RU2777606C2 - Compositions and methods for fusarium control - Google Patents

Abstract

FIELD: biotechnology; agriculture.

SUBSTANCE: isolated strain of Bacillus amyloliquefaciens NRRL B-50760, having suppression activity against fusarium, compositions, and methods using the specified strain are proposed. A method for the prevention of fusarium development includes growing a microbial strain or its culture in a growth medium or plant soil, and includes application to the plant or the medium surrounding it of the effective amount of the microbial strain or its culture. Compositions for fusarium suppression, containing a microbial strain or its culture, and a seed resistant to Fusarium fungus infection are also proposed.

EFFECT: inventions are effective in control of fusarium of cereal plants, such as wheat and barley.

19 cl, 7 tbl, 11 ex

Description

[0001] The contents of the attached Sequence Listing are incorporated herein in their entirety by reference. The attached file named "SGI1520-lWO_ST25.txt" was created on July 24, 2012 and is 55 KB in size. This file is accessible with Microsoft Word on a computer running Window OS.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The present invention relates to biological methods for combating phytopathogenic diseases. In particular, the invention relates to compositions and methods that can be used in the control of Fusarium on cereals such as wheat and barley.

BACKGROUND OF THE INVENTION

[0003] Fusarium, also known as scab, pink mold and Fusarium, is a disease that devastates the agricultural land of wheat, barley and many other cereals around the world and especially in the United States, Europe and China. This disease can reach epidemic proportions and cause extensive damage to cereals, especially wheat and barley, in humid and semi-humid cereal growing areas, including India, Russia, France, Germany and the United Kingdom. In particular, fusarium or Fusarium wheat is one of the most detrimental wheat diseases in the United States. Nationwide, this disease, which has developed in the Midwest and the High Plains, has cost the wheat industry millions of dollars, and has thus become a major obstacle to the successful functioning of this industry in recent years. Fusarium, in addition to wheat, also affects barley, oats, corn and many other crops, and reproduces on them.

[0004] This disease can be caused by many different plant pathogens and, first of all, by several species of fungi of the genus Fusarium. Potential causative agents of wheat fusarium include many different Fusarium species, such as F. culmorum , F. graminearum (teleomorph, Gibberella zeae) , F. avenaceum (teleomorph, G. avenacea) , F. poae , as well as pathogens other than genus Fusarium , such as Microdochium nivale (teleomorph, Monographella nivalis) , and Microdochium majus. In the United States, Europe and other most important agronomic areas of the world, Fusarium graminearum (literally Gibberella zeae ) is the dominant causative agent of Fusarium.

[0005] These pathogens typically survive on plant debris. They fall on the spikelets of cereals and infect them during flowering, stopping or partially delaying the development of grain in the cereal spike. As a result, a pathogen that has entered the plant can kill part of the ear or the entire ear. Some infected seeds have such low germination vigor that they often fail to germinate. Germinated infected seeds often die early in the germination phase due to pellicularia or root rot causing poor growth of the grown cereal. Healthy seedlings can also become infected during the emergence phase. In addition to poor, uneconomical growing, crop losses due to pathogen attack can be quite high if conditions are favorable for disease development.

[0006] Fungal pathogens of the genus Fusarium spread throughout cereal cultivation areas throughout the world and cause particularly great damage in areas of high rainfall between flowering and grain filling. If the causative agent is Fusarium graminearum , then the disease requires priority intervention, not only because it reduces the commercial performance of the affected grain, in addition to direct yield losses, but also because Fusarium infection can lead to the accumulation of mycotoxins, trichothecenes, in grains, thus endangering human and livestock health. Trichothecenes are the major mycotoxin contamination of cereals worldwide, capable of causing food refusal, vomiting, diarrhea and weight loss in non-ruminant animals and posing a health risk to other animals and humans when exposed to these toxins at high levels. In the United States, this threat has increased even more due to the recent shift in F. graminearum strains towards greater toxin production and potency. The most frequently detected mycotoxins are deoxynivalenol (DON, also known as vomitoxin) and zearalenone (ZEA). Deoxynivalenol is a particularly virulent toxin that causes gastrointestinal disturbances, which are accompanied by bleeding and other serious conditions in humans and animals that have eaten infected grains, and in some cases can lead to death. Because deoxynivalenol is generally resistant to pH changes and high temperatures, detoxification can be very difficult. Thus, grains contaminated above a certain level cannot be used in brewing, processing, livestock feed and therefore must be disposed of.

[0007] To date, many different strategies have been developed to control Fusarium in crops. The most promising among them are chemical methods, the development of resistant crop varieties, as well as traditional methods of crop rotation and field cultivation. Among these avenues, some effectiveness in reducing Fusarium infection can be obtained from the use of chemical pesticides, however, fungicide residues used in the late stages of crop growth, usually during flowering periods, just a few weeks before harvest, reduce the attractiveness of these methods. On the other hand, an alternative approach to combating this disease is represented by traditional breeding and genetic engineering methods, thanks to which there is a noticeable progress in the development of resistant varieties of agricultural crops. Genetic engineering allows you to change the production of phytohormone and to manipulate its signaling pathway. The progress achieved in recent years in the field of traditional breeding has made it possible to make significant progress in understanding the genetic basis of resistance to Fusarium and to obtain information about the many genes and quantitative trait loci (QTL: quantitative trait loci) that confer this resistance. However, progress in increasing the resistance of agricultural crops to Fusarium has been slow, which is explained, first of all, by the difficulty of studying this disease. In fact, relatively little is currently known about the mechanisms that determine plant resistance or susceptibility to Fusarium. In addition, the genetic diversity of Fusarium species, which are the predominant pathogens, often creates problems in determining how long the effectiveness of chemical fungicides should be and how resistant the crops to be protected should be. As a result, at present, almost all varieties of wheat in agro-industrial production remain vulnerable to infection.

[0008] In addition, despite some success in the fight against Fusarium, which can give traditional methods of arable tillage with burying of crop residues infected with the pathogen, for example, F. graminearum , conventional plowing of the soil after stubble is inconsistent with the established practice of soil conservation, then there is with the condition of minimum arable cultivation. Given the potential for spreading inoculum over long distances and the potential for different crops to become alternative hosts for pathogens, crop rotation is often unacceptable. In addition to polluting the environment with pesticides, their use can give rise to pesticide resistance problems. In addition, registered cases of an increase in the content of DON in grains may also be associated with their use. In addition, the costs and growing problems in both the public and private sectors associated with pesticide pollution and food safety requirements make this method of controlling such diseases less attractive and force the use of pesticides in crop care as little as possible. .

[0009] Summing up, we can say that, despite significant progress in the development of ways to combat Fusarium, reducing the impact of this devastating disease on the production and quality of grain remains an unresolved problem. Therefore, in order to increase the productivity and quality of many grain crops, it is important to search for and develop new ways to combat Fusarium. These problems need to be urgently addressed not only in the United States, but throughout the globe, including Asia and Europe.

