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    • 专利摘要:
    Process for imparting protection against a pathogen by elicitation to a plant by means of an aqueous composition whose active agents are extracted from edible mushrooms (coulemelle, oyster mushroom, Paris mushroom). Alkaline extraction followed by enzymatic hydrolysis to obtain a fraction less than 100 kDa. The process consists in spraying the aerial parts of the plant with the composition thus obtained; it acts to fight against fungal diseases of plants such as vines, fruit trees, vegetable crops and cereals.
    公开号:FR3057438A1
    申请号:FR1660000
    申请日:2016-10-14
    公开日:2018-04-20
    发明作者:Celine Faugeron；Vincent Gloaguen；Cedric Astier；Charlotte MOINE
    申请人:Covertis；Universite de Limoges；
    IPC主号:
    专利说明:

    (54) PROCESS FOR ELICITATION OF AN EDIBLE MACROSCOPIC.
    FR 3 057 438 - A1
    Process for conferring by elicitation on a plant protection against a pathogenic agent by means of an aqueous composition whose active agents are extracted from edible fungi (coulemelle, oyster mushroom, button mushroom). An alkaline extraction is carried out then an enzymatic hydrolysis to obtain a fraction of less than 100 kDa. The method consists in spraying the aerial parts of the plant with the composition thus obtained; this is done to fight against cryptogamic diseases of plants such as vines, fruit trees, vegetable crops and cereals.
    Time Course)
    Method of elicitation of a plant by means of extracts of edible macroscopic fungi
    The subject of the invention is a method for stimulating the natural defenses of plants of the genus which are agronomically useful. These are in particular, the vine, fruit trees, such as the apple tree, vegetable plants and in particular tomatoes and potatoes, or cereals, such as wheat.
    Stimulating the natural defenses of plants is a very current problem, which has been and still is the subject of much research. In fact, plants are capable of developing physiological or metabolic responses allowing them to defend themselves during attacks by pathogenic agents such as: viruses, bacteria, fungi or insects. These natural defenses are of several types:
    the strengthening of pre-existing cell barriers by stimulating the lignification of the walls of plant cells;
    the synthesis by the plant of compounds with antibiotic activity such as phytoalexins;
    the synthesis of enzymatic proteins that can attack the wall of pathogens such as chitinases or glucanases.
    These natural defenses can be triggered by elicitor molecules, even at low concentration. Among these elicitors are, for example, oligosaccharides, in particular oligogalacturonates, oligoglucans or oligochitines. These molecules can be produced in the context of an attack by a pathogen and are perceived by the attacked plant as a signal to which it responds by stimulating its natural defenses.
    An exogenous supply of a solution of elicitor (s), for example in the form of a spray, can also stimulate the natural defenses of the treated plant and thus make it possible to reduce the symptoms of the diseases caused by these pathogens.
    International application WO 2007/042557 relates to phytopharmaceutical compositions intended to stimulate the natural defenses of plants, in particular the vine, the tomato and the potato, thus making it possible to combat plant diseases, in particular powdery mildew and downy mildew. This application describes a composition characterized in that it comprises, as active principle, at least one oligosaccharide secreted by or obtained from a strain of pathogenic or non-pathogenic fungus of plants. However, the only fungi described in this application are plant pathogenic fungi.
    International application WO 2004/082381 describes a process for stimulating the natural defenses of monocots (cereals) and dicots (vines, tobacco, potatoes and tomatoes) in which the leaves or seeds of the plant are applied. treated plant an effective amount of at least one of the products of the pentosan group in the form of a composition, the concentration of at least one of the products of the group is between 50 mg / L and 5000 mg / L. The process uses arabinoxylans and optionally hydrolyzed xylans, but the applicant of the present application wishes to avoid elicitors of this type, which have low efficiencies for the protection of the treated plants.
    International application WO 2004/082380 describes a process for potentiating and simulating the natural defenses of monocots and dicots, in particular of the vine, the potato and the tomato. The active ingredient used in the process described is xanthan obtained from a bacterial process, but the depositor wishes to avoid elicitors from sources involving pathogenic organisms.
    In international application WO 1999/053761, the use of one or more antifungal and / or antibacterial and / or antiviral compounds has been described for amplifying (potentiating) the responses of plant defenses to pathogens, obtained by use in isolation from elicitor compounds. The antifungal and / or antibacterial and / or antiviral compounds (designated by “compounds B” in the aforementioned patent and used as potentiators) can be, for example, derivatives of phosphorous acid and the elicitor compounds (designated by “compounds A” in the aforementioned patent) can be, for example, oligosaccharides or oligopectins which can be extracted from algae or yeasts. In this document, the lists given for the compounds to be used for elicitors A, cite non-phytotoxic products; but the applicant considers that compounds B, in addition to being synthetic compounds, can exhibit phytotoxicity and that, if they are excluded, there is no reason to suppose that the results announced for non-phytotoxic elicitors would actually be achieved.
    In all the documents of the prior art cited above, one certainly obtains an improvement in the defenses of the plants to be treated compared to a control. However, the applicant of the present application considers that the processes described are not satisfactory because he wishes there to be no phytotoxicity of the active principle and that the reduction of the needs for additional treatment is not carried out by pathogenic fungi synthetic plants or fungicides.
    International application WO 9745018 relates to an agent for inducing resistance against phytopathogenic microorganisms in plants. This agent is an extract of biomass from micro-organisms that are not pathogenic for plants. In a described embodiment, the agent is a constituent of fungal biomass, derived from biotechnological waste from a fermentation process; this agent can be a fungus. However, the applicant considers that this document is not relevant to the state of the art of the invention targeted by the present application, since the composition used comes from a microbial biomass.
