Composition for immunization in plants

FIELD: biotechnology.

SUBSTANCE: the present innovation deals with immunization in plants, particularly, compositions and methods for inducing plant resistance to phytopathogenic organisms, such as phytopathogenic fungi. An agent indicated for inducing plant resistance to phytopathogenic microorganisms is being an extract out of biomass of non-phytopathogenic microorganisms. The method to obtain the above-mentioned agent includes the following stages: a) resuspending against 50-200 g (dry weight) biomass of non-phytopathogenic microorganisms in 1 l either inorganic or organic solvent, b) mixing at room temperature for 1-12 h, c) incubating, d) resuspending, e) cooling up to room temperature at maturing and f) filtrating, not obligatory. The innovation enables to induce plant resistance to phytopathogenic microorganisms.

EFFECT: higher efficiency of immunization.

12 cl, 6 ex, 5 tbl

 

The present invention relates to immunization of plants, in particular to compositions and methods for the induction of plant resistance to phytopathogenic organisms, such as phytopathogenic fungi.

Background of invention

Various studies have shown that the sensitivity of the plants to certain diseases does not correlate with lack of genetic potential contributing mechanisms of resistance to these diseases. It is known that resistance can be induced in plants that seem sensitive, by inoculation of avirulent forms of phytopathogens, hypovirulence phytopathogens or limited by inoculation with pathogenic fungi. The resulting induced resistance is stable and generally non-specific for a pathogen.

This protective system is a complex interaction of cases, early detection of the pathogen causing the signals that spread from the inoculation intra - and intercellular throughout the plant. These signals trigger a series of induced defensive reactions aimed at blocking or even to the destruction of penetrating pathogen. Many defenses are controlled at the level of transcription of a gene.

Recognition of the pathogen occurs in the most close to the surface of the plant months is E. The products resulting from the decomposition of the cell wall of the attacking pathogen ("retrieve" glucan pathogens), as well as fragments of plant cells, resulting from the attack ("retrieve" oligosaccaride pathogens), are among the most well-studied alarms. Secondary signals are propagated from the place of attack throughout the plant. The most well-known compound in this circuit signals is salicylic acid, but as signals that induce a protective response, has also been described electrical signals.

Protective reactions that are activated upon receipt of alarms, represent a wide range of chemical, biochemical and mechanical protection. In monocotyledonous plants is often observed strengthen cell membranes by deposition of callus in front of the place in which the pathogen is trying to penetrate. The two - and monocotyledonous plants observed induction of hydrolytic enzymes (e.g., chitinases with lysozyme activity, β-1,3-glucanase, protease). Plants can also respond by local synthesis is greatly increased concentrations of toxic secondary metabolites, the so-called phytoalexins that can destroy penetrate the organism. One of the earliest reactions attacked plant cells is the generation who tion of active oxygen radicals, that is often the beginning of a complete destruction of a limited number of cells around the affected places.

Summary of the invention

According to the invention it has been unexpectedly found that the extract of the biomass of microorganisms that normally do not cause disease no plants (neutopenia microorganisms)can be used to induce in plants resistance to phytopathogenic microorganisms.

Thus, the invention relates to an agent, intended to induce in plants resistance to phytopathogenic microorganisms, where the agent (button in this description referred to as "the agent according to the invention") is an extract of biomass neutopenia microorganisms, a process which involves:

a) re-suspension from 50 g to 200 g (dry weight) biomass neutopenia microorganisms in 1 l of inorganic or organic solvent,

b) stirring at room temperature for 1-12 h,

C) incubation,

g) re-suspension

d) cooling to room temperature during maturation and

e) optionally filtering.

The invention also relates to agricultural compositions comprising the agent obtained by the above process, in combination with acceptable from selskokhozyaistv is authorized perspective carriers (diluents) and optionally with one or more pesticides, designed to protect plants and is able to induce in plants resistance to phytopathogenic microorganisms.

The present invention also relates to an extract of Penicillium chrysogenum, is able to induce in plants resistance to phytopathogenic microorganisms.

The invention also relates to a method for induction of plant resistance to phytopathogenic microorganisms by treating the plants, the soil or seeds with the agent according to the invention. The agent can be applied individually, i.e. without including the preparative form or in the form of agricultural composition.

The invention also relates to a method for obtaining agent, intended to induce in plants resistance to phytopathogenic microorganisms, where the agent is an extract of biomass neutopenia microorganisms obtained by:

a) resuspendable from 50 g to 200 g (dry weight) biomass neutopenia microorganisms in 1 l of inorganic or organic solvent,

b) stirring at room temperature for 1-12 h,

C) incubation

g) resuspendable,

d) cooling to room temperature during maturation and

e) optionally filtering.

Detailed description of the invention

In the context of the present description under "plants" is usually understood ku is tiberuim plants, grown/apply for a good crop. Target plants to be protected through the introduction of sustainability in the scope of the present invention are, for example, the following species of plants: cereals (wheat, barley, rye, oats, rice, sorghum and related crops); beet (sugar beet and fodder beet); stone fruit, pome fruit and berries (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries), leguminous plants (beans, lentils, peas and soybeans), oil plants (rape, mustard, poppy, olive, sunflower, coconut the castor-oil plant, cocoa beans and groundnuts), cucumber plants (cucumber, pumpkin and melons), fibre plants (cotton, flax, hemp and jute), citrus crops (oranges, lemons, grapefruit and mandarins), vegetables (spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes, peppers), Laurel crops (avocado, cinnamon tree and camphor tree), or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, vines, hops, bananas and natural rubber plants, as well as ornamental plants, plants of meadows and pastures (dermoabrasion cereals) and embankments.

