The strain of the fungus agent pityrodes montagne used as biofungicide (options), biofungicide, its production method (variants), the method of suppressing fungal infection in plants, the method of screening of fungal microorganisms

 

(57) Abstract:

The invention relates to biological methods of combating plant diseases. Received new strains of fungi species Agent pityrodes Montagne DSM 7522, 8805, 8806, 8807, 8808, used as biofungicide. Based on the strains created biofungicide containing the above-mentioned strains and filler. The last chosen from the group comprising silicon dioxide, dry milk, carboxymethylcellulose, sucrose, ascorbic acid. A method for screening fungal microorganisms, namely, that plant seeds are sown in sand, seed and sand cause the mycelium and spores of the pathogen, the mixture is covered with sand, stand to receive sprouts plants. Explore the sprouts on the intensity of their exposure to phytopathogens and rejected fungi that do not affect the development of the disease themselves or find themselves pathogenic. Seeds of grain crops infecting pathogenic fungi and inoculated selected at the first stage of the fungus. Treated seeds are sown in peat, grow, and then examine the sprouts and selected isolates that suppress the development of pathogenic fungi. The invention allows to obtain an effective means of combating plant diseases. 10 S. and 2 C.p. f-crystals, 3 ill., 20 table. the particular new microorganisms belonging to the genus Agent, and their use for combating fungal infections in plants. In addition, this invention relates to compositions, including new strains of the genus Nictria and used for this purpose. The present invention provides a method of screening test is effective organisms in the microbial strains isolated from the soil.

Crops susceptible to various diseases caused by fungi, bacteria and viruses, and can be struck by several insect pests. To combat such diseases developed methods of cultivation, chemical and biological methods. These methods are aimed at preventing the qualitative and quantitative losses of crops due to disease and destruction by pests.

As a rule, the term "biological control of plant diseases" means the destruction of pathogens of plants with other organisms, which can be called iocontrolcode agents (BKA). Products produced iocontrolcode agents, often referred to as biopesticides. Mechanisms of biological control of plant diseases can be very different, and the desired effect often dostigaeytsya metabolites, formed iocontrolcode agents, which are sometimes parasitic on the pathogen or compete with them for space and/or nutrients.

The need for development biocontrolled agents has grown even more due to the fact that many traditional chemical control agents were harmful to the environment and people. The lack of chemicals is also something that many pests quickly become immune to one or even more of the controlling means. Development of immunity to biopesticides unlikely, as the basis of their actions are the mechanisms of different types. Chemicals usually have a faster and more effective action than biopesticides. On the other hand, the effect of biopesticides is often longer than the chemicals, as they produce the effect creates a viable and reproducible microorganisms.

The most important group of biopesticides are bacterial products, which have a targeted effect on insect pests. The most common are bioinsecticide based on the bacteria Bacillus thuringiensis. Finland is biofungicide based actinomycete Streptomyces, to the CT, which prevents the spread of root rot (caused by the fungus Heterobasidion annosum) in coniferous forests, was derived from harmless forest rot fungus Phlebia gigantea.

Were comprehensively studied bacteria of the genus Pseudomonas, in particular of the species Pseudomonas fluorescens, and currently there are a large number of strains of P. fluorescens with fungicidal action. See, for example, published patent application WO 92/18613, FI 921722 and WO 90/01327, a European patent N 228457.

In the search for microorganisms that are suitable for biological control is usually done by testing a large number of microbial strains in order to identify their iocontrolcode activity or other desired properties. In the published patent discusses some methods of screening test.

In U.S. patent N 4647533 is considered a three-stage method of screening test, according to which the bacterium first isolated from soil containing many spores of pathogenic fungi Pythium. In the second stage, the selected bacteria are subjected to a screening test in the greenhouse by the germination of seeds of grain crops in soil that contains a large number of Pythium spores, while testing each type of bacteria and conduct a control test without those who have the most Mature leaves. At the third stage, the selected bacteria again qualify in the field when conducting the same tests as in the greenhouse. In addition, in the third step, select those bacteria, in the presence of which plants grow best.

In the patent Finland N 921722 describes the following method. First, in an appropriate environment cultivated mycelium of the strain of Pythium. This mycelium is covered with a layer of sterile soil in which add the studied microorganisms, and then estimate their impact on the growth of the fungus Pythium. In the second stage, the soil sample inoculant strain of Pythium causing Rhizoctonia. In the soil sow the seeds of a plant susceptible to this fungal infection, and determine the effect of the test organism on plant growth. For subsequent tests are selected such substances that have an inhibitory effect on the fungus Pythium in both of the above tests.

