EP4068965A1 - Utilisation de saccharides de dattes seuls ou en melange avec des polyphenols pour proteger les plantes contre des pathogenes - Google Patents
Utilisation de saccharides de dattes seuls ou en melange avec des polyphenols pour proteger les plantes contre des pathogenesInfo
- Publication number
- EP4068965A1 EP4068965A1 EP20819668.3A EP20819668A EP4068965A1 EP 4068965 A1 EP4068965 A1 EP 4068965A1 EP 20819668 A EP20819668 A EP 20819668A EP 4068965 A1 EP4068965 A1 EP 4068965A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- extract
- plant
- use according
- polyphenols
- plants
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 230000002132 lysosomal effect Effects 0.000 description 1
- 150000002703 mannose derivatives Chemical class 0.000 description 1
- 150000002704 mannoses Chemical class 0.000 description 1
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000009329 organic farming Methods 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 235000009048 phenolic acids Nutrition 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000024428 response to biotic stimulus Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009919 sequestration Effects 0.000 description 1
- 235000021309 simple sugar Nutrition 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000000956 solid--liquid extraction Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 150000004044 tetrasaccharides Chemical class 0.000 description 1
- 239000000892 thaumatin Substances 0.000 description 1
- 235000010436 thaumatin Nutrition 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
- A01N43/08—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N2300/00—Combinations or mixtures of active ingredients covered by classes A01N27/00 - A01N65/48 with other active or formulation relevant ingredients, e.g. specific carrier materials or surfactants, covered by classes A01N25/00 - A01N65/48
Definitions
- the present invention belongs to the technical field of agronomic or agricultural protection products and relates to the use of an extract of saccharides, or of a mixture of extracts of saccharides and polyphenols, obtained from fruits of the date palm Phoenix dactylifera for protect plants against pathogens, in particular to stimulate the natural defenses of plants and develop their resistance against pathogens.
- Grapevine downy mildew is a disease caused by an oomycete, Plasmopara viticola, which causes many losses every year and which is likely to appear all over the world. Prevention is necessary and requires the establishment of regular treatments throughout the year. Other pathogens, such as fungi or insects, are also feared in agronomic and horticultural environments.
- substances of natural origin as protective agents for plants against pathogens has been the subject of research.
- These substances can be of plant, animal or mineral origin. They generally have a short lifespan, which limits their presence in food products and in the environment. They can act in different ways, for example by stimulating the natural defenses of the plant or by acting directly on a population of pathogens to inhibit its growth and development.
- Plants do not have an immune system equivalent to that of humans and animals, however they are able to set up defense mechanisms which can be induced by compounds acting as signals on the defense genes to trigger the synthesis of anti-pathogenic molecules or molecules capable of structurally strengthening the plant. Such compounds are said to be elicitors.
- the present invention relates to the use of an extract of water-soluble saccharides obtained from fruits of the date palm Phoenix dactylifera for the protection of plants against pathogens.
- such an extract is used for stimulating the defense and resistance reactions of the plant for treatment or prevention, in particular for inducing an eliciting activity of the defense mechanisms.
- Dates can be considered as an interesting source of compounds with various biological activities.
- a significant part of the production of dates is transformed into derived products, such as jams, juices and syrups. Therefore, a large amount of date kernels and unmarketed fruit is produced as waste which can be recovered on the basis of its high content of bioactive compounds such as polysaccharides.
- a saccharide extract in the context of the invention can comprise a quantity of saccharides greater than or equal to 40%, 50% or even 60%, by weight of the extract.
- the saccharides contain rhamnose, arabinose, fucose, xylose, mannose, galactose, galacturonic acid, glucose and / or glucuronic acid.
- Saccharides within the meaning of the invention, are monosaccharides, oligosaccharides or polysaccharides. Preferably, they will be polysaccharides.
- Polysaccharides are organic compounds that are abundant in nature. They are homo or heteropolymers formed by the linear or branched chain of oses.
- the polysaccharides found in dates are cellulose, pectin and hemicelluloses. These polysaccharides are part of the category of dietary fiber and are divided into two groups: soluble fibers, or water soluble, including pectins and some hemicelluloses, and insoluble fibers, insoluble in water, including cellulose and certain other hemicelluloses .