Preferred plants to be protected according to the invention include Solanaceae such as tomato and potato, beans, cucumber, pepper, tobacco, peanuts, and cultural varieties of vineyards is Yes.

Particularly preferred plants to be protected according to the invention are the Poaceae. Solanaceae.

Under "pathogenic microorganisms" in the context of the present description mean fungi, bacteria and viruses that attack plants and cause damage to plants. The agent according to the invention is particularly effective in inducing resistance to phytopathogenic fungi. Such phytopathogenic fungi include, for example, Phytophtora infestants, Cladosporium fulvum, Plasmopora viticola, Colletotrichum lagenarium, Pseudomonas lachrymans and Puccinia tritici.

When the use of an agent according to the invention are especially good results can be achieved in respect of the protection of Solanaceae such as tomato and potato, from Phytophtora infestants and Cladosporium fulvum, the protection of cultural grapes from Plasmopora viticola and protection of cucumber against Colletotrichum lagenarium and Pseudomonas lachrymans.

Under "nefitepoegono microorganisms" in the context of the present description involves microorganisms of these genera and belonging to this species, which do not cause disease in plants. Preferably neutopenia microorganisms are also non-specific for plants, microorganisms, i.e. they do not grow on trees.

Examples of genera of microorganims, which can be used according to the invention, are the following:

- bacteria: Acetobacter, Achromobacter, Actinoplanes,Aerobacter, Alcaligenes, Arthrobacter, Bacillus, Brevibacterium, Cephalosporium, Clostridium, Corynebacterium, Cryptococcus, Escherichia, Flavobacterium, Gluconobacter, Lactobacillus, Leuconostoc, Methanobacillus, Methanomonas, Methylovibrio, Microbacterium, Micrococcus, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhodopseudomonas, Saccharopolyspora, Sarcina, Sporotrichum, Streptococcus, Streptomyces, Thermomonospora, Thiobacillus, Xanthomonas,

- fungi and yeast: Acremonium, Aschersonia, Ashbya, Aspergillus, Aureobasidium, Beaveria, Candida, Claviceps, Clitopilus, Curvularia, Cyclindrocarpon, Eremothecium, Erwinia, Fusarium, Fusidium, Gibberalla, Hansenula, Hirsutella, Klyveromyces, Metarhizium, Mucor, Myocandida, Neocosmospora, Phaecilomyces, Penicillium, Pericularia, Phanerochaete, Phycomyces, Pichia, Pullularia, Rhizopus, Saccharomyces, Schizosaccharomyces, Sclerotium, Sesquicilliopsis, Streptomyxa, Tolypocladium, Torula, Torulopsis, Trametes, Trichoderma, Trigonopsis.

Examples of particularly preferred species, which can be used according to the invention, are the following:

- bacteria: Acetobacter aceti, Acetobacter suboxydans, Acetobacter xylinum, Achromobacter obae, Actinoplanes missouriensis, Aerobacter aerogenes, Alcaligenes faecalis, Arthrobacter hyalinus, Arthrobacter paraffineus, Arthrobacter simplex, Bacillus acidocaldarius, Bacillus amyloliquefaciens, Bacillus amylosolvens, Bacillus brevis, Bacillus caldolyticus, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus firmus, Bacillus lentus, Bacillus licheniformis, B. megaterium, Bacillus, Bacillus moritae, Bacillus polymyxa, Bacillus popiliae, Bacillus pumilis, Bacillus subtilis, Brevibacterium amylolyticum, Brevibacterium flavum, Brevibacterium lactofermentum, Clostridium acetobutylicum, Clostridium butyricum, Corynebacterium gelatinosum, Corynebacterium glutamicum, Corynebacterium guanofaciens, Corynebacterium hydrocarboclastus, Corynebacterium petrophilum, Cryptococcus laurentii, Escherichia coli, Flavobacterium aminogenes, Gluconobacter melanogenus, Lactobacillus bulgaris, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus leichmanii, Lactobacillus pentosus, Leuconostoc brevis, Leuconotoc dextranicum, Leuconostoc mesenteroides, Methanobacillus omelianski, Methanobacillus soenhngenii, Methanomonas margaritae, Methanomonas capsulatus, Methanomonas methanica, Methylovibrio soehngenii, Microbacterium ammoniaphilum, Micrococcus glutamicus, Micromonospora carbonaceae, Micromonospora echinospora, Micromonospora inyoensis, Micromonospora olivoasterospora, Micromonospora purpurea, Mycobacterium phlei, Mycobacterium smegmatis, Nocardia alkanoglutinosa, Nocardia gardneni, Nocardia mediterranei, Nocardia uniformis, Propionibacterium freudenreichii, Propionibacterium shermanii, Protaminobacter ruber, Proteus rettgeri, Pseudomonas amyloderamosa, Pseudomonas aureofaciens, Pseudomonas dacunhae, Pseudomonas denitrificans, Pseudomonas methylotrophus, Pseudomonas ovalis, Pseudomonas pyrrocinia, Rhodopseudomonas spheroides, Saccharopolyspora erythraea, Sarcina lutea, Sporotrichum pulverulentum, Streptococcus cremoris, Streptococcus fradiae, Streptococcus lactis, Streptococcus mutants, Streptococcus thermophilus, all species of Streptomyces, Thermomonospora curvata, Thermomonospora fusca, Thiobacillus ferroxidans, Thiobacillus thiooxidans,