The object of the present invention are microorganisms of the genus Agent, in particular Agent pityrodes Montagne, which was very effective against the fungus Fusarium.

In addition, this invention relate to biofungicides composition on the basis of a microbial strain that contains casesi or fillers. Examples of such drugs are compounds that are suitable for seed, powdered or granular formulations applied to the substrates required for growth, or liquid formulations intended for tillage.

The present invention also provides a new and effective way of screening test fungal strains representing the three-step test, which includes testing in sand, peat and field soil. At each stage of the test fungal strains proved ineffective, exclude from subsequent tests. Fungal strains, showed the best results in the greenhouse, then experience in the field.

Fig. 1 - experiment to identify the response to the dose when using strain J76 in the summer of 1992, the Percentage of diseased sprouts when insulinopenia seed fungus F. culmorum.

K = processing has not been performed,

B = the etching composition "Baytan",

J76-0 = spore suspension J76, 1,2107CFU/ml

J76-1 = spore suspension J76, 1,2106CFU/ml

J76-2 = spore suspension J76, 1,2105CFU/ml

J76-3 = spore suspension J76, 1,4104CFU/ml

Fig. 2 - experiment to identify the response to the dose when using strain J76 in the summer of 1992, the Percentage of pores is, 3a - 3d histogram representing the effects of fungal isolates rejected at various stages of tests conducted in the greenhouse, on the state of the sprouts in the field.

K = processing has not been performed,

B = the etching composition "Baytan 1",

Detailed description of the invention.

The following is a detailed description of fungal strains of the present invention. Provides data about their selection, detail strains and indicates their effectiveness in the field. Here are ways to get biofungicide compositions of these strains, characteristics of the compositions and test the effectiveness of these compounds. In addition describes a method of screening test fungal strains isolated from soil samples.

Selection of microorganisms.

Soil samples, which were isolated fungal strains of the present invention, were collected in 1989, 1990 and 1991, Their total number was 190. Soil samples of different types and from different crop rotations rotation were taken in different parts of Finland, mainly with research stations, Agricultural research center. Samples were taken from Korn is 1 to 2 liters

The selection of the microorganisms produced according to the method of dilution (selection from the soil) or by the method of cultivating plants (selection from the roots). The results obtained are described in detail below in the section "Methods".

Characterization of microorganisms

Fungal strains isolated from soil samples was tested using the method of screening test of the present invention, which is described below in the Methods section. We found five strains, which allowed us to obtain very good results. Strains, named J76, J1431, J1432, MOS1 and ROS2, were sent for analysis to the Central management of agricultural crops, Baarn, the Netherlands. In accordance with the Budapest Treaty, these strains were included in the data Bank of the DSM (Deutsch Sammlung von Microorganismes und Zellkulturen GmbH) under the numbers DSM 7522 (15 March 1993), DSM 8805 (10 December 1993), DSM 8806 (10 December 1993), DSM 8807 (10 December 1993) and DSM 8808 (10 December 1993).

These microorganisms have the following morphological characteristics.

Morphology: sparkadia without boundary sterile hyphae. Periodic branch conidiophore if there are multiple branches at each node. End branches contain whorls of failed formed in the article what does mucous balls ellipsoidal or drop shaped with short hilus, 7-8x4 µm, smooth, without appendages.

The character development of the colony: the colony on agar oat flakes increased in diameter up to 40 cm within 7 days at a temperature of 22oC; Gielen mycelium, spore-forming areas are covered green, concentric rings sparkadia. The smell is not present, the colony bleached, bleed and have exudate limited light.

Structure conidiophore strain J76 similar to the structure of Myrothecium verrucaria, but differs from this species by the formation of negrinho sparkadia, the presence of conidia in dry chains and without the typical fan-shaped ridge.

Representatives of the genus Gliocladium differ from new strains of the fact that for the genus Gliocladium typical education individual cystopathy conidiophores.

Thus, strains J76, J1431, J1432, MOS1 and ROS2 were identified as species of Agent pityrodes Montagne.

Of these strains is possible to obtain stable formulations that are easy to apply to the affected crops. The composition can be obtained in powder form. Cultivation of microbes can be started with inoculum, which is a containing spores of the pellets (pellet) potato agar with glucose, maintained at a temperature of -80oC. Microbes can chazot, and small quantities of other nutrients. For cultivation of microbes can be used non-specific nutrient, such as corn extract, or a specific nutrient, such as SDS (glucose 4 g/l, yeast extract 4 g/l, malt extract 10 g/l) or potato dextrose broth. A pH of approximately 6.0 and regulated in the beginning prior to sterilization. Cultivation is done by shaking for one to two weeks. Biomass can be separated from the broth by filtration of the casings filter paper or centrifugation. If cultivation is produced in a liquid medium, the biomass is mixed into the filler, for example silica, powdered milk and/or carboxymethyl cellulose (CMC). This mass is dried at room temperature and pulverized into powder.

