Preparation for plant protection from diseases

 

(57) Abstract:

The invention relates to the microbiological industry, production of plant protection products. Preparation for plant protection against diseases contains, wt%: the biomass of the strain of the fungus Trichoderma viride Pers ex S. F. Gray 16 SMC F-59M 80-90; microcrystalline cellulose 5-10; salts of magnesium 5-10. The drug has antagonistic activity against a broad spectrum of pathogens of agricultural crops and can be used in conjunction with chemical pesticides. 8 table.

The invention relates to the microbiological industry, production of plant protection products.

One of the main sources of plant pathogens is the soil, which develops and stored in the form of spores of phytopathogenic fungi such as Fusarium, Helminthosporium, Rhisoctonia, Pythium, Sclerotium, Alternaria, Verticillium. To combat soil-borne pathogens are mainly used chemical fungicides. However, the intensive use of chemicals leads to environmental violations, to the emergence of more resistant strains of pathogenic microorganisms and other undesirable consequences.

Increasingly in agricultural practice PRECI drugs including on the basis of antagonists that suppress the growth of phytopathogens.

Known strain of Trichoderma harzianum, which has high biological activity against Sclerotium rolfsii causing root rot of vegetable crops (1). However, it is effective against pathogens only vegetable crops. Also known strain of Trichoderma harzianum, which inhibits the growth of pathogens causing grey mould vineyards and the milky sheen of fruit trees (2). The disadvantage of this strain is that it is only active against pathogens vineyards and fruit trees.

Closest to the proposed invention is a strain of Trichoderma viride-AL, effective against pathogens of barley, oats, rye, wheat, beets and potatoes (3). Its disadvantage is also narrow spectrum of antagonistic activity.

Known drug Trichodermin-10, dry powder, recommended against root and stem rot of vegetable and grain crops, and cotton (3). The drug is produced, increasing the biomass of the fungus on solid media (grain, peat, straw) for a long time (up to 14 days). The disadvantage of this drug is inconvenient commodity form, as if the red is from the media with water.

Known drug on the basis of a strain of Trichoderma harzianum, with the commercial name Fior II, which is used to protect vineyards and fruit trees from some of the diseases caused by fungal pathogens (3). The drug is a spore mass of the fungus and produced by the fungus peptides. Get the drug, cultivating the fungus in a liquid medium by forming a thin layer on trays, to the complete maturation of the dispute. Then all the biomass is dried, finely ground and sieved. The powder contains live spores, soluble in water, however, this method of production is time-consuming and laborious.

Known liquid-based preparation Trichoderma viride-AL (3). The drug presents mycelial form of the fungus, which is more effective due to better survival rate than conidia. As the drug is used in the culture fluid, which effectively defended barley, oats, rye, wheat, beets and potatoes from the root and stem rot. However, the drawback of this drug is the lack of the commodity form, which does not allow for storage of the drug.

Closest to the proposed invention is a paste-like preparation based on fungus Trichoderma har, growing biomass on solid substrate (barley, wheat) for 5-7 days, then dried and milled. For cooking pasta in a 3-5% solution of adhesive, which is used as carboxymethylcellulose (CMC), add 30-50 wt. % dry ground biomass of the fungus. CMC in this preparation enhances sporulation of the fungus and its survival after the deposition of the stems, stimulates the growth of fungus, as well as the germination of seeds and development of seedlings of cucumbers and tomatoes. The disadvantages of this product are multi-stage, long-term and the complexity of its preparation.

The objective of the invention is to obtain a strain of Trichoderma viride Pers ex S. F. Gray, which has antagonistic activity against a broad spectrum of fungal pathogens and the use of this strain for protection of plants from diseases.

The task is solved in that the proposed strain of the fungus Trichoderma viride Pers ex S. F. Gray 16, are highly active against a broad spectrum of fungal pathogens, and based on it to get the product containing wt.%:

The biomass of the strain of the fungus Trichoderma viride Pers ex S. F. Gray 16 - 85-92

Carboxymethylcellulose - 3-5

Magnesium - 5-10

The drug has antagonistic activity against pathogens CE is at. With the natural strain was carried out selection on the basis of productivity and hyperparasitism activity against the causative agents of various diseases. Selected active clone is described as a strain of Trichoderma viride Pers ex S. F. Gray at number 16. The strain deposited with the Central collection of microorganisms of Public concern "BIOLOGICAL product" (CCMC B) registration number - SMC F-59M.

