WO2022074176A1 - Method for eliciting at least one defense mechanism in plants against plant pests and plant diseases - Google Patents

Method for eliciting at least one defense mechanism in plants against plant pests and plant diseases Download PDF

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Publication number
WO2022074176A1
WO2022074176A1 PCT/EP2021/077812 EP2021077812W WO2022074176A1 WO 2022074176 A1 WO2022074176 A1 WO 2022074176A1 EP 2021077812 W EP2021077812 W EP 2021077812W WO 2022074176 A1 WO2022074176 A1 WO 2022074176A1
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plant
fatty acid
spp
plants
composition
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PCT/EP2021/077812
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French (fr)
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Marie-Laure FAUCONNIER
Estelle DEBOEVER
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Université de Liège
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/42Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing within the same carbon skeleton a carboxylic group or a thio analogue, or a derivative thereof, and a carbon atom having only two bonds to hetero atoms with at the most one bond to halogen, e.g. keto-carboxylic acids

Definitions

  • the present invention relates to a method and a composition for eliciting at least one defense mechanism in plants against plant pests and plant diseases.
  • the present invention is also suitable for protecting plants against plant pests and plant diseases.
  • the present invention also relates to the use of such a composition.
  • Elicitors are one of the emerging alternatives and are defined as molecules able to stimulate defense responses in a host plant. They can have a biological origin, derived from plants or microbes, or even being synthetized. In the past decades, different types of elicitors have been reported, including carbohydrate polymers, lipids, and peptides. Their recognition by plants results in the activation of various signal transduction or early defense mechanisms. Indeed, even if they do not have an advanced immune system as animals, plants show resistance against diseases and damages caused by various organisms. This resistance is mainly based on innate cell immunity and systemic signaling from the site of infection. Like for animals, plant immune responses are divided into two components: the innate immunity or pre-existing basal defense, and the plant-induced immunity.
  • MAMPs microbial- or pathogen-associated molecular patterns
  • PAMP-triggered immunity prevents further pathogen invasion and activates many signaling cascades.
  • the second step happens in the cell and rely on the fact that certain pathogens manage to bypass this first line of defense. They produce effectors (virulence proteins) which are indicators of potential pathogens recognized by R proteins in the plant cell and activate the effector- triggered immunity (ETI).
  • ETI effector- triggered immunity
  • ETI hypersensitive response
  • SAR systemic acquired resistance
  • ISR induced systemic resistance
  • ROS superoxide anion
  • H2O2 hydrogen peroxide
  • hydroxyl radical •OH
  • ROS oxidative hydrolysis
  • RBOH respiratory burst oxidase homologs
  • cell wall peroxidases are the main producer of apoplastic ROS in early stages of biotic interactions. ROS production is proof of successful recognition of infection or elicitors, and activation of plant defenses, especially the PTI.
  • compositions for eliciting at least one defense mechanism in plants against plant pests and plant diseases as an efficient alternative to eliciting compositions or elicitors used nowadays, while being non-phytotoxic.
  • compositions for eliciting at least one defense mechanism in plants against plant pests and plant diseases and for protecting said plants against said plant pests and plant diseases are also an object of the invention to provide a composition for eliciting at least one defense mechanism in plants against plant pests and plant diseases and for protecting said plants against said plant pests and plant diseases.
  • a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases comprising:
  • composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
  • an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates
  • a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases and for protecting said plants against said plant pests and plant diseases comprising: - applying to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant, an effective and substantially non-phytotoxic amount:
  • composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
  • an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates
  • the present invention is also about a method suitable for protecting plants against plant pests and plant diseases, said method comprising:
  • composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
  • an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates
  • the present invention is also about a method suitable for eliciting at least one defense mechanism in plants against plant pests and plant diseases, said method comprising:
  • composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
  • an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates
  • the terms "effective and non- phytotoxic amount” mean an amount of the elicitor or an amount of the composition according to the present invention that is sufficient to induce control or destruction of the plant pests present or liable to appear on the plants, and that does not have a phytotoxicity impact for said plants.
  • the method according to the invention allows obtaining elicitation of at least one defense mechanism in plants against plant pests selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants.
  • the method according to the invention allows obtaining protection of plants against plant pests selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants
  • composition and/or the elicitor according to the invention in particular for the method according to the invention, is applied/is used before harvest or post-harvest to the whole plant, the leaves, the flowers, fruits, seeds on the plant, seedlings pricking out, plants pricking out, by spraying, drenching, soaking, dipping, injection or administration through fertilizing or irrigation systems.
  • said composition and/or said elicitor is/are exogenous.
  • exogenous refers to the fact that the composition and/or the elicitor has/have an origin external from the plant meaning that said composition and/or said elicitor are external facto rs/agents regarding the plant.
  • said composition and/or said elicitor is/are applied exogenously.
  • this means the composition and/or the elicitor has/have an origin external from the plant meaning that said composition and/or said elicitor are external facto rs/agents regarding the plant, said external factors/agents being applied to the plant, in particular being applied to an aerial part of said plant, more particularly being applied to leaves of said plant.
  • the terms “aerial part of said plant” as used herein refer to the structures of a plant that are above ground, including but not limited to the stems, leaves, petioles, flowers, fruits and seeds on the plants. In other words, the terms “aerial part of said plant” as used herein refer to parts of plant which are partially or completely exposed in air.
  • said composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or of said elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates is applied in agricultural and horticultural applications (for example in fields/cultures), to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant.
  • said composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or of said elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates is actively applied, in particular actively applied in agricultural and horticultural applications (for example in fields/cultures), to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant.
  • compositions for eliciting at least one defense mechanism in plants against plant pests and plant diseases comprising at least one fatty acid hydroperoxide and/or at least one of its derivates.
  • compositions for eliciting at least one defense mechanism in a plant against plant pests and plant diseases comprising at least one fatty acid hydroperoxide and/or at least one of its derivates.
  • compositions for eliciting at least one defense mechanism in plants against plant pests and plant diseases and for protecting said plants against said plant pests and plant diseases comprising at least one fatty acid hydroperoxide and/or at least one of its derivates.
  • elicitor refers to an inducer of the plant immune system, i.e. to molecules able to stimulate defense responses in a (host) plant against plant pests and plant diseases.
  • fatty acid hydroperoxides and their derivates for example their degradation products such as hydroxy fatty acids
  • elicitors i.e. are molecules able to stimulate defense responses in a (host) plant against plant pests and plant diseases.
  • plant pests mean any species, strain or biotype of plant, animal, or pathogenic agent injurious to plants, for example fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants.
  • plant pests include all plant pathogens, i.e. all biological organisms that can cause disease symptoms and/or plant diseases and/or significantly reduce the productivity, quality, and even cause the death of plants.
  • plant diseases mean anything that prevents a plant from performing to its maximum potential, notably in terms of development and productivity.
  • the term “derivates” means any molecules derivatising from a fatty acid hydroperoxide, for example any degradation products derivatising from a fatty acid hydroperoxide, in particular a hydroxy fatty acid derivatising from a fatty acid hydroperoxide.
  • the term "for protecting plant” refer to the activation of mechanisms aimed at controlling or reducing the pests and/or to minimize their effects on the plant. Plant protection can be for example achieved by killing the pests, by delaying their growth and/or reproduction or by reducing sporulation.
  • HPOs Fatty acid hydroperoxides
  • HPOs have emerged as a promising (bio-)pesticides against plant pests (including plant pathogens)
  • Plant pathogens including plant pathogens
  • biocide have been studied on plant pathogenic bacteria
  • Deboever, E. et al. Linolenic fatty acid hydroperoxide acts as biocide on plant pathogenic bacteria : biophysical investigation of the mode of action. Bioorg. Chem. 2020, 100, 1-25) and on fungal pathogens (Graner, G. et al., Screening of oxylipins for control of oilseed rape (Brassica napus) fungal pathogens. Phytochemistry 2003, 63, 89-95).
  • the present invention is about a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases, meaning that the method allows obtaining elicitation/stimulation of at least one defense response in a (host) plant against plant pests and plant diseases, i.e. allows obtaining elicitation/stimulation of at least one defense mechanism in a (host) plant against plant pests and plant diseases.
  • the method according to the invention comprises applying at least one molecule able to elicit/stimulate defense responses in a (host) plant against plant pests and plant diseases.
  • said at least one molecule able to elicit/stimulate defense response(s) in a host plant against plant pests and plant diseases is a fatty acid hydroperoxide and/or at least one of its derivates.
  • the present invention is about an eliciting or elicitor composition meaning that the composition is able to stimulate at least one defense response in a (host) plant against plant pests and plant diseases, i.e. to stimulate at least one defense mechanism in a (host) plant against plant pests and plant diseases, more particularly that the composition comprises at least one molecule able to stimulate defense responses in a (host) plant against plant pests and plant diseases.
  • said at least one molecule able to stimulate defense response(s) in a host plant against plant pests and plant diseases is a fatty acid hydroperoxide and/or at least one of its derivates.