Methods

The selection of microbes.

a). Method dilution (selection from the soil).

10 g of soil was mixed with 100 ml of 0.5% aqueous solution of agar (baktagir). From this mixture was selected three 10 ml samples from each of them were prepared by two dilutions (10-1and 10-2) in 0.5% aqueous solution of agar. The diluted sample was dissolved (in water bath) in a shaker for 20 to 30 minutes 1 ml of diluted what was inkubirovali at room temperature for 3-7 days then in pure culture on potato agar with glucose was administered individual fungi.

b). Method of cultivating plants (selection from the roots).

15 pots in the form of a matrix with a consistent layout (matrix with successive 50 ml pots Vefi-VP) were filled with soil of the same sample. In one pot were sown four seed wheat, barley, or approximately 15 seeds turnip, and for each plant species used five pots. Seeds were covered with sand, watered and incubated in the chamber for cultivation at a temperature of +15oC with a 14-hour light period. After two weeks of cultivation the plants were taken out and washed in running water or cleaned with a dry brush. From the roots cut off small pieces and put on a flat Cup. Used media: agar from bull bile of Litman (10 g peptone, 10 g dextrose, 15 g bullish baccarelli, 0.01 g B-crystal violet dye, 0.03 g of streptomycin, 20 g of agar and 1000 ml of water), PCNB (15 g peptone, 1 g KH2PO4, 0.5 g MgSO47H2O, 2 g of Avicola, 3 parts per million of streptomycin, 20 g of agar and 1000 ml of water), potato-dextrose agar with streptomycin (39 mg potato agar with dextrose, 300 hours per million to the Kie Cup potato agar with dextrose. The strains were kept on the pellets potato agar with dextrose in vials (Nalgene 5000-0012, sterile pyrophosphate 1.2 ml) at a temperature of -80oC.

Testing in the greenhouse according to the method of the screening test

The test in the sand.

Seeds of grain crops were sown in the sand layer. On these seeds and substrate necessary for growth, with a pipette and put first the mycelium and spores of the pathogen in water suspension, and then the spores of the test fungus, after which the seeds were covered with sand. After two and a half weeks was checking the intensity of symptoms of the disease. Subsequent trials were excluded fungi, which did not affect the intensity of the disease, or they found themselves pathogenic.

Test in peat.

The seeds of cereal soaked in a suspension of spores of the pathogen and dried. After that, seeds were dipped in a suspension of spores of the test fungus, dried and sown. As a substrate for growth, used steamed peat. Casings two and a half weeks of cultivation checked the status of sprouts. Strains of fungi, which hampered the emergence of the disease, were selected for testing in field soil.

Tested in field soil.

nowych cultures. Before sowing, seeds were treated the same way when tested in peat, and used 3 replicate pot for each treatment. The symptoms of the disease germs tested casings four weeks of cultivation.

The strains that showed good results in these tests were selected for field trials, which made it possible to definitively determine biopesticidal efficiency of microbial isolates.

The pathogenicity of fungal strains.

Experimentally examined the possible harmful effects of the fungal strain J76. The results showed that this fungus does not pathogenic to plants. The tests were carried out in respect of the 33 species of plants.

The experimental part.

The following experiments illustrate possible applications of the present invention. In section (A) discusses the use of suspensions then Agent pityrodes strains J76, J1431, J1432, MOS1 and ROS2 according to this invention for combating plant diseases, caused mainly by species of Fusarium spp. In section (B) describes the method of preparation and use of compositions on the basis of these strains. In section (C) deals with the experiments, which investigated the effect of the present invention.

(A) the Use of fungal strains in the form of suspensions of spores

Experiments with a spore suspension of the strain J76.

In the summer of 1992 at three test sites were examined the impact of a suspension of spores of the strain J76. In these trials used the seeds of two different grain crops: wheat, artificial inoculated with Fusarium culmorum, and seeds of barley infected Finivale natural way.

The test results for wheat are shown in table. 1. Yields artificially inoculated wheat are given in table. 2.

The test results sprouts of barley are presented in table. 3. This disease had no statistically significant impact on the yield of barley.

