RU2724464C1 - Strains, biopreparation, biopreparation production method and method for biological protection of crops against fusariosis - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: group of inventions relates to biotechnology. There are presented strains of spore-forming antagonist bacteria with antifusary activity (versions), a biopreparation for protection of agricultural crops against fusariosis, a method for making a biopreparation and a method of protecting agricultural crops from fusariosis. Biopreparation includes a combination of the presented strains, wherein it contains not less than 10CFU/g of viable antagonist bacteria and has residual moisture content of not more than 10 %. Method for preparing the biopreparation involves solid-phase cultivation of the presented strains on a nutrient substrate. Method of protecting crops includes isolating fungi of the genus Fusarium from samples collected on fields, selecting the strains of spore-forming bacteria-antagonists most actively suppressing strains of fungi of the genus Fusarium, preparing the biopreparation and treating the biopreparation with sowing material. Invention provides expansion of arsenal of strains of spore-forming bacteria-antagonists with antifusary activity and biopreparations aimed at effective fight against fusariosis, as well as provide during manufacture of biopreparation possibility of using a medium with a simple composition with high output of the finished product and stability of the obtained biopreparation during storage.EFFECT: what is presented are strains, a biopreparation, a biopreparation production method and a method for biological protection of crops against fusariosis.13 cl, 28 dwg, 30 tbl

Description

FIELD OF TECHNOLOGY

The present group of inventions relates to biological methods of combating phytopathogenic diseases. In particular, the invention relates to strains, biological products, methods for their preparation and methods that can be used in the fight against fusariosis of crops.

BACKGROUND

Constant population growth causes a number of different social and economic problems. In particular, one of such problems is considered to be providing the population with high-quality agricultural products. At the same time, one of the main agricultural crops cultivated in the Russian Federation and in the Southern Federal District (SFD), in particular, is winter wheat.

Among the urgent problems in the agricultural sector, fungal diseases occupy a prominent place, the main one in the Southern Federal District of which is fusarium spike of winter wheat. The disease is observed in most regions where wheat is grown and causes losses of 25-30% of the crop [Gagkaeva T.Yu., Hannibal FB, Gavrilova O.P. Infection of wheat grain with Fusarium and Alternaria mushrooms in southern Russia in 2010 // Protection and Plant Quarantine. 2012. No. 1 .; Gagkaeva T.Yu., Gavrilova O. P. Contamination of grain with Fusarium fungi in the Krasnodar and Stavropol Territories // Plant Protection and Quarantine. 2014. No. 3].

The causative agents are fungi of the genus Fusarium, which are located in various parts of the plant, both in the spikelet stem and scales, and in the seeds, especially in the germinal part of the seeds.

At the moment, the infection of fungi of the Fusarium genus with crops has acquired the character of a pandemic and, under favorable conditions, develops in most cases and poses a serious danger to public health.

Fungi of this genus are particularly dangerous because of the production of mycotoxins of the trichothecin series, which accumulate in the seeds of plants and pose a threat to both humans when eating and in agriculture when feeding livestock [Gagkaeva T.Yu., Gavrilova O.P. ., Levitin M.M. Biodiversity and habitats of the main toxin-producing fungi of the genus Fusarium // Biosphere. 2014. No. 1.].

According to the chemical composition, this group of mycotoxins is divided into 2 groups: A (T-2 and NT-2 toxins, diacetoxystsirpinol and their derivatives); B (deoxynivalenol (DON), nivalenol (NIV) and their derivatives.

There are various ways to protect against phytopathogenic fungi, while the most commonly used of them is the use of fungicides. However, the effectiveness of the use of such substances is constantly decreasing. So, for example, in a number of works it was shown that the effectiveness of the use of chemicals to control phytopathogenic fungi is significantly reduced due to the gradual development of resistance to the action of various fungicidal substances. [Becker R., Hettwer U., Karlovsky P., Deising H. B. et al. Adaptation of Fusarium graminearum to tebuconazole yielded descendants diverging for levels of fitness, fungicide resistance, virulence, and mycotoxin production // Phytopath., 2010, No. 100, p. 444-453; Chekmarev V.V., Kobylskaya G.V., Buchneva G.N., Korabelskaya O.I. Resistance of fungi of the genus Fusarium to seed dressers // Protection and Plant Quarantine. 2011. No. 3]. According to other authors, it follows that the maximum effect that can be achieved with the use of fungicides is not more than 7 5%, but this can be achieved if complexes of several fungicidal preparations are used [Sanin S. S., Motovilin A. A., Korneva L. .G., Zhokhova T.P., Polyakova T.M., Akimova E.A. Chemical protection of wheat from diseases during intensive grain production // Protection and Plant Quarantine. 2011. No8; Gasich E.L., Khlopunova L.B., Gagkaeva T.Yu., Dmitriev A.P. The effect of fungicides on the development of wheat gibellinosis during artificial infection in laboratory conditions // Plant Protection and Quarantine. 2015. No. 1].

At present, the vector of research has shifted from the search for chemicals to the search and use of antagonist bacteria and their metabolites that exhibit antagonistic properties to various fungal pathogens of agricultural diseases [Oleg Sidorenko, Prospects for the use of biological preparations based on microorganisms // Proceedings of TLCA. 2012. No. 6].

One of the promising groups of microorganisms, which differ in their ability to effectively resist phytopathogens and exhibit phytostimulating properties, is a group of soil bacteria-antagonists of the Bacilli class.

Recently, bacteria of this family are considered as promising agents for the biological control of plant diseases, as well as organisms that exhibit phytostimulating activity due to their wide distribution, natural antagonism to many phytopathogenic fungi and the ability to produce substances of a hormonal nature.

Foreign experience indicates the high efficiency of the use of biological products based on bacilli for the biological control of pathogenic fungi of the genus Fusarium. In particular, their effectiveness on corn, cucumbers, and potatoes has been shown [Cavaglieri, L., Orlando, J. R. M. I., Rodriguez, M. I., Chulze, S., & Etcheverry, M. (2005). Biocontrol of Bacillus subtilis against Fusarium verticillioides in vitro and at the maize root level. Research in Microbiology, 156 (5), 748-754 .; Sadfi, N., Cherif, M., Fliss, I., Boudabbous, A., & Antoun, H. (2001). Evaluation of bacterial isolates from salty soils and Bacillus thuringiensis strains for the biocontrol of Fusarium dry rot of potato tubers. Journal of Plant Pathology, 101-117 .; Chen, F., Wang, M., Zheng, Y., Luo, J., Yang, X., & Wang, X. (2010). Quantitative changes of plant defense enzymes and phytohormone in biocontrol of cucumber Fusarium wilt by Bacillus subtilis B579. World Journal of Microbiology and Biotechnology, 26 (4), 675-684].

The mechanism of action is the colonization of plant roots by bacteria during seed germination and the suppression of pathogenic fungi due to the isolation of chitinolytic enzymes, siderophores and specific antimicrobial peptides.

However, soil bacteria are highly sensitive to local soil and climatic conditions - soil type, temperature, humidity, composition of local microbial communities. This is due to the inefficiency or poor efficiency of the use of strains isolated in certain areas outside these regions. If the introduced strains are foreign, they are quickly suppressed by the microbial community already in the soil and lose the ability to influence pathogenic fungi. In addition, a method based on the use of strains and types of antagonist bacteria that are not characteristic of these bio- and agrocenoses is environmentally unsafe because the natural balance between different groups of soil microorganisms can be upset, which in turn can lead to a decrease soil fertility and crop yields.

S.,

R., Baldrian P. Drivers of microbial community structure in forest soils // Applied microbiology and biotechnology. - 2018. - T. 102. - №. 10. - C. 4331-4338. и др.).Thus, it is advisable to isolate the strains from the soils of the regions where they are planned to be used (see, for example, Pii Y. et al. The interaction between iron nutrition, plant species and soil type shapes the rhizosphere microbiome // Plant Physiology and Biochemistry. - 2016 . - T. 99. - C. 39-48. Ma J. et al. Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations // Science of the Total Environment. -2016. - T. 563. - C. 199-209.

S.,

R., Baldrian P. Drivers of microbial community structure in forest soils // Applied microbiology and biotechnology. - 2018. - T. 102. - No. 10 .-- C. 4331-4338. and etc.).

The prior art invention is known which relates to biotechnology, microbiology and agriculture and can be used to obtain a bacterial preparation against plant diseases caused by phytopathogenic fungi of the genus Fusarium, Microdochium, Pyrenophora and Puccinia. The strain Bacillus subtilis BZR 517 has fungicidal activity against phytopathogenic bacteria and fungi. The strain of Bacillus subtilis is deposited in the Collection of the GNU VNIISCH of the Russian Academy of Agricultural Sciences under registration number RCAM01728 (RF Patent No. 2552146 C1, publ. 06/10/2015).

Also known from the prior art is an invention that relates to agriculture. The method of producing suppressive compost with respect to the causative agent of plant fusarium Fusarium oxysporum consists in the preparation of compost obtained from agricultural waste, isolation of microorganism strains from compost, preparation of a biological product on the basis of the isolated strains, introduction of a biological product in compost, and evaluation of the suppressive properties of the compost; strains according to 4 mechanisms of suppressiveness, assessment of the mutual antagonistic activity of the strains, two-time introduction of the biological product into the compost, assessment of the fertilizing properties of the suppressive compost. The invention allows to obtain composts having both fertilizing properties and stable suppressive properties in relation to the pathogen of plant fusarium Fusarium oxysporum (RF Patent No. 2629776 C1, publ. 04.09.2017).

In addition, the invention is known from the prior art that relates to compositions and methods provided for the combination of a new strain of Bacillus amyloliquefaciens RTI301 and a new strain of Bacillus subtilis. RTI477, a combination having growth promoting activity and activity against plant pathogens. Compositions containing strains RTI301 and RTI477 are useful for improving plant growth and / or providing protection against pathogenic infection when applied to plant roots, seeds, callus tissue, grafts and cuttings. Synergistic results are observed for the combination of strains, and the combination of strains is useful for increasing crop yields, including soy and corn. Compositions containing a combination of strains can be used alone or in combination with other microbial, biological or chemical insecticides, fungicides, nematicides, bactericides, herbicides, plant extracts, plant growth regulators and fertilizers. (Patent US 9622484 B2, publ. 04/18/2017)

The solutions described in the sources RU 2552146 C1, RU 2629776 C1, US 9622484 B2 are the closest solutions to the studied object, however, the developed object differs in such features as strains of antagonist bacteria, method of cultivation, form of storage of the drug, culture in relation to which is possible application, in addition, the claimed group of inventions has a number of significant advantages.

Patent RU 2478290 C2 is the closest to the studied object. It proposed a method for producing a biological product that contains Bacillus amyloliquefaciens biomass All-Russian collection of industrial microorganisms (hereinafter VKPM) B-11008 and humates in the following ratio of components, vol. %: biomass of Bacillus amyloliquefaciens VKPM B-11008 - vegetative cells and spores 1.24 ÷ 1.30 × 10 ¹⁰ CFU / ml of culture fluid and spore content 94% of the total number of CFU - 99.0, humates - 1.0. As a nutrient medium for obtaining the claimed biological product using cheap environment based on waste grain production of the following composition, g / l: wheat bran - 40.0; corn extract - 0.5; MgSO ₄ - 0.9; KH ₂ PO ₄ - 0.5; CaCO ₃ - 1.0; pH 7.0 ± 0.2. The specified composition of the medium provides a high level of biomass accumulation of B. amyloliquefaciens VKPM B-11008 (5.6 ÷ 5.9 g / l), a high titer of viable cells and spores (1.24 ÷ 1.28 × 10 ¹⁰ CFU / ml) and the intensity of spore formation (spore content - 94.0% of the total number of CFU). The strain of this species was tested on the following representatives of the genus: Fusarium: F. graminearum, F. solani, F. oxysporum, F. sambucinum. This strain showed a high level of antagonistic activity. The verification of the effect of this strain on phytopathogenic fungi remains unclear. So the following method is proposed: crops with a strain of bacteria are incubated at 28 ° C for 3 days. To the grown culture of B. amyloliquefaciens VKPM B-11008, daily suspensions of test cultures of phytopathogenic bacteria are streaked with a stroke (suspension titer 10 ⁸ CFU / ml). Then the plates are incubated at a temperature of 28 ° C for 24 hours and the results are recorded. In real conditions, these groups are in competition for the colonization of rhizoplanes, and the following method for assessing antagonistic activity is more correct: simultaneous culture of the fungus and bacteria on a dense medium. Also, the possibility of using this drug on winter wheat has not been verified. In addition, phytopathogenic fungi in this method are not isolated during work, and already known cultures of phytopathogenic fungi are taken from various collections, which can greatly affect the effectiveness of this drug in various regions and countries.