  • a method according to the invention comprising applying to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant, an effective and substantially non-phytotoxic amount: • of a composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
  • an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates; allows obtaining elicitation of at least one defense mechanism in said plant against plant pests and plant diseases, even if said composition and/or said elicitor is applied to at least one aerial part of said plant, in particular to leaves of said plant. Indeed, even if applied to at least one aerial part of said plant, in particular to leaves of said plant, said composition and/or said elicitor demonstrates an efficient eliciting activity (i.e.
  • the method according to the present invention allows an effective elicitation/stimulation of at least one defense mechanism in plants against plant pests and plant diseases, said method allowing a large-scale treatment in fields, meaning that the method allows an easy, massive, and rapid treatment of plants in fields while reducing at the maximum the quantity (volume) of the composition or of the compound to be applied.
  • the method according to the invention comprising applying to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant, an effective and substantially non-phytotoxic amount:
  • composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
  • an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates
  • ISR induced systemic resistance
  • ROS production oxidative burst peaks
  • composition according to the invention comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, demonstrates an efficient eliciting activity (i.e. eliciting properties) on/in plants against plant pests and plant diseases, meaning that the composition according to the present invention acts as an effective elicitor by eliciting at least one defense mechanism in plants against plant pests and plant diseases, while being not phytotoxic.
  • compositions according to the invention comprising at least one fatty acid hydroperoxide and/or at least one of its derivates:
  • ISR induced systemic resistance
  • ROS production oxidative burst peaks
  • said at least one fatty acid hydroperoxide is chosen in the group consisting of 9-hydroperoxy-10,12,15-octadecatrienoic acid (9-HPOT), 13-hydroperoxy- 9,1 1 ,15-octadecatrienoic acid (13-HPOT), 9-hydroperoxy- 10,12-octadecadienoic acid (9-HPOD), 13-hydroperoxy- 9,1 1 -octadecadienoic acid (13-HPOD), and mixtures thereof.
  • said at least one fatty acid hydroperoxide is of general formula (I):
  • R2 is C8H15O2 or C12H19O2
  • said at least one of the derivates of said at least one fatty acid hydroperoxide is a hydroxy fatty acid.
  • said hydroxy fatty acid is chosen in the group consisting of 9-hydroxy-10,12,15-octadecatrienoic acid (9-HOT), 13-hydroxy- 9,1 1 ,15-octadecatrienoic acid (13-HOT), 9-hydroxy-10,12-octadecadienoic acid (9-HOD), 13-hydroxy-9,1 1 -octadecadienoic acid (13-HOD), and mixtures thereof.
  • said hydroxy fatty acid is of general formula (II):
  • R2 is C8H15O2 or C12H19O2
  • said at least one fatty acid hydroperoxide and/or said at least one of its derivates, in particular said hydroxy fatty acid is/are present in the composition at a concentration ranging from 0.01 pM (0.32 ppm) to 10000 pM (3200 ppm), preferably at a concentration of 100 pM (32 ppm).
  • said at least one fatty acid hydroperoxide and/or said at least one of its derivates, in particular said hydroxy fatty acid is/are present in the composition at a concentration ranging from 20 pM to 5000 pM, preferably at a concentration ranging from 50 pM to 1000 pM, more preferably at a concentration ranging from 75 pM to 200 pM.
  • the composition according to the invention further comprises a solvent and/or a co-formulant selected from the group comprising detergents, emulsifiers, dispersing agents, anti-foaming agents, penetration enhancers, humectants, ionic or non-ionic wetting agents, anti-freeze agents, preservative agents such as antioxidants (for example carotenoids and/or polyphenols and/or vitamin E), absorbent agents, thickeners, buffers, sticker agents, diluents, and mixtures thereof, preferably a surfactant selected from the group comprising: detergents, emulsifiers, dispersing agents, anti-foaming agents, penetration enhancers, humectants or ionic or non-ionic wetting agents, and mixtures thereof.
  • a solvent and/or a co-formulant selected from the group comprising detergents, emulsifiers, dispersing agents, anti-foaming agents, penetration enhancers, humectants
  • composition according to the invention in particular for the method according to the invention, can contain additional components, such as co-formulants, to obtain a product with good handling, efficacy and stability properties.
  • co-formulant designates any substance other than the elicitor component according to the invention, that is intentionally added to the elicitor/eliciting composition according to the invention.
  • the composition according to the invention in particular for the method according to the invention, can comprise a surfactant, i.e. a compound that lowers the surface tension of a liquid, allowing easier spreading.
  • the surfactant can be a detergent, an emulsifier (including alkyl polyglucosides glycerol ester or polyoxyethylene (20) sorbitan monolaurate), a dispersing agent (including sodium chloride, potassium chloride, potassium nitrate, calcium chloride or starch of corn), a foaming agent (including derivates of tartric acid, malic acid or alcohols), a penetration enhancer, a humectant (including ammonium sulfate, glycerin or urea) or a wetting agent of ionic or non-ionic type or a mixture of such surfactants.
  • a surfactant i.e. a compound that lowers the surface tension of a liquid, allowing easier spreading.
  • the surfactant can be a detergent,
  • penetration enhancer mean a compound that accelerates the uptake of active ingredient through the cuticle of a plant into the plant, i.e. the rate of uptake, and/or increases the amount of active ingredient absorbed into the plant.
  • Classes of substances known as penetration enhancers include alkyl phosphates, such as tributyl phosphate and tripropyl phosphate, and naphthalenesulphonic acid salts.
  • dispersing agent means a substance added to a suspension, usually a colloid, to improve the separation of particles and to prevent settling or clumping.
  • emulsifier means a substance that stabilizes an emulsion, i.e. a mixture of two or more liquids. Mention can be made of the emulsifier sold under the trade names Tween® 20, which essentially comprises polyoxyethylene (20) sorbitan monolaurate (polysorbate 20).
  • Tween® 20 which essentially comprises polyoxyethylene (20) sorbitan monolaurate (polysorbate 20).
  • the composition according to the invention in particular for the method according to the invention, can comprise one or more other active compounds/or substances selected from the group comprising herbicides, insecticides, plant growth regulators or other plant immune system elicitors, and mixtures thereof.
  • the composition according to the invention in particular for the method according to the invention, is under the form of granules, tablets, powders, liquids, (micro-) emulsions, nano-formulations, (micro-) encapsulates, (water-soluble) concentrates, (concentrated) suspensions, (concentrated) dispersions, wettable granulates and powders or aerosols.
  • the present invention is also about an elicitor for eliciting at least one defense mechanism in plants against plant pests and plant diseases , said elicitor being a fatty acid hydroperoxide and/or a derivate of said fatty acid hydroperoxide.
  • the present invention is also about an elicitor for eliciting at least one defense mechanism in a plant against plant pests and plant diseases, said elicitor being a fatty acid hydroperoxide and/or a derivate of said fatty acid hydroperoxide.
  • the present invention is also about an elicitor suitable for protecting plants against plant pests and plant diseases.
  • said at least one fatty acid hydroperoxide is chosen in the group consisting of 9-hydroperoxy- 10,12,15-octadecatrienoic acid (9-HPOT), 13-hydroperoxy-9,1 1 ,15- octadecatrienoic acid (13-HPOT), 9-hydroperoxy-10,12-octadecadienoic acid (9- HPOD), 13-hydroperoxy-9,1 1 -octadecadienoic acid (13-HPOD), and mixtures thereof.
  • said at least one fatty acid hydroperoxide is of general formula (I):
  • R2 is C8H15O2 or C12H19O2
  • said at least a derivate of said at least one fatty acid hydroperoxide is a hydroxy fatty acid.
  • said hydroxy fatty acid is chosen in the group consisting of 9-hydroxy-10,12,15- octadecatrienoic acid (9-HOT), 13-hydroxy-9,1 1 ,15-octadecatrienoic acid (13- HOT), 9-hydroxy-10,12-octadecadienoic acid (9-HOD), 13-hydroxy-9,1 1 - octadecadienoic acid (13-HOD), and mixtures thereof.
  • said hydroxy fatty acid is of general formula (II):
  • R2 is C8H15O2 or C12H19O2
  • the elicitor according to the invention is under the form of granules, tablets, powders, liquids, (micro-) emulsions, nanoformulations, (micro-) encapsulates, (water-soluble) concentrates, (concentrated) suspensions, (concentrated) dispersions, wettable granulates and powders or aerosols.
  • the present invention is also about the use of a composition and/or the use of an elicitor according to the invention to elicit at least one defense mechanism in plants against plant pests and plant diseases, in particular in agricultural and horticultural applications.
  • the present invention is also about the use of a composition and/or the use of an elicitor according to the invention to elicit at least one defense mechanism in a plant against plant pests and plant diseases, in particular in agricultural and horticultural applications.
  • composition and/or the elicitor according to the invention is used to elicit at least one defense mechanism in plants against plant pests and plant diseases.
  • composition and/or the elicitor according to the invention is used against plant pests selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants.