Test for detection of a reaction to a dose of strain J76, conducted in the summer of 1992

Because the strain J76 showed really good results during the experiments conducted in the early summer of 1992, examined the impact of this strain, depending on the dose of grain sown in early summer. Used as a healthy wheat seeds, and seeds that were artificially inoculated with Fusarium culmorum.

This experiment was carried out on small plots with an area of 2 m2. Nerazvitija disease took over four equal period of time. Samples for each treatment were taken from the four plots, seeding, which was made separately for each time of sampling. The number of sprouts are shown in table. 4, and the percentage of affected shoots are given in table. 5 separately for affected and healthy seeds. The data presented in the table. 5, graphically shown in Fig. 1 and 2.

Field experiments with suspensions of spores J76, conducted in the summer of 1993

These experiments were conducted on research stations Jokioinen, Metionin and Alkane. In the experiments used samples of seeds of six types:

wheat Luja" infected by the fungus F. culmorum in a natural way;

wheat Luja" artificially inoculated with the fungus F. cilmorum;

wheat Luja", uninfected;

wheat "Laari", uninfected;

barley "Kustaa, infected with various fungi Fusarium and Bipolaris sorokiniana in a natural way;

oats "Yty" infected by the fungus F. avenaccum natural way.

Healthy seeds were sown only in Jokioinen; for the other four samples of seeds of the experiments were conducted in each of the three test areas. For conducting experiments plots 10 m2planted six duplicate samples intended for each breemen;

B = chemically treated seeds, etched "Baytan 1";

J76S = spore suspension J76 of culture on flat cups (8,4109CFU/kg of seeds).

In table. 6 shows the intensity of disease separately for each treatment and samples of seeds. The yields are summarized in table. 7.

The test strain J76 against impact on hard smut of wheat and root collar rot of barley.

In the summer of 1993, the strain J76 was included in the field experiments, in which ten of chemical fungicides used for seed treatment of cereals, experienced in relation to the impact on a solid smut of wheat (caused by the fungus Tilletia caries). Strain J76 used in the form of a suspension of conidia and chemicals in accordance with instructions for use. The experiments were carried out on plots with area of 0.1 m2when five-time duplication (table. 8).

The "He" of the active ingredient was triadimenol. The composition of "Baytan 1" was a mixture that included triadimenol, imazalil. The composition of Tisato S" contained carboxyl, imazalil. The active ingredient in the composition "Panontin" was Kazatin.

During the field experiments to test the chemist is the seed spores J76. The experiments were carried out on plots measuring 10 m2with four-fold redundancy. There were no differences in the emergence of seedlings under different treatments. Strain J76 allowed to largely eliminate the symptoms of the disease (table. 9), although this pathogen is very different from the fungi Fusarium, against which he was selected.

(B) Products obtained from fungal strains, the efficiency of their application in field experiments

Powder compositions of the fungal strain J76 were obtained as follows.

Part 1

The cultivation was conducted in Erlenmeyer flask with a capacity of 1 l, which was placed 0.5 liters of a nutrient medium containing 4 g/l sucrose, 4 g/l yeast extract and 10 g/l malt extract. The pH is brought to 6.0 before sterilization in an autoclave. As inoculum used pellets agar with disputes, which had been stored at a temperature of -80oC (potato dextrose agar). The rotation speed of the shaker was equal to 150 rpm, the culture was grown at room temperature (22oC) and the period of cultivation ranged from 7 to 12 days. The cells were separated by filtration through filter paper. Biomass (cell) mixed SiO2, The BR>
Milk powder - 15

Carboxymethylcelluose (7% aqueous solution) - 10

This mixture over the 2 days was dried in open Petri dishes at room temperature in a sterile atmosphere. The layer thickness was 1-2 see the Dried mixture was crushed into powder. The viability of the preparation was equal to 107CFU/g .

SOME (colonialera unit) is a unit, which is used to determine the viability of microbes.

A diluted suspension of microbes were placed on a flat Cup of agar and after a few days counted colonies. If you know the level of dilution, it is possible to calculate the number of colonies or the number of microbial cells in the original sample.