    International application WO 99-033346, which gave rise to European patent 1001678, describes a process capable of conferring on an agronomically useful plant acquired systemic resistance, that is to say immunity with respect to an agent. pathogen, immunity which results when the plant is in contact with said pathogen, by potentiating the natural defenses; in this process, said plant is treated before contact with said pathogenic agent, by application in particular foliar, of a liquid composition comprising an oligo-β-1,3-glucan composed of 3 to 250 saccharin units. The concentration of the liquid composition in oligo-β1,3-glucans is lower than the concentration at which said oligo-β-1,3-glucan acts as an elicitor, i.e. at which it directly induces defense reactions. It is indicated that the oligo-β-1,3-glucans can be extracted from various fungi, a list of which is provided in the aforementioned European patent 1001678 (see the passage on page 4, line 54 to page 5, line 4). Example 1 indicates that the β-1,3-glucans used are extracted from a bacterium; Example 2 indicates that the raw material is a seaweed; Example 3 indicates that the raw material is zymosan; Example 4 indicates that the β-1,3-glucan consists of lichenan, which is a hydrolyzate extracted from the fungus Cetraria islandica. However, the applicant of the present application proposes to preferably use non-phytotoxic extracts of macroscopic fungi rather than extracts of algae or microorganisms. Furthermore, those skilled in the art know that the molecular organization of the cell wall of macroscopic fungi comprises a large number of layers forming a skeletal structure in which there are mannoproteins linked via β1,6-glucans to β-1,3-glucans; this complex skeletal structure is reinforced by a small amount of chitin covalently linked to the deepest β-Ι ^ Ιυοθηθε of said structure (see in particular the publication Smits et al. 2001). Therefore, the applicant of the present application proposes, in an innovative way and through the use of mushrooms as sources, the use of oligoglucan and oligochitine / oligo-chitosans complexes (publication Desaki et al. Biosci. Biotechnol Biochem 2011) and / or mannoproteins (publication Basse et al. J. Biol. Chem. 1992) in order to take advantage of the synergy of eliciting effects between these different families of molecules.
    The aim pursued by the applicant is, therefore, to propose a new process for stimulating the natural defenses of plants and in particular an active process for tomatoes, potatoes, vines, wheat and apple trees. The method according to the invention allows an improvement of the defenses compared to an untreated control with a reduction in the number of successive treatments to be carried out; the composition to be used is obtained by a process which results in a non-phytotoxic product and the use of this composition results in a reduction, or even a suppression, of the needs for additional treatments with synthetic fungicides.
    This composition is obtained from coulemelle, oyster mushroom or button mushroom; it therefore comprises a whole series of products extracted from mushrooms serving as raw material and in particular, as might be expected by studying the state of the art, β-Ι, β-glucans. The composition obtained according to the invention corresponds to a particular formulation of the various products present, which makes it possible to obtain, when applied to the plant to be treated, results superior to those obtained in the state of the technique using only β1,3-glucans. The tests carried out on the tomato show a 35% increase in defense activities, following treatment with the composition obtained by the process according to the invention; tests on potatoes show a reduction of around 40% of blemish spots on the foliage compared to an untreated control. The tests carried out on the vine show a 100% increase in the defense activity following the treatment according to the invention, when compared with an untreated control. This demonstrates the interest and the patentability of the process, which proposes the implementation of the new composition according to the invention.
    For purely indicative purposes and without this in any way constituting a limitation as to the composition used by the method according to the invention, it is possible to indicate, as a preliminary, that the composition according to the invention generally comprises a rate of proteins of approximately 10% relative to the dry matter, a total sugar content of approximately 75% relative to the dry matter, and a monosaccharide composition of approximately 70 to 76 mol% of glucose, approximately 13% galactose molar and about 11% molar mannose; its β-glucan level is generally around 54% relative to the dry matter and its polysaccharide fraction is around 40 to 75% of the dry extract, this fraction having a molecular mass greater than 10 kDa. It is all of these characteristics which make it possible, when the composition applied by the process according to the invention is applied to plants, to obtain new and particularly advantageous treatment results.
    The present invention therefore has as its object a method capable of conferring by elicitation on an agronomically useful or ornamental plant, protection against a pathogenic agent, said method consisting in treating at least some of the aerial parts of said plant by an aqueous composition, the essential active agents of which are obtained by extraction from fungi, characterized in that the active agents of said composition are obtained:
    a) by subjecting a macroscopic mushroom powder to an alkaline extraction and then neutralizing the extract obtained before carrying out its filtration;
    b) by diluting the filtered extract and subjecting it to a glycosidase between 10 and 65 ° C, the hydrolysis thus practiced being stopped by inactivating the enzyme at a temperature above 65 ° C;
    c) by subjecting the hydrolyzate obtained to a filtration step in order to isolate an active fraction thereof with a molecular weight of less than 100 kDa.
    According to one embodiment, the method according to the invention is characterized in that the alkaline extraction step is carried out at a temperature between 20 and 100 ° C, while maintaining a suspension of 5 to 15 parts with stirring by weight of mushroom powder in an alkaline aqueous solution having a pH greater than 10; the neutralization of the solution obtained by the alkaline extraction step can be carried out with an organic or mineral acid, the precipitate obtained being removed; the product obtained after neutralization of the alkaline extract can be subjected to several filtrations, to separate from the liquid phase the acid salt (s) and the simple sugars.
    In one embodiment of the method according to the invention, the enzymatic hydrolysis carried out with glycosidase is maintained for 24 hours with stirring.
    In one embodiment of the method according to the invention, the edible mushrooms subjected to an alkaline extraction are species chosen from the group formed by oyster mushrooms, coulemelles and button mushrooms.
    In one embodiment of the process according to the invention, the aerial parts of the plants to be treated are sprayed with a composition having a pH of between 4 and 8, the concentration of active agents of which is 1.5 to 4 % in weight.
    In one embodiment of the method according to the invention, the composition, with which the aerial parts of the plant to be treated are sprayed, comprises a protein content of 5 to 15% by weight relative to its dry matter.
    In one embodiment of the method according to the invention, the composition, with which the aerial parts of the plant to be treated are sprayed, comprises a total sugar content of 60 to 80% by weight relative to the dry matter.
    In one embodiment of the method according to the invention, the composition, with which the aerial parts of the plant to be treated are sprayed, has a monosaccharide formulation corresponding to a weight proportion of 38 to 50% of glucose, from 18 to 28% galactose, 20 to 30% mannose and 0.5 to 4% glucosamine and / or acetylated glucosamine.