  • Non-limiting examples of phytopathogenic fungi and fungal-like organisms that can be targeted by the compositions according to the present invention include Pyricularia spp.; Puccinia spp.; Erysiphe spp.; Cochliobolus spp.; Helminthosporium spp.; Drechslera spp.; Rhynchosporium spp.; Cercospora spp.; Botrytis spp.; Alternaria spp.; Venturia spp.; Cladosporium spp.; Monilinia spp.; Didymella spp.; Phoma spp.; Aspergillus spp., Aureobasidium spp.; Ascochyta spp.; Stemphylium spp.; Pleospora spp.; Peronospora spp.; Pythium spp.; Phytophthora spp.;
  • Plant diseases caused by fungi that can be targeted by the compositions according to the present invention include notably yeasts, rusts, smuts, mildews, molds, mushrooms and toadstools.
  • Non-limiting examples of phytopathogenic bacteria that can be targeted by the compositions according to the present invention include the genii Erwinia, Pseudomonas, Xanthomonas, and Ralstonia.
  • Non-limiting examples of phytopathogenic viruses that can be targeted by the compositions according to the present invention include Cucumber Mosaic Virus, Barley Yellow Mosaic Virus, Strawberry Mild Yellow Edge Virus, Strawberry Latent Ringspot Virus, Beet Necrotic Yellow Vein Virus and Potato Virus Y.
  • Non-limiting examples of phytopathogenic insects that can be targeted by the compositions according to the present invention include notably aphids, beetles, bugs, hoppers, locusts, mites, ants, ticks, trips, whiteflies, rootworms, maggots, weevils, (stem)borers, caterpillars, butterflies, leaf-rolers, and leaf-miners.
  • the present invention is also about a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases comprising:
  • the present invention is also about a method for eliciting at least one defense mechanism in a plant against plant pests and plant diseases comprising:
  • the present invention is also about a method suitable for protecting plants against plant pests and plant diseases comprising:
  • the terms "effective and non- phytotoxic amount” mean an amount of the elicitor or an amount of the composition according to the present invention that is sufficient to induce control or destruction of the plant pests present or liable to appear on the plants, and that does not have a phytotoxicity impact for said plants.
  • the method according to the invention allows obtaining elicitation of at least one defense mechanism in plants against plant pests selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants.
  • the method according to the invention allows obtaining protection of plants against plant pests selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants
  • composition and/or the elicitor according to the invention in particular for the method according to the invention, is applied/is used before harvest or post-harvest to the whole plant, the leaves, the flowers, fruits, seeds on the plant, seedlings or seedlings pricking out, propagation material such as tubers or rhizomes, plants pricking out, by spraying, drenching, soaking, dipping, injection or administration through fertilizing or irrigation systems.
  • composition according to the invention in particular for the method according to the invention, can be ready to be applied to the plant by means of a suitable device, such as a spraying device, or can be the commercial concentrated compositions which have to be diluted before application to the plant.
  • the composition and/or the elicitor according to the invention in particular for the method according to the invention, is/are from natural, synthetic, or biosynthetic origin. More specifically, said at least one fatty acid hydroperoxide and/or said at least one derivate of said at least one fatty acid hydroperoxide is/are from natural, synthetic, or biosynthetic origin.
  • Fig.1 shows the distribution of qualitative scores of lesions on HPO- treated and DMSO-treated control plants inoculated with Botrytis cinerea. 1 , no symptoms; 2, lesions smaller than 0.5 cm; 3, lesions larger than 0.5 cm; 4, beginning of sporulation on lesions.
  • Fig.2 shows the early defense responses detection induced by HPOs in A. thaliana cells suspensions.
  • A, B, C Production of ROS by controls and HPOs treated cells.
  • A Kinetics of ROS production for 13-HPOT (grey), 13- HPOD (dark grey) and control (black).
  • B Mean area under H2O2 production curves for 90 min measurements.
  • C Production of ROS by positive control (COS-OGA 25 ppm), 13-HPOD (100 pM) and 13-HPOT (100 pM) treated cells in presence of DPI and SHAM inhibitors.
  • Fig.3 shows the early defense responses detection induced by HPOs in planta.
  • B Kinetics of ROS production for 13- HPOT (dark grey), 13-HPOD (light grey), FytoSave® 25 ppm (black) and control (dashed).
  • HPOs fatty acid hydroperoxides
  • 13-HPOs was enzymatically synthetized from the reaction of LOX- 1 on linoleic (13-HPOD) or linolenic acid (13-HPOT) as described in Deboever, E. et al. (Linolenic fatty acid hydroperoxide acts as biocide on plant pathogenic bacteria : biophysical investigation of the mode of action. Bioorg. Chem. 2020, 100, 1-25), in Deleu, M. et al. (Linoleic and linolenic acid hydroperoxides interact differentially with biomimetic plant membranes in a lipid specific manner. Colloids Surfaces B Biointerfaces 2019, 175, 384-391 ) and in Fauconnier, M.L.
  • ISR was induced in A. thaliana according the procedure described in Ongena, M. et al. (Systemic induction of phytoalexins in cucumber in response to treatments with fluorescent pseudomonads. Plant Pathol. 2000, 49, 523-530). Seeds were sterilized with ethanol and bleach solutions before being rinsed with sterile water multiple times. Afterwards, seeds were dispersed in square Petri Dish filled with agar medium (6-8 g/L) and left overnight in the dark before placing them in growing chambers under lights. After one week, seedling was transferred in sterilized araponics filled with hydroponic solution (5 mL/10 L) and placed in growing chamber under lights.
  • Photoautotrophic cell suspensions from A. thaliana strain Landsberg erecta ecotype were cultured on a rotary shaker at 100 rpm, in MS medium (4.4 g/L) with 0.5 mg/L naphthalene acetic acid, 0.05 mg/L kinetin, pH 5.7 and maintained with approximately 2% CO2 in the atmosphere under a 16/8 h light/dark photoperiod, at 24°C. H2O2 production was assessed using luminol-dependent-chemiluminescence on seven-day-old autotrophic A.
  • thaliana cell suspensions directly after the addition of the elicitors (FytoSave® as a positive control; 13-HPOD; and 13-HPOT) in the growing medium using the method described by Baker and Mock (see Baker et al., A method to detect oxidative stress by monitoring changes in the extracellular antioxidant capacity in plant suspension cells. Physiol. Mol. Plant Pathol. 2004, 64, 255-261 ). Luminescence was monitored for 1 h30 with relative light unit (RLU) measurements every 3 min. 8 technical replicates were carried out for each treatment tested and two independent measurements were carried out.
  • RLU relative light unit
  • A. thaliana plants were grown as described by Smith and Heese (Smith, J.M. et aL, Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae. Plant Methods 2014, 10, 1-9).
  • measurements were performed on 5-mm disks prepared from leaves of 4-weeks plants using a method adapted from Smith and Heese. The day before the experiment, disks were placed in water in a 96 wells plate. The day of measurements, the water was replaced by 150pL of treatment solutions (20 pg.rnL’ 1 horseradish peroxidase, 0.2 mM luminol and HPO) were added to the plate.
  • THSD Tukey Honest Significant Differences
  • ROS production is a biphasic process with apparition of a first transient phase within minutes after the infection and, then, a second phase more intense and sustained that can last for many hours/days.
  • This first wave linked to the activation of the early defense responses was the target in order to investigate whether HPOs are perceived by plants and could induce an immune response.
  • the elicitor FytoSave® was used as positive control as COS-OGA (active substance) is known to induce a significant production of ROS at a concentration of 25 ppm mainly via the activation of parietal peroxidases (see van Aubel, G. et al., Plant immunity induced by COS-OGA elicitor is a cumulative process that involves salicylic acid. Plant Sci. 2016, 247, 60-70; van Aubel, G. et al., Oligosaccharides successfully thwart hijacking of the salicylic acid pathway by Phytophthora infestans in potato leaves. Plant Pathol. 2018, 67, 1901-191 1 ; Ledoux, Q. et al., Specific localization and measurement of hydrogen peroxide in Arabidopsis thaliana cell suspensions and protoplasts elicited by COS-OGA. Plant Signal. Behav. 2014, 2324).

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Abstract

The present invention relates to a method and a composition for eliciting at least one defense mechanism in plants against plant pests and plant diseases.

Description

METHOD FOR ELICITING AT LEAST ONE DEFENSE MECHANISM IN PLANTS AGAINST PLANT PESTS AND PLANT DISEASES
Field of the invention
The present invention relates to a method and a composition for eliciting at least one defense mechanism in plants against plant pests and plant diseases. The present invention is also suitable for protecting plants against plant pests and plant diseases. The present invention also relates to the use of such a composition.
