Bacterial strains of bacillus amyloliquefaciens species and applicaiton of strains bacillus amyloliquefaciens species in plant cultivation

Abstract

The object of the invention are strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B, deposited in Polish Collection of Microorganisms PCM in Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw. The invention relates also to the application of strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B as means of biostimulation of plant growth and development, and pest management. Strains Bacillus amyloliquefaciens 8 / 6 or Bacillus amyloliquefaciens A12B or their derivatives, such as spores or metabolites, are applied: in the form of aqueous suspension via fertigation, foliar spraying, soil spraying, used for seed treatment or in the solid form for soil application.

Description

[0001] Bacterial strains of Bacillus amyloliquefaciens species and application of strains Bacillus amyloliquefaciens species in plant cultivation

[0002] The object of the present invention are bacterials strains of Bacillus amyloliquefaciens species and the application of strains of Bacillus amyloliquefaciens species in vegetable, fruit plants and crop cultivation, for biostimulation of plant growth and development, and for pest management, including fungal and bacterial pathogens affecting crops both during vegetation period and crop yields storage, consequently decreasing crop yields.

[0003] One of the main objectives of agriculture in highly developed countries is reducing the use of chemical pesticides by introducing environmentally friendly systems of production. The spread and implementation of these systems allows to restore environmental sustainability and to take advantage of its natural immunity against pest and other harmful factors limiting crop production efficiency. There is a growing widespread expectation within the society to develop methods alternative to chemical methods. One of the solutions allowing to limit chemicalization of agriculture is the use of bioproducts - pesticides based on microorganisms enabling plant protection from diseases and simultaneously production of fruits and vegetables safe for consumers with lower chemical pesticide residue.

[0004] The common fungal disease of crops is root rot caused by Pythium ultimum fungus, which attacks roots, notably of the seedlings. Infected plants are characterised by failure to thrive and seedlings die back. Pythium genus may survive in soil as oospores, that is, spores resistant to unfavourable conditions, notably dehydration. Oospores are spread by land particles moved by people or machines, and the disease spreads the fastest in humid conditions at high water content (>70%) in the ground. Spores infect roots through small lesions, at point of lateral roots formation or through other similarly damaged lesions. Pythium genus may survive in soil or the ground even in the absence of plants without difficulty.

[0005] In the recent years sharp eyespot, a disease caused by Rhizoctonia cerealis, commonly called cereal rhizoctoniose, begins to pose a serious problem in Poland. Rhizoctonia cerealis is a common pathogen of winter crops, mainly wheat and triticale, and its presence has been found in 80% of soils. This pathogen may also affect other cereal and grass species. The fungus is controlled by using fungicides used in cereal crop protection against other stalk base pathogens, however the problem intensifies due to the increased resistance of the sharp eyespot pathotypes to used active ingredient.

[0006] Tan spot is a disease of great commercial importance in majority of wheat cultivation regions in the world, until recently occurring mainly in wheat cultivation areas across North America and Australia. From the mid-eighties it is also observed in Europe. Wheat diseases are cause by many different organisms. Causative factor in tan spot is Pyrenophora tritici-repentis (Died.) Drechsler , conidial stage Drechslera tritici-repentis (Died.) Drechsler. This fungus is a polyphage, affects a plurality of species of Gramineae family, including wheat and triticale. In cultivation of mentioned cereals disease occurs annually, widespread and presents an essential element of disease complex with spot-typical symptoms. Wheat as well as triticale are subject to infection at every developmental stage. Not only leafs are affected, as one could suppose considering disease name, but also stalks, rhizomes and caryopsis. This pathogen survives as dormant mycelium in stubble field and crop residues.

[0007] Other more serious fungal diseases of many plants, called fusariosis, are caused by Fusarium oxysporum fungus, widespread in greenhouse cultivation. It poses a problem mainly in tomato, cucumber, paprika, pot plants and cut flowers crops, yet it may affect significantly wider spectrum of host plants. Main symptoms of fusarium are withering and wilting of leaves and rotting of roots. Fusarium oxysporum fungi penetrate plant tissues, affecting its vascular bundles, which in result turn brown. The result of the infestation, besides leaves wilting, is also growth inhibition which is caused by obstructed, and in further stages of disease development, entirely suppressed water flow through a plant. Mycelium, which in advanced stage may turn pink due to massive production of Fusarium spores, can be observed on the surface of affected plant. Fusarium oxysporum fungi may spread onto neighbouring plants, so the affected plant may even pose a significant threat to the whole crop. Chemical control of fusariosis on affected plants is not feasible. Therefore, preventing pathogen emergence is of particular significance. Though the disease is soil borne, it may also afflict plants grown in non-soil bound growing systems, e.g. in mineral wool.

[0008] Gray mold caused by Botrytis cinerea fungus affects multiple plant species, whereby it may occur throughout the vegetation period, affecting all epigeal parts. The causer of disease affects crop, fruit and ornamental plants. Berries are particularly susceptible to infections. The pathogen may inflict damage already at the germination stage, causing damping-off and dying of young seedlings. Gray mold also affects vegetables during storage period. The fungus requires high relative humidity and strongly damp leaves to infect and develop. Little amount of light, weakening of plants by other diseases as well as calcium and potassium deficiency in soil promote the development of the disease.

[0009] Mildew is a collective name of a group of diseases caused by fungi affecting epigeal parts of plants. These fungi are highly specialized and often affect one selected plant species. Therefore in every crop there might be a different fungus species that causes the disease. Thus, for example, Podosphaera pannosa generally causes the disease on roses, Erysiphe graminis or Blumeria graminis on grasses and cereals, Uncinula necator on grapevines, Erysiphe heraclei on carrots, and Podosphaera leucotricha on apple trees. These fungi may cause symptoms on all epigeal parts: leaves, stems, fruits and flowers. Besides vegetable and ornamental plant crops, mildew is also found in berry crops. Despite that almost every crop is affected by different species of pathogenic fungus, the common trait of mildews are white, powdery stains, appearing most often on upper side of leaves. Typically, they start to appear on lower part of the plant. Once the fungus spread, it may cover the whole surface of the leaf, causing yellowing and eventually drying out. In case of very severe infestation, symptoms may also appear on the lower side of the leaf and, depending on the species, on other epigeal parts of the plant.