    In one embodiment of the method according to the invention, the composition, with which the aerial parts of the plant to be treated are sprayed, contains 35 to 45% by weight of glucans relative to its dry matter.
    In one embodiment of the method according to the invention, the composition, with which it is sprayed on the aerial parts of the plant to be treated, contains from 30 to 50% by weight of a dry matter, the molecular mass of which is greater than 10 kDa.
    In an embodiment of the method according to the invention, said method is associated with a treatment of the aerial parts of the treated plant with a conventional phytosanitary agent applied simultaneously or sequentially or alternately.
    In one embodiment of the method according to the invention, said method constitutes a preventive treatment against cryptogamic diseases, in particular those chosen from the group formed by diseases of the vine, fruit trees, vegetable crops and cereals, and in particular against downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator), gray rot (Botrytis cinerea) in the vine, downy mildew (Phytophthora infestans) of potatoes, yellow rust (Puccinia striiformis), Septoria leaf spot (Septoria sp.), Fusarium head blight (Fusarium graminearum) in wheat and apple scab (Venturia inaequalis).
    In one embodiment of the method according to the invention, said implementation is carried out at the early vegetative stage and / or at the adult and reproductive vegetative stages.
    In one embodiment of the method according to the invention, said implementation is carried out at repeated time intervals, as often as necessary, and preferably every eight to fifteen days.
    The subject of the invention is also a composition for implementing the method defined above, characterized in that it is obtained:
    a) by subjecting an edible macroscopic mushroom powder to an alkaline extraction and then neutralizing the extract obtained before carrying out its filtration;
    b) by diluting the filtered extract and subjecting it to a glycosidase between 10 and 65 ° C, the hydrolysis thus practiced being stopped by inactivating the enzyme at a temperature above 65 ° C;
    c) by subjecting the hydrolyzate obtained to a filtration step in order to isolate an active fraction thereof with a molecular weight of less than 100 kDa.
    In a preferred embodiment, the composition according to the invention is characterized in that the alkaline extraction step is carried out at a temperature between 20 and 100 ° C, while maintaining under suspension a suspension of 5 to 15 parts in weight of mushroom powder in an alkaline aqueous solution having a pH greater than 10.
    In one embodiment of the composition according to the invention, the product obtained after neutralization of the alkaline extract is subjected to several filtrations, in order to separate the acid salt (s) and the sugars from the liquid phase simple.
    In one embodiment, the composition according to the invention is obtained by carrying out the enzymatic hydrolysis with glycosidase for 24 hours with stirring.
    In one embodiment of the composition according to the invention, the edible mushrooms subjected to an alkaline extraction, are species chosen from the group formed by oyster mushrooms, coulemelles and button mushrooms.
    In one embodiment of the composition according to the invention, the concentration of said composition in active agents is from 150 to 400 mg / L.
    In one embodiment, the composition according to the invention comprises a protein level of 5 to 15% by weight relative to its dry matter.
    In one embodiment, the composition according to the invention comprises a total sugar content of 60 to 80% by weight relative to its dry matter.
    In one embodiment, the composition according to the invention has a monosaccharide formulation corresponding to a weight proportion of 38 to 50% of glucose, from 18 to 28% of galactose, from 20 to 30% of mannose and from 0.5 to 4 % of glucosamine and / or acetylated glucosamine.
    In one embodiment, the composition according to the invention contains 35 to 45% by weight of glucans relative to its dry matter.
    In one embodiment, the composition according to the invention contains from 30 to 50% by weight of a dry matter, the molecular mass of which is greater than 10 kDa.
    In one embodiment, the composition according to the invention contains at least one compatible formulation agent.
    In one embodiment, the composition according to the invention comprises at least one anti-phytopathogenic agent chosen from the group formed by a fungicidal agent, an antibacterial agent, an antiviral agent, a pesticidal agent and a biocontrol agent.
    In one embodiment, the composition according to the invention contains at least one nutrient for plants with a view to its simultaneous application.
    To illustrate the embodiment and the implementation of the invention, three examples of the manufacture of dry extracts of three compositions according to the invention and seven examples of implementation of the treatment method according to the invention will be given below. invention. These examples are, of course, provided for purely illustrative and not limiting.
    EXAMPLES
    Example 1:
    100 g of dried and finely crushed coulelles (<1mm) are incorporated into 1 liter of 8% sodium hydroxide supplemented with 0.1% sodium borohydride. The suspension is kept under stirring at room temperature for 24 hours. By adding a solution of glacial acetic acid, the medium is brought to pH 6.5. A glucanase is added, at a rate of 0.1% m / V and the whole is kept under stirring at room temperature for 20 hours. The hydrolyzate is centrifuged and then filtered. The solution is then ultrafiltered in a tangential ultrafiltration system of the Pellicon mini type sold by the company Millipore equipped with a membrane with a porosity of 100 kDa. Then the permeate obtained is subjected to an ultrafiltration on a 1 kDa membrane to desalt the solution. The solution is concentrated and then lyophilized. 7 g of a beige powder are thus obtained which constitutes the hydrolyzate EX1.
    Example 2:
    The raw material consists of the feet of Pleurotus Ostreatus mushrooms. 200 g of dried and finely ground oyster mushroom stalks (<1mm) are dispersed in 2L of 8% sodium hydroxide, in the presence of 0.1% sodium borohydride. The extraction is carried out at room temperature, for 24 hours, with mechanical stirring. By centrifugation, the fraction insoluble in NaOH / NaBH 4 is removed. The supernatant obtained is then neutralized with glacial acetic acid. The chosen enzymatic system is a βglucanase from Aspergillus niger, and added to the extract at a rate of 0.1% m / V. The whole is kept at room temperature for 3 hours. After denaturation at 100 ° C for 10 min, the medium is centrifuged. The extract obtained is clarified by filtration. The resulting filtrate is subjected to microfiltration on a membrane with a porosity of 0.1 μm and then on a membrane of 100 kDa at a pressure of 1 Bar. The system used is a tangential ultrafiltration system of the Pellicon mini type sold by the company Millipore. This permeate is subjected to ultrafiltration on a 1 kDa membrane at a pressure of 2 bars to desalt the solution. A retentate is thus obtained, which is then lyophilized. 15 g of a beige powder constituting the EX2 extract are obtained.