Background of the invention
Since chemical pesticides have been shown to be harmful to human health and biodiversity in general, considerable research has been done to find more environmentally friendly plant protection solutions. Elicitors are one of the emerging alternatives and are defined as molecules able to stimulate defense responses in a host plant. They can have a biological origin, derived from plants or microbes, or even being synthetized. In the past decades, different types of elicitors have been reported, including carbohydrate polymers, lipids, and peptides. Their recognition by plants results in the activation of various signal transduction or early defense mechanisms. Indeed, even if they do not have an advanced immune system as animals, plants show resistance against diseases and damages caused by various organisms. This resistance is mainly based on innate cell immunity and systemic signaling from the site of infection. Like for animals, plant immune responses are divided into two components: the innate immunity or pre-existing basal defense, and the plant-induced immunity.
In the first part, plants perceive and recognize molecules shared by many microbes even non-pathogens, so called microbial- or pathogen- associated molecular patterns (MAMPs or PAMPs). This specific process, also known as the PAMP-triggered immunity (PTI), prevents further pathogen invasion and activates many signaling cascades. The second step happens in the cell and rely on the fact that certain pathogens manage to bypass this first line of defense. They produce effectors (virulence proteins) which are indicators of potential pathogens recognized by R proteins in the plant cell and activate the effector- triggered immunity (ETI). Afterwards, cell death around the infected area is generally observed in order to preserve the spread of the infection to other parts of the plant, also known as the hypersensitive response (HR) which is characteristic of ETI. While PTI can be compared to the first line of a battlefield, ETI acts as a second phase of the plant immunity, referring to the well-known “zigzag model” of the plant defense system. In both cases, the receptor and effector molecules play key roles in their activation and execution.
Once the plant survives infection at a site, it usually acquires increased resistance to future attacks and is transmitted to other parts and the whole plant through the phloem via signal molecules. This phenomenon is known as the systemic acquired resistance (SAR). On the other hand, plants can also exhibit this type of systemic resistance when facing non-pathogenic microorganisms such as rhizobacteria. This is called induced systemic resistance (ISR) which is initiated by the microorganism itself and activates a protective mechanism over time rather than an immediate defense mechanism in plants.
Over the last decades, characteristic physiological phenomenon, known as early events, were shown to be involved in plant immunity, particularly during PTI. These multiple and complex pathways lead to the development of an effective response. One of the earliest defense responses is the quick production of ROS (superoxide anion (02* ), hydrogen peroxide (H2O2) and hydroxyl radical (•OH)) at the pathogen infection site, also known as the oxidative “burst”. In general, membrane-bound NADPH oxidases also called respiratory burst oxidase homologs (RBOH) and cell wall peroxidases are the main producer of apoplastic ROS in early stages of biotic interactions. ROS production is proof of successful recognition of infection or elicitors, and activation of plant defenses, especially the PTI. For instance, it has been shown that when the elongation factor Tu and flagellin (flg22), two well-known elicitors, are recognized by their associated PRRs (pathogen related proteins), RBOHD (respiratory burst oxidase homolog form D) is directly activated to ensure a rapid extracellular oxidative burst.
In view of the above, it is of interest to stimulate/elicit the plant immune system against plant pests and plant diseases. In particular, it is of interest to stimulate/elicit the plant immune system in order to control diseases notably caused by plant pests such as fungi, oomycetes, bacteria, viruses, nematodes and insects.
More particularly, it is of interest to have available a method for stimulating/eliciting the plant immune system against plant pests and plant diseases. Even more particularly, it is of interest to have available a method for stimulating/eliciting the plant immune system in order to control diseases notably caused by plant pests such as fungi, oomycetes, bacteria, viruses, nematodes and insects.
Summary of the invention
It is an object of the invention to provide a composition for eliciting at least one defense mechanism in plants against plant pests and plant diseases.
It is an object of the invention to provide a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases, said method being intended for a large-scale treatment in fields, meaning that the method allows an easy, massive, and rapid treatment of plants in fields while reducing at the maximum the quantity (volume) of the composition or of the compound to be applied.
It is also an object of the invention to provide a composition suitable for protecting plants against plant pests and plant diseases.
It is also an object of the invention to provide a method suitable for protecting plants against plant pests and plant diseases, said method being intended for a large-scale treatment in fields, meaning that the method allows an easy, massive, and rapid treatment of plants in fields while reducing at the maximum the quantity (volume) of the composition or of the compound to be applied.
More particularly, it is an object of the invention to provide a composition for eliciting at least one defense mechanism in plants against plant pests and plant diseases, as an efficient alternative to eliciting compositions or elicitors used nowadays, while being non-phytotoxic.
More particularly, it is an object of the invention to provide a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases, as an efficient alternative to methods used nowadays, while being non-phytotoxic, said method being intended for a large-scale treatment in fields, meaning that the method allows an easy, massive, and rapid treatment of plants in fields while reducing at the maximum the quantity (volume) of the composition or of the compound to be applied.
More particularly, it is also an object of the invention to provide a composition for eliciting at least one defense mechanism in plants against plant pests and plant diseases and for protecting said plants against said plant pests and plant diseases.
More particularly, it is also an object of the invention to provide a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases and for protecting said plants against said plant pests and plant diseases, said method being intended for a large-scale treatment in fields, meaning that the method allows an easy, massive, and rapid treatment of plants in fields while reducing at the maximum the quantity (volume) of the composition or of the compound to be applied.
To this end, according to the invention, there is provided a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases, the method comprising:
- applying to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant, an effective and substantially non-phytotoxic amount:
• of a composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
• of an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates; and
- obtaining elicitation of at least one defense mechanism in said plant against plant pests and plant diseases.
More particularly, according to the invention, there is provided a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases and for protecting said plants against said plant pests and plant diseases, the method comprising: - applying to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant, an effective and substantially non-phytotoxic amount:
• of a composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
• of an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates; and
- obtaining elicitation of at least one defense mechanism in said plant against plant pests and plant diseases and obtaining protection of said plants against said plant pests and plant diseases.
The present invention is also about a method suitable for protecting plants against plant pests and plant diseases, said method comprising:
- applying to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant, an effective and substantially non-phytotoxic amount:
• of a composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
• of an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates; and
- obtaining protection of said plants against said plant pests and plant diseases.
The present invention is also about a method suitable for eliciting at least one defense mechanism in plants against plant pests and plant diseases, said method comprising:
- applying to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant, an effective and substantially non-phytotoxic amount:
• of a composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
• of an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates; and
- obtaining elicitation of at least one defense mechanism in said plant against plant pests and plant diseases. In the context of the present invention, the terms "effective and non- phytotoxic amount" mean an amount of the elicitor or an amount of the composition according to the present invention that is sufficient to induce control or destruction of the plant pests present or liable to appear on the plants, and that does not have a phytotoxicity impact for said plants.
Preferably, the method according to the invention allows obtaining elicitation of at least one defense mechanism in plants against plant pests selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants.
Preferably, the method according to the invention allows obtaining protection of plants against plant pests selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants
Advantageously, the composition and/or the elicitor according to the invention, in particular for the method according to the invention, is applied/is used before harvest or post-harvest to the whole plant, the leaves, the flowers, fruits, seeds on the plant, seedlings pricking out, plants pricking out, by spraying, drenching, soaking, dipping, injection or administration through fertilizing or irrigation systems.
Advantageously, for the method according to the invention, said composition and/or said elicitor is/are exogenous.
The term “exogenous” as used herein refers to the fact that the composition and/or the elicitor has/have an origin external from the plant meaning that said composition and/or said elicitor are external facto rs/agents regarding the plant.
In this sense, preferably and according to the method of the present invention, said composition and/or said elicitor is/are applied exogenously. In the sense of the present invention, this means the composition and/or the elicitor has/have an origin external from the plant meaning that said composition and/or said elicitor are external facto rs/agents regarding the plant, said external factors/agents being applied to the plant, in particular being applied to an aerial part of said plant, more particularly being applied to leaves of said plant. The terms “aerial part of said plant” as used herein refer to the structures of a plant that are above ground, including but not limited to the stems, leaves, petioles, flowers, fruits and seeds on the plants. In other words, the terms “aerial part of said plant” as used herein refer to parts of plant which are partially or completely exposed in air.
Advantageously, according to the present invention, said composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or of said elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates is applied in agricultural and horticultural applications (for example in fields/cultures), to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant.
Advantageously, according to the present invention, said composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or of said elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates is actively applied, in particular actively applied in agricultural and horticultural applications (for example in fields/cultures), to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant.
According to the invention, there is provided a composition for eliciting at least one defense mechanism in plants against plant pests and plant diseases, said composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates.
According to the invention, there is provided a composition for eliciting at least one defense mechanism in a plant against plant pests and plant diseases, said composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates.
More particularly, according to the invention, there is provided a composition for eliciting at least one defense mechanism in plants against plant pests and plant diseases and for protecting said plants against said plant pests and plant diseases, said composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates.
The term "elicitor" as used herein refers to an inducer of the plant immune system, i.e. to molecules able to stimulate defense responses in a (host) plant against plant pests and plant diseases. In the context of the present invention, fatty acid hydroperoxides and their derivates (for example their degradation products such as hydroxy fatty acids) are elicitors, i.e. are molecules able to stimulate defense responses in a (host) plant against plant pests and plant diseases.
