WO2023062025A1

WO2023062025A1 - Use of an extract of part of a plant for stimulating plant defenses against viruses, and associated composition and methods

Info

Publication number: WO2023062025A1
Application number: PCT/EP2022/078282
Authority: WO
Inventors: Christelle MARTINEZ-BARBREAU
Original assignee: Elicir
Priority date: 2021-10-11
Filing date: 2022-10-11
Publication date: 2023-04-20
Also published as: CA3234149A1; AU2022362673A1

Abstract

The elicitor composition which stimulates plant or tree defences, reducing the effects of an attack by a virus, comprises an extract of at least one part of at least one of the following plants: rocket salads, including of the genera Eruca sativa, Diplotaxis, Erucastrum and Bunias, Cakile, plants of the genera Allium, mustard (Sinapis alba, Brassica nigra, Sinapis arvensis, Brassica juncea), wasabi (Eutrema japonicum), horseradish (Armoracia rusticana), watercress (Nasturtium officinale), plants of the species Brassica rapa, Brassica ruvo, Brassica napus, Raphanus sativus, Barbarea verna, Erysimum allionii, Erysimum cheiri, Tropaeolum majus L, Alliaria petiolata, Salvadora persica, Carica papaya and Brassica oleracea.

Description

USE OF A PLANT PART EXTRACT TO BOOST PLANT DEFENSES AGAINST VIRUSES, COMPOSITION AND ASSOCIATED METHODS

Technical field of the invention

The present invention relates to a use of an extract of particular plants to stimulate the defenses of plants or trees against viruses, and to a composition and associated methods. The present invention thus aims to reduce the effects of a virus attack.

State of the art

In their constant struggle for survival, plants, like animals, have developed a wide range of protection and defense systems, which allow them to resist infectious diseases and parasites (fungi, bacteria, viruses, etc.). ). Sometimes, protection systems exist before any contact between the pathogenic agent and the host, but more often than not, resistance (induced resistance) sets in after the plant has encountered its aggressor.

For a number of plants there are pre-existing defensive means before attack by a potential pathogen or pest. For example, plants have morphological barriers. For example, it can be thick cuticles covered with hydrophobic substances (wax, cutin, suberin, etc.) preventing pathogens from entering or developing. We can observe the presence of thorns on the surfaces of the leaves and stems having a repellent power against animals (in particular herbivorous mammals), or the closure of the stomata in the leaves and the lenticels of the stems to prevent the penetration of fungal spores, bacteria, etc.

Plants also produce biologically active secondary metabolites and inert precursors to protect against pests. These "secondary metabolites" are not necessary for essential biological activity, such as growth and reproduction, but help the plant to adapt to its environment, which transform into active molecules during attack by a pest, an agent pathogen or after injury.

In response to attack by a pathogen, the plant also deploys induced metabolic changes in the plant which very often cause either late and discreet defense or active and rapidly induced resistance.

Late defense is often linked to receptors present in the plant membrane or cell, capable of recognizing molecular signals associated with microbes or pathogens. For example, fragments of cell walls, chitin or peptide motifs from the flagellum of bacteria (exogenous elicitor), proteins (endogenous elicitor) are among the motifs, which, once perceived by the plant, can initiate a basic immune response. (PAMP-Triggered Immunity or “PTI”), in order to limit the invasion and delay the advance of the pathogen.

On the other hand, specific recognition of pathogen attack inducing a rapid host response can also restrict or halt invasion and allow defense to kick in very quickly. Recognition of foreign components in plants is coordinated by specialized receptors, which trigger host defense responses upon detection of the pathogen. In this case of specific perception, the elicitor is encoded in the pathogenic agent by genes called avirulence genes (“AVR”). The plant receptors then recognizing the product of the avirulence gene are encoded by the resistance (R) genes of the plant. The interaction between the product of the avirulence gene of the parasite and the Resistance gene of the plant is commonly referred to as a "gene for gene relationship".

R genes have been isolated from various plant species (e.g. tomato, flax, rice, tobacco, Arabidospsis, sugar beet). Despite the significant diversity of the parasites to which they confer resistance (fungi, bacteria, viruses or even nematodes), their comparison reveals a strong homology of sequences as well as the conservation of structural motifs:

The majority of proteins deduced from R genes isolated from plants share different structural elements characterized within protein domains involved in defense mechanisms in yeast, Drosophila or vertebrates. By analogy, most R products would combine a receptor domain and an effector domain respectively ensuring two major functions: the recognition of elicitor molecules by mechanisms of protein-protein interactions and the direct or indirect activation of transduction signals.

Five main structural domains conserved within R genes have been distinguished:

1/ The majority of the products deduced from the sequences of R genes cloned to date present in the C-terminal position an intra- or extracytoplasmic LRR (“Leucine-Rich Repeats”) domain. The LRR domains correspond to the repetition of a motif of variable size comprising leucines. They would be involved in the mechanisms of protein-protein and protein-polysaccharide interactions

2/ The NBS (“Nucleotide Binding Site”) or NB domain, associated with the LRR domain (NBS-LRR), is widely distributed within the cloned R genes. This domain, composed in particular of various conserved kinase-type motifs, corresponds to a site for binding and hydrolysis of the nucleotide triphosphates ATP and GTP. It presents analogies with animal proteins potentially involved in the phenomena of apoptotic cell death, in particular APAF-1 identified in humans and CED-4 identified in Caenorhabditis elegans. All of these areas are grouped under the terminology NB-ARC (Van der Biezen and Jones, 1998).

3 and 4/ Two other domains, called “TIR” and “CC” (or even “LZ”), less frequent within the identified R genes, can be associated, in the Nterminal position, with the NBS-LRR products. The TIR (Toll Interleukin Receptor) domain has significant sequence homologies with the intracellular domains of protein receptors isolated from Drosophila (Toll receptor) and from humans (Interleukin-1 receptor). Based on these analogies, a role in the cell signaling cascade is attributed to the TIR domain. The CC (“Coiled-Coil”) or LZ “(Leucine-Zipper”) domain, which can vary in size and position, is known to play a role in the homodimerization or heterodimerization of proteins. 5/ Finally, the Serine/Threonine kinase domain is present alone (for example product of the Pto gene) or, associated with an LRR domain (for example, product of the Xa21 gene). It would be involved in phosphorylation reactions during signaling cascades.

In the majority of cases, during an incompatible interaction, the very active defense, leading to a situation of resistance, a whole series of events takes place at the end of the perception of the pathogenic agent by the plant. .

The entire metabolism of infected cells is solicited and involves many mechanisms whose function is to destroy and trap the parasites: G proteins, ion fluxes, in particular of Ca2 ⁺ , H ⁺ , K ⁺ and Cl', activated forms of oxygen (or ROS for "Reactive Oxygen Species") and a whole cascade of phosphorylation/dephosphorylation of proteins such as MAPKs ("Mitogen-ActivatedProtein-Kinase").

Most often, this signal surge results phenotypically in the rapid death of cells infected by the pathogenic agent and the formation of localized necrotic lesions around the site of parasite penetration. This genetically determined reaction is called a hypersensitivity reaction (or “HR”) or apoptosis (programmed cell death).

The significant metabolic modifications allowing the aggressor to be confined to its site of penetration are the result of the expression of numerous defense genes in the infected zone. Cell death (“HR”) also generates signals that activate local acquired resistance (“LAR”), in contact with lesions, and systemic acquired resistance (SAR), remote from the site of infection.

During the establishment of the LAR, the defense responses are intense and particularly localized at the level of the ring of cells surrounding the HR zone. These responses include the synthesis of a broad spectrum of anti-microbial compounds such as "PR" proteins (Pathogenesis Related Protein), certain secondary metabolites with antibiotic properties, for example phytoalexins, as well as the accumulation of molecules involved in signaling pathways, including ROS and various hormones including salicylic acid. These two levels of local resistance (HR and LAR) are accompanied by an established resistance at the plant level, SAR, whose expression, later and less intense, can persist for several weeks after infection. SAR keeps the plant in a dormant state that enables it to resist not only the original aggressor, but also a wide range of other pests that may intervene later.