The strain is characterized by the following features.

Cultural and morphological characteristics.

Strain Trichoderma viride Pers ex S. F. Gray 16 is a saprophytic fungus green color found in nature in soil and on dead wood. Grows well on standard media (čapek, potato-dextrose agar, wort-agar). In a standard environment of čapek after 5 days the fungus in the form of dark green grass covers the entire Petri dish. Surface colonies ragged, in the centre colour is dark green, gradually lightening to the periphery to white. Color the back side of grayish-green. Conidiophores smooth, branching in the form of loose bundles. For conidiophores and twigs are tiality in the form of whorls. Fieldy straight or slightly bent over the neck, forming a globose head of conidia. Conidia broadly ova, for 3 days reaches a size of 80x80 mm Sporulation observed on the third day of growth and development of the mycelium reaches its maximum on the 5th day. By this time the colonies of the fungus become green color. The color of the reverse side whitish-green.

Depending on the nutrient medium composition and cultivation conditions the fungus can form deep conidia and chlamydospores.

The optimal temperature for growth is 30oC. However, the strain grows in a wide range of temperatures from 20 to 35oC. Under 5 and 40oC spores germinate, but the mycelium does not develop, sporulation absent.

The proposed strain grows in the range of pH values from 2.5 to 9.5. The optimum pH value of 5.5 to 6.5. In more acidic conditions (pH of 2.5 to 5.0) and more alkaline (pH 7.0 - 9.5) is changed morphological characteristics of culture: slow the sporulation, the structure of the surface of the lawn becomes krupeninoy (at alkaline pH) or large-ragged (at acidic pH values).

Physiological and biochemical characteristics.

The sources of carbon. Well metabolizes glucose, glycerol, sucrose. Can grow on media containing galactose, lactose, maltose, starch. Not absorbed Cellulosics, Restores the nitrates.

Biological activity.

Strain Trichoderma viride Pers ex S. F. Gray 16 has a high hyperparasitism activity against a broad spectrum of plant pathogens belonging to the genera Fusarium, Helminthosporium, Rhisoctonia, Pythium, Sclerotium, Alternaria, Verticillium. These fungal pathogens cause such plant diseases as Fusarium head blight and Helminthosporium crops rot and mold of table grapes during storage, rot of potato.

The fungus strain Trichoderma viride Pers ex S. F. Gray 16 acts on pathogenic fungi, covering their their hyphae mycelium and then entering by enzymatic lysis.

The proposed strain resistant to antibiotics: ristomycin, penicillin, tetracycline, chloramphenicol, neomycin, streptomycin, gentamicin, polymyxin, ampicillin.

Strain Trichoderma viride Pers ex S. F. Gray 16 is resistant to chemical insecticides to karbofos, vomerine, talstar, applaud, and fungicides, which limit the growth of phytopathogenic fungi: TMTD, baytan, balitono.

High degree of resistance to chemical pesticides allows the use of the proposed strain in an integrated system of plant protection owls the different animals.

Biotechnological characteristics.

In submerged cultivation on different media (synthetic, organic, organo-synthetic) biomass yield ranges from 16 to 60 g/DM3.

After 20 hours of cultivation in liquid nutrient medium 90-100% of all surface conidia made on Wednesday, sprout. Mycelium is a at this time, a thin transparent deceptively hyphae. 35-40 hours culture of the fungus is a Mature mycelium, which becomes granular, formed deep conidia and chlamydospores. Cell membranes thicken, they are formed intercalary (internal) and terminal (end) chlamydospores, which ripen to a 40-50 hour. Intercalary disputes connected with 3-5 pieces together, oval-oblong. Terminal chlamydospores - single, rounded. 40-50 hours begins lysis of the mycelium.

Deep spores begin to germinate after 12 hours, 21 hours, almost all conidia germinate. Chlamydospores as well as the surface of the spores begin to germinate later - only after 16 -18 hours.

Deep spores are less resistant to high temperatures. They cannot stand the heat etc is good conidia. When 40oC parastemal conidia depends on processing time: the longer, the more disastrous the effects of temperature.

Example 1. Strain Trichoderma viride Pers ex S. F. Gray isolated from soils contaminated with fungi of the genus Fusarium.