SUMMARY OF THE INVENTION

The problem solved by the claimed group of inventions is to obtain new strains of spore-forming antagonist bacteria with antifusar activity, to develop a biological product based on these strains and the method for its manufacture, as well as a method for biological protection of crops from fusarium infection.

The technical result of the claimed group of inventions is to expand the arsenal of strains of spore-forming bacteria-antagonists with antifusar activity, as well as compositions of biological products aimed at a more effective fight against fusarium. In addition, the technical result of the claimed method of manufacturing a biological product is the ease of its reproducibility, manufacturability and stability, the possibility of using a simple composition, inexpensive environment, obtaining a high yield of the finished product, the stability of the obtained biological product during storage.

The indicated technical results are achieved by isolating 10 new strains of spore-forming antagonist bacteria with antifusar activity. New strains were deposited on November 7, 2017 (national patent deposition) and November 18, 2019 (international patent deposition) at the Bioresource Center of the All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the Kurchatov Institute Research Center - State Research Institute of Genetics (address: 117545, Moscow , 1st Road passage, 1) - further VKPM.

VKPM makes deposits for the purposes of the patent procedure in the Russian collection authorized for deposits for the purposes of the patent procedure and satisfies the requirements for such collections, and also guarantees the viability of the deposited objects for at least the duration of the patent, including the International Authority for the Deposit according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms.

1. The strain Paenibacillus jamilae K 1.14 (VKPM B-13016).

2. The strain Paenibacillus peoriae O 1.27 (VKPM B-13017).

3. The strain Paenibacillus peoriae O 2.11 (VKPM B-13018).

4. The strain Paenibacillus peoriae R 3.13 (VKPM B-13019).

5. The strain Paenibacillus peoriae R 4.5 (VKPM B-13020).

6. Strain Bacillus amyloliquefaciens R 4.6 VKPM B-13021).

7. Strain Paenibacillus jamilae R 4.24 (VKPM B-13022).

8. The strain Paenibacillus polymyxa R 5.31 (VKPM B-13023).

9. Strain Paenibacillus peoriae R 6.14 (VKPM B-13048).

10. Strain Bacillus amyloliquefaciens V 3.14 (VKPM B-13024).

In addition, technical results are achieved due to the composition of the biological product and the developed method for producing a biological product containing 10 of the above strains of spore-forming antagonist bacteria with antifusar activity.

A biological product for protecting crops from Fusarium, including a combination of 10 of the above strains, containing at least 10 ⁸ CFU / g of viable antagonist bacteria, while the residual moisture of the dry biological product is not more than 10%.

A method of manufacturing a biological product based on a combination of strains of spore-forming antagonist bacteria with antifusar activity by solid-phase cultivation on a nutrient substrate, including preparing the substrate by hydration in water, heat treatment of the substrate, inoculation of the heat-treated hydrated substrate, incubation, homogenization of the substrate during the processing, softening and subsequent drying.

A method of protecting crops from Fusarium, includes the selection of Fusarium mushrooms from samples taken in fields exposed to the danger of Fusarium; selection of strains of spore-forming bacteria-antagonists, the most actively suppressing strains of fungi of the genus Fusarium, distributed in the target territory; preparation of the aforementioned biological product by the above method, processing of the seed with a biological product.

The developers produced batches of biological product weighing 10 and 50 kg and tested it under production conditions on wheat of the Thunder variety on an area of 30 hectares using the PS10 plant, as well as on corn on an area of 50 hectares. In the fall of 2018, a full-scale experiment on winter wheat of the Thunder and Tanya varieties was conducted on an area of 200 hectares. Based on the first two experiments, a series of field and laboratory analyzes were carried out for the entire growing season, and studies of winter wheat crops in the fall of 2018 were carried out before the stage of autumn tillering. According to the results of laboratory studies, a decrease in the number of phytopathogenic fungi p. Fusarium:

1) in the rhizosphere of winter wheat during the tillering phase (a decrease of 1.98 times);

2) in the grain of winter wheat (a decrease of more than 5 times);

3) in the rhizosphere of corn in the seedling phase (2.64 times);

4) in the rhizosphere of corn in the phase of milk ripeness (1.82 times);

5) in the stems of winter wheat in the seedling phase (2.28 times in irrigated fields and 1.41 times in fields without irrigation);

6) in the rhizosphere of winter wheat in the seedling phase (5 times in irrigated fields and 1.26 times in fields without irrigation);

7) in the stems of winter wheat during the tillering phase (1.83 times in irrigated fields and 2.25 times in fields without irrigation).

At the early stages of plant development, the treatment of winter wheat seeds with a biological product leads to a protective effect both in plants and in soil adjacent to the roots, which indicates colonization of the root surface by bacteria that impede the introduction of the pathogen. By the tillering phase, the percentage of plants in the tissues of which p. Fusarium is reduced compared to the seedling phase. Thus, the drug retains its protective effect to the tillering phase, which speaks in favor of the active colonization of wheat plants with biological preparations. This provides long-term active protection of plants, and upon the occurrence of adverse external conditions (wetting, snow retention in depressions), it can prevent the development of root rot.

The most powerful effect was recorded for the most valuable parameter - in grain of winter wheat obtained from plants grown from seeds treated with a biological product, a more than five-fold decrease in infection with fusariosis was observed. A significant decrease in the number of phytopathogenic fungi p. Fusarium is also noted in the rhizosphere of winter wheat in the seedling phase. The most active penetration of fungi into plants occurs during the earliest stages of wheat development - immediately after germination. The use of a biological product makes it possible to control the pathogen abundance precisely during this critical period, and the experimental results show that in the variants where the number of fungi in the rhizosphere was reduced during the seedling phase, the percentage of plants affected by fungi subsequently turns out to be significantly lower.

BRIEF DESCRIPTION OF THE DRAWINGS

The figure 1 shows the primary selection of bacteria, while the highlighted areas show the absence of growth of the fungus.

The figure 2 shows the definition of antagonism between the studied bacteria (along the edges) and the fungus p. Fusarium (in the center), where wide zones of growth inhibition are visible.

The figure 3 shows the definition of antagonism between the studied bacteria (along the edges of the glass) and the fungus p. Fusarium (center), where the fungus grows on a thin layer of agar deposited on a glass slide, but cannot approach the bacteria colonies.

The figure 4 shows the study of the ability to biofilm formation in strains of bacilli with high fungicidal activity.

The figure 5 shows the growth of wheat in the control (A) and after treatment with strains Ra 3.12 (B) and Ra 3.13 (C).

Figure 6 shows the germination of wheat seeds in the control (top) and on the lawn Ra 2.6 (bottom) on KSA medium.

The figure 7 shows the change in shoot length in plants growing from infected seeds when they are treated with a chemical disinfectant and biological product.

The figure 8 shows the growth of phytopathogenic microflora in the control (A) and its suppression by drugs (B, C, D, D).

The figure 9 shows the colony of strains from a mixture of preparations on the roots of wheat seedlings.

The figure 10 shows the results of PCR analysis of infected samples. G is the result of amplification with primers G1 (Table 7), O is the result of amplification with primers 02 (Table 8).

The figure 11 shows the number of mushrooms p. Fusarium in the rhizosphere of winter wheat in the tillering phase.

The figure 12 shows the number of mushrooms p. Fusarium in the rhizosphere of winter wheat in the ripening phase.

The figure 13 shows the number of mushrooms p. Fusarium in the grain of winter wheat.

The figure 14 shows the effect of the biological product F-1 on the infection of winter wheat grain with fungi p. Fusarium Only one of the experimental samples shows infection. (characteristic colonies of pink Fusarium graminearum are visible).

The figure 15 shows the number of mushrooms p. Fusarium in the rhizosphere of corn in the seedling phase.

The figure 16 shows the number of mushrooms p. Fusarium in the rhizosphere of corn in the phase of milk ripeness.

The figure 17 shows the verification of the effectiveness of PCR with the given amplification parameters.

The figure 18 shows the results of PCR analysis of selected samples with genus-specific primers F2.

Samples 1-8 - Analysis of 8 colonies from wheat crops

Samples 9-15 - Analysis of 7 colonies from corn crops

16 - negative control, 17 - positive control (pure culture of the fungus from the collection), 18 - 50 bp + DNA length marker (Eurogen).

The figure 19 presents the results of amplification of samples with species-specific primers for F. culmorum (A) and F.graminearum (B).

The figure 20 presents the results of amplification of samples with species-specific primers for F. oxysporum (A) and F. sporotrichoides (B).

The figure 21 shows the results of amplification of samples with species-specific primers on F. roae (A) and F. avenaceum (B).

The figure 22 shows the percentage of plant damage by phytopathogenic fungi p. Fusarium in irrigated fields (seedlings).

The figure 23 shows the percentage of plant damage by phytopathogenic fungi p. Fusarium in irrigated fields (seedlings).

The figure 24 shows the number of mushrooms p. Fusarium in rhizospheric soil in irrigated fields, CFU / g of soil.

The figure 25 shows the number of mushrooms p. Fusarium in rhizospheric soil in fields without irrigation, CFU / g of soil.

Figure 26 shows the percentage of plants affected by phytopathogenic fungi p. Fusarium in irrigated fields (autumn tillering).

The figure 27 shows the percentage of plants affected by phytopathogenic fungi p. Fusarium in fields without irrigation (autumn tillering).

The figure 28 shows the number of mushrooms p. Fusarium in rhizospheric soil during the phase of autumn tillering, CFU / g of soil.

DETAILED DESCRIPTION OF THE INVENTION

Materials for the study were obtained during two field expeditions. During the expeditions, samples of soil and plants were taken from the Volia farm of the Kanevsky district of the Kranodar Territory, as well as the farms of the Labinsky district of Rassvet (including the sections Upornaya, Kaladzhinsky, Crossing), Otradnensky, " Mostovsky "," Kurganinsky "and" Homeland ". 30 soil samples were taken from these fields, as well as plant samples (winter wheat) and crop residues, which are the reservoir of infection (corn, sugar beets).

During the development of a method for producing a biological product from these materials, 5 strains of p mushrooms were isolated. Fusarium, as well as 1040 strains of bacteria that are antagonistic to these strains. These cultures served as material for further studies of antagonistic activity and selection of the most active strains.

Isolation of cultures of phytopathogenic fungi p. Fusarium was produced by sowing samples of affected plants onto solid nutrient wort-agar and Saburo agar with streptomycin.

The cultures of antagonist bacteria were isolated by sowing previously pasteurized samples of affected plants and soil onto wort agar with preliminary sowing of p. Fusarium Only those colonies that showed visible antagonism with respect to the phytopathogen culture were selected.