Part 2

Cells were cultured in the same way as when receiving structure 1, after which the biomass was mixed SiO2powder , milk powder, carboxymethyl cellulose and ascorbic acid in the following quantities,%:

Biomass - 60

SiO2- 20

Dry milk - 14

Carboxymethylcellulose (7%) - 3

Ascorbic acid - 3

This mixture was dried in the same way as when receiving structure 1, and crushed into powder. The viability of this drug was 107CFU/g

Part 3

Cells is small in the following quantities,%:

Biomass - 20

Sucrose - 25

Starch - 55

This mixture was dried in the same way as when receiving structure 1, and crushed into powder. The viability of this drug was 107CFU/g

Part 4

Strain J76 cultured in solid nutrient medium with filler made of SiO2. As the nutrient broth used 8% malt extract (Maltex MP10, Lahden Polttimo). 120 g of nutrient broth was mixed in a beaker with 50 g of powdered silica gel and 20 min were placed in an autoclave at a temperature of 120oC. Chilled environment was inoculable 10 g of a suspension of spores J76, which was obtained by scraping the dispute with a Cup of potato agar with glucose in sterile water. The medium was incubated for 20 days at a temperature of 16oC, after which it 2 days was dried at room temperature. The viability of the dry preparation was 107CFU/g

Other strains of the present invention can be obtained similarly.

The efficacy of powdered formulations in field experiences.

The following experiments were conducted by the Institute of plant protection in the Agricultural research centre of Finland is MD research center in Jokioinen, Mietoinen and Alkane. When conducting these experiments used samples of seeds of six species:

wheat Luja" infected by the fungus F. culmorum in a natural way;

wheat Luja" artificially inoculated with the fungus F. culmorum;

wheat Luja", uninfected;

wheat "Laari", uninfected;

barley "Kustaa", infected with various fungi and Fusarium fungus Bipolaris sorokiniana in a natural way;

oats "Yty" infected by the fungus F. avenaceum in a natural way.

Samples of healthy wheat was sown only in Jokioinen, and experiments with four other samples of seeds were carried out on all three test sites. During the experiments were seeded plots measuring 10 m2with six duplicate for each treatment.

All samples of seeds were subjected to the same processing:

K = untreated control seeds;

B = chemically treated control seeds, etched "Baytan 1";

J76PK = powdery composition J76 used as a dry Etchant (8,4108CFU/kg = the highest amount of absorbed seeds);

J76PN = powdery composition J76 used in the form of a liquid Etchant (8,4108CFU/kg).

In table. 10 shows the values of the intensity of disease th, in 1993, conducted testing the efficacy of compounds derived from strain J76, on different fungi. The results of these experiments are given in table. 12 - 18.

The results of experiments to identify the impact of the five strains Agent pityrodes of the present invention (J76, J1431, MOS1 and ROS2) on the fungus Fusarium culmorum affecting wheat are shown in table. 18. The results are given as average values obtained in the two experiments, one of which as a substrate used peat, and the other field soil.

(C) the Nature of the impact of strain J76

A preliminary study of the impact of strain J76 as an antagonist of other fungi was made using a microscope and laboratory tests.

Observations under the microscope allowed us to establish that the hyphae J76 interact with the fungus F. culmorum and the first distinct reactions occur very quickly. In places of contact of the mycelium cells of the hyphae of the fungus Fusarium begin to decompose after about an hour after the interaction. First, the cell walls lose their shape, then disappears contents of the cells and, finally, fully decompose the cell walls. The decomposition of the hyphae of the fungus Fusariu the STU disappear. Under the influence of strain J76 decompose also the spores of this fungus (as conidia and chlamydospores), but slower than hyphae. Usually hyphae of strain J76 loosely envelop the spores of the fungus Fusarium up to their decomposition. Penetration strain J76 in hyphae of the fungus Fusarium was not observed.

On the basis of microscopic observations it was concluded that the strain J76 likely emit into the environment of biologically active substances. By their nature they can be like enzymes or antibiotics. The effect of strain J76 may be associated with development of such compounds, as it was not detected signs of parasitism on other fungi, and due to slow growth, it could not effectively compete for nutrients. When performing tests on the cellophane were also made assumptions about the allocation of this fungus metabolites acting on the growth of other fungi.

When growing strain J76 and F. culmorum in close proximity to each other on a very thin substrate is formed a zone of inhibition, which stops the growth of the fungus Fusarium. On the substrate normal thickness, this effect has not been observed. This is probably due to diffusion into the substrate of emitted substances in small chibka F. culmorum.

Test cellophane: on a substrate, which is necessary for growth, put a plastic film on which the cultivated strain J76. After culturing for 10 days film together with the strain J76 removed. Components isolated strain J76 and passed through the film remained in the substrate. As control samples used cups, which placed the cellophane film without strain J76. The test results on the cellophane is given in table. 19.

(D) the Execution of a method of screening test and evaluation of the obtained results

Tests in sand.

The substrate for planting.