In the context of the present invention, based on the FAO definition of “pest” in terms of the International Plant Protection Convention and phytosanitary measures worldwide (FAO, 1990; revised FAO, 1995; IPPC, 1997), the terms “plant pests” mean any species, strain or biotype of plant, animal, or pathogenic agent injurious to plants, for example fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants. In this sense, the terms “plant pests” include all plant pathogens, i.e. all biological organisms that can cause disease symptoms and/or plant diseases and/or significantly reduce the productivity, quality, and even cause the death of plants.
In the context of the present invention, the terms “plant diseases” mean anything that prevents a plant from performing to its maximum potential, notably in terms of development and productivity.
In the context of the present invention, the term “derivates” means any molecules derivatising from a fatty acid hydroperoxide, for example any degradation products derivatising from a fatty acid hydroperoxide, in particular a hydroxy fatty acid derivatising from a fatty acid hydroperoxide.
In the context of the present invention, the term "for protecting plant" refer to the activation of mechanisms aimed at controlling or reducing the pests and/or to minimize their effects on the plant. Plant protection can be for example achieved by killing the pests, by delaying their growth and/or reproduction or by reducing sporulation.
Fatty acid hydroperoxides (HPOs) are amphiphilic molecules naturally produced by plants among (a)biotic stress by the oxidative catabolism of polyunsaturated fatty acids. They belong to the large family of plant oxylipins.
Recently, HPOs have emerged as a promising (bio-)pesticides against plant pests (including plant pathogens) (Prost, I. et al., Evaluation of the Antimicrobial Activities of Plant Oxylipins Supports Their Involvement in Defense against Pathogens. Plant Physiol. 2005, 139, 1902-1913) and their exogenous applications as biocide have been studied on plant pathogenic bacteria (Deboever, E. et al., Linolenic fatty acid hydroperoxide acts as biocide on plant pathogenic bacteria : biophysical investigation of the mode of action. Bioorg. Chem. 2020, 100, 1-25) and on fungal pathogens (Graner, G. et al., Screening of oxylipins for control of oilseed rape (Brassica napus) fungal pathogens. Phytochemistry 2003, 63, 89-95).
In other words, the present invention is about a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases, meaning that the method allows obtaining elicitation/stimulation of at least one defense response in a (host) plant against plant pests and plant diseases, i.e. allows obtaining elicitation/stimulation of at least one defense mechanism in a (host) plant against plant pests and plant diseases.
More particularly, the method according to the invention comprises applying at least one molecule able to elicit/stimulate defense responses in a (host) plant against plant pests and plant diseases. Specifically, in the context of the present invention, said at least one molecule able to elicit/stimulate defense response(s) in a host plant against plant pests and plant diseases is a fatty acid hydroperoxide and/or at least one of its derivates.
The present invention is about an eliciting or elicitor composition meaning that the composition is able to stimulate at least one defense response in a (host) plant against plant pests and plant diseases, i.e. to stimulate at least one defense mechanism in a (host) plant against plant pests and plant diseases, more particularly that the composition comprises at least one molecule able to stimulate defense responses in a (host) plant against plant pests and plant diseases. Specifically, in the context of the present invention, said at least one molecule able to stimulate defense response(s) in a host plant against plant pests and plant diseases is a fatty acid hydroperoxide and/or at least one of its derivates.
In the context of the present invention, it was surprisingly determined that a method according to the invention comprising applying to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant, an effective and substantially non-phytotoxic amount: • of a composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
• of an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates; allows obtaining elicitation of at least one defense mechanism in said plant against plant pests and plant diseases, even if said composition and/or said elicitor is applied to at least one aerial part of said plant, in particular to leaves of said plant. Indeed, even if applied to at least one aerial part of said plant, in particular to leaves of said plant, said composition and/or said elicitor demonstrates an efficient eliciting activity (i.e. eliciting properties) on/in plants against plant pests and plant diseases, meaning that the method according to the present invention allows an effective elicitation/stimulation of at least one defense mechanism in plants against plant pests and plant diseases, said method allowing a large-scale treatment in fields, meaning that the method allows an easy, massive, and rapid treatment of plants in fields while reducing at the maximum the quantity (volume) of the composition or of the compound to be applied.
Indeed, in the context of the present invention, it was shown unexpectedly that the method according to the invention comprising applying to at least one of a part of a plant being an aerial part of said plant, in particular to leaves of a plant, an effective and substantially non-phytotoxic amount:
• of a composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
• of an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates;
- allows to obtain a significant disease reduction following the treatment with fatty acid hydroperoxides or with their derivates as they could decrease the size of the lesions;
- allows to obtain induced systemic resistance (ISR) effect;
- induce oxidative burst peaks (ROS production) occurring later and being longer and more intense than elicitors used nowadays. Knowing that this ROS production is one of the ways of quantifying the level of perception of an elicitor by plants, it reflects that plants largely perceive the stimuli induced by fatty acid hydroperoxides and their derivates as elicitors.
It was surprisingly determined that a composition according to the invention, comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, demonstrates an efficient eliciting activity (i.e. eliciting properties) on/in plants against plant pests and plant diseases, meaning that the composition according to the present invention acts as an effective elicitor by eliciting at least one defense mechanism in plants against plant pests and plant diseases, while being not phytotoxic.
Indeed, in the context of the present invention, it was shown unexpectedly that compositions according to the invention comprising at least one fatty acid hydroperoxide and/or at least one of its derivates:
- allow to obtain a significant disease reduction following the treatment with fatty acid hydroperoxides or with their derivates as they could decrease the size of the lesions;
- have an induced systemic resistance (ISR) effect;
- induce oxidative burst peaks (ROS production) occurring later and being longer and more intense than elicitors used nowadays. Knowing that this ROS production is one of the ways of quantifying the level of perception of an elicitor by plants, it reflects that plants largely perceive the stimuli induced by fatty acid hydroperoxides and their derivates as elicitors.
The invention is defined by the independent claims. The dependent claims define advantageous embodiments.
Preferably, according to the invention, in particular for the method according to the invention, said at least one fatty acid hydroperoxide is chosen in the group consisting of 9-hydroperoxy-10,12,15-octadecatrienoic acid (9-HPOT), 13-hydroperoxy- 9,1 1 ,15-octadecatrienoic acid (13-HPOT), 9-hydroperoxy- 10,12-octadecadienoic acid (9-HPOD), 13-hydroperoxy- 9,1 1 -octadecadienoic acid (13-HPOD), and mixtures thereof.
In particular, according to the invention, in particular for the method according to the invention, said at least one fatty acid hydroperoxide is of general formula (I):
R1-CHOOH-R2 (I) wherein Ri is C5H11 or C5H9 or C9H15 or C9H13
R2 is C8H15O2 or C12H19O2
Advantageously, according to the invention, in particular for the method according to the invention, said at least one of the derivates of said at least one fatty acid hydroperoxide is a hydroxy fatty acid.
Preferably, according to the invention, in particular for the method according to the invention, said hydroxy fatty acid is chosen in the group consisting of 9-hydroxy-10,12,15-octadecatrienoic acid (9-HOT), 13-hydroxy- 9,1 1 ,15-octadecatrienoic acid (13-HOT), 9-hydroxy-10,12-octadecadienoic acid (9-HOD), 13-hydroxy-9,1 1 -octadecadienoic acid (13-HOD), and mixtures thereof.
In particular, according to the invention, in particular for the method according to the invention, said hydroxy fatty acid is of general formula (II):
R1-CHOH-R2 (II) wherein R1 is C5H11 or C5H9 or C9H15 or C9H13
R2 is C8H15O2 or C12H19O2
Advantageously, in particular for the method according to the invention, said at least one fatty acid hydroperoxide and/or said at least one of its derivates, in particular said hydroxy fatty acid, is/are present in the composition at a concentration ranging from 0.01 pM (0.32 ppm) to 10000 pM (3200 ppm), preferably at a concentration of 100 pM (32 ppm).
Preferably, in particular for the method according to the invention, said at least one fatty acid hydroperoxide and/or said at least one of its derivates, in particular said hydroxy fatty acid, is/are present in the composition at a concentration ranging from 20 pM to 5000 pM, preferably at a concentration ranging from 50 pM to 1000 pM, more preferably at a concentration ranging from 75 pM to 200 pM.
Eventually, in particular for the method according to the invention, the composition according to the invention further comprises a solvent and/or a co-formulant selected from the group comprising detergents, emulsifiers, dispersing agents, anti-foaming agents, penetration enhancers, humectants, ionic or non-ionic wetting agents, anti-freeze agents, preservative agents such as antioxidants (for example carotenoids and/or polyphenols and/or vitamin E), absorbent agents, thickeners, buffers, sticker agents, diluents, and mixtures thereof, preferably a surfactant selected from the group comprising: detergents, emulsifiers, dispersing agents, anti-foaming agents, penetration enhancers, humectants or ionic or non-ionic wetting agents, and mixtures thereof.