[0010] Leaf blight caused by Zymoseptoria tritici fungus is currently the most important wheat disease in Europe and one of the most economically detrimental disease of this crop in the United States. The disease is characterized by necrotic changes on leaves and stems which develop after collapse of infected cells and spread during cold, damp weather. Currently several fungicides are used to control this disease, however multiple Zymoseptoria tritici populations have developed fungicide resistance quickly. The studies on the use of biological pesticides are conducted, but none is yet available for commercial production.

[0011] Clubroot causing Plasmodiophora brassicae protozoon can be found across the world in all temperate zones. It infects over 300 species of 64 genera of cruciferous plants, such as, among others, head cabbage, savoy cabbage, leaf mustard, brussels sprouts, small radish, radish, turnip, cauliflower, broccoli, rapeseed and kohlrabi. His detrimental effect depends on soil pH: the lower it is, the more favourable the conditions for development of the pathogen. The infection occurs the fastest at pH of 5, 3-5, 7, soil temperature of 18-26°C and humidity above 70% of maximum water holding capacity. The type of nitrogen fertilizers that are used during vegetable cultivation have also influence on the speed of plant infestation. Liming and increasing soil pH to minimum 7 does not cause the destruction of clubroot spores in soil, but only their germination is suppressed. At the moment there are no registered chemical pesticides for direct control of clubroot. The Institute of Plant Protection in Poznan has conducted a research on different methods of soil sterilization in order to inhibit Plasmodiophora brassicae which showed that the most effective method for destroying spores is soil combustion, autoclaving and exposure to high temperature, while deep freezing of infected soul was entirely ineffective.

[0012] In recent years there have been multiple reports on using in plant cultivation a bacteria of Bacillus amyloliquefaciens species that is found in soils across the world. As described, among others, in the paper of Helg George “Use Bacillus Amyloliquefaciens to Organically Control Plant Disease” (2019, gardenerspath.com), these bacteria inhibit the development of many plant pathogens. In the paper the plant pathogens controlled by Bacillus amyloliquefaciens, such as, among others soil fungal pathogens - Fusarium, Pythium, Rhizoctonia, Phytophtora and Verticillium, foliar - several rusts, powdery mildew, downy mildew, Botrytis cinerea and Allernaria, are enumerated. On the other hand, identified bacterial foliar pathogens were Pseudomonas syringae pv. tomato and Xanthomonas. In Canada a strain of Bacillus amyloliquefaciens has been registered as a preventive for control of fire blight.

[0013] The invention described in EP3383183 Bl relates to pesticidal mixtures comprising as active ingredients strain MBI600 of Bacillus amyloliquefaciens ssp. plantarum deposited in American Type Culture Collection (ATCC) nr SD-1414 (component A) and cis-jasmone (component B), whereby the ratio of strain MBI600 of Bacillus amyloliquefaciens ssp. plantarum to cis-jasmone is from 1016CFU / 1 g to 105CFU / 1 g. The invention relates also to a method for pest control, notably nematodes.

[0014] A composition including a new strain RTI301 of Bacillus amyloliquefaciens, deposited in American Type Culture Collection (ATCC) nr PTA-121165, of growth enhancement activity and plant pathogen control, is known from patent application EP3240405 Al. The new strain may be used in mixture with other microbiological or chemical insecticides, fungicides, herbicides, vegetal extracts and plant growth regulators. The composition is in the form of fluid or oil dispersion and the strain RTI301 of Bacillus amyloliquefaciens, is present in the concentration of about 1,0* 108CFU / ml to 1,0* 1012CFU / ml. The composition may also have a form of dust, dry wettable powder or granule, and Bacillus amyloliquefaciens RTI301is present in the concentration of about l,0x 108CFU / g to l,0x l012CFU / g.

[0015] A new strain of bacteria Bacillus amyloliquefaciens deposit number DSMZ 29231, which was deposited in Leibniz-Insti tut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, is known from the patent description of EP3197280 Bl. The strain is used in prevention and / or treatment of plant diseases caused by pathogens such as fungi or bacteria. Among the most effectively controlled pathogens is fungus species Ascomycetes Sclerotinia minor and bacteria species of Ralstonia solanacearum. In the description the results of the greenhouse study of the lettuce infected with Ascomycetes Sclerotinia minor and tomatoes infected with Ralstonia solanacearum were shown.

[0016] In the patent description CN10498809 IB B a new strain Fl of Bacillus amyloliquefaciens, deposited in China General Microbiological Culture Collection Center (CGMCC) under number 10942, of high inhibitory activity against Aspergillus niger, Aspergillus albicans, Aspergillus parasiticus and Asepergillus flavus, was disclosed. Antifungal proteins produced by the new strain are notably resistant to infections caused by black pepper mold during its storage. The invention described in CN10663592 IB discloses a new strain Y15 of bacteria Bacillus amyloliquefaciens. desposited China General Microbiological Culture Collection Center (CGMCC) under number 13327 that prevents diseases and promotes plant growth. The new strain is preferably used to prevent and treat frogeye leaf spot in watermelon, tomato yellow leaf curl virus and promoting growth of watermelon, tomato, cucumber, rice, chrysanthemum and others.

[0017] On the other hand, a new strain N22 of Bacillus amyloliquefaciens. desposited China General Microbiological Culture Collection Center (CGMCC) under number M2020256 is known from the patent description CN112011477B, as well as its use in prevention and treatment of brown rot in peaches caused by Monilinia fructicola. In order to prolong the storage period and retain freshness of peaches after harvest, fluid inoculum comprising Bacillus amyloliquefaciens N22 at 109~ 1010cfu / mL is used.

[0018] As was described in another publication Lin Luo et al., “ Bacillus amyloliquefaciens as an excellent agent for biofertilizer and biocontrol in agriculture: An overview for its mechanisms". Microbiological Research, Vol. 259, June 2022, bacteria has an ability to promote plant growth through different mechanisms, among others, improving nutrients availability in soil, including nitrogen supply, solubilization of organic and non-organic phosphates, providing potassium from its insoluble forms, chelation of iron ions as well as increasing plant resistance to biotic stress from soil pathogens.