[0010] Fighting fusarium with biological agents has received attention since the mid-1990s. Biological agents for such control (BCA: Biological control agents), despite the fact that their number is currently very limited, can, in their hostility to the environment, be very effective in reducing the level of morbidity initiated by pathogens of the genus Fusarium. Public acceptance, compatibility with other crop disease control agents, longevity and persistence, along with other positive factors, all point to the need for development of Fusarium biological control strategies. Means of such biological control can play an important role in the organic grain industry. In conventional grain production, such products can extend the protection of spikelets beyond the flowering phase, when chemical fungicides can no longer be applied. To date, significant progress has already been made in the field of biological control. For example, some strains of spore-producing bacteria (eg Bacillus and Pseudomonas spp .) and yeasts (eg Cryptococcus spp .) exhibit the qualities required to control Fusarium and reduce mycotoxin contamination. However, despite this progress, there remains an unmet need for improved microorganisms for use in Fusarium control. Although BCA agents themselves are already recognized as a more suitable tool for combating plant pathogens, and BCA products are on the market much more widely than ever before, still very few attempts have been made to date to develop strategies and an antagonistic microorganism. for biological control of Fusarium. In addition, the life cycle of the genus Fusarium and other Fusarium pathogens indicates that these pathogens may potentially be suitable for use in biocontrol methods using antagonistic microorganisms in various phases of growth and development. Thus, there is a need to find new biological control agents, preferably with different methods of activity, as well as biocontrol methods that would effectively prevent or suppress the development of Fusarium.

SUMMARY OF THE INVENTION

[0011] The invention relates to compositions containing microbiological strains and cultures. Some strains, cultures and their compositions can be used to control Fusarium, for example, various crops, including wheat and other cereals. The invention also relates to biological control compositions and methods of using these compositions to prevent, control, or treat a disease caused by pathogens and to preserve crops. The invention also relates to methods for using such compositions as biological control agents in combination with other agriculturally effective compounds or compositions for controlling harmful plant pathogens.

[0012] In one aspect, the present invention relates to isolated microbial strains with suppressive activity against Fusarium. The microbial strains according to this aspect are selected from the group consisting of Microbacterium , Bacillus , Mycosphaerella and Variovorax species. In some preferred embodiments, the microbial strains are selected from the group consisting of Mycosphaerella species strain SGI-010-HI 1 (deposit NRRL 50471), Microbacterium species strain SGI-014-C06 (deposit NRRL B-50470), Variovorax species strain SGI- 014-G01 (deposit NRRL B-50469), Bacillus species strain SGI-Q15-F03 (deposit NRRL B-50760), Bacillus species strain SGI-015-H06 (deposit NRRL B-50761) and variants thereof having pesticidal activity. The microbial strain according to other preferred embodiments may contain a DNA sequence showing at least 85% identity to any of the nucleotide sequences shown in the accompanying Sequence Listing. The invention also relates to biologically pure cultures and enriched cultures of the microbial strains described here.

[0013] According to another aspect, the present invention relates to compositions comprising a microbial strain of the invention or a culture thereof and an agriculturally effective amount of a compound or composition selected from the group consisting of an acaricide, a bactericide, a fungicide, an insecticide, a microbicide, a nematicide, pesticide and fertilizer. These compositions in some embodiments of this aspect can be obtained in the form of a preparation selected from the group consisting of an emulsion, colloid, pulverulent preparation, particles, granules, powder, spray and solution; in some other embodiments, these compositions may contain a carrier. In one preferred embodiment, the carrier is agriculturally acceptable. In one particularly preferred embodiment of the invention, the carrier is the seed of a plant. In other preferred embodiments, the composition is a seed coating preparation. In addition, the invention relates to seeds coated with a composition in accordance with the present invention.

[0014] In another aspect, the present invention relates to methods for preventing, inhibiting or treating the development of a plant pathogen. These methods include growing the microbial strain of the invention or a culture thereof in the nutrient medium or soil of the host plant before or simultaneously with the cultivation of the host plant in the nutrient medium or soil, where in some preferred embodiments of this aspect of the invention, the phytopathogen causes fusarium. In one particularly preferred embodiment, the phytopathogen is Fusarium graminearum.

[0015] In another aspect, the present invention relates to methods for preventing, inhibiting or treating the development of plant Fusarium. These methods include the delivery to the plant or to the environment surrounding the plant, an effective amount of the microbial strain of the invention or its culture. In one of the embodiments of the invention, such fusarium is caused by the fungus Fusarium graminearum. In some embodiments of this aspect, the microbial strain or culture thereof is provided to soil, seed, roots, flower, leaf, plant part, or entire plant, where in a preferred embodiment, the plant is susceptible to Fusarium graminearum. In some other preferred embodiments of the invention, the plant is wheat, corn, barley or oats, in another preferred embodiment, the microbial strain of the invention or its culture is introduced into the plant as an endophyte.

[0016] In the following aspect, the invention relates to plants of non-natural origin. Plants of non-natural origin are artificially infected with the microbial strain according to the invention or its culture. In addition, in some preferred embodiments of this aspect, the invention relates to seed, reproductive tissue, vegetative tissue, plant parts and plant progeny of non-natural origin.

[0017] In another aspect, the invention relates to a method for producing an agricultural composition. This method includes inoculating a microbial strain according to the present invention or a culture thereof into or onto a substrate and growing it at a temperature of 1-37°C to produce cells or spores in an amount of at least 10 ² -10 ³ per milliliter or per gram.

[0018] These and other objects and features of the present invention are more fully explained in the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0019] Unless otherwise indicated, all technical terms, conventions, and other scientific terms used herein have their conventional meanings known to those skilled in the art to which the present invention pertains. Certain terms with commonly known meanings are defined here for the purposes of avoidance of confusion and/or for ease of reference, and the inclusion of such definitions here should not be construed as representing a material difference from the meanings generally accepted among those skilled in the art. Many of the methods and procedures that are described here or to which reference is made should be well known to those skilled in the art and widely used by them in accordance with conventional methodologies.

[0020] Unless the context of this description clearly implies otherwise, the singular number of nouns also assumes their meanings in the plural. For example, the term "cell" can mean either one or multiple cells, including mixtures thereof.

[0021] The expressions "antagonistic microorganism" and "microbial antagonist" both mean a microorganism whose strain shows a degree of inhibition of Fusarium that is at a statistically significant level greater than the degree of inhibition from an untreated control.

[0022] Antibiotic: The term "antibiotic" as used herein means a substance capable of killing or inhibiting the growth of a microorganism. Antibiotics can be produced by at least one of these sources: 1) a microorganism, 2) a synthetic process, and 3) a semi-synthetic process. The antibiotic may be a microorganism that produces a volatile organic compound. In addition, the antibiotic may be a volatile organic compound released by the microorganism.

[0023] Bactericidal: The term "bactericidal" as used herein means the ability of a composition or substance to increase the mortality of bacteria or to inhibit their rate of growth. Inhibition of the growth rate of bacteria in most cases can be measured by the decrease in the number of viable bacterial cells over time.

[0024] Biological control: The term "biological control" and its abbreviated form "bio control" as used herein means combating a pathogen, or an insect, or any other undesirable organism with the help of at least one other non-human organism. An example of a known biological control mechanism is the use of microorganisms that fight root rot by outcompeting fungi for space on the root surface, or microorganisms that either inhibit the growth of the pathogen or kill it. A "host plant" in the context of biological control is a plant susceptible to a disease caused by a given pathogen. A "host plant" in the context of separating an organism belonging to a fungal species from its natural environment is a plant that supports the growth of the fungus, that is, for example, a plant of a species for which the fungus is an endophyte.

[0025] The term "cereal" as used herein means any cereal species that may be susceptible to Fusarium. Such susceptible crops include wheat, barley, oats and triticale; however, wheat and barley are two crops for which the disease is a particularly significant economic problem.