- fungi and yeast: Acremonium chrysogenum, Aschersonia aleyrodis, Ashbya gossypii, Aspergillus awarnori, Aspergillus flavus, Aspergillus itaconicus, Aspergillus oryzae, Aspergillus sojae, Aspergillus terreus, Aspergillus wentii, Aureobasidium pullulans, Beauveria bassiana, Beauveria inflatum, Candida flareri, Candida lipolytica, Candida oleophila, Candida periculosa, Candida tropicalis, Candida utilis, Cephalosporium acremonium, Claviceps paspali, Claviceps fusiformis, Clitopilus passeckerianus, Curvularia lunata, Cyclindrocarpon radicicola, Eremothecium ashbyii, Hansenula anomala, Hirsutella thompsonii, Klyveromyces fragilis, Klyveromyces lactis, Metharhizium anisophae, Mucor miehei, Mucor pusillus, Myocandida riboflavina, Neocosmospora vasinfecta, Phaecilomyces varioti, Penicillium chrysogenum, Penicillium camemberti, Penicillium griseofulvum, Penicillium roqueforti, Penicillium patulum, Phanerochaete chrysosporium, Phycomyces blakesleanus, Pichia guilliermondi, Pichia stiptis, Pullularia pullulans, Rhizopus delemar, Rhizopus formosaensi, Rhizopus japanicus, Rhizopus nigricans, Rhizopus niveus, Saccharomyces cerevisae, Saccharomyces carisbergiensis, Saccharomyces rouxii, Saccharomyces lipolytica, Schizosaccharomyces pobe, Sclerotium glutanicum, Sesquicilliopsis rosariensis, Streptomyxa affinis, Tolypocladium inflatum, Tolypocladium terricola, Torula cremonis, Torulopsis magnoliae, Torulopsis utilis, Trametes sanguinea, Trigonopsis variabilis.

Neutopenia microorganisms preferably represent fungi and yeast, especially mushrooms.

The most preferred species of fungi belonging to the following genera and species: Acremonium spp., such as Acremonium chrysogenum, Aspergillus spp., such as Aspergillus awamori, Aspergillus itaconicus, Aureobasidium spp., such as Aureobasidium pullulans, Beauveria spp., such as Beauveria bassiana, Beauveria inflatum, Clitopilus spp., such as Clitopilus passeckerianius, Mucor spp., such as Mucor miehei, Mucor pusillus, Neocosmospera spp., such as Neocosmospera vasinfecta, Phaecilomyces spp., such as Phaecilomyces varioti, Penicillium spp., such as Penicillium chrysogenum, Penicillium camemberti, Penicillium citrinum, Penicillium griseofulvum, Penicillium roqueforti, Penicillium urticae, Penicillium patulum, Phanerochaete spp., such as Phanerochaete chrysosporium, Pullularia spp., such as Pullularia pullulans, Schizosaccharomyces spp., such as Schizosaccharomyces pombe, Tolypocladium spp., such as Tolypocladium inflatum, Tolypocladium terricola, Trametes spp., such as Trametes sanguinea, Trichoderma spp., such as Trichoderma koningii, Trichoderma reseei, Trichoderma viride.

Even more preferred are microorganisms that can be used in accordance with the invention, belong to the genera Penicillium and Cephalosporium, among which particularly preferred are the species Penicillium chrysogenum and Cephalosporium acremonium.

Under "biomass" in the context of the present description mean dried organic waste produced in about the Jesse biotechnological fermentation, for example, in the manufacture of pharmaceuticals, such as antibiotics. In the process of collecting product wet microbial biomass, such as phase containing an antibiotic, is separated by filtration from the liquid and dried, for example, for 4-6 h at a temperature of +130°C to +150°C. These dried organic waste can serve as a further source material for the production of the agent according to the invention.

Preferably the starting material is the biomass of the fungus (mycelium), obtained from waste products of biotechnological processes of fermentation, preferably by fermentation of Penicillium chrysogenum and Cephalosporium acremonium.

Preferred examples of inorganic solvent suitable for use in the extraction process stages (a) and (d)is water. Preferred examples of organic solvents suitable for use in the extraction process stages (a) and (g)are alcohols, such as isopropanol, ethanol or methanol.

Typically used concentrations from 50 to 200 g (dry weight) biomass neutopenia microorganisms per 1 l of solvent. Preferably approximately 150 g (dry weight) biomass suspended in 1 l of solvent.

The suspension obtained after stage (a)typically has a pH value of from about 2.8 to about 5,6, preferably about is about 3.3 to about 3.6, and does not need additional regulation of pH values. This suspension is typically stirred at room temperature (+20°C to +25°C) for 1-12 h (stage (b)).

Typical conditions incubation stage (C) are, for example, 1 hour at +120°C, 2 h at +80°or 12 h at +20°C. the maximum temperature is +120°C and the minimum temperature is +20°C. At +120°With the incubation time should not exceed 2 hours and should not be less than 0.5 h and at + 20° the minimum incubation time should be 8 hours, and the maximum time is 70 hours For a specialist in this area it is obvious how to determine the minimum and maximum period of time of incubation at the given temperatures from +120°C to +20°C. Preferably, the incubation is conducted while heating or autoclaving, for example, for 1 h at +120°With (extract, obtained as a result of this process, hereinafter designated as the extract of the PEN-A), or for 2 h at +80°C. the Extract obtained by incubation for 12 h at +20°later in the present description designated as PEN-B.