The establishment of this systemic resistance, at the level of uninfected tissues, depends on an elaborate network of intercellular communication. Signals released by cells expressing HR diffuse and reach other cells which in turn trigger a specific response. Salicylic acid, ethylene, jasmonic acid or even systemin have been recognized for several years as chemical messengers. These messengers alert uninfected cells and direct their metabolism towards the establishment of defense responses, in particular, the strengthening of the cell wall, the stimulation of secondary metabolic pathways (enzymes of phenylpropanoid metabolism, of the biosynthetic pathway of ethylene and jasmonic acid) and protein accumulation. The special case of viruses:

A phytovirus, or plant virus, is a virus attacking plant organisms. These viruses have the particularity of penetrating the plant cell of their host in order to divert the mechanisms of the cell to their advantage and allow them to reproduce.

Among the viruses infecting plants, those whose genome consists of one or more single-stranded RNA molecules of messenger ("positive") polarity are the most important both in number (85 to 90% of known viruses) than by their economic impact. Although showing various morphologies, these viruses possess a very simple structure, the non-enveloped particles being made up of a single or a few (2 or 3) types of capsid protein (CP) subunits.

For example, plants use RNA interference, or RNAi, to defend themselves against many viruses. During their replication phase, RNA viruses pass through a double-stranded RNA (dsRNA) stage which is recognized by the plant as a warning signal and allows the initiation of its defense system which can lead to the elimination of the virus. For most RNA viruses, an effective antiviral RNAi response relies primarily on the activity of the DICER-LIKE4 ("DCL4") protein to produce the bulk of antiviral "interfering" RNAs from dsRNA ( siRNA). The latter are associated with an ARGONAUTE protein (“AGO”), in order to guide it towards the viral RNA, which leads to its degradation.

Plant viruses have four essential characteristics compared to other pathogens:

1/ Unlike other pathogens, viral populations have a very high evolutionary potential. This rapid evolution leads to the appearance of viral variants capable of circumventing and adapting very quickly to the genetic resistance of plants.

2/ Plant viruses are chemically incurable once the disease has taken hold in a field. No effective remedy exists against plant viruses. Infected susceptible plants will retain the virus in their tissues until they die.

3/ It is the host cells that are responsible for multiplying the viruses that parasitize them. Plant viruses can infect all parts of a plant. Only the meristems (undifferentiated cells) of the buds escape their invasion. They are most often the cause of generalized diseases.

4/ Viruses can only survive in a living plant. They are destroyed as soon as the plant dies. They also require external agents to spread from one plant to another.

Within the virus/plant interaction itself, very specific mechanisms are put in place, different from the metabolic events involved in other plant/pathogen interactions (bacteria, fungi, etc.).

Thus, plant viruses are major pathogens found in crops around the world, and will represent an increasingly serious threat. Among emerging infectious diseases, viruses alone account for almost half of the pathogens involved, i.e. 47% against 30% for fungi, and only 16% for bacteria (Anderson et al., 2004).

In this context, nowadays, the deployment of plant varieties carrying resistance genes against phytoviruses remains the most effective approach in the control of these viral pathogens, in order to use the existing plant defense machinery already in place. . One of the major criteria for breeders remains to obtain varieties with durable resistance, i.e. which remains effective for a long period when the variety is cultivated intensively in an environment conducive to the development of disease.

Presentation of the invention

The present invention aims to remedy all or part of these drawbacks.

To this end, according to a first aspect, the present invention relates to an elicitor composition according to claim 1.

The action of this composition bypasses viral resistance. Bypassing a resistance results temporally in the emergence and spread of a virulent variant at the level of the cell, then of the plant, and finally at the level of the entire plot. The virulence of a pathogen, according to the definition of plant pathologists, refers to the qualitative component of pathogenicity, i.e. the ability or not to infect a genotype carrying a given resistance. Evolutionary forces will be strongly involved in the appearance of virulent variants, which do not naturally preexist in an agroecosystem. The genetic variations will therefore be linked to the variations/mutations of the AVR genes: The first stage corresponds to the appearance at the cellular level of a virulent variant capable of infecting a plant possessing a resistance gene in the case of an interaction of type gene for gene. This means that it accumulates the necessary mutations in its avirulence factor to become virulent.

It has been demonstrated that the composition which is the subject of the invention, preferably in its form extracted from a rocket plant, and particularly from Eruca Sativa and from Diplotaxis tenuifolia, acts very early after its spraying, demonstrating that this composition mimics the relationship gene for gene. For example, thirty minutes after being sprayed, the preferential composition triggers a series of metabolic events which show that the defense mechanisms triggered resemble those described in a gene for gene relationship:

1/ The production of reactive oxygen species (FAO or ROS), within 30 minutes of leaf spraying on Arabidopsis thaliana. The transient production of reactive oxygen species or forms (ROS) constitutes one of the earliest responses following the gene-for-gene recognition of a pathogenic agent by the plant. These activated forms of oxygen are produced within the first few minutes or even hours after relicitation. They consist essentially of (O2), (OH) and (H2O2) The significant accumulation of these molecules, commonly called "oxidative burst", is often correlated with the control of cell proliferation and cell death, the development of plants and to the induction of defense responses. In the case of the response to pathogens, these reactive oxygen species play a role both as antimicrobial compounds, in strengthening the cell wall and in signaling. Potential changes redox are integrated by the cell, which results in the activation of genetic programs resulting in the establishment of HR.

2/ The release of intercellular signals (phytohormones such as salicylic acid, jasmonic acid, ethylene) to the cells adjacent to the infection sites conditioning for immunization and an increase in the resistance of the latter (LAR for "Local Acquired Resistance”) in peach trees infected with bacteriosis and treated with the preferential composition.

3/ immunization of the whole plant (SAR for “Systemic Acquired Resistance”) and potentiation phenomenon during a second attack by a parasite when the plant has been previously treated with this composition.

4/ production of B-1,3 glucanase (PR2 Proteins) from ten hours after spraying of the preferred composition.

This composition object of the invention is therefore capable of acting independently of the structure of the products of Resistance and Avirulence genes, making it possible to overcome any mutation of the virus.

In conclusion, while the interaction between plants and viruses is very specific and different from other plant/pest interactions (no chemical solution possible), it has been demonstrated that the composition which is the subject of the invention acts effectively against viruses , by acting in very early times by simulating a gene for gene interaction, which allows the plant to overcome all the weaknesses of a plant vis-à-vis a virus (too rapid appearance of variants, replication of the viruses, etc).

Given the rapid evolution of viruses, this composition puts plants in a state of resistance, by acting downstream of the interaction between the R and AVR genes, in order to bypass any virus mutations. Thus, the composition that is the subject of the invention has the capacity to trigger all the defense mechanisms appearing “after” a “gene for gene” recognition phenomenon, both for viruses and for other pathogens, in particular bacteria and fungi.

The effectiveness of the present invention reduces the effects of viruses on plants, plants, or trees, so as to allow the plant, in particular a tree, to continue to grow correctly despite this attack, by overcoming the consequences of this attack, it that is, allowing the plant or tree to grow despite the virus, reducing or eliminating the impact of the virus. The plant extract can also, in certain uses, allow the plant to eradicate certain viruses. This use can be curative or preventive.

It is noted that the reduction of the effects of the viruses on the plants or trees attacked by these viruses comprises, in certain cases, the total or partial reduction of the symptoms. Indeed, plants and trees have, when their defense system is functional (in particular thanks to the stimulation obtained by the implementation of the invention), the capacity to defeat a virus. The use of the composition that is the subject of the present invention aims to stimulate what plants already know how to do, but which they do not do in cases of “sensitivity”, because they do not recognize their aggressor. The decline of the pathology thus appears in certain examples of the description. In preferred embodiments, the extract is obtained from at least one of the following plants: Arugula, including Eruca sativa, Diplotaxis, Erucastrum and Bunias, Cakile, broccoli, plants of the Allium genera (garlic, onion, shallot , leeks and chives), common cabbage, cauliflower, Brussels sprouts, kale, mustard, wasabi, watercress, horseradish, white cabbage, field cabbage (Brassica rapa), including bok choy cabbage, Chinese cabbage and turnip, kohlrabi, collard greens, red cabbage, Chinese broccoli, broccoli rave, rapeseed, radish, Siberian wallflower, wallflower, Indian watercress, garlic grass, pilu tree, papaya tree, including its fruit.