[0026] The term "effective amount" means an amount sufficient to obtain useful or intended results. With respect to controlling a disease, treating, inhibiting or protecting a disease, an effective amount is an amount sufficient to suppress, stabilize, reverse the course of the disease, slow or delay the development of the target infection or disease state. Thus, the expression "effective amount" in this description means that amount of antagonistic treatment, which is necessary to reduce the level of development of the pathogen and/or the level of disease caused by the pathogen compared to the level observed in the untreated control. Typically, an effective amount of a given antagonistic treatment results in a reduction of at least: 20%; as a rule, from 30 to 40% and more; as a rule, from 50 to 60% and more; as a rule, from 70 to 80% and more; or, as a rule, from 90 to 95% compared with the level of disease and/or the level of pathogen development observed in the untreated control under the corresponding treatment conditions. An effective amount may be delivered in one or more doses. The actual delivery rate of the liquid preparation usually lies in the range of at least 1x10 ³ to 1x10 ¹⁰ viable cells/ml and preferably from about 1x10 ⁶ to 5x10 ⁹ viable cells/ml. Under most conditions, the antagonistic microbial strains of the invention described in the Examples below will be optimally effective at a delivery rate in the range of about 1×10 ⁶ to 1×10 ⁹ viable cells/mL, if delivery provides substantially uniform contact of at least about 50 % plant tissues. If the antagonists are supplied in the form of a solid formulation, then the rate of delivery should be controlled to provide a number of viable cells per unit area of plant tissue surface comparable to that obtained at the above liquid treatment rates. Generally, the biological control agents of the invention are biologically effective when supplied at concentrations above 10 ⁶ cfu/g (colony forming units per gram), preferably above 10 ⁷ cfu/g, even more preferably above 10 ⁸ cfu/g , and most preferably, if at a concentration of 10 ⁹ CFU / g.

[0027] Further, the expression "effective microbial antagonist" when used with respect to a microorganism means that a given microbial strain exhibits a degree of inhibition of Fusarium that is statistically significantly greater than that observed in an untreated control. Typically, an effective microbial antagonist is capable of producing a reduction of at least: 20% or more; as a rule, from 30 to 40% and more; as a rule, from 50 to 60% and more; as a rule, from 70 to 80% and more; typically 90 to 95% relative to the level of disease and/or the level of pathogen development present in the untreated control under appropriate treatment conditions.

[0028] Composition: The term "composition" means the combination of an active agent with at least another compound, carrier or composition, inert(s) (e.g., detectable agent or label, liquid carrier, etc.) or active, for example, pesticide.

[0029] Isolated microbial strain, isolated culture, biologically pure culture, and enriched culture: As used herein, the term "isolated" when applied to a microorganism (e.g., bacteria or microfungus) means a microorganism that has been isolated and/or purified from a medium where it comes naturally. Similarly, the term "isolated strain" of a microbe means a strain that has been isolated and/or purified from its natural environment. Thus, the term "isolated microorganism" does not include in its meaning a microorganism present in an environment in which it normally occurs. In addition, the term "isolated" does not necessarily reflect the extent to which a given microbe has been purified. The term "substantially pure culture" of a microbial strain means a culture that is substantially free of other microbes other than the target microbial strain or strains. In other words, a "substantially pure culture" of a microbial strain is essentially free of contaminants, which may include microbial contaminants as well as unwanted chemical contaminants. In addition, as used herein, the term "biologically pure" strain means a strain isolated from materials with which it is normally associated in nature. It should be noted that a strain associated with other strains or compounds or materials that do not normally occur under natural conditions is also defined as "biologically pure". It is quite clear that the monoculture of a particular strain is "biologically pure". The term "enriched culture" of an isolated microbial strain as used herein means a microbial culture containing more than 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain.

[0030] As used herein, the term "endophyte" means an endosymbiont that lives within a plant for at least part of its life without causing obvious disease. Transmission of endophytes can occur either vertically (directly from parent to offspring) or horizontally (from individual to unrelated individual). Vertically transmitted fungal endophytes are usually asexual and are transferred from the mother plant to the offspring by penetrating the host seeds through the fungal hyphae. Bacterial endophytes can also be transferred vertically from seeds to seedlings (Ferreira et al. , 2008). In contrast, horizontally transmitted endophytes are generally sexed and carried by wind-borne spores and/or insect vectors. Endophytes in cultivated cereals have received much attention due to their ability to fight both disease and insect infestation, as well as to promote plant growth.

[0031] Functionally comparable protein: As used herein, the term "functionally comparable protein" refers to proteins that have at least one common characteristic. Such characteristics can be sequence similarity to each other, biochemical activity, transcription pattern similarity to each other, and phenotypic activity. As a rule, functionally comparable proteins have some sequence similarity among themselves or are similar in at least one biochemical trait. Within the framework of this definition, homologs, orthologs, paralogs, and analogs are considered functionally comparable. In addition, functionally comparable proteins, in General, have similar at least one biochemical and/or phenotypic activity. Functionally comparable proteins confer similarity on the same characteristic, but not necessarily to the same extent. As a rule, functionally comparable proteins give the same characteristics, which, in quantitative terms, in one of the compared proteins, are at least 20% of those of the other, more often from 30 to 40%; more often from 50 to 60%, even more often from 70 to 80%, even more often from 90 to 95% and, as a rule, from 98 to 100% of those of another.

[0032] Fungicidal: As used herein, the term "fungicidal" refers to the ability of a composition or substance to reduce the growth rate of fungi or increase the mortality of fungi.

[0033] Fusarium fungus: In the context of this invention, the term " Fusarium fungus" includes both the sexual (teleomorphic) phase of this organism and its asexual (anamorphic) phase, also called perfect and imperfect fungal phases, respectively. For example, the anamorphic phase of the fungus Fusarium graminearum is Gibberella zeae , i.e. the causative agent of Fusarium. This disease usually occurs when a flower or seedcap is inoculated with conidia released by the imperfect form or ascospores released by the perfect form of this fungus.

[0034] Mutant: As used herein, the term "mutant" or "variant" in relation to a microorganism means a modification of the parent strain in which the target biological activity is similar to that expressed by the parent strain. For example, in the case of Microbacterium , the "parent strain" refers to the original strain of Microbacterium prior to mutagenesis. Mutants or variants can occur naturally without human intervention. They can also be obtained by processing one or more methods and compositions known to experts in this industry. For example, the parent strain may be treated with a chemical such as N-methyl-N'-nitro-N-nitrosoguanide, ethyl methanesulfone, or with gamma, x-ray, or ultraviolet radiation, or any other means well known to those skilled in the art.

[0035] Nematicidal: The term "nematicidal" as used herein means the ability of a substance or composition to increase mortality or inhibit the growth rate of nematodes.

[0036] Pathogen: The term "pathogen" as used herein means an organism, such as an algae, arachnid, bacterium, fungus, insect, nematode, parasitic plant, yeast, protozoan, or virus capable of producing a disease in a plant or animal. The term "phytopathogen" as used herein means a pathogenic organism that infects a plant.