    Example 3:
    The raw material consists of button mushrooms. 100 g of dried and finely ground mushrooms (<1mm) are dispersed in 1.5L of 8% sodium hydroxide, in the presence of 0.1% sodium borohydride. The extraction is carried out at room temperature for 24 hours, with magnetic stirring. The medium is then neutralized using acetic acid. After centrifugation, the supernatant is ultrafiltered to 1 kDa. A β-glucanase is added to the extract at the rate of 0.1 m / V. The whole is kept under stirring at room temperature for 24 hours. After denaturation at 100 ° C for 10 min, the medium is centrifuged. This permeate is subjected to ultrafiltration on a 1 kDa membrane. A retentate is thus obtained, which is then lyophilized. 1 g of a beige powder constituting the EX3 extract is obtained.
    Example 4:
    Study showing the stimulation of defenses in tomatoes by oyster mushroom EX2 extract (see Figures 1A to 1 D)
    - Tomato plants of the Marmande variety grown in a greenhouse are used at the 2-leaf stage (BBCH 12 102). The control batch consists of 24 plants distributed in 12 pots; the same for the batch treated.
    -A t = 0, each plant is treated with oyster extract EX2 prepared at a concentration of 0.4 g / L, by foliar spray until runoff.
    -every day and for 7 days, plants are harvested, then separated into roots and aerial parts. The proteins are extracted in a suitable buffer and the phenylalanine ammonia lyase (PAL) and peroxidase enzymatic activities are measured in the corresponding extracts. These activities constitute plant defense markers: PAL is involved in the biosynthesis of phytoalexins, peroxidase is involved in the formation of lignins constituting the wall of plant cells.
    -The specific enzymatic activities are expressed as a percentage of the value obtained for the treated plants reduced to those of the untreated control plants.
    Figure 1 shows the evolution of phenylalanine ammonia lyase and peroxidase activities at the roots and aerial parts of tomato plants sprayed with oyster mushroom extract. Data are expressed as% of the untreated control. The representations provided in Figures 1A-1D are as follows:
    Fig. 1A: PAL roots activity
    Fig. 1B: PAL aerial parts activity
    Fig. 1C: Root peroxidase activity
    Fig. 1D: Peroxidase activity in aerial parts
    FIGS. 1A-1D show that between one and two days after application of the extract, an increase in PAL activity is observed both in the roots and in the aerial parts, which suggests a systemic elicitor action of the oyster extract. Regarding the peroxidase activity, it appears more intense between 3 and 7 days suggesting a later systemic response in this case.
    Example 5:
    Study showing the stimulation of defenses in tomatoes by coulemelle extract in the presence of Botrytis cinerea (see Figure 2)
    - Tomato plants of the Marmande variety grown in a greenhouse are used at the 2-leaf stage (BBCH 12 102). Each batch of plants consists of 6 plants distributed in 3 pots.
    -A t = 0, each plant is treated with EX1 coulemelle extract in the form of a 0.4 g / L solution by foliar spray until runoff. The operation is repeated at t = 2 days and t = 5 days. Control plants are treated at the same frequency but with an aqueous solution of surfactant sold under the brand "Tween" at 0.5% by weight.
    -A t = 7 days, the plants are inoculated with the pathogenic fungus (Botrytis cinerea, strain UBOCC-A101100 from the University of Western Brittany). A suspension of conidia (approximately 10 5 conidia / ml) in aqueous solution of surfactant sold under the brand name “Tween” at 0.5% by weight is deposited on several sheets in the form of regularly distributed droplets (100 μL / plant).
    -At = 14 days, the plants are harvested by removing leaf discs from each plant, which are frozen at -80 ° C. The NRAs are extracted from the leaf samples.
    -The expression levels of defense genes are quantified by amplification techniques by RT-PCR (Reverse Transcription Polymerase Chain Reaction) using specific molecular probes of known and chosen genes.
    -These genes correspond to that coding for PAL and to several pathogenesis proteins (PR proteins): PR1 (protein marker of plant systemic response), PR2 (protein of glucanase type), and PR3 (protein of chitinase type).
    -The gene expression levels are quantified by reference to a constituent gene, that of tubulin. The results are expressed as a ratio between the level of expression of the gene linked to defense processes and the level of expression of tubulin.
    FIG. 2 shows the expression of the genes associated with the defense reactions (PAL: phenylalanine ammoni lyase, PR1, PR2 and PR3: Pathogenesis Related) at the level of the leaves of tomato plants which have been treated either with an aqueous solution at 0, 5% by weight of the surfactant sold under the brand name "Tween" (control), either by the coulemelle extract, then inoculated with Botrytis cinerea. Data are expressed as a ratio to the expression of the tubulin gene.
    The control shows activation of the defense genes, particularly PAL and PR1 (PR3 and PR2 being less strongly activated) due to the presence of the pathogen (Figure 2). However, pretreatment with coulemelle extract causes greater expression of the four defense genes studied, in particular PR1, described as associated with the systemic response of plants. Thus, the stimulating activity of natural defenses is well proven for this extract.
    Example 6:
    Study showing stimulation of defenses in apple trees with oyster mushroom extract (see Figure 3)
    - Apple plants (Malus domestica, var. Golden delicious) were obtained in a greenhouse (20-25 ° C, natural photoperiod) from germination. They are used at the 4-6 leaf stage and arranged in blocks of 30 plants per test modality.
    -Each block receives the treatment by foliar spray until runoff. Oyster mushroom extract is applied at a concentration of 0.7 g / L of dry matter.
    -The blocks designated by “+ H 2 0 2 blocks” are treated at D = 1 by a spraying of hydrogen peroxide to simulate an attack by a pathogen (patent WO 2011/161388) .- At D3, leaf discs are taken from plants (10 discs per method). After freezing in liquid nitrogen, the RNAs are extracted, and the expression of the defense genes (the list of which is presented in Table 1) is measured by quantitative PCR using the qPFD® tool (patent mentioned above ).
