ES2575728T3 - Composition comprising an elicitor of the plant immune system - Google Patents

Description

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ELISA test with the monoclonal antibody 2F4 (refer to Cabrera et al., 2008, Glycobiology 18 (6): 473-482) but also with the help of bioassays that should be optimized for each target plant species. Too much calcium displaces the chitosan from the complex. Too much monovalent cation displaces calcium from the pectin egg carton conformation and can be phytotoxic. A small amount of copper can replace calcium in the egg carton conformations and is beneficial for the supramolecular conformation of the elicitor. NaCl is preferably used as a monovalent cation salt. KNO3 is also suitable, but less preferred due to the possible effect on the opening of stomata, possibly allowing the entry of parasites into the leaf or plant. CaCl 2 is preferably used as the divalent cation salt, but CuCl 2 or ZnCl 2 are also suitable. More preferably, CaCl 2 is used from about 0.2 to 1 mM, most preferably 0.5 mM, in combination with NaCl from about 5 to 100 mM, most preferably from 20 to 50 mM.

Sucrose may be added to the composition as defined herein. Preferably, sucrose is added in a concentration of from about 1 mM to 20 mM, most preferably sucrose of about 5 to 10 mM. Sucrose triggers signaling through hexokinase and is also a wetting agent.

Preferably, both salts and sucrose can be added to the compositions as defined herein.

The term "fungicide" encompasses chemical or biological substances or compositions used to destroy or inhibit fungi or oomycetes, for example by preventing sporulation, or their spores. Fungicides can exert their biological effect through different modes of action, for example, but not limited to, by interference in the synthesis of nucleic acids, mitosis and cell division, respiration, synthesis of amino acids and proteins, signal transduction, lipids and synthesis of membrane, sterol biosynthesis, glucan biosynthesis in the pathogen or by induction of the defense of the host plant.

Said fungicide may be, for example, a fungicide selected from: acylalanines (benalaxyl), anilinopyrimidines (cyprodinil or pyrimethanil), benzamides (fluopicolide or zoxamide), benzimidazoles (fuberidazole, thiabendazole or metrafenone), benzothiadiazoles (acibenzolar-S-methyl), carbamates (bentiavalicarb, iprovalicarb or propamocarb), carboxamides (boscalid), chloronitriles (chlorothalonil), chlorophenyl (tolclofos-methyl), cyanoacetamide oximes (cymoxanil), cyanoimidazoles (cysophamide), dicarboximides (iprodione), dithiocarbamates (thiram, metiram, mancozeb, maneb or propineb), guanidines (dodine), hydroxyanilides (fenhexamide), imidazoles (fenamidone, imazalil or triflumizol), morpholines (dimetomorf, fenpropimorf, spiroxamina or dodemorf), phosphonates (fosetil), oxatiinas (flutolanil), oxazoles (famoxadona or himexazol ), phenylamides (metalaxyl or metalaxyl-M), phenylpyridinamides (fluazinam), phenylpyrroles (fludioxonil), phthalimides (captan or folpet), quinazolinones (proquinazide), quinolines ( quinoxifene), strobilurins (dimoxystrobin, fluoxastrobin, cresomin-methyl, pyraclostrobin, trifloxystrobin or picoxystrobin), thiophenes (siltiofam), triazoles (diphenoconazole, epoxiconazole, fenbuconazole, flusilazole, metconazole, myclobutanil, penconazole, propiconazole, tebuconazole, tetraconazole, triadimenol, triticonazole or protioconazole), copper derivatives (copper oxychloride, copper hydrochloride, copper oxide or copper sulfate) and sulfur.

Preferably, the fungicide is selected from the list comprising: phosphonates, benzamides, carbamates, dithiocarbamates, phthalimides, triazoles, quinolines, sulfur and cyanoimidazoles.

 Phosphonates: The mode of action of phosphonates is largely unknown but could involve the inhibition of mitochondrial ATP synthase. Suitable examples of phosphonates are phosphorous acid derivatives, including the phosphorous acid itself and its alkali metal or alkaline earth metal salts. In a preferred example, the fungicides are ethyl hydrogen phosphonates such as fosetyl-Al, fosetyl-K and fosetyl-Na. Mention may be made of the phosphonates sold under the trade names of Aliette, Autograph, Avalon, Flanker, Legion, Linebacker, Novasource, Prodigy Signature and Quali-Pro, all of which comprise fosetyl-Al as an active component, and Magellan and Phostrol, comprising phosphorous acid as an active component.

 Benzamides interfere with mitosis and cell division. In a preferred example, the benzamides contain 2,6-dichloro-N- [3-chloro-5- (trifluoromethyl) -2-pyridinyl] benzamide (fluopicolide) as the active component. A mention of Infinity can be made.