[0037] Percent identity: "Percent sequence identity", as used herein, is determined by locally matching two optimally aligned sequences in an alignment region determined by the length of the local alignment of the two sequences. When two sequences are optimally aligned, a given amino acid sequence at the alignment site may contain additions or deletions (eg, gaps or overhangs) compared to a reference sequence (which contains no additions or deletions). Local alignment of two sequences is carried out only on those segments of each sequence that are supposed to be sufficiently similar according to a certain criterion, selected in accordance with the algorithm used for alignment (for example, the BLAST program). Percent identity is calculated by determining the number of matches at positions where identical nucleic acid bases or amino acid residues are located in both sequences, dividing the resulting match by the total number of positions in a given alignment region, and multiplying the result by 100. Optimal sequence alignment can be performed using the algorithm Smith-Waterman local homology (Smith and Waterman, Add. APL. Math. 2:482, 1981) or Needleman-Wunsch global homology algorithm (Needleman and Wunsch, J. Mol. Biol. 48:443, 1970), similarity study method Pearson-Lipman (Pearson and Lipman, Proc. Natl Acad. Sci. USA 85: 2444, 1988), heuristic implementation of these algorithms (NCBI BLAST, WU-BLAST, BLAT, SIM, BLASTZ) or through visual studies. If two sequences have been chosen for alignment, then it is preferable to use the GAP and BESTFIT algorithms to perform their optimal alignment. Typically, the default values are 5.00 for the weight of spaces and 0.30 for the length of the weight of spaces. The term "substantial sequence identity" between polynucleotide or polypeptide sequences refers to a polynucleotide or polypeptide containing a sequence that has at least 50% identity, preferably at least 70%, preferably at least 80%, more preferably at least 85% , more preferably at least 90%, even more preferably at least 95%, and most preferably at least 96%, 97%, 98% or 99% identity compared to a reference identity determined by a computer program.

[0038] The analysis of nucleic acid and amino acid sequences can be carried out by comparing them with the corresponding nucleic acid or amino acid sequences stored in public or private databases. Such studies can be carried out using the NCBI BLAST v 2.18 program of the National Center for Biotechnology Information Basic Local Alignment Search Tool. The NCBI BLAST program is available online at blast.ncbi.nlm.nih.gov/Blast.cgi (National Center for Biotechnology Information). In the NCBI BLAST program, the following parameters can generally be used: Filter options set to "default" by default; The alignment matrix (Comparison Matrix) is set to "BLOSUM62"; Gap Costs are set to "Existence: 11, Extension: 1", Word Size is set to 3, Expect Threshold (E threshold) is set to 1e-3, and Local Alignment Minimum Length is set to 50 % length of the analyzed sequence. Sequence identity and similarity can also be determined using the GenomeQuest™ program (Gene-IT, Worcester Mass. USA).

[0039] The term "pesticidal" as used herein means the ability of a given substance or composition to reduce the growth rate of a pest, ie an unwanted organism, or to increase the mortality of the pest.

[0040] By "suppressive activity" of a biological control agent against a given plant pathogen is meant the ability of the agent to suppress, inhibit, stabilize, reverse, slow or delay the development of the pathogen itself or the progression of the infection or disease state caused by the pathogen.

[0041] Variant: The term "variant" as used herein in relation to a microorganism means a strain having the identifying characteristics of the species to which it belongs, and at least one nucleotide sequence variation or identifiable distinguishing feature related to the parent strain and being genetically (inherited). For example, strain SGI-SGI-014-CQ6 of the species Microbacterium , which has fungicidal activity and has identifiable features, including 1) the ability to suppress the growth of the fungus Fusarium graminearum and its teleomorph Gibberella zeae , 2) the ability to suppress the development of fusarium, 3) the presence of mandatory genes with identity Greater than 95%, Greater than 96%, Greater than 97%, Greater than 98%, or Greater than 99% with mandatory SGI-014-C06 Microbacterium species genes can be used to confirm that the analyzed variant is Microbacterium SGI-014-C06 species.

[0042] With respect to nucleic acids and polypeptides, the term "variant" is used herein to refer to a polypeptide, protein, or polynucleotide molecule with some artificial or natural difference in its amino acid or nucleic acid sequences from reference polypeptides or polynucleotides, respectively. These differences may be, for example, substitutions, insertions, deletions, or any targeted combination of such changes in the reference polypeptide or polypeptide. Polypeptide and protein variants may also consist of changes in charge and/or post-translational modifications (eg, glycosylation, methylation, phosphorylation, etc.).

[0043] All publications and patent applications mentioned in this specification are incorporated herein by reference as if each of the publications or patent applications were individually incorporated by reference.

[0044] References do not imply recognition of prior art or prior art. By discussing the references, it is stated that their authors declare, and the applicants have the right, to challenge the accuracy and relevance of the cited documents. It is understood that although references are made herein to numerous prior art publications, these references do not constitute an acknowledgment that any of these documents form part of the general knowledge in the art.

Taxonomic identification methods

[0045] Microorganisms can often be distinguished by direct microscopic examination (because all cells in a sample look different under a microscope), by staining characteristics, by simple molecular analysis (for example, simply by determining restriction fragment length polymorphism (RFLP), etc. In addition to the illustrative examples of such taxonomic analysis methods described in Examples 2-3 below, up to several different levels of analysis can be used in the taxonomic identification of a particular microorganism, and each analysis can be based on distinct characteristics of that organism. such taxonomic analyzes may include nucleic acid-based analysis (for example, analysis of individual specific genes by their level of presence or by their exact sequence, or by the expression of a particular gene or family of genes), protein analysis (for example, at a functional level using direct and whether indirect enzymatic analysis or at the structural level using immunodetection methods), etc.

[0046] a. Analysis by nucleic acids. It is well known to those skilled in the art that a wide variety of nucleic acid analysis methods can be used in the taxonomic identification of a given microorganism. These methods can be used to identify cells by the nucleotide sequence of a gene, or to identify a cell that has specific genes or gene families. Common gene families suitable for taxonomic studies may be the 16S gene family, the actin gene family, and the recombinase A ( recA) gene family. These methods typically involve the amplification and sequencing of genes from very small numbers of cells, which often overcomes the problems associated with concentrating cells and their DNA from dilute suspensions. The term "nucleic acid amplification" generally refers to methods for increasing the copy number of a nucleic acid molecule in a sample or preparation. Nucleic acid amplification techniques are well known. Amplification of nucleic acids can be carried out, for example, using the polymerase chain reaction (PCR), in which a biological sample taken from a subject is brought into contact with a pair of oligonucleotide primers under conditions that allow the primers to hybridize with a nucleic acid template in this sample. Primers, under the right conditions, elongate, detach from the template, and then anneal, elongate, and detach to increase the copy number of the nucleic acids. In vitro amplification can also be carried out by the strand displacement method, the isothermal method without transcription, the amplification method with the chain repair reaction, the ligase chain reaction method, the ligation chain reaction method with gap filling, the coupled method of ligase detection and PCR; and an amplification method without RNA transcription.

[0047] In addition to the primers shown in illustrative Examples 2-3 and the accompanying Sequence Listing, other primers have also been designed by the authors during laboratory experiments, and, in addition, the design of new primers for individual biological species and phylogenetic groups of microorganisms is ongoing. Such highly targeted primers may find use in the methods described herein for the selection and/or specific identification of specific, target microorganisms.