Indeed, the composition according to the invention, in particular for the method according to the invention, can contain additional components, such as co-formulants, to obtain a product with good handling, efficacy and stability properties. The term "co-formulant" designates any substance other than the elicitor component according to the invention, that is intentionally added to the elicitor/eliciting composition according to the invention.
The composition according to the invention, in particular for the method according to the invention, can comprise a surfactant, i.e. a compound that lowers the surface tension of a liquid, allowing easier spreading. The surfactant can be a detergent, an emulsifier (including alkyl polyglucosides glycerol ester or polyoxyethylene (20) sorbitan monolaurate), a dispersing agent (including sodium chloride, potassium chloride, potassium nitrate, calcium chloride or starch of corn), a foaming agent (including derivates of tartric acid, malic acid or alcohols), a penetration enhancer, a humectant (including ammonium sulfate, glycerin or urea) or a wetting agent of ionic or non-ionic type or a mixture of such surfactants.
The terms "penetration enhancer" mean a compound that accelerates the uptake of active ingredient through the cuticle of a plant into the plant, i.e. the rate of uptake, and/or increases the amount of active ingredient absorbed into the plant. Classes of substances known as penetration enhancers, include alkyl phosphates, such as tributyl phosphate and tripropyl phosphate, and naphthalenesulphonic acid salts.
The term "dispersing agent" means a substance added to a suspension, usually a colloid, to improve the separation of particles and to prevent settling or clumping.
The term "emulsifier" means a substance that stabilizes an emulsion, i.e. a mixture of two or more liquids. Mention can be made of the emulsifier sold under the trade names Tween® 20, which essentially comprises polyoxyethylene (20) sorbitan monolaurate (polysorbate 20). Eventually, the composition according to the invention, in particular for the method according to the invention, can comprise one or more other active compounds/or substances selected from the group comprising herbicides, insecticides, plant growth regulators or other plant immune system elicitors, and mixtures thereof.
Advantageously, the composition according to the invention, in particular for the method according to the invention, is under the form of granules, tablets, powders, liquids, (micro-) emulsions, nano-formulations, (micro-) encapsulates, (water-soluble) concentrates, (concentrated) suspensions, (concentrated) dispersions, wettable granulates and powders or aerosols.
The present invention is also about an elicitor for eliciting at least one defense mechanism in plants against plant pests and plant diseases , said elicitor being a fatty acid hydroperoxide and/or a derivate of said fatty acid hydroperoxide.
The present invention is also about an elicitor for eliciting at least one defense mechanism in a plant against plant pests and plant diseases, said elicitor being a fatty acid hydroperoxide and/or a derivate of said fatty acid hydroperoxide.
The present invention is also about an elicitor suitable for protecting plants against plant pests and plant diseases.
Preferably, for an elicitor according to the invention, said at least one fatty acid hydroperoxide is chosen in the group consisting of 9-hydroperoxy- 10,12,15-octadecatrienoic acid (9-HPOT), 13-hydroperoxy-9,1 1 ,15- octadecatrienoic acid (13-HPOT), 9-hydroperoxy-10,12-octadecadienoic acid (9- HPOD), 13-hydroperoxy-9,1 1 -octadecadienoic acid (13-HPOD), and mixtures thereof.
In particular, for an elicitor according to the invention, said at least one fatty acid hydroperoxide is of general formula (I):
R1-CHOOH-R2 (I) wherein R1 is C5H11 or C5H9 or C9H15 or C9H13
R2 is C8H15O2 or C12H19O2 Advantageously, for an elicitor according to the invention, said at least a derivate of said at least one fatty acid hydroperoxide is a hydroxy fatty acid.
Preferably, for an elicitor according to the invention, said hydroxy fatty acid is chosen in the group consisting of 9-hydroxy-10,12,15- octadecatrienoic acid (9-HOT), 13-hydroxy-9,1 1 ,15-octadecatrienoic acid (13- HOT), 9-hydroxy-10,12-octadecadienoic acid (9-HOD), 13-hydroxy-9,1 1 - octadecadienoic acid (13-HOD), and mixtures thereof.
In particular, for an elicitor according to the invention, said hydroxy fatty acid is of general formula (II):
R1-CHOH-R2 (II) wherein R1 is C5H11 or C5H9 or C9H15 or C9H13
R2 is C8H15O2 or C12H19O2
Advantageously, the elicitor according to the invention is under the form of granules, tablets, powders, liquids, (micro-) emulsions, nanoformulations, (micro-) encapsulates, (water-soluble) concentrates, (concentrated) suspensions, (concentrated) dispersions, wettable granulates and powders or aerosols.
The present invention is also about the use of a composition and/or the use of an elicitor according to the invention to elicit at least one defense mechanism in plants against plant pests and plant diseases, in particular in agricultural and horticultural applications.
The present invention is also about the use of a composition and/or the use of an elicitor according to the invention to elicit at least one defense mechanism in a plant against plant pests and plant diseases, in particular in agricultural and horticultural applications.
Preferably, the composition and/or the elicitor according to the invention is used to elicit at least one defense mechanism in plants against plant pests and plant diseases.
Advantageously, the composition and/or the elicitor according to the invention is used against plant pests selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants. Non-limiting examples of phytopathogenic fungi and fungal-like organisms that can be targeted by the compositions according to the present invention include Pyricularia spp.; Puccinia spp.; Erysiphe spp.; Cochliobolus spp.; Helminthosporium spp.; Drechslera spp.; Rhynchosporium spp.; Cercospora spp.; Botrytis spp.; Alternaria spp.; Venturia spp.; Cladosporium spp.; Monilinia spp.; Didymella spp.; Phoma spp.; Aspergillus spp., Aureobasidium spp.; Ascochyta spp.; Stemphylium spp.; Pleospora spp.; Peronospora spp.; Pythium spp.; Phytophthora spp.; Rhizoctonia spp.; Sclerotinia spp.; Sclerotium spp.; Colletotrichum spp.; Mycosphaerella spp.; Diaporthe spp.; Elsinoe spp.; Verticillium spp.; Pyrenopeziza spp.; Fusarium spp.; Typhula spp.; Ustilago spp.; Urocystis spp.; Tilletia spp.; Ramularia spp.; Penicillium spp.; Acremoniella spp.; Allomyces spp.; Amorphothec spp.; Aspergillius spp.; Blastocladiella spp.; Candida spp.; Chaetomium spp.; Coccidioides spp.; Conidiobolus spp.; Coprinopsis spp.; Corynascus spp.; Cryphonectria spp., Cryptococcus spp.; Cunninghamella spp.; Curvularia spp.; Debarymyces spp.; Diplodia spp; Emericella ssp.; Encephalitozoon spp.; Eremothecium spp.; Gaeumanomyces spp.; Geomyces spp.; Gibberella spp.; Gloeophyllum spp.; Glomus spp.; Hypocrea spp.; Kluyveromyces spp.; Lentinula spp.; Leucosporidium spp.; Macrophomina spp.; Magnaportha spp.; Metharhizium spp.; Mucor spp.; Neurospora spp.; Nectria spp.; Paracocidioides spp.; Phaeopsheria spp.; Phanerochaete spp.; Phakopsora spp.; Phymatotrichum spp.; Pneumocystis spp.; Pyronema spp.; Rhizoctonia spp.; Rhizopus spp.; Saccharomyces spp.; Scerotium spp.; Spizellomyces spp.; Thermomyces spp.; Thielaviopsis spp.; Trametes spp.; Trichophyton spp.; or Yarrowia spp..
Plant diseases caused by fungi that can be targeted by the compositions according to the present invention include notably yeasts, rusts, smuts, mildews, molds, mushrooms and toadstools.
Non-limiting examples of phytopathogenic bacteria that can be targeted by the compositions according to the present invention include the genii Erwinia, Pseudomonas, Xanthomonas, and Ralstonia.
Non-limiting examples of phytopathogenic viruses that can be targeted by the compositions according to the present invention include Cucumber Mosaic Virus, Barley Yellow Mosaic Virus, Strawberry Mild Yellow Edge Virus, Strawberry Latent Ringspot Virus, Beet Necrotic Yellow Vein Virus and Potato Virus Y.
Non-limiting examples of phytopathogenic insects that can be targeted by the compositions according to the present invention include notably aphids, beetles, bugs, hoppers, locusts, mites, ants, ticks, trips, whiteflies, rootworms, maggots, weevils, (stem)borers, caterpillars, butterflies, leaf-rolers, and leaf-miners.
The present invention is also about a method for eliciting at least one defense mechanism in plants against plant pests and plant diseases comprising:
- applying an effective and substantially non-phytotoxic amount of the composition and/or of the elicitor according to the invention to at least one of a part of a plant; and
- obtaining elicitation of at least one defense mechanism in said plant against plant pests and plant diseases.
The present invention is also about a method for eliciting at least one defense mechanism in a plant against plant pests and plant diseases comprising:
- applying an effective and substantially non-phytotoxic amount of the composition and/or of the elicitor according to the invention to at least one of a part of a plant; and
- obtaining elicitation of at least one defense mechanism in said plant against plant pests and plant diseases.