 Carbamates act by interfering with the synthesis of lipids and membrane. In a preferred example, the carbamates contain propamocarb, preferably propamocarb hydrochloride (propyl [3 (dimethylamino) propyl] carbamate hydrochloride) as the active component. Mention may be made of the carbamates sold under the tradenames of Infinito and Stellar (comprising fluopicolide and propamocarb hydrochloride), Banol, Previcur, Proplant (which comprise propamocarb hydrochloride) and Previcur Energy (which comprises propamocarb and fosetyl-Al) .

 Dithiocarbamates show contact activity at multiple sites. In a preferred example, dithiocarbamates containing (polymeric) complex of manganese ethylenebis (dithiocarbamate) with zinc salt (mancozeb) are used as the active component. Mention may be made of the dithiocarbamates sold under the trade names Acrobat MZ, Clevis, Cuprofix MZ, Dithane, Evolve, Fore, Gaucho, Gavel, Junction,

Mancozide, Manhandle, Manzate, Maxim, Moncoat, Nubark, Penncozeb, Pentathlon, Potato Seed Treater, Protect, Ridomil Gold MZ, SA-50, Stature, MZ Tops, Wingman and Zyban.

 Phthalimides also show contact activity at multiple sites. In a preferred example, the phthalimides comprise N- (trichloromethylthio) phthalimide or 2 - [(trichloromethyl) thio] -1H-isoindole-1,3 (2H) -dione (folpet) as the active component. Mention may be made of the phthalimides sold under the trade names of

Folpet and Fungitrol.

 Triazoles act by interfering in the biosynthesis of sterol in membranes. In a preferred example, the triazoles contain (2RS, 3SR) -1- [3- (2-chlorophenyl) -2,3-epoxy-2- (4-fluorophenyl) propyl] -1H-1,2,4-triazole (epoxiconazole) as an active component. A mention may be made of the triazole fungicide sold under the trade name

10 of Opus.

 Cyanoimidazoles act by interfering with the chain of electronic transport at the level of complex III in the inner membrane of mitochondria, which blocks oxidative phosphorylation driven by electronic transfer. In a preferred example, the cyanoimidazoles contain 4-chloro-2-cyano-N, N-dimethyl-5- (4-methylphenyl) -1H-imidazole-1-sulfonamide (cysoxamide) as the active component. A mention of the

15 cyanoimidazole sold under the trade name Ranman.

 Quinolines act by interfering in, for example blocking, signal transduction. In a preferred example, the quinolines contain 5,7-dichloro-4-quinolyl 4-fluorophenyl ether (quinoxifene) as the active component. A mention may be made of the quinoline fungicide sold under the trade names of Legend or Quintec.

20  Sulfur-containing fungicides show contact activity at multiple sites and contain sulfur as an active component. A mention may be made of the sulfur-containing fungicide sold under the trade name Thiovit®.

The compositions as defined herein may typically contain additional components, known as co-formulants or adjuvants, to obtain a product with good properties of

25 manipulation, efficiency and stability. As used herein, the terms "coformulant" or "adjuvant" denote any substance other than the elicitor component of the principal oligosaccharide complex defined herein, which is deliberately added to the elicitor composition as defined in the present document.

The composition as defined herein may further comprise a co-formulant or

Adjuvant selected from the group comprising: surfactants, antifreeze agents (including urea, ethylene glycol, propylene glycol or glycerol), preserving agents (including potassium sorbate, paraben and its derivatives, 1,2-benzisothiazolin-3 (2H) -one or oils essential), absorbent agents (including corn cobs)

or sawdust), thickeners (including clays or xanthan gum), buffers, adhesive agents (including latex, silicon or

alkoxylated alkyl), diluents (including rapeseed methyl ester) or any usual inert component used in conventional manner agricultural compositions, or a mixture thereof.

The composition as defined herein may further comprise a surfactant.

By "surfactant" is meant herein a compound that decreases the surface tension of a liquid, allowing an easier extension. The surfactant can be a detergent, an emulsifier (including glycerol ester of alkyl polyglycosides or polyoxyethylene (20) -sorbitan monolaurate), a dispersing agent

40 (including sodium chloride, potassium chloride, potassium nitrate, calcium chloride or corn starch), a foaming agent (including tartaric acid derivatives, malic acid or alcohols), a penetration enhancer, a humectant (including ammonium sulfate, glycerin or urea) or an ionic or nonionic wetting agent or a mixture of such surfactants. The surfactants used in the composition as defined herein are penetration enhancers, dispersing agents or emulsifiers.

The term "penetration enhancer" is understood herein as a compound that accelerates the uptake of the active component through the cuticle of a plant inside the plant, i.e. the rate of uptake, and / or increases the amount of active component absorbed in the plant. The classes of substances known as penetration enhancers include alkyl phosphates, such as tributyl phosphate and tripropyl phosphate, and salts of naphthalenesulfonic acid. Mention can be made, for example, of surfactants

50 sold under the tradename Dehscofix®, comprising castor oil and ethoxylated fatty acids, such as Dehscofix CO 95 ® (available from Huntsman, USA), comprising C18 ethoxylated fatty acids from castor oil.