- the saccharides are naturally water-soluble polysaccharides or a mixture of such polysaccharides with hydrolysates of naturally non-water-soluble polysaccharides.
- the extraction carried out on the date fruits makes it possible to obtain a fraction enriched in soluble and insoluble polysaccharides.
- the initially insoluble polysaccharides can be upgraded within the framework of the invention once made water-soluble, in particular after the implementation of a method of enzymatic or chemical hydrolysis, for example by acid hydrolysis.
- the saccharide extract contains, by weight: at least 20% mannose, at least 7% arabinose, at least 6% glucose, at least 5% galactose.
- This saccharide extract is generally obtained from the kernel of date fruits.
- the saccharide extract contains, by weight: at least 40% galacturonic acid, at least 7% arabinose, at least 3% xylose.
- This saccharide extract is obtained from the skin and pulp tissues of date fruits.
- the polysaccharides extracted from dates have a very high inducing effect on the natural defenses of plants.
- they make it possible to induce the genes most representative of the PR (Pathogenesis Related) proteins involved in plant defense reactions. They are therefore good agents for stimulating the natural defense reactions of plants.
- PR proteins accumulate in the tissues of a plant in response to biotic or abiotic stress, or following the application of a product that induces the defenses. Each PR protein has its own function. They accumulate at a local and systemic level and their presence is an indicator of the establishment of defense mechanisms by the plant.
- PR1 proteins would be involved in the sequestration of sterols. They are thought to have an anti-fungal and anti-oomycete action, by inhibiting the growth and germination of spores, in particular against downy mildew and gray rot in tomatoes (Niderman et al., 1995; Garnish et al., 2016). They would also have a favorable action in drought resistance (Liu et al., 2013).
- PR2 proteins have a glucanase (b-1,3-glucanase) action, responsible for the degradation of glucan, a major polysaccharide constituent of the wall of fungi (Benhamou, 2009). PR2 is induced in response to the salicylic acid pathway. PR2 is a protein that may also be involved in tolerances to abiotic stresses such as drought (Liu et al., 2013).
- PR3 proteins have chitinase activity. They have a direct antimicrobial effect through the degradation of the fungal wall and an indirect effect through the possible release of fragments inducing defense genes (Benhamou, 2009).
- PR4 proteins showed in particular ribonuclease and chitinase activity.
- Studies in wheat have suggested anti-fungal activity against Fusarium culmorun (Bertini et al., 2009; Caruso et al., 2001; Filipenko et al., 2013).
- Another study reported possible antifungal activity in corn (Bravo et al., 2003).
- PR4 is a protein that appears to be activated by both SA and JA pathways (Bertini et al., 2003; Wang et al., 2011).
- PR5 proteins are “Thaumatin like” proteins with activity against oomycetes but also against Verticillium, Fusarium, and necrotrophic fungi. This protein also helps induce drought tolerance (Liu et al., 2013).
- PR8 proteins exhibit antibacterial action through lysosomal and antifungal activity, catalyzing hydrolysis of chitin from fungal walls and inhibiting hyphal growth. By virtue of their degradation action, they are thought to be at the origin of the production of endogenous elicitors, thus triggering the establishment of defense reactions within the attacked plant (Métraux et al., 1988).
- the PR14 proteins are lipid transfer proteins expressed in young leaves and appear to have a role in the transport of cutin monomers; they are associated with the assembly of cutin and wax (Sels et al., 2008). They have antimicrobial properties, for example against Pseudomonas, Fusarium, Pythium and Botrytis (Kido et al., 2010).
- the extract also contains at least 5%, by weight, of proteins, preferably at least 10%, more preferably between 5 and 15%.
- the extract of water-soluble saccharides is a mixture with an extract of polyphenols also obtained from fruits of the date palm Phoenix dactylifera.
- the mixture will be in the form of a composition.
- Polyphenols are antioxidant compounds specific to the plant kingdom, and endowed with various biological activities demonstrated by numerous studies described in the scientific literature. Mention will in particular be made of their antioxidant, anti-inflammatory, antimicrobial, antiviral or even anticancer activity.
- the saccharides / polyphenols ratio is between 30/70 and 70/30, expressed by weight.