Samples of the earth in quantities of 1 gram suspended in 100 grams of water and homogenized at 400 rpm./minutes After deposition of soil particles of a portion of the supernatant was diluted by a series of sterile saline. 0.1 ml of these serial dilutions were sown on Petri dishes with the environment of čapek or potato-dextrose agar. Net clones fungi of the genus Trichoderma were evaluated for their antagonistic activity against different strains of phytopathogenic fungi (test object) method "counter-cultures". For this purpose, a dense nutrient medium (čapek or potato agar) loop parallel to each other at a distance of 2 cm diameter Petri dishes were seeded analyzed strains and the test object. The Cup was kept in a thermostat at a temperature of 28oC for 7 days. Then evaluated visually, the area occupied by each crop. If the strain Trichoderma took more than half of the Petri dishes, he was considered to be active with respect to the test object.

The most active strain would be settled in a liquid medium.

3-5 million surface of conidia of the fungus is placed in a flask with a volume of 750 ml with 150 ml of organic-synthetic medium of the following composition (per 1 liter of medium):

Sulfuric acid hydrolysate BVK - 70 ml;

Glycerin 30 ml

Disubstituted phosphate potassium - 0.5 g

One-deputizing potassium phosphate, 0.5 g

Sulfate magnesium 7-water and 0.25 g

Ammonium sulphate - 3.0 g

Drinking water Up to 1 liter

Cultivated on a shaker at 180 rpm./min at 30oC for 40-60 hours. In regularly taken samples of the culture fluid control pH and dry weight biomass of the fungus. After 20 hours of cultivation in liquid nutrient medium 90-100% of all surface conidia made on Wednesday, sprout. Mycelium is a at this time, a thin transparent deceptively hyphae. 35-40 hours culture of the fungus is a Mature mycelium, which becomes granular, formed deep conidia and chlamydospores. When using the microscope culture of the fungus in this period is represented mainly by the mycelium containing up to 10% of chlamydospores. the pH decreases from 6.9 to 2.5. The maximum biomass yield was observed to 40 hours of cultivation and is 68 g/l Biomass of fungus get, separating the remains of nutrient Wed is

Antagonistic activity of a strain of Trichoderma viride Pers ex S. F. Gray 16 was determined by the method of "counter-cultures", as described in example 1.

Cells of the fungus strain Trichoderma viride Pers ex S. F. Gray 16 have high antagonistic activity to a wide range of fungal phytopathogens, related to the genera Fusarium, Rhizoctonia, Helminthosporium, Colletotrichum, Alternaria, Sclerotinia, Verticillium, Botrytis, Phytophtora, Actinomyces, Cladosporium, Aspergillus, Risopus, Epicoccum and Penicillium (tables 1-4). These pathogens are diseases of cereal crops, in particular Fusarium head blight and root rot. Characteristic lesions in tables 1, 2, 3 and 4 given by Industry classification of pests diseases of agricultural crops, developed by the Central Institute of Agrochemical service of agriculture.

Thus, a strain of Trichoderma viride Pers ex S. F. Gray 16 has a high hyperparasitism activity to a wide range of fungal pathogens belonging to the genera Fusarium, Rhizoctonia, Helminthosporium, Colletotrichum, Alternaria, Sclerotinia, Verticillium, Botrytis, Phytophtora, Actinomyces, Cladosporium, Aspergillus, Risopus, Epicoccum and Penicillium.

Example 4. The study of the compatibility of the strain Trichoderma viride Pers ex S. F. Gray 16 with chemical pesticides.

Compatibility of strain Trichoderma viride Pers ex S. F. Gray 16 with chemical pesticides studied, raising greatech, used in field conditions (table 5).

As can be seen from table 5, the strain Trichoderma viride Pers ex S. F. Gray 16 is compatible with all of the above chemical pesticides at concentrations recommended for use. Karbofos, vimetin and talstar slightly delay the growth of cultures of the fungus.

Example 5. Getting medication on the basis of a strain of Trichoderma viridePers ex S. F. Gray 16.