[0048] Methods for the preparation and use of nucleic acid primers are described, for example, in [Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989], Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998]. Amplification primer pairs can be obtained from a known sequence, for example, using a special computer program "Primer", developed by the Fusarium Institute, Cambridge, Massachusetts, USA (Whitehead Institute for Biomedical Research, Cambridge, Mass.). It is well known to a person skilled in the art that the specificity of a probe or primer increases with its length.For example, a primer containing 30 consecutive nucleotides of its rRNA-coding nucleotide or flanking region will anneal to the target sequence with higher specificity than a corresponding primer consisting of only 15 nucleotides.Thus , probes and primers containing at least 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of the target nucleotide sequence, such as 16S rRNA.

[0049] Nucleic acids suitable for nucleic acid manipulation (eg, PCR) can be obtained using well-known technologies such as phenol-chloroform extraction, or using one of the many commercial DNA extraction kits. An alternative way of DNA amplification can be to attach cells directly to the reaction mixture for nucleic acid amplification and carry out all other manipulations at the stage of denaturation with cell lysis and DNA isolation.

[0050] The resulting product of nucleic acid amplification reactions is analyzed using standard methods, including electrophoresis, restriction endonuclease digestion patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing. If hybridization methods are used for all purposes of cell identification, a wide variety of probe labeling methods may be suitable, including fluorescent labeling, radioactive labeling, and non-radioactive labeling. When using nucleic acid sequencing techniques, the homology of the nucleotide sequences of the amplified nucleic acid molecules can be conducted using various databases of known sequences, including, for example, the DDBJ/GenBank/EMBL databases.

[0051]b. Protein-mediated analysis. In addition to nucleic acid analysis, microorganisms can be characterized by taxonomy and identified directly by the presence (or absence) of specific proteins. Such an assay may be based on the activity of a particular protein, such as in an enzyme assay, or the response of co-cultured organisms, or simply the presence of a specified protein (which may be determined, for example, by immunological methods such as in situ antibody immunofluorescence staining).

[0052] Enzyme analysis. To perform such an assay, fluorescent or chromogenic substrate analogs are included in the nutrient medium (eg, cultures on a microtiter plate), and then incubated and screened for reaction products, thus identifying the cultures by their enzymatic activity.

[0053] Co-cultivation response. In some embodiments of the present invention, the activity of an enzyme carried by a microbial isolate can be determined by the response (or rate of response) of a co-cultured organism (eg, a reporter organism).

[0054] A variety of methods can also be used to identify microorganisms selected and isolated from the source environment by binding at least one antibody or one molecule derived from an antibody to a specific molecule, and more precisely, to an epitope of a molecule, a microorganism.

[0055] Antibodies to microbial proteins can be prepared using standard techniques described in many literature sources, for example, (Harlow and Lane, (Antibodies, A Laboratory Manual , CSHL, New York, 1988). Determine if a given agent binds essentially only with the protein of the target microorganism can easily be done using conventional or adapted techniques.One such technique, suitable for in vitro analysis, is the Western blotting procedure described in many well-known works, including (Harlow and Lane, Antibodies, A Laboratory Manual , CSHL, New York, 1988).

[0056] Shorter fragments of antibodies (molecules derived from antibodies, for example, FAbs, Fvs and single chain Fvs (SCFvs)) can also serve as specific binding agents. Methods for preparing such fragments are widely known.

[0057] The detection of antibodies that bind to cells in accordance with the invention can be carried out using standard methods, for example, enzyme immunoassay (ELISA), which allows you to get a detectable, for example, fluorescent or luminescent signal.

Isolated cultures according to the invention

[0058] As described in more detail in the EXAMPLES section, the authors have discovered several new microorganisms useful for agriculture, capable of, for example, effectively suppressing Fusarium. These new antagonistic microorganisms are particularly effective in reducing the severity of Fusarium disease and in inhibiting the growth of the fungus Fusarium graminearum , the main causative agent of Fusarium in wheat. These microbial antagonists were identified from a pool of approximately 5,000 microbial strains obtained from wild plant samples collected from several locations throughout the United States. The initial selection of the antagonistic microorganism was based on the ability of the microorganism to inhibit the development of the pathogen F. graminearum and its teleomorph Gibberella zeae in in vitro antagonism tests. The selected microbial antagonists were then subjected to greenhouse biological testing on wheat seedlings, where the seedlings were inoculated with microbial strains and then re-inoculated with F. graminearum spores, for the ability of these microbial strains to reduce the severity of infection by the fungus and for their ability to maintain seed yield. The antagonistic microorganisms thus selected have been shown in greenhouse trials to be effective in reducing the severity of Fusarium.

[0059] In addition, taxonomic analysis has shown that each of the antagonistic microorganisms described herein is closely related to either the bacterial genus Microbacterium , the bacterial genus Bacillus , the bacterial genus Variovorax , or the fungal genus Mycosphaerella.

[0060] The genus Microbacterium , an exemplary genus of the family Microbacteriaceae , generally refers to opportunistic, Gram-positive, non-spore-forming, rod-shaped bacteria that were originally isolated from lactic acid-producing bacteria in early research. Members of the genus Microbacterium were initially widely characterized by their marked heat tolerance, presence in dairy products, and production of small amounts of L(+) lactic acid from glucose. In contrast to these genera of the family Microbacteriaceae , whose species are distinguished by a coherent peptidoglycan type, the species Microbacterium has ornithine or lysine either in the interpeptide bridge or in position 3 of the B-type peptidoglycan. In other chemotaxonomic properties, such as isoprenoid quinones (MK-11, MK-12, MK-13), polar lipids, fatty acids, and core DNA composition, members of this family show the usual range of diversity seen in other Microbacteriaceae genera. Based on the results of some taxonomic studies, the two bacterial genera Microbacterium and Aureo bacterium can be combined. To date, the genus Microbacterium includes at least 33 species that have been isolated from a wide range of habitats, including soil, dairy products, plant galls, insects, and clinical specimens. Various aspects of their ecology, phylogeny, taxonomy, cultivation, and long-term storage conditions have recently been summarized by Evtushenko and Takeuchi (Evtushenko and Takeuchi, 2006). It is quite clear that a microorganism of the genus Microbacterium can be quite reliably identified by any of the taxonomic methods described above, including chemotaxonomic analysis of cell wall peptidoglycans and sequence analysis by the 16S rDNA alignment method, as described (Evtushenko and Takeuchi, 2006) and the references cited by them, as well as in the references cited in Examples 2-3 of this description. As discussed in more detail below, the discovery of antagonistic activity against Fusarium in several microorganisms of natural origin has previously been reported. However, such antagonistic activity was not reported before the present invention by a microorganism of the genus Mycobacterium . In addition, prior to the present invention, methods and processes for using a bacterial strain of the genus Mycobacterium as a biocontrol agent to prevent, inhibit, or treat the development of a Fusarium pathogen were not known to the present inventors.