Re-suspension at the stage (g) is usually carried out by shaking or mixing of the suspension, which could be divided into solid and liquid components at the stage (b).

Stage (d), obviously, is necessary is th only when the temperature used for incubation, greater than room temperature.

The filtering stage (e) is usually carried out using paper filters and the result is a clear solution of a brownish color, usually with an odor characteristic of fermentation products. Stage filtering can also be carried out on an industrial scale by periodic or continuous centrifugation or using a filter working under pressure. Stage filtering is used, for example, to reduce the risk of phytotoxicity. Preferred agents according to the invention be obtained by using the stage of filter (s).

After stage (d) or preferably after stage filtration (e) the extract is usually dried so that it can be packaged and shipped in powdered form and to direct the consumer could resuspendable end use according to the invention. Drying conditions are not critical, and can be used any known methods, such as lyophilization, spray drying or drying on a rotary evaporator.

Changes in the extraction process can lead to serious losses of the desired activity in relation to the protection of plants and/or cause significant side effects of the extract, such as phytotoxicity,

The agent is obreteniyu typically comprises as active ingredients the following components:

1) a branched or unbranched oligosaccharides with degree of polymerization from 2 to 30 or more,

2) monosaccharides and

3) proteins, glycoprotein and/or lipoproteins. Preferably the agent according to the invention can contain:

1) 0.5 to 8.0 g/l branched or unbranched oligosaccharides with degree of polymerization from 2 to 30, preferably with 1-6 β-links and 1-3 β-links

2) 0.1 to 4.0 g/l of monosaccharides and

3) 0.1 to 1.5 g/l of proteins, glycoprotein and/or lipoprotein.

The monomers obtained by hydrolysis of these oligosaccharides, mainly represent mannose, galactose and glucose in a ratio of, for example, 1:1:1 (for Penicillium chrysogenum), 2:1:2, 1:1:2 or 2:1:1, and represent N-acetylglucosamine, glucosamine and chitin.

The most pronounced activity against plant protection corresponds to molecules with a molecular weight <3000 Yes.

The invention also relates to compositions for the induction of plant resistance to phytopathogenic microorganisms, comprising as active ingredient an agent according to the invention in combination with an acceptable agricultural point of view diluent (hereinafter in the present description is designated as a diluent and optionally with one or more applicable for plant protection pesticides. The compositions have common met the house, for example, by mixing the agent according to the invention with a diluent and optionally with additional ingredients such as surfactants.

The concept of "thinner" in the context of the present description refers to liquid or solid with acceptable agricultural point of view, the product that can be added to existing stuff to make it simpler or more convenient to use forms, such as dilution of the active substance with the purpose of obtaining used or the level of activity required. Examples of such diluents is talc, kaolin, diatomaceous earth, xylene or water.

Particularly preferred formulation, such as dispersible in water concentrates or wettable powders, may contain surfactants such as wetting and dispersing agents, for example condensation products of formaldehyde and naphthalenesulfonate, alkylarylsulfonate, ligninsulfonate, fatty alkylsulfate, ethoxylated alkylphenol and an ethoxylated fatty alcohol. Typically, the formulation contains from 0.01 to 90 wt.% active substance (agent according to the invention and optional pesticides), from 0 to 20% is acceptable from an agricultural point of view surfactant and 10 to 99.99% of diluent (s). Conc the private form of the composition, for example, emulsion concentrates usually contain from about 2 to 90 wt.%, preferably from 5 to 70 wt.% the active substance. Suitable for application form of the composition typically contains from 0.0005 to 10 wt.% the active agent according to the invention as active ingredient, and the usual suspension for spraying can, for example, contain from 0.0005 to 0.05, for example about 0.001, 0.002 or 0.005 wt.% the active substance.

In addition to the usual diluents and surfactant agent according to the invention may contain additional excipients intended for special purposes, such as stabilizers, decontamination officers (solid preparative forms or media, with an active surface), adhesives, improves adhesion to plants, corrosion inhibitors, antispyware and dyes.

Suitable for plant pesticides that can be used in combination with the agent according to the invention include fungicides, herbicides, bactericides, insecticides, etc. of the Agent according to the invention is preferably used in combination with fungicides, such as sulfur, CHLOROTHALONIL, euphranor with fungicides from a class of guanidine such as guazatine, dithiocarbamate, such as MANCOZEB, MANEB, zineb, propineb, trichlorocarbanilide and their analogs, such as Captan, captafol and fo is IPET, the benzimidazole, such as carbendazim, benomyl, azoles, such as difenoconazol, tsyprokonazolu, flusilazol, flutriafol, hexaconazole, propiconazole, penconazole, tebuconazole, metconazole, epoxiconazole, tetraconazole, triticonazole, provenzal, tricyclazole, fluquinconazole, prochloraz, morpholine, such as fenpropimorph, fenpropidin, dimethomorph, or otherwise suitably activity products such as fungicides type jenoxifen, famoxadone, spiroxamine, fenhexamid, 2-(2-phenoxyphenyl)-(E)-2-methoxyimino-N-methylacetamide, [2-(2,5-dimethylphenoxy)phenyl]-(E)-2-methoxyimino-N-methylamide, (1R,3S/1S,3R)-2,2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclohexanecarboxylic, ethoxyacrylate and methoxykynuramine as described for formula I in WO 97/00011, AZOXYSTROBIN, kresoxim-methyl, cymoxanil, cyprodinil, piroximone, oxadixyl, metalaxyl or R-metalaxyl, or such insecticides as furathiocarb, or compounds described for the formula I in EP-A-0580553, and preferred are combinations with cyproconazole, propiconazole, R-metalaxyl or oxadixyl.