Evaluation of the antagonistic activity of the selected cultures was carried out by co-cultivation under growth conditions on Petri dishes and in microculture on glasses.

Isolated phytopathogenic fungi were identified in the VKPM.

Ten selected most active strains of antagonist bacteria were identified based on DNA analysis in VKPM, and deposited in this collection.

Testing the effect of strains on plants was carried out by inoculating seeds with the culture of the test strain, followed by determination of germination, germination energy, as well as growth and development indicators of plants in the presence and absence of phytopathogenic fungi. Vegetation experiments to study the stimulating effect of antagonist bacteria were carried out in a climate chamber and greenhouse, and experiments with phytopathogens were carried out under controlled phytotron conditions.

Obtaining strains of microorganisms.

Isolation and selection of phytopathogenic fungi and antagonist bacteria exhibiting antagonistic effects to them.

Mushrooms of the genus Fusarium were isolated from the following fields: Farm "Will" - mushroom Fus 1, Kalajinskaya (field No. 67, 39 Ha), a mushroom with an index of Ras 6.1; Persistent (field No. 154, 43 Hectares), a mushroom with an index of Ras 6.2.1; Persistent (field No. 154, 43 Hectares), a mushroom with an index of Ras 6.2.2; 154, 43 Ha), a mushroom with an index of Ras 6.2.2; Otradnenskoe (field No. 68, 68 ha) mushroom with the index OTR 1, Crossing (field No. 6, 117 ha) mushroom Fus 5.

The primary screening aimed at isolating antagonistic strains was the culture of the fungus Fusarium graminearum (Fus1).

The selection of antagonist strains was carried out according to the following scheme: 10 g of soil was ground in a porcelain mortar with a rubber pestle, then 100 ml of sterile water was added.

To isolate antagonist strains belonging to the Bacilli class from soil samples, pasteurization of this soil suspension was carried out for 20 minutes at a temperature of 90 ° C. In this case, only spores of antagonist bacteria of this class remained.

A fungal culture was planted on the dense nutrient medium of Wort agar with a lawn, in relation to which strains of antagonist bacteria were selected. The soil suspension obtained (in a volume of 50 μl) was applied on top of the fungus culture and ground using a Drigalski spatula until the medium surface completely dried. Incubated for 3 days at a temperature of 25 ° C.

The selection was made according to the principle there is antagonism / no antagonism. All bacteria were selected that showed a zone of inhibition of fungal growth around their colonies (Fig. 1). All strains were assigned names based on the soil sample from which the strain was isolated.

The further stage of the work was to determine the severity of 1040 strains isolated at the first stage in relation to the other 4 species of the genus Fusarium.

The biomass of the studied fungus was crocheted in the center of the cup with a dense nutrient medium of Wort agar, and the studied bacteria were placed along the edges of the cup-loop. Incubated for 7 days at a temperature of 25 ° C. After incubation, the size of the growth suppression zone between the fungus and the bacilli was measured (Fig. 2).

As a result of this stage of work, it was revealed that out of 1040 initially isolated strains, 212 strains showed the greatest degree of antagonism. In assessing the degree of growth inhibition of all 5 fungal strains, out of 212 strains, 37 strains with the highest antagonistic activity were obtained. The degree of antagonistic activity of these strains.

Studies were also carried out using the method of microculture on glasses (Fig. 3), confirming the high antagonistic activity of the selected cultures.

Selection of strains most promising for biotechnological use.

After isolating 37 of the most active cultures, they were examined for the presence of properties that allow these strains to be used to develop a method for producing a complex antifusarious biological product.

These properties included:

A) The ability of the strains to form stable biofilms, which is important for the efficiency of colonization of the roots of wheat seedlings, and is also important for the technological process of solid-phase fermentation during production of the formulation.

B) The ability of cultures to grow at elevated temperatures, which allows to suppress the development of extraneous antagonist bacteria during production of the formulation.

Biofilm formation is one of the most important genetically determined processes in the life of antagonist bacteria. In the course of many human diseases involved bacteria that are in the state of biofilms. Thanks to the “sense of quorum” in this structure, they solve vital tasks for their survival in harsh environmental conditions. Horizontal gene transfer is carried out, antibiotic resistance is increased, and cooperation with other species occurs. All this helps them in a constant tough fight, both in the human body and in the soil, on the roots of plants. The search for active biofilm strains can be promising for use in biotechnology, since biofilm formation is closely related to the colonization of the plant root system. The identification of the genetic patterns of this process in microbiota, as well as the study of the morphology, diversity and specificity of the properties of biofilms in individual species and strains, can play a key role both in the development of drugs aimed against them and in the creation of biological products for plants using these formations.

Bacillus bacteria isolated from winter wheat rhizosphere were grown on LB agar medium. Pure cultures were obtained that were tested for biofilm ability. A standard technique for culturing and staining with gentian violet cells was used (Stepanovic et al., 2000).

The control is a non-inoculated medium.

Cultivation and staining was carried out according to the following method:

1. 200 μl of bacterial suspension were added to 3 wells per culture. Negative control - nutrient broth without culture.

2. The plate was incubated for 24 hours at 30 ° C.

3. Remove the broth, rinse 3 times with 250 μl of sterile saline.

4. The biofilm was fixed with 200 μl of absolute ethanol.

5. After 15 minutes, ethanol was removed and allowed to dry.

6. Stained for 5 minutes, 200 μl of 1% crystal violet.

7. Washed under running water.

8. It was dried, 200 μl of ethanol was added, the plate was incubated for 5 minutes.

9. The optical density was measured at a wavelength of 57 0 nm on a FLUOstarOmega.

Statistical processing was performed according to student criterion. All results presented are statistically significant.

As a result of the screening, it was possible to establish the most promising strains for further research on the genetic basis of biofilm formation (Fig. 4.).

To assess the ability to grow at elevated temperatures, the tested strains were seeded on a dense LB medium and cultivated at temperatures from 25 to 42 ° C. To do this, the studied strains were seeded on a solid mustow agar medium and placed for 3 days in thermostats with a temperature of 25, 36 and 42 ° C. Once a day, data were collected on the degree of growth of the strains (Fig. 5). The data are presented in table 1.

At the final selection of the 10 most effective strains, the degree of antagonistic activity with respect to different strains of phytopathogenic fungi p was taken into account. Fusarium, as well as the level of biofilm formation and resistance to high temperatures.

Identification of selected strains of antagonist bacteria and phytopathogenic fungi used to test them.

The top 10 strains of antagonist bacteria and phytopathogenic fungi culture p. Fusarium were transferred to the VKPM for identification to the species level.

The following information was obtained:

By bacteria:

K 1.14, R 4.24 - Paenibacillus jamilae

O 1.27, O 2.11, R 3.13, R 4.5, R 6.14 - Paenibacillus peoriae 18

R 5.31 - Paenibacillus polymyxa

R 4.6, V 3.14 - Bacillus amyloliquefaciens

Thus, molecular genetic identification of 10 strains of spore-forming antagonist bacteria and 6 strains of phytopathogenic fungi was carried out, of which 5 belong to the genus Fusarium.

Bacterial cultures were deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) according to the procedure of national patent deposit.

Verification of fungicidal activity of 10 selected best crops in model experiments on plants.

The fungicidal activity of both individual preparations and a mixture of 10 preparations was tested in the wheat / mushroom / bacterium system in a greenhouse.

In the presence of plants, the interaction of phytopathogens and their antagonists may differ from their direct interaction in a microbiological experiment.

For the protective effect on plants to be successful, antagonist bacteria must possess a number of properties, the most important of which are:

- the absence of phytopathogenic effects on plants of the antagonist bacteria themselves;

- the ability to effectively suppress phytopathogenic flora on the surface of germinating seeds

- the ability to colonize the root system of producer plants

- the ability to endophytic growth (optional, but desirable condition).

Testing the effect of strains of antagonist bacteria on germination, germination energy, as well as indicators of plant growth and development in the presence and absence of phytopathogenic fungi.

At the first stage of the research, the task was set to check the absence of a phytopathogenic effect on plants of the antagonist bacteria themselves (in the absence of phytopathogenic fungi).

Phytopathogenic organisms are those that suppress the normal development of the plant and lead to its death. Phytopathogenicity may not be absolute and not lead to the death of the plant, but significantly weaken and lead to a loss or a significant decrease in productivity.

To assess the effect of antagonist bacteria on the initial stages of plant growth and development, surface-sterilized seeds were treated with a bacterial suspension at the rate of 3 × 10 ⁷ CFU per 10 g of seeds and placed on Petri dishes and in plastic containers. A double layer of filter paper moistened with tap water and plastic sand containers moistened with a special solution for growing wheat were used as a substrate for germinating seeds in Petri dishes.

In relation to the tested strains (Table 2), there were no signs of phytopathogenicity in the early stages of development. None of the tested strains showed a sharp suppression of development, although for some there was a slight growth retardation on Petri dishes, while growth in the sand did not show visible differences from the control. In no case were foci of tissue necrosis noted.

Some strains showed growth stimulation effects. Moreover, for some, an increase in the vegetative mass of shoots was noted, for others, in the root system (Fig. 6).

To ensure the absence of negative influence of antagonist bacteria on seed germination, the method of sowing seeds on the lawn of the tested microorganisms was used. In this case, the concentration of antagonist bacteria was many times higher than that usually used and made it possible to assess the possible negative effect in case of an overdose of the drug.

Germination on a bacterial lawn for the tested strains showed that germination in most cases is delayed, however, germination does not decrease, and seedlings do not show signs of developmental disruption.

At the next stage of work, experience was set in a greenhouse. Plants were grown from seeds treated with test bacterial strains and their mixture.

It was found that in a greenhouse, plants treated with a mixture of strains demonstrate normal growth and development, which indicates the absence of inhibitory effect of bacilli on wheat plants.

At the final stage of research, a study was made of germination, germination energy, and growth and development indicators of plants in the presence of phytopathogens. To do this, wheat seeds of the Thunder cultivar were used, treated with a mixture of 10 of the above strains (a biological product with the working name F-1) in combination with the standard Chansil-Trio dressing, as well as seeds treated only with Chansil-Trio 23 and untreated seeds as a control. As shown by microbiological analysis, these seeds are largely infected with p. Fusarium (Table 3).

For this, an experiment was set up on wheat plants in a sandy culture (N = 210 plants per one experiment variant). The data are shown in table 3.

As can be seen from table 3, both options for seed treatment with drugs led to an increase in germination energy, as well as a decrease in plant length compared to control. This is due to the inhibition of phytopathogenic fungi p. Fusarium, which are known to produce gibberelins, which leads to the rapid extension of plants.

However, treatment with a biological product led to a more significant effect, significantly different from the control (Fig. 7.).

In addition, the percentage of normally sprouted and developed plants became much larger, both in comparison with the untreated control and with chemically treated seeds (4th column).

Evaluation of the fungicidal activity of drugs in seed treatment.

Ten tons of Thunder wheat seeds previously treated with Chansil Trio were treated with 100 L of an aqueous suspension of the F-1 biological product using PS-10 treater. Untreated seeds served as a control sample, and seeds treated only with the Chansil Trio preparation by standard technology were also examined using a PS-10 treater.

About 1 gram of seeds (21 seeds) was placed in a test tube and filled with 10 ml of sterile water. Shaken vigorously for 10 minutes. The aqueous phase is superficially on plates with KSA solid nutrient medium, without the addition of ciprofloxacin antibiotic and with the addition of ciprofloxacin at a concentration of 10 μg / ml in order to inhibit the growth of antagonist bacteria.

The ability to effectively suppress the phytopathogenic flora on the surface of germinating seeds can significantly reduce the inhibitory effect of phytopathogens and give an advantage to the treated seeds already at the initial stages of growth.