As a substrate for seeding used the sand with a grain size of 0.2-0.7 mm (Kauniston Sora Oy, Loimaa). The sand was moistened by mixing 4 hours sand with 1 tsp water. From the matrix of consecutive pots (Vefi-VP 96) cut plate size 5x7 pots (capacity 50 ml), which was placed in a plastic box. Pots filled with moist sand so that it does not reach the upper edge of 1-1,5 cm In each pot were sown on three grain spring wheat "Luja".

Processing

Infection by the fungus Fusarium culmorum. The fungus F. culmorum were grown on plates of potato agar with dextrose at room temperature for 1 IU is using a homogenizer Ultra-Turrax". The number of spores brought up to 106spores/ml of This solution was suspended in 30 ml servings in bags "Mini-DOF" -20oC. For testing the frozen solution was thawed and re-mixed. This solution was used as is (basic solution) and dilute to 10-2. Pathogenicity of Fusarium strains were maintained by serial infection of plants (wheat seeds were inoculable suspension of the fungus Fusarium, after which the pathogen was again isolated from diseased sprouts).

Suspension of the antagonist. The pellets potato agar with dextrose containing antagonist, was taken from the freezer, was divided into three parts and was placed on three cups of potato agar with dextrose. The cups were incubated at room temperature (in the dark) for three weeks. The main solution of suspension of the antagonist was prepared by scraping the contents of the two cups with the antagonist in 50 ml of distilled water. The resulting mixture was stirred using a homogenizer Ultra-Turrax". From the basic solution was prepared by two solution is diluted to 10-1and 10-3.

Treatment of seeds. On sown seeds in pots using a pipette was applied first, 1 ml of suspension of F. culmorum, and then 1 ml susp the ability of spores (two breeding suspension of F. culmorum x three breeding suspension antagonist).

The control treatment. For testing of the same fungus used five pots, which additionally were planted 15 seeds and processed only the main solution of the test fungus.

In tests on sand simultaneously tested from 15 to 30 fungal strains. On the first day of testing also produced sowing in pots, which were used to check the status of the seeds and pathogenicity inoculum of F. culmorum. Control seeds sown in a separate matrix pots were subjected to three treatments, which included: without inoculation (only water), inoculation main solution F. culmorum and inoculation basic diluted solution of F. culmorum (10-2). Each of these three treatments were made in relation to 30 seeds planted in 10 pots.

Growth conditions.

After applying with a pipette and fungal suspensions seeds were covered with moist sand. The crates were wrapped in transparent plastic and transferred into the chamber to grow (temperature 10-15oC, 14-hour light period).

Monitoring.

After growing seedlings for 16-18 days, they are thoroughly washed in running tap water and examined the symptoms of the disease. Sprouts have kartki with distinct symptoms and non-seeds, which were soft, were considered sick.

The first investigated plants exposed to the control treatment. If from seeds treated with water, developed only healthy plants, and seeds treated with pathogen was observed distinct symptoms of the disease, the test results are considered valid and the check was made of plants treated with fungal strains.

When tested in the sand the score assigned to each selected fungal strain was determined based on the number of treated plants that were sick. If germination of plants treated with the test fungus was significantly less than in control healthy plants, or were discovered by the symptoms of another disease, the fungus was considered unsuitable for further research. If the lesion was absent, each of the six combinations of the density of spores of the pathogen and spores of the test fungus was evaluated separately according to the following scale:

0 = all 15 plants healthy

1 = no more than 2 infected plants

2 = 3-5 infected plants

3 = 6-9 infected plants

4 = 10-13 infected plants

5 = not more than one healthy plants

On the basis of six above the s in the peat. For subsequent tests were also selected strains, which are not less than three times received a score of 0 and 1. If the fungus has not received a single score of 0 was selected for further studies, if he received no less than four times - a score of 1.

Tests in peat

The substrate for growing.

As a substrate for growing used steamed, fertilized and izvestkovyy peat. Until the fall of 1992, he applied the whole of untreated peat from Torronsuo, and then sifted untreated peat from Aragaki. (Fertilization: 800 g dolomitic lime and 100 g fertilizer Y-lannos /100 l of peat (Y-lannos = trademark of Finnish universal fertilizer). Moist peat was placed in plastic boxes (28,h,h,4 see the box "Hammut" company Weibulls Robusta, Muoviyhtyma Oy) in a layer thickness of 5 cm At the bottom of the box put plastic wrap.

Processing.

T = Healthy, pinacolborane seeds of spring wheat "Luja". Irrigation was made with distilled water.