An eliciting composition which is the subject of the invention may comprise a total raw extract obtained by grinding and extracting the plant, in a fraction enriched in active compound(s) of such a total extract, or in one or more compounds ( s) active(s) in mixture. Such a composition advantageously makes it possible, in an effective amount in a composition, to combat the symptoms of the attack of a plant or of a tree mentioned above by a virus mentioned above.

In preferred embodiments, the extract is obtained from at least one of the following plants: Arugula, including Eruca sativa, Diplotaxis, Erucastrum and Bunias, Cakile, broccoli, common cabbage, cauliflower, Brussels sprouts , kale, mustard, watercress, white cabbage, field cabbage (Brassica rapa), including bok choy, Chinese cabbage and turnip, kohlrabi, collard greens, red cabbage, Chinese broccoli, broccoli rabe, radish , and Siberian wallflower.

In preferred embodiments, the extract is obtained from at least one of the following plants:

- Rockets, including Eruca sativa, Diplotaxis, Erucastrum and Bunias,

- Cakile.

In preferred embodiments, the extract is obtained from at least one of the plants of the species Brassica oleracea.

In preferred embodiments, the extract is obtained from at least one plant not containing Methyl-isothiocyanate and/or Propenyl isothiocyanate.

The inventor found that these isothiocyanates could have harmful effects on plant growth while reducing the effects of viruses.

In embodiments, the extract is obtained from at least one plant containing at least one butyl-isothiocyanate. The inventor found that these isothiocyanates have favorable effects on plant growth while reducing the effects of viruses.

In embodiments, the extract is obtained from at least one plant containing 1,3-thiazepane-2-thione. The inventor found that this compound which, due to its structure, is not an isothiocyanate has favorable effects on plant growth while reducing the effects of viruses.

The inventor discovered that these extracts have at least one active substance which reduces the effects of viruses, the presence of their vector of diffusion and/or their colonization by insects.

In embodiments, the extract of at least one plant part is an extract obtained from a ground material of said plants, and: - said extract of at least one plant part comprises at least leaves of said plants, preferably essentially leaves and flowers, and

- the method for obtaining said liquid extract comprises the following steps: a) a step of grinding said plants; b) filtration of the ground material obtained; and c) recovering the liquid extract obtained after filtration.

The term “essentially comprises” is intended here to denote by comprising at least 75%, preferably at least 95%, of leaves and flowers of said plants by weight, for example dry, relative to the total weight of the plant, before mixing with the solvent aqueous.

In some embodiments, the extract is obtained by a method comprising, in addition, a step of nebulizing the liquid extract and passing the nebulized liquid extract through a stream of hot air.

According to a second aspect, the present invention relates to a process for reducing the effects of viruses on plants, including trees, comprising a step of applying the eliciting composition which is the subject of the invention.

In embodiments, the method is applied to reducing the effects of an attack by one of the following viruses:

- Beet yellows viruses,

- Tobacco mosaic virus,

- Cassava mosaic virus,

- Banana Bunchy Top Virus (BBTV),

- Banana streak virus (BSV),

- Barley yellow dwarf virus (JNO or BYDV),

- Cucumber mosaic virus,

- Sugarcane mosaic virus (SCMV),

- Maize lethal necrosis disease viruses (MLN, MCMV, SCMV),

- The Potyviridae family of viruses,

- Sweet potato feathery mottle virus (Potyvirus), or SPFMV,

- Sweet potato chlorotic stunting virus (Crinivirus), or SPCSV and SPVD,

- Sweet potato moderate mottle virus, or SPMMV,

- Sweet potato latent virus, or SPLV,

- Sweet potato chlorotic spot virus or SPCFV,

- Sweet potato virus G, or SPVG,

- Sweet potato leafroll virus, or SPLCV,

- Tomato brown rugose fruit virus (ToBRFV),

- Tomato spotted wilt virus (TSWV),

- Tomato mosaic virus (Tomato mosaic virus, ToMV),

- Zucchini yellow mosaic virus, or

ZYMV), - rose mosaic virus.

In embodiments, the method is applied to reducing the effects of an attack of at least one of the beet yellows viruses.

In embodiments, the method is applied to reducing the effects of attack by at least one of the cucumber mosaic viruses.

In embodiments, the application of the eliciting composition is a foliar application to the plants.

According to a third aspect, the invention relates to a use of the eliciting composition which is the subject of the invention to reduce, on plants, including trees, the effects of an attack by one of the viruses listed above.

In embodiments, the application to the plant or tree is by foliar spraying, ground irrigation, drip, use in hydroponics, seed treatment and/or seed coating.

In some embodiments, the application to the plant or the tree is done with a dilution in water of the composition between 2 g/L and 2000 g/L expressed in grams of plants on which the extraction per liter of product applied.

In some embodiments, the application to the plant or the tree is done with a dilution in water of the composition between 5 g/L and 200 g/L expressed in grams of plants on which the extraction per liter of product applied.

In embodiments, said extract of at least one part of plants is a liquid extract of said plants.

In embodiments, application to the plant or tree is by foliar spray, soil drench, soil irrigation, drip, hydroponic cultivation, seed treatment and/or seed coating.

In embodiments, at least one active principle is obtained from leaves of said plants.

In embodiments, at least one active principle is obtained from flowers of said plants.

In embodiments, at least one active principle is obtained by grinding at least a part of said plants.

In embodiments, at least one active principle is obtained by aqueous extraction.

In embodiments, at least one active principle is obtained, by oil extraction, by solvent extraction, or by extraction of cakes or pastes.

In embodiments, the composition is formulated as a powder, soluble powder, wettable powder, granules, dispersible granules, or wettable or slow-release granules, to be diluted in water at the time of use.

In embodiments, the composition is formulated as a liquid, liquid soluble concentrate, emulsifiable concentrate, suspension concentrate, or ready to use. According to a fourth aspect, the present invention relates to a process for the production of a composition which is the subject of the invention, which comprises a step of grinding at least a part of said plants, to provide a ground material, and a filtering step to extracting solid parts from said ground material and obtaining a liquid.

The particular advantages, aims and characteristics of this composition and of this process being similar to those of the composition which is the subject of the present invention, they are not repeated here.

According to a fifth aspect, the present invention relates to a method for the physical simulation of gene for gene interaction, a composition capable of being extracted by aqueous extraction from a plant given in the claimed lists, in particular from an arugula plant, and particularly of the Eruca Sativa or Diplotaxis type or of plants genetically modified to produce this composition.

Thus, the process and the composition that is the subject of the invention perform an action on the plant within hours, or even in less than one hour, after the gene-for-gene interaction with the virus or its variant, thus avoiding the problem of mutations , and providing a response immediately strengthening the resistance of treated plants against viruses, bacteria, fungi and other pathogens.

While the interaction between plants and viruses is very particular and different from other plant/parasite interactions, since there is no possible chemical solution, it is demonstrated that the composition which is the subject of the invention, in particular in its form extracted from arugula plants, and particularly from Eruca Sativa or Diplotaxis Tenuifolia, acts effectively against viruses, by acting in very early times by simulating a gene for gene interaction, which allows the plant to overcome all the weaknesses of a plant vis-à-vis a virus (too rapid appearance of variants, replication of the virus, etc.) and, more generally, of a pathogen.

The advantages, aims and particular characteristics of the other aspects of the invention, in particular those which are claimed, also apply to this method and this composition and to the use of this composition to simulate, in a plant whose resistances must be stimulate, a gene for gene interaction.

Brief description of figures

Other advantages, objects and characteristics of the present invention will emerge from the description which follows, given for the purpose of explanation and in no way limiting, with reference to the appended drawing, in which:

FIG. 1 represents, in the form of a flowchart, steps of an embodiment of a process for producing and using a ground material, which is a preferred example of production of the composition which is the subject of the invention,

Figure 2 represents dates of evaluations of beet treatments,

Figure 3 represents numbers of Myzus persicae insects,

Figure 4 shows incidences of the pest Myzus persicae, Figure 5 represents numbers of Aphis fabae insects,

Figure 6 represents incidences of the pest Aphis fabae

Figure 7 shows assessments of areas infected with jaundice,

Figure 8 shows a photograph of the untreated modality (control),

Figure 9 represents a photograph of the modality treated with the product PP1,

Figure 10 represents a photograph of the modality treated with the product PP1, and

FIG. 11 represents a descriptive table of the processing methods of the first demonstration.