[0019] Phosphorus is an important macronutrient necessary to growth, improving healthiness and high crop yield. Phosphorus in soul may occur in the form of organic or mineral compounds. Particular phosphorus compounds are characterized by greatly varying water solubility and weak acids, and consequently a different availability to plants. The content of phosphorus compounds soluble in weak acids in soil determines the availability of this mineral to plants. The phosphorus available to plants account is present at only 2 to 10% of general phosphorus contained in soil. Phosphorus- based fertilized are routinely used in soul in farming practice. Used phosphorus fertilizer may be however quickly immobilized in result of precipitation with aluminium and calcium contained in soil, whereby it becomes unavailable to plants, constituting the reserve of this nutrient ingredient in soil, of highly limited availability to plants. Soil erosion leads also to the loss of phosphorus in the soil which typically accumulates in lakes and rivers, causing eutrophication. Deploying microorganisms capable of phosphorus solubilization, that is improving its solubility, and consequently its availability in soil to plants, seems to be a perfect alternative to phosphorus-based mineral fertilizers. Potassium has a very important part in crop management. The crucial role of this element is a responsibility for water management of the plant which is essential during droughts. Besides, it also takes part in regulating nitrogen uptake and is an important element as well in building stress resistance. However, soils in Poland are not rich in available potassium that is ranked in second place after acidification as a factor decreasing soil fertility and also influencing on crop yield. According to the 2020 data provided by GIOS (Glowny Inspektorat Ochrony Srodowiska - Chief Inspectora for Environmental Protection), described in “Monitoring chemism of arable soils in Poland” programme, available potassium content in soils in Poland is average or low. Potassium fulfils many important functions for plants, among which the most important one is its participation in water management of the plant which is very important in periods of water shortages, and proper nutrition of plants with this element improves tolerance to water stress. Plants that are well nourished with this element uses less water to produce yield which is particularly important during drought periods. The higher osmotic pressure in cells occurs in plants well nourished with this element which is preferable regarding frost resistance, and thanks to thicker cell walls of plant stem they are less susceptible to failover. Potassium is also responsible for transport of reserve substances to storage organs which is very important for forming reserve organs: roots, bulbs and seeds. It also activates enzymes in mitochondria which is important with regard to biotic and abiotic stresses. Potassium is necessary ingredient in protein synthesis, because it is responsible for correct water management that is a condition for effective utilization of nitrogen.

[0020] In farming practice silicon-based fertilizers are exploited more and more often. Silicon is one of the most common elements on the Earth. It constitutes about 20-odd per cent of lithosphere, it is comprised in more than 300 minerals occurring in nature. It is one of the fundamental soil constituents, because it is component of nearly all bedrocks from which soil forms. In soils of temperate zones silicon typically occurs in soil solid phase in the form of primary and secondary minerals (silicates and quartz), small amount of silicon is present in soil in the form of plant debris and microorganisms. The content of total silicon in soils is very high, typically about 30%. The original source of silicon available to plants in soils are weathering processes of primary and secondary silicates and aluminosilicates, rocks comprising in their composition minerals rich in silicon compounds being subjected to weathering under environmental conditions which leads to formation of silicon compounds with different type crystallization and free silica. Silicon compounds soluble in water play an important role in the accessibility process of certain elements from soil to plants. Deploying microorganisms capable of solubilizing silicon that is increasing its solubility, and consequently availability to plants in soil, seems to be a valuable addition to silicon content in soil and plants.

[0021] In practice it is often the case that there are several pathogens on the plant. In such case, the use of available solutions may require the use of a few different formulations in pest management, each designed to control different pathogen. In addition, further products intended for biostimulation of plant growth and development are also used. Consequently, it leads to increased costs of pest management and also excessive chemicalization of natural environment. Surprisingly, this problem has been solved in the present invention.

[0022] The object of the invention is isolating new strains of bacteria of Bacillus amyloliquefaciens species of such broad spectrum that its deployment in crop cultivation not only protects plants from many common pests, but also lead to biostimulation of plant growth and development.

[0023] Core inventive advance is Bacillus amyloliquefaciens 8 / 6 strain, deposited in Polish Collection of Microorganisms PCM in Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw, under number B / 00390, on October 6, 2021.

[0024] Core inventive advance is Bacillus amyloliquefaciens A12B strain, deposited in Polish Collection of Microorganisms PCM in Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw, under number B / 00391, on October 6, 2021.

[0025] Core inventive advance is also the use of strains Bacillus amyloliquefaciens species, deposited in Polish Collection of Microorganisms PCM in Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw on October 6, 2021, selected from among strains of Bacillus amyloliquefaciens 8 / 6, deposition number B / 00390, or Bacillus amyloliquefaciens A12B, deposition number B / 00391, as a means of biostimulation of plant growth and development, and of pest management.

[0026] Preferably, strains Bacillus amyloliquefaciens 8 / 6 or Bacillus amyloliquefaciens A12B, or derivatives thereof, such as spores or metabolites, are applied: in the form of aqueous suspension via fertigation, foliar spraying, soil spraying, used for seed treatment or in the solid form for soil application.

[0027] Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B that are object of the invention stand out due to broad spectrum of action. These strains show biostimulation properties, among others, via silicon, potassium and phosphorus solubilization. Each strain is characterized by simultaneous antagonistic effect against many pests that are causing diseases such as root rot, sharp eyespot, tan spot, fusariosis, gray mold, leaf blight as well as clubroot. Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B may be used in pest management of multiple plant species, notably such as, among others, cabbage, paprika, strawberry, wheat. The studies has shown that both Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have a exceptionally strong antagonistic effect and efficiency in suppressing development of mycelium in comparison to at least 15 tested fungal pathogens of plants. Their use allows to limit costs of pest management through decreasing the number of formulations controlling different pathogens occurring simultaneously during the same period on plants and thereby also allows to avoid excessive chemicalization of plants. Coupling above mentioned features with high durability and stability of spores-forming bacteria of Bacillus amyloliquefaciens species allow to obtain a formulation which apart from broad spectrum of action is characterized by resistance to transport conditions and long shelf life.

[0028] In following examples the characteristics of strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B are presented, as well as study results confirming their capacity of phosphorus, potassium and silicon solubilization. Study results confirming antagonistic properties against selected fungal pathogens are also presented, in exemplary studies conducted in laboratory tests and greenhouse and field experiments, on cabbage, paprika, strawberry and wheat crops. These examples do not restrict core inventive advance or limit the scope of the invention, since they are only illustrative.

[0029] Example 1

[0030] Identification of strain Bacillus amyloliquefaciens 8 / 6

[0031] Strain Bacillus amyloliquefaciens 8 / 6 was deposited in accordance with the Budapest Treaty in Polish Collection of Microorganisms PCM in Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw. The deposit was made on October 6, 2021. The deposit was assigned number B / 00390.

