mg) was added to one litre of half-strength Hoagland solution at 50°C. Then, 5 ml of these solutions were mixed with 20 ml of half-strength Hoagland solution (35°C) and added throughout the first five days of the trial to seedling.

The experimental design involved 50 plants in each control and treatment. After trial, the plants were cut at soil level, dried in an oven at 75°C for 4 days and weighed. The statistical average of seedlings weight was compared.

3.3. Results.

Figure 2 and figure 3 show the results for the efficacy of the assay. Figure 2 shows that CNT-W seedlings reached 306,1 mg of average weigh, while CNT-D plants reached 224,5 mg. It means a reduction of biomass average weight about 27% regards the CNT-W due to drought conditions. All the treated trials showed more biomass that CNT-D, therefore it suggested a greater efficiency in managing drought conditions.

As Figure 3 displays, the highest concentration soil application (40 mg/L) reduced 11 % the seedling biomass weight compared to CNT-W, so it means almost 21% more biomass than CNT-D. The second monolaurin application (20 mg/L) followed this tendency and the most diluted application (5 mg/L) still showed an enhancement regarding to CNT-D.

Furthermore, foliar treatment also demonstrated some efficiency against drought stress in seedlings. The highest concentration foliar application (1 %) increased about 12% the biomass weight with regards to CNT-D. The second and third monolaurin foliar applications (0,5% and 0,1 %) still showed an enhancement regarding to CNT-D.

3.4. Conclusions.

These results confirm the stimulation of the immune system in tomato plants to elicit a defensive response against drought by means of monolaurin application. The defensive strategies of treated plants, triggered by monolaurin application, display more efficient results against drought stress than natural strategies in untreated plants. Therefore, the genetic overexpression caused by monolaurin biostimulation results in an enhanced defensive capacity against abiotic stresses.

4. Compositions

4.1. Liquid compositions

4.1.1. Foliar liquid compositions comprising monolaurin.

Reference liquid compositions for the foliar application comprising monolaurin are indicated below.

Table 4 discloses the ingredients, their function and their amount expressed in percent by weight in relation to the total weight of the composition.

Table 4. Example of foliar liquid composition comprising monolaurin. (1) Monolaurin 90% contains 10% of free glycerol, diglyceride, and triglyceride

(2) Agri-pure™ AP-406: plant-based rapeseed oil methyl ester marketed by Cargill; any other vegetable oil suitable for agriculture could be used instead.

This formulation was dispersed in water at a ratio of 0.5 litre formulation/hectolitre of water (0.5% v/v) and, after stirring at 1000 rpm, kept stable for more than 120 minutes. The mixture was carried out at 50°C.

Concentrated foliar liquid composition

A concentrated dispersion in water comprising the formulation in Table 4 was prepared by, firstly, mixing all the ingredients listed in the table above; and secondly, dispersing the resulting mixture in water at a ratio of 0.1-5 litre of formulation per hectolitre of water (resulting in a final concentration of 0.1-5% v/v) and, after strong stirring at 1000 rpm for 5 minutes, kept stable for more than 120 minutes. The mixture was carried out at 50°C.

4.1.2. Foliar liquid compositions comprising monolaurin and other active ingredients.

Reference liquid compositions for foliar application comprising monolaurin and additional active active ingredients are indicated below.

Liquid compositions comprising monolaurin and herb essential oil (bactericidal).

A reference liquid composition for foliar application comprising monolaurin and at least one essential oil is indicated below. Origanum essential oil can be replaced with any other essential oil.

Table 5 indicates the ingredients, their function and their amount expressed as percent by weight in relation to the total weight of the composition.

Table 5. Example of foliar liquid composition comprising monolaurin and Origanum essential oil as bactericidal active ingredient.

(1) Monolaurin 90% contains 10% of free glycerol, diglyceride, and triglyceride

(2) Agri-pure™ AP-406: plant-based rapeseed oil methyl ester marketed by Cargill; any other vegetable oil suitable for agriculture could be used instead. A diluted aqueous dispersion comprising the formulation in Table 5 was prepared following the processes disclosed in the previous section adding the herb essential oil for preparing the first mixture of ingredients.

Liquid compositions comprising monolaurin and l-a-amino acid liquid biostimulant. A reference liquid composition for foliar application comprising monolaurin and at least one liquid biostimulant compound is indicated below. Any other biostimulant disclosed in the state of the art can be used.

Table 6 discloses the ingredients, their function and their amount expressed as percent by weight in relation to the total weight of the composition.

Table 6. Example of foliar liquid composition comprising monolaurin and l-a-amino acid as biostimulant active ingredient.

(1) Monolaurin 90% contains 10% of free glycerol, diglyceride, and triglyceride

(3) TerraSorb®: l-a-amino acid_biostimulant for foliar application marketed by Bioiberica; any other liquid biostimulant suitable for agriculture could be used instead.

A diluted aqueous dispersion comprising the formulation in Table 6 was prepared following the processes disclosed in the previous section adding the antifungal agent for preparing the first mixture of ingredients.