    -The relative expression levels were calculated with the so-called 2 _ΔΔ0 'method: these are relative expressions compared to the EAU control, normalized by the geometric mean of the relative expressions of 3 reference genes (TuA, Actin, GAPDH). These relative expressions are transformed into Iog2 to give the same weight to inductions and repressions of genes.
    Table 1: Names of defense genes and defense pathways with which they are associated
    Types of defense Gene names PR proteins PR4 Pathogenesis-related protein 4(heve-like) PRM Pathogenesis-related protein 14(lipid transfer protein) PR15 Pathogenesis-related protein 15(oxalate oxidase) Phenylpropanoid route PAL Phenylalanine ammonia-lyase PPO Polyphenol oxidase Cysteine pathway CSL alliinase Oxidative stress Apox Ascorbate peroxidase POX Peroxidase Salicylic acid route EDS1 Disease resistant protein 1 WRKY WRKY transcription factor 30 Jasmonic acid pathway JAR Resistant Jasmonate 1 Ethylene route ACCO 1 -aminocyclopropene-1 -carboxylateoxidase EIN3 EIN3-binding F box protein 1
    FIG. 3 shows the expression of the genes associated with defense reactions at the level of apple leaves which have been pretreated with oyster mushroom extract (JO) and then treated with H 2 O 2 (J1). The data are expressed as a ratio relative to the expression of reference genes (tubulin, Actin, ...).
    Pretreatment with oyster mushroom extract stimulates the expression of many genes associated with different defense pathways (phenylpropanoid pathway, oxidative stress, hormone signaling pathway, etc.), which confirms the eliciting effect of the extract. allowing to increase the defense responses in the presence of pathogens.
    Example 7:
    Study showing the stimulation of the defenses in the vine by the coulemelle extract in the presence of Botrytis cinerea (see Figure 4)
    -Young vine plants (Cabernet Sauvignon grape) from nurseries are grown in a greenhouse (20-25 ° C, natural photoperiod). They are used when they have formed at least one branch carrying 6 well-developed leaves (stage BBCH 16).
    At t = 0, the aerial shoots are treated with a foliar spray of the coulemelle extract (1 spray of 200 μΙ_ of an aqueous solution at 0.4 g / L (dry matter) per spread sheet). The operation is repeated at t = 2 days and t = 5 days. Cookies are not processed.
    At t = 7 days, all the plants are inoculated with the pathogenic fungus (Botrytis cinerea, strain UBOCC-A101100 from the University of Western Brittany). A suspension of conidia (approximately 10 5 conidia / ml) in an aqueous solution at 0.5% by weight of the surfactant sold under the brand "Tween" is deposited on several sheets in the form of regularly distributed droplets (100 μL / plant) .
    At t = 14 days, the treated and inoculated leaves are harvested and undergo a protein extraction in a suitable buffer in order to measure the enzymatic activities phenylalanine ammonia lyase (PAL) and peroxidase in the corresponding extracts.
    -The specific enzymatic activities are expressed as a percentage of the value obtained for the treated plants reduced to those of the untreated control plants.
    FIG. 4 shows the phenylalanine ammonia lyase and peroxidase activities in the vine leaves treated by spraying with the extract of coulemelle or not (control) and then inoculated with Botrytis cinerea. Data are expressed as% of the untreated control.
    Strong stimulation of the PAL and peroxidase enzymatic activities is observed following the pre-treatment of vine plants with coulemelle extract; these activities are increased by 25% for PAL and multiplied by 5 for peroxidase.
    Example 8:
    Effectiveness of coulemelle extract on the reduction of symptoms linked to downy mildew on potatoes during field trials:
    -A full field trial was conducted on the potato (Solanum tuberosum, var. Ditta) during 2014 in Auchy-les-Mines (62, France) in order to determine the effectiveness of the extract protection for downy mildew (Phytophthora infestans). The plantation was carried out on 04/18/14.
    On the experimental plot, 4 micro-plots per treatment condition were distributed statistically;
    - Five treatments were carried out with the coulemelle extract: June 11 (40 cm stage), 06/18/14 (BBCH 60 stage), 06/25/14 (BBCH 70 stage) and 07/09/17 14 (stage BBCH 74).
    -In early season, the pressure of mildew was important which required the application of a synthetic fungicide, dithane (between June 1, stage 5 cm and 7 June 2014, stage 20 cm).
    -Then, given the less favorable weather conditions for the development of the disease thereafter, two artificial contaminations of the plot were carried out: the first by a suspension of spores from contaminated leaves from a pile of waste (30 / 05/14), the second on 02/06/14 by an inoculum from a laboratory (Eurofins, Loos en Gohelle). These two contaminations made it possible to obtain a uniform downy mildew pressure on the plot.
    The ratings presented in Figure 5 were made on July 4 at the BBCH 70 stage corresponding to the average number of spots per plant on the central rows. FIG. 5 shows the counting of spots appearing on the foliage of the potato plants having or not having undergone a pretreatment with the coulemelle extract (counting of 04/07/14). The trial was carried out in the open field after contamination with downy mildew (Phytophthora infestans). Pre-treatment with coulemelle extract (application of the equivalent of 35 g of dry extract per hectare) reduces the symptoms associated with mildew. The number of leaf spots is reduced by an average of 39%. The coulemelle extract has therefore shown its effectiveness in the open field in the event of medium downy mildew pressure.
    Example 9:
    Effectiveness of oyster mushroom extract on reducing symptoms of downy mildew (Plasmopara viticola) on the vine during full-field trials (see Figure 6)
    A full field trial was conducted on the vine (Vitis vinifera), Grenache noir grape variety) during 2015 in Villelongue del Monts (66, France) in order to determine the effectiveness of the oyster mushroom extract in protection vis-à-vis mildew (Plasmopara viticola), alone or alternately with Bordeaux mixture (BB) (750 g Cu / ha).
    -On the experimental plot, 4 micro-plots per treatment condition were distributed statistically; the test was conducted under controlled conditions with artificial contamination on May 17 limited to the 1st strain of each elementary plot and the general humidity was maintained by regular spraying.