Such combinations are particularly effective for treating or preventing outbreaks of late blight (Phytophtora infestants), Anthracnose (Colletotrichum lagenarium), rust (Puccinia tritici), mildew (Erysiphe graminis), bacterial wilt (Erwinia tracheiphia) and grey mould (Pseudomonas lachrymans).

Examples of acceptable for plants formulations of fungicides, intended for protection of plants, are the following.

A. Preparative Form wetting powder

10 parts of the agent according to the invention in dried form are mixed and milled with 4 parts of synthetic fine silica, 3 parts of sodium lauryl, 7 parts ligninsulfonate sodium and 66 parts of fine kaolin and 10 parts of diatomaceous earth as long as the average particle size will be approximately 5 μm. Obrazovavshijsya wettable powder is diluted before use with water as a liquid for spraying, which can be used for spraying the leaves, and also be used for the impregnation zone, the location of the roots.

B. Granules

To 94.5 Mascali quartz sand in the drum mixer spray of 0.5 Mascali binding agent (nonionic surfactant) and all the components are thoroughly mixed. Then add 5 moschata agent according to the invention in dried form and thorough mixing continued until obtaining a granular formulation with a particle size in the range from 0.3 to 0.7 mm (if necessary, the granules can be dried by the addition of 1-5 wt.% talc). Granules can be applied by soil around the plants, the underlying processing.

century Emulsion concentrate

10 moschata agent according to the invention are mixed with 10 mascectomy emulsifier and 80 mascectomy xylene. The resulting concentrate before use, dilute with water to obtain an emulsion of the desired concentration.

, seed Dressing

45 parts of the agent according to the invention are mixed with 1.5 parts of adduct diarylpropionitrile ether/ethylene oxide, 2 parts of spun oil, 51 part finely ground talc and 0.5 parts of the dye rhodamine C. the Mixture is milled in contraplex mill at 10,000 rpm to obtain particles of average size not exceeding 20 microns. The resulting dry powder has good adhesion and can be used for seed treatment, for example, by mixing within 2-5 min in a slowly rotating vessel.

The agent according to the invention can be applied to plants by spraying the surface of the leaves and/or stems, it can be used for soil treatment by soaking or by soil granules or capsules and can be applied to seeds as an agent for treating.

The preferred method of treatment is to spray the surface of the leaves and/or stems, or a combination of the intermittent spraying the surface of the leaves and/or stems and soil treatment by soaking.

When the agent according to the present invention is applied by spraying the surface of leaves and stems, it may also contain additional ingredients such as adjuvants, stabilizers, surfactants and adhesives known in this field.

When applying the composition for seed treatment may be performed in combination with adhesives, or the active agent can be used in encapsulated form, which may be obtained using known methods of encapsulation.

The applied amount of the agent according to the invention, generally, is 0.005 to 1.0 g glucose equivalents at the plant or, in other words, 0.05 to 2 kg per ha for processing. May require re-treatment. Glucose equivalents are determined according to the method "Anthron using glucose as standard [Dische Z. (1962) Color Reactions of Carbohydrates, including "Methods in Carbohydrate Chemistry", (Ed. by R.L. Whistler, Wolfrom M.L) Academic Press Inc. New York)].

Concentration, which used the extract to induce resistance in plants usually ranges from 0.5 to 3.0 g/l glucose equivalents.

The degree of protection is calculated relative to control plants according to the following formula:

Below the invention is illustrated by examples. Assume that the examples are illustrative and do not limit the scope of the invention.

Example 1: Obtaining an extract of Penicillium

300 g (dry weight) of waste mi is the men Penicillium chrysogenum, obtained in the production of penicillin, transferred into a glass vessel type Duran® volume 2000 ml, which add distilled water to a final volume of 2 l (pH of 3.2). The resulting suspension is stirred at room temperature for 1 h at 700 rpm and then autoclave for 1 h at temperatures of +121°and a pressure of 1 bar. Then the hot vessel gently shaken and allowed to cool and stand overnight. Then all the contents of the vessel is filtered through a paper coffee filters 1×10 type Melitta® and collect past through the filter of a liquid containing the active extract (hereinafter in the present description designated as PEN-extract). The sugar content (glucose equivalents) of extracts are determined according to the method "Anthron using glucose as standard [Dische Z. (1962) Color Reactions of Carbohydrates, including "Methods in Carbohydrate Chemistry" (edited by R.L. Whistler, Wolfrom M.L) Academic Press Inc. New York)].

Usually PEN-extract contains approximately 5 g of glucose equivalents per 1 liter and usually before applying to the pilot plant it is diluted in 2-3 times.

Example 2: Processing tomato for protection against Phytophtora infestants

Plant and fungal material

Tomato plants (Lycopersicum esculentum) cultivar Baby F1 grown in the greenhouse consists of 120 holes a bed for the seeds in a mixture of 1/3 sand and 2/3 soil type TKS1®, at 25°when Svetova the regime of 16 h light/8 h darkness. After 14 days separate the seedlings are transferred into pots with a diameter of 10 cm and incubated in the same conditions of cultivation for another 3 weeks.