Seeds were treated either with the Chansil trio preparation or with the Chansil trio preparation and with the preparation based on the obtained antagonist bacteria, or left without treatment.

1 gram of seeds (21 seeds) from each group was placed in a test tube and filled with 10 ml of sterile water. Shaken vigorously for 10 minutes. The aqueous phase was surface plated on plates with KSA solid nutrient medium, without the addition of ciprofloxacin and with the addition of ciprofloxacin at a concentration of 10 μg / ml in order to inhibit the growth of antagonist bacteria.

The plates were incubated for 2 days at a temperature of 25 ° C, then bacterial colonies were counted. After the plate, they were incubated for another 5 days at the same temperature to detect the growth of fungi.

Mushroom colonies were microscopic to determine the genus of the fungus.

The data obtained are presented in table 4.

In total, three morphologically distinct colony types were distinguished on the plates. The first type included yellowish mucous colonies, bacteria belong to non-spore-forming microorganisms. White mucous and beige non-mucous colonies belonged to spore-forming bacteria of the river. Bacillus of different species. Seeding by bacteria of seeds without treatment and treated with Chansil trio did not differ significantly from each other, however, on seeds treated with Chansil trio and bacilli, a significantly larger number of bacteria of the r. Bacillus, which is associated with treatment with a bacillus preparation.

On cups with the combined growth of antagonist bacteria and fungi, a single growth of p mushrooms is noted. Fusarium (according to microscopy). On plates with an antibiotic that suppresses the growth of antagonist bacteria, a significant number of bacterial colonies are noted, and on the treated seeds the number of CFU of the fungus is much less, more than twice.

It should be noted that the flush from seeds treated only with the Chansil Trio preparation contained a significant amount of spores of non-phytopathogenic fungi p. Mucor

Thus, studies of microflora of seeds showed the following:

1. On the surface of the untreated seeds, a sufficient number (about 500 CFU / seed) of “wild” spore-forming antagonist bacteria, mainly bacilli, is contained.

2. These bacilli are able to inhibit the growth of p. Fusarium, as in the case of their destruction with the antibiotic of fungi grows about 40 times more. That is, when the fungus seeds and living antagonist bacteria are flushed from the surface with water, and then sown on a cup, the bacteria kill most of the fungi.

3. Chansil Trio standard etch kills approximately half of the mushrooms (see column 5).

4. The addition of bacilli grown by us (these are essentially the same as on grain, but we selected and grew strains with maximum antifungal activity) significantly, by about 10 times, reduces the number of fungi (see column 4).

5. Processing with our preparation in a dose of 7 kg per 10 tons of wheat increases the number of bacilli located on the surface of the seeds by about three times. Obviously, 2/3 of the bacilli on the surface of the treated seeds belong to the strains of the claimed preparation.

It should be noted that seed treatment with a biological product reduced the number of fungi on the surface of seeds by 2.6 times (see column 5). With joint growth on a nutrient medium, the number of fungal colonies decreases significantly more (see column 4). This is due to the fact that the bacteria of the biological product are active in the vegetative state and begin to work actively only when the seeds germinate, which means the germination of fungi and bacilli and their interaction.

This can be observed in the following experiment, when the seeds were treated with preparations of antagonist bacteria and laid on a solid nutrient medium KSA, allowing them to germinate.

The efficiency of suppressing fungi was evaluated by the number of foci of natural phytopathogenic flora.

In all cases, the number of foci of mold decreased sharply compared with the control. Under growing conditions on the bacterial lawn, a sharp inhibition of the growth of the existing phytopathogenic microflora was observed (Fig. 8).

Thus, it is proved that the claimed biological product works. It is not alien to the agroecosystem of the wheat field and enhances the natural fungicidal activity of the bacterial microflora of wheat.

The ability to colonize the root system of producer plants is desirable to ensure the continued presence of protective microorganisms in the growth zone of the root system.

Not all bacteria can survive on or near the root system of a plant. The cause of the death of microorganisms can be a reaction of the plant's immune system. For a number of antagonist strains, good root survival is shown (Fig. 9).

During colonization of the roots, bacteria displace fungi from the rhizosphere and from the rhizoplane, which complicates their penetration into the plant. Active counteraction of bacteria-antagonists of plant colonization by phytopathogenic fungi is considered as an additional advantage, since it allows us to hope for suppression of the growth of fungi p. Fusarium in the internal cavities of the plant. It is known that the main site of development of Fusarium graminearum, the causative agent of hard smut of wheat, is the hollow stalks of cereals. A large mass of fungus can accumulate inside the stem, which after harvesting remains on the crop residues and is the main reason for the spread of the disease next year. Reducing the number of mushrooms p. Fusarium inside the stem along with the work of neutralizing crop residues will help reduce the overall contamination of the territories.

Wheat seeds were treated simultaneously with a suspension of p mushrooms. Fusarium and antagonist bacteria. Plants treated only with p. Fusarium Plants were grown in greenhouses in vessels with natural soil for 4 weeks. After this, the plants were removed from the vessels, washed from the remains of the soil. The stems, leaves and roots were superficially sterilized and placed on a medium conducive to the growth of fungi.

Of the explants of all plant organs - roots, stems and leaves treated only with p. Fusarium, intensive fungal germination was observed. In the case of plants treated simultaneously with p. Fusarium and strain antagonist, fungal growth p. Fusarium has not been reported.

Thus, at this stage, it was found that both individual strains of antagonist bacteria that form the basis of the drug being developed, and a mixture of 10 selected most promising strains, do not exhibit phytopathogenic effects on plants, exhibit antagonistic activity against phytopathogenic Fusarium sp., And also inhibit plant colonization by pathogens.

Development of a method of manufacturing a biological product with antifusarium activity based on solid-phase fermentation of spore-forming bacilli on a plant substrate.

Below is a description of the low-cost method of manufacturing small batches of a biological product of spore-forming antagonist bacteria with antifusar activity on the basis of solid-phase fermentation of spore-forming bacilli on a plant substrate using standard laboratory equipment.

Solid-phase fermentation makes it possible to achieve high titers of the target strains without the use of complex specialized equipment. Previous studies have shown that hydrated soybeans that have undergone heat treatment by autoclaving at 121 ° C are optimal substrates for spore-forming bacilli. The maximum titer of the target strains of antagonist bacteria was achieved when grown at temperatures of 37 ° C - 42 ° C and a pH of 6.8. The best way to dry solid-phase biological products is air drying. In addition to soybeans, peas and sunflower meal can be used as a substrate.

The results served as the basis for the development of low-cost technology for the production of small quantities of the drug based on spore-forming bacilli with the help of non-specialized household and standard laboratory equipment by personnel who do not have special education.

The main advantage of this method is its easy reproducibility, manufacturability and stability, the use of a simple composition, inexpensive environment, high yield of the finished product, the stability of the obtained preparation during storage.

Experimental evaluation of the effectiveness of growing spore-forming antagonist bacteria with antifusarium activity using laboratory and household equipment.

Soybeans were selected as a substrate for solid-phase fermentation, the effectiveness of which for the cultivation of bacilli was shown earlier (Chistyakov et al., 2015). By the content of protein, fat, phosphatides and some other nutrients, soybeans are significantly superior to many oilseeds and cereals. Depending on the place and conditions of soybean growth, the nutrient content 30

can vary significantly (protein from 29 to 50.3%; fat from 13.5 to 25.4%, and the sum of protein and fat from 52 to 65%).

The essence of the developed method of solid-phase cultivation is to exploit the ability of technologically significant strains of spore-forming antagonist bacteria to form biofilms on the surface of nutrient substrates. Forming biofilms, bacteria form a quasi-multicellular community, which has an increased, compared with the planktonic form, resistance to destructive environmental factors. In addition, the bacteria of the target strains, forming a biofilm uniformly covering the substrate, inhibit the development of putrefactive microflora. These features create the conditions for technological plasticity for solid-phase fermentation, unattainable with liquid-phase fermentation.

However, when developing a real solid-phase cultivation technology, it must be borne in mind that under production conditions, the risk of contamination by pathogenic microflora is significantly higher than under laboratory conditions. An effective way to prevent contamination is the cultivation of antagonist bacteria at elevated temperatures.

In the course of the research, the possibility of reproducing the optimal growth parameters of the antagonist bacteria used in the invention at a temperature of 42 ° C was tested. Based on the data obtained, an adjustment was made to the method for culturing the target strains of antagonist bacteria, as well as a set of equipment.

To develop the technology used 10 selected the most effective strains of spore-forming bacteria-antagonists (Table 5).

According to the conclusion of the VKPM, working with these strains does not require special precautions. Preparation of raw materials.

The substrate for solid-phase cultivation was soybean (variety "Doe"), hydrated in water at room temperature for 12 hours. The volume of water required for washing soybeans was determined empirically, visually checking the absence of dust and dirt on their surface. The optimal volume of water for soaking 1 kg of substrate is 1 liter.

Heat treatment of the substrate.

Heat treatment of the substrate for growing the biological product was carried out in an autoclave (Sanyo 3781 (b), Japan) for 20 minutes at 120 ° C. The final moisture content of the substrate was 60% when autoclaved.

In heat treatment of soybeans, the key factor is the ratio of temperature and processing time. The temperature and duration of heat treatment of the substrate, which ensures complete sterilization during heat treatment, was selected based on preliminary experiments with substrate quantities of 1 kg each and amounted to 20 minutes at 120 ° C. The heat treatment efficiency was evaluated by the absence of mesophilic aerobic and facultative anaerobic microorganisms in the center of the sample. Three samples weighing 10 g were taken from the center of the sample (GOST 26669-85). The determination of KMAFAnM was carried out according to GOST 10444.15-94.

The initial pH of the soy substrate was 7.0-7.2.

Inoculation.

To inoculate 1 kg of heat-treated hydrated soybeans, one Petri dish with LB agar or Saburo with the lawn of the target strain grown on its surface was used. At the same time, more than 1.0 - 3.0 × 10 ⁸ CFU of the targeted strain per 1 kg of substrate. When selecting the inoculation time, the main parameter is the uniformity of mixing of the inoculum and the substrate.

Incubation.

The use of solid phase fermentation allows to achieve high technological flexibility. While maintaining the above inoculation parameters, fermentation can be carried out on any pallets made of food materials, including plastic, enameled trays and trays and on disposable pallets made of plastic film.

In a series of experiments, solid-phase growth was carried out in household plastic trays and pallets made of corrugated cardboard, lined with plastic wrap, in a layer of a substrate 2-3 cm thick, the growing time was 4-8 hours.

Growth was carried out in a thermostat (TS-80, Russia), which allows incubation at a temperature of 42 ± 0.2 ° С.

Culture growth was assessed by the content of viable cells (CFU), determined by seeding serial dilutions of the culture on a solid medium (MP7A). The experiments were performed in triplicate. Homogenization.

Homogenization of soybeans, softened by heat treatment, and becoming softer during the fermentation process, does not represent a technological problem. A 2000W Kenwood MG 700 household meat grinder allows you to homogenize up to 3 kg of substrate in a minute. Thus, the rate of homogenization is limited only by the speed of the staff. The most simple and low-cost version of homogenization can be carried out using a manual meat grinder, for example, Manual meat grinders MA-S, RPP (Rivne, Ukraine).

Drying.

Drying of the biological product was carried out at a temperature of 45 ° C.

After homogenization, the crushed preparation was distributed in a thin layer (1-3 cm) on plastic trays and dried to a moisture content of 10%.

Determination of the viability of antagonist bacteria.

The survival of the cultures was evaluated by the content of viable cells (CFU). To calculate the number of CFU / g (colony forming units / g), the standard method of parallel dilutions in physiological saline and deep plating on solid agar media was used.