    -The treatments carried out between 12/05/15 and 06/07/15 at a dose of 200 L / ha are presented in Table 2. The witness received no treatment; the “copper” treatment consists in applying between 12/05/15 and 06/07/15 Bordeaux mixture at the dose equivalent to 750 g Cu / ha (seven applications in total). EX2 oyster extract is applied in the form of an aqueous solution at 35 g / ha, either alone or alternately with Bordeaux mixture (oyster extract / Cu). A method intended to evaluate the effectiveness of treatments based on Bordeaux mixture at the same dose as the copper treatment but applied 3 times instead of 7, was also evaluated (Cu 14).
    Table 2 shows the treatment schedule for the different modalities (vineyard / late blight field trial).
    BB: Bordeaux mixture; Pie: oyster mushroom extract; -: no treatment.
    Table 2:
    Dates 05/12/15 5/18/15 05/28/15 06/05/15 06/16/15 6/26/15 07/06/15 BBCH stadiums 55 57 63 73 75 79 79 Tratitetenotts Witness Copper BB BB BB BB BB BB BB Extract ofoyster Magpie Magpie Magpie Magpie Magpie Magpie Magpie Extract ofoyster mushroom / Cu BB Magpie BB Magpie BB Magpie BB Cu 1/2 BB BB BB
    The ratings presented in Figure 6 were made on July 20 at stage 81 (start of veraison). The frequency of attacks is estimated by the percentage of clusters attacked by the disease.
    Figure 6 shows the surface area of the grapes attacked (%) on vines after treatment (or not) with oyster mushroom extract, as well as alternating with Bordeaux mixture (oyster mushroom extract / Cu). The trial was carried out in the open field after contamination with downy mildew (Plasmopara viticola). The efficiency expressed as a% of the control is indicated in the table appended to the figure.
    Oyster mushroom extract therefore reduces the symptoms of mildew attack. The efficacy rate of 57% is not as high as that of the copper treatment (89%) used here as a fungicide. However, the alternate treatment (oyster mushroom extract / Cu cuivre) has a higher level of efficacy (82%): it therefore makes it possible to offer an effective crop protection solution allowing a reduction in the use of the fungicide. The oyster mushroom extract applied alternately with copper shows a much higher level of efficacy (82%) than that of the 1 / a dose of copper, thus confirming the stimulation of the plant's defenses by oyster mushroom extract.
    Example 10:
    Effectiveness of oyster mushroom extract on reducing symptoms linked to powdery mildew (Erysiphe necator) on vines during full-field trials (see Figure 7).
    A full field trial was conducted on the vine (Vitis vinifera, Chardonnay grape) during 2015 in Alénya (66, France) in order to determine the effectiveness of the oyster extract EX2 in protection against - powdery mildew (Erysiphe necator), alone or alternately with Thiovit (8 kg / ha).
    -On the experimental plot, 4 micro-plots per treatment condition were distributed statistically;
    - Powdery mildew appeared relatively late on clusters after a very discreet phase on leaves. At the first count on June 25 (vegetative stage BBCH 77), a correct level of disease was noted in the untreated control plots with an attack of 70.50% in frequency and 30.25% in intensity. At the second count on July 23 (vegetative stage BBCH 85), an attack of 77.50% in frequency and 40.60% in intensity was observed.
    -The treatments were carried out between 04/17/15 and 06/26/15 at a dose of 180 L / ha and are presented in Table 3. The witness received no treatment. The "sulfur" treatment consists in applying, between 14/04/15 and 26/06/15, the product sold under the trade name "Thiovit" at a dose equivalent to 8 kg / ha (eight applications in total). The oyster mushroom extract EX2 is applied in the form of a 35 g / ha solution, either alone or alternately with the above-mentioned "Thiovit" (oyster mushroom / sulfur extract). A method intended to evaluate the effectiveness of treatment based on the aforementioned product "Thiovit" at the same dose as the sulfur treatment, but applied 4 times instead of 7 was also evaluated (S 14).
    The different methods are presented in Table 3 below.
    Table 3: Treatment schedule for the different methods (full-field vine / powdery mildew test)
    T1 T2 T3 T4 T5 T6 T7 T8 Dates 05/12/15 5/18/15 05/28/15 06/05/15 06/16/15 6/26/15 07/06/15 6/26/15 BBCH stadiums 15 53 55 61 73 75 77 77 Trattemenotts Witness - - - - - - - - sulfur S S S S S S S S Extract ofoyster Magpie Magpie Magpie Magpie Magpie Magpie Magpie Magpie Extract ofoyster mushroom S Magpie S Magpie S Magpie S Magpie S 1/2 S S S S
    FIG. 7 shows the surface area of the grape attacked (%) by powdery mildew on vines (Chardonnay grape variety) treated (or not) with oyster mushroom extract, as well as alternating with the above-mentioned "Thiovit" product (extract of Oyster / S). The efficiency is expressed in% of the control and indicated in the table attached to Figure 7.
    Oyster mushroom extract helps reduce symptoms of powdery mildew attack. The 65% efficacy rate is not as high as that of the sulfur treatment (92%) used here as a fungicide. However, the alternate treatment (oyster mushroom / sulfur extract) has a higher level of effectiveness (96%): it therefore makes it possible to offer an effective crop protection solution, which allows a reduction in the use of the fungicide. The oyster mushroom extract applied alternately with sulfur shows a much higher level of efficiency (96%) than that of the 1 / a dose of sulfur (87%), thus confirming the stimulation of the plant's defenses by oyster extract.
    It can therefore be seen that, in order to combat mildew or powdery mildew, the alternating fungicide / oyster mushroom program has shown a sufficient level of protection for the safe conduct of vine growing in the open field.
    Example 11:
    Study showing defense stimulation by oyster mushroom extract and button mushroom extract in Brachypodium distachyon in the presence of Fusarium graminearum.
    A model plant of temperate cereals, Brachypodium distachyon, was cultivated in a greenhouse and used for this test 4 weeks after sowing. A batch of 10 plants per repetition was formed and two biological repetitions were carried out.
    The pretreatments consisted in applying by spraying the oyster mushroom extract and the button mushroom extract of examples 2 and 3 at a concentration of 0.35 g / L, on all the ears on days 0, 2 and 5, this until runoff. Inoculation of Fusarium graminearum was carried out on day 7 (= mid anthesis stage, BBCH65), by spraying all the ears with a suspension of spores at 10 5 spores / ml.