[0061] Bacillus is a genus of Gram-positive variable, spore-forming, rod-shaped bacteria. Like other genera associated with the early history of microbiology, such as Pseudomonas or Vibrio , about 266 species members of the genus Bacillus are found throughout, and it is widely recognized as one of the genera with the greatest 16S and environmental diversity. Bacillus species can be obligatory aerobes or facultative anaerobes, and test positive for catalase. Ubiquist in nature, the genus Bacillus includes both free-living and pathogenic species. Under severe environmental conditions, these cells produce oval endospores, which can remain dormant for a long time. These characteristics originally defined the genus, however, not all such species are closely related, and many have moved to other genera. In fact, several attempts have been made to reconstruct the phylogeny of this genus. Bacillus -specific studies with the greatest diversity were carried out in (Xu and Cote, Intl. J. of Syst. Evol. Microbiol. 53 (3): 695-704; 2003) using 16S and the ITS region (internal transcribed spacer), where the authors divided the genus into 10 groups which include the nested genera Paenibacillus, Brevibacillus, Geobacillus, Marinibacillus and Virgibacillus. However, according to more recent studies, the genus Bacillus contains a very large number of nesting taxa, and especially in both 16S and 23S, it is considered paraphyletic to Lactobacillales (Lactobacillus, Streptococcus, Staphylococcus, Listeria, etc.) due to Bacillus coahuilensis , etc., for example , (Yarda et al , Syst. Appl. Microbiol. 31 (4): 241-250, 2008; Yarda et al , Syst. Appl. Microbiol. 33 (6): 291-299, 2010). One of the clades, formed by the species B. anthracis , B. cereus , B. mycoides , B. pseudomycoides , B. thuringiensis and B. weihenstephanensis according to modern classification standards, should be a single species (with 97% 16S identity), however, according to medical reasons, they are considered separate species. In addition to the taxonomic analysis methods described in Examples 2-3, phylogenetic and taxonomic analyzes of the Bacillus species can be carried out using a number of different methods, including those detailed in (Xu and Cote, 2003; Yarda et al. , 2008; Yarda et al. , 2010).

[0062] The genus Variovorax was originally created by the reclassification of the genus Alcaligenes paradoxus to Variovoraxparadoxus (Willems et al. , 1991), which is considered to be the typical species of the genus. V. paradoxus has been extensively studied as a model for novel biodegradation agents as well as microbe-microbe and microbe-plant interactions. Other species in this genus are V. dokdonensis , V. soli (Kim et al. 2006), and V. boronicumulans (Miwa et al. 2008). Variovorax species are very diverse in terms of catabolism and have mutually beneficial interactions with other bacterial species in many biodegradation processes and are therefore ecologically significant and have a high potential for practical applications. For example, a soil methanotroph, only when co-cultivated with a V. paradoxus strain, exhibits a high affinity for methane (a potent greenhouse gas), a feature that is not commonly observed in laboratory cultures. Similarly, its close relative Variovorax was found to be a central, non-photosynthetic partner within the phototrophic consortium "Chlorochromatium aggregatum". Some species of the genus Variovorax also have the ability to interfere with the communication of other bacteria. In addition, some species of the genus Variovorax may interact closely with other biota (eg plants) in different ecosystems. Moreover, some species of Variovorax , when found outside the plant roots and/or inside the plant, have shown the ability to promote plant growth by reducing ethylene levels, repressing quorum sensing pathogenesis, and increasing heavy metal tolerance, which is very beneficial for phytoremediation. In addition to the methods of taxonomy analysis described in Examples 2-3, phylogenetic and taxonomic analyzes of the species Variovorax can be carried out by a variety of methods, including those discussed in detail in this description.

[0063] Mycosphaerella is a very large fungal genus with over 2,000 species names and at least 500 species associated with over 40 anamorphic genera (specifically, Cercospora , Pseudocercospora , Septoria , Ramularia , and others ). In addition, several thousand anamorphs do not have telemorphs. The genus Mycosphaerella includes species that are pathogenic, saprobic, endophytic, or mutualistic associations. Various aspects of their ecology, origin, taxonomy, cultivation methods and long-term storage conditions have recently been described in (Crous et al. , Persoonia , 23:119-146, 2009). Taxonomic identification of microorganisms of the genus Mycosphaerella can be made by any of the above-described appropriate methods or a combination thereof. The most common of these methods are sequence analysis by alignment with 16S rDNA sequences and internal transcribed spacer regions, as described, for example, in (Crous et al. , Studies in Mycology , 55:235-253,2006; Crous et al , 2009, supra; Goodwin et al , Phytopathology 91: 648-658, 2001), as well as the methods described in Examples 2-3 below.

Deposits of biological materials

[0064] Purified cultures of microbial strains identified as having suppressive activity against Fusarium were deposited with the Agricultural Research Service Culture Collection located at 1815 N. University Street, Peoria, IL 61604, USA (NRRL) ) in accordance with the "Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure" and the regulations arising from it (Budapest Treaty). These deposits are placed under the following numbers:

Identification
SGI strain Deposit number Preliminary taxonomy of species SGI-005-G08 NRRL- Microbacterium SGI-010-H11 NRRL 50471 Mycosphaerella SGI-014-C06 NRRL B-50470 Microbacterium SGI-014-G01 NRRL B-50469 Variovorax SGI-015-F03 NRRL B-50760 bacillus SGI-015-H06 NRRL B-50761 bacillus

[0065] These microbial strains were deposited with the condition of providing access to this culture during this application is in the process of consideration and decision on it by the head of the US Patent Office in accordance with 37 C.F.R. §1.14 and 35 U.S.C. §122. These deposits represent essentially pure cultures of the deposited strains. Access to deposits is provided in accordance with the patent laws of the countries where the opposing parties to this application or their heirs are registered. It is quite clear that access to deposits does not mean permission to practice the subject matter of the invention in violation of patent rights guaranteed by the state.

[0066] Preferred microorganisms in accordance with the invention have all the identification characteristics deposited by the strain and, in particular, the identification characteristics regarding the ability to inhibit the development of Fusarium, as described herein, and the ability to inhibit the development of the pathogen Fusarium graminearum and its teleomorph Gibberella zeae . In particular, preferred microorganisms according to the invention relate to the deposited microorganisms described above and their mutants.

Microbiological compositions

[0067] Microbiological compositions in accordance with the invention, containing isolated microbial strains or cultures thereof, can take a wide variety of forms, including, but not limited to, stationary culture forms, whole culture forms, stored cell strain, mycelium and/or hyphae (especially, initial glycerol solutions), agar strips, agar complexes in water-glycerol mixture, freeze-dried mother liquors, and dried mother liquors such as lyophilisates or mycelia dried on filter paper or seed. In accordance with the definition given in the present description, the term "isolated culture" or its grammatical equivalents as used herein means that this culture is a culture liquid, pellet, scraping, dried sample, lyophilate or section (for example, hyphae or mycelium); or a support, container, or medium, such as a plate, paper, filter, matrix, straw, pipette or pipette tip, fiber, needle, gel, swab, tube, vial, particle, etc., that contains one type of organism. In the present invention, an isolated culture of a microbial antagonist is a culture fluid or scraping, pellet, dried preparation, lyophilate, or slice of a microorganism, or a support, container, or medium that contains the microorganism in the absence of other organisms.

[0068] The present invention also provides compositions comprising at least one isolated microbial strain or culture thereof in accordance with the invention and a carrier. The carrier may be any one carrier or a plurality of carriers, satisfying a variety of properties, such as increased stability, wettability, dispersibility, etc. The composition according to the invention may include wetting agents, such as natural or artificial surfactants, which may be non-ionic or ionic surfactants or combinations thereof. The composition may also include water-in-oil emulsions that contain at least one isolated microorganism in accordance with the invention (U.S. Patent No. 7485451), incorporated here by reference. Desired formulations may be prepared from wettable powders, granules, gels, agar strips or granules, thickeners, and the like, microencapsulated particles, and the like, liquids such as aqueous fluids, aqueous suspensions, emulsions such as water- in oil, etc. The preparation may contain grains or legumes (for example, crushed grains or beans, broth or flour made from grains or beans), starch, sugar or vegetable oil. The carrier may be an agricultural carrier. In some preferred embodiments, the carrier is a seed and the composition can be applied to, or coated or impregnated with, the seed.