Description of embodiments

The eliciting composition stimulating the defenses of plants or trees reducing the effects of an attack of a virus comprises an extract of at least a part of at least one of the following plants: Rockets, including genera Eruca sativa, Diplotaxis, Erucastrum and Bunias, Cakile, plants of the genera Allium, mustard (Sinapis alba, Brassica nigra, Sinapis arvensis, Brassica juncea), wasabi (Eutrema japonicum), horseradish (Armoracia rusticana), watercress (Nasturtium officinale), plants of Brassica species rapa, Brassica ruvo, Brassica napus, Raphanus sativus, Barbarea verna, Erysimum allionii, Erysimum cheiri, Tropaeolum majus L, Alliaria petiolata, Salvadora persica, Carica papaya and Brassica oleracea.

Preferred embodiments and certain viruses are listed above.

Before presenting the various aspects of the present invention, the viruses on which the eliciting composition which is the subject of the invention have been successfully tested are described below:

For the purpose of clarity and conciseness, the demonstrations of the following description do not cover all the combinations of plants indicated above, but illustrate the effectiveness of the present invention in all these combinations.

First demonstration: Effect of the eliciting composition object of the invention against the beet yellows virus.

Beets are the first reservoir of jaundice viruses. It is therefore important to remove all crop residues (digging lines, trailing beets) because leaf regrowth can become sources of infection. It is also important to manage weeds well, in the plots and at the edges of the fields, because a certain number of species are hosts to vector aphids and sometimes also to jaundice viruses.

To fight against this virus, the following techniques are used:

Preventive control by using seeds treated with a systemic insecticide in the coating (imidacloprid). This technique offers by far the best results in terms of efficiency and persistence. However, it is important to note that in 2018 neonicotinoids were banned. 2020 was the second year without a neonicotinoid (NNI) on seed since 1993. Imidacloprid was authorized as a seed treatment that year. It is a very effective, neurotoxic insecticide. As a result, the aphid only bites once and there is therefore no more contamination per circle (INRAE, 2020).

In Europe, imidacloprid will no longer be approved from 31/7/2022, but some countries have kept it or granted exemptions, when ANSES in France withdrew all marketing authorizations for agricultural purposes. Two other NNIs were withdrawn at the end of 2019 (thiamethoxam, clothianidin). Only thiamethoxam and imidacloprid were used in our territory as a coating for beet seeds.

Preventive control by the application, during sowing, of long-lasting micro-granular insecticides, suitable for the control of aphids vectors of jaundice.

Curative control by spraying with aphicide products.

Currently, the products available are:

Teppeki (50% Flonicamide), registered trademark, has been approved since December 21, 2018 with the following conditions of use (marketing authorization, or "AMM", no. 2050046):

Approval dose of 0.14 kg/ha,

One application per year from the six true leaf stage,

Effective only on aphids (selective of auxiliaries),

Possible mixture with herbicides,

Remanence of two weeks minimum,

Add a liter of oil according to regulations.

Movento (Spirotetramate at 100 g/l), registered trademark, obtained a derogatory marketing authorization for 120 days for use in 2020 with the following conditions of use:

Approval dose of 0.45 l/ha,

Two applications per year (minimum interval of 14 days) from the two-leaf stage until cover of the beets,

Effective only on aphids (selective of auxiliaries),

Do not use in a mixture because of the risk of reduced efficiency and

Remanence of two weeks minimum.

However, the use of these two active substances in 2020 did not sufficiently control aphid populations throughout the territory. These two substances are not a lasting solution. At this stage, no chemical or non-chemical solution comes close in effectiveness to NNI-based chemical treatments and does not make it possible to deal with an exceptional situation (INRAE, 2020)

Given the urgent need to find an effective solution to combat beet yellows, the fully biosourced elicitor composition that is the subject of the invention is capable of significantly reducing, or even eliminating, the effects caused by serious plant viral diseases and up to then incurable. Toxicity testing of this composition has shown that it is non-toxic. A trial has been set up with the company Ephydia (registered trademark, BPE approved company for good environmental practices) against the beet yellows virus, the results of which are presented below.

Material and methods

Plant material: Beta vulgaris is a species of plants in the family of

Amaranthaceae. It is cultivated around the world for sugar production, and in an ancillary way to make ethanol or baker's yeast from molasses made from white sugar manufacturing residues.

Cultivation conditions

Planting date: 03/25/2021

Depth: two meters

Row spacing: 45 cm

Row spacing: 18.5 cm

Planting rate: 100,000 plants / ha

Planting method: seedlings

Test carried out by: Ephydia

Width of the plot treated: 2.7 m

Length of the plot treated: 9.25 m

Area of the plot treated: 24,975 m ²

Number of repetitions: four

Soil Texture: Loamy

Place of the test:

City / Municipality: Combles (80)

Country: France

Postcode: 80360

Description of treatments:

Control (untreated plot),

Application of PP1, at 50% V/V,

Application of PP1, at 50% V/V and Teppeki, at 0.14 kg/ha,

Application of Teppeki, at 0.14 kg/ha.

In this first demonstration, extracts of Eruca sativa and Diplotaxis tenuifolia were used. As the results are very similar, these two extracts have been grouped under the generic term “PP1” below.

Description of processing methods

Type of application: Foliar application

Table 1 in figure 11: Description of treatment methods (or “Trt”)

Table 2: Date and stage of application of treatments

Description of the Teppeki product:

MA No.: 2050046

Date of 1st product authorization: 21/04/2005

Active substance: Flonicamid 500 g/kg Function: Insecticide

Usage: 15053106 Industrial and fodder beet*Trt Part.Aer.*Aphids

Date of authorization for use: 08/12/2021

Maximum application rate: 0.14 kg/ha

Maximum number of applications: One

Stage of application: Minimum: BBCH 12

Max: BBCH 49

Pre-harvest time: 60 days

It is important to note that the Teppeki product is only allowed to be applied once on the beet crop, however during the trial the program received three applications of the Teppeki product and six applications of the Teppeki product alone for the reference modality. So the results that will follow will be nuanced knowing that these levels of efficiency with the reference are never achieved in the fields in real conditions.

Description of the evaluations carried out during the trial.

Aphid rating at each application and five days after each treatment:

Myzus persicae: green peach aphid

Aphis fabae: black bean aphid

Scoring at the onset of symptoms, during the summer and before harvest

Harvest gross weight and saccharimetry

Figure 2: Date of assessments.

Results and discussion.

In Figures 3 to 6, each set of four vertical bars represents, from left to right, the untreated control, the treatment with the composition that is the subject of the invention, the combination of this composition and the Teppeki and the Teppeki alone.

Evaluation of the number of aphids and the incidence of the pest - Myzus Persicae.

Figure 3: Number of Myzus persicae insects.

Figure 4: Incidence of the pest - Myzus persicae.

Note: "DA - A" stands for "Day after treatment A", etc.

The green peach aphid, Myzus persicae is the main vector of beet yellows. It has very good capacities for transmitting moderate jaundice viruses (BChV and BMYV) as well as severe jaundice (BYV).

The results illustrated in FIG. 3 show that the PP1 treatment makes it possible to statistically reduce the number of aphids vectors of jaundice (except for the notation 31 DA-A, associated letter “a”) in comparison with the untreated modality. All treated modalities are statistically identical up to 14 DA-A and 41 DA-A notation (associated letters "b") and different from 21 DA-A to 35 DA-A of the modality treated with Teppeki in large overdose .

In addition, the results illustrated in FIG. 4 show that the PP1 treatment allows a reduction in the incidence compared to the control except for the notations 10 DA-A, 21 DA-A and 31 DA-A. These results show that PP1 has an effect on the presence of the beet yellows vector - Myzus persicae. Aphid presence values are directly correlated with the appearance of jaundice symptoms. Therefore, the results obtained show that PP1 allows the reduction of symptoms.