By "dispersing agent" is meant a substance added to a suspension, usually a colloid, to improve the separation of particles and prevent sedimentation or agglutination. A mention of the

55 dispersing agent sold under the tradename Tensiofix Dp400 (available from Ajinomoto OmniChem), which essentially comprises organic sulfonate and 2-methylpentane-2,4-diol.

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other guests; Cladosporium spp. (for example, Cladosporium fulvum) in a range of hosts including cereals (for example, wheat) and tomatoes; Monilinia spp. in stone fruit, nuts and other hosts; Didymella spp. in tomatoes, turf, wheat, cucurbits and other hosts; Phoma spp. (for example, Phoma betae in sugar beet and Phoma lingam in rapeseed), in turf, rice, potatoes, wheat and other hosts; Aspergillus spp. and Aureobasidium spp. in wheat, wood and other guests; Ascochyta spp. (for example, Ascochyta pisi) in peas, wheat, barley and other hosts; Stemphylium spp. (Pleospora spp.) In apples, pears, onions and other hosts; summer diseases (for example, bitter rot (Glomerella cingulata), black rot or cercosporiosis (Botryosphaeria obtusa), Brooks fruit stains (Mycosphaerellapomi), apple-cedar rust (Gymnosporangium juniper-ivirginianae), sooty mold (Gloeodespomigena), stippled (Schizothyrium pomi) and white rot (Botryosphaeria dothidea)) in apples and pears; Plasmopara viticola on vines; other lanuginous moulders, such as Bremia lactucae in lettuce, Peronospora spp. (for example, Peronospora manshurica or Peronospora tabacina) in soybeans, tobacco, onions and other hosts, Pseudoperonospora humuli in hops and Pseudoperonospora cubensis in cucurbits; Pythium spp. (including Pythium ultimum) on the lawn and other hosts (for example, Pythium aphanidermatum); Phytophthora infestans in potatoes and tomatoes and other Phytophthora spp. in vegetables, strawberries, avocado, pepper, ornamental plants, tobacco, cocoa and other hosts (for example, Phytophthora cinnamomi, Phytophthora cactorum, Phytophthora phaseoli, Phytophthora parasitica, Phytophthora porri, Phytophthora citrophthora, Phytophthora megasperma f.sp.soiae or Phytophthora infestans ); Thanatephorus cucumeris in rice and turf and other Rhizoctonia spp. in various hosts such as wheat and barley, peanuts, vegetables, cotton and turf; Sclerotinia spp. on the lawn, peanuts, potatoes, rapeseed and other hosts (for example, Sclerotinia sclerotiorum); Sclerotium spp. on the lawn, peanuts and other guests; Gibberella fujikuroi in the rice; Colletotrichum spp. (for example, Colletotrichum lindemuthianum) in a range of guests including turf, coffee and vegetables; Laetica fuciformis on the lawn; Mycosphaerella spp. in bananas, peanuts, citrus fruits, pecans, papaya and other guests; Diaporthe spp. in citrus, soy, melon, pears, lupine and other guests; Elsinoe spp in citrus fruits, vines, olives, pecans, roses and other guests; Verticillium spp. (for example, Verticillium dahliae or Verticillium alboatrum) in a range of hosts including hops, potatoes and tomatoes; Pyrenopeziza spp. in rapeseed and other guests; Oncobasidium theobromae in cocoa that causes vascular regressive death; Fusarium spp. (for example, Fusarium nivale, Fusarium sporotrichioides, Fusarium oxisporum, Fusarium graminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum), Typhula spp., Microdochium nivale, Ustilago spp. for example Ustilago maydis (for example, corn charcoal), Urocystis spp., Tilletia spp. and Clavicepspurpurea in a variety of hosts but particularly wheat, barley, turf and corn; Ramularia spp. in sugar beet, barley and other hosts; diseases after harvest particularly of fruits (for example, Penicillium expansum, Penicilliumn digitatum, Penicillium italicum and Trichoderma viride in oranges, Colletotrichum musae and Gloeosporium musarum in bananas and Botrytis cinerea in grapes); other pathogens on vines, especially Eutypa lata, Guignardia bidwellii, Phellinus igniarus, Phomopsis viticola, Pseudopeziza tracheiphila and Stereum hirsutum; other tree pathogens (eg, Lophodermiunm seditiosum) in wood, especially Cephaloascus fragrans, Ceratocystis spp., Ophiostoma piceae, Penicillium spp., Trichoderma pseudokoningii, Trichoderma viride, Trichoderma harzianum, Aspergillus niger, Leptographium liindbergi and Aureobasidium pullulans; and fungal vectors of viral diseases (for example, Polymyxa graminis in cereals as the vector of yellow barley mosaic virus (BYMV) and Polymyxa betae in sugar beet as the vector of rhizomania), Acremoniella spp., Allomyces spp., Amorphothec spp., Aspergillius spp., Blastocladiella spp., Candida spp., Chaetomium spp., Coccidioides spp., Conidiobolus spp., Coprinopsis spp., Corynascus spp., Cryphonectria spp., Cryptococcus spp., Cunninghamella spp., Curvularia spp. , Debarymyces spp., Diplodia spp. (for example, Diplodia maydis), Emericella ssp., Encephalitozoon spp., Eremothecium spp., Gaeumanomyces spp. (for example, Gaeumanomyces graminis f.sp. tritici), Geomyces spp., Gibberella spp. (for example, Gibberella zeae), Gloeophyllum spp., Glomus spp., Hypocrea spp., Kluyveromyces spp., Lentinula spp., Leptosphaeria salvinii, Leucosporidium spp., Macrophomina spp. (for example, Macrophomina phaseolina), Magnaportha spp. (for example Magnaporthe oryzae), Metarhizium spp., Mucor spp., Neurospora spp., Nectria spp. (for example, Nectria heamatococca), Paracocidioides spp., Phaeopsheria spp., Phanerochaete spp., Phakopsora spp. (for example, Phakopsora pachyrhizi), Phymatotrichum spp. (for example, Phymatotrichum omnivorum), Pneumocystis spp., Pyronema spp., Rhincosporium secalis, Rhizoctonia spp. (for example, Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis), Rhizopus spp. (for example, Rhizopus chinensid), Saccharomyces spp., Scerotium spp. (for example, Scerotium rolfsii), Spizellomyces spp., Thermomyces spp., Thielaviopsis spp. (for example, Thielaviopsis basicola), Trametes spp., Trichophyton spp., or Yarrwia spp.