    Harvesting of the plants takes place at t = 0, t = 24h or t = 48h. Several analyzes are implemented:
    Fusarium wilt symptoms were observed 10 days post-inoculation;
    The fungal biomass was quantified and expressed in ng of F. graminearum genomic DNA per 10 ng of total genomic DNA (plant + fungus) determined by quantitative PCR. Primers specific for the F. graminearum TUB2 gene were used to determine the amount of fungal genomic DNA by reference to a standard range.
    The expression of two B. distachyon defense genes was followed: the PAL gene coding for phenylalanine ammonialyase (1st enzyme in the phenylpropanoid biosynthesis pathway) and the PR9 gene coding for a “pathogenesis-related” protein (PR of peroxidase type). Two reference genes were used, ACT7 and UBC18. The expression of these two genes was determined in ears treated beforehand with the extract of oyster mushroom or button mushroom relative to control ears, inoculated with the PH-1 strain of F. graminearum.
    FIG. 8 represents the rating of the symptoms at 10 days post-inoculation in% of symptomatic spikelets. A spikelet is considered symptomatic if 50% or more of the floral cavities show symptoms of Fusarium wilt. The witness is an untreated ear of corn; the ears are treated prior to the inoculation of F. graminearum with extracts of oyster mushroom or button mushroom.
    The results show a significant reduction in the symptoms of Fusarium wilt on plants treated with mushroom extracts. Thus, oyster mushroom and button mushroom extracts provide protection against B. distachyon infection by F. graminearum by 77% and 94.7%, respectively.
    FIG. 9 represents the quantification of the fungal biomass expressed in ng of genomic DNA from F. graminearum per 10 ng of total genomic DNA (plant + fungus) determined by quantitative PCR. Primers specific for the TUB2 gene from F. graminearum were used to determine the amount of fungal genomic DNA by reference to a standard range. The asterisks indicate the significantly different values of the control (Student's f test, p-value <0.05).
    The quantification of the biomass of F. graminearum on the different ears indicates a negligible amount of fungal genomic DNA within the same amount of total genomic DNA (plant + fungus) on the ears pretreated with each of the two extracts by comparison with the control not treated (Figures 10A and 10B).
    FIGS. 10A and 10B represent the relative expression of the PAL (A) and PR9 (B) genes in ears of B. distachyon pretreated with oyster mushroom extracts (dot pattern bars), or button mushroom extract ( hatched bars) and inoculated with F. graminearum, then harvested 48 hours after post-inoculation. Relative expression was determined in relation to ears of B. distachyon not previously treated with the extracts, using the AACt method. Two reference genes of B. distachyon, constitutively expressed were used: ACT7 and UBC18.
    The results indicate that the relative expression of the PAL and PR9 genes is more induced 48 hours after inoculation with F. graminearum in the ears treated with oyster mushroom and button mushroom extracts than in the non-pretreated ears (FIGS. 10A and 10B). . In fact, the expression of the PAL gene is induced 5.2 times more following the application of the oyster mushroom extract and almost 3.5 times more following the application of the button mushroom extract. The results are even more pronounced for the PR9 gene, which has a 10.2 and 15.4 times greater induction following the application of extracts of Pleurote and button mushroom, respectively, compared to untreated ears.
    权利要求:
    Claims (30)
    [1]
    1. Method suitable for conferring by elicitation on an agronomically useful or ornamental plant protection against a pathogenic agent, said method consisting in treating at least some of the aerial parts of said plant with an aqueous composition, including the active agents essentials are obtained by extraction from fungi, characterized in that the active agents of said composition are obtained:
    a) by subjecting an edible macroscopic mushroom powder to an alkaline extraction and then neutralizing the extract obtained before carrying out its filtration;
    b) by diluting the filtered extract and subjecting it to a glycosidase between 10 and 65 ° C, the hydrolysis thus practiced being stopped by inactivating the enzyme at a temperature above 65 ° C;
    c) by subjecting the hydrolyzate obtained to a filtration step in order to isolate an active fraction thereof with a molecular weight of less than 100 kDa.
    [2]
    2. Method according to claim 1, characterized in that the alkaline extraction step is carried out at a temperature between 20 and 100 ° C, maintaining with stirring a suspension of 5 to 15 parts by weight of mushroom powder in an alkaline aqueous solution having a pH greater than 10.
    [3]
    3. Method according to one of claims 1 or 2, characterized in that the neutralization of the solution obtained by the alkaline extraction step, is carried out with an organic or mineral acid and that the precipitate obtained is removed .
    [4]
    4. Method according to one of claims 1 to 3, characterized in that the product obtained after neutralization of the alkaline extract is subjected to several filtrations, to separate from the liquid phase the acid salt (s) and simple sugars.
    [5]
    5. Method according to one of claims 1 to 4, characterized in that the enzymatic hydrolysis carried out with glycosidase is maintained for up to 24 hours with stirring.
    [6]
    6. Method according to one of claims 1 to 5, characterized in that the edible mushrooms subjected to an alkaline extraction are species chosen from the group formed by oyster mushrooms, coulemelles and button mushrooms.
    Ί. Method according to one of claims 1 to 6, characterized in that it consists in spraying the aerial parts of the plants to be treated with a composition having a pH between 4 and 8, the concentration of active agents of which is 1 , 5 to 4% by weight.
    [7]
    8. Method according to one of claims 1 to 7, characterized in that the composition, with which the aerial parts of the plant to be treated are sprayed, comprises a protein content of 5 to 15% by weight relative to its material dried.
    [8]
    9. Method according to one of claims 1 to 8, characterized in that the composition, with which the aerial parts of the plant to be treated are sprayed, comprises a total sugar content of 60 to 80% by weight relative to the dry matter.
    [9]
    10. Method according to one of claims 1 to 9, characterized in that the composition, with which the aerial parts of the plant to be treated are sprayed, has a monosaccharide formulation corresponding to a weight proportion of 38 to 50% of glucose, 18 to 28% galactose, 20 to 30% mannose and 0.5 to 4% glucosamine and / or acetylated glucosamine.