F = Seeds inoculated with the fungus F. culmorum. Seeds were soaked in a basic solution of F. culmorum (solution used in excessive quantities), containing about 106spores/ml (cultivation of the fungus Fusarium, compare with the test in section the Finance fungus F. culmorum was produced in the same way, as in the case of processing F. When the seeds were dried, they were moistened the main solution antagonist. The basic solution was obtained by scraping the mycelium and spores from one Cup antagonist in 25 ml of distilled water. The processing was made by shaking the seeds and mortar antagonist in a small plastic bottle. After treatment the seeds were dried on paper.

F2 = Insulinopenia fungus F. culmorum was produced in the same way as in the case of treatment f Treatment antagonist was performed in the same way as in the case of the processing of F0, using the main solution antagonist, diluted to 10-2.

In peat did 10 rows, each of which were sown on 30 seeds. Sowing was made in the following sequence: protective number, F, F0, F2, T, F, F0, F2, T, protective number. After sowing, the seeds were covered with peat, which was moistened.

Growth conditions.

The seeds were grown in the greenhouse at a temperature of approximately 15oC. In the dark time of the year using a lamp with multiple filaments created additional lighting for 12 hours/day. If necessary, the seeds were moistened with water. The cultivation period was 18 days.

Sort.

Rodnoverie observations, obtained after treatments T and F, were decided on the reliability of the test. In healthy control plants (T) is not allowed more than 12 infected plants (60 sown seeds), and in the control group (F), infected with a pathogen, should be not less than 52 diseased plants (60 seeds). The tested fungal strain was considered acceptable for the next stage of trials (experiments in field soil), if from seeds (60 seeds), solution treated with one of two concentrations, developed no more than 19 of the diseased plants.

Tested in field soil

The substrate for planting.

Used soil (sandy clay) were taken from experimental fields in Jokioinen. The soil was crushed by hand or in winter 1992-1993 gg) was sifted through a screen with holes of size 1x1 see Plastic pots with a capacity of 1.5 liters (diameter 14 cm) filled with soil so that the top edge of the blank to a height of 3-4 cm At the bottom of the pot put the filter paper.

Processing.

1. Healthy seeds. Moistened only with distilled water.

2. The control group treated with the fungus Fusarium. Seeds were moistened with inoculants Fusarium (a drug used in the tests in the sand) containing 106the dispute is to the control group, processed by the fungus Fusarium. After drying, seeds were treated with suspension of the antagonist, which was prepared by mixing mycelium and spores from the same Cup with 25 ml of distilled water. The processing was made in the plastic bottle, which was placed 130 seeds (120-150 seeds) and 1 ml (1.5 ml) suspension of the antagonist.

4. The control group, treated with a fungicide. Insulinopenia produced as well as in the control group, treated with Fusarium. After drying, the seeds were treated with 2 g of etching powder "Baytan 1" for one kg of seeds. In addition, produced previously used processing compositions "Ceresan and Tisato S".

In pots sown on 36 treated seeds, three duplicate for each treatment. The seeds were covered with field soil. The growth conditions were the same as when tested in the peat. The cultivation period was four weeks.

Checking and sorting.

The shoots were washed and assessed their symptoms:

0 = completely healthy plants,

1 = slight fungus Fusarium;

2 = medium to strong disease;

3 = sprouts completely poorely - plant death.

Efficiency tests in Teplitskaya invention. This trial tested whether were selected for further testing fungal strains isolated during a series of experiments conducted between October 1991 and February 1993, For seed treatment were randomly selected 60 strains from those that were rejected when tested in the sand, but had no pathogenic effects on wheat. Of strains defective during testing in peat, took 92 strain. Were selected all 58 fungi used during tests in soil. 43 fungus were rejected during this test and 15 were selected for field experiments.

In addition to the above 210 treatments test included 6 control treatments: untreated seeds (K), protravlena "Baytan 1" (B) and four fungal strains investigated in previous tests, including J76.

This test used the wheat "Luja" infected by the fungus F. culmorum in a natural way. Observations of near 1.4 m long, which was sown 5.5 g of seeds. For all 216 treatments were sown six duplicate rows. Randomization was achieved with the help of the cubic lattice experimental design. Thus, reduced erroneous variations associated with pocva disease.

The test results illustrate four histogram shown in Fig. 3a - 3d. Between isolates defective during testing in peat and non-pathogenic isolates defective during testing in the sand, there was no difference. Test in peat gave very good results. The strains selected after him for further tests were, on average, much better than rejected during the test fungi.