Diseases of plants caused by fungi including yeasts, rusts, coals, moldy mildews, molds, mushrooms and poisonous fungi that can be treated using the compositions as defined herein are for example:

"Roya" is a fungal disease in plants, which produces an alteration of the reddish brown color of the stems and leaves.

"Black rot" is characterized by the darkening and rotting of leaves of fruits and vegetables.

"Black spot" is one of the many fungal diseases in plants. It is called "black spot" because it produces small black spots on plants.

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which is sufficient to induce the control or destruction of the pathogens of plants present or susceptible to appear in the plants, and which does not entail any symptom of appreciable phytotoxicity for said plants. Such amount may vary within a wide range depending on the pathogen of plants to be controlled, the type of plant, the climatic conditions and the compounds included in the composition according to the invention. This amount can be determined by systematic field tests that are within the capabilities of a person skilled in the art.

As an example, the fungicide in the composition as defined herein is applied at a reduced rate. Preferably, the fungicide rate is reduced by at least a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 40, 50, 60, 70, 80, 90 or 100 in comparison with the recommended rate, or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or even 95% or more of the recommended dosage for that plant and / or conditions. More preferably, the fungicide rate is reduced by 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 60% to 80%, or 60% to 70% of the recommended rate for said plant and / or conditions.

The application of the composition as defined herein can be carried out according to techniques well known to those skilled in the art. The composition as defined herein can be applied to the whole plant, or to leaves, flowers, fruits, seeds and / or roots of the plant, as well as to the soil or inert substrate in which the plant is growing or in the that you want to grow (for example, inorganic substrates such as sand, rock wool, glass wool, expanded minerals such as perlite, vermiculite, zeolite or expanded clay), pumice, materials or tuff, synthetic organic substrates (for example, polyurethane), organic substrates (eg, peat, compost, residual tree products such as coconut fiber, fiber or wood chips, tree bark) or a liquid substrate (eg, hydroponics systems by flotation, nourishing film technique, aeroponic system). The application can be done by spraying, spraying, soaking, immersion, injection, etc., or by fertigation systems.

It may also be useful to apply the compositions as defined herein to propagation material such as tubers or rhizomes, but also seeds, seedlings or replicing of seedlings and plants or repotting of plants. Compositions as defined herein may also be applied after harvest to control rotting.

Among the plants that can be protected by the method as defined herein, mention may be made of cotton; linen; vine; fruit or vegetable crops such as Rosaceae sp. (for example, fruits of pips such as apples and pears, but also stone fruits such as apricots, almonds and peaches), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp. , Oleaceae sp., Actinidaceae sp., Lauraceae sp., Musaceae sp. (for example, bananas and bananas), Rubiaceae sp., Theaceae sp., Sterculiceae sp., Vitaceae sp., Rutaceae sp. (for example, lemons, oranges and grapefruit); Solanaceae sp. (for example, tomatoes), Liliaceae sp., Asteraceae sp. (eg, lettuce), Umbelliferae sp., Cruciferae sp., Chenopodiaceae sp., Cucurbitaceae sp., Papilionaceae sp. (for example, peas), Rosaceae sp. (for example, strawberries); major crops such as Graminae sp. (for example, corn, grass or cereals such as wheat, rice, barley and triticale), Asteraceae sp. (for example, sunflower), Brassicaceae sp. (for example, canola and rapeseed), Fabacae sp. (for example, peanuts), Papilionaceae sp. (for example, soybean), Solanaceae sp. (for example, tomatoes and potatoes), Chenopodiaceae sp. (for example, beets); horticultural and forest crops; as well as genetically modified homologs of these crops.