[0069] In some embodiments, the agricultural carrier may be soil or a plant growing medium. Other suitable agricultural carriers are water, fertilizers, vegetable oils, humectants, and combinations thereof. Alternatively, the agricultural carrier may be a solid material such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, seed capsules, other plant and animal products, or combinations thereof, including granules, pellets, and suspensions. The carriers may also be mixtures of any of the above ingredients, such as, for example, pesta (pesta: flour and kaolin clay), agar or flour granules in loam, sand or clay, etc. Formulations may include food sources for cultured organisms, such as barley, rice, or other biological materials, such as seeds, plant parts, sugarcane bagasse, husks or stalks from grain processing, crushed plant material (such as “stockpiling waste”) or wood from construction waste, sawdust or fine fibers from paper recycling , woven fabrics or wood. Other suitable materials for this purpose may also be known to those skilled in the art.

[0070] In liquid form, such as solutions or suspensions, microorganisms can be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers are water, aqueous solutions, petroleum distillates and other liquid carriers.

[0071] Solid compositions can be prepared by dispersing antagonistic microorganisms in or on an appropriately divided solid carrier such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, fuller, pasteurized soil, and the like. When such formulations are used as wettable powders, biocompatible dispersants such as non-ionic, anionic, amphoteric or cationic dispersants and emulsifiers may be used.

[0072] In a preferred embodiment of the invention, the compositions herein prevent Fusarium damage to plants and especially cereals such as wheat, barley, oats and corn, and when used in sufficient amounts act as microbial antagonists. Like other biological control agents, these compositions have a high margin of safety because they generally do not burn or damage the plant.

[0073] As detailed herein, Fusarium can be controlled by applying one or more of the microbiological compositions of the invention to, or parts of, the host plant. These compositions can be applied in an amount effective to reduce the level of fusarium compared to its level in the untreated control. The active ingredients are used at a concentration sufficient to inhibit the development of the target phytopathogen when they are applied to the crop. It will be apparent to those skilled in the art that effective concentrations may vary and depend on the following factors: (a) the type of plant or agricultural product; (b) the physiological state of the plant or agricultural product; (c) concentrations of pathogens affecting the plant or agricultural product; (d) the type of disease affecting the plant or agricultural product; (e) weather conditions (eg temperature, humidity); and (f) stages of development of the plant disease. According to the invention, typical concentration values lie above the level of 1×10 ² cfu/ml of carrier. Preferred concentration levels are in the range of approximately 1×10 ⁴ to 1×10 ⁹ cfu/ml, and even better in the range of 1×10 ⁶ to 1×10 ⁸ cfu/ml. More preferred is a concentration range of approximately 35 to 150 mg dry microbial mass per gram of carrier (dry preparation) or per milliliter of carrier (liquid composition). For solid preparations, the delivery rate should be adjusted to obtain a number of viable cells per unit area of plant tissue surface comparable to that obtained from the above liquid treatment concentrations. Generally, the biological control agents of the invention are biologically effective when they are supplied at a concentration above 10 ⁶ cfu/g (colony forming units per gram), preferably above 10 ⁷ cfu/g, even more preferably at a concentration of 10 ⁸ cfu /g, and most preferably at a concentration of 10 ⁹ CFU/g.

[0074] In some embodiments of the invention, the amount of one or more biological control agents in the microbial composition in accordance with the invention may vary depending on the final preparation, as well as the size and type of plant or seeds used. In a preferred embodiment, one or more biological control agents in the microbial composition is in an amount of from about 2% (wt.) to 80% (wt.) of the total amount of the drug. In a preferred embodiment, one or more biological control agents in the microbial composition is in an amount of from about 5% (wt.) to 65% (wt.), and most preferably from about 10% (wt.) to 60% (wt.) ) of the total amount of the drug.

[0075] The microbiological compositions of the invention may be applied to wheat or other cereal crops by a variety of methods well known to those skilled in the art, such as dusting, coating, pouring, rubbing, rolling, dip dressing, spraying, brushing, and other suitable methods, which do not cause significant damage to wheat and other cereals during their processing. Particularly suitable is the spray method.

[0076] Compositions in accordance with the invention, as a rule, are chemically inert; therefore, they are substantially compatible with any other components of the spray pattern. They can also be used in combination with biologically compatible pesticidal active agents, eg herbicides, nematicides, fungicides, insecticides and the like. They can also be used in combination with plant growth regulating substances such as fertilizers, plant growth regulators and the like, provided that such compounds or substances are biologically compatible.

[0077] If pesticides or fungicides are used in their commercial preparations and in forms prepared from these preparations, then active microbial antagonists and compositions in accordance with the present invention can, moreover, be used together with them in the form of a synergistic mixture. The components of such a mixture, being synergists, increase the effectiveness of the active composition without the need to add special synergists to the mixture.

[0078] Used as pesticides in their commercial preparations and in forms prepared from these preparations, active microbial antagonists and compositions in accordance with the present invention can, moreover, be used together with them in the form of a mixture with inhibitors that reduce the degradation of active compositions after their application in the place where the plant grows, on the surface of parts of the plant or in its tissues.

[0079] Active microbial antagonists and compositions according to the invention, in their original state or in preparations, can also be used in the form of a mixture with known acaricides, bactericides, fungicides, insecticides, microbicides, nematicides, pesticides or combinations thereof, for example, to expand their spectrum of action or to prevent the development of resistance in this way. In many cases, synergy gives a positive result, that is, the activity of such a mixture may exceed the activity of its individual components. A mixture with other known active compounds such as fertilizers, growth regulators, protective agents and/or signaling chemicals are also within the scope of the invention.

[0080] In a preferred embodiment, the compositions may also contain at least one chemical or biological pesticide. The amount of at least one chemical or biological pesticide used in a given composition may vary depending on the final preparation, as well as the size of the treated plant and seed. In a preferred embodiment, the amount of at least one used chemical or biological pesticide is from about 0.1% (wt.) to 80% (wt.) of the total amount of the drug. More preferably, the amount of at least one chemical or biological pesticide used is from about 1% (wt.) to 60% (wt.), and most preferably from about 10% (wt.) to 50% (wt.). .).

[0081] Any pesticide known to those skilled in the art can be used in the invention. These can be, for example, chemical pesticides belonging to the classes of carbamates, organophosphates, organochlorines and pyrethroids. The invention also provides for chemical biocontrol agents such as, for example: benomyl; borax; captafol; captan; chlorthalonil; copper-containing preparations; preparations containing dichlon, dichloran, iodine, zinc; fungicides that inhibit the biosynthesis of ergosterol, such as e.g. sodium arsenite, sodium DNOC (dinitroocresol), sodium hypochlorite, sodium phenylphenate, streptomycin, sulfur, tebuconazole, terbutrazol, thiabendozole, thiophanate-methyl, triadimefon, tricyclazole, triforin, validimycin, vinclozolin, cineb and cyram. A wide variety of insecticidal compounds can be used in the compositions of the invention, including, for example, those specified in the application (U.S. Pat. Appl. No. 20110033432A1).