Three such crops were made from each dilution. Cups with crops were incubated in an incubator at 37 ° C for 48 hours. For each series of crops, the average number of colonies grown on three plates was calculated.

One of the properties of bacilli is the ability to grow in the form of a biofilm. According to modern concepts, biofilms consist of living and dead cells, cell fragments and extracellular polymers attached to the surface (Bridier et al., 2017). A feature of biofilms is the formation of mucus by bacteria. The extracellular polymeric substances that form it provide protection against aggressive agents, dehydration, and ultraviolet radiation (Ngo Thi Na, Naumann, 2009; Ippolitov et al., 2002). All these features provide high environmental and technological plasticity of the cultures of bacilli obtained by solid-phase fermentation.

The research results confirm that the use of soybean nutrient substrates in combination with an economical method of solid-phase cultivation can become the basis for creating low-cost technologies for the production of biological products.

The results of the growth rate of the studied microorganisms after solid-phase fermentation on soy substrate for 24 hours are presented in table b. The optimal growth temperatures of various strains were selected earlier in experiments on Petri dishes.

An example of the method that provides using standard laboratory equipment for seven days 0.1 kg of dry biological product (residual moisture content of not more than 10%) with a titer of antagonist bacteria of at least 10 ⁸ CFU / g based on 10 declared strains of spore-forming antagonist bacteria .

The required amount of soybeans is weighed.

The beans are washed with running tap water.

Hydration - soybeans are soaked for 12 hours in water in an enameled container at a temperature of (22 ± 1) ° С.

Soybeans are autoclaved at 120 ° C for 20 minutes.

To obtain the inoculum, a uterine culture is used. Dry uterine culture should be obtained from a specialized microbiological laboratory. The culture is revived by the standard method of transferring to Saburo broth, BCH or LB ..

The autoclaved beans are transferred to an inoculum container and cooled to 60 ° C. To inoculate 1 kg of heat-treated hydrated soybeans, one Petri dish with LB agar or Saburo with the lawn of the target strain grown on its surface was used. At the same time, more than 1.0 - 3.0 × 10 ⁸ CFU of the targeted strain per 1 kg of substrate.

Inoculated soybeans are laid out on pallets (layer thickness 10 cm), covered with plastic wrap. Trays with inoculated soybeans are placed in an incubator. The incubation process should last for 24-48 hours at a temperature of 37-42 ° C.

After each incubation, the incubator thermostat is wet-cleaned with detergents and then treated with 3% hydrogen peroxide and dried. After incubation, plastic trays are treated in the same way.

The fermented substrate is crushed using an electric or manual meat grinder.

The crushed preparation is distributed in a thin layer on plastic trays and dried at a temperature of 450 ° C to a moisture content of 8-10%.

The dried preparation is crushed using a household mill. A complex preparation is obtained by mixing equal weights of individual drugs.

Storage conditions for biological products significantly affect the viability of the bacterial cells that make up the drug, and, accordingly, its shelf life. The moisture content in the preparation should be minimal, which is of paramount importance for maintaining the viability of antagonist bacteria.

The finished biological product should be stored in the refrigerator at a temperature of (4 ± 2) ° С and humidity not more than 10%.

The development of molecular genetic methods for analyzing the results with the aim of subsequent screening of the effectiveness of the biological product in field experiments.

For a quantitative analysis of the degree of infection of samples with pathogenic fungi, an analysis of the polymerase chain reaction (PCR analysis) in real time was carried out. At the first stage of the work, genus-specific and species-specific primers were selected. Then, a PCR analysis was carried out, followed by electrophoresis and visualization in an agarose gel, to confirm the presence of target fragments in the samples, as well as preliminary identification of Fusarium species. Then a real-time PCR analysis was performed.

Primer selection.

Highly sensitive detection of a pathogen before infection of plants or before the development of symptoms of the disease is important to prevent their infection or to inhibit the development and spread of the disease and, therefore, to reduce economic losses. Diagnostic procedures should have a sufficiently high sensitivity. Before the discovery of nucleic acid amplification methods, in particular PCR, methods based on the use of specific hybridization probes were used to detect a specific organism. However, due to their low sensitivity, such methods often led to “false negative” results (Ward et al., 2004). Due to its high sensitivity, the PCR method makes it possible to detect even trace amounts of pathogen DNA (Ward et al., 2004). However, the high sensitivity of the method can lead to a “false positive” result. Therefore, to ensure the reliability of the results obtained, strict observance of the reaction formulation rules, the use of controls, the protection of samples and reagents from possible DNA contamination through aerosols, as well as the presence of separate zones for sample preparation procedures and analysis of PCR products (Kwok, Higushi, 1989) are necessary.

In practice, to make decisions on crop protection measures, methods that are more sensitive than traditional PCR are unlikely to be required, since the threshold level of infection can be easily detected using PCR.

For the molecular diagnosis of phytopathogens, conservative genes are often used as targets, the nucleotide sequences of which in closely related microorganisms often differ only in a few nucleotide substitutions (single-nucleotide polymorphism, SNP).

The nucleotide sequences for the studied strains obtained by sequencing the variable regions of genes encoding 16S rRNA are presented in the sequence listing SEQ ID NO 1 - SEQ ID NO 10.

Currently, the most commonly used tables are:

1) ITS regions of ribosomal RNA (rRNA). In the fungal genome, each copy of rRNA is represented by three genes separated by internal transcribed spacers (ITS),

According to it, there are extensive databases; the high copy number of rRNA genes in any genome provides high sensitivity of PCR. ITS contain conservative and variable regions, which allows classification of fungi at different taxonomic levels.

However, the rRNA nucleotide sequences do not always have a sufficient level of variability to distinguish between individual species (Tooleyetal., 1996).

2) “housekeeping” genes: genes of beta-tubulin, actin, 1-alpha translation elongation factor (Abramova, 2013); phosphate permease (RNO) genes; gene of the large subunit of ATP-citrate lyase (acl1).

The 1-alpha translation elongation factor gene (tef1α) is widely used in phylogenetic studies of fusarium fungi.

The advantages are that its nucleotide sequences are highly informative at the species level, its non-orthological copies were not found for species of the Fusarium genus, the FusariumID database was created on the basis of the sequences of this gene, the tef1α gene is represented in the genome as a single copy (Kristensen et al., 2005 , 2007).

Based on the analysis of the literature, the following primers were selected for analysis (Table 7).

Testing the effectiveness of PCR to identify pathogens.

By PCR, it was found that two species of Fusarium are present in the infected samples: F. graminearum (samples 2 and 3, Fig. 20) and F. oxysporum (samples 4-9, Fig. 20). Therefore, in a quantitative analysis, species-specific primers for these two species were further used, as well as genus-specific primers for determining the presence of pathogens of other species.

Thus, work was carried out on the selection of primers to assess the presence of phytopathogens in plant tissues by molecular genetic methods.

Below are the experimental tests of the claimed biological product.

Experience No. 1. The effect of the drug on winter wheat crops, spring-summer 2018.

Accounting for infection of rhizosphere soil with fungi p. Fusarium In order to study the effect of winter wheat seed treatment with the F-1 biological product in the fall of 2017, 10 tons of seeds were processed using PS-10. Sampling was carried out during the tillering phase in April 2018. The experimental and control fields were divided into 30 sectors under GPS control, and plant samples were taken in each sector. The selection was made by the method of an envelope of 5 points of the sector to obtain a representative picture. Under laboratory conditions, wheat roots with rhizospheric soil were sown on Chapek's medium, followed by registration and identification of p. Fusarium Pathogens were identified by colony morphology, confirmed by light microscopy. Pathogen biomass was also selected to confirm p. Fusarium using molecular genetic methods. Data on the number of phytopathogenic fungi are presented in table 9.

As you can see, the number of phytopathogenic fungi in the experimental field was almost halved.

It is also seen that in the treated area there are no samples with the content of Fusarium mushrooms of more than 9000 CFU / g. This suggests that the test drug, although it does not completely destroy the phytopathogen, allows it to be effectively controlled.

For statistical data processing, the Pearson test (X-square) was used. As a result, the value p = 0.03501 was obtained, which indicates the statistical significance of the differences. A graphical representation of the data is shown in FIG. eleven.

The number of phytopathogenic fungi in the rhizospheric soil of winter wheat in the ripening phase is presented in table 10.

As you can see, the number of phytopathogenic fungi in the rhizosphere of winter wheat in the experimental and control field by the ripening phase turned out to be approximately at the same level, which may be due to the cessation of plant vegetation and low rainfall. The data are presented in FIG. 12.

Evaluation of the impact of biological treatment on the natural microbial community of the soil.

In the spring, a study was conducted of the effect of treatment with the drug on the total number of molds in the studied samples. The data for this experiment are shown in table 11.

From the data of table 11 it can be seen that treatment with the drug reduces the number of molds, that is, the fungicidal effect of the biological product F-1 is not limited to p. Fusarium and potentially can serve as a tool for controlling other fungal infections.

Work on the study of the effect of seed treatment of winter wheat with biological product F-1 on the infection of plants with fungi of the river. Fusarium

In the course of studying the effect of the F-1 biological product on winter wheat and its affection with Fusarium, wheat ears were collected to determine the morphometric parameters of plants (described in the description below). The content of p. Fusarium in the grain of winter wheat. For this, a microbiological analysis of swabs from wheat grains selected in the experimental and control field was performed. For flushing, 10 g of grain were taken, which were treated with 10 ml of sterile water to flush the fungal spores, then these flushes were sown on a nutrient medium. Data on the number of mushrooms on wheat grain are shown in table 12.

As you can see, the grain in the experimental version was much less infected with p. Fusarium compared to control (FIG. 13).

Differences with the control amounted to 5.28 times, in addition, in many grain samples from the experimental field, phytopathogenic fungi were completely absent, which was not observed in the grain from the control field. The total number of mushrooms also decreased (Fig. 14).

Work on the study of the effect of the biological product F-1 on the morphometric parameters of wheat plants.

To assess the effect of the F-1 biological product on the growth and development of wheat plants in the field, the analysis of the main morphobiometric indicators of wheat plants was carried out. The results are summarized in table 13.

According to the results of the analysis and statistical processing of the obtained data, it was established that the processing with a biological product did not significantly affect any of the indicators of plant development, and also did not affect the yield (the weight of seeds per linear meter was estimated). These data coincide with the data obtained during preliminary experiments in 2017 under the conditions of a greenhouse and phytotron.

Experience No. 2. The effect of the drug on corn crops, spring-summer 2018.

Work on the study of the effect of corn seed treatment with the biological product F-1 on the infection of soils and plants with fungi of the river Fusarium

In the spring of 2018, experience was laid on the effect of corn seed treatment on the damage of plants by fungi p. Fusarium Corn seeds were manually processed with F-1 biological product, after which sowing was carried out. Sampling was carried out in the seedling phase and in the phase of milk ripeness before harvesting corn for silage.

Rhizospheric soil was sampled from plant roots. Microbiological soil analysis data are presented below.

Accounting for infection of rhizosphere soil with fungi p. Fusarium is presented in table 14.

As can be seen from the data in table 14, in the seedling phase, the number of mushrooms p. Fusarium in the experiment was 2.64 times lower than in the control. Pathogenic fungi were not found in 37% of the experimental sites on the experimental field, and in 16% of the sites in the control variant. Also, in the experimental version, 2 sites with the number of phytopathogenic fungi above 10,000 CFU / g of soil were observed, while in the control version, 31 sites. In a complex, this indicates effective control over the number of phytopathogenic fungi in the root zone when using the biological product F-1.

Abundance data is also presented in diagram form in FIG. 15.

Thus, one can see statistically significant differences in the number of phytopathogenic fungi between control and experiment.

The next sampling was carried out in the phase of milk ripeness of plants, immediately before harvesting corn for silage.