The invention is further exemplified by the following non-limiting examples.

Examples

Example 1. Comparison of the efficacy of a fungicide applied alone or mixed with an elicitor in the control of Phytophthora infestans in potato (field test).

Potato plants (Solanum tuberosum) of the Bintje variety sensitive to Phytophthora infestans near Gembloux, Belgium were grown in the field. 28 days after planting, the first application of the treatments was performed. The treatments were applied 10 times at intervals of 7 days between applications. The plants were treated by spraying 0.4 L of a test solution per hectare. The test solutions were composed of elicitor alone, fungicide alone or a mixture of both. The active component in the elicitor was an oligosaccharide complex consisting of negatively charged oligo-galacturonans stabilized by positively charged quitooligosaccharides. The elicitor was provided as a powder comprising 70.4% active component and applied to 50 g active component / ha (71 g powder). The fungicide used was Infinito (Bayer, Germany), which was chosen for its high level of potato protection against P. infestans. Infinity comprises the active components propamocarb and fluopicolid at 625 g / L and 62.5 g / L, respectively. The fungicide was applied at 0.4 L / ha, which is one third of the recommended rate. The summary of the test solutions is detailed in Table 1.

Table 1. Compositions of the test solutions applied in potato against P. infestans.

Test solution

Rate Unit of the rate Active components (p / ha)

fungicide (Infinity)

0.4 L / ha propamocarb (250) + fluopicolid (25)

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(http://www.faizyme.com/assaperg.htm). The frozen samples were ground in liquid nitrogen to a fine powder with a MM 400 grinder from RETSCH. The proteins were extracted using acetate buffer (0.1 M, pH 5.2) containing -mercaptoethanol (0.014 M). Briefly, 0.5 g of leaf powder was homogenized with an Ultra-Turrax (IKA) apparatus and centrifuged at 4000 rpm (4 ° C) for 10 min. The supernatant was collected and filtered through Miracloth (Calbiochem) and frozen directly with liquid nitrogen. The protein content was measured at 630 nm using the Bio Rad protein assay reagent with bovine serum albumin as standard. Peroxidase activity assays were performed with fresh extracts. Guaiacol (0.17 M) and H2O2 (0.19 M) were used as substrates. The kinetics of tetraguayacol formation was determined by measuring the absorbance every minute at 420 nm. The slope of the data allowed to express the activity peroxidase in "mmol H2O2.h-1.mg of protein-1" and the results were normalized adjusting the treatment with adjuvant (control) to 100%. All measurements were made using the ELx800 Universal microplate reader spectrophotometer (Bio-Tek, instruments, INC) and statistical analyzes were performed with Minitab software (Minitab, 2007).

Figure 8 shows the effect of the different test solutions on the peroxidase activity of the tomato leaves.

The fungicide (Aliette) applied alone did not significantly modify the peroxidase activity of the tomato leaves. The addition of the elicitor statistically increased the peroxidase activity. Therefore, mixing the elicitor with Aliette confers peroxidase activity to the plant in addition to the intrinsic fungicidal effect. Unexpectedly, the fungicide used seems to have a negative effect on the effect of the elicitor. By reducing the concentration of the fungicide, the induction of the plant immune system by the elicitor could be increased.

Example 6. Comparison of tomato peroxidase activity treated with an elicitor alone or combined with a fungicide (greenhouse assay).

Tomato plants (Lycopersicon esculentum, variety Saint Pierre) were treated as in example 4. The composition of the test solutions is detailed in table 6.

Table 6. Compositions of test solutions applied in tomato.

Test solution

Rate Unit of the rate Active components (mg / L)

adjuvant (Dehscofix CO 95) (control)

0.1% v / v -

elicitor + adjuvant (Dehscofix CO 95)

25 0.1% mg / L v / v Oligosaccharide complex (25) -

elicitor + adjuvant (Dehscofix CO 95)

50 0.1% mg / L v / v Oligosaccharide complex (50) -

elicitor + adjuvant (Dehscofix CO 95)

75 0.1% mg / L v / v Oligosaccharide complex (75) -

fungicide (Aliette) + elicitor + adjuvant (Dehscofix CO 95)

400 25 0.1% mg / L mg / L v / v Fosetyl-Al (320) oligosaccharide complex (25) -

fungicide (Aliette) + elicitor + adjuvant (Dehscofix CO 95)