During tests in sand and peat was wrongly rejected only a few fungi. On the other hand, when tested in soil rejected a sufficiently large number of very good isolates, but from those isolates that were selected for the field experiments, only two of the 15 showed negative results in vivo.

On the basis of the obtained results we can conclude that the test in sand and peat can reliably select the best antagonists for further research, but during testing in field soil can be rejected even good antagonists.

Qualifying test fungal strain J1431

Of fungal strains of the present invention, the strain J1431 investigated in all three qualifying sobran for subsequent tests.

When tested in peat using strain J1431 were obtained the following results:

T: 3 infected plants

F: 36 infected plants

F0: 3 infected plants

F2: 7 infected plants

During tests in field soil using the strain J1431 when performing different treatments were obtained the following values of the affected sprouts,%:

Healthy plants - 66

The control group treated with the fungus Fusarium - 87

Protravlena "Baytan 1" - 31

J1431 - 19

On the basis of selection test conducted in the summer of 1993, the strain J1431 was selected for field experiments along with 61 other fungal strain. This test used the spring wheat "Luja" artificially inoculated with spore suspension of the fungus F. culmorum. Seeds were sown in single row plots (1.4 m) at six duplicate this experience. After 34 days after planting the sprouts were dug from the soil, washed and identified the symptoms of the disease. When different treatments were received the following number of healthy shoots per meter row:

The control group treated with the fungus Fusarium - 5,9

Protravlena "Baytan 1" - 56

J76 - 61

J1431 - 65

Other tested strains 30-38

Etbonoc what's in the greenhouse.

When tested in the sand strain J1432 scored 0, 0, 0, 1, 1, and 2 and was chosen for further testing.

When tested in peat using strain J1432 were obtained the following results:

T: 10 infected plants

F: 60 diseased plants

F0: 10 infected plants

F2: 37 diseased plants

During the test in field soil using the strain J1432 when different treatments were obtained the following values of the affected sprouts,%:

Healthy plants - 56

The control group treated with the fungus Fusarium - 98

Protravlena "Baytan 1" - 18

J1432 - 38

Based on the results of tests in field soil strain J1432 was removed from the field experiments.

Microorganisms introduced into the database

On the basis of the Budapest Treaty, the following microorganisms (see table. 20) were included in the data Bank at the address of the DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Maseheroder Weg 1b, D-38124 Braunschweig, Germany.

1. The strain of the fungus Agent pityrodes Montagne DSM 7522 used as biofungicide.

2. The strain of the fungus Agent pityrodes Montagne DSM 8805 used as biofungicide.

3. The strain of the fungus Agent pityrodes Montagne DSM 8806 used as biofungicide.

6. Biofungicide containing the microorganism, characterized in that it contains fungal strain selected from the group including fungal strains Agent pityrodes Montagne DSM 7522, or DSM 8805, or DSM 8806, or DSM 8807, or DSM 8808.

7. Biofungicide under item 6, characterized in that it further comprises fillers and/or additives customary in this field.

8. Biofungicide under item 7, characterized in that the fillers and additives selected from the group including silicon dioxide, dry milk, carboxymethylcellulose, sucrose, ascorbic acid and starch.

9. The method of producing biofungicide under item 7 or 8, characterized in that it includes the cultivation of the fungal strain according to any one of paragraphs.1-5 in acceptable growth environment, Department of cell mass, adding to her fillers and/or additives, drying and grinding the mass to powder.

10. The method of producing biofungicide under item 7 or 8, characterized in that it includes the cultivation of the fungal strain according to any one of paragraphs.1-5 in an acceptable environment together with silicon dioxide and adding, if desired, fillers and/or additives, drying and grinding the mass to powder.

11. The method of suppressing fungal the Oia biofungicide, characterized in that use biofungicide on PP.6-8.

12. Method of screening fungal microorganisms in samples of microbes, comprising the following steps: (a) the test in sand, under which the seeds of grain crops are sown in the sand, seed and substrate pipette put the mycelium and spores of the pathogen, and then the spores of the test fungus in an aqueous suspension, after which the seeds are covered with sand, after a certain period of time check the intensity of the symptoms sprouts and rejected fungi, which did not affect the development of the disease or themselves pathogenic, and (b) testing of selected fungi in peat, in accordance with which cereals seeds are soaked in a suspension of spores of the pathogen and dried, and then moisten with spore suspension of the test fungus, dried and sown in steamed peat grow within a certain period of time, to monitor the state of sprouts and selected for subsequent testing of the isolates, clearly suppressing disease development.

 

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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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