Evaluation of the number of aphids and the incidence of the pest - Aphis Fabae.

Figure 5: Number of Aphis fabae insects

Figure 6: Pest incidence - Aphis fabae

The black bean aphid, Aphis fabae, is a secondary vector of BYV (severe jaundice), but does not transmit BChV or BMYV (moderate jaundice virus). Jaundice is never transmitted to the offspring of infected aphids.

The results illustrated in FIG. 5 show that the PP1 treatment makes it possible to statistically reduce the number of aphids vector of jaundice in comparison with the untreated modality, except at 41 DA-A (associated letters “a”). In addition, we can observe that from 31 DA-A, the PP1 product is statistically equivalent to the Teppeki reference, even applied in a large overdose (associated letters "b").

In addition, the results illustrated in Figure 6 show that the PP1 treatment allows a reduction in incidence compared to the control except for the notation 31 DA-A (associated letters "a").

These results show that PP1 has an effect on the presence of the beet yellows vector Aphis fabae. In addition, aphid presence values are directly correlated with the appearance of jaundice symptoms. Therefore, these results show that PP1 allows the reduction of symptoms.

Areas infected with beet yellows (Figure 7). Figure 7 represents an evaluation of the area infected by jaundice according to the treatments. The risk period begins as soon as the first aphids appear in the plots at the earliest at the end of April at the beginning of May, i.e. from the two-leaf stage until the ground cover at the end of June. The latency period is generally two to four weeks but would be shorter for severe jaundice (one to two weeks) than for moderate jaundice (four to six weeks).

In the case of this trial, Myzus persicae aphids were detected in the trial from 05/14/2021 to 06/24/2021 and Aphis fabae aphids were detected in the trial from 06/09/2021 to 24 /06/2021 . The first symptoms of the disease were observed on 3/09/2021 as shown in Figure 7. These results demonstrate that the plots are starting to show symptoms of beet yellows and that the PP1 product is as effective as the reference TEPPEKI (which was applied six times whereas it is applied only once in normal condition) and allows to decrease the symptoms compared to the untreated modality.

Thus, PP1 could prove to be much more effective than the Teppeki reference product under normal application conditions. PP1 thus makes it possible to reduce the presence of vector aphids but also to limit the appearance of symptoms.

Figure 8: Photograph of the untreated modality - plot 401

Figure 9: Photograph of the modality treated with the product PP1 - plot 101

Figure 10: Photograph of the modality treated with the product PP1 - plot 403 Conclusions of the first demonstration.

The data from this trial indicate that the application of the PP1 plant extract by foliar application makes it possible to reduce the presence of the two varieties of aphid vectors of severe jaundice and moderate jaundice. These data are related to the fact that PP1 reduces the symptoms of jaundice. Plants that have not been bitten by aphids continue their photosynthesis, have good vigor and continue their development cycle.

Beet harvest according to the modalities:

Witness: 0.8 Ton

PP1: 1.2 tons

PP1 + Teppeki: 1.3 Tons

Teppeki: 1.2 tons

Thus PP1 would prove to be a solution to the problem of jaundice and a replacement solution for neonicotinoids. The PP1 product is able to significantly reduce the incidence of beet yellows virus.

Second demonstration: Effect of PP1 against the cucumber mosaic virus.

This test is carried out in an area known for its contamination by the cucumber mosaic virus.

Control methods are generally as follows:

- Limit the presence of reservoir plants,

- Eliminate and immediately destroy plants that show symptoms,

- Limit the presence of aphids,

- Use clean tools when pruning and gardening work,

- Choose seeds or seedlings that are healthy and

- Choose resistant cucumber varieties.

A trial is carried out to evaluate the effectiveness of PP1 against cucumber mosaic.

Plant material:

STYX variety, organic plants,

Calendar: planting April 11, 2021,

Harvest from May 23, 2021 to July 15, 2021,

Device: Eight-meter tunnel, opaque micro-perforated thermal mulching, four double rows (distance 0.35 m),

Irrigation: one boom per crop row

Modalities: Untreated controls (T) and plants treated with PP1

Block trial with four repetitions

Treatments: six treatments were carried out at a rate of 14 days.

Symptoms :

Chlorotic spots (more or less marked mosaic) appear on the young leaves; these can warp, wrinkle, or even dry out in severe cases. In the plots, circular disease foci are observed, which gradually spread. An early attack causes complete dieback of young plants. Affected plants have reduced growth and an altered habit.

A plant infected with this virus remains a carrier of the virus until it dies.

Observations and measurements taken: - Observations: Agronomic measurements from May 23, 2021 to July 15, 2021:

The incidence represents the percentage of contaminated leaves or fruits.

The severity represents the percentage of surface covered by the symptoms of the disease.

Table 3: Incidence and severity on cucumber leaves - May 23, 2021

Severity and incidence were measured on 20 randomly sampled leaves for each modality.

Table 4: Incidence and severity on cucumber leaves - July 15, 2021

Table 5: Incidence and severity on cucumbers (Fruits) - July 15, 2021

The severity and incidence were measured on 20 fruits sampled at random, for each modality. Analyzes were performed according to Newman and Keuls. The different letters express significantly different results at the 5% level.

It is observed, both for the leaves and for the fruits, the eliciting composition which is the subject of the invention (PP1) significantly reduces both the incidence and the severity of the effects of the viruses and significantly increases the quantity of beets harvested.

The results show the efficacy of PP1 against cucumber mosaic virus. Indeed, the notations on leaves and on fruits show that the treated plants have significantly fewer symptoms than the control plants.

Third demonstration: B1 / Broccoli extract (“PP2”) against the cucumber mosaic virus (CMV). An extract of broccoli leaves is produced according to the protocol illustrated in FIG. 1, in liquid form, without concentration. This test is carried out in an area known for its contamination by the cucumber mosaic virus (presence of the vector). No biocontrol solution is known to date to eradicate the disease. In this context, a trial is carried out to evaluate the efficacy of PP2 against the cucumber mosaic virus (CMV).

Symptoms :

Chlorotic (mosaic) spots appear on young leaves, which may become distorted, puckered, or even dry out in severe cases. An early attack causes complete dieback of young plants. Affected plants have reduced growth and an altered habit. A plant infected with this virus remains a carrier of the virus until it dies.

Material and method :

Cucumber variety: TYRIA, organic plants, not tolerant to mosaic virus. Calendar: planting 08 April 2020

Harvest: from May 28, 2020 to July 30, 2020

Device: Eight-meter tunnel, trellising on string, two double rows (distance 0.35 m). Irrigation: one boom per crop row

Modalities: Untreated controls (T) and treated plants (PP2)

Design: Block trial with four randomized repetitions

Treatments: six treatments were carried out at a rate of 14 days.

Results :

Table 6: Incidence and severity on cucumber leaves - May 30, 2020

Table 7: Incidence and severity on cucumber leaves - July 20, 2020

Table 8: Incidence and severity on cucumbers (Fruits) - July 20, 2020

The severity and incidence were measured on 20 fruits sampled at random, for each modality. Analyzes were performed according to Newman and Keuls. The different letters express significantly different results at the 5% level. The results show the efficacy of PP2 against cucumber mosaic virus. Indeed, the notations on leaves and on fruits show that the treated plants have significantly fewer symptoms than the control plants.

Fourth demonstration: Brussels sprout extract (PP3) on cucumber mosaic virus (CMV). An extract of Brussels sprouts leaves is produced according to the protocol illustrated in FIG. 1, in liquid form, without concentration.

This test is carried out in an area known for its contamination by the cucumber mosaic virus (presence of the vector).

No biocontrol solution is known to date to eradicate the disease.

In this context, a trial is performed to evaluate the efficacy of PP3 against cucumber mosaic virus.

Material and method :

Cucumber variety: TYRIA, organic plants, not tolerant to mosaic virus.

Calendar: planting 08 April 2020

Harvest: from May 28, 2020 to July 30, 2020

Modalities: Untreated controls (T) and treated plants (PP3)

Design: Block trial with four randomized repetitions

Treatments: six treatments were carried out at a rate of 14 days.