[0082] Microbiological compositions in accordance with the invention preferably contain at least one biological pesticide. Typical biological pesticides that are suitable for use in accordance with the invention and may be included in the microbiological composition in accordance with the invention for the prevention of phytopathogenic disease are microbes, animals, bacteria, fungi, genetic material, plant and natural products of living organisms. In these compositions, the microorganism according to the invention is isolated prior to formulation by the additional organism. In the composition with antagonistic microorganisms according to the invention, microbes can be used, for example, species of Ampelomyces , Aureobasidium , Bacillus , Beauveria , Candida , Chaetomium , Cordyceps , Cryptococcus , Dabaryomyces , Erwinia , Exophilia , Gliocladium , Mariannaea , Paecilomyces , Paenibacillus , Pantoea , Pichia , Pseudomyces , Sporobolomyces , Talaromyces and Trichoderma , where fungal strains of the genus Muscodor are particularly preferred. Particularly preferred is also the use of microbiological compositions in accordance with the present invention in combination with microbial antagonists described in (US Patent No. 7518040; US Patent No. 7601346; US Patent No. 6312940).

[0083] Examples of fungi that can be used in combination with microbial antagonists and compositions according to the invention include Muscodor species, Aschersonia aleyrodis , Beauveria bassiana ("white muscarine"), Beauveria brongniartii , Chladosporium herbarum , Cordyceps clavulata , Cordyceps entomorrhiza , Cordyceps fads , Cordyceps gracilis , Cordyceps melolanthae , Cordyceps militaris , Cordyceps myrmecophila , Cordyceps ravenelii , Cordyceps sinensis , Cordycepshecocephala , Cordyceps subsessilis , Cordyceps unilateralis , Cordyceps variabilis , Cordyceps washingtonensis , Culicinomyces clavosporus , Entomophaga grylli , Entomophaga maimaiga , Entomophaga muscae , Entomophaga praxibulli , Entomophthora plutettae , Fusarium lateritium , Hirsutella citriformis , Hirsutella thompsoni , Metarhizium anisopliae ("green muscarine"), Metarhizium flaviride , Muscodor albus , Neozygitesfloridana , Nomuraea rileyi , Paecilomyces farinosus , Paecilomyces fumoso roseus , Pandora neoaphidis , Tolypocladium cylindrosporum , Verticillium lecanii , Zoophthora radicans , and mycorrhizal fungi such as Laccaria bicolor. Other mycopesticidal species known to those skilled in the art may also be suitable.

[0084] The present invention also provides methods for treating a plant by applying any of a variety of conventional formulations in an effective amount to either the soil (i.e. furrows), parts of the plant (i.e. spraying), or seeds prior to planting (i.e. coating or etching). Common formulations can be solutions, emulsifiable concentrate, wettable powders, suspension concentrate, soluble powders, granules, suspension emulsion concentrate, natural and artificial materials impregnated with the active compound, and very small controlled release polymer capsules. In some embodiments, biological control compositions are formulated in powders that are either marketed as ready-to-use formulations or mixed together at the time of use. In both cases, the powder can be added to the soil before planting or during planting. Alternatively, the biocontrol agent and the insect control agent may be both, or one of them, liquid preparations and mixed with each other during treatment. It will be understood by those skilled in the art that the effective amount of the composition according to the invention depends on the final preparation of the composition, as well as on the size of the plants or seeds being treated.

[0085] Depending on the final preparation and method of its application, the composition according to the invention may include one or more suitable additives. Such additives in these compositions can be, for example, adhesives such as carboxymethyl cellulose and natural or artificial polymers in the form of powders, granules or latexes, such as gum arabic, chitin, polyvinyl alcohol and polyvinyl acetate, as well as natural phospholipids such as cephalins and lecticins, and artificial phospholipids.

[0086] In one of the preferred embodiments of the invention, microbiological compositions are prepared in a single stable solution, emulsion or suspension. To make solutions, active chemicals (ie, biocontrol agents) are typically dissolved in solvents prior to addition of the biocontrol agent. Suitable liquid solvents can be, for example, petroleum aromatics such as xylene, toluene or alkylnaphthalenes, aliphatic hydrocarbons such as cyclohexane or paraffins, for example petroleum fractions, mineral and vegetable oils, alcohols such as butanol or glycol, as well as their ethers and esters, ketones such as methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, strong polar solvents such as dimethylformamide and dimethyl sulfoxide. The liquid medium for the emulsion or suspension is water. In one embodiment of the invention, the chemical control agent and the biological control agent are suspended in liquids separately from each other and mixed at the time of consumption. In a preferred embodiment of the suspension, the insect control agent and the biological agent are combined in a ready-to-use formulation having a shelf life of at least two years. When used, the liquid can be sprayed or applied in the form of a film with a sprayer, or in the furrow during crop sowing. The liquid composition may be applied to the soil prior to seed germination, or directly to the soil in contact with the roots, by a variety of means and methods well known in the art, including, for example, drip irrigation, sprinklers, soil injection, or soil soaking.

[0087] Stabilizers and buffers may be added as needed, including alkali and alkaline earth metal salts and organic acids such as citric acid and ascorbic acid, inorganic acids such as hydrochloric acid or sulfuric acid. Biocides such as formaldehyde or formaldehyde releasing agents and benzoic acid derivatives such as p-hydroxybenzoic acid may also be added.

Seed Coating Products

[0088] In some particularly preferred embodiments of the invention, the biocontrol compositions of the invention are formulated for therapeutic seed treatment. The therapeutic seed treatment composition contains at least one insect control agent and at least one biological control agent. It is contemplated that seeds can be substantially uniformly coated with one or more layers of the biocontrol compositions described herein, using conventional mixing and spraying techniques, or combinations thereof, and specially designed and manufactured coating devices to provide the desired accuracy, safety, and efficiency of seed application. processing products. Such devices use a variety of coating technologies, such as rotary type, drum type, fluidized ball, gushing ball, rotary spray, or combinations thereof. The liquid seed treatment according to the invention can be carried out using either a centrifugal disc “sprayer” or a spray that provides a uniform distribution of the coating on the seeds as they move through the spray pattern. After that, the seeds are preferably mixed or stirred for some time in order to further even out the distribution and dried. Prior to coating with the composition according to the invention, the seeds may be primed (germination stimulation) to increase the uniformity of germination and emergence. Alternatively, the dry powder preparation may be dosed to the seeds being stirred and mixed with them until a homogeneous distribution is obtained.

[0089] In another aspect, the invention provides seeds treated with the microbiological compositions of the invention. In one embodiment, at least a portion of the seed surface is coated with the microbiological composition of the invention. In one specific embodiment, the microorganism-treated seeds have a concentration of spores or microbial cells of about 10 ⁶ to 10 ⁹ per seed. Seeds may also have higher numbers of spores or microbial cells, such as 10 ¹⁰ , 10 ¹¹ or 10 ¹² spores per seed. The microbial spores and/or cells may coat the seeds in a free state or, preferably, be applied to them in a pre-formulated liquid or solid composition. A solid composition containing microorganisms can be prepared by mixing the solid carrier with the spore suspension until the solid carriers are impregnated with the spore or cell suspension. After that, the mixture is dried, resulting in the desired particles.

[0090] In some other embodiments, it is contemplated that the solid or liquid biocontrol compositions of the invention further comprise functional agents capable of protecting seeds from the harmful effects of selective herbicides, such as activated charcoal, nutrients (fertilizers), and others