    [10]
    11. Method according to one of claims 1 to 10, characterized in that the composition, with which the aerial parts of the plant to be treated are sprayed, contains 35 to 45% by weight of glucans relative to its dry matter.
    [11]
    12. Method according to one of claims 1 to 11, characterized in that the composition, with which one sprays on the aerial parts of the plant to be treated, contains from 30 to 50% by weight of a dry matter, the molecular mass is greater than 10kDa.
    [12]
    13. Method according to one of claims 1 to 12, characterized in that it is associated with a treatment of the aerial parts of the treated plant with a conventional phytosanitary agent applied simultaneously or sequentially or alternately.
    [13]
    14. Method according to one of claims 1 to 13, characterized in that it constitutes a preventive treatment against cryptogamic diseases in particular those chosen from the group formed by diseases of the vine, fruit trees, vegetable crops and cereals, and in particular against downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator), gray rot (Botrytis cinerea) in the vine, downy mildew (Phytophthora infestans) in potatoes, yellow rust (Puccinia striiformis) , septoria leaf spot (Septoria sp.), Fusarium head blight (Fusarium graminearum) in wheat and apple scab (Venturia inaequalis).
    [14]
    15. Method according to one of claims 1 to 14, characterized in that it is implemented in the early vegetative stages and / or in the adult and reproductive vegetative stages.
    [15]
    16. Method according to one of claims 1 to 15, characterized in that it is implemented at repeated time intervals, as often as necessary, preferably every 8 to 15 days.
    [16]
    17. Composition for implementing the method according to one of claims 1 to 16, characterized in that it is obtained:
    a) by subjecting an edible macroscopic mushroom powder to an alkaline extraction and then neutralizing the extract obtained before carrying out its filtration;
    b) by diluting the filtered extract and subjecting it to a glycosidase between 10 and 65 ° C, the hydrolysis thus practiced being stopped by inactivating the enzyme at a temperature above 65 ° C;
    c) by subjecting the hydrolyzate obtained to a filtration step in order to isolate an active fraction thereof with a molecular weight of less than 100 kDa.
    [17]
    18. Composition according to claim 17, characterized in that the alkaline extraction step is carried out at a temperature between 20 and 100 ° C, while maintaining with stirring a suspension of 5 to 15 parts by weight of mushroom powder in an alkaline aqueous solution having a pH greater than 10.
    [18]
    19. Composition according to one of claims 17 or 18, characterized in that the neutralization of the solution obtained by the alkaline extraction step is carried out with an organic or mineral acid and that the precipitate obtained is removed .
    [19]
    20. Composition according to one of claims 17 to 19, characterized in that the product obtained after neutralization of the alkaline extract is subjected to several filtrations, in order to separate the acid salt (s) from the liquid phase and simple sugars.
    [20]
    21. Composition according to one of claims 17 to 20, characterized in that the enzymatic hydrolysis carried out with glycosidase is maintained for 24 hours with stirring.
    [21]
    22. Composition according to one of claims 17 to 21, characterized in that the edible mushrooms subjected to an alkaline extraction are species chosen from the group formed by oyster mushrooms, coulemelles and button mushrooms.
    [22]
    23. Composition according to one of claims 17 to 22, characterized in that its concentration of active agents is 150 to 400 mg / L.
    [23]
    24. Composition according to one of claims 17 to 23, characterized in that it comprises a protein content of 5 to 15% by weight relative to its dry matter.
    [24]
    25. Composition according to one of claims 17 to 24, characterized in that it comprises a total sugar content of 60 to 80% by weight relative to its dry matter.
    [25]
    26. Composition according to one of claims 17 to 25, characterized in that it has a monosaccharide formulation corresponding to a weight proportion of 38 to 50% of glucose, of 18 to 28% of galactose, of 20 to 30% of mannose and 0.5 to 4% glucosamine and / or acetylated glucosamine.
    [26]
    27. Composition according to one of claims 17 to 26, characterized in that it contains 35 to 45% by weight of glucans relative to its dry matter.
    [27]
    28. Composition according to one of claims 17 to 27, characterized in that it contains from 30 to 50% by weight of a dry matter, whose molecular mass is greater than 10kDa.
    [28]
    29. Composition according to one of claims 17 to 28, characterized in that it contains at least one compatible formulation agent.
    [29]
    30. Composition according to claim 29, characterized in that it comprises at least one anti-phytopathogenic agent chosen from the group formed by a fungicidal agent, an antibacterial agent, an antiviral agent, a pesticidal agent and a biocontrol agent.
    [30]
    31. Composition according to one of claims 17 to 29, characterized in that it contains at least one nutrient for plants for its simultaneous application.
    1/3
    PAL PR1 PR2 PR3
    2/3
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    同族专利:
    公开号 | 公开日
    WO2018069497A1|2018-04-19|
    US20190254288A1|2019-08-22|
    EP3525593A1|2019-08-21|
    FR3057438B1|2020-10-02|
    JP2020500209A|2020-01-09|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1660000|2016-10-14|
    FR1660000A|FR3057438B1|2016-10-14|2016-10-14|PROCESS FOR ELICITATION OF A PLANT BY MEANS OF EXTRACTS OF EDIBLE MACROSCOPIC FUNGI|FR1660000A| FR3057438B1|2016-10-14|2016-10-14|PROCESS FOR ELICITATION OF A PLANT BY MEANS OF EXTRACTS OF EDIBLE MACROSCOPIC FUNGI|
    US16/342,107| US20190254288A1|2016-10-14|2017-10-13|Process for eliciting a plant by means of edible macroscopic fungal extracts|
    JP2019541867A| JP2020500209A|2016-10-14|2017-10-13|Steps for inducing plants with extracts of edible macroscopic fungi|
    EP17780779.9A| EP3525593A1|2016-10-14|2017-10-13|Process for eliciting a plant by means of edible macroscopic fungal extracts|
    PCT/EP2017/076174| WO2018069497A1|2016-10-14|2017-10-13|Process for eliciting a plant by means of edible macroscopic fungal extracts|
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