Results :

Table 9: Incidence and severity on cucumber leaves - May 30, 2020

Table 10: Incidence and severity on cucumber leaves - July 20, 2020

Table 11: Incidence and severity on cucumbers (Fruits) - July 20, 2020

The severity and incidence were measured on 20 fruits sampled at random, for each modality.

Conclusion :

Analyzes were performed according to Newman and Keuls. The different letters express significantly different results at the 5% level. The results show the efficacy of PP3 against cucumber mosaic virus. Indeed, the notations on leaves and on fruits show that the treated plants have significantly fewer symptoms than the control plants.

Fifth demonstration: Arugula extract (“PP1”) on the tomato spotted wilt virus (“Tomato spotted wilt virus”, TSWV).

Tomato spotted wilt virus (TSWV) is widely distributed throughout the world in temperate and subtropical zones where it has been on the rise since the early 1980s. Emerging in France since 1987, it has a wide range of potential hosts. It is transmitted by at least nine species of thrips.

Symptoms of Tomato spotted wilt virus (TSWV) can take on various appearances on tomato foliage, such as leaf deformities with apical curvature of the apex, vegetation blockage, more or less contrasting mosaic, chlorotic spots and lesions becoming necrotic, more or less marked chlorosis and bronze coloration of the leaf blade or veins, accompanied by rings, small dark lesions becoming necrotic also visible on petioles and stem , anthocyaninization of the lamina.

Fruits are also affected. They can be "tanned" and present large arabesques and more or less concentric chlorotic rings. Dry necrotic alterations, cracks are sometimes visible. Early contamination leads to a reduction in the number and size of fruits; if they are late, the fruits grow normally but are badly colored and more or less deformed.

No biocontrol solution is known to date to eradicate the disease.

In this context, a test is carried out to evaluate the effectiveness of the eliciting composition which is the subject of the invention against the tomato TSWV virus. The extracts of rocket leaves (Diplotaxis) are obtained according to the protocol illustrated in FIG. 1 in their non-concentrated liquid form (extracts called “PP1”).

Material and method :

The experiment takes place in a 250m ² rigid greenhouse equipped with openings and side vents.

Experimental device: Type “complete blocks with four repetitions”. Elementary plot of 10 plants.

Technical itinerary: Sowing on December 30, 2021 for planting on January 26, 2022. Harvest over four months from the beginning of March to the end of June 2022.

Modalities: Controls (untreated), PP1 (foliar spray application)

Treatments: Foliar spray

Six applications of PP1, at a rate of 14 days. Analysis method: Analysis of variance with a risk threshold of 5%. Ratings with the same letters are not significantly different.

Results :

Table 12: Incidence and severity on tomato leaves - March 20, 2022

Table 13: Incidence and severity on tomatoes (Fruits) - March 20, 2022

Table 14: Incidence and severity on tomato leaves - April 25, 2022

Table 15: Incidence and severity on tomatoes (Fruits) - April 25, 2022

Table 16: Incidence and severity on tomato leaves - June 5, 2022

Table 17: Incidence and severity on tomatoes (Fruits) - June 5, 2022

The results show the efficacy of PP1 against tomato virus disease (TSWV). Indeed, the notations on leaves and on fruits show that the treated plants have significantly fewer symptoms than the control plants. The progression of the disease is very slowed down by foliar treatments of PP1.

Sixth demonstration: Arugula extract on tomato mosaic virus (ToMV). Tomato mosaic virus (ToMV) is present on all continents. It is frequently found on tomato and pepper. It is serious both in open field cultivation and under cover. While its incidence has decreased significantly with the spread of resistant tomato varieties, the recent marketing of new susceptible varieties has shown how much ToMV is always ready to attack susceptible plant material.

No biocontrol solution is known to date to eradicate the disease.

In this context, a trial is carried out to evaluate the efficacy of PP1 against the tomato ToMV virus.

The symptoms caused by the presence of this virus are very varied and are quite comparable. A slowdown in plant growth can be observed, as well as coloring anomalies that can appear on the leaflets and leaves. Other symptoms can also be expressed on the leaves, such as thinning of the veins, mottling, a mosaic of green or yellow patches with the leaf blade wrinkling and contracting.

You can also see the fall of flowers. When the fruits reach maturity, they are reduced in size and sometimes more or less bumpy. They also express yellow discolorations, sometimes in rings. These symptoms may be present on green or ripe fruit when the plant appears healthy. Late infections have no effect on production.

The extracts of rocket leaves (Diplotaxis) are obtained according to the protocol illustrated in FIG. 1 in their non-concentrated liquid form (extracts called “PP1” below).

The experiment takes place in a greenhouse, above-ground cultivation.

Technical itinerary: planting the buckets on February 5, 2020. Harvest over five months from the beginning of March to the end of July 2020.

Modalities: Controls (untreated), PP1 (foliar spray)

Treatments: Foliar spray - Six applications of PP1, at a rate of 14 days.

Analysis method: Analysis of variance with a risk threshold of 5%. Ratings with the same letters are not significantly different.

Results :

Table 18: Incidence and severity on tomato leaves - March 5, 2020

Table 19: Incidence and severity on tomatoes (Fruits) - March 5, 2020

Table 20: Incidence and severity on tomato leaves - June 30, 2020

Table 21: Incidence and severity on tomatoes (Fruits) - June 30, 2020

The results show the efficacy of PP1 against tomato mosaic virus (ToMV). Indeed, the notations on leaves and on fruits show that the treated plants have significantly fewer symptoms than the control plants. The progression of the disease is very slowed down by foliar treatments of PP1.

Seventh demonstration: Arugula extract on Zucchini yellow mosaic virus, or ZYMV.

ZYMV is a non-persistent potyvirus transmitted by aphids. It is one of the best examples of viruses emerging in plants. Isolated for the first time in Italy and then in France in the 1970s, it spread throughout the world in a few years, sometimes causing epidemics of exceptional gravity. This recent and rapid dissemination in various types of cultivation (intensive, extensive, under shelter, open field) and very varied ecosystems (temperate, tropical, Sahelian, island) is attested by the fact that ZYMV causes very strong symptoms. ZYMV is now reported on cucurbits in virtually all of their production areas worldwide. However, its frequency can vary greatly depending on the region. Regularly encountered in tropical or subtropical regions, its epidemics are more irregular in temperate countries such as France. A survey carried out from 2004 to 2008 in the main French production areas showed that ZYMV was only present in 11% of 2660 samples analyzed, mainly on squash (23% of the samples tested), zucchini (14%) and melon. (8%), and to a lesser extent on cucumber (3%). In the areas where this virus was detected, the epidemics were generally severe, with a strong impact on yield. ZYMV causes very severe symptoms of mosaic, yellowing, stunting and distortion on the foliage of virtually all cucurbits. It also causes discoloration and spectacular deformations of the fruits which are then unmarketable. Early attacks can result in total crop loss.

The experiment takes place in open fields.

Technical itinerary: planting the buckets on April 15, 2022. Harvest on June 30, 2022.

Modalities: Controls (untreated), PP1 (foliar spray)

Treatments: Foliar spray - Six applications of PP1, at a rate of 14 days. Analysis method: Analysis of variance with a risk threshold of 5%. Ratings with the same letters are not significantly different.

Results :

Table 22: Incidence and severity on courgette leaves - June 30, 2022

Table 23: Incidence and severity on courgettes (Fruits) - June 30, 2022

The results show the efficacy of PP1 against ZYMV zucchini yellow mosaic virus. Indeed, the notations on leaves and on fruits show that the treated plants have significantly fewer symptoms than the control plants. The progression of the disease is very slowed down by foliar treatments of PP1. Eighth demonstration: Radish extract (“PP4”) on rose mosaic virus.

Rose mosaic is a viral disease that affects roses (Rosa sp.). It is caused by several viruses of the llarvirus and Nepovirus genera which occur separately or more often in combination, which has led some authors to speak of the “rose mosaic viral complex”. In some cultivars, these viruses can cause flower variegation. Other infected cultivars may remain symptomless.

The disease is not lethal to roses, but infection has the effect of reducing plant vigor and weakening them, making them more vulnerable to transplant stress or winter injury.

This disease causes various symptoms on the leaves: ringspots, chlorotic lines, watermarks, mottling of the leaves, as well as yellow mosaic patterns.