[0032] Isolating of strain Bacillus amyloliquefaciens 8 / 6 culture

[0033] Strain Bacillus amyloliquefaciens 8 / 6 has been isolated from a soil acquired from a farmland. Soil samples of 10 g have been suspended in 90 ml of sterile distilled water and afterwards stirred vigorously. The sample was heated at 70°C for 15 minutes. A dilution series of tested sample (to 10'9) was prepared from obtained suspension. 0.1ml of every dilution was sampled and spread plated on solidified substrate 1 / 5 NPT. Composition of NPT culture medium is shown in Table 1.

[0034] Table 1

[0035] All culture medium components have been dissolved in H2O, brought to pH of 5.75 and autoclaved at 121 °C for 15 minutes.

[0036] Plates have been incubated at 28°C for 72 hours. Individual cultures have been streak plated on suitable solid substrate. Plates have been incubated at 28°C for 72 hours. In order to obtain pure cultures the procedure was repeated several times.

[0037] Isolated pure cultures of Bacillus amyloliquefaciens have been transferred to test tubes containing fluid culture medium NPT. They have been incubated for 24 hours at 28°C. The cells have been stored using off-the-shelf (Microbank, Biorcorp) cryovials designed to store bacterial and fungal cultures.

[0038] Species identification of strain Bacillus amyloliquefaciens 8 / 6

[0039] Species identification of strain have been determined by means of phylogenetic analysis conducted on the basis of sequence:

[0040] A. 16S rRNA encoding gene

[0041] B. By analysing whole genome sequence of the strain.

[0042] A, Analysing sequence of 16S rRNA gene

[0043] Genomic DNAhas been isolated from bacteria using commercial DNA isolation kit Genomic Mini AX Bacteria Kit (A&A Biotechnology, Poland) in accordance with the manufacturer’s instructions. The quality of obtained eluates comprising DNA has been assessed spectrophotometrically using NanoDrop ND-1000 (NanoDrop Technologies, USA).

[0044] The polymerase chain reaction (PCR) has been conducted in 10 pl reaction mixture, comprising 0.2 pl RUN polymerase along with 1 pl ten times concentrated polymerase buffer provided by the manufacturer (A&A Biotechnology), 0.2 pl of every starter (Genomed S.A.), 0.2 pl comprising a mixture of 2 mM of every deoxynucleotide triphosphate (dATP, dGTP, dCTP i dTTP) (A&A Biotechnology), 50 ng of genomic DNA template. The content of reaction mixture has been supplemented with ultrapure water up to 10 pl of volume. PCR reaction has been performed in T Gradient Thermocycler (Biometra, Germany) apparatus. Amplification conditions have been performed in further steps, starting with 10 minutes denaturation at 94°C, then 35 cycles of 94°C for 40 seconds, 50°C for 40 seconds and 72°C for 2 minutes, and final step of elongation (72°C for 5 minutes).

[0045] Obtained amplification products and DNA mass pattern (GeneRuler™ Express DNA Ladder; Thermo Scientific) have been applied onto 1.0% (w / v) agarose gel comprising ethidium bromide. Sizing has been performed in buffer 1 x TBE (89 mM Triborate, 2 mM EDTA, pH 8.3) at 100 mA. PCR products have been exposed by illuminating gel with UV lamp.

[0046] DNA has been isolated from gel using commercial agarose gel-based DNA isolation kit. For this purpose, a piece of gel containing the right PCR product has been cut out, suspended in 0.4 ml of R7S and incubated at 50°C until the complete dissolution of agarose. Subsequently, 0.2 ml of isopropanol has been added and mixed. The whole has been transferred to minicolumn for purifying DNA and centrifuged (12000*g, 30 s), then washed two times with rinsing solution Al. DNA has been eluted using 30 pl of water. The quality of obtained eluates comprising DNA has been assessed spectrophotometrically using NanoDrop ND-1000 (NanoDrop Technologies, USA). Sequencing of PCR products has been conducted at Genomed S.A. (Warsaw). Obtained DNA sequences have been compared with 16S rRNA sequence base using BLAST software (https: / / blast.ncbi.nlm.nih.gov). Identification conducted on the basis of 16S rRNA gene sequence enabled classifying tested isolates as belonging to Bacillus genus.

[0047] B, Genome sequencing and analysis

[0048] Analysis of whole genome sequence has been performed by Genomed S.A. company. For this purpose, material from 18h bacterial culture on culture medium NB (Nutrient Bulion; BTL sp. z o.o.) has been sampled. DNA has been isolated by A&A Biotechnology company with Genomic Mini AX Bacteria+ kit for isolating DNA from bacteria in accordance with the manufacturer's instructions (A&A Biotechnology, Poland).

[0049] Genomic DNA has been fragmented via sonication using Covaris E210 (Covaris), in accordance with parameters recommended for generating sequencing libraries using Illumina technology. The libraries have been generated using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, E7645L) in accordance with the manufacturer's recommendations. MiSeq sequencing has been performed using paired-end (PE) technology, 2x300 nt, using MiSeq Reagent Kit v3 (600-cycle) (Illumina), in accordance with test protocol. MiSeq reads have been filtered by version 3.0 of Cutadapt software. Quality control of sequencing results has been conducted using FastQC. Analyses resulted in 1 194 104 raw reads and 1 179 656 sequences after read trimming. Bioinformatic analysis has been conducted using version 2.1.0. of Bactopia pack. De novo assembly has been performed using Shovill software with Skesa software as main assembly software. It allowed to assemble de novo sequences of 3 863 919 bp. Selection of reference genome has been performed on the basis of MinHash using sourmash pack.

[0050] Strain Bacillus amyloliquefaciens CC178 (NC 022653.1) from NCBI data base has turned out to be the most similar to the 8 / 6 sample. Similarity between these strains is 99.99%.

[0051] Example 2

[0052] Identification of strain Bacillus amyloliquefaciens A12B

[0053] Strain Bacillus amyloliquefaciens A12B was deposited in accordance with the Budapest Treaty in Polish Collection of Microorganisms PCM in Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw. The deposit was made on October 6, 2021. The deposit was assigned number B / 00391.

[0054] Isolating of strain / A / cv7 / / / .s amyloliquefaciens A12B culture

[0055] Strain Bacillus amyloliquefaciens A12B has been isolated from a soil acquired from a farmland in the same manner as strain Bacillus amyloliquefaciens 8 / 6 that was described in Example 1.

[0056] Species identification of strain Bacillus amyloliquefaciens A12B

[0057] Species identification of strain have been determined by means of phylogenetic analysis conducted on the basis of sequence:

[0058] A. 16S rRNA encoding gene

[0059] B. By analysing whole genome sequence of the strain.