The biomass obtained as described in example 2, add sodium salt of carboxymethyl cellulose (CMC) as the adhesive in an amount of 5% of the total biomass and calcined magnesium chloride (stabilizer) in the amount of 10%. The mixture was mixed thoroughly and leave for a day to swell the cellulose. Mass periodically stirred. The product has the following composition wt%:

The biomass of the strain of the fungus Trichoderma viride Pers ex S. F. Gray 16 - 85

CMC - 5

Magnesium chloride - 10

Thus obtained the drug from the strain of Trichoderma viride Pers ex S. F. Gray 16 can be stored up to 3 months at a temperature of +15 to +30oC and up to 6 months at +4oC. the Influence of the composition of the drug on the preservation of biological activity of the fungus are shown in table 6.

As follows from the data table. 6, CMC promotes the conservation of biological Activ biomass obtained as described in example 2, add CMC (adhesive) in an amount of 3% of the total biomass and calcined magnesium sulfate (stabilizer) in the amount of 5%. The mixture was mixed thoroughly and leave for a day to swell the cellulose. Mass periodically stirred. The product has the following composition wt%:

The biomass of the strain of the fungus Trichoderma viride Pers ex S. F. Gray 16 - 92

CMC - 3

Magnesium sulfate - 5

Thus obtained the drug from the strain of Trichoderma viride Pers ex S. F. Gray 16 can be stored up to 3 months at a temperature of +15 to +30oC and up to 6 months at +4oC.

Example 7. Test the fungicidal activity of the drug on the basis of the strain Trichoderma viride Pers ex S. F. Gray 16.

Drug-based strain Trichoderma viride Pers ex S. F. Gray 16 was prepared as described in example 5. Effectiveness was assessed by pre-treatment of seeds of spring barley varieties Abava and prima against root rot caused by fungi of the genera Fusarium and Helminthosporium. The tests were carried out on plots measuring 10 m2and 25 m2experimental fields of the Institute of agricultural use of reclamation lands and the Belarusian Institute of plant protection. Experiments were performed in a 4-fold repetition. The results are shown in tables 6 and the military infection fungi of the genera Fusarium, Helminthosporium-level chemical disinfectants panorama and baytan. Marked bio-stimulating effect of the drug (higher tillering productive stems and number of grains per ear). Due to the large number of productive stems per 1 m2yield increase relative to the control was 3.5 t/ha, which is 1.8 t/ha greater than that of chemical disinfectants.

Thus, on the basis of the strain Trichoderma viride Pers ex S. F. Gray 16, which has antagonistic activity to a wide range of fungal pathogens (related to the genera Fusarium, Rhizoctonia, Helminthosporium, Colletotrichum, Alternaria, Sclerotinia, Verticillium, Botrytis, Phytophtora, Actinomyces, Cladosporium, Aspergillus, Risopus, Epicoccum and Penicillium) received the drug, effective against plant diseases. The product can be stored up to 3 months at a temperature of +15 to +30oC and up to 6 months at +40oC. the product is compatible with chemical pesticides (TMTD, Bayleton, Baytan, karbofos, Vimetin, Talstar, Applaud) at concentrations recommended for use. The drug can be used by pre-sowing treatment of seeds of grain crops at a dose of 15-30 grams per tonne of seed.

Sources of information

1. Wells, H. D., Bell, D. K., Jaworski, C. A. Efficacy of Trichoderma harzianum as a biocontrol Sclerotium rolfsii, Phytopathology, 62, 442, 1972.

2.dummy this strain, and the application of strain and new peptides or products obtained in the process of cultivation, as a biological control agent in the form of phytosanitary products - 1984.

3. The apsita A. F., Svinka Y. E., Strakowski S. C., Viesturs U. E., Lisowska A. J. , Bicevskis E. J., Swoka I. M., Benik R., the Use of trichodermin for protecting plants from phytopathogenic micromycetes, West. C. agricultural science N 9(397), S. 114-118, 1989.

4. RF patent N 2094991, A 01 N 63/04, A 01 G 7/00. Method of protecting plants cucumber and tomato against pathogenic fungi.

Preparation for plant protection from diseases, containing the active principle on the basis of a strain of the fungus Trichoderma and carboxymethyl cellulose, characterized in that it further comprises a magnesium salt, as active principle - the biomass of the strain of the fungus Trichoderma viride Pers ex S. F. Gray 16 SMC F-59M in the following ratio, wt.%:

The biomass of the strain of the fungus Trichoderma viride Pers ex S. F. Gray 16 SMC F-59M - 85 - 92

Carboxymethylcellulose - 3 - 5

Magnesium salt of 5 - 10

 

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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

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