400 50 0.1% mg / L mg / L v / v Fosetyl-Al (320) oligosaccharide complex (50) -

fungicide (Aliette) + elicitor + adjuvant (Dehscofix CO 95)

400 75 0.1% mg / L mg / L v / v fosetyl-Al (320) oligosaccharide complex (75) -

The elicitor was applied as a formulation with the adjuvant Dehscofix CO 95. The active component in the elicitor was an oligosaccharide complex consisting of oligo-galacturonans (with degrees of polymerization between 9 and 20) and quito-oligosaccharides (with degrees of polymerization of between 5 and 10 and with an acetylation degree of approximately 25%) in equal proportions. The elicitor also contained the CaCl 2 (0.5 mM) and NaCl (50 mM) salts to ensure good ionic conditions for the stability of the oligosaccharide and sucrose complex (10 mM). The elicitor was applied at 25, 50 or 75 mg of active component / L. The fungicide used was Aliette, which was chosen because of its favorable ecotoxicological profile and the versatile effect of plant protection against oomycetes. Aliette was provided as a powder comprising 80% of its active component fosetyl-Al. Aliette was applied at 400 mg / L, while the recommended rate is 8000 g / L. Control plants were sprayed with Dehscofix CO 95, the adjuvant used in the elicitor formulation. 24 h after the treatment, samples of the leaves of each plant were taken individually, immediately frozen in liquid nitrogen and stored at -80 ° C until use. All samples were tested to determine peroxidase activity according to experiment 5.

Figure 9 shows the effects of the elicitor applied alone or in combination with fungicide on the peroxidase activity of the tomato leaves.

The peroxidase activity increased significantly by applying the elicitor to tomato. In the elicitor range tested alone (0 mg / L, 25 mg / L, 50 mg / L, 75 mg / L) the peroxidase activity seems to increase linearly with the elicitor concentration. The R2 (coefficient of determination) of this relationship is

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approximately 87%, which is good enough for a biological response. The fungicide (Aliette) applied alone did not significantly modify the peroxidase activity of the tomato leaves. When applied in combination with the elicitor, the peroxidase activity of the leaves also increases with the concentration of elicitor. However, in combination with the fungicide, the concentration of elicitor must be higher to produce an activity similar to that obtained with the pulverized elicitor only at the same rate. Mixing the elicitor with the fungicide confers peroxidase activity to the plant in addition to the intrinsic fungicidal effect of Aliette.

Example 7. Comparison of tomato peroxidase activity treated with an elicitor alone or formulated with different surfactants (greenhouse assay).

Tomato plants (Lycopersicon esculentum, variety Saint Pierre) were treated as in example 4. The composition of the test solutions is detailed in table 7.

Table 7. Compositions of test solutions applied in tomato.

Test solution

Rate Unit of the rate Active components (mg / L)

elicitor

fifty mg / L Oligosaccharide complex (50)

elicitor + Dehscofix CO 95

50 0.1% mg / L v / v Oligosaccharide complex (50) -

elicitor + Tensiofix Dp400

50 0.1% mg / L v / v Oligosaccharide complex (50) -

elicitor + Tensiofix D33

50 0.1% mg / L v / v Oligosaccharide complex (50) -

The elicitor was either applied alone or formulated with different surfactants. The surfactants were added at 0.1 percent by volume to the elicitor formulation. Dehscofix CO 95 is an ethoxylated castor oil, a non-ionic vegetable oil. Tensiofix D33 (Ajinomoto OmniChem, Belgium) is a nonionic wetting extender and Tensiofix Dp400 (Ajinomoto OmniChem, Belgium) is an anionic salt that has dispersion properties. The active component in the elicitor was an oligosaccharide complex consisting of oligo-galacturonans (with polymerization degrees of between 9 and 20) and quito-oligosaccharides (with polymerization degrees of between 5 and 10 and with a degree of acetylation of approximately 25%) in equal proportions. The elicitor also contained the CaCl2 (0.07 mM) and KNO3 (8 mM) salts to ensure good ionic conditions for the stability of the oligosaccharide and sucrose complex (10 mM). The elicitor was applied to 50 mg of active component / L. 24 h after the treatment, samples of the leaves of each plant were taken individually, immediately frozen in liquid nitrogen and stored at -80 ° C until use. All samples were tested to determine peroxidase activity according to experiment 5.

Figure 10 shows the effect of the surfactants on the peroxidase activity of the tomato leaves induced by the elicitor.

Dehscofix CO 95, a penetration enhancer, and Tensiofix Dp 400, a dispersing agent, both in combination with the elicitor gave a double increase in peroxidase activity compared to the elicitor alone.

Example 8. Comparison of the activity of peroxidase of potato treated with combinations of elicitor (greenhouse test).