The present invention is also about a method suitable for protecting plants against plant pests and plant diseases comprising:
- applying an effective and substantially non-phytotoxic amount of the composition and/or of the elicitor according to the invention to at least one of a part of a plant; and
- obtaining protection of said plant against plant pests and plant diseases.
In the context of the present invention, the terms "effective and non- phytotoxic amount" mean an amount of the elicitor or an amount of the composition according to the present invention that is sufficient to induce control or destruction of the plant pests present or liable to appear on the plants, and that does not have a phytotoxicity impact for said plants.
Preferably, the method according to the invention allows obtaining elicitation of at least one defense mechanism in plants against plant pests selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants.
Preferably, the method according to the invention allows obtaining protection of plants against plant pests selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants
Advantageously, the composition and/or the elicitor according to the invention, in particular for the method according to the invention, is applied/is used before harvest or post-harvest to the whole plant, the leaves, the flowers, fruits, seeds on the plant, seedlings or seedlings pricking out, propagation material such as tubers or rhizomes, plants pricking out, by spraying, drenching, soaking, dipping, injection or administration through fertilizing or irrigation systems.
The composition according to the invention, in particular for the method according to the invention, can be ready to be applied to the plant by means of a suitable device, such as a spraying device, or can be the commercial concentrated compositions which have to be diluted before application to the plant.
Preferably, the composition and/or the elicitor according to the invention, in particular for the method according to the invention, is/are from natural, synthetic, or biosynthetic origin. More specifically, said at least one fatty acid hydroperoxide and/or said at least one derivate of said at least one fatty acid hydroperoxide is/are from natural, synthetic, or biosynthetic origin.
Short description of the drawings
These and further aspects of the invention will be explained in greater detail by way of examples and with reference to the accompanying figures in which: Fig.1 shows the distribution of qualitative scores of lesions on HPO- treated and DMSO-treated control plants inoculated with Botrytis cinerea. 1 , no symptoms; 2, lesions smaller than 0.5 cm; 3, lesions larger than 0.5 cm; 4, beginning of sporulation on lesions.
Fig.2 shows the early defense responses detection induced by HPOs in A. thaliana cells suspensions. (A, B, C) Production of ROS by controls and HPOs treated cells. (A) Kinetics of ROS production for 13-HPOT (grey), 13- HPOD (dark grey) and control (black). (B) Mean area under H2O2 production curves for 90 min measurements. (C) Production of ROS by positive control (COS-OGA 25 ppm), 13-HPOD (100 pM) and 13-HPOT (100 pM) treated cells in presence of DPI and SHAM inhibitors.
Fig.3 shows the early defense responses detection induced by HPOs in planta. (A) Production of ROS by controls and HPOs treated foliar disks of A. thaliana. Results are expressed as mean ± standard deviation (n = 6) of the area under the H2O2 production curves. Bars with different letters are statistically different (ANOVA, Tukey’s HSD p < 0.05). (B) Kinetics of ROS production for 13- HPOT (dark grey), 13-HPOD (light grey), FytoSave® 25 ppm (black) and control (dashed).
Detailed description of embodiments of the invention and examples
In order to demonstrate fatty acid hydroperoxides (HPOs) are efficient elicitors, several experiments were performed to determine if plants are able to recognize exogenously applied HPOs via induction of plant early defense responses. Particularly, ROS production was monitored and was studied at different physiological levels since, as stated above, ROS production is proof of activation of plant defenses, especially the PTI.
The different experimental procedures and the obtained results are described hereafter.
Experimental procedures
1 . Materials
13-HPOs was enzymatically synthetized from the reaction of LOX- 1 on linoleic (13-HPOD) or linolenic acid (13-HPOT) as described in Deboever, E. et al. (Linolenic fatty acid hydroperoxide acts as biocide on plant pathogenic bacteria : biophysical investigation of the mode of action. Bioorg. Chem. 2020, 100, 1-25), in Deleu, M. et al. (Linoleic and linolenic acid hydroperoxides interact differentially with biomimetic plant membranes in a lipid specific manner. Colloids Surfaces B Biointerfaces 2019, 175, 384-391 ) and in Fauconnier, M.L. et al. (An efficient procedure for the production of fatty acid hydroperoxides from hydrolyzed flax seed oil and soybean lipoxygenase. Biotechnol. Tech. 1996, 10, 839-844). The purity (higher than 98%) was checked by high-performance liquid chromatography. Nuclear magnetic resonance and mass spectrometry were used for a full chemical characterization of the sample.
2. Induction of IS R in Arabidopsis thaliana
ISR was induced in A. thaliana according the procedure described in Ongena, M. et al. (Systemic induction of phytoalexins in cucumber in response to treatments with fluorescent pseudomonads. Plant Pathol. 2000, 49, 523-530). Seeds were sterilized with ethanol and bleach solutions before being rinsed with sterile water multiple times. Afterwards, seeds were dispersed in square Petri Dish filled with agar medium (6-8 g/L) and left overnight in the dark before placing them in growing chambers under lights. After one week, seedling was transferred in sterilized araponics filled with hydroponic solution (5 mL/10 L) and placed in growing chamber under lights. After approximately 5 weeks, the plants were transferred in 10 mL vials containing 10 mL hydroponic solutions and surrounded with aluminum foil. All plants in vials were put in small greenhouses and left for one day of adaptation before elicitation. The next day, half the plants were elicitated by putting 10 mL of hydroponic solution containing HPOs (20 mM in DMSO 1 %) in their vials. The other half were elicitated with 10 mL of hydroponic solution with 1 % DMSO. After 24 h, 4 leaves of each plant were infected with B. cinerea. One 3-pl droplet containing 2500 spores was deposited on the adaxial face of each leaf.
To determine the infection level, we used a very-used and reproducible phenotypic method (see Ongena, M. et al., Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environ. Microbiol. 2007, 9, 1084-1090; Ongena, M. et al., Systemic induction of phytoalexins in cucumber in response to treatments with fluorescent pseudomonads. Plant Pathol. 2000, 49, 523-530): 4 days after inoculation (dai) of the pathogen, the disease level was determined as the percentage of B. cinerea lesions having extended beyond the inoculum drop zone to produce spreading lesions. Three independent experiments were carried out, with 8 plants per treatment.
3. Production of H2O2 in Arabidopsis thaliana
3.1. Measurements in cell suspensions
Photoautotrophic cell suspensions from A. thaliana strain Landsberg erecta ecotype were cultured on a rotary shaker at 100 rpm, in MS medium (4.4 g/L) with 0.5 mg/L naphthalene acetic acid, 0.05 mg/L kinetin, pH 5.7 and maintained with approximately 2% CO2 in the atmosphere under a 16/8 h light/dark photoperiod, at 24°C. H2O2 production was assessed using luminol-dependent-chemiluminescence on seven-day-old autotrophic A. thaliana cell suspensions directly after the addition of the elicitors (FytoSave® as a positive control; 13-HPOD; and 13-HPOT) in the growing medium using the method described by Baker and Mock (see Baker et al., A method to detect oxidative stress by monitoring changes in the extracellular antioxidant capacity in plant suspension cells. Physiol. Mol. Plant Pathol. 2004, 64, 255-261 ). Luminescence was monitored for 1 h30 with relative light unit (RLU) measurements every 3 min. 8 technical replicates were carried out for each treatment tested and two independent measurements were carried out.
3.2. Inhibition of hydrogen peroxide production in A. thaliana cell suspensions
Like previously, photoautotrophic cell suspensions from A. thaliana were used. 20 pM DPI, an inhibitor of NADPH oxidases and of other flavin- containing oxidases that bind flavoproteins close to their NAD(P)H-binding site or 2 mM SHAM, an inhibitor of peroxidases, were added to MS medium (Murashige and Skoog medium) just before re-suspension. H2O2 production was assessed using luminol-dependent-chemiluminescence on seven-day-old autotrophic A. thaliana cell suspensions directly after the addition of the inhibitors and elicitors in the growing medium using the method described by Baker and Mock see Baker et aL, A method to detect oxidative stress by monitoring changes in the extracellular antioxidant capacity in plant suspension cells. Physiol. Mol. Plant Pathol. 2004, 64, 255-261 ).
3.3. Measurements in foliar disks
A. thaliana plants were grown as described by Smith and Heese (Smith, J.M. et aL, Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae. Plant Methods 2014, 10, 1-9). For all ROS experiments, measurements were performed on 5-mm disks prepared from leaves of 4-weeks plants using a method adapted from Smith and Heese. The day before the experiment, disks were placed in water in a 96 wells plate. The day of measurements, the water was replaced by 150pL of treatment solutions (20 pg.rnL’1 horseradish peroxidase, 0.2 mM luminol and HPO) were added to the plate.