[0060] A, Analysing sequence of 16S rRNA gene

[0061] Genomic DNA has been isolated from bacteria using commercial DNA isolation kit Genomic Mini AX Bacteria Kit (A&A Biotechnology, Poland) in accordance with the manufacturer’s instructions. The quality of obtained eluates comprising DNA has been assessed spectrophotometrically using NanoDrop ND-1000 (NanoDrop Technologies, USA).

[0062] The polymerase chain reaction (PCR) has been conducted in 10 pl reaction mixture, comprising 0.2 pl RUN polymerase along with 1 pl ten times concentrated polymerase buffer provided by the manufacturer (A&A Biotechnology), 0.2 pl of every starter (Genomed S.A.), 1 pl comprising a mixture of 2 mM of every deoxynucleotide triphosphate (dATP, dGTP, dCTP i dTTP) (A&A Biotechnology), 50 ng of genomic DNA template. The content of reaction mixture has been supplemented with ultrapure water up to 10 pl of volume. PCR reaction has been performed in T Gradient Thermocycler (Biometra, Germany) apparatus. Amplification conditions have been performed in further steps, starting with 10 minutes denaturation at 94°C, then 35 cycles of: 94°C for 40 seconds, 50°C for 40 seconds and 72°C for 2 minutes, and final step of elongation (72°C for 5 minutes).

[0063] Obtained amplification products and DNA mass pattern (GeneRuler™ Express DNA Ladder; Thermo Scientific) have been applied onto 1.0% (w / v) agarose gel comprising ethidium bromide. Sizing has been performed in buffer 1 x TBE (89 mM Triborate, 2 mM EDTA, pH 8.3) at 100 mA. PCR products have been exposed by illuminating gel with UV lamp.

[0064] DNA has been isolated from gel using commercial agarose gel-based DNA isolation kit. For this purpose, a piece of gel containing the right PCR product has been cut out, suspended in 0.4 ml of R7S and incubated at 50°C until the complete dissolution of agarose. Subsequently, 0.2 ml of isopropanol has been added and mixed. The whole has been transferred to minicolumn for purifying DNA and centrifuged (12000*g, 30 s), then washed two times with rinsing solution Al. DNA has been eluted using 30 pl of water. The quality of obtained eluates comprising DNA has been assessed spectrophotometrically using NanoDrop ND-1000 (NanoDrop Technologies, USA). Sequencing of PCR products has been conducted at Genomed S.A. (Warsaw). Obtained DNA sequences have been compared with 16S rRNA sequence base using BLAST software (https: / / blast.ncbi.nlm.nih.gov). Identification conducted on the basis of 16S rRNA gene sequence enabled classifying tested isolates as belonging to Bacillus genus.

[0065] B, Genome sequencing and analysis

[0066] Analysis of whole genome sequence has been performed by Genomed S.A. company. For this purpose, material from 18h bacterial culture on culture medium NB (Nutrient Bulion; BTL sp. z o.o.) has been sampled. DNA has been isolated by A&A Biotechnology company with Genomic Mini AX Bacteria+ kit for isolating DNA from bacteria in accordance with the manufacturer's instructions (A&A Biotechnology, Poland).

[0067] Genomic DNA has been fragmented via sonication using Covaris E210 (Covaris), in accordance with parameters recommended for generating sequencing libraries using Illumina technology. The libraries have been generated using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, E7645L) in accordance with the manufacturer's recommendations. MiSeq sequencing has been performed using paired-end (PE) technology, 2x300 nt, using MiSeq Reagent Kit v3 (600-cycle) (Illumina), in accordance with test protocol. MiSeq reads have been filtered by version 3.0 of Cutadapt software. Quality control of sequencing results has been conducted using FastQC. Analyses resulted in 1 027 332 raw reads and 1 013 656 sequences after read trimming. Bioinformatic analysis has been conducted using version 2.1.0. of Bactopia pack. De novo assembly has been performed using Shovill software with Skesa software as main assembly software. It allowed to assemble de novo sequences of 4 035 409 bp. Selection of reference genome has been performed on the basis of MinHash using sourmash pack.

[0068] Strain Bacillus amyloliquefaciens CC178 (NC 022653.1) from NCBI data base has turned out to be the most similar to the A12B sample. Similarity between these strains is 100%.

[0069] Example 3

[0070] Evaluation of capacity of strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B to solubilize undissolved forms of phosphorus has been conducted using plate count test on solid substrate NBRIP with calcium phosphate (pH of 7.0). Tests have been performed in parallel for each strain. Qualitative and quantitative information on the composition of NBRIP solid substrate is shown in Table 2.

[0071] Table 2

[0072] Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have been multiplied separately on TSB substrate (soya casein broth containing casein peptone - 17 g / 1, soya peptone - 3g / l, NaCl 5g / l, KH2PO4 - 2,5g / l, glucose and H2O - 2,5g / l) at 30°C for 24h. lOpl of inoculum with standarised absorbance of IxlO9CFU / ml has been applied on NBRIP solidified substrate. The halo diameter (clear zone) of culture was measured after 14 days of plate incubation at 30°C. The apparition of halo diameter around culture testified the capacity of the microorganism to solubilize phosphorus.

[0073] Quantitative determination of capacity of strains to solubilize phosphorus has been conducted on NBRIP fluid substrate. Qualitative and quantitative information on this substrate is shown in Table 3.

[0074] Table 3

[0075] Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have been multiplied separately on TBS substrate at 30°C for 24h. Subsequently, 5 ml of inoculation culture has been centrifuged and obtained biomass has been suspended in 0.85% NaCl so as to obtain absorbance of about IxlO9CFU / ml. 50 ml of sterilized substrate has been inoculated with 500 pl of ionoculum. In addition, control sample (i.e. non-inoculated culture) has been prepared. Cultures have been incubated at 30°C with shaking for 48 hours. Subsequently cultures have been centrifuged (4600 rpm, 20 minutes), sediment has been discarded and ortophospates (PO43-) have been identified in supernatant using Merck cuvette tests for phosphate identification (no.1.00673). PO43' concentration has been measured using Spectroquant Pharo 100 / 300 intended for research conducted using cuvette tests. In result of conducted experiment it was observed that strain 8 / 6 had solubilized 52.3 mg (+ / - 0.6) PO43', whereas strain A12B had solubilized 45.2 mg (+ / - 0.6) PO43' in 1 litre of culture medium.