4.1.3. Nanoemulsion liquid foliar composition

Monolaurin can be presented in nanoemulsions as well as other kinds of nano-formulation. The nano-formulation is in the form of an oil-in-water nanoemulsion comprising monolaurin. The droplet size was between 200-400 nm measured using nanoparticle tracking analysis (NTA). Table 7 indicates the ingredients, their function and their amount expressed in grams.

Table 7. Example of liquid nanocomposition comprising monolaurin.

(1) Monolaurin 90% contains 10% of free glycerol, diglyceride, and triglyceride

Monolaurin was mixed with the soy lecithin at 50°C by way of strong stirring (4000 rpm) for 5 minutes. On the other hand, water was mixed with the surfactant at 50°C by way of strong stirring (4000 rpm) for 5 minutes. Both solutions were mixed at 50°C by way of strong stirring (4000 rpm) for 10 minutes. After that, the mixture was sonicated with an ultrasound probe (Hielscher UP200st, 26 kHz) for 12 minutes at 50°C. The temperature was controlled during the ultrasound step using a cooling jacket, and the resulting mixture in nano dispersion form was stable for at least three days.

4.2. Solid compositions

4.2.1. Solid compositions for soil sprinkling application

Exemplary compositions of a mineral solid absorption composition for soil sprinkling application is disclosed below. Instead of silica, other minerals can be used such as bentonite, sepiolite, active carbon, diatomaceous earth and/or combinations thereof.

Table 8 discloses the ingredients, their function and their amount expressed as percent by weight in relation to the total weight of the composition.

Table 8. Example of solid composition for soil sprinkling comprising monolaurin. (1) Monolaurin 90% contains 10% of free glycerol, diglyceride, and triglyceride Monolaurin was added as spray at 70°C to a rotary drum where the mixture of silica (IQE D300/IQE Ibersil D100) was already placed. The drum rotated at 30 rpm.

4.2.3. Compositions in combination with solid active compound for soil sprinkling application.

Exemplary composition of solid absorption composition with other active compounds for sprinkling application is indicated below. Instead of humic-fulvic acids, other compounds can be used as fertilizers, manure, dry seaweed, compost and/or combinations thereof, among others.

Table 9 discloses the ingredients, their function and their amount expressed as percent by weight in relation to the total weight of the composition.

Table 9. Example of solid composition for soil sprinkling comprising monolaurin in combination with solid biostimulant compound.

(1) Monolaurin 90% contains 10% of free glycerol, diglyceride, and triglyceride.

(2) Kation™ H-65: solid humic-fulvic acid concentrated marketed by Nutrelic with 60% by weight of humic acid and 5% by weight of fulvic acids; any other humic and/or fulvic acid compound suitable for agriculture could be used instead.

Monolaurin was added as a spray at 70°C to a rotary drum where the mixture of silica (IQE D300/IQE Ibersil D100) and Kation H-65 was already placed. The drum rotated at 30 rpm.

Claims

1 . Use of an effective amount of monolaurin for stimulating the plant immune system against biotic and abiotic stress.

2. The use according to claim 1 , wherein the abiotic stress is caused by drought, salinity, heat, cold, chilling, freezing, nutrient, high light intensity, ozone, heavy metal stresses and combinations thereof.

3. The use according to any one of claims 1 to 2, wherein the biotic stress is caused by bacteria, viruses, fungi, parasites, nematodes, beneficial and harmful insects, herbivorous animals and combinations thereof.

4. The use according to any one of claims 1 to 3, wherein the plants belong to the solanaceae family.

5. The use according to any one of claims 1 to 4, wherein the effective amount of monolaurin is from 0,001 mg to 5.000 mg per plant.

6. The use according to any one of claims 1 to 5, wherein the effective amount of monolaurin is from 0,01 mg to 2.000 mg per plant or, optionally, from 0,1 to 1.000 mg per plant.

7. The use according to any one of claims 1 to 6, wherein monolaurin forms part of an agrochemical composition, comprising a biostimulant effective amount of monolaurin and one or more agriculture acceptable excipients and/or carriers.

8. The use according to claim 7, wherein the agrochemical composition comprises from 0,01 to 99% by weight of monolaurin.

9. The use according to any one of claims 7 to 8, wherein the agrochemical composition comprises from 15-75% by weight of monolaurin, optionally, from 20-50% by weight of monolaurin; further optionally from 35-45%.

10. The use according to any one of claims 7 to 9, wherein the agrochemical composition further comprises at least one additional active ingredient.

11. The use according to claim 10, wherein the at least one additional active ingredient is selected from the group consisting of pesticides, antifungal agents, antibacterial agents, essential oils, amino acid-based compounds, fertilizers, biostimulants and mixtures thereof.

12. The use according to any one of claims 1 to 11 , wherein the stimulation of the plant immune system comprises applying monolaurin, or alternatively the agrochemical composition, in a form selected from the group consisting of liquids, solids, and gels.

13. The use according to any one of claims 1 to 12, wherein the stimulation of the plant immune system comprises applying monolaurin, or alternatively the agrochemical composition, in a foliar application, a stem application, a stem injection, a root application, a root injection, a soil application, or a combination thereof.

14. The use according to any one of claims 1 to 13, wherein the stimulation of the plant immune system involves at least one application of a biostimulant effective amount of monolaurin and/or an agrochemical composition comprising it.