In this phase of plant development, there was a significant increase in the number of phytopathogens in the basal zone, compared with the seedling phase. However, in the control variant, the number of phytopathogenic fungi p. Fusarium was approximately two times higher than in the experiment.

These differences were also statistically significant (Fig. 16).

As in the case of wheat, after a period of drought, the effect of the use of the biological product was smoothed out, but not so much, which is associated with a more powerful root system of corn and the preservation of moisture in deeper layers of the soil. This allowed differences to persist in the summer.

Investigation of the effect of the biological product F-1 on the morphometric parameters of corn plants.

To assess the effect of the F-1 biological product on the growth and development of corn plants in the field, the analysis of the main morphobiometric indicators of corn plants was carried out. Among these parameters, the length and weight of the cobs at the time of selection were taken into account, and the height of 5 plants was measured at each site. The results obtained are summarized in tables 16 and 17.

As can be seen from the data given in tables 16 and 17, differences in the main morphobiometric parameters of maize plants do not reach the level of statistical significance, that is, the biological product F-1 does not adversely affect the growth and development of plants.

Studies confirming the affiliation of phytopathogenic fungi to p. Fusarium using molecular genetic methods.

To accurately identify the cultures whose growth was observed in Petri dishes, the polymerase chain reaction (PCR) method was also used.

DNA was isolated using an M-SorbTube magnetic isolation kit (Synthol, Russia). A mushroom culture was grown on solid nutrient agar for 15 hours. The biomass was washed from the plates with sterile saline, centrifuged at 3000 rpm for 15 minutes. The precipitate was frozen in liquid nitrogen and mechanically ground in a mortar to destroy the cell walls of the fungi. The procedure was repeated three times. Then, a lysis buffer solution was added and the tubes were shaken and incubated at 75 ° C for 10 minutes. After lysis, the tubes were centrifuged at 3000 rpm for 1 min and the liquid fraction containing DNA was transferred to a tube containing magnetic sorbent particles. Then, 500 μl of the precipitating solution was added, the contents of the tubes were thoroughly mixed and left for 5 minutes at room temperature. The tubes were centrifuged at 16,000 rpm for 5 minutes and placed in a magnetic tripod. The liquid fraction was removed. The precipitate of magnetic particles with sorbed DNA was washed 3 times with a precipitating solution by resuspension and centrifugation. The tubes were then left open to dry the pellet. After this step, DNA was eluted from the magnetic particles using an elution buffer and stored at −80 ° C. until analysis.

The total amount of DNA in the sample (total DNA) was evaluated using a NanoDrop 2000 spectrophotometer with Thermo Scientific (USA).

Amplification of DNA.

For each sample, a mixture was prepared for PCR: H2O (DD): 18.3 μl; 25 mM 10xdNTP nucleotide solution - 2 μl; 10x DreamTaq PCR buffer (20 mM MgC12) (ThermoFisher) - 2.5 μl; DreamTaq polymerase - 0.2 μl (ThermoFisher), 1 μl DNA matrix (5 ng μl-1) and 0.5 μl of the forward (F) primer and reverse (R) primer (0.5 μM).

The amplification protocol was as follows:

1. The first denaturation at 95 ° C for 5 min;

2. Denaturation at 95 ° C for 30 seconds;

3. Annealing of the primer at 58-70 ° С - 30 sec (the optimum temperature was selected for each pair of primers separately);

4. Elongation at 72 ° C - 1.5 min .;

5. Cycle 2-4 was repeated 35 times;

6. Final elongation at 72 ° C for 3 minutes.

The effectiveness of PCR was tested using a PCR Control Set reagent kit (Eurogen, Russia). All reagents and primers, except for the DNA template, were added to the samples for negative control.

To establish the generic affiliation of the fungi, gender-specific primers (Table 18) for the 1-alpha translation elongation factor gene (tef1α) and the ITS region gene (internal transcribed region) of ribosomal RNA (rRNA) were used. Primers were selected at the previous stage of the study.

To establish species specificity, species-specific primers were used (Table 19) for the main species of representatives of the genus Fusarium, affecting the studied fields.

Primers were selected and tested at the previous stage of the study.

In FIG. Figure 17 shows that the reaction for strain Fus1 proceeds with G1 primers for F. graminearum at a temperature of 62 ° C, the reaction for strain Ras 6.2.1 - with O2 primers for F. oxysporum, which corresponds to the data on their genetic identification carried out in the first stage works.

In FIG. 18 it can be seen that in samples containing fungi of the genus Fusarium, a specific amplification product of 340 nucleotide pairs is formed. Of the 15 colonies, morphologically similar to colonies of fungi of the genus Fusarium, only 9 turned out to be such according to DNA analysis. Only such colonies were taken into account in statistical calculations. This makes tests more cumbersome, but greatly improves their accuracy.

In addition, species identification of pathogenic fungi isolated from crops was also performed by PCR.

As a result of amplification with species-specific primers, the following results were obtained (Table 20):

In FIG. 19-21 show the results of agarose gel electrophoresis performed to visualize the results of PCR amplification.

In these figures, one can see pronounced fluorescence bands corresponding to amplified fragments of 250-400 bp in length. Less pronounced bands of smaller size refer to the so-called non-specific PCR products and are not taken into account.

Thus, we can conclude that:

• Strain TO-12 belongs to the species F. culmorum;

• Strain TO-7 refers to the species F. graminearum;

• Strains TO-7.2 and TO-10 belong to the species F. poae;

• Strain KG11 demonstrates a reaction to species-specific primers for F. culmorum, F. oxysporum and F. poae.

For further identification, additional studies are needed. However, it can be assumed that since the primers for F. oxysporum in our test system, unlike the other two, which also caused the amplification reaction, are complementary to the calmodulin gene, this strain probably carries a mutation in the tef1α gene or received a significant portion of it from a closely related species by horizontal transport, but refers, nevertheless, to the species F.oxysporum. No representatives of the species F. avenaceum and F. sporotrichoides were found among the isolated strains.

Experience No. 3. The effect of the drug on winter wheat crops, autumn 2018.

Work on the study of the effect of seed treatment of winter wheat with biological product F-1 on the damage of plants by fungi of the river. Fusarium in the fall of 2018 (shoots).

To study the impact of winter wheat seed treatment with the F-1 biological product, in the fall of 2018, 61 tons of seeds were processed using PS-10.

These seeds were used for sowing on irrigated fields (field 40, brigade 3, 92 ha, precursor - potato), as well as on fields without irrigation (field 94, 90 ha, brigade 6), precursor - corn. Fields were selected as control, where wheat seeds treated only with chemical fungicides were used for sowing - an irrigated field of 39, 87 ha, a team of 3, a predecessor of sugar beets and a field without irrigation of 91, 91 ha, a team of 6, a predecessor of corn.

Sampling was carried out in the seedling phase in November 2018 and in the tillering phase in December 2018. Sampling was carried out according to the envelope method, in 5 independent replicates. Each field was divided into 12 sectors and 5 samples were taken in each sector, a total of 60 samples from the field.

In the laboratory conditions, fragments of wheat plants were sown (sections of the stem without root system) on Chapek’s environment, followed by registration and identification of the fungi of the river. Fusarium 10 fragments of stem 1 cm long were placed on each plate. Pathogens were identified by colony morphology, confirmed by light microscopy. Pathogen biomass was also selected to confirm p. Fusarium using molecular genetic methods. The data on the affection of wheat plants with phytopathogenic fungi are presented as% of plant fragments that gave rise to growth of p. Fusarium and are shown in tables 21 and 22 as well as in figures 22 and 23.

According to the analysis, there is a significant decrease in the damage to wheat plants by phytopathogenic fungi of the river. Fusarium 2.28 times. Apparently, the treatment of wheat seeds with a biological product makes it difficult and slows the penetration of the fungus into the plant tissue due to the formation of a biofilm of antagonist bacteria on the surface of the germinating root.

Data on the average plant damage are presented graphically in FIG. 22

As can be seen from table 22, in fields without irrigation, a decrease in the degree of infection of plants with fungi p. Fusarium when using the biological product F-1 was lower (1.41 times), but the differences were also statistically significant. Apparently, this difference can be explained by differing precursor crops in fields 4 0 and 39, whereas in fields without irrigation No. 94 and No. 91, the predecessor was the same (corn).

The precursor culture significantly affects the initial infectious background, and this may affect the observed efficacy of the drug. Data on the average plant damage are presented graphically in FIG. 23.

Study of the effect of winter wheat seed treatment with the F-1 biological product on the infection of rhizospheric soil with fungi of the river Fusarium in the fall of 2018 (shoots).

The selection was made by the method of an envelope of 5 points of each sector of the field to obtain a representative picture. Received a mixed sample to assess the number of mushrooms p. Fusarium in soil adjacent to the roots. In total, 12 mixed soil samples were taken from each field. In the laboratory conditions, wheat roots with rhizospheric soil were sown on Chapek’s environment, followed by registration and identification of p. Fusarium The data are shown in tables 23 and 24, as well as in figures 24 and 25.

The data shown in table 23 show the presence of a very significant difference in the number of mushrooms p. Fusarium in the soil. Despite the large scale of variation in the number of fungi in the soil of the experimental field, their number is noticeably lower compared to the control. This indirectly shows that antagonist bacteria colonize plant roots and control the number of fungi p. Fusarium in a layer of soil directly adjacent to the roots. Data is presented in diagram form in FIG. 24.

A similar pattern was observed in fields without irrigation, albeit on a smaller scale (Table 24).

In fields without irrigation, the number of fungi p. Fusarium in rhizospheric soil in the experiment was 22% lower than in control. These differences were statistically significant (p = 0.02). The average abundance of phytopathogenic fungi is graphically presented in FIG. 25.

In general, the data obtained in the analysis of the rhizospheric soil are in good agreement with the data obtained for fragments of stems of wheat plants. This indicates that the bacteria that make up the biological product actively colonize the root surface and prevent the penetration of infection into plants.

Study of the effect of the biological product F-1 on the morphometric parameters of wheat plants (seedlings).

To assess the effect of the F-1 biological product on the growth and development of wheat plants in the field in the fall of 2018, an analysis of the main morphobiometric indicators of wheat plants was carried out. The results obtained are summarized in table 25.

In fields with irrigation, the differences between experience and control were also significant and more pronounced in comparison with fields without irrigation. This could be affected by a higher infectious background associated with high humidity during the previous growing season.

Investigation of the effect of winter wheat seed treatment with the F-1 biological product on plant damage by fungi p. Fusarium in autumn 2018 is presented in table 26.

According to the analysis of the occurrence of fungi p. Fusarium in plant tissues, a significant decrease in the affection of wheat plants with phytopathogenic fungi p. Fusarium 1.83 times. It should be noted that compared with the seedling phase (45.57% ± 3.58% in the control, and 19.95% ± 1.29% in the experiment), the occurrence of phytopathogens in the tillering phase became lower. Apparently, during the development of wheat plants, part of the penetrating fungi is eliminated due to the active immunity of plants. In this case, treatment with a biological product can contribute to this process. It is important that the percentage of infected plants is even lower during the germination process, and this trend continues as the plants develop and transition to the tillering phase. Graphically the occurrence of fungi p. Fusarium in plant tissues is shown in the diagram (Fig. 26).

In fields without irrigation, a similar pattern is observed (Table 27).

In fields without irrigation, the occurrence of fungi p. Fusarium in plant tissues during the tillering phase in the experiment was 2.25 times lower than in the control. As in irrigated fields, by the tillering phase, the percentage of plants in the tissues of which there were p. Fusarium is declining. In the control, the decrease is from 50.49% to 32.81%, and in the experiment from 35.63% to 14.58%.