[0076] Example 4

[0077] Evaluation of capacity of strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B to solubilize immobilised forms of potassium has been conducted using qualitative test on solid substrate. Tests have been performed in parallel for each strain. The apparition of halo diameter around grown culture testified the capacity of the tested strain to provide insoluble forms of potassium. Musocvite Mica, a mineral from the group of silicates, basic potassium and aluminium aluminosilicate, has been used as a source of insoluble potassium.

[0078] Qualitative and quantitative information on this substrate is shown in Table 4. Table 4

[0079] Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have been multiplied separately on TBS substrate at 30°C for 24h. After incubation period cultures have been concentrated 2 times and obtained bacterial suspension (10 pl) has been applied on solidified substrate. Plates have been incubated at 30°C. The apparition of clear zone around grown culture testified the capacity of the microorganism to solubilize potassium.

[0080] Quantitative determination of potassium solubilization has been conducted on Aleksandov fluid substrate. Qualitative and quantitative information on this substrate is shown in Table 5.

[0081] Table 5

[0082] Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have been multiplied separately on TBS substrate at 30°C for 24h. Subsequently, 5 ml of inoculation culture has been centrifuged and obtained bacterial has been suspended in 0.85% NaCl so as to obtain absorbance of about IxlO9CFU / ml. 50 ml of sterilized substrate has been inoculated with 2.5 ml of inoculum. In addition, control sample (i.e. non-inoculated culture) has been prepared. Cultures have been incubated at 30°C with shaking for 5 days. Subsequently cultures have been centrifuged (4600 rpm, 20 minutes), sediment has been discarded and potassium has been determined in supernatant using Merck cuvette tests for determining potassium (no. 1.14562). K+concentration has been measured using Spectroquant Pharo 100 / 300 intended for research conducted using cuvette tests. In result of conducted experiment it was observed that strain 8 / 6 had solubilized 2.67 mg (+ / - 0.058) K+, whereas strain A12B had solubilized 2.37 mg (+ / - 0.058) K+in 1 litre of culture medium.

[0083] Example 5

[0084] Evaluation of capacity of strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B to solubilize immobilised forms of silicon has been conducted using plate count test on solid substrate with magnesium trisilicate. Tests have been performed in parallel for each strain. Qualitative and quantitative information on solid substrate with magnesium trisilicate is shown in Table 6.

[0085] Table 6

[0086] Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have been multiplied separately on TBS substrate at 30°C for 24h. After incubation period cultures have been concentrated 2 times and obtained bacterial suspension (10 pl) has been applied on solidified substrate. Plates have been incubated at 30°C for 96h. The apparition of clear zone around grown culture testified the capacity of the microorganism to solubilize silicon.

[0087] Quantitative determination of silicon solubilization has been conducted on fluid substrate with magnesium trisilicate. Qualitative and quantitative information on this substrate is shown in Table 7.

[0088] Table 7

[0089] Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have been multiplied separately on TBS substrate at 30°C for 24 hours. Subsequently, 5 ml of inoculation culture has been centrifuged and obtained bacterial has been suspended in 0.85% NaCl so as to obtain absorbance of about IxlO9CFU / ml. 100 ml of sterilized substrate has been inoculated with 2.5 ml of ionoculum. In addition, control sample (i.e. non-inoculated culture) has been prepared. Cultures have been incubated at 30°C with shaking for 5 days. Subsequently cultures have been centrifuged (4600 rpm, 20 minutes), sediment has been discarded and silicon has been determined in supernatant using Merck cuvette tests for determining silicic acid (no. 1.14562). SiCh concentration has been measured using Spectroquant Pharo 100 / 300 intended for research conducted using cuvette tests.

[0090] In result of conducted experiment it was observed that strain 8 / 6 had solubilized 41.3 mg (+ / - 0.2) SiCh, whereas strain A12B had solubilized 39.3 mg (+ / - 0.2) SiCh in 1 litre of culture medium.

[0091] Example 6

[0092] Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have been used in cabbage, paprika and wheat crops, and tests for properties biostimulating growth and development of these plants have been performed.

[0093] Greenhouse experiment on cabbage and paprika - application via fertigation

[0094] White head cabbage var. Jaguar Fl seeds have been planted in multiceullular trays. The plants have been watered 2 times with suspension containing: one thereof strain Bacillus amyloliquefaciens 8 / 6, the other Bacillus amyloliquefaciens A12B, each of 108CFU / ml, on day of planting and 7 days before transplanting the plants to ground artificially populated with Plasmodiophora brassicae. Control combinations have been watered with water. Tests have been performed in parallel for each strain.

[0095] The experiment was established in 4 replications, one replication included 5 plants. After 7 and 11 weeks since transplanting plants to infected substrate, fresh weight of epigeal part and roots has been evaluated (Table 8). Standard IR is a commercial pesticide. The results presented in Table 8 indicated that 8 / 6 and A12B have an influence on increased cabbage weight. 8 / 6 and A12B strains have increased the cabbage biomass by 25%.

[0096] Table 8

[0097] Paprika var. Robertina seeds have been planted in seed boxes and subsequently bedded out into multicellular trays. The plants have been watered with suspension containing: one thereof strain Bacillus amyloliquefaciens 8 / 6, the other Bacillus amyloliquefaciens A12B, each of 108CFU / ml, 10 days before transplanting the plants to ground infected with Fusarium oxysporum (artificial inoculation). Subsequently, 2 further applications have been performed: 24 hours and 12 days after planting in the substrate. The experiment was established in 4 replications, one replication included 5 plants. After a month since transplanting plants to infected substrate, the experiment was ended, and fresh weight and dry weight of epigeal part and roots has been evaluated. Biometric parameters of paprika plants that have been treated with strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B, on the day of termination of the experiment, are presented in Table 9. Water was the control and Standard IR is a commercial pesticide.

[0098] The experiment results confirm that the use of Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B strains have contributed to increasing fresh and dry weight of paprika epigeal part and roots.

[0099] Field experiment on spring wheat - application via foliar spraying

[0100] Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have been tested in plot experiment. Spring wheat has been seeded in experimental plots of surface 15 m2. The experiments has been conducted in randomized blocks design in 4 replications. The suspension containing: one thereof Bacillus amyloliquefaciens 8 / 6, another Bacillus amyloliquefaciens A12B, 108CFU / ml each, has been applied on the leaves using 4 ml / ha of working liquid. Tests have been conducted in parallel for each strain. The control has been sprayed with water. The evaluation of retained assimilation surface of spring wheat leaves and crop yield have been presented in Table 10. The control was water and Standard I is a commercial pesticide.