Clues of the disease are: bright yellow zigzag patterns on the leaves, arranged symmetrically to the midrib; yellow to cream stains may be diffuse and marbling; localized browning may be reminiscent of leaf desiccation.

The radish extracts are obtained according to the protocol illustrated in FIG. 1 in their non-concentrated liquid form (extracts called “PP4” below).

The experiment takes place in a heated greenhouse.

Technical itinerary: experimentation carried out on four-year-old rose-producing roses. Six Applications, at a rate of 14 days.

Modalities: Controls (untreated), PP4 (foliar spray)

Results :

Table 24: Incidence and severity on leaves - June 30, 2022

The results show the effectiveness of the extract against rose mosaic virus. Indeed, the notations on leaves and on fruits show that the treated plants have significantly fewer symptoms than the control plants. The progression of the disease is very slowed down by foliar treatments of PP4.

The composition that is the subject of the present invention, hereinafter called “CEI” (including in particular PP1 to PP4) does not correspond to what the literature describes 1/ The CEI composition is extracted from leaves, stems, flowers, seeds and/or roots , according to a mode preferential extraction, with or without added water, according to the method described with reference to Figure 1. When used in the fields, the composition is preferably diluted in the spray tanks (tanks) to be sprayed at the foliar level (or other application methods described in the description of the uses)

2/ The CEI composition, obtained under these extraction conditions, has no direct antimicrobial activity.

In the process for producing CEI, the leaves, stems, seeds, roots and/or flowers are subject to an extraction of compounds, by a known technique, for example by pressing, by ultrasound, and/or by the use of solvents, in particular oily or aqueous.

In embodiments of this method, plant parts are ground up and heavily diluted in water. In embodiments of this method, plant parts are ground without adding water. Optionally, the filtered ground material is then formulated in the form of a powder, by nebulization in an ascending stream of hot dry air, preferably at a temperature below 60°C. Optionally, the extract in liquid form is sterilized by passage at high temperature for a very short time, according to known techniques.

The eliciting composition that is the subject of the invention is in particular used, by application, to stimulate the defenses of plants or trees and reduce the effects of viruses, in particular the beet yellows virus and the cucumber mosaic virus.

CEI acts by stimulating the defenses of the plants, and by allowing the treated plants to defend themselves against these viruses.

CEI can be defined as an elicitor, given that the molecules possessing the property of inducing within the plant a cascade of defense reactions against pathogenic agents are called elicitors.

The demonstration of the eliciting activity of defense mechanisms is also demonstrated at several levels: The demonstration of the production of defense molecules, such as jasmonic acid, salicylic acid, or even peroxidases, was carried out after treatment with CEI, under infection conditions on beet (Beta vulgaris subsp. vulgaris)

CEI has the particularity of stimulating the defenses of plants, and allowing them to react effectively, even in the case of invasive viruses, which are difficult to fight.

As illustrated in FIG. 1, in one embodiment, the method for manufacturing and using the composition that is the subject of the present invention comprises a step 105 of extracting an extract from said plants.

In embodiments, the extract is obtained from at least one plant containing at least one butyl-isothiocyanate. The inventor has observed that these isothiocyanates have favorable effects on plant growth while reducing the effects of viruses. In embodiments, the extract is obtained from at least one plant containing 1,3-thiazepane-2-thione. The inventor has observed that this compound which, due to its structure, is not an isothiocyanate has favorable effects on the growth of the plant while reducing the effects of viruses.

For example, this extraction is performed according to the following procedure:

- During a grinding step 110, the leaves, roots, stems, seeds and/or flowers of said plants are finely ground with running water, for fifteen minutes, in a suitable mixer device, in order to obtain a homogeneous ground material;

- During a filtration step 115, the ground material is filtered to separate the debris from the organs used and to obtain a green liquid without residue (the filtrate), which is the basis of the composition that is the subject of the invention or constitutes it.

Alternatively, no water is added before grinding the source plant parts. In a variant, at least one of the active principles of the ground material is obtained by oil extraction. In a variant, at least one of the active principles of the ground material is obtained by solvent extraction by mechanical extraction or by microwaves, or by extraction of cakes or pastes. In a variant, at least one of the active principles is obtained by mechanical extraction or microwave extraction.

As a variant, the extraction step 105 includes a step of compressing the leaves, roots, stems, seeds or flowers of said plants and collecting the extracted liquid, by simple gravity or by centrifugation. As a variant, a simple centrifugation is implemented during the extraction step 105, to extract the liquid from the parts of said plants used.

As set forth in the description which follows, the inventor has discovered that the use of this composition has a significant effect on the trees and plants mentioned above infected by the viruses mentioned above. The inventor has also discovered that the eliciting composition that is the subject of the invention has biostimulating effects on the growth of the treated plants without constituting, at the doses used, a fertilizer or nourishing the treated plants.

It is noted that the liquid composition obtained at the end of step 105 can be formulated to make it easier to use. For example, it can be used in the form of powder, soluble powder, wettable powder, granules, dispersible granules, or wettable or slow-release granules, to be diluted in water at the time of use, liquid, liquid soluble concentrate, emulsifiable concentrate, suspension concentrate, or ready to use, depending on the formulation chosen and the intended use or infused on a substrate dispersed in the soil of the culture. The formulations are made from the product of the extraction step 105 according to techniques known to those skilled in the art.

Active moieties can potentially be purified, by any means, to facilitate formulation. Different extraction steps can be added to improve its quality. The composition that is the subject of the invention can be diluted in water, depending on the dose required, at the time of its use. During an optional step 120, volatile extracts are removed from the extract obtained. For example, this extract is transformed into a powder, for example by nebulization and passage of the nebulized extract in a flow of hot air, preferably ascending.

With regard to the use, during step 125, the biostimulant is applied in any form whatsoever (liquid formulation, powder, soluble powder, granules, dispersible granules, slow-dispersing granules, and all formulations) according to the uses and the formulation envisaged. The use of the biostimulant object of the invention is preferably carried out by foliar application or foliar spraying. Other modes of use of the biostimulant that is the subject of the invention are soil watering, soil irrigation, drip, hydroponic crops, or else in seed treatment and/or seed coating.

Preferably, the leaves and flowers of said plants represent at least 75%, preferably at least 95%, of the parts of said plants on which the extraction is carried out, percentage by dry weight, relative to the total weight of these plants.

The composition that is the subject of the invention can be used for a single application or at a rate of between one day and one hundred and twenty days, or continuously, or according to the key stages of plant development, or in accordance with good agricultural and the scheduled treatment schedule for each plant species. The composition of the present invention can be mixed with other products (phytosanitary products, crop supports and fertilizing materials, fertilizers, fertilizers, biocides, or any other product intended for agriculture).

Application doses and application rates are adapted to plant uses and models. The application doses are included, for example, between 0.001 g/L and 2000 g/L of extracted plants, preferably between 2 g/L and 2000 g/L of extracted plants and, more preferably, between 5 g/ L and 200 g/L of plants extracted, expressed in grams of plants on which the extraction was carried out per liter of product applied.

The doses per liter or per hectare can be adapted to the types of plants infected, the level of infection and the level of symptoms caused by the virus. The doses and the rates of treatment with the composition that is the subject of the invention will also be adapted to the strategy of preventive or curative action against these viruses.

Concerning the plants from which the extracts used in the present invention are taken, they are preferably freshly picked. Alternatively, the plants or the parts of interest are suitably dried, in a manner known to those skilled in the art. The crushing can be carried out with two crushers which are used with different blade speeds. The first ground product obtained in 10 min of grinding is then poured into the second grinder having a faster blade speed. The ground material is homogeneous, with no visible residue of parts of leaves, stems or flowers. The amount of water added during the grinding is between 0 and 200 mL of water, preferably between 20 and 150 mL of water, and, even more preferably, between 50 and 120 mL of water, at room temperature per 100 g of leaves, stem, root, flower or seed.

Two successive filtrations are carried out, with a nylon filtration cloth (Dutcher, registered trademark) 1000 μm then 500 μm. The filtration is carried out at room temperature, without pressure. For the recovery of the filtrate which is active, depending on the quantity to be sprayed, adapts the dilution (dose per hectare). According to usage, between 5 g of plants extracted per liter of mixture to be sprayed and 2000 g of plants extracted per liter of mixture to be sprayed, as described opposite the examples.