Potato plants (Solanum tuberosum, variety Bintje) were grown under controlled conditions in a greenhouse (24 ° C, 16 hr day / 8 hr night regime) for 6 weeks.

The plants were treated twice: a first application of the treatments was carried out 7 days before harvesting the leaves, a second application was made 3 days before harvesting. For each treatment, 8 potato plants were sprayed with 200 mL of a test solution on both sides of the leaves until drained. The composition of the test solutions is detailed in Table 8.

Table 8. Compositions of test solutions applied in potato.

Test solution

Rate Unit of the rate Active components (mg / L)

adjuvant (Dehscofix CO 95) (control)

0.1% v / v -

elicitor + adjuvant (Dehscofix CO 95)

50 0.1% mg / L v / v Oligosaccharide complex (50) -

elicitor + adjuvant (Dehscofix CO 95) + elicitor Vacciplant Fruit

50 0.1% 0.25% mg / L v / v v / v oligosaccharide complex (50) laminarin (45000)

elicitor Vacciplant Fruit

0.25% v / v laminarin (45000)

The elicitor was applied as a formulation with the adjuvant Dehscofix CO 95. The active component in the elicitor was an oligosaccharide complex consisting of oligo-galacturonans (with degrees of polymerization between 9 and 20) and quito-oligosaccharides (with degrees of polymerization between 5 and 10 and with a degree of acetylation of

5

10

fifteen

twenty

25

30

35

40

Four. Five

approximately 25%) in equal proportions. The elicitor also contained the CaCl2 (0.07 mM) and KNO3 (14 mM) salts to ensure good ionic conditions for the stability of the oligosaccharide complex. The elicitor was applied to 50 mg of active component / L. In one treatment, the elicitor was combined with a second elicitor, Vacciplant Fruit. Vacciplant Fruit was obtained from Belchim (Belgium) and contained laminarin (a  (1 → 3) -glucan linear with  (1 → 6) bonds) as an active component. Vacciplant Fruit was applied at the recommended rate of 0.25% (v / v). Control plants were sprayed with Dehscofix CO 95, the adjuvant used in the elicitor formulation.

3 days after the second application of the treatments, samples of 3 leaves were taken in the same physiological phase per plant. The leaves of 2 plants were combined, immediately frozen in liquid nitrogen and stored at -80 ° C until use.

All the samples were tested to determine the peroxidase activity as detailed in example 5.

Figure 11 shows the effect of combining the elicitor of quito-oligosaccharide -oligo-galacturonan (COS-OGA) with a second elicitor (Vacciplant Fruit) in the induction of peroxidase activity.

When applied alone, the second elicitor, Vacciplant Fruit, did not improve the peroxidase activity compared to the control. Spraying plants with the COS-OGA elicitor produced a significant increase in peroxidase activity. A synergistic effect was obtained when the COS-OGA elicitor was combined with the elicitor Vacciplant Fruit.

Example 9. Efficiency of the oligosaccharide elicitor in the control of Chaetosiphon fragaefolii (aphids) in strawberry.

Strawberries (cultivar Anaïs) were grown in cages. Three cages were used, each containing 3 strawberries in this experiment.

On days D-7 and D-1 the strawberry plants were treated with an elicitor. The elicitor was provided as a liquid formulation comprising 10 g / L of active component, which was an oligosaccharide complex consisting of negatively charged oligogalacturonans stabilized by positively charged quito-oligosaccharides. After dilution (200 times), the elicitor was applied at 50 ppm. Six adult aphids (Chaetosiphon fragaefolii) were placed in each strawberry on day D-0.

Evaluations of the numbers of aphids in 3 randomly selected plants (one in each of 3 replications) were performed on days D + 19 and D + 26.

Figure 12 shows the effects of the elicitor on the development of aphids in strawberry plants 19 days after inoculation. The number of aphids was 139 per plant for the untreated control, while the number of aphids was reduced to 114 per plant after elicitor applications. This corresponds to a reduction of aphids by 18% in the presence of the elicitor.

Figure 13 shows the effects of elicitor on the development of aphids in strawberry trees 26 days after inoculation. The number of aphids was 315 per plant for the untreated control, while the number of aphids was reduced to 260 per plant after elicitor applications. This corresponds to a reduction of the aphids in 17.5% in the presence of the elicitor. That means that the protection obtained after only two applications of the elicitor lasts at least one month.

Example 10. Comparison of the efficacy of a fungicide applied alone or mixed with an elicitor in the control of Sphaerotheca fuliginea (downy mildew) in cucumber seedlings (greenhouse test).

Cucumber seedlings were grown in a greenhouse.

Seedlings were treated with fungicide alone, elicitor alone or a combination of both as summarized in the table

9. The test solutions were applied once on day 0 and a second time on day +7 (phase of 2/4 leaves).

Table 9. Compositions of test solutions applied in cucumber.