Strain Phytophtora infestants cultivated on biconcave slices of potato tuber (sort Bintije obtained from the farm for the production of biological material) in closed plastic plates in the dark at a temperature of +12°C up to +16°and 60-80%relative humidity for 6-7 days.

Suspension inoculum receive, washing the slices of potato tuber, which is the most heavily covered sporliuk mycelium Phytophtora infestants, 100 ml chilled on ice distilled water. Mitilary debris removed by filtration and determine the number of sporangia in flushing, producing estimates using the microscope in the chamber for counting type "Neubauer".

Immediately before inoculation, the inoculum density was adjusted to 30-40000 of sporangia in ml.

Processing

Tomato plants at the stage 5 true leaves treated by soaking the soil and/or sprayed with a PEN-extract for 7 and 3 days before inoculation with a suspension of sporangia Phytophtora infestants.

Soaking: the Plants are kept dry for 1 day before treatment and flower pots are placed in a tablet size 7×7 cm for collecting excess solution to soak. Put 60 ml PEN-extract containing 1-2 g glucose ek is ivalent 1 L. After treatment, the plants kept in the greenhouse at room temperature without watering for 1 day.

Spraying: the whole plant is sprayed with a PEN-extract containing 1-2 g glucose equivalent, until the moisture (approximately 10-15 ml per plant) using a pneumatic gun for spraying model JATO 232 FR pressure of 0.5 bar.

After treatment, the plants kept in the greenhouse until the infection inoculum. Control plants to simulate processing sprayed with distilled water.

Infection inoculate

Treated or control plants were sprayed with approximately 10 ml of chilled on ice, the suspension of sporangia in a ventilated chamber for inoculation using a pneumatic gun for spraying model JATO 232 FR pressure of 0.2 bar, covering mainly the lower surface of leaves homogeneous layer of small droplets.

Immediately after inoculation the plants are transferred to a Plexiglas boxes and incubated in the dark at a temperature of +12°C up to +16°C and 100%relative humidity for 24 hours and Then returned to the greenhouse and maintained under standard conditions described above.

Assessment

After 5-6 days after inoculation, when the control plants become noticeable typical symptoms, the degree of destruction is determined visually as the percentage of Pora is authorized five leaf surface of the lower leaves of the experimental plants.

The degree of protection is calculated relative to control plants according to the following formula:

The results are shown in table Ia. Best results are obtained when combining processing by soaking and spraying.

Table Ia

Induced protection of tomato plants against Phytophthora infestants using PEN when processed by soaking and spraying and when combined processed by soaking/spraying
TreatmentaboutThe concentration of PEN% affected leaf surface% protection
P+O1.5 g/l 2.0 g/l491
P+O1.5 g/l, 1.0 g/l1075
P+O1.5 g/l, 0.5 g/l23,350,1
P+*2.0 g/l, 1.5 g/l889
2S1.5 g/l4014,3
1P1.5 g/l18,360,8
21.5 g/l1078,6
1B1.5 g/l16,764,2
control, water  47 
TreatmentaboutThe concentration of PEN% affected leaf surface% protection
* control water 73 

Data represent average values obtained in 4 independent experiments. The average error ≤9.5 per cent. "P" denotes a saturated, "About" refers to spraying.aboutThe first treatment 7 days before inoculation, the second processing for 3 days before inoculation. * This value is obtained in a different experiment, and so it also uses a different reference value shown in the last row of table Ia.

Example 3: Processing of potatoes for protection against Phytophthora infestants

Similar to the method described in example 2, the protection of plants against Phytophtora infestants induce in potato plants grown from tubers of the cultivar Bintje"on stage 4 leaves. The results are shown below in table IB. Best results are obtained when processing by spraying.

Table IB

Induced protection of potato against Phytophthora infestants using PEN when processed by spraying and when combined processed by soaking/spraying
Processing *The concentration of PEN % affected leaf surface% protection
1A+1B2 g/l 2 g/l1083
1A+1B1 g/l 1 g/l2067
23 g/l789
22 g/l395
21G/l886
20.5 g/l2756
BMC500 ppm million2361
control, water 610

Data represent average values obtained in 3 independent experiments. The average error ≤11%. "P" denotes a saturated, "About" refers to spraying. *The first treatment 7 days before inoculation, the second processing for 3 days before inoculation. BMC indicates beta-butyric acid, which was used as a standard chemical inducer.

Example 4: Treatment of plants beans against Uromyces appendiculatus

Plant and fungal material

Two seed bean Golden (Phaseolus aureus) grades of "Musica" is grown in flower pots with a diameter of 8 cm in a mixture of 1/3 sand and 2/3 soil type TKS1®, at +25°With a light regime of 16 h light/8 h those whom the notes within 16 days. For the study from each pot select one sprout before disclosing second leaf.

Spores of strain Uromyces appendiculatus take after storage in cryogenic conditions (CRYO) in liquid nitrogen. From 1 mg dispute receive a 1 ml suspension for inoculation.

Processing

Bean plant at the stage of the second sheet or treated by soaking the soil and/or by spraying PEN-extract for 7 and 3 days before inoculation with a suspension of sporangia Uromyces appendiculatus.