The inventor has observed that the filtrate obtained can be kept for at least six days in a container at room temperature, without losing its activity in stimulating the defenses of plants and trees.

The extract of at least part of said plants can thus be a liquid extract obtained from a ground material of said plants, and:

- said extract of at least one plant part comprises at least leaves of said plants, preferably essentially leaves, and

- the method for obtaining said liquid extract comprises the following steps: a) a step of grinding said plants in an aqueous medium; b) filtration of the ground material obtained; and c) recovering the liquid extract obtained after filtration.

Concerning the formulation in the form of powder, granules, dispersible granules, or granules with slow diffusion, a drying temperature is used, and, in embodiments, coatings of the particles with other natural molecules (preferably very hydrophilic ) which allow a very good dissolution in water. The formulations are conventional formulations in agriculture, in particular for phytosanitary products, intended to be transported and stored in the form of powder, etc... and to be, just before application, diluted in water. The present invention relates to the use of an eliciting composition comprising a plant extract obtained as described above to stimulate the defenses of plants or trees and reduce the effects of viruses on these plants.

In some embodiments, the eliciting composition that is the subject of the invention also comprises at least one of the following substances, obtained by synthesis or by extraction from plants, in particular the plants mentioned above:

- 1,3-thiazepane-2-thione, and/or

- a brassinosteroid.

Claims

1. Eliciting composition stimulating the defenses of plants or trees reducing the effects of an attack by a virus, characterized in that it comprises an extract of at least a part of at least one of the following plants:

- Rockets, including genera Eruca sativa, Diplotaxis, Erucastrum and Bunias,

- Cakile,

- plants of the Brassica oleracea species,

- plants of the Allium genera,

- mustard (Sinapis alba, Brassica nigra, Sinapis arvensis, Brassica juncea),

- wasabi (Eutrema japonicum),

- horseradish (Armoracia rusticana),

- watercress (Nasturtium officinale),

- plants of the Brassica rapa species,

- Plants of the Brassica ruvo species,

- Plants of the Brassica napus species,

- Plants of the species Raphanus sativus,

- Plants of the Barbarea verna species,

- Plants of the species Erysimum allionii,

- Plants of the species Erysimum cheiri,

- Plants of the species Tropaeolum majus L,

- Plants of the species Alliaria petiolata,

- Plants of the species Salvadora persica,

- Plants of the species Carica papaya.

2. Eliciting composition stimulating the defenses of plants or trees according to claim 1, in which the extract is obtained from at least one of the following plants: Arugula, including Eruca sativa, Diplotaxis, Erucastrum and Bunias, Cakile, broccoli , plants of the Allium genera (garlic, onion, shallot, leek and chives), common cabbage, cauliflower, Brussels sprouts, kale, mustard, wasabi, watercress, horseradish, white cabbage, field cabbage (Brassica rapa) , including bok choy cabbage, Chinese and turnip cabbage, kohlrabi, collard greens, red cabbage, Chinese broccoli, broccoli rabe, rapeseed, radish, Siberian wallflower, wallflower, Indian watercress, garlic grass, pilu tree, papaya tree, in particular its fruit.

3. Eliciting composition stimulating the defenses of plants or trees according to one of claims 1 or 2, in which the extract is obtained from at least one of the following plants: Arugula, including Eruca sativa, Diplotaxis, Erucastrum and Bunias, Cakile, broccoli, common cabbage, cauliflower, Brussels sprouts, kale, mustard, watercress, white cabbage, field cabbage (Brassica rapa), including bok choi, Chinese and turnip cabbage, turnip, collard greens, red cabbage, Chinese broccoli, broccoli rave, radish, and Siberian wallflower.

4. Eliciting composition stimulating the defenses of plants or trees according to one of claims 1 to 3, in which the extract is obtained from at least one of the following plants:

- Rockets, including Eruca sativa, Diplotaxis, Erucastrum and Bunias,

- Cakile.

5. Elicitor composition stimulating the defenses of plants or trees according to one of claims 1 to 3, in which the extract is obtained from at least one of the plants of the species Brassica oleracea.

6. Eliciting composition stimulating the defenses of plants or trees according to one of claims 1 to 5, in which the extract is obtained from at least one plant not containing Methyl-isothiocyanate and/or Propenyl isothiocyanate.

7. Elicitor composition stimulating the defenses of plants or trees according to one of claims 1 to 6, in which the extract is obtained from at least one plant containing at least one butyl-isothiocyanate.

8. Elicitor composition stimulating the defenses of plants or trees according to one of claims 1 to 7, in which the extract is obtained from at least one plant containing 1, 3-thiazepane-2-thione.

9. Eliciting composition stimulating the defenses of plants or trees according to one of claims 1 to 8, in which said extract of at least one part of plants is an extract obtained from a ground material of said plants, and:

- said extract of at least one plant part comprises at least leaves of said plants, preferably essentially leaves and flowers, and

10. Eliciting composition according to claim 9, the extract is obtained by a process comprising, in addition, a step of nebulization of the liquid extract and of passage of the nebulized liquid extract in a flow of hot air.

11 . Method for reducing the effects of viruses on plants, including trees, comprising a step of applying the eliciting composition according to one of Claims 1 to 10.

12. Method according to claim 11, for reducing the effects of an attack by one of the following viruses:

- Beet yellows viruses,

- Tobacco mosaic virus,

- Cassava mosaic virus,

- Banana Bunchy Top Virus (BBTV),

- Banana streak virus (BSV),

- Barley yellow dwarf virus (JNO or BYDV),

- Cucumber mosaic virus,

- Sugarcane mosaic virus (SCMV),

- Maize lethal necrosis disease viruses (MLN, MCMV, SCMV),

- The Potyviridae family of viruses,

- Sweet potato feathery mottle virus (Potyvirus), or SPFMV,

- Sweet potato chlorotic stunting virus (Crinivirus), or SPCSV and SPVD,

- Sweet potato moderate mottle virus, or SPMMV,

- Sweet potato latent virus, or SPLV,

- Sweet potato chlorotic spot virus or SPCFV,

- Sweet potato virus G, or SPVG,

- Sweet potato leafroll virus, or SPLCV,

- Tomato brown rugose fruit virus (ToBRFV),

- Tomato spotted wilt virus (TSWV),

- Tomato mosaic virus (Tomato mosaic virus, ToMV),

- Zucchini yellow mosaic virus, or ZYMV,

- rose mosaic virus.

13. Process according to claim 11, for reducing the effects of an attack by at least one of the beet yellows viruses.

14. Method according to claim 11, for reducing the effects of an attack by at least one of the cucumber mosaic viruses.

15. Method according to one of claims 11 to 14, in which the application of the eliciting composition is a foliar application to the plants.

16. Use of the eliciting composition according to one of claims 1 to 10, to reduce, on plants, including trees, the effects of an attack by one of the following viruses:

- Beet yellows viruses,

- Tobacco mosaic virus,

- Cassava mosaic virus, - Banana Bunchy Top Virus (BBTV),

- Banana streak virus (BSV),

- Barley yellow dwarf virus (JNO or BYDV),

- Cucumber mosaic virus,

- Sugarcane mosaic virus (SCMV),

- Maize lethal necrosis disease viruses (MLN, MCMV, SCMV),

- The Potyviridae family of viruses,

- Sweet potato feathery mottle virus (Potyvirus), or SPFMV,

- Sweet potato chlorotic stunting virus (Crinivirus), or SPCSV and SPVD,

- Sweet potato moderate mottle virus, or SPMMV,

- Sweet potato latent virus, or SPLV,

- Sweet potato chlorotic spot virus or SPCFV,

- Sweet potato virus G, or SPVG,

- Sweet potato leafroll virus, or SPLCV,

- Tomato brown rugose fruit virus (ToBRFV),

- Tomato spotted wilt virus (TSWV),

- Tomato mosaic virus (Tomato mosaic virus, ToMV),

- Zucchini yellow mosaic virus, or ZYMV,

- Rose mosaic virus.