Patent Grant
9474273
The present invention generally relates to new compositions for protecting plants against plant pathogens, essentially comprising a particular plant elicitor, and the use of these compositions in agricultural applications, more particularly to protect plants against (infection by) plant pathogens and the corresponding methods of, and uses in, the protection of plants and crops by application of these compositions.
Plant pathogens can cause serious damage in agriculture, resulting in critical losses of yield, quality and profit. Examples are phytopathogenic fungi the majority of which belong to the Ascomycetes, including the causal agents of powdery mildew of various plants, the Basidiomycetes, including the causal agents of severe rusts of virtually all cereal grains and cultivated grasses and the Deuteromycetes (Fungi imperfecti), including Fusarium, Botrytis and Verticillium. Further pathogens are Oomycetes, which are not true fungi but fungal-like organisms that use the same mechanisms as fungi to infect plants. They include some of the most destructive plant pathogens including Phytophthora infestans, the causal agent of potato late blight and Plasmopara viticola, the causal agent of grapevine downy mildew. Besides fungi, also bacteria including the genii Erwinia, Xanthomonas, Pseudomonas and Raistonia, viruses including Cucumber Mosaic Virus, Barley Yellow Mosaic Virus, Strawberry Mild Yellow Edge Virus, Strawberry Latent Ringspot Virus, Beet Necrotic Yellow Vein Virus and Potato Virus Y, insects including beetles, rootworms, hoppers, locusts, (stem)borers, aphids, mites, thicks, ants, whiteflies, maggots, weevils, midges, caterpillars, butterflies, leaf miners, leaf rolers, bugs, and plasmodiophorid protists including the genii Polymyxa, Plasmodiophora and Spongospora are important plant pathogens.
Plants respond to infection by pathogens by activating their innate immune system. The plant defense systems recognize molecular patterns that are common to many classes of pathogens, e.g. fungal chitin, and may respond to pathogen-specific virulence factors (effectors). Pathogen recognition triggers ion channel gating, oxidative burst, cellular redox changes, protein kinase cascades and other responses that either directly activate cellular changes such as cell wall reinforcement, or activate changes in gene expression that lead to the formation of defensive compounds, e.g. directed to fight infection or to make the plant less attractive to pathogens.
The role of peroxidases in plant protection against pathogens is of the utmost importance. Peroxidases undergo two possible catalytic cycles involving either the consumption or the release of H2O2 and reactive oxygen species. Reactive oxygen species can act directly on pathogens and H2O2 is further known to be a mediator of signal transduction in the establishment of plant defense. Moreover, peroxidases can control the availability of H2O2 in the cell wall, which is a prerequisite for the crosslinking of phenolic groups in the cell wall. The peroxidase mediated crosslinking of different compounds in the cell wall ensures reinforcement of the barrier against pathogen penetration. Peroxidases are also known to be involved in the production of phytoalexins, which are antibiotic compounds produced by plants under stress conditions.
Thus, it is of interest to stimulate the plant immune system in order to control diseases caused by parasites or pathogens such as fungi, oomycetes, bacteria, viruses, nematodes and insects.
Fungal diseases and diseases caused by oomycetes can also be controlled through the use of fungicides in agriculture. Fungicides are chemical compounds or biological substances used to kill or inhibit fungi or oomycetes or their spores. Fungicides sometimes also have an effect on other plant pathogens such as bacteria, viruses, nematodes or insects. A drawback of using certain fungicides is that fungicide residues can be found on food for human consumption, sometimes posing a danger to human or animal health.
Therefore, it is of high-interest in agriculture to improve the performances of chemical fungicides, in particular in terms of biological activity, with the aim of decreasing the amounts of active ingredient to be used.
The inventors have now unexpectedly found that by adding an oligosaccharidic plant immune system elicitor comprising oligo-galacturonans stabilized by chito-oligosaccharides, to certain fungicides, the efficacy of the fungicide is amplified. This implies that using the elicitor composition of the present invention results in a high reduction in the amount of fungicides needed due to the enhanced potency thereof. Applying the fungicide at a reduced rate, leads to a reduced amount of fungicidal residues on the plants, which is beneficial for human and animal health and the environment in general. Furthermore, by addition of the elicitor, the plant immune system is triggered, particularly reflected by its increased peroxidase activity, which is not achieved through application of the fungicide alone. This synergistic effect of the elicitor on the fungicide renders the plant more resistant to attacks of pathogens and will in the long run thus provide for an increased yield of the crop.
Thus, in a first aspect the present invention relates to a composition for protecting plants against (infection by) plant pathogens comprising:
a) an elicitor comprising: one or more oligo-galacturonan(s) with a degree of polymerization between 9 and 20, and one or more chito-oligosaccharide(s) with a degree of polymerization higher than 5 and a degree of acetylation lower than 50%, in proportions ranging from 1:50 to 50:1, 1:40 to 40:1, 1:30 to 30:1, 1:20 to 20:1, 1:10 to 10:1, 1:5 to 5:1, 1:2 to 2:1, or in equal amounts, and
b) a fungicide, preferably a fungicide selected from the group comprising: phosphonates, benzamides carbamates, dithiocarbamates, phtalimides, triazoles, quinolines, sulphur, and cyanoimidazoles,
c) optionally salts and/o sugar,
wherein said fungicide is present in a concentration which is reduced by at least a factor 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 40, 50, 60, 70, 80, 90, 100 or 200 when compared to the recommended concentration for said plant and/or conditions. More preferably, said elicitor comprises: one or more oligo-galacturonan(s) with a degree of polymerization between 9 and 20, and one or more chito-oligosaccharide(s) with a degree of polymerization between 5 and 10 and a degree of acetylation about 25%, in proportions ranging from 1:50 to 50:1, 1:40 to 40:1, 1:30 to 30:1, 1:20 to 20:1, 1:10 to 10:1, 1:5 to 5:1, 1:2 to 2:1, or in equal amounts.
In an embodiment, the composition of the invention comprises a phosphonate fungicide. In another embodiment, the composition of the invention comprises a benzamide fungicide. In another embodiment, the composition of the invention comprises a carbamate fungicide. In another embodiment, the compostion of the invention comprises a dithiocarbamate fungicide. In another embodiment, the compostion of the invention comprises a phtalimide fungicide. In another embodiment, the compostion of the invention comprises a triazole fungicide. In another embodiment, the compostion of the invention comprises a quinoline fungicide. In another embodiment, the compostion of the invention comprises a sulphur-containing fungicide. In another embodiment, the compostion of the invention comprises a cyanoimidazole fungicide.
The composition according to the invention may further comprise a co-formulant selected from the group comprising: surfactants, anti-freeze agents (urea, ethylene glycol, propylene glycol or glycerol), preservative agents (potassium sorbate, paraben and its derivates, 1,2-benzisothiazol-3(2H)-one or essential oils), absorbent agents (including raids of corn or sawdust), thickeners (including clays or xanthane gum), buffers, sticker agents (including latex, silicon or alkoxylated alkyl), diluents (including rapeseed methyl ester) or a mixture thereof.
In a preferred embodiment, said co-formulant is a surfactant selected from the group comprising: detergents, emulsifiers (alkyl polyglucosides, glycerol ester or polyoxyethylene (20) sorbitan monolaurate (polysorbate 20)), dispersing agents (sodium chloride, potassium chloride, potassium nitrate, calcium chloride or starch of corn), anti-foaming agents (derivates of tartric acid, malic acid or alcohols), penetration enhancers, humectants (ammonium sulfate, glycerin or urea) or wetting agents of ionic or non-ionic type, or a mixture thereof. More preferably, said surfactant comprises one or more of the following components: castor oil ethoxylate, rapeseed methyl ester, alkyl phosphates, tributyl phosphate, tripropyl phosphate, naphthalenesulphonic acid salts, organic sulfonate/2-methylpentane-2,4-diol, alkylpolyglucoside, siloxanes derivates, alkylsulfonates, polycarboxylates, lignosulfonates, alkoxylated triglycerides, fatty amines polymers, dioctylsulfosuccinates or polyoxyethylene (20) sorbitan monolaurate. Most preferably said surfactant is C18-castor-oil-ethoxylate (Dehscofix®), organic sulfonate/2-methylpentane-2,4-diol (Tensiofix Dp400) or polyoxyethylene (20) sorbitan monolaureate (Tween®20-).
In a further embodiment of the invention, the fungicide in the composition of the invention can be selected from the group comprising acylalanines (benalaxyl), anilinopyrimidines (cyprodinil or pyrimethanil), benzamides (fluopicolide or zoxamide), benzimidazoles (fuberidazole, thiabendazole or metrafenone), benzothiadiazoles (acibenzolar-5-methyl), carbamates (benthiavalicarb, iprovalicarb or propamocarb), carboxamides (boscalid), chloronitriles (chlorothalonil), chlorophenyls (tolclophos-methyl), cyanoacetamide oximes (cymoxanil), cyanoimidazoles (cyazofamid), dicarboximides (iprodione), dithiocarbamates (thiram, metiram, mancozeb, manebe or propineb), guanidines (dodine), hydroxyanilides (fenhexamid), imidazoles (fenamidone, imazalil or triflumizole), morpholines (dimethomorph, fenpropimorph, spiroxamine or dodemorph), phosphonates (fosetyl), oxathiins (flutolanil), oxazoles (famoxadone or hymexazol), phenylamides (metalaxyl or metalaxy-M), phenylpyridinamides (fluazinam), phenylpyrroles (fludioxonil), phtalimides (captan or folpet), quinazolinones (proquinazide), quinolines (quinoxyfen), strobilurins (dimoxystrobin, fluoxastrobin, kresomin-methyl, pyraclostrobin, trifloxystrobin or picoxystrobin), thiophenes (silthiofam), triazoles (difenoconazole, epoxyconazole, fenbuconazole, flusilazole, metconazole, myclobutanil, penconazole, propiconazole, tebuconazole, tetraconazole, triadimenol, triticonazole or prothioconazole), copper derivates (copper oxychloride, copper hydrochloride, copper oxide or copper sulphate) and sulphur.
In a preferred embodiment of the composition according to the invention, the fungicide is selected from the group comprising: phosphonates, benzamides, carbamates, dithiocarbamates, phtalimides, triazoles, quinolines, sulphur, and cyanoimidazoles. More preferably, said fungicide comprises ethylhydrogenphosphonate (fosetyl), preferably fosetyl-Al, fosetyl-Na or fosetyl-K, 2,6-dichloro-N-[3-chloro-5-(trifluoromethyl)-2-pyridinylmethyl]benzamide (fluopicolid), propyl 3-(dimethylamino)propylcarbamate hydrochloride (propamocarb), manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt (mancozeb), N-(trichloromethylthio)phthalimide (folpet), (2RS,3SR)-1-[3-(2-chlorophenyl)-2,3-epoxy-2-(4-fluorophenyl)propyl]-1H-1,2,4-triazole (epoxyconazole), 5,7-dichloro-4-quinolyl 4-fluorophenyl ether (quinoxyfen), sulphur, 4-chloro-2-cyano-N,N-dimethyl-5-(4-methylphenyl)-1H-imidazole-1-sulfonamide (cyazofamid) or a combination thereof.
In a preferred embodiment, said composition according to the invention comprises a further plant immune system elicitor selected from the group comprising: silica, copper, sulfur, aluminium, vanadium, cobalt, nickel, iron, silver, salicylic acid and its derivates (including acetyl-salicylic acid, isonicotinic acid, acibenzolar-S-methyl), jasmonic acid and its derivates (including methyl jasmonate), ethylene and its derivates, polysaccharides (including glucans, xyloglucans, chitin, chitosans, fucans, galactofucans, xylans, galactans, alginates, galacturonans, apiogalacturonans, fructans including inulin, mannans, xylomannans, galactomannans, glucomannans and galactomannans), algae extracts (green algae extracts including ulvans, brown algae extracts including laminarin, and red algae extracts including carragenans), oligosaccharides (including trehalose), peptides (including systemin, 13-pep, flg-22, glutathion), amino acids, proteins (including harpin and flagellin), peptone, beef extract, essential oils (including cumin, anise, mint, cinnamon, thyme, basil, cardamom, coriander, oregano, manzanilla, clove, jojoba and tea tree oils), lipids (including ergosterol, amphotericin, sphingolipids, cerebrosides), glycolipids (including syringolids), glycoproteins (including cryptogeins), lipopeptides, lipoproteins (including volicitin), yeast extracts (including extracts from Saccharomyces, Candida, Pichia, Aureobasidium and more particularly Saccharomyces cerevisiae, Candida famata, Candida oleophila, Pichia guilliermondii, Aureobasidium pullulans), fungal extracts (including extracts from Trichoderma, Megasperma, Pyricularia, Alternaria, Pythium, Puccinia, Colletottichum, Verticillium, Magnaporthe), bacterial extracts (including extracts from Escherichia, Rhyzobia, Pseudomonas), BABA, probenazole, isothianil, phosphorous acid and its derivates (including aluminium, sodium and potassium fosetyl), horsetail extracts, potassium iodide and potassium thiocyanate, Citrus extracts, Yucca extracts Salix extracts and plant decoctions (including nettle decoction). More preferably, said further plant immune system elicitor comprises laminarin.
In another preferred embodiment, said elicitor composition according to the invention comprises a further plant immune system elicitor that contains silicon, preferably said further plant immune system elicitor is a silicate, more preferably sodium silicate.
The composition according to the present invention can also be composed as follows:
a) an elicitor comprising: one or more oligo-galacturonan(s) with a degree of polymerization between 9 and 20, and one or more chito-oligosaccharide(s) with a degree of polymerization higher than 5 and a degree of acetylation lower than 50% (preferably with a degree of polymerization between 5 and 10 and a degree of acetylation of about 25%), in proportions ranging from 1:50 to 50:1, 1:40 to 40:1, 1:30 to 30:1, 1:20 to 20:1, 1:10 to 10:1, 1:5 to 5:1, 1:2 to 2:1, or in equal amounts, and
b) an adjuvant, preferably a surfactant selected from the group comprising: detergents, emulsifiers (alkyl polyglucosides, glycerol ester or polyoxyethylene (20) sorbitan monolaurate), dispersing agents (sodium chloride, potassium chloride, potassium nitrate, calcium chloride or starch of corn), anti-foaming agents (derivates of tartric acid, malic acid or alcohols), penetration enhancers, humectants (ammonium sulfate, glycerin or urea) or wetting agents of ionic or non-ionic type. More preferably, said surfactant comprises one or more of the following components: castor oil ethoxylate, rapeseed methyl ester, alkyl phosphates, tributyl phosphate, tripropyl phosphate, naphthalenesulphonic acid salts, organic sulfonate/2-methylpentane-2,4-diol, alkylpolyglucoside, siloxanes derivates, alkylsulfonates, polycarboxylates, lignosulfonates, alkoxylated triglycerides, fatty amines polymers, dioctylsulfosuccinates or polyethylene (20) sorbitan monolaureate, even more preferably said surfactant is Dehscofix®, Tensiofix Dp400 or Tween®20.
Additionally, other co-formulants may be present in the composition, such as anti-freeze agents (urea, glycol ethylene, glycol propylene or glycerol), preservative agents (potassium sorbate, paraben and its derivates, 1,2-benzisothiazol-3(2H)-one or essential oils), absorbent agents (including raids of corn or sawdust), thickeners (including clays or xanthane gum), buffers, sticker agents (including latex, silicon or alkoxylated alkyl), diluents (including rapeseed methyl ester) or a mixture thereof.
Said composition comprising the elicitor and an adjuvant as defined above, may also comprise additionally a fungicide, further elicitor and/or further adjuvants as defined herein.
Alternatively, the composition according to the present invention can also be composed as follows:
a) an elicitor comprising: one or more oligo-galacturonan(s) with a degree of polymerization between 9 and 20, and one or more chito-oligosaccharide(s) with a degree of polymerization higher than 5 and a degree of acetylation lower than 50% (preferably with a degree of polymerization between 5 and 10 and a degree of acetylation of about 25%), in proportions ranging from 1:50 to 50:1, 1:40 to 40:1, 1:30 to 30:1, 1:20 to 20:1, 1:10 to 10:1, 1:5 to 5:1, 1:2 to 2:1, or in equal amounts, and
b) a further elicitor selected from the group comprising: silica, a silicate, sodium silicate, copper, sulfur, aluminium, vanadium, cobalt, nickel, iron, silver, salicylic acid and its derivates (including acetyl-salicylic acid, isonicotinic acid, acibenzolar-S-methyl), jasmonic acid and its derivates (including methyl jasmonate), ethylene and its derivates, polysaccharides (including glucans, xyloglucans, chitin, chitosans, fucans, galactofucans, xylans, galactans, alginates, galacturonans, apiogalacturonans, fructans including inulin, mannans, xylomannans, galactomannans, glucomannans and galactomannans), algae extracts (green algae extracts including ulvans, brown algae extracts including laminarin, and red algae extracts including carragenans), oligosaccharides (including trehalose), peptides (including systemin, 13-pep, flg-22, glutathion), amino acids, proteins (including harpin and flagellin), peptone, beef extract, essential oils (including cumin, anise, mint, cinnamon, thyme, basil, cardamom, coriander, oregano, manzanilla, clove, jojoba and tea tree oils), lipids (including ergosterol, amphotericin, sphingolipids, cerebrosides), glycolipids (including syringolids), glycoproteins (including cryptogeins), lipopeptides, lipoproteins (including volicitin), yeast extracts (including extracts from Saccharomyces, Candida, Pichia, Aureobasidium and more particularly Saccharomyces cerevisiae, Candida famata, Candida oleophila, Pichia guifliermondii, Aureobasidium pullulans), fungal extracts (including extracts from Trichoderma, Megasperma, Pyricularia, Alternaria, Pythium, Puccinia, Colletotrichum, Verticillium, Magnaporthe), bacterial extracts (including extracts from Escherichia, Rhyzobia, Pseudomonas), BABA, probenazole, isothianil, phosphorous acid and its derivates (including aluminium, sodium and potassium fosetyl), horsetail extracts, potassium iodide and potassium thiocyanate, Citrus extracts, Yucca extracts Salix extracts and plant decoctions (including nettle decoction). More preferably, said further plant immune system elicitor comprises laminarin.
Said composition comprising the two different elicitors as defined above, may also comprise additionally a fungicide, a further elicitor and/or an adjuvant or surfactant as defined herein.
In yet another alternative, the composition according to the present invention can be composed as follows:
Said composition comprising the two different elicitors as defined above, may also comprise additionally a fungicide, a further elicitor and/or an adjuvant or surfactant as defined herein.
In a preferred embodiment, said composition comprises:
The invention further provides for the use of any one of the compositions according to the present invention in agricultural applications, particularly to protect plants against (infection by) plant pathogens, preferably phytopathogenic fungi, oomycetes, bacteria, viruses, nematodes, insects, and/or aphids, more preferably to enhance the efficacy of the fungicide or to stimulate the plant innate immune system.
The invention additionally provides a method for protecting plants against (infection by) plant pathogens such as phytopathogenic fungi, oomycetes, bacteria, viruses, nematodes, insects, and/or aphids, comprising applying an effective and substantially non-phytotoxic amount of any one of the compositions according to the invention to said plants.
The invention further provides a method for enhancing the fungicidal activity of a fungicide, comprising adding any any one of the compositions according to the invention comprising one or more oligo-galacturonan(s) with a degree of polymerization between 9 and 20 and one or more chito-oligosaccharide(s) with a degree of polymerization higher than 5 and a degree of acetylation lower than 50% (preferably with a degree of polymerization between 5 and 10 and a degree of acetylation of about 25%) in varying proportions to a fungicidal composition.
In a preferred embodiment of said method, the composition of the invention is applied before harvest or post harvest to the whole plant, the leaves, the flowers, fruits, seeds, seedlings or seedlings pricking out, propagation material such as tubers or rhizomes, plants pricking out, and/or to the soil or inert substrate wherein the plant is growing or in which it is desired to grow, by spraying, drenching, soaking, dipping, injection or administration through fertilising or irrigation systems.
Preferably, the plant is selected from the group comprising: cotton, flax, vine, fruit, vegetable, major horticultural and forest crops such as: Rosaceae sp., Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp., Actimidaceae sp., Lauraceae sp., Musaceae sp., Rubiaceae sp., Theaceae sp., Sterculiceae sp., Rutaceae sp., Solanaceae sp., Vitaceae sp. Liliaceae sp., Asteraceae sp., Umbelliferae sp., Cruciferae sp., Chenopodiaceae sp., Cucurbitaceae sp., Papilionaceae sp., such as Graminae sp., Fabacae sp., as well as genetically modified homologues of these crops.
Preferably, said plant pathogen is selected from the group comprising: fungi, oomycetes, bacteria, viruses, nematodes and insects such as aphids.
The invention also provides for the use of an elicitor comprising: one or more oligo-galacturonan(s) with a degree of polymerization between 9 and 20, and one or more chito-oligosaccharide(s) with a degree of polymerization higher than 5 and a degree of acetylation lower than 50% (preferably with a degree of polymerization between 5 and 10 and a degree of acetylation of about 25%), in proportions ranging from 1:50 to 50:1, 1:40 to 40:1, 1:30 to 30:1, 1:20 to 20:1, 1:10 to 10:1, 1:5 to 5:1, 1:2 to 2:1, or in equal amounts, for enhancing the efficacy of a fungicide.
In a preferred embodiment of said use, the fungicide is selected from the group comprising: phosphonates, benzamides, carbamates, dithiocarbamates, phtalimides, triazoles, quinolines, sulphur, and cyanoimidazoles. More preferably, said fungicide comprises ethylhydrogenphosphonate (fosetyl), preferably fosetyl-Al, fosetyl-Na or fosetyl-K, 2,6-dichloro-N-[3-chloro-5-(trifluoromethyl)-2-pyridinylmethyl]benzamide (fluopicolid), propyl 3-(dimethylamino)propylcarbamate hydrochloride (propamocarb), manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt (mancozeb), N-(trichloromethylthio)phthalimide (folpet), (2RS,3SR)-1-[3-(2-chlorophenyl)-2,3-epoxy-2-(4-fluorophenyl)propyl]-1H-1,2,4-triazole (epoxyconazole), 5,7-dichloro-4-quinolyl 4-fluorophenyl ether (quinoxyfen), sulphur, 4-chloro-2-cyano-N,N-dimethyl-5-(4-methylphenyl)-1H-imidazole-1-sulfonamide (cyazofamid) or a combination thereof.
In a particularly preferred embodiment, said composition additionally comprises an adjuvant such as a surfactant selected from the group comprising: detergents, emulsifiers (alkyl polyglucosides, glycerol ester or polyoxyethylene (20) sorbitan monolaureate), dispersing agents (sodium chloride, potassium chloride, potassium nitrate, calcium chloride or starch of corn), anti-foaming agents (derivates of tartric acid, malic acid or alcohols), penetration enhancers, humectants (ammonium sulfate, glycerin or urea) or wetting agents of ionic or non-ionic type.
Additionally, other co-formulants may be present, such as anti-freeze agents (urea, glycol ethylene, glycol propylene or glycerol), preservative agents (potassium sorbate, paraben and its derivates, 1,2-benzisothiazolin-3(2H)-one or essential oils), absorbent agents (including raids of corn or sawdust), thickeners (including clays or xanthane gum), buffers, sticker agents (including latex, silicon or alkoxylated alkyl), diluents (including rapeseed methyl ester) or a mixture thereof.
In a preferred embodiment, said surfactant comprises one or more of the following components: castor oil ethoxylate, rapeseed methyl ester, alkyl phosphates, tributyl phosphate, tripropyl phosphate, naphthalenesulphonic acid salts, organic sulfonate/2-methylpentane-2,4-diol, alkylpolyglucoside, siloxanes derivates, alkylsulfonates, polycarboxylates, lignosulfonates alkoxylated triglycerides, fatty amines polymers, dioctylsulfosuccinates or polyethylene (20) sorbitan monolaureate.
In a preferred embodiment, said fungicide is applied in a concentration which is reduced by at least a factor 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 40, 50, 60, 70, 80, 90, 100 or 200 when compared to the recommended concentration for said plant and/or conditions.
The invention further provides a method for enhancing the efficacy of a fungicide, comprising adding an elicitor comprising one or more oligo-galacturonan(s) with a degree of polymerization between 9 and 20 and one or more chito-oligosaccharide(s) with a degree of polymerization higher than 5 and a degree of acetylation lower than 50% (preferably with a degree of polymerization between 5 and 10 and a degree of acetylation of about 25%), in varying proportions ranging from 1:50 to 50:1, 1:40 to 40:1, 1:30 to 30:1, 1:20 to 20:1, 1:10 to 10:1, 1:5 to 5:1, 1:2 to 2:1, or in equal amounts, to a fungicidal composition.
The invention further provides a composition for protecting plants against (infection by) plant pathogens comprising:
a) an elicitor comprising one or more oligo-galacturonan(s) with a degree of polymerization between 9 and 20 and one or more chito-oligosaccharide(s) with a degree of polymerization higher than 5 and a degree of acetylation lower than 50% in proportions ranging from 1:50 to 50:1, preferably from 1:10 to 10:1, more preferably from 1:5 to 5:1, and
b) a surfactant selected from the group comprising: castor oil ethoxylate, rapeseed methyl ester, alkyl phosphates, tributyl phosphate, tripropyl phosphate, naphthalenesulphonic acid salts, organic sulfonate/2-methylpentane-2,4-diol, alkylpolyglucoside, siloxanes derivates, alkylsulfonates, polycarboxylates, lignosulfonates, alkoxylated triglycerides, fatty amines polymers, dioctylsulfosuccinates or polyoxyethylene (20) sorbitan monolaurate, preferably wherein said surfactant is C18-castor-oil-ethoxylate (Dehscofix®), organic sulfonate/2-methylpentane-2,4-diol (Tensiofix Dp400), and
c) optionally polyoxyethylene (20) sorbitan monolaurate (Tween® 20). The invention also provides for the use of any of said compositions as defined herein in agricultural applications, more particularly for protecting plants against (infection by) plant pathogens preferably against phytopathogenic fungi, oomycetes, bacteria, viruses, nematodes and insects, or for stimulating the plant immune system. The invention further provides a method for protecting plants against (infection by) plant pathogens comprising applying an effective and substantially non-phytotoxic amount of the composition as defined herein to said plants.
Alternatively, the invention provides a composition for protecting plants against (infection by) plant pathogens comprising:
a) an elicitor comprising one or more oligo-galacturonan(s) with a degree of polymerization between 9 and 20 and one or more chito-oligosaccharide(s) with a degree of polymerization higher than 5 and a degree of acetylation lower than 50% in proportions ranging from 1:50 to 50:1, preferably from 1:10 to 10:1, more preferably from 1:5 to 5:1, and
b) a second elicitor, preferably a second elicitor comprising sodium silicate, and
c) optionally a surfactant, preferably a surfactant comprising polyoxyethylene (20) sorbitan monolaurate. The invention also provides for the use of any of said compositions as defined herein in agricultural applications, more particularly for protecting plants against (infection by) plant pathogens preferably against phytopathogenic fungi, oomycetes, bacteria, viruses, nematodes and insects, or for stimulating the plant immune system. The invention further provides a method for protecting plants against (infection by) plant pathogens comprising applying an effective and substantially non-phytotoxic amount of the composition as defined herein to said plants.
The compositions of the present invention can be provided in a single mixture, or can be a combination of two or more separate mixtures or compositions, which can be applied to the plant or substrate simultaneously, or sequentially. For example, a composition comprising an elicitor and a fungicide according to the present invention can be administered simultaneously, or the elicitor can be applied first, followed by the fungicide, or vice versa.
The present invention will be further exemplified by the following description, drawings and examples, which are not to be seen as limiting. The skilled person will be capable of designing alternative embodiment using the general concept of the present invention. Said alternative concepts also form part of the invention.
As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The term also encompasses “consisting of” and “consisting essentially of”.
The term “about” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, in particular variations of +/−10% or less, preferably +/−5% or less, more preferably +/−1% or less, and still more preferably +/−0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed.
All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. When specific terms are defined in connection with a particular aspect or embodiment, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments, unless otherwise defined.
The present invention advantageously provides a composition which is more active as regards its efficacy against phytopathogenic fungi, oomycetes, bacteria, viruses, nematodes and insects by adding an elicitor of the plant immune system to a fungicide applied at a reduced rate. This enables to apply the fungicide at the reduced rate, which in turn will reduce the amount of fungicidal residues found on plants.
A typical composition according to the invention comprises the following components:
a) an elicitor characterized in that it comprises one or more oligo-galacturonan(s and one or more chito-oligosaccharide(s) in proportions ranging from 1:50 to 50:1, preferably from 1:40 to 40:1, more preferably from 1:30 to 30:1, even more preferably from 1:20 to 20:1, most preferably from 1:10 to 10:1 and for instance is 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1, and
b) a fungicide, wherein said fungicide is preferably selected from the group comprising: phosphonates, benzamides, carbamates, dithiocarbamates, phtalimides, triazoles, quinolines, sulphur, and cyanoimidazoles.
The term “elicitor” as used herein refers to an inducer of the plant immune system. When plants are attacked by pathogens, they defend themselves with an arsenal of mechanisms directed to fight infection or make the plant less attractive to pathogens. As in most cellular responses to the environment, defense mechanisms are activated when receptors directly or indirectly come in contact with pathogens. The ligands of these plant receptors are elicitors of the plant immune system. There is a wide variety of elicitors, including so-called non-specific elicitors or PAMPs (pathogen associated molecular patterns) e.g. degradation products of cell wall components of pathogens or derived from a plant cell wall, and pathogen-specific elicitors or effectors e.g. avirulence gene products of pathogens such as AVR 9 (Avr gene products). Elicitors of the plant immune system comprise proteins, oligosaccharides, polysaccharides, lipids, glycolipids, glycoproteins, peptides of diverse origin, lipopeptides, algal extracts, extracts from the walls of plant material and/or fungal material, fungi, bacterial material and viral material, or yeast material and/or extracts. Elicitors also comprise salicylic acid, jasmonic acid, lipid peroxidation products and/or one or more of their esters. The elicitor preferably used in the present invention is an oligosaccharidic complex of oligo-galacturonans stabilized by chito-oligosaccharides, as e.g. specified in document WO 2008/065151.
The term “oligo-galacturonan” encompasses herein a chain of a-(1-4)-linked D-galacturonic acids. Oligo-galacturonans are derived from pectin, which is a major constituent of plant cell walls. Pectin consists of a complex set of polysaccharides, including homogalacturonans, which are linear chains of α-(1-4)-linked D-galacturonic acids. Oligo-galacturonans are released from these galacturonans through the action of pectolytic enzymes.
The oligo-galacturonans present in the composition according to the invention have a degree of polymerization higher than 8, preferably comprised between 9 and 20 or between 9 and 15.
The terms “chito-oligosaccharide” and “chitosan oligosaccharide” are used interchangeably herein and refer to a linear oligosaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitosan is naturally found in few organisms, but is mostly produced industrially by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans (crabs, shrimp, etc.), insects, and in the cell walls of some fungi and other organisms.
The chito-oligosaccharides used in the composition according to the invention have a degree of acetylation lower than 50%, lower than 40%, or lower than 30%, preferably about 25% and a degree of polymerization higher than 5, preferably comprised between 5 and 10.
In a preferred embodiment, the elicitor used in the compositions according to the present invention comprises:
In a preferred embodiment, salts are added to the composition according to the invention to ensure good ionic conditions for the stability of the oligosaccharidic complex, in particular to obtain the so-called “egg-box” conformation of the oligo-galacturonan therein. Preferably, a combination of both monovalent and divalent cation salts is preferably added. Ideally, both calcium and monovalent cations such as sodium are added. The best composition can be determined with the help of the ELISA test with the 2F4 Monoclonal Antibody (cf. Cabrera et al., 2008, Glycobiology 18 (6): 473-482) but also with the help of bioassays that must be optimized for each plant species targeted. Too much calcium displaces chitosan from the complex. Too much monovalent cation displaces calcium from the pectin egg box and can be phytotoxic. A small amount of copper can replace calcium in the egg boxes and is beneficial for the elicitor supramolecular conformation. NaCl is preferably used as monovalent cation salt. KNO3 is also suitable, but less preferred due to possible effect on opening of stomata, possibly enabling entry of parasites into the leaf or plant. CaCl2 is preferably used as divalent cation salt, but CuCl2 or ZnCl2 are also suitable. More preferably, from about 0.2 to 1 mM, most preferably 0.5 mM CaCl2 is used, in combination with from about 5 to 100 mM, most preferably 20 to 50 mM NaCl.
In another preferred embodiment, sucrose is added to the composition according to the invention. Preferably, sucrose is added in a concentration of from about 1 mM to 20 mM, most preferably about 5 to 10 mM sucrose. Sucrose triggers signaling through hexokinase and is also a wetting agent.
In a most preferred embodiment, both salts and sucrose are added to the compositions of the invention.
The term “fungicide” encompasses chemical or biological substances or compositions used to kill or inhibit fungi or oomycetes, e.g. by preventing sporulation, or their spores. Fungicides can exert their biological effect by different modes of action, for example, but not limited to, by interference with nucleic acid synthesis, mitosis and cell division, respiration, amino acids and protein synthesis, signal transduction, lipids and membrane synthesis, sterol biosynthesis, glucan synthesis in the pathogen or by inducing host plant defense.
Any fungicide can be included in the composition of the invention, such as, 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 (benthiavalicarb, iprovalicarb or propamocarb), carboxamides (boscalid), chloronitriles (chlorothalonil), chlorophenyls (tolclophos-methyl), cyanoacetamide oximes (cymoxanil), cyanoimidazoles (cyazofamid), dicarboximides (iprodione), dithiocarbamates (thiram, metiram, mancozeb, manebe or propineb), guanidines (dodine), hydroxyanilides (fenhexamid), imidazoles (fenamidone, imazalil or triflumizole), morpholines (dimethomorph, fenpropimorph, spiroxamine or dodemorph), phosphonates (fosetyl), oxathiins (flutolanil), oxazoles (famoxadone or hymexazol), phenylamides (metalaxyl or metalaxy-M), phenylpyridinamides (fluazinam), phenylpyrroles (fludioxonil), phtalimides (captan or folpet), quinazolinones (proquinazide), quinolins (quinoxyfen), strobilurins (dimoxystrobin, fluoxastrobin, kresomin-methyl, pyraclostrobin, trifloxystrobin or picoxystrobin), thiophenes (silthiofam), triazoles (difenoconazole, epoxyconazole, fenbuconazole, flusilazole, metconazole, myclobutanil, penconazole, propiconazole, tebuconazole, tetraconazole, triadimenol, triticonazole or prothioconazole), copper derivates (copper oxychloride, copper hydrochloride, copper oxide or copper sulphate) and sulphur.
Preferably, the fungicide is chosen from the list comprising: phosphonates, benzamides, carbamates, dithiocarbamates, phtalimides, triazoles, quinolines, sulphur and cyanoimidazoles.
The compositions of the invention will typically contain additional components, known as co-formulants or adjuvants, to obtain a product with good handling, efficacy and stability properties. As used herein, the terms “co-formulant” or “adjuvant” designate any substance other than the main oligosacharidic complex elicitor component defined herein, that is intentionally added to the elicitor composition of the invention.
In a preferred embodiment, the composition according to the invention further comprises a co-formulant or adjuvant selected from the group comprising: surfactants, anti-freeze agents (including urea, ethylene glycol, propylene glycol or glycerol), preservative agents (including potassium sorbate, paraben and its derivates, 1,2-benzisothiazolin-3(2H)-one or essential oils), absorbent agents (including raids of corn or sawdust), thickeners (including clays or xanthane gum), buffers, sticker agents (including latex, silicon or alkoxylated alkyl), diluents (including rapeseed methyl ester) or any standard inert ingredient conventionally used in agricultural compositions, or a mixture thereof.
In a particularly preferred embodiment, the composition according to the invention further comprises a surfactant.
With “surfactant” is meant herein a compound that lowers the surface tension of a liquid, allowing easier spreading. The surfactant can be a detergent, an emulsifier (including alkyl polyglucosides glycerol ester or polyoxyethylene (20) sorbitan monolaurate), a dispersing agent (including sodium chloride, potassium chloride, potassium nitrate, calcium chloride or starch of corn), a foaming agent (including derivates of tartric acid, malic acid or alcohols), a penetration enhancer, a humectant (including ammonium sulfate, glycerin or urea) or a wetting agent of ionic or non-ionic type or a mixture of such surfactants. The surfactants used in the present invention are penetration enhancers, dispersing agents or emulsifiers.
The term “penetration enhancer” is understood herein as a compound that accelerates the uptake of active ingredient through the cuticle of a plant into the plant, i.e. the rate of uptake, and/or increases the amount of active ingredient absorbed into the plant. Classes of substances known as penetration enhancers, include alkyl phosphates, such as tributyl phosphate and tripropyl phosphate, and naphthalenesulphonic acid salts. Mention may be made, for example, of surfactants sold under the trade name 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.
With “dispersing agent” is meant a substance added to a suspension, usually a colloid, to improve the separation of particles and to prevent settling or clumping. Mention can be made of the dispersing agent which is sold under the trade name Tensiofix Dp400 (available from Ajinomoto OmniChem), essentially comprising organic sulfonate and 2-methylpentane-2,4-diol.
The term “emulsifier” as used herein refers to a substance that stabilizes an emulsion, i.e. a mixture of two or more liquids. Mention can be made of the emulsifiers sold under the trade names Tween® 20, which essentially comprises polyoxyethylene (20) sorbitan monolaurate (polysorbate 20), and Radia®, which essentially comprises alkyl polyglycosides.
In a preferred embodiment, said surfactant comprises one or more of the following components: castor oil ethoxylate, rapeseed methyl estr, alkyl phosphates, tributyl phosphate, tripropyl phosphate, naphthalenesulphonic acid salts, organic sulfonate/2-methylpentane-2,4-diol, alkylpolyglucoside, siloxanes derivates, alkylsulfonates, polycarboxylates, lignosulfonates, alkoxylated triglycerides, fatty amines polymers, dioctylsulfosuccinates or polyoxyethylene (20) sorbitan monolaurate (polysorbate 20), more preferably said surfactant is C18-castor-oil-ethoxylate (Dehscofix®), organic sulfonate/2-methylpentane-2,4-diol (Tensiofix Dp40) or polyoxyethylene (20) sorbitan monolaurate (Tween®20).
The present invention also discloses a composition comprising:
In another embodiment, the compositions according to the invention also comprise one or more other active compounds selected from the group comprising: herbicides, insecticides, plant growth regulators or other plant immune system elicitors.
In a preferred embodiment, said compositions according to the invention further comprises a further plant immune system elicitor chosen among silica, copper, sulfur, aluminium, vanadium, cobalt, nickel, iron, silver, salicylic acid and its derivates (including acetyl-salicylic acid, isonicotinic acid, acibenzolar-S-methyl), jasmonic acid and its derivates (including methyl jasmonate), ethylene and its derivates, polysaccharides (including glucans, xyloglucans, chitin, chitosans, fucans, galactofucans, xylans, galactans, alginates, galacturonans, apiogalacturonans, fructans including inulin, mannans, xylomannans, galactomannans, glucomannans and galactomannans), algae extracts (green algae extracts including ulvans, brown algae extracts including laminarin, and red algae extracts including carragenans), oligosaccharides (including trehalose), peptides (including systemin, 13-pep, flg-22, glutathion), amino acids, proteins (including harpin and flagellin), peptone, beef extract, essential oils (including cumin, anise, mint, cinnamon, thyme, basil, cardamom, coriander, oregano, manzanilla, clove, jojoba and tea tree oils), lipids (including ergosterol, amphotericin, sphingolipids, cerebrosides), glycolipids (including syringolids), glycoproteins (including cryptogeins), lipopeptides, lipoproteins (including volicitin), yeast extracts (including extracts from Saccharomyces, Candida, Pichia, Aureobasidium and more particularly Saccharomyces cerevisiae, Candida famata, Candida oleophila, Pichia guifliermondii, Aureobasidium pullulans), fungal extracts (including extracts from Trichoderma, Megasperma, Pyricularia, Alternaria, Pythium, Puccinia, Colletotrichum, Verticillium, Magnaporthe), bacterial extracts (including extracts from Escherichia, Rhyzobia, Pseudomonas), BABA, probenazole, isothianil, phosphorous acid and its derivates (including aluminium, sodium and potassium fosetyl), horsetail extracts, potassium iodide and potassium thiocyanate, Citrus extracts, Yucca extracts Salix extracts and plant decoctions (including nettle decoction).
Preferably, said further plant immune system elicitor contains laminarin (a linear β(1→3)-glucan with β(1→6)-linkages) such as, for example Vacciplant Fruit®.
The present invention also discloses a composition comprising:
In another preferred embodiment, said compositions according to the invention further comprise a further plant immune system elicitor that contains silicon or silicium (Si), such as, for example, silica (SiO2) or silicates, including sodium silicate (Na2SiO3). Preferably, said further plant immune system elicitor is a silicate, more preferably sodium silicate.
The present invention also discloses a composition comprising:
In a preferred embodiment, said compositions of the invention comprising an elicitor and a second elicitor further comprise an an adjuvant, preferably a surfactant, more preferably a surfactant comprising polyoxyethylene (20) sorbitan monolaurate such as Tween® 20.
The compositions according to the present invention encompass not only compositions which are ready to be applied to the plant by means of a suitable device, such as a spraying device, but also the commercial concentrated compositions which have to be diluted before application to the plant.
The compositions according to the invention are themselves in quite diverse, solid or liquid forms. As solid composition forms, mention may be made of powders for dusting and granules, in particular those obtained by extrusion, by compacting, by impregnation of a granulated support or by granulation from a powder, tablets or effervescent lozenges. As liquid composition forms or forms intended to constitute liquid compositions when applied, mention may be made of solutions, in particular water-soluble concentrates, emulsions, concentrated suspensions, dispersions, aerosols and wettable granules and powders (or powders for spraying), pastes, gels and water soluble packagings.
In another aspect, the present invention relates to the use of the compositions of the invention in agricultural applications, more particularly for protecting plants against (infection by) plant pathogens.
The present invention not only provides in the simultaneous use of the different components of the compositions, i.e. the use of the compostions, but also provides in the sequential use of the different components of the compositions. For instance, the inventors have found that the sequential use of the oligosaccharidic plant immune system elicitor comprising oligo-galacturonans stabilized by chito-oligosaccharides and a fungicide also results in enhanced efficacy of the fungicide. By “sequential use” is meant herein that first the oligosaccharide elicitor is added and subsequently the fungicide, adjuvant, surfactant or other elicitor is applied to the plant, or vice versa.
“Plant pathogens” refer to organisms that cause infectious diseases in plants and include fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protozoa, nematodes and parasitic plants. In a preferred embodiment, the plant pathogens are fungi, oomycetes, bacteria, viruses, nematodes and insects.
The majority of phytopathogenic fungi belong to the Ascomycetes and the Basidiomycetes, reproducing both sexually and asexually via the production of spores that can be spread through air (wind) or water, or can be soil borne such as zoospores that are capable of living saprotrophically, carrying out the first part of their lifecycle in the soil. Deuteromycetes (Fungi imperfecti) are fungi from which only the asexual form of reproduction is known, meaning that this group of fungus produces their spores asexually. The Oomycetes are not true fungi but are fungal-like organisms that use the same mechanisms as fungi to infect plants.
Fungal and fungal-like organisms are heterotrophic, i.e. they need an external source of nutrients for growth, development and reproduction. An understanding of other key features of these organisms can assist in their identification:
Plant diseases caused by fungi including yeasts, rusts, smuts, mildews, molds, mushrooms and toadstools that can be treated using the elicitor compositions according to the present invention are for example:
“Rust” is a fugal diseases in plants, which produces reddish-brown discoloration of the stems and leaves.
“Black Rot” is characterized by the darkening and decaying of leaves of fruit and vegetable plants.
“Black Spot” is one of the many fungal diseases in plants. It is named “black spot” because it produces small black spots on plants.
“Bottom Rot” is a fungal disease found on lettuce plants. The characteristic of this fungus is that it first affects the leaves on the lower part of the plant and then moves upward to affect the upper part.
“Canker” affects the roots and bark, is found on woody trees and is notorious for causing localized damage to the bark of trees.
“Cotton Ball” is notorious for attacking cranberry plants.
“Crown Wart” like canker attacks on the barks of woody trees, this fungus attacks the stem of the alfalfa plants. It forms white protrusions at the base of the stem of the plant.
“Potato Wart” is a fungal disease that causes dark, warty, spongy excrescences in the eyes of potato tubers, similar to the crown wart in alfalfa plants.
“Damping Off” causes excessive moisture conditions of the seedlings.
“Dry Rot” causes the drying and crumbling of timber, bulbs, potatoes or fruits.
“Rhizoctinia Disease” is caused by fungi called Pellicularia and Corticium. It is often seen to affect small potatoes.
“Root Rot” infects the roots causing root decay, eventually causing the plant to die.
“Sclerotium Rot” is caused by Fungus of the genus Sclerotium causing the formation of sclerotia on plants.
“Dutch Elm Disease” is a fungal disease affecting Elms. It spreads from one plant to another through root grafts or by the elm beetles that feed on small twigs.
“Pinkroot” attacks onion plants and makes them unsuitable for consumption.
“Soft Rot” is a slimy, mushy decay caused by fungi.
“Yellow Spot” is characterized by a yellow spotting on the leaves of plants.
“Powdery Mildews” is often specific to the host that it invades. It is normally seen on roses, lilac, English oak, zinnias, etc.
“Plant Wilting” gets it name because it causes the plant it infects to wilt. The fungus invasion starts in the roots and then slowly makes its way into the stem and plugs the vascular system of the plant.
“Decay” is decomposition of wood that is caused by fungi. When it attacks living plant tissue, it kills the plants.
Non-limiting examples of phytopathogenic bacteria include the genii Erwinia (including Erwinia amylovora, causing fire blight on pears), Pseudomonas (including Pseudomonas syringae), Xanthomonas (including Xanthomonas orizae, Xanthomonas citri, Xanthomonas fuscans (citrus cancer) and Xanthomonas fragariae) and Ralstonia.
Non-limiting examples of phytopathogenic viruses include Cucumber Mosaic Virus, Barley Yellow Mosaic Virus, Strawberry Mild Yellow Edge Virus, Strawberry Latent Ringspot Virus, Beet Necrotic Yellow Vein Virus and Potato Virus Y.
Phytopathogenic insects that can be targeted by application of the compositions according to the invention include aphids, beetles, bugs, hoppers, locusts, mites, ants, ticks, trips, whiteflies, rootworms, maggots, weevils, (stem)borers, caterpillars, butterflies, leaf-rolers, leaf-miners, etc.
“Plant protection” as used herein refers to the activation of mechanisms aimed at controlling or reducing the pathogens and/or to minimize their effects on the plant. Plant protection can be achieved by killing the pathogens, by delaying their growth and/or reproduction, by reducing sporulation, etc.
According to another aspect of the present invention, there is provided a method for protecting plants against (infection by) plant pathogens, characterized in that an effective and substantially non-phytotoxic amount of a composition according to the invention is applied to the plants. The expression “effective and non-phytotoxic amount” means an amount of elicitor composition according to the invention that is sufficient to induce control or destruction of the plant pathogens present or liable to appear on the plants, and that does not entail any appreciable symptom of phytotoxicity for said plants. Such an amount can vary within a wide range depending on the plant pathogen 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 trials that are within the capabilities of a person skilled in the art.
In a particularly preferred embodiment, the fungicide in the composition of the invention is applied at a reduced rate. Preferably, the rate of the fungicide is reduced by at least a factor 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 40, 50, 60, 70, 80, 90, or 100 when compared to the recommended rate, or is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, or even 95% or more of the recommended dosage for said plant and/or conditions. More preferably, the rate of the fungicide 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.
Application of the composition according to the invention can be carried out in accordance with techniques well known to persons skilled in the art. The composition according to the invention 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 wherein the plant is growing or in which it is desired to grow (e.g. inorganic substrates like sand, rockwool, glasswool; expanded minerals like perlite, vermiculite, zeolite or expanded clay), Pumice, Pyroclastic materials or stuff, synthetic organic substrates (e.g. polyurethane), organic substrates (e.g. peat, composts, tree waste products like coir, wood fibre or chips, tree bark) or to a liquid substrate (e.g. floating hydroponic systems, Nutrient Film Technique, Aeroponics). The application can be done by spraying, drenching, soaking, dipping, injection, etc., or via fertigation systems.
It can also be useful to apply the compositions according to the invention to propagation material such as tubers or rhizomes, but also seeds, seedlings or seedlings pricking out and plants or plants pricking out. The compositions according to the invention can also be applied post-harvest to control decay.
Among the plants that can be protected by the method according to the invention, mention can be made of cotton; flax; vine; fruit or vegetable crops such as Rosaceae sp. (for instance pip fruit such as apples and pears, but also stone fruit such as apricots, almonds and peaches), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp., Actimidaceae sp., Lauraceae sp., Musaceae sp. (for instance banana trees and plantins), Rubiaceae sp., Theaceae sp., Sterculiceae sp., Vitaceae sp., Rutaceae sp. (for instance lemons, oranges and grapefruit); Solanaceae sp. (for instance tomatoes), Liliaceae sp., Asteraceae sp. (for instance lettuces), Umbelliferae sp., Cruciferae sp., Chenopodiaceae sp., Cucurbitaceae sp., Papilionaceae sp. (for instance peas), Rosaceae sp. (for instance strawberries); major crops such as Graminae sp. (for instance maize, lawn or cereals such as wheat, rice, barley and triticale), Asteraceae sp. (for instance sunflower), Brassicaceae sp. (for instance rapeseed and colza), Fabacae sp. (for instance peanuts), Papilionaceae sp. (for instance soybean), Solanaceae sp. (for instance tomatoes and potatoes), Chenopodiaceae sp. (for instance beetroots); horticultural and forest crops; as well as genetically modified homologues of these crops.
The invention is further exemplified by the following non-limiting examples.
Potato plants (Solanum tuberosum) of the Phytophthora infestans sensitive variety Bintje were field grown near Gembloux, Belgium.
28 days after planting, the first application of the treatments was performed. The treatments were applied 10 times at 7 days interval 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 ingredient in the elicitor was an oligosaccharidic complex consisting of negatively charged oligo-galacturonans stabilized by positively charged chito-oligosaccharides. The elicitor was provided as a powder comprising 70.4% active ingredient and was applied at 50 g active ingredient/ha (71 g powder). The fungicide used was Infinito (Bayer, Germany), which was chosen for its high level of protection of potato against P. infestans. Infinito comprises the active ingredients 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.
The percentage of sporulation on leaves (sporulation area compared to leaf area) was evaluated every 3-4 days from the appearance of the disease in the untreated plants (58 days after planting), and during 46 days, thus, until 104 days after planting. These values were transformed into protection values by a comparison of the symptoms with the untreated plants.
During the whole experiment, the elicitor applied alone did not offer any protection of potato plants against P. infestans compared to untreated plants.
The fungicide applied alone offered a total protection during the first two observations. After that a reduction of the protection—from 100 to 95%—was observed between days 61 and 71 after planting. A steeper decrease was then noted between days 71 and 104 after planting. At the end of the experiment, the protection fell down to 57%.
With the mixture of the fungicide and the elicitor, a total protection was also observed during the first two observations. After that the protection reached 95% and this plateau was maintained until day 78 after planting, which represents the same level of protection as that offered by the fungicide alone one week before. The level of 75% of protection was reached at day 89 after planting for the fungicide alone while this threshold was only reached at day 96 when mixed with the elicitor. This delay of one week corresponds to two P. infestans developmental cycles. It is therefore possible to delay the infection thanks to the addition of the elicitor to the fungicide. At the end of the experiment, the protection offered by the mixture corresponded to 66.2%, which represents a 16% increase of the protection compared to the fungicide alone. This is unexpected, since application of the elicitor alone didn't have any effect on the infection of the potato plants by P. infestans.
Grape seedlings of the variety Marselan were grown under controlled conditions in a greenhouse.
The seedlings were treated by spraying a test solution on both faces of the leaves. The test solutions were composed of elicitor alone, fungicide alone or a mixture of both. The active ingredient in the elicitor was an oligosaccharidic complex consisting of negatively charged oligo-galacturonans stabilized by positively charged chito-oligosaccharides. The elicitor was provided as a concentrated solution, which was diluted in order to obtain a test solution comprising 20 mg active ingredient/L. The fungicide used was Aliette (Bayer, Germany), which was chosen for its high level of protection of grape against P. viticola, despite the absence of registration on this crop. Aliette was provided as a powder comprising 80% of its active ingredient fosetyl. Aliette was applied at 1.73 g/L, which is about one fourth of the recommended rate applied on apple against Erwinia amylovora. The summary of the test solutions is detailed in Table 2.
The treatments were applied 3 days before the seedlings were artificially inoculated (by brumisation).
After inoculation, the seedlings were incubated in a chamber with high degree of humidity for 6 days after inoculation.
The percentage of sporulation on leaves (sporulation area compared to leaf area) was evaluated 7 days after inoculation. These values were transformed into protection values by a comparison of the symptoms with the untreated seedlings.
The elicitor applied alone offered a 64.8% protection against P. viticola on grape seedlings. The protection offered by the fungicide alone was higher (90.6%) but not total. Once in mixture, these two compounds offered a total protection (99.7%) against P. viticola.
Certified healthy microtubers of potato (Solanum tuberosum) of the Phytophthora infestans sensitive variety Bintje were grown on compost in individual containers under controlled conditions in a greenhouse (24° C., 16 h day/8 h night regime) for 6 weeks. 6 weeks after planting, the 3 last leaves of each plant were labeled just before the first treatment, ensuring to have leaves of the same physiological age for further inoculation.
The first application of the treatments was performed 6 weeks after planting and 7 days before planned inoculation. A second application was made 3 days before inoculation. The treatments were applied by spraying 50 mL of a test solution on the whole plant and on both leaves faces. 8 plants were selected for each test solution to assess. The summary of the test solutions is detailed in Table 3.
The elicitor was applied as a formulation with the adjuvant Dehscofix CO 95 (Huntsman, Belgium). The active ingredient in the elicitor was an oligosaccharidic complex consisting of oligo-galacturonans (with degrees of polymerization between 9 and 20) and chito-oligosaccharides (with degrees of polymerization between 5 and 10 and with a degree of acetylation aroud 25%) in equal proportions. The elicitor further contained the salts CaCl2 (0.5 mM) and NaCl (50 mM) to ensure good ionic conditions for the stability of the oligosaccharidic complex. The elicitor was applied at 50 mg active ingredient/L. The fungicide used was Aliette, which was chosen for its favorable ecotoxicological profile and versatile plant protection effect against oomycetes. Aliette was provided as a powder comprising 80% of its active ingredient fosetyl-Al. Aliette was applied at 800 mg/L, which is about one tenth of the recommended rate. Control plants were sprayed with Dehscofix CO 95, the adjuvant used in the elicitor formulation.
3 days after the second treatment, the 3 labeled leaves of each plant were collected in individual transparent plastic boxes on filter paper with a small amount of water. P. infestans inoculum was collected from potato leaves and spore concentration was adjusted to 104 spores/mL prior to inoculation. 2 droplets of 10 μL were put on five leaflets of the potato leaves yielding 10 inoculation points per leaf for further assessments. All the boxes containing the leaves were incubated in a growth cabinet at 90% relative humidity and 24° C. with a 16 h day/8 h night regime.
6 and 8 days after inoculation, the leaves were evaluated for disease occurrence, sporulation occurrence and sporulation area.
Disease occurrence and sporulation occurrence are evaluated by first attributing a score, corresponding to the stage of the observed lesion, to each inoculation point.
Disease occurrence is then calculated for each leaf as the number of observed scores equal or higher than 2 divided by the number of inoculation points. It yields a value comprised between 0 and 1. The value 0 of disease occurrence is considered for a leaf as no disease development for all the inoculation points and the value 1 as a disease development on all inoculation points.
Sporulation occurrence is calculated for each leaf as the number of observed scores equal or higher than 3 divided by the number of inoculation points. It yields also a value comprised between 0 and 1. The value 0 of sporulation occurrence is considered for a leaf as no late blight's sporulation for all the inoculation points and the value 1 as presence of late blight's sporulation on all inoculation points.
Sporulation area is a visual estimate of the leaf surface under sporulation expressed.
The fungicide Aliette, applied at a rate 10 times lower than recommended, confers partial protection. This protection is especially marked on sporulation occurrence and percentage sporulation. The elicitor also reduces disease occurrence and sporulation, but the reduction of the symptoms conferred by the fungicide is higher than the one obtained after elicitor application. More interesting, these data highlight the fact that the symptoms reduction of the mixture of fungicide and elicitor is higher than the one of the two test compounds alone. Thus, the addition of the elicitor to a low rate of the fungicide improves the control of disease development.
Winter wheat of the Septoria tritici very sensitive variety Istabraq were field grown near Namur, Belgium.
The plants were treated with either fungicide alone, elicitor alone or a combination of both. The fungicide used was Opus (BASF, Germany), which was chosen for its high level of protection of winter wheat against S. tritici. Opus comprises the active ingredient epoxyconazole at 125 g/L. The fungicide was applied at 0.25 L/ha, which is one fourth of the recommended rate. The fungicide was applied alone at stages 32 (second node detectable) and 55 (emergence of inflorescence at 50%).
The active ingredient in the elicitor was an oligosaccharidic complex consisting of negatively charged oligo-galacturonans stabilized by positively charged chito-oligosaccharides. The elicitor was provided as a concentrated solution comprising 10 g/L of active ingredient and was applied alone at 1 L/ha alone at stages 30 (ear at 1 cm), 32 (second node detectable), 39 (flag leaf ligule just visible) and 55 (emergence of inflorescence at 50%).
The fungicide and the elicitor were also applied in combination as a program: the fungicide was applied at stages 32 and 55 whereas the elicitor was applied at stages 30, 32, 39 and 55. The treatments are summarized in Table 4.
Assessments of leaf diseases were made on 15 plants randomly selected in each plot from four replicates. The percentage of leaf area (pest severity) colonized by the disease was visually assessed on the different leaf layers. The percentages of leaves with symptoms of the disease were also calculated (pest incidence).
Synergistic effects were assessed by applying the method defined by Colby (1967, “Calculation of the synergistic and antagonistic responses of herbicide combinations”, Weeds, 15, pages 20-22) using the following formula, which is also referred to as the Colby formula:
wherein E represents the expected percentage of inhibition of the pest for the combination of the two compounds at defined doses, X is the percentage of inhibition observed for the pest by compound (A) at a defined dose, Y is the percentage of inhibition observed for the pest by compound (B) at a defined dose. When the percentage of inhibition observed for the combination is greater than E, there is a synergistic effect.
The percentages of pest severity with the two compounds alone were 18.1 (for the fungicide) and 18.8% (for the elicitor), whereas the same parameter was assessed at 24.7% for the untreated control. The reduction of the severity compared to the untreated control corresponds to 26.8 and 23.9% in presence of the fungicide and the elicitor, respectively. Once in combination these two treatments present a percentage of severity of 9.1%, which corresponds to a reduction of the symptoms of 63.1% compared to the untreated control. The Colby analysis demonstrates a synergistic effect concerning pest severity between the fungicide and the elicitor. The observed percentage of protection (63.1%) is greater than the expected percentage of protection E (44.3%), wherein E was calculated as:
The percentages of pest incidence with the two compounds alone were 83.3 (for the fungicide) and 78.3% (for the elicitor), whereas the same parameter was assessed at 95.0% for the untreated control. The reduction of the incidence compared to the untreated control corresponds to 12.3 and 17.5% in presence of the fungicide and the elicitor, respectively. Once in combination these two treatments present a percentage of incidence of 66.7%, which corresponds to a reduction of the symptoms of 29.8% compared to the untreated control.
The Colby analysis also demonstrates a synergistic effect concerning pest incidence between the fungicide and the elicitor. The observed percentage of protection (29.8%) is greater than the expected percentage of protection E (27.6%), wherein E was calculated as:
Tomato plants (Lycopersicon esculentum, variety Saint Pierre) were grown under controlled conditions in a greenhouse (24° C., 16 h day/8 h night regime) for 4 weeks. 1 day before treatment, 4 weeks old plants were placed in a growth cabinet (Weiss) at 18° C. with a 16 h/8 h day/night regime and 90% relative humidity. The plants were watered 2 h before treatment. For each treatment, 12 tomato plants were sprayed with 200 mL of a test solution on both sides of the leaves until run off. The composition of the test solutions is detailed in Table 5.
The elicitor was applied as a formulation with the adjuvant Dehscofix CO 95. The active ingredient in the elicitor was an oligosaccharidic complex consisting of oligo-galacturonans (with degrees of polymerization between 9 and 20) and chito-oligosaccharides (with degrees of polymerization between 5 and 10 and with a degree of acetylation aroud 25%) in equal proportions. The elicitor further contained the salts CaCl2 (0.5 mM) and NaCl (50 mM) to ensure good ionic conditions for the stability of the oligosaccharidic complex and sucrose (10 mM). The elicitor was applied at 50 mg active ingredient/L. The fungicide used was Aliette, which was chosen for its favorable ecotoxicological profile and versatile plant protection effect against oomycetes. Aliette was provided as a powder comprising 80% of its active ingredient fosetyl-Al. Aliette was applied at 400, 800 or 1600 mg/L, whereas the recommended rate is 8000 g/L. Control plants were sprayed with Dehscofix 0095, the adjuvant used in the elicitor formulation.
24 h after treatment, the leaves were sampled from each plant individually, immediately frozen in liquid nitrogen and stored at −80° C. until use.
All samples were tested for peroxidase activity according to the assay procedure (Ref. No. FGAP001) for peroxidase (Guaiacol Units) provided by Faizyme Laboratories (http://www.faizyme.com/assaperg.htm). Frozen samples were ground in liquid nitrogen to fine powder with a RETSCH MM 400 crusher. Proteins were extracted using acetate buffer (0.1M, pH 5.2) containing β-mercaptoethanol (0.014 M). Briefly, 0.5 g leaf powder was homogenized with an Ultra-Turrax (IKA) and centrifuged at 4000 rpm (4° C.) for 10 min. The supernatant was collected and filtered through Miracloth (Calbiochem) and directly frozen with liquid nitrogen. Protein contents were measured at 630 nm using Bio Rad protein assay reagent with bovine serum albumin as standard. The peroxidase activity assays were made on fresh extracts. Guaiacol (0.17 M) and H2O2 (0.19 M) were used as substrates. Kinetics of tetraguaiacol formation was determined by measuring absorbance every minute at 420 nm. The slope of the data allowed to express peroxidase activity in “μmol H2O2.h−1.mg protein−1” and results were normalized by setting treatment with adjuvant (control) at 100%. All measurements were performed using ELx800 Universal Microplate Reader spectrophotometer (Bio-Tek, instruments, INC) and statistic analyses were made with Minitab software (Minitab, 2007).
The fungicide (Aliette) applied alone did not significantly modify the peroxidase activity of the tomato leaves. Addition of the elicitor statistically increased the peroxidase activity. Thus, mixing the elicitor with Aliette confers peroxidase activity to the plant on top of intrinsic fungicide effect. Unexpectedly, the fungicide used appears to negatively influence the elicitor effect. By reducing the concentration of the fungicide, we could increase induction of the plant immune system by the elicitor.
Tomato plants (Lycopersicon esculentum, variety Saint Pierre) were treated as in example 4. The composition of the test solutions is detailed in Table 6.
The elicitor was applied as a formulation with the adjuvant Dehscofix CO 95. The active ingredient in the elicitor was an oligosaccharidic complex consisting of oligo-galacturonans (with degrees of polymerization between 9 and 20) and chito-oligosaccharides (with degrees of polymerization between 5 and 10 and with a degree of acetylation around 25%) in equal proportions. The elicitor further contained the salts CaCl2 (0.5 mM) and NaCl (50 mM) to ensure good ionic conditions for the stability of the oligosaccharidic complex and sucrose (10 mM). The elicitor was applied at 25, 50 or 75 mg active ingredient/L. The fungicide used was Aliette, which was chosen for its favorable ecotoxicological profile and versatile plant protection effect against oomycetes. Aliette was provided as a powder comprising 80% of its active ingredient fosetyl-Al. Aliette was applied at 400 mg/L, whereas 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 treatment, the leaves were sampled from each plant individually, immediately frozen in liquid nitrogen and stored at −80° C. until use.
All samples were tested for peroxidase activity according to experiment 5.
Peroxidase activity is significantly increased by application of the elicitor to tomato. In the elicitor range tested alone (0 mg/L, 25 mg/L, 50 mg/L, 75 mg/L) peroxidase activity appears to increase in a linear way with the concentration of the elicitor. The R2 (determination coefficient) of this relationship is about 87% which is quite good 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 elicitor concentration. However, in combination with the fungicide, the elicitor concentration must be higher to yield similar activity than the one obtained with the elicitor sprayed alone at the same rate. Mixing the elicitor with the fungicide confers peroxidase activity to the plant on top of intrinsic fungicide effect of Aliette.
Tomato plants (Lycopersicon esculentum, variety Saint Pierre) were treated as in example 4. The composition of the test solutions is detailed in Table 7.
The elicitor was either applied alone or formulated with different surfactants. The surfactants were added at 0.1 volume percent to the elicitor formulation. Dehscofix CO 95 is an ethoxylated castor oil, a non-ionic plant oil. Tensiofix D33 (Ajinomoto OmniChem, Belgium) is a non-ionic wetter spreader agent and Tensiofix Dp400 (Ajinomoto OmniChem, Belgium) is an anionic salt that exhibits dispersing properties. The active ingredient in the elicitor was an oligosaccharidic complex consisting of oligo-galacturonans (with degrees of polymerization between 9 and 20) and chito-oligosaccharides (with degrees of polymerization between 5 and 10 and with a degree of acetylation around 25%) in equal proportions. The elicitor further contained the salts CaCl2 (0.07 mM) and KNO3 (8 mM) to ensure good ionic conditions for the stability of the oligosaccharidic complex and sucrose (10 mM). The elicitor was applied at 50 mg active ingredient/L.
24 h after treatment, the leaves were sampled from each plant individually, immediately frozen in liquid nitrogen and stored at −80° C. until use.
All samples were tested for peroxidase activity according to experiment 5.
Dehscofix CO 95, a penetration enhancer, and Tensiofix Dp 400, a dispersing agent, both in combination with the elicitor gave a twofold increase of the peroxidase activity in comparison with the elicitor alone.
Potato plants (Solanum tuberosum, variety Bintje) were grown under controlled conditions in a greenhouse (24° C., 16 h day/8 h night regime) for 6 weeks.
The plants were treated twice: a first application of the treatments was performed 7 days before harvest of the leaves, a second application was made 3 days before harvest. For each treatment, 8 potato plants were sprayed with 200 mL of a test solution on both sides of the leaves until run off. The composition of the test solutions is detailed in Table 8.
The elicitor was applied as a formulation with the adjuvant Dehscofix CO 95. The active ingredient in the elicitor was an oligosaccharidic complex consisting of oligo-galacturonans (with degrees of polymerization between 9 and 20) and chito-oligosaccharides (with degrees of polymerization between 5 and 10 and with a degree of acetylation around 25%) in equal proportions. The elicitor further contained the salts CaCl2 (0.07 mM) and KNO3 (14 mM) to ensure good ionic conditions for the stability of the oligosaccharidic complex. The elicitor was applied at 50 mg active ingredient/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 linear β(1→3)-glucan with β(1→6)-linkages) as active ingredient. 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, 3 leaves at the same physiological stage were sampled per plant. The leaves of 2 plants were combined, immediately frozen in liquid nitrogen and stored at −80° C. until use.
All samples were tested for peroxidase activity as detailed in example 5.
When applied alone, the second elicitor, Vacciplant Fruit, did not improve peroxidase activity as compared with the control. Spraying plants with the COS-OGA elicitor yielded a significant increase of peroxidase acitivity. A synergistic effect was obtained when the COS-OGA elicitor was combined with the Vacciplant Fruit elicitor.
Strawberries (cultivar AnaIs) were grown in cages. Three cages, each containing 3 strawberry plants were used in this experiment.
At day 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 ingredient, which was an oligosaccharidic complex consisting of negatively charged oligo-galacturonans stabilized by positively charged chito-oligosaccharides. After dilution (200 fold), the elicitor was applied at 50 ppm.
Six adult aphids (Chaetosiphon fragaefolii) were put on each strawberry plant at day D-0.
Assessments of the aphids' numbers were made on 3 plants randomly selected (one in each from 3 replicates) at days D+19 and D+26.
The number of aphids was 139 per plant for the untreated control, whereas the number of aphids was reduced at 114 per plant following elicitor applications. This corresponds to a reduction of the aphids by 18% in the presence of the elicitor.
The number of aphids was 315 per plant for the untreated control, whereas the number of aphids was reduced at 260 per plant following elicitor applications. This corresponds to a reduction of the aphids by 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.
Cucumber seedlings were grown in a greenhouse.
The seedlings were treated with fungicide alone, elicitor alone or a combination of both as summarized in Table 9. The test solutions were applied once at day 0 and a second time at day +7 ( 2/4 leaves stage).
The fungicide used was LEGEND (Dow Agrosciences), which was chosen for its high level of protection of cucumber against S. fuliginea and because its mode of action consists in blocking the early signal transduction pathway. LEGEND comprises the active ingredient quinoxyfen at 250 g/L. The fungicide was applied at 0.001 L/hL, which is one twentieth of the maximal recommended rate.
The elicitor was applied as a formulation with the adjuvant Dehscofix CO 95. The active ingredient in the elicitor was an oligosaccharidic complex consisting of negatively charged oligo-galacturonans stabilized by positively charged chito-oligosaccharides in presence of recommended salts. The elicitor was provided as a concentrated solution comprising 6.25 g/L of active ingredient. The elicitor was applied at 0.8 L diluted in 100 L. The elicitor was applied with 0.08 L adjuvant Dehscofix CO 95 in 100 L, which is required for the penetration of the elicitor into leaves.
The fungicide and the elicitor (+0.08 L/hL Dehscofix CO 95) were also applied in mixture. Natural infestation occurred (from day +7) and additional artificial inoculation was carried out at day +10.
Assessments of leaf disease were made on 16 plants from 4 replicates. The percentages of leaves with symptoms of the disease (pest frequency) and the percentages of leaf area colonized by the disease (pest severity) were visually assessed. These assessments were performed at day +7 (only natural infestation) and +18 (also artificial inoculation) and the protection calculated with reference to the untreated control.
Colby analysis shows that a synergistic effect exists between the fungicide and the elicitor concerning pest frequency. The observed percentage of protection (100%) is greater than the expected percentage of protection (90.5%), wherein E was calculated as:
The percentage of protection (expressed as reduction of pest severity) was 82% (for the fungicide) and 93% (for the elicitor), whereas the same parameter was assessed at 100% for the mixture of both fungicide and elicitor.
Colby analysis shows a slight synergistic effect exists between the fungicide and the elicitor regarding pest severity. The observed percentage of protection (100%) is greater than the expected percentage of protection E (98.7%), wherein E was calculated as:
The percentage of protection (expressed as reduction of pest frequency) was 53% for the fungicide and 9% for the elicitor (oligosaccharidic complex+Dehscofix CO 95), whereas protection was assessed at 88% for the mixture of both (fungicide and elicitor).
Colby analysis shows that a synergistic effect exists between the fungicide and the elicitor concerning pest frequency. The observed percentage of protection (88%) is much higher than the expected percentage of protection E (57.2%), wherein E was calculated as:
The percentage of protection (expressed as reduction of pest severity) was 89% (for the fungicide) and 24% (for the elicitor), whereas the same parameter was assessed at 99% for the mixture of both (fungicide and elicitor).
Colby analysis again shows a clear synergistic effect between the fungicide and the elicitor regarding pest severity. The observed percentage of protection (99%) is greater than the expected percentage of protection E (91.6%), wherein E was calculated as:
In conclusion, whatever the parameter (pest frequency and pest severity) and the assessment (at day +7 and day +18), a synergistic effect is observed between the elicitor and a very low rate of the fungicide LEGEND (quinoxyfen).
The field trial was carried out in a vineyard in France.
The grapevines were treated with fungicide alone, elicitor alone or a combination of both as summarized in Table 10. The total spray volume was 400 L/ha.
The applied fungicide was THIOVIT, which contains 80% sulfur as active ingredient. THIOVIT was chosen because sulfur is widely used to preventively protect grapevine against E. necator (powdery mildew) and especially because it is a multisite fungicide along with compounds such as copper, dithiocarbamates, phthalimides, chloronitriles and triazines (http://frac.info/frac/publication/anhang/FRAC %20Code %20List %202011-final.pdf). THIOVIT was applied at 25 kg/ha/season, which is about one third of the recommended rate. The active ingredient in the COS-OGA elicitor is an oligosaccharidic complex consisting of negatively charged oligo-galacturonans stabilized by positively charged chito-oligosaccharides. The elicitor was provided as a concentrated solution comprising 6.25 g/L of active ingredient. The elicitor was applied at 4 L/ha as a formulation with 0.4 L/ha adjuvant Dehscofix CO 95, which is required for the penetration of the elicitor into the leaves. THIOVIT and the elicitor (with 0.4 L/ha Dehscofix CO 95) were also applied in combination.
Assessment of leaves was made six weeks after appearance of infestation on fruits. The percentages of leaves with symptoms of the disease (pest frequency) and the percentages of leaf area colonized by the disease (pest intensity) were visually assessed and protection calculated with respect to untreated control.
Frequency reduction for the THIOVIT fungicide alone was 60.6% whereas this parameter was assessed at 57.6% for the elicitor (oligosaccharidic complex+Dehscofix CO 95) alone. Frequency reduction for the mixture of both THIOVIT and elicitor was 93.1%.
Colby analysis demonstrates that a synergistic effect exists between the COS-OGA elicitor and the multisite fungicide THIOVIT concerning pest frequency. The observed percentage of protection (93.1%) is greater than the expected percentage of protection E (83.3%), which was calculated as:
Tomato plants (Solanum lycopersicum, var. Moneymaker) were grown for four weeks at 24° C. with a 16 h/8 h day/night regime and were well watered every two to three days.
Twenty four hours before treatment, well watered plants were transferred in a growth cabinet at constant temperature (18° C.) and humidity (90%) under the same photoperiod. For each treatment, 12 plants were sprayed until run off on both faces of the leaves with 200 mL of a test solution and returned to the incubator for one more day. The compositions of the test solutions are detailed in Table 11.
Twenty four hours after treatment, the leaves were sampled, frozen in liquid nitrogen and stored at −80° C. until grinding in liquid nitrogen, protein extraction and peroxidase quantitation. All samples were tested for peroxidise activity according to example 5.
The peroxidase activity increase observed with the combination of the COS-OGA elicitor and the adjuvant Dehscofix CO 95 (225% as compared to water) is much greater than the expected increase E (87.2%), which was calculated as:
Consequently, a synergistic effect exists between the adjuvant Dehscofix CO 95 and the COS-OGA elicitor regarding peroxidase activity.
Tomato plants (Solanum lycopersicum, var. Moneymaker) were grown for four weeks at 24° C. with a 16 h/8 h day/night regime and were well watered every two to three days.
Twenty four hours before treatment, well watered plants were transferred in a growth cabinet at constant temperature (18° C.) and humidity (90%) under the same photoperiod. For each treatment, 12 plants were sprayed until run off on both faces of the leaves with 200 mL of a test solution and returned to the incubator for one more day. The compositions of the test solutions are detailed in Table 12.
Twenty four hours after treatment, the leaves were sampled, frozen in liquid nitrogen and stored at −80° C. until grinding in liquid nitrogen, protein extraction and peroxidase quantitation. All samples were tested for peroxidise activity according to example 5.
Colby analysis demonstrates that there is a synergistic effect between the COS-OGA elicitor and 2 mM Na2SiO3 regarding peroxidase activity. The observed peroxydase activity increase (209%) is much greater than the expected increase E (154%), which was calculated as:
Colby analysis shows that there is a synergistic effect between the COS-OGA elicitor in combination with 5 mM Na2SiO3 regarding peroxidase activity. The increase of peroxidase activity observed with the combination of COS-OGA elicitor and 5 mM Na2SiO3 (308% with respect to water) is much greater than the increase expected from the components alone (170%), which was calculated as:
In conclusion, a synergistic effect exists between the COS-OGA elicitor in combination with the Dehscofix CO 95 adjuvant and the silicium elicitor (Na2SiO3) regarding peroxidase activity.
Potato plants (Solanum tuberosum, variety Bintje) were grown under controlled conditions. The plants were treated twice at days −7 and day −1 before artificial inoculation of the potato leaves (day 0) by spraying a test solution on the plants. Artificial inoculation was carried out by spotting droplets of 10 μl containing 5.104 P. infestans spores per ml on the lower leaf sides. The test solutions were composed of elicitor alone, fungicide alone or a combination of both. The fungicides tested were Ranman component A, Previcur N and Signum, which were chosen because they are widely used to protect potato against P. infestans (downy mildew). Ranman component A contains 400 g/L cyazofamid (an inhibitor of complex II of the electron transport chain of the mitochondria) as active ingredient, Previcur N contains 722 g/L propamocarb (a cell membrane permeability disruptor) as active ingredient and Signum contains 26.7% (w/w) pyraclostrobin and 6.7% (w/w) boscalid as active ingredients. The Ranman component A fungicide was applied at one thousandth of its recommended rate. The Previcur N and Signum fungicides were applied at one hundredth of their recommended rate. The fungicides were also applied in combination with the elicitor formulation. The active ingredient in the elicitor was an oligosaccharidic complex consisting of negatively charged oligo-galacturonans stabilized by positively charged chito-oligosaccharides. The elicitor was provided as a ready to use solution containing 50 mg/L of active ingredient and 0 1% (v/v) of the adjuvant Tensiofix DP400.
Assessment of the sporulation area on leaves was made each day between the third and the sixth day after artificial inoculation in order to calculate the Area Under Disease Progression Curve (AUDPC). By the end of the test, 100% of the leaf area of controls (untreated plants) were covered with sporulation.
The fungicides applied alone offered a protection of 81.3% (Ranman component A), 39.3% (Previcur N) and 99.2% (Signum).
The elicitor applied alone offered a protection of 63.1%.
The observed percentage of protection (97.5%) offered by the combination of the Ranman component A fungicide and the elicitor is greater than the expected percentage of protection (93.1%), which was calculated as:
Consequently a synergistic effect exists between the COS-OGA elicitor and the fungicide Ranman component A regarding protection of potato against P. infestans.
The observed percentage of protection (80.7%) offered by the combination of the Previcur N fungicide and the elicitor is greater than the expected percentage of protection (77.6%), which was calculated as:
Consequently a synergistic effect also exists between the COS-OGA elicitor and the fungicide Previcur N regarding protection of potato against P. infestans.
The observed percentage of protection offered by the combination of the Signum fungicide and the elicitor was only 79.8%, which indicates that there was no synergy between the COS-OGA elicitor and this fungicide. In contrast, addition of the COS-OGA elicitor to the Signum fungicide had a clear antagonistic effect regarding protection of potato against P. infestans. An antagonistic effect on plant protection of adding the COS-OGA elicitor to a fungicide was also observed with other fungicides (Table 13).
| Number | Date | Country | Kind |
|---|---|---|---|
| 11151836 | Jan 2011 | EP | regional |
This application is a continuation of U.S. patent application Ser. No. 13/980,432, filed Jul. 18, 2013, now allowed, which is a U.S. national stage entry under 35 U.S.C. §371 of PCI International Patent Application No. PCT/EP2012/051018, filed Jan. 24, 2012, which claims priority to European Patent Application No. EP 11151836.1, filed Jan 24, 2011, the contents of all of which are hereby incorporated by reference in their entirety into the subject application.
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| Number | Date | Country | |
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| 20150045221 A1 | Feb 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13980432 | US | |
| Child | 14511272 | US |