Soaking: the Plants are kept dry for 1 day before treatment and flower pots are placed in a tablet size 7×7 cm for collecting excess solution impregnation. In greenhouse at room temperature inflict 40 ml PEN-extract containing 1-2 g glucose equivalents at 1 HP After treatment of the plants kept in the greenhouse for 1 day at a temperature of +20...+25°without irrigation.

Spraying: the whole plant is sprayed with a PEN-extract containing 1-2 g glucose equivalents until the moisture using a pneumatic gun for spraying model JATO 232 FR pressure of 0.5 bar.

After treatment, the plants kept in the greenhouse until the infection inoculum. Control plants as simulation processing sprayed with distilled water.

Infection inoculate

Treated or control plants were sprayed with approximately ml suspension of sporangia in a ventilated chamber for inoculation using a pneumatic gun for spraying model JATO 232 FR pressure of 0.2 bar, covering the lower surface of leaves homogeneous layer of small droplets.

Immediately after inoculation the plants are transferred to a Plexiglas boxes and incubated in the dark at a temperature of +20°C and 60%relative humidity for 24 h, and then turn on the light and the plants kept in the same conditions for another 4 days. The plants back into the greenhouse and maintained under standard conditions described above. Raw inoculate the leaves are removed.

Assessment

2 weeks after inoculation, when the control plants found sporulation of the fungus, the degree of destruction is determined visually as the percentage of affected leaf surface treated inoculate leaves. The degree of protection is calculated relative to control plants according to the following formula:

The results are shown in table II. Best results are obtained when two-time processing by saturating concentration of 2 g/l or 0.5 g/l

Table II

Induced plant protection beans from Uromyces appendiculatus using PEN when processed by soaking and spraying and when combined processed by soaking/spraying
Processing *The concentration of PEN% affected the earnest surface % protection
2S2.0 g/l595
2S1.0 g/l6037
2S0.5 g/l397
1A+1B2.0 g/l1782
1A+1B1.0 g/l3365
1A+1B0.5 g/l5047
22.0 g/l2772
21.0 g/l905,3
20.5 g/l6037
control, water 879

Data represent average values obtained in 2 independent experiments using each of 4 individual plants. The average error ≤8.5 percent. "P" denotes a saturated, "About" refers to spraying. *The first treatment 7 days before inoculation, the second processing for 3 days before inoculation.

Example 5: Processing of wheat for protection against Puccinia recondita spp. tritici

Plant and fungal material

Ten wheat seeds (Triticum arvense) varieties "Anna" is grown in flower pots with a diameter of 8 cm in a mixture of 1/3 sand and 2/3 soil type TKS1® at +25 With a light regime of 16 h light/8 h dark for 7 days before full opening of the first sheet.

The strain of brown rust Puccinia recondita spp. tritici propagated on the same variety of wheat. Fully affected leaves with spores cut and spores dispersed in water containing 0.05% Tween 20®. The inoculum density was adjusted to 100,000 spores/ml

Processing

Wheat plants at the stage of just-opened second sheet sprayed for 7 and 3 days before inoculation. All the plants are sprayed with a PEN-extract containing 1-2 g glucose equivalents and 0.05% Tween 20 until the moisture using a pneumatic gun for spraying model JATO 232 FR pressure of 0.5 bar.

After treatment, the plants kept in the greenhouse until the infection inoculum. Control plants as simulation processing sprayed with distilled water.

Infection inoculate

Treated or control plants were sprayed with approximately 3 ml of the suspension of sporangia in a ventilated chamber for inoculation using a pneumatic gun for spraying model JATO 232 FR pressure of 0.2 bar, to obtain a homogeneous layer of small droplets throughout the shoot.

After drying drops of plants transferred in Plexiglas boxes and incubated at a temperature of +20°C and 60%relative humidity for 24 h in the dark, then include Sveti plants incubated under the same conditions for another 4 days. The plants back into the greenhouse and maintained under standard conditions described above.

Assessment

10 days after inoculation, when the control plants found sporulation of the fungus, the degree of destruction is determined visually as the percentage of affected leaf surface treated inoculate leaves. The degree of protection is calculated relative to control plants according to the following formula:

The results are shown in table III. Best results are obtained when processed by a fraction PEN having a molecular weight of 2-3 kDa at a concentration of 2 g/L.

Table III

Induced plant protection of wheat against Puccinia recondita spp. tritici using PEN and fractions PEN with different molecular weight during processing by spraying
ProcessingConcentration% of lesions% protection
PEN2.0 g/l15,973,9
PEN1.0 g/l42,230,9
PEN >30 kDa4.0 g/l21,465
PEN >10 kDa2.0 g/l33,345,5
PEN >10 kDa1.0 g/l 2559
PEN 2-3 kDa2.0 g/l8,985,4
PEN 2-3 kDa1.0 g/l2067,3
PEN >300 Yes1.0 g/l11,181,3
PEN >300 Yes0.5 g/l33,345,5
CGA 24570430 part./million31,348,8
CGA 24570460 part./million6020
the control (composition) 42,929,8
control (water) 61,10

Data represent average values obtained in 4 independent experiments. The average error ≤10%. CGA 245704, the active ingredient of the drug BION®, was used as a standard chemical inducer.