[0101] Table 10

[0102] The experiment results confirm that the use of Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B strains have limited infestation of plants caused by fungal disease, whereby leaf assimilation surface (conducting photosynthesis) was the largest. Further, foliar application of strains has contributed to increasing spring wheat yield by 3% in comparison with the control. Tests have confirmed that Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B strains have properties biostimulating growth and development of crops.

[0103] Example 7

[0104] Determining antagonistic properties of Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens strains relative to selected fungal pathogens Antagonism against fungal pathogens has been performed in laboratory tests. Tests have been conducted in parallel for each strain. A mycelium disk, cut with corkborer, has been applied on scale pans with PDA agar (Potato-Dextrose Agar) at a distance of 2 cm from the edge. Fungal isolates have been incubated at standard temperature for a period of 1-5 days, until the moment when mycelium was reaching inside scale pan. Subsequently, at a distance of 5 cm from the mycelium disk, a fresh bacterial suspension of 10 pl has been spread. The scale pans have been incubated at ambient temperature for 14 days, taking a reading after 7 and 14 days. Antagonistic effect has been determined on the basis of the apparition (or lack thereof) of zone of suppressed spore growth in bacterial multiplication area. 15 pathogen isolated have been deployed in tests:

[0105] 1. IOR collection number: 1089 Pathogen species: Mycosphaerella fragariae

[0106] 2. IOR collection number: 59 Pathogen species: Rhizoctonia cerealis

[0107] 3. IOR collection number: 2029 Pathogen species: Fusarium oxysporum

[0108] 4. IOR collection number: 2160 Pathogen species: Fusarium solani

[0109] 5. IOR collection number: 2105 Pathogen species: Fusarium culmorum

[0110] 6. IOR collection number: 1715 Pathogen species: Phytophtora cactorum

[0111] 7. IOR collection number: 2207 Pathogen species: Phytophthora nicotianae

[0112] 8. IOR collection number: 1664 Pathogen species: Helminthosporium solani

[0113] 9. IOR collection number: 2369 Pathogen species: Phytium ultimum

[0114] 10. IOR collection number: 2227 Pathogen species: Botrytis allii

[0115] 1 1 . IOR collection number: 2299 Pathogen species: Botrytis cinerea

[0116] 12. IOR collection number: 2233 Pathogen species: Alternaria brassicae

[0117] 13. IOR collection number: 1548 Pathogen species: Alternaria dauci

[0118] 14. IOR collection number: 2046 Pathogen species: Alternaria solani

[0119] 15. IOR collection number: 2157 Pathogen species: Colletotrichum acutatum

[0120] In result of conducted experiments, antagonistic effect of both strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B has been demonstrated relative to every of above mentioned 15 tested fungal pathogens. Mycelium growth inhibition in every case has occurred a dozen or so, or even tens of mm before the bacterial growth spot, which testifies exceptionally strong antagonistic effect of Bacillus amyloliquefaciens 8 / 6 and A12B strains. Example 8

[0121] Use of Bacillus amyloliquefaciens 8 / 6 to protect winter wheat from root rot caused by Pythium ultimum and sharp eyespot caused by Rhizoctonia cerealis

[0122] In pot experiments of treating winter wheat var. Fidelius seed, the effect of its treatment against Pythium ultimum and Rhizoctonia cerealis have been evaluated. Wheat seed has been treated with Bacillus amyloliquefaciens 8 / 6 strain (aqueous suspension containing 108CFU / ml of Bacillus amyloliquefaciens 8 / 6). The substrate before seeding had been artificially inoculated with fungal pathogens. Every combination had been drawn in 4 replications, one replication constitutes 1 pot, in which 50 wheat seeds had been seeded. The effect of treating wheat seeds with Bacillus amyloliquefaciens 8 / 6 on limiting infestation caused by Pythium and Rhizoctionia was illustrated in Table 11. The control was water and Standard I is a commercial pesticide.

[0123] Table 11

[0124] It was observed that the new strain 8 / 6 has a high efficiency (above 70%) in limiting winter wheat seedling infestation by Pythium ultimum and Rhizoctonia cerealis, in comparison to the control object.

[0125] Example 9

[0126] Use of Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B to protect paprika from fusariosis caused by Fusarium oxysporum

[0127] The evaluation of efficiency Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B strains in limiting paprika fusariosis has been conducted in greenhouse experiment. Paprika var. Robertina plants have been watered with 5 ml of aqueous suspension containing: one thereof Bacillus amyloliquefaciens 8 / 6, another Bacillus amyloliquefaciens A12B, 108CFU / ml each, for 10 days before transplanting them to the substrate artificially infected with Fusarium oxysporum pathogen. Subsequently, two further applications have been performed using 1 ml of aqueous suspension containing Bacillus amyloliquefaciens 8 / 6 or 1 ml of aqueous suspension containing Bacillus amyloliquefaciens A12B, at 7-14 day intervals. The experiment was established in four replications with 5 plants in randomized blocks design. The effect of Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B strains on infestation of paprika plants by Fusarium oxysporum, was shown in Table 12. The control was water and Standard I is a commercial pesticide.

[0128] Table 12

[0129] Obtained results confirm that strain Bacillus amyloliquefaciens 8 / 6 had efficiently limited plant infestation by Fusarium oxysporum - 14 days after 3rdapplication the efficiency had exceeded 90%, significantly more than used reference pesticide Standard I. It was the highest efficiency in the entire experiment. Further, strain Bacillus amyloliquefaciens 8 / 6 had caused an increase of fresh weight and dry weight of paprika epigeal part and roots what had been already shown in Table 8 of Example 6. On the other hand, strain Bacillus amyloliquefaciens A12B had turned out to be not as efficient as strain 8 / 6 and in the first evaluation it had presented efficiency of about 40% and efficiency has decreased in further evaluations, however, similarly to strain Bacillus amyloliquefaciens 8 / 6, strain A12B also increased weight of paprika plants in comparison to the control.

[0130] Example 10

[0131] Use of Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B to protect strawberry from gray mold caused by Botrytis cinerea

[0132] The evaluation of efficiency Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B strains in limiting strawberry gray mold has been conducted in field experiment via foliar application. Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B, in field experiment on strawberry crop, have been applied on leaves four times from the start of florescence phase every 7-10 days. In every application 4 1 rate of aqueous suspension containing: one thereof Bacillus amyloliquefaciens 8 / 6, another Bacillus amyloliquefaciens A12B, 108CFU / ml on 1 ha each, was used. The experiment was established in randomizes block design in four replications.