Thus, the biological product retains its protective effect to the tillering phase, which speaks in favor of the active colonization of wheat plants by the biological product bacteria (Fig. 27).

Study of the effect of winter wheat seed treatment with the F-1 biological product on the infection of rhizospheric soil with fungi of the river Fusarium in autumn 2018 (autumn tillering) is presented in table 28.

The study of rhizospheric soil during the tillering phase showed (Fig. 28) that at this stage of plant development, the number of fungi p. Fusarium did not differ in the soils of the control and experimental fields. In addition, compared with the seedling phase, there was a significant drop in their numbers in the rhizosphere of wheat. One explanation may be the establishment of low temperatures and snowfall in November (the selection was carried out in December, after the snow melted).

Apparently, the most active penetration of fungi into plants occurs during the earliest stages of wheat development - immediately after germination, and at later stages the number of mushrooms in the rhizosphere decreases. At the same time, it is important to ensure control of the pathogen abundance precisely in this critical period, and the results of the experiment show that in the variants where the number of mushrooms in the rhizosphere was reduced during the seedling phase, the percentage of plants into which the fungi successfully introduced subsequently turns out to be lower.

Study of the effect of the biological product F-1 on the morphometric parameters of wheat plants (autumn tillering).

In the phase of autumn tillering, the following morphobiometric indicators of plants were determined: stem length, root length, wet and dry mass of the plant, and the number of stems per plant. The data are shown in tables 2 9 and 30.

By the tillering phase, the plants in the experimental and control fields without irrigation were aligned along the length of the shoots and roots, the differences in mass remained, but also became less pronounced. This is due to the fact that even a small amount of phytohormones produced by mushrooms has a significant effect on the plant during the seedling phase, and as the plants grow and develop, the contribution of exogenous phytohormones decreases.

Moreover, the control and experimental fields turned out to be exactly the same in terms of the number of stems on the plant; treatment did not affect this feature.

In fields with irrigation, the difference between the control and the experimental field is also smoothed out, but is still more noticeable in comparison with irrigated fields (Table 30).

Plants in the control and experimental fields differed in root length and dry weight (in the control somewhat more than in the experiment). However, there was a significant difference in the number of shoots per plant - in the experiment 3.6 shoots per plant, in the control 2.9. This indicator can potentially have a positive effect on productivity, however, account must be taken of the productivity of the lateral shoots of wheat.

Thus, as a result of research work, 104 0 cultures of spore-forming bacteria p. Bacillus from soil fields belonging to the Customer and root systems of winter wheat plants. 5 strains of fungi of the river were isolated. Fusarium, dominating the Customer’s fields, of which 3 strains belonged to the species Fusarium graminearum and 2 to the species Fusarium oxysporum, and in addition, the phytopathogenic fungus Plectosphaerella cucumerina was isolated.

At the same time, the antagonistic activity of the isolated cultures was evaluated and was tested by co-cultivation under growth conditions on Petri dishes and in microculture on glasses. As a result, 212 antagonistic strains were first selected, then 37 of the most active cultures were selected. Of these cultures, 10 strains were selected that best meet the goals of research, that is, the development of a biological product and a method for producing a biological product.

At the same time, strains of antagonist bacteria and phytopathogenic fungi that were identified by VKPM specialists were isolated. The strains of antagonist bacteria were transferred to the VKPM for deposition according to the procedure of national patent deposition.

At the same time, the effect of the strains on plants was tested, which was carried out by treating the seeds with the culture of the test strain, followed by determination of germination, germination energy, as well as growth and development indicators of plants in the presence and absence of phytopathogenic fungi. Vegetative experiments to study the stimulating effect of antagonist bacteria were carried out in a climate chamber and a greenhouse, in a sandy and soil culture. As a result, the absence of negative effects for plants, a neutral and positive effect on their growth and development indicators was shown.

At the same time, a method was developed for the preparation of a biological product containing 10 strains of spore-forming antagonist bacteria with antifusar activity.

A trial batch of the preparation was tested in real conditions for 10 tons of Grom wheat using the PS10 plant.

Grain processing showed the effectiveness of the developed biological product in comparison with the chemical fungicide "Chansil Trio" in the laboratory.

The developed biological product F-1 showed the ability to reduce the number of fungi of the genus Fusarium in the following experimental variants:

1) in the rhizosphere of winter wheat in the tillering phase (a decrease of 1.98 times)

2) in the grain of winter wheat (a decrease of more than 5 times)

3) in the rhizosphere of corn in the seedling phase (2.64 times)

4) in the rhizosphere of corn in the phase of milk ripeness (1.82 times)

5) in the stems of winter wheat in the seedling phase (2.28 times in the irrigated willow fields 1.41 times in the fields without irrigation)

6) in the rhizosphere of winter wheat in the seedling phase (5 times in irrigated fields and 1.26 times in fields without irrigation)

7) in the stems of winter wheat during the tillering phase (1.83 times in irrigated fields and 2.25 times in fields without irrigation)

At the same time, the most powerful effect was registered for the most valuable parameter - in the grain of winter wheat treated with the biological product F-1, a more than five-fold decrease in infection with fusarium was observed.

In the early stages of plant development, the treatment of winter wheat seeds with the F-1 biological product leads to a protective effect both in plants and in the rhizospheric soil adjacent to the roots, which indicates the colonization of rhizoplane by bacteria that impede the introduction of the pathogen. The consequence of colonization of the root system by bacteria is that in the tillering phase, the percentage of plants in the tissues of which were present p. Fusarium in the experiment is significantly lower than in the control: 1.83 times in irrigated fields and 2.25 times in fields without irrigation.

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Sequence listing

<110> Agrofirm Urozhaynaya Limited Liability Company

(Obshchestvo s ogranichennoi otvetstvennostiu "Agrofirma" Urozhainaia ")

<120> Strains, biological product, a method of obtaining a biological product and a method of biological protection of crops from fusarium