- Polyphenols are present in all organs of the plant. They present a very wide variety of structures. Thus, they are divided into different families:
- Phenolic acids such as derivatives of hydroxy-benzoic acid and derivatives of cinnamic acid
- Flavonoids such as flavan-3-ols, including catechins and tannins, and flavones, including flavonols.
- the date palm fruit consists of three tissues: the stone, the pulp and the skin. It also evolves according to four stages of maturity: Hababouk, Blah (Kimri), Besser (Khalal) and Tamar. The selection of the fruit according to one of these stages, and according to the tissues, makes it possible to obtain different results in terms of polyphenol content.
- the polyphenol extract contains up to 80% polyphenols, by weight relative to the total weight of the purified dry extract.
- the polyphenol extract contains at least 95%, more preferably at least 97%, even more preferably at least 99% of condensed tannins, by weight relative to the total weight of polyphenols.
- the tannins condensed in the extract according to the invention are oligomers or polymers of pro-anthocyanidins.
- an effective amount of the extract of saccharides, or of the mixture of extracts of saccharides and of polyphenols, supplied to the plants is at least 0.01 g per liter, preferably of at least 0.1 g per liter, more preferably at least 0.7 g per liter, for a contribution in liquid form, or at least 10 g per hectare, preferably at least 100 g per hectare for a intake in solid form.
- the effective amount should be sufficient to induce defense and resistance mechanisms and will vary from plant to plant or depending on the method of treatment. This amount can be easily determined by a person skilled in the art.
- the saccharide extract or the mixture of extracts of saccharides and polyphenols, is applied to the whole plant, to the leaves, to the flowers, to the roots, to the fruits, to the seeds, to seedlings, soil, solid or liquid culture medium, culture material.
- the application is carried out by sprinkling, irrigation, spraying, soaking or injection.
- the plants are agronomic, ornamental, aromatic or medicinal plants, in particular, the plants are fruit plants, in particular vines, vegetable plants, flowers, trees, shrubs.
- the plant pathogens are fungi, bacteria, viruses, nematodes, parasitic plants, protozoa or insects, in particular Plasmopora viticola.
- the invention also relates to a method of protecting a plant against pathogens, in particular for activating defense and resistance reactions of the plant, the method comprising application to said plant, or in the environment of the plant. , an effective amount of an extract of water-soluble saccharides obtained from fruits of the date palm Phoenix dactylifera alone or as a mixture with an extract of polyphenols obtained from this same date palm, the extracts being as described above and applied in the same way as mentioned above previously.
- the invention also relates to a plant protection product containing an effective amount of a saccharide extract obtained from fruits of the Phoenix dactylifera date palm alone or as a mixture with a polyphenol extract from this same date palm, the extracts being as described above.
- the plant protection product further comprises at least one other component such as a solvent, a surfactant, an emulsifier, a dispersing agent, a filler, a fertilizing compound or a plant protection compound.
- at least one other component such as a solvent, a surfactant, an emulsifier, a dispersing agent, a filler, a fertilizing compound or a plant protection compound.
- the plant protection product is in liquid form, in gel form, in powder or granule form.
- Fruits can be selected according to one of the following four stages of maturity: Hababouk (stage 1), Blah (Kimri) (stage 2), Besser (Khalal) (stage 3), Tamar (stage 4).
- stage 1 the first stage of maturity
- stage 2 the second stage
- Stage 3 the second stage
- Tamar stage 4
- the tissues that is, the skin, the pulp and the nucleus, are separated from each other. After a grinding operation, the fabrics are dried and powdered. It should be noted that in stage 1 of ripening, the fruit is studied entirely without separation of the tissues because the stone is not yet formed.
- Saccharide extracts can be obtained from tissue powders according to a process consisting in first extracting the alcohol-insoluble matter (MIA) and then, in a second step, in purifying the water-soluble saccharides, in particular the polysaccharides , from this material.
- MIA alcohol-insoluble matter
- other extraction methods can be implemented.
- MIA is obtained using the process below:
- the determination of the mass composition of neutral and acidic monosaccharides is carried out by gas chromatography coupled with mass spectrometry (in English Gas chromatography-mass spectrometry or GC-MS). This analysis is carried out on the MIAs obtained from the nuclei and on the soluble fractions obtained from the MIAs of skin, pulp and nucleus tissues as well as on the insoluble fractions obtained from the MIAs of nuclei.