Luminescence was monitored for 1 h30 with RLU measurements every 3 min. Data corresponded to a mean of two independent biological repetitions obtained from six foliar disks (6 technical replicates/treatment). Results are then expressed as mean ± standard deviation (n = 6) of the area under the H2O2 production curves. ROS production values were analyzed using Tukey Honest Significant Differences (THSD) test for multiple comparisons (p< 0.05).
Results
1 . In planta protective effect of HPOs against phytopathogens
Protective effects of 13-HPOD and 13-HPOT on infected plants were tested. Bioassays were conducted under controlled conditions in order to test the induction of ISR by HPOs on A. thaliana infected by B. cinerea. After 4 days, disease severity on plants roots treated with the HPO solution before B. cinerea inoculation was compared with disease severity on plants treated with DMSO (dimethylsulfoxide).
Results are presented on Figure 1 and reveal significant disease reduction following the treatment with both HPOs as they could decrease the size of the lesions. Moreover, around 40% of the plants no longer had any symptoms of the disease. This clearly shows that HPOs have an ISR effect without there being any major differences between the two forms, although structurally different.
2. Perception of HPOs and early defense responses activation
Generally, ROS production is a biphasic process with apparition of a first transient phase within minutes after the infection and, then, a second phase more intense and sustained that can last for many hours/days. This first wave linked to the activation of the early defense responses was the target in order to investigate whether HPOs are perceived by plants and could induce an immune response.
Cultured cell suspensions have been demonstrated to be a valuable model system for studying elicitor-induced defense reactions in plants and they offer the possibility to study easily early signaling events like oxidative burst see Jogaiah, S. et al., Role of Oomycete Elicitors in Plant Defense Signaling. In Bioactive Molecules in Plant Defense; Springer International Publishing, 2019; pp. 59-74 ISBN 9783030271657). Here, A. thaliana cell suspensions were used to detect the production of H2O2 in plants pretreated with HPOs. The monitoring of extracellular ROS accumulation was performed using the luminol-based experiment (see Monnier, N. et al., Rhamnolipids From Pseudomonas aeruginosa Are Elicitors Triggering Brassica napus Protection Against Botrytis cinerea Without Physiological Disorders. Front. Plant Sci. 2018, 9, 1-14; van Aubel, G. et al., Plant immunity induced by COS-OGA elicitor is a cumulative process that involves salicylic acid. Plant Sci. 2016, 247, 60-70).
The elicitor FytoSave® was used as positive control as COS-OGA (active substance) is known to induce a significant production of ROS at a concentration of 25 ppm mainly via the activation of parietal peroxidases (see van Aubel, G. et al., Plant immunity induced by COS-OGA elicitor is a cumulative process that involves salicylic acid. Plant Sci. 2016, 247, 60-70; van Aubel, G. et al., Oligosaccharides successfully thwart hijacking of the salicylic acid pathway by Phytophthora infestans in potato leaves. Plant Pathol. 2018, 67, 1901-191 1 ; Ledoux, Q. et al., Specific localization and measurement of hydrogen peroxide in Arabidopsis thaliana cell suspensions and protoplasts elicited by COS-OGA. Plant Signal. Behav. 2014, 2324).
A range of six different concentrations was tested (0,5pM to 100 pM) on the cell suspensions and monitored for the production of ROS during 90 min.
Results are presented on Figures 2A, 2B and 2C. As it can be seen, both 13-HPOD and 13-HPOT showed a significantly higher ROS production than the positive control, with RLU (relative light unit) values two to ten times higher (Figures 2A and 2B). A strong concentration effect is also observed as shown in Figure 2B, with a complete loss of ROS production at 5 pM and 1 pM for 13- HPOD and 13-HPOT respectively. Also, a quicker (30 min instead of 40 min) and stronger oxidative burst peak was observed for 13-HPOT (Figure 2A). Comparatively to COS-OGA, the kinetics of HPOs is slower, within 5 min only for COS-OGA, but it lasts longer with a total duration of half an hour before returning to basal level.
To better understand the potential sources of hydrogen peroxide after elicitation, different treatments (SHAM and DPI) were used while performing the same previous luminol-test on cell suspensions. This time, only the concentration of 100 pM, which produced the most hydrogen peroxide, was studied for both molecules. As shown on Figure 2C, in the presence of DPI or SHAM, the control cells (COS-OGA treatment) lost nearly all fluorescence compared to cells without inhibitors. This is consistent with previous results on A. thaliana cells treated with COS-OGA. It was shown that ROS were produced through both pathways as both DPI and SHAM inhibit H2O2 production (see Parasassi, T. et al., Membrane aging during cell growth ascertained by laurdan generalized polarization. Exp. Cell Res. 1992, 202, 432-439). Similarly, in presence of 13-HPOD and 13-HPOT, hydrogen peroxide generation by cell suspensions was strongly decreased with SHAM and DPI in both cases. According to this, peroxidases and RBOH seem to be mainly responsible for the production of extracellular H2O2 in this case, indicating that both HPOT and HPOD act on membranes as well as on cell walls.
The defense reactions activated in cell cultures have been shown to represent closely the reactions that occur in the plant cells surrounding the site of pathogen attack (see Jogaiah, S. et al., Role of Oomycete Elicitors in Plant Defense Signaling. In Bioactive Molecules in Plant Defense; Springer International Publishing, 2019; pp. 59-74 ISBN 9783030271657). To assess the early elicitation of A. thaliana by HPOs, foliar disks were treated with HPO solutions and monitored for the production of ROS over 120 min.
Results are presented on Figures 3A and 3B and, as it can be seen, like for measurements performed in cells suspensions, a strong oxidative burst is observed after both treatments, this being all the more pronounced in the case of 13-HPOT (Figure 3A). These results confirm the fact that exogenously applied HPOs are perceived by plants as they also induce the production of hydrogen peroxide in treated plants, this being significantly different from the controls in both cases. Here again, the oxidative burst peaks of the HPOs were observed later and longer than for the COS-OGA, those being all the more similar to those observed for the rhamnolipids (Figure 3B).
The present invention has been described in terms of specific embodiments, which are illustrative of the invention and not to be construed as limiting. More generally, it will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and/or described hereinabove.
Use of the verbs "to comprise", "to include", "to be composed of", or any other variant, as well as their respective conjugations, does not exclude the presence of elements other than those stated.
Use of the article "a", "an" or "the" preceding an element does not exclude the presence of a plurality of such elements.

Claims

26 Claims
1 . A method for eliciting at least one defense mechanism in plants against plant pests and plant diseases, the method comprising:
- applying to at least one of a part of a plant being an aerial part of said plant an effective and substantially non-phytotoxic amount:
• of a composition comprising at least one fatty acid hydroperoxide and/or at least one of its derivates, and/or
• of an elicitor being at least one fatty acid hydroperoxide and/or at least one of its derivates; and
- obtaining elicitation of at least one defense mechanism in said plant against plant pests and plant diseases.
2. The method according to claim 1 , wherein said at least one fatty acid hydroperoxide is chosen in the group consisting of 9-hydroperoxy-10,12,15- octadecatrienoic acid (9-HPOT), 13-hydroperoxy-9,1 1 ,15-octadecatrienoic acid (13-HPOT), 9-hydroperoxy-10,12-octadecadienoic acid (9-HPOD), 13- hydroperoxy-9,11 -octadecadienoic acid (13-HPOD), and mixtures thereof.
3. The method according to claim 1 or 2, wherein said at least one of the derivates of said at least one fatty acid hydroperoxide is a hydroxy fatty acid.
4. The method according to claim 3, wherein said hydroxy fatty acid is chosen in the group consisting of 9-hydroxy-10,12,15-octadecatrienoic acid (9-HOT), 13-hydroxy-9,1 1 ,15-octadecatrienoic acid (13-HOT), 9-hydroxy-10,12- octadecadienoic acid (9-HOD), 13-hydroxy-9,1 1 -octadecadienoic acid (13- HOD), and mixtures thereof.
5. The method according to any claims 1 to 4, wherein said composition further comprises a solvent and/or a co-formulant selected from the group comprising detergents, emulsifiers, dispersing agents, anti-foaming agents, penetration enhancers, humectants, ionic or non-ionic wetting agents, anti- freeze agents, preservative agents such as antioxidants, absorbent agents, thickeners, buffers, sticker agents, diluents, and mixtures thereof.
6. The method according to any claims 1 to 5, wherein said composition is under the form of granules, tablets, powders, liquids, (micro-) emulsions, nanoformulations, (micro-) encapsulates, (water-soluble) concentrates, (concentrated) suspensions, (concentrated) dispersions, wettable granulates and powders or aerosols.
7. The method according to any claims 1 to 6, wherein said plant pests are selected from the group comprising fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protists, protozoa, nematodes, insects and parasitic plants.
8. The method according to any claims 1 to 7, wherein the composition is applied before harvest or post-harvest to the whole plant, the leaves, the flowers, fruits, seeds on the plant, seedlings or seedlings pricking out, propagation material such as tubers or rhizomes, plants pricking out, by spraying, drenching, soaking, dipping, injection or administration through fertilizing or irrigation systems.
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