<140> RU 2019139147

<141> 2019-12-02

<160> 10

<210> 1

<211> 844

<212> DNA

<213> Paenibacillus peoriae R 3.13

<400> 1

cccaggcgaa tcttaatgta gttaacttcg gcaccaaggg tatcgaaacc cctaacacct 60

agcattcatc gtttacggcg tggactacca gggtatctaa tcctgtttgc tccccacgct 120

ttcgcgcctc agcgtcagtt acagcccaga gagtcgcctt cgccactggt gttcctccac 180

atatctacgc atttcaccgc tacacgtgga attccactct cctcttctgc actcaagctc 240

cccagtttcc agtgcgaccc gaagttgagc ctcgggatta aacaccagac ttaaagagcc 300

gcctgcgcgc gctttacgcc caataattcc ggacaacgct tgccccctac gtattaccgc 360

ggctgctggc acgtagttag ccggggcttt cttctcaggt accgtcactc ttgtagcagt 420

tactctacaa gacgttcttc cctggcaaca gagctttacg atccgaaaac cttcatcact 480

cacgcggcgt tgctccgtca gctttcgccc attgcggaag attccctact gctgccyccc 540

gtagagtctg ggccgtgtct cagtcccagt gtggccgatc accctctcag gtcggctacg 600

catcgtcgcc ttggtaggcc tttaccccac caactagcta atgcgccgcm gccatccaca 660

agtgacagat tgctccgtct ttcctccttc tcccatgcag gaaaaggatg tatcgggtat 720

tagctaccgt ttccggtagt tatccctgtc ttgtgggcag gttgcctacg tgttactcac 780

ccgtccgccg ctagttrttt agaagcaagc twtctaatya ccccgckcga cttgcatgya 840

ttag 844

<210> 2

<211> 701

<212> DNA

<213> Paenibacillus peoriae R 4.5

<400> 2

agttaacttc ggcaccaagg gtatcgaaac ccctaacacc tagcattcat cgtttacggc 60

gtggactacc agggtatcta atcctgtttg ctccccacgc tttcgcgcct cagcgtcagt 120

tacagcccag agagtcgcct tcgccactgg tgttcctcca catctctacg catttcaccg 180

ctacacgtgg aattccactc tcctcttctg cactcaagct ccccagtttc cagtgcgacc 240

cgaagttgag cctcgggatt aaacaccaga cttaaagagc cgcctgcgcg cgctttacgc 300

ccaataattc cggacaacgc ttgcccccta cgtattaccg cggctgctgg cacgtagtta 360

gccggggctt tcttctcagg taccgtcacy yywrkagcag ttactctmym arrcgttctt 420

ccctggcaac agagctttac gatccgaaaa ccttcatcac tcacgcggcg ttgctccgtc 480

agctttcgcc cattgcggaa gattccctac tgctgcctcc cgtagagtct gggccgtgtc 540

tcagtcccag tgtggccgat caccctctca ggtcggctac gcatcgtcgc cttggtagsc 600

ctttacccca ccaactagct aatgcgccgc mgccatccca caagtgacag attrctcccs 660

ctttcctcct tctcccatgc aggaaaarga tgtatcgggt a 701

<210> 3

<211> 867

<212> DNA

<213> Bacillus amyloliquefaciens R 4.6

<400> 3

agmcggatgc ttaatgcgtt agctgcagca ctaaggggcg gaaaccccct aacacttagc 60

actcatcgtt tacggcgtgg actaccaggg tatctaatcc tgttcgctcc ccacgctttc 120

gctcctcagc gtcagttaca gaccagagag tcgccttcgc cactggtgtt cctccacatc 180

tctacgcatt tcaccgctac acgtggaatt ccactctcct cttctgcact caagttcccc 240

agtttccaat gaccctcccc ggttgagccg ggggctttca catcagactt aagaaaccgc 300

ctgcgagccc tttacgccca ataattccgg acaacgcttg ccacctacgt attaccgcgg 360

ctgctggcac gtagttagcc gtggctttct ggttaggtac cgtcaaggtg ccgccctatt 420

tgaacggcac ttgttcttcc ctaacaacag agctttacga tccgaaaacc ttcatcactc 480

acgcggcgtt gctccgtcag actttcgtcc attgcggaag attccctact gctgcctccc 540

gtaggagtct gggccgtgtc tcagtcccag tgtggccgat caccctctca ggtcggctac 600

gcatcgtcgc cttggtgagc cgttacctca ccaactagct atgcgccgcg ggtccwtctg 660

taagtggtac cgaagccacc ttttatgtct gaccatgcgg ttcaacaacc atccggtatt 720

agccccggtt tcccggagtt atcycrkctt acaggcaggt tacccacgtg ttactcaccc 780

gtcgcgctta catcaggagy agcttccatc tgwccgctcg acttgcatgt attaggcacg 840

ccgcagcgtt ysgtccttga ccgggat 867

<210> 4

<211> 770

<212> DNA

<213> Paenibacillus jamilae R 4.24

<400> 4

tccccaggcg gaatcttaat gtgttaactt cggcaccaag ggtatcgaaa cccctaacac 60

ctagcattca tcgtttacgg cgtggactac cagggtatct aatcctgttt gctccccacg 120

ctttcgcgcc tcagcgtcag ttacagccca gagagtcgcc ttcgccactg gtgttcctcc 180

acatctctac gcatttcacc gctacacgtg gaattccact ctcctcttct gcactcaagc 240

tccccagttt ccagtgcgac ccgaagttga gcctcgggat taaacaccag acttaaagag 300

ccgcctgcgc gcgctttacg cccaataatt ccggacaacg cttgccccct acgtattacc 360

gcggctgctg gcacgtagtt agccggggct ttcttctcag gtaccgtcac yyywrkagca 420

gttactctmy marrcgttct tccctggcaa cagagcttta cgatccgaaa accttcatca 480

ctcacgcggc gttgctccgt cagctttcgc ccattgcgga agattcccta ctgctgcctc 540

ccgtagagtc tgggccgtgt ctcagtccca gtgtggccga tcaccctctc aggtcggcta 600

cgcatcgtcg ccttggtags ctttacccca ccaactagct aatgcgccgc mgccatccac 660

aagtgacaga ttrctccgcc tttcctcctt ctcccatgca ggaaargatg tatcgggtat 720

tagctaccgt ttccggtagt tatccctgtc ttgtrggcag rttgcctacg 770

<210> 5

<211> 467

<212> DNA

<213> Paenibacillus polymyxa R 5.31

<400> 5

tccccaggcg atsttaatgt gttaacttcg gcaccaaggg tatcgaaacc cctaacacct 60

agcattcatc gtttacggcg tggactacca gggtatctaa tcctgtttgc tccccacgct 120

ttcgcgcctc agcgtcagtt acagcccaga gagtcgcctt cgccactggt gttcctccac 180

atctctacgc atttcaccgc tacacgtgga attccactct cctcttctgc actcaagctc 240

cccagtttcc agtgcgaccc gaagttgagc ctcgggatta aacaccagac ttaaagagcc 300

gcctgcgcgc gctttacgcc caataattcc ggacaacgct tgccccctac gtattaccgc 360

ggctgctggc acgtagttag ccggggcttt cttctcaggt accgtcacyy ctagagcagt 420

tactctctma gacgttcttc cctggsaaaa magctttacg atcggaa 467

<210> 6

<211> 857

<212> DNA

<213> Paenibacillus peoriae R6.14

<400> 6

atgtgttaac ttcggcacca agggtatcga aacccctaac acctagcatt catcgtttac 60

ggcgtggact accagggtat ctaatcctgt ttgctcccca cgctttcgcg cctcagcgtc 120

agttacagcc cagagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca 180

ccgctacacg tggaattcca ctctcctctt ctgcactcaa gctccccagt ttccagtgcg 240

acccgaagtt gagcctcggg attaaacacc agacttaaag agccgcctgc gcgcgcttta 300

cgcccaataa ttccggacaa cgcttgcccc ctacgtatta ccgcggctgc tggcacgtag 360

ttagccgggg ctttcttctc aggtaccgtc actcttgtag cagttactct acaagacgtt 420

cttccctggc aacagagctt tacgatccga aaaccttcat cactcacgcg gcgttgctcc 480

gtcaggcttt cgcccattgc ggaagattcc ctactgctgc ctcccgtagg agtctgggcc 540

gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc gtcgccttgg 600

taggccttta ccccaccaac tagctaatgc gccgcaggcc catccacaag tgacagattg 660

ctccgtcttt cctccytctc ccatgcagga aaggatgtat cgggtattag ctaccgtttc 720

cggtagttat ccctgtcttg tgggcaggtt gcctacgtgt tactcacccg tccgccgcta 780

ggttrtttag aagcaagctt ctamayaacc ccgctcgact tgcatgtatt aggcacgccg 840

ccgcgttcgt cctgagc 857

<210> 7

<211> 843

<212> DNA

<213> Bacillus amyloliquefaciens V 3.14

<400> 7

gcgttagctg cagcactaag gggcggaaacc ccctaacact tagcactcat cgtttacggc 60

gtggactacc agggtatcta atcctgttcgc tccccacgct ttcgctcctc agcgtcagtt 120

acagaccaga gagtcgcctt cgccactggtg ttcctccaca tctctacgca tttcaccgct 180

acacgtggaa ttccactctc ctcttctgcac tcaagttccc cagtttccaa tgaccctccc 240

cggttgagcc gggggctttc acatcagactt aagaaaccgc ctgcgagccc tttacgccca 300

ataattccgg acaacgcttg ccacctacgta ttaccgcggc tgctggcacg tagttagccg 360

tggctttctg gttaggtacc gtcaaggtgcc gccctatttg aacggcactt gttcttccct 420

aacaacagag ctttacgatc cgaaaaccttc atcactcacg cggcgttgct ccgtcagact 480

ttcgtccatt gcggaagatt ccctactgctg cctcccgtag gagtctgggc cgtgtctcag 540

tcccagtgtg gccgatcacc ctctcaggtcg gctacgcatc gtcgccttgg tgagccgtta 600

cctcaccaac tagctaatgc gccgcgggtcc atctgtaagt ggtagccgaa gccacctttt 660

atgtctgaac catgcggttc agacaaccatc cggtattagc cccggtttcc cggagttatc 720

ccagtcttac aggcaggtta cccacgtgtta ctcacccgtc cgccgctcac atcaggagca 780

agctcccatc tgtccgctcg acttgcatggt attaggcacg ccgccagcgt tcgttctgag 840

cag 843

<210> 8

<211> 682

<212> DNA

<213> Paenibacillus peoriae O 1.27

<400> 8

ttaatgyagt taacttcggc accaagggta tcgaaacccc taacacctag cattcatcgt 60

ttacggcgtg gactaccagg gtatctaatc ctgtttgctc cccacgcttt cgcgcctcag 120

cgtcagttac agcccagaga gtcgccttcg ccactggtgt tcctccacat ctctacgcat 180

ttcaccgcta cacgtggaat tccactctcc tcttctgcac tcaagctccc cagtttccag 240

tgcgacccga agttgagcct cgggattaaa caccagactt aaagagccgc ctgcgcgcgc 300

tttacgccca ataattccgg acaacgcttg ccccctacgt attaccgcgg ctgctggcac 360

gtagttagcc ggggctttct tctcaggtac cgtcactctt rtagcagtta ctctacaaga 420

cgttcttccc tggcaacaga gctttacgat ccgaaaacct tcatcactca cgcggcgttg 480

ctccgtcagc tttcgcccat tgcggaagat tccctactgc tgcctcccgt agagtctggg 540

ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca tcgtcgcctt 600

ggtagscctt taccccacca actagctaat gcgccgcmgs catccmcaag tgacagattg 660

ctccgtcttt cctccttctc cc 682

<210> 9

<211> 851

<212> DNA

<213> Paenibacillus jamilae K 1.14

<400> 9

atgtgttaac ttcggcacca agggtatcga aacccctaac acctagcatt catcgtttac 60

ggcgtggact accagggtat ctaatcctgt ttgctcccca cgctttcgcg cctcagcgtc 120

agttacagcc cagagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca 180

ccgctacacg tggaattcca ctctcctctt ctgcactcaa gctcyccagt ttccagtgcg 240

acccgaagtt gagcctcggg attaaacacc agacttaaag agccgcctgc gcgcgcttta 300

cgcccaataa ttccggacaa cgcttgcccc ctacgtatta ccgcggctgc tggcacgtag 360

ttagccgggg ctttcttctc aggtaccgtc acyyywrkag cagttactct mymarrcgtt 420

cttccctggc aacagagctt tacgatccga aaaccttcat cactcacgcg gcgttgctcc 480

gtcaggcttt cgcccattgc ggaagattcc ctactgctgc ctcccgtagg agtctgggcc 540

gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc gtcgccttgg 600

taggccttta ccccaccaac tagctatgcg ccgcaggccc atccacaagt gacagattgc 660

tccgcctttc ctccttctcc catgcaggaa aggatgtatc gggtattagc taccgtttcc 720

ggtagttatc cctgtcttgt gggcaggttg cctacgtgtt actcacccgt ccgccgctag 780

gttarttaga agcaagcttc taatyaaccy ccgctcgact tgcatgtata acacgccgcc 840

gcgtcgtctg a 851

<210> 10

<211> 852

<212> DNA

<213> Paenibacillus jamilae O 2.11

<400> 10

atgtgttaac ttcggcacca agggtatcga aacccctaac acctagcatt catcgtttac 60

ggcgtggact accagggtat ctaatcctgt ttgctcccca cgctttcgcg cctcagcgtc 120

agttacagcc cagagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca 180

ccgctacacg tggaattcca ctctcctctt ctgcactcaa gctccccagt ttccagtgcg 240

acccgaagtt gagcctcggg attaaacacc agacttaaag agccgcctgc gcgcgcttta 300

cgcccaataa ttccggacaa cgcttgcccc ctacgtatta ccgcggctgc tggcacgtag 360

ttagccgggg ctttcttctc aggtaccgtc actcttgtag cagttactct acaagacgtt 420

cttccctggc aacagagctt tacgatccga aaaccttcat cactcacgcg gcgttgctcc 480

gtcaggcttt cgcccattgc ggaagattcc ctactgctgc ctcccgtagg agtctgggcc 540

gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc gtcgccttgg 600

taggccttta ccccaccaac tagctatgcg ccgcaggccc atccacaagt gacagattrc 660

tccgtctttc ctccytctcc catgcaggaa aggatrtatc gggtattagc taccgtttcc 720

ggtagttatc cctgtcttgt ggcaggttgc ctacgtgtta ctcacccgtc cgccgctggr 780

ttrtktagaa gcaagcttct amayaayccc gctcgacttg catgtattaa cacgccgtag 840

cgttcgtcct ga 852

<---

Claims (13)

1. The strain Paenibacillus jamilae K 1.14 spore-forming antagonist bacteria with antifusar activity, deposited in the All-Russian collection of industrial microorganisms under the number B-13016. 2. The strain Paenibacillus peoriae O 1.27 spore-forming antagonist bacteria with antifusar activity, deposited in the All-Russian collection of industrial microorganisms under the number B-13017. 3. The strain Paenibacillus peoriae O 2.11 spore-forming antagonist bacteria with antifusar activity, deposited in the All-Russian collection of industrial microorganisms under the number B-13018. 4. The strain Paenibacillus peoriae R 3.13 spore-forming antagonist bacteria with antifusar activity, deposited in the All-Russian collection of industrial microorganisms under the number B-13019. 5. The strain Paenibacillus peoriae R 4.5 spore-forming antagonist bacteria with antifusar activity, deposited in the All-Russian collection of industrial microorganisms under the number B-13020. 6. The strain Bacillus amyloliquefaciens R 4.6 spore-forming antagonist bacteria with antifusar activity, deposited in the All-Russian collection of industrial microorganisms under the number B-13021. 7. The strain Paenibacillus jamilae R 4.24 spore-forming antagonist bacteria with antifusar activity, deposited in the All-Russian collection of industrial microorganisms under the number B-13022. 8. The strain Paenibacillus polymyxa R 5.31 spore-forming antagonist bacteria with antifusar activity, deposited in the All-Russian collection of industrial microorganisms under the number B-13023. 9. The strain Paenibacillus peoriae R 6.14 spore-forming antagonist bacteria with antifusar activity, deposited in the All-Russian collection of industrial microorganisms under the number B-13048. 10. The strain Bacillus amyloliquefaciens V 3.14 spore-forming antagonist bacteria with antifusar activity, deposited in the All-Russian collection of industrial microorganisms under the number B-13024. 11. Biological product for protecting crops from Fusarium, including a combination of strains according to claims. 1-10, containing at least 10 ⁸ CFU / g of viable antagonist bacteria, while the residual moisture of the dry biological product is not more than 10%. 12. A method of manufacturing a biological product based on a combination of strains of spore-forming bacteria-antagonists with antifusarium activity according to claims. 1-10 by their solid-phase cultivation on a nutrient substrate, including preparation of the substrate by hydration in water, heat treatment of the substrate, inoculation of the heat-treated hydrated substrate, incubation, homogenization of the substrate, softened during heat treatment and during fermentation and subsequent drying. 13. A method of protecting crops from Fusarium infection, comprising isolating Fusarium fungi from samples taken in fields at risk of Fusarium disease; selection of strains of spore-forming bacteria-antagonists, the most actively suppressing strains of fungi of the genus Fusarium, distributed in the target territory; preparation of a biological product according to claim 11, the method according to claim 12, processing of the seed with a biological product.

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