Test solution

Rate Unit of the rate Active components (mg / L)

fungicide (LEGEND)

0.001 L / hL Quinoxifene (2.5)

elicitor + adjuvant (Dehscofix CO 95)

0.8 0.08 L / hL L / hL Oligosaccharide complex (50) -

fungicide (LEGEND) + elicitor + adjuvant (Dehscofix CO 95)

0.001 0.8 0.008 L / hL L / hL L / hL Quinoxifene (2.5) Oligosaccharide complex (50) -

The fungicide used was LEGEND (Dow Agrosciences), which was chosen for its high level of cucumber protection against

S. fuliginea and because its mode of action consists of blocking the transduction path of early signals. LEGEND comprises the active component quinoxifene at 250 g / L. The fungicide was applied at 0.001 L / hL, which is one twentieth of the maximum recommended rate. The elicitor was applied as a formulation with the adjuvant Dehscofix CO 95. The active component in the elicitor was an oligosaccharide complex consisting of oligo

5

10

fifteen

twenty

25

30

35

40

Four. Five

negatively charged galacturonans stabilized by positively charged quito-oligosaccharides in the presence of recommended salts. The elicitor was provided as a concentrated solution comprising 6.25 g / L of active component. The elicitor was applied at 0.8 L diluted in 100 L. The elicitor was applied with 0.08 L of Dehscofix CO 95 adjuvant in 100 L, which is required for the penetration of the elicitor in the leaves. The fungicide and the elicitor (+ 0.08 L / hL Dehscofix CO 95) were also applied in the mixture. Natural infestation occurred (from day +7) and additional artificial inoculation was carried out on day + 10.

Disease assessments of the leaves in 16 plants were made from 4 replicas. The percentages of leaves with symptoms of the disease (frequency of pests) and the percentages of the foliar surface colonized by the disease (severity of the pest) were visually evaluated. These evaluations were performed on day +7 (only natural infestation) and +18 (also artificial inoculation) and the protection was calculated with reference to the untreated control.

Figure 14 shows the effects of treatments on the frequency of the disease caused by S. fuliginea in cucumber seedlings on day +7. At this time, only natural infestation occurred. The protection (expressed as the reduction of the frequency of pests) was 50% for the fungicide and 81% for the elicitor (oligosaccharide complex + Dehscofix CO 95), while protection reached 100% for the mixture of both ( fungicide and elicitor). Colby's analysis shows that there is a synergistic effect between the fungicide and the elicitor regarding the frequency of pests. The observed percentage of protection (100%) is greater than the expected percentage of protection (90.5%), in which E was calculated as:

50 x 81

E  50  81 90.5

100

Figure 15 shows the effects of treatments on the severity of the disease caused by S. fuliginea in cucumber seedlings on day +7. The percentage of protection (expressed as the reduction in severity of the pest) was 82% (for the fungicide) and 93% (for the elicitor), while the same parameter was evaluated at 100% for the mixture of both fungicide as elicitor. Colby's analysis shows that there is a slight synergistic effect between the fungicide and the elicitor regarding the severity of the pest. The observed percentage of protection (100%) is greater than the expected percentage of protection E (98.7%), in which E was calculated as:

82 x 93

E  82  93  98.7

100

Figure 16 shows the effects of the treatments on the frequency of the disease caused by S. fuliginea in cucumber seedlings on day +18. At this time, the symptoms also resulted from artificial inoculation. The percentage of protection (expressed as the reduction in the frequency of pests) was 53% for the fungicide and 9% for the elicitor (oligosaccharide complex + Dehscofix CO 95), while protection was evaluated at 88% for the mixture of both (fungicide and elicitor). Colby's analysis shows that there is a synergistic effect between the fungicide and the elicitor regarding the frequency of pests. The observed percentage of protection (88%) is much higher than the expected percentage of protection E (57.2%), in which E was calculated as:

53 x 9

E  53  9  57.2

100

Figure 17 shows the effects of treatments on the severity of the disease caused by S. fuliginea in cucumber seedlings at the same time (day +18). The percentage of protection (expressed as the reduction of severity of the pest) was 89% (for the fungicide) and 24% (for the elicitor), while the same parameter was evaluated at 99% for the mixture of both ( fungicide and elicitor). The Colby analysis again shows a clear synergistic effect between the fungicide and the elicitor regarding the severity of the pest. The observed percentage of protection (99%) is greater than the expected percentage of protection E (91.6%), in which E was calculated as:

89 x 24

E  89  24  91.6

100

In conclusion, whatever the parameter (frequency of pests and severity of the pest) and the evaluation (on day +7 and day +18), a synergistic effect is observed between the elicitor and a very low LEGEND fungicide rate (quinoxifene)

Example 11. Comparison of the efficacy of a sulfur-containing fungicide applied alone or mixed with an elicitor in the control of Erysiphe necator (downy mildew) (field test).

The field trial was carried out in a vineyard in France.

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Claims (1)

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