[0133] Tests have been conducted in parallel for each strain. The average percentage of infestation of strawberry fruit surface and an efficiency of strain 8 / 6 and A12B in pest management of strawberry against Botrytis cinerea was shown in Table 13. The control was water and Standard I is a commercial pesticide.

[0134] Table 13

[0135] An efficiency of strain 8 / 6 for first 17 days of experiment amounted to approximately 30%. Its highest efficiency against Botrytis cinerea was found on the 7thday after 3rdapplication and it was 52%. An efficiency of strain A12B 10 days after 1stapplication was the highest efficiency in the whole experiment in the further two evaluations the efficiency has decreased to 20%, however, 14 days after 3rdapplication the efficiency of the strain was 62% and was the highest in the whole experiment.

[0136] In addition, during the experiment, fruits have been collected 3 times, subsequently they were placed at 5°C for 5 days. After that period, strawberries have been removed and damage to the fruits caused by gray mold was evaluated in 5-point scale (0 - no damage, 1 - single brown stains, 2 - visible multiple brown stains, 3 - wet stains, 4 - visible mold, 5 - 50% of fruit covered with mold). Every time the evaluation was conducted on 2 kg sample of fruits. The effect of application of aqueous suspensions containing strains Bacillus amyloliquefaciens 8 / 6 or Bacillus amyloliquefaciens A12B on shelflife of strawberries was shown in Table 14. In each of 3 harvests after 5 days of storage of fruits at 5°C, the highest number of strawberries without or with insignificant damage symptoms (qualified in 5 -point scale as 0 and 1) was in a combination treated on leaves with aqueous suspension containing 108CFU / ml of a strain Bacillus amyloliquefaciens. For a combination treated with strain Bacillus amyloliquefaciens 8 / 6 the number of fruits that were covered with gray mold in 50%, was the smallest. The control was water and Standard I is a commercial pesticide.

[0137] Table 14

[0138] Example 11 Use of Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B to protect spring wheat from tan spot caused by Pyrenophora tritici-repentis, powdery mildew caused by Blumeria graminis fungus and leaf blight caused by Zymoseptoria tritici

[0139] Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have been foliar applied on spring wheat crop, with rate of 4 1 of aqueous suspension containing: one thereof Bacillus amyloliquefaciens 8 / 6, another Bacillus amyloliquefaciens A12B, 108CFU / ml on 1 ha each. An experiment has been conducted on plots with 4 replications. Tests have been conducted in parallel for each strain. As a reference product Standard II with a rate of 11 / ha was used. Formulations have been applied in plant development phase BBCH 21-29, BBCH 31-32, BBCH 37-39 and BBCH 51-59. An analysis of infestation with Pyrenophora tritici-repentis fungus, conducted after last application, showed, that strain Bacillus amyloliquefaciens and A12B protect plants against this pathogen and an efficiency of strains 8 / 6 and A12B on lower leaves was respectively 60% and 49%, while on sub-flag leaves above 70% (Table 15). An analysis of the occurrence of the two other important pathogens in wheat crop, Blumeria gramnis and Zymoseptoria tritici, showed that strains 8 / 6 and A12B limit infestation of plants with Blumeria gramnis by 70% (Table 16) and in the case of infestation with Zymoseptoria tritici by 61% (8 / 6) and 72% (A12B) (Table 17). The control was water and Standard II is a commercial pesticide.

[0140] Table 15 Table 16

[0141] Table 17

[0142] The results of conducted experiments confirm that strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B efficiently protect wheat plants against fungal disease what was confirmed by an analysis of retaining assimilation surface of flag leaves LI. Strains 8 / 6 and A12B protect assimilation surface of wheat leaves at above 90%. Simultaneously, strains Bacillus amyloliquefaciens 8 / 6 and A12B have caused an increase in spring wheat yield by 3% in comparison to the control, what had been already shown in Table 10 of Example 6.

[0143] Example 12

[0144] Use of Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B to protect winter wheat from tan spot caused by Pyrenophora tritici-repentis and leaf blight caused by Zymoseptoria tritici Strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B have been applied separately via foliar spraying with rate of 51 of aqueous suspension on ha containing 1 • 108CFU / ml of Bacillus amyloliquefaciens 8 / 6 or Bacillus amyloliquefaciens A12B.

[0145] An experiment has been conducted on plots with 4 replications. Standard II with rate 1 kg / ha was used as a reference product. The control was water. Formulations have been applied in wheat development phase BBCH 30, BBCH 39, BBCH 57-59. The analysis of results presented in Table 18 showed that strains Bacillus amyloliquefaciens 8 / 6 and A12B efficiently protect winter wheat against Zymoseptoria tritici. The efficiency on flag leaves was high, of about 70% for 8 / 6 and of about 90% for A12B, and on lower leaves, including sub-flag leaves, the efficiency of 8 / 6 is 23% and of strain A12B is 34%. On the other hand, the analysis of results presented in Table 19 showed that strains efficiently protect winter wheat against Pyrenphora tritici-repentis . The efficiency on flag leaves is high and it is above 60%, and on lower leaves, including sub-flag leaves, the efficiency is about 20%.

[0146] Table 18

[0147] Table 19

[0148] The results of conducted experiments confirm that strains Bacillus amyloliquefaciens 8 / 6 and Bacillus amyloliquefaciens A12B efficiently protect wheat plants against fungal diseases what was confirmed by an analysis of retaining assimilation surface of flag leaves LI. New strains 8 / 6 and A12B protect assimilation surface of wheat leaves in 95% (Table 20).

[0149] Table 20

Claims

Claims1. Strain Bacillus amyloliquefaciens 8 / 6, deposited in Polish Collection of Microorganisms PCM in Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw, under number B / 00390, on October 6, 2021.

2. Strain Bacillus amyloliquefaciens A12B, deposited in Polish Collection of Microorganisms PCM in Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw, under number B / 00391, on October 6, 2021.

3. The application of strains of Bacillus amyloliquefaciens species, deposited in Polish Collection of Microorganisms PCM in Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw on October 6, 2021, chosen among strains Bacillus amyloliquefaciens 8 / 6, deposit number B / 00390, o Bacillus amyloliquefaciens A12B, deposit number B / 00391, as a means of biostimulation of plant growth and development and pest management.

4. The application according to claim 3, characterized in that strain Bacillus amyloliquefaciens 8 / 6 ox Bacillus amyloliquefaciens A12B or derivatives thereof, such as spores or metabolites, are applied: in the form of aqueous suspension via fertigation, foliar spraying, soil spraying, used for seed treatment or in the solid form for soil application.

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