Example 6: Treatment of cucumber against Colletotrichum lagenarium and Pseudomonas lachrymans

Flora and pathogenic material

Plants of cucumber (Cucumis sativus) cultivars "Wisconsin" grown in the greenhouse for 10 days in flower pots with a volume of 40 ml of Colletotrichum lagenarium grown in Petri dishes on agar vegetable juice type V8 within 7 days at +20°C. Pseudomonas lachrymans grown on medium MCM (yeast-dextrose-calcium carbonate) for 24 h at +30&x000B0; C in Erlenmeyer flasks.

Processing

Solution for spraying, containing 1.5 or 3 g/l glucose equivalent extract of the PEN-A PEN or-B, sprayed to wet the leaves, using a special cap for spraying. After treatment, all plants incubated in the greenhouse at + 22°for 3 or 7 days. Control plants treated with water.

Infection inoculate

Spore suspension of C. lagenarium (1,2×105spores/ml) spray the leaves of the plant using a fire hose to spray type Velbiss. The plants are incubated for 30 h at a relative humidity (RH) of 95% in the dark at +23°C. Then the plants transferred to the greenhouse with temperatures ranging from +22°C up to +23°With normal S.

A suspension of P. lachrymans (108cells/ml) spray the leaves with a fire hose for spraying type Velbiss under a pressure of 2 bar. Before inoculation the plants incubated at 100% RH for 4 hours After inoculation the plants again incubated in the greenhouse at RH 100% and a temperature of +23°C up to +24°C.

Assessment

After 6 days (.lachrymans) or 7-8 days (.lagenarium) the disease can be detected visually, and in this time estimate % diseased leaf area. The results are summarized in the table below.

Treatment before inoculation for: (days) % protective activity
PEN-APEN-B
1.5 g/l3.0 g/l1.5 g/l3.0 g/l
Colletotrichum/cucumber 3 days.96929896
Colletotrichum/cucumber 7 days.09500
Pseudomonas/cucumber 3 days.10108035
Treatment before inoculation for: (days)% protective activity
PEN-APEN-B
1.5 g/l3.0 g/l1.5 g/l3.0 g/l
Pseudomonas/cucumber 7 days.0202020

1. The agent intended to induce in plants resistance to phytopathogenic microorganisms, where the agent is an extract of biomass, which is obtained in the process of biotechnological fermentation neutopenia microorganisms, with the exception of microorganisms of the genus Saccharomyces and the process of obtaining which provides:

a) re-suspension from 50 g to 200 g (dry weight) biomass neutopenia microorganisms in 1 l reorganize the who or organic solvent

b) stirring at room temperature for 1-12 h,

C) incubation,

g) re-suspension

d) cooling to room temperature during maturation and,

e) optionally, filtering.

2. The agent according to claim 1, where the biomass neutopenia microorganisms is the biomass of the fungus obtained from waste processes biotechnological fermentation.

3. The agent according to claim 1, where neutopenia the microorganism is a non-specific for a plant organism.

4. The agent according to any one of the preceding paragraphs, where the biomass is obtained by fermentation Acremonium spp., Aspergillus spp., Aureobasidium spp., Beauveria spp., Clitopilus spp., Mucor spp., Neocosmospera spp., Phaecilomyces spp., Penicillium spp., Phanerochaete spp., Pullularia spp., Schizosaccharomyces spp., Tolypocladium spp., Trametes spp. and Trichoderma spp.

5. The agent according to any one of the preceding paragraphs, where the biomass is obtained by fermentation of Penicillium chrysogenum and Cephalosporium acremonium.

6. The agent according to any one of the preceding paragraphs, comprising as active ingredients 1) a branched or unbranched oligosaccharides with degree of polymerization 2-30, 2) monosaccharides and 3) proteins, glycoprotein and/or lipoprotein with a molecular weight <3000 Yes.

7. Composition for the induction of plant resistance to phytopathogenic microorganisms, comprising an effective amount to induce plant resistance agent Liu the WMD from the preceding paragraph in combination with acceptable agricultural point of view diluent.

8. The composition according to claim 7, which further comprises one or more pesticides for plant protection.

9. Extract of Penicillium chrysogenum, is able to induce in plants resistance to phytopathogenic microorganisms, obtained by using the following method:

a) re-suspension from 50 g to 200 g (dry weight) of biomass of Penicillium chrysogenum in 1 l of inorganic or organic solvent,

b) stirring at room temperature for 1-12 h,

C) incubation,

g) re-suspension

d) cooling to room temperature during maturation and

e) optionally filtering.

10. The extract according to claim 9, where the extract induces resistance in plants when applied in a concentration of 0.5-3.0 g/l glucose equivalents.

11. Method of inducing resistance in plants by phytopathogenic microorganisms, capable of handling plants, soil or seeds with an effective amount of an agent according to any one of claims 1 to 6.

12. The method of receiving agent, intended to induce in plants resistance to phytopathogenic microorganisms, where the agent is an extract from biomass neutopenia microorganisms, comprising the following stages:

a) re-suspension from 50 g to 200 g (dry weight) biomass neutopenia microorganisms in 1 l neorganicheskoi is or organic solvent

b) stirring at room temperature for 1-12 h,

C) incubation,

g) re-suspension

d) cooling to room temperature during maturation and,

e) optionally, filtering.



 

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SUBSTANCE: method involves treating potato with specific composition before laying for storage. Said composition includes suspension of Steinernema feltiae and immune stimulant produced by sequential extracting of Mortierella pusilla micromycet biomass with the use of non-polar extractant in above-critical state, water, alkaline, water, acid, water, alkaline, and water, with following joining of first extract with solid residue.

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