- the analyzes by GC-MS of the simple sugars constituting the polysaccharide structures of AIMs show us a great variability of the quantities of polysaccharides according to the fractions resulting from pulp, skin or stones.
- Rha rhamnose; Macaw: arabinose; Fuc: fucose; Xyl: xylose; Man: mannose; Gai: galactose; GalA: galacturonic acid, GIc: glucose, GIcA: glucuronic acid.
- MIA fraction of Alcohol-Insoluble Matter from the nucleus
- FSN fraction of soluble polysaccharides obtained from MIA of nuclei
- FISN fraction of insoluble polysaccharides obtained from MIA of nuclei.
- fraction of soluble polysaccharides extracted from nuclei is characterized by a polysaccharide content of nearly 50%, by weight.
- the fraction of soluble polysaccharides extracted from pulps and skins is for its part characterized by a polysaccharide content of more than 66%, by weight.
- polysaccharides are composed of monosaccharides of rhamnose, arabinose, fucose, xylose, mannose, galactose, galacturonic acid, glucose and glucuronic acid.
- mannose is the monosaccharide which is found in greater quantity. There is also a significant amount of glucose. This testifies to the presence of polysaccharides from the glucomannan family. In the fraction derived from FSC tissues and skin, it is galacturonic acid which is in significant quantity. The presence in this fraction of galactose and xylose testifies to the presence of pectin-type polysaccharides.
- the fraction of insoluble polysaccharides obtained from MIA of FISN cores is also rich in monosaccharides, in particular in mannose. Mannose forms about 90% of the weight by weight of the insoluble fiber fraction. This testifies to the presence of polysaccharides of the galactomannan family. Thus, it may be advantageous, from this fraction of insoluble fibers, to prepare a soluble fraction of monosaccharides (see Example 4) which can be used in the context of the applications targeted by the invention. d) Determination of the mass protein composition of the soluble and insoluble polysaccharide fractions obtained from the AIMs of nuclei and tissues (pulp and skin):
- the method for determining the protein concentration of the MIA, FSC, FISN and FSN fractions is based on the determination of nitrogen and carbon by combustion of samples of each of these fractions, in the form of lyophilized powder, according to the method well known to Dumas. The results are reported in Table 2.
- the contents vary from 40 to 50% for carbon and from 1.0 to 2.2% for nitrogen. These data make it possible to estimate protein contents varying from 6 to 14% depending on the samples.
- the FISN fraction with an estimated protein content of almost 14% is significantly richer in protein than the other fractions. There is no significant difference between the estimated protein contents for the FSN and FSC fractions.
- Polyphenols can be obtained from tissue powders according to a process for extracting and purifying polyphenols essentially comprising the following steps: solid-liquid extraction using a solvent, preferably polar, and recovery of an extract containing polyphenolic compounds, purification of the polyphenolic compounds of the extract by a solid phase chromatography method, for example in reverse phase or by ion exchange, recovery of a purified extract concentrated in polyphenolic compounds.
- the implementation is as follows:
- the polar solvent is composed of ethanol / water / acetic acid in a ratio of 50 to 80 ethanol: 1 to 10 acetic acid: qs in water (V / V / V) (hydroalcoholic solvent).
- a ratio of 80: 19: 1 Vol. Ethanol / Vol. Water / Vol. Acetic acid /
- a ratio of 50: 49: 1 can be used.
- the extract obtained, named El is stored, and the residue obtained is subjected to a second extraction by bringing into contact with 30 ml of the same solvent for 15 min., With stirring and then filtered.
- the extract obtained, named E2 is stored and the residue obtained is recovered and subjected to a third extraction by bringing into contact with 30 ml of the same solvent for 15 min., With stirring and then filtered.
- the extract obtained, named E3, is kept.
- the extracts E1, E2 and E3 are mixed. Filtration is carried out in order to recover the supernatant and then the phenolic compounds in the extract are concentrated by evaporation under vacuum.
- the extracts are centrifuged at 4500 rev / min for 10 min at 4 ° C in order to recover the supernatant which will be used for the Sep Pack C18 cartridge (sold by the company Waters) for the implementation of a solid phase extraction ( SPE).
- the purification can be carried out using an FPX 66 ion exchange column (Rohm & Haas France SAS).
- Concentration of the extracts is carried out by evaporation of the solvent and a purified fraction of polyphenols is obtained. The fraction is then dried to obtain a polyphenol powder.
- the extracts obtained contain up to 80% polyphenols, by weight relative to the total weight of the purified dry extract.
- the extracts contain at least 95%, more preferably at least 97%, even more preferably at least 99% of condensed tannins, by weight relative to the total weight of polyphenols.
- 70 mg of soluble fibers previously extracted are dissolved in 200 ml of water acidified with 0.1% formic acid.
- 70 mg of previously extracted polyphenols are dissolved in 50 mL of water acidified with 0.1% formic acid.
- the two solutions are mixed, stirred and then centrifuged at 4500 rpm for 10 min. The supernatant is recovered and then dried.
- the mixtures to be studied are prepared by soluble fiber-tannin fractions (50/50 w: w).
- the objective is to prepare fractions of soluble saccharides rich in oligomeric mannoses from the fraction containing insoluble polysaccharides FISN obtained from MIA.
- beta-mannanase bacterial or fungal in particular beta-mannanase derived from Aspergillus Niger including beta-mannanase purified û'Aspergillus Niger marketed under the tradename Gamanase ® (NOVO-NORDISK, Denmark).
- the unit of beta-mannanase is defined as being the quantity of beta-mannanase which releases, from locust bean gum, a quantity of reducing sugars equivalent to 1 micromole of mannose per minute, at pH5 and at 30C.
- Beta-mannanase can be immobilized by covalent bonding to the surface of a traditional support, or else immobilized by polymerization after having been adsorbed to the surface of a traditional support.
- the liquid extract can then be hydrolyzed at a temperature of 40-70 ° C with the immobilized enzymes.
- the analyzes by GC-MS of the polysaccharide structures of the hydrolyzed and solubilized insoluble fractions show the richness of this fraction in mannose.
- This mannose obviously comes from polysaccharides of the galactomannan and glucomannan family since this soluble part is rich in glucose and galactose.
- the sugars identified are mannose (Man); galactose (Gai); a trisaccharide with a galactose / mannose ratio of 1: 2 (Gal-Man2); a tetrasaccharide with a galactose / mannose ratio of 1: 3 (Gal-Man3); and a pentasaccharide with a galactose / mannose ratio of 1: 4 (Gal-Man4).
- Man mannose
- Gai galactose
- Gal-Man2 trisaccharide with a galactose / mannose ratio of 1: 2
- Gal-Man3 tetrasaccharide with a galactose / mannose ratio of 1: 3
- Gal-Man4 pentasaccharide with a galactose / mannose ratio of 1: 4
- the objective is to evaluate, under semi-controlled conditions, the inducing effect of the extract containing water-soluble polysaccharides (obtained according to Example 1 and named hereafter “Polysaccharides”) on the expression of 7 genes of PR proteins, markers of defense mechanisms, using the qPFD ® tool (described in the patent application WO2011 / 161388) on wheat plants.
- Polysaccharides obtained according to Example 1 and named hereafter “Polysaccharides”
- the wheat plants were produced from seeds of the soft wheat variety ALIXAN. For each modality, 30 grains of wheat were sown in 3 pots (2 repetitions / modality) then placed under controlled conditions for 1 week (25 ° C constant, 16h photoperiod), until the emergent F2 leaf stage. The young plants were then transferred to a climatic chamber dedicated to experimentation.
- Tween 20 was added to each candidate product, at a concentration of 0.05%, ie 25 ⁇ l per 50 ml of product.
- the 7 genes of the PR (Pathogenesis Related) proteins below have been selected as being representative of the major PR proteins involved in plant defense.
- PR-1 Pathogenesis-related protein 1
- PR-2 Pathogenesis-related protein 2 (glucanases)
- PR-4 Pathogenesis-related protein 4 (hevein-like)
- PR-5 Pathogenesis-related protein 5 (thaumatin-like, osmotin)
- PR-8 Pathogenesis-related protein 8 (class III chitinase)
- PR-14 Pathogenesis-related protein 14 (lipid transfer protein)
- PR-15 Pathogenesis-related protein 15 (oxalate oxidase)
- the tests are carried out in a climatic chamber, under controlled conditions (21 ° C day / 19 ° C night, 16h photoperiod) on wheat plants (2/3 leaf stage), randomized in blocks of 7 pots.
- each modality (2 pots) are applied twice, with the exception of the WATER control which is applied once only (OJ).
- the candidate product and the SDP control are applied 4 days apart, on D-4 and on D0.
- Each modality is then treated on D1 with hydrogen peroxide (H202) to simulate a pest attack. All treatments are carried out up to the limit of runoff, with a sprayer connected to compressed air.
- H202 hydrogen peroxide
- RNAs were extracted from the leaf tissues collected, using the NucleoSpin RNA Plant kit (Macherey-Nagel). The yield and the quality of the extracted RNAs were evaluated using a spectrophotometer (Nanodrop ND-1000). The RNAs were then reverse-transcribed into cDNA and the expression levels of 7 PR protein genes were monitored (3 technical repeats) by quantitative PCR (SYBR Green intercalating agent) using the qPFD ® tool ( “Low Quantitative Density Chip” developed by INRA).
- the relative expression levels were calculated using the 2 DDa method: these are expressions relative to the OJ sample (before treatment) at each sample time, normalized by the geometric mean of the relative expressions of 3 genes reference (TuB, Actin, GAPDH). These relative expressions are transformed into log2 to give the same weight to the inductions and repressions of the genes.
- Relative expression density map of PR protein genes Table 5 below shows the density map of the relative expressions of the 7 genes of the PR proteins.
- the expression levels of the 7 genes, transformed into log2 are represented by a density map. This makes it possible to have a global view of the induction profile and to directly visualize the differences in the levels of expression of the marker genes between the treated samples, after having subtracted the variations in expression of the EAU control.
- Log2 variation scale (relative expression): the more the value extends in the positive, the stronger the induction.
- the SDP control has a very strong capacity for inducing the PR1, PR4 and PR5 genes, 2 and 3 days after the second treatment (D2). It also shows a moderate induction of the PR8 gene on date D2.
- the induction profile of the SDP control in this test corresponds to the known effects of this product. The test is therefore validated.
- the polysaccharides show a strong capacity for inducing the PR1, PR4 and PR5 genes, 2 days after the second treatment (D2).
- the PR14 gene is also induced at a high level, 2 days after the last treatment (D2).
- the Fold change corresponds to the ratio of the level of expression of a gene for a treatment modality (SDP control, candidate product) compared to the EAU control, without log2 transformation and averaged for the 2 repeats. Fold change values greater than or equal to 2 represent moderate to strong inductions.
- the SDP control very strongly induces the PR1, PR4 and PR5 genes, at a level 253 to 1515 times higher than the WATER control.
- the PR8 and PR14 genes are induced respectively 4 and 3 times more than with the EAU control.
- the polysaccharides exhibit a strong capacity for inducing the PR1, PR4, PR5 and PR14 genes, at a level 7 to 40 times higher than the EAU control.
- the SDP control exhibits a strong capacity for inducing the PR1, PR4 and PR5 genes, 100 to 170 times higher than the EAU control.
- the polysaccharides strongly induce the PR1, PR4 and PR5 genes, respectively 18, 78 and 85 times more than the EAU control.
- the polysaccharides derived from the fruits of the date palm Phoenix dactylifera have a high capacity for inducing PR protein genes in wheat. They therefore have an inducing effect of the defense genes on the wheat crop and can be used as elicitors.
- Aim The objective is to confirm on vine plants what was observed on wheat plants in the previous example.
- the vines were produced from seeds of the Chardonnay grape variety. They were cultivated for 9 to 10 weeks in a greenhouse under semi-controlled conditions (19 ° C, 16 hours of daylight) then selected at the 4 - 6 leaf stage and transferred to a greenhouse dedicated to experimentation.
- Tween 20 was added to each candidate product, at a concentration of 0.05%, ie 25 ⁇ l for 50 ml of product.
- Each block of each modality is treated twice, with the exception of the WATER indicator which is applied once only (J0).
- the candidate products are applied 4 days apart (D-4 / D0) and the SDP control at 6-day intervals (D-6 / D0).
- Each modality is then treated on D1 with hydrogen peroxide (H202) to simulate an attack by pests and diseases. All the treatments are carried out on the 2 leaf faces, up to the limit of runoff, with a sprayer connected to compressed air.
- H202 hydrogen peroxide
- RNA extraction, retro-transcription and quantitative PCR The procedure is identical to that of Example 4.
- Table 9 below shows the density map of the relative expressions of the 7 genes of the PR proteins.
- the SDP control exhibits a strong capacity for inducing PR protein genes, with the exception of the PR14 gene, 2 days after the second treatment (D2). On the other hand, it no longer shows induction of the PR protein genes, 3 days after the second treatment (D3).
- the profile and the high level of induction on D2 of the SDP control in this test correspond to the known effects of this product under our conditions. The test is therefore validated.
- the polysaccharides show a strong inducing capacity of the PR protein genes, 2 days after the second application (D2).
- the SDP control induces the PR2, PR3, PR4, PR5, PR8 and PR14 genes, at a level 2 to 27 times higher than the WATER control.
- the PR1 gene is the most induced, 158 times more than with the EAU control.
- the polysaccharides exhibit a capacity for inducing the PR5 and PR14 genes, at a level 3 times higher than the EAU control. They strongly induce the PR1, PR3, PR4 and PR8 genes, at a level 5 to 36 times higher than the EAU control.
- the PR2 gene is the most induced, 50 times more than with the EAU control.
- the SDP control exhibits a low capacity for inducing the PR14 gene, at a level 1.5 times higher than the EAU control. It does not induce the other PR protein genes.
- the polysaccharides induce the PR3 gene at a level 3 times higher than the EAU control.
- polysaccharides derived from the fruits of the date palm Phoenix dactylifera have a high capacity for inducing PR protein genes in grapevine. They therefore have an inducing effect of the defense genes on the wheat crop and can be used as elicitors.
- Example 7 The polysaccharides derived from the fruits of the date palm Phoenix dactylifera have a high capacity for inducing PR protein genes in grapevine. They therefore have an inducing effect of the defense genes on the wheat crop and can be used as elicitors.
- Aim The study is being carried out in order to determine the effectiveness of protection of a mixture of water-soluble polysaccharides and polyphenols against downy mildew (Plasmopara viticola) of the vine (Vitis vinifera), at three different doses, in preventive or curative application .
- the tests are carried out on vine discs under controlled conditions.
- water and Bordeaux mixture are also applied 24 hours before or 24 hours after contamination of the plant by Plasmopara viticola.
- Table 12 illustrates the application modalities.
- Plant material vine seedlings are obtained from seeds of the Chardonnay grape variety.
- Plasmopara viticola Leaves 5 cm in diameter with large sporulation spots were frozen. The strain is then multiplied just before the experiment on vine plants, in order to have a fresh inoculum.
- Culture the seedlings are cultivated for 6 weeks under controlled conditions (25 ° C, 16h photoperiod) then selected at the 4-leaf stage and transferred to a dedicated climatic module for experimentation at the time of inoculation (21 ° C day, 19 ° C night, 16h photoperiod).
- Treatment the products are applied by spraying using a sprayer connected to compressed air up to the point of runoff on both leaf faces. The treatments were applied as a preventive measure, at -24h before inoculation, on plants, or curative, at +24h on discs.
- Tinoculum production and inoculation the leaves with dense sporulation are rinsed with reverse osmosis water and the suspension is filtered. The final concentration of the inoculum is adjusted to the concentration of 5.10 4 in sp / ml.
- Leaf discs are made using a cookie cutter at the rate of 8 discs per Petri dish. The inoculum is then applied to the underside of the discs with a fine droplet sprayer. The dishes are kept at room temperature until reading.
- Method of analysis the results are read between 6 and 9 days after inoculation using a quantitative scale for the severity of the disease varying from 0 to 100% of sporulation covering the surface of the leaf discs.
- the severity (intensity) of the disease is calculated by averaging the scores (%) obtained by modality.
- the incidence (frequency) of the disease is calculated and corresponds to the percentage of plants affected by late blight.
- the percentage of protection is calculated by the formula ((Water witness score-X score / Water witness score) x 100) from the severity scores.
- the data were analyzed with the XLSTAT software with an ANOVA.
- the statistical analysis used to differentiate the means is the Fisher Minimum Significant Difference (LSD) procedure. It indicates statistically significant differences at the 95% confidence level.
- a severity score is also observed which is significantly lower than the corresponding treated water control for the doses at 0.01%, 0.1% and 0.7% of the mixture according to the invention, which are respectively 79 , 80 and 71%, with a positive dose effect.
- the degree of protection obtained for the mixture at the dose of 0.7% is 30%.
- the mixture has the ability to induce natural defense reactions in the plant and to protect it before it is attacked by the pathogen but also to induce defense mechanisms in treatment when the plant is in contact with the pathogen.
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- Pest Control & Pesticides (AREA)
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Abstract
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CH15212019 | 2019-12-02 | ||
PCT/EP2020/084058 WO2021110648A1 (fr) | 2019-12-02 | 2020-12-01 | Utilisation de saccharides de dattes seuls ou en melange avec des polyphenols pour proteger les plantes contre des pathogenes |
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US (1) | US20230015964A1 (fr) |
EP (1) | EP4068965A1 (fr) |
CN (1) | CN115151134A (fr) |
CA (1) | CA3160242A1 (fr) |
MX (1) | MX2022006625A (fr) |
WO (1) | WO2021110648A1 (fr) |
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FR2814471B1 (fr) * | 2000-09-27 | 2004-12-17 | Centre Nat Rech Scient | Utilisation de polymeres et d'oligomeres de xyloglucane, et de composes derives, en tant que produits phytosanitaires et biofertilisants |
FR2837668B1 (fr) * | 2002-03-27 | 2005-04-22 | Centre Nat Rech Scient | Utilisation de composes comprenant une structure osidique contenant x, f, et g, et de composes derives, en tant que produits phytosanitaires et biofertilisants |
FR2852202B1 (fr) * | 2003-03-14 | 2006-08-11 | Nouveau procede pour la potentialisation et la simulation des defenses naturelles des plantes | |
WO2005112638A1 (fr) * | 2004-05-24 | 2005-12-01 | National University Corporation Kagawa University | Utilisation de sucres rares chez les plantes ou les microorganismes |
FR2891993B1 (fr) * | 2005-10-13 | 2009-10-23 | Univ Victor Segalen Bordeaux 2 | Composition pour application phytopharmaceutique pour stimuler les defenses naturelles des plantes |
FR2918842B1 (fr) * | 2007-07-19 | 2012-08-03 | Elicityl | Compositions contenant un melange synergique de polyols et de xyloglucanes en tant que produits phytosanitaires et biofertilisants |
FR2922412B1 (fr) * | 2007-10-23 | 2011-06-17 | Ard Sa | Nouvelles molecules pour la stimulation des defenses naturelles des plantes et leurs formulations |
FR2934943B1 (fr) * | 2008-08-12 | 2011-06-17 | Algieplus | Utilisation d'apiogalacturonanes et de ses derives pour la stimulation des reactions de defense et de resistance des plantes contre le stress biotiques et abiotiques |
FR2961826B1 (fr) | 2010-06-24 | 2012-08-03 | Agronomique Inst Nat Rech | Dispositif pour determiner ou etudier l'etat de stimulation des defenses naturelles de plantes ou parties de plantes |
CN103284320B (zh) * | 2013-06-09 | 2015-02-18 | 红云红河烟草(集团)有限责任公司 | 一种椰枣提取物及其在卷烟中的应用 |
WO2019188773A1 (fr) * | 2018-03-30 | 2019-10-03 | パナック株式会社 | Agent d'induction de résistance pour des plantes |
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- 2020-12-01 CN CN202080084132.0A patent/CN115151134A/zh active Pending
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- 2020-12-01 CA CA3160242A patent/CA3160242A1/fr active Pending
- 2020-12-01 US US17/781,991 patent/US20230015964A1/en active Pending
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US20230015964A1 (en) | 2023-01-19 |
MX2022006625A (es) | 2022-08-04 |
CA3160242A1 (fr) | 2021-06-10 |
CN115151134A (zh) | 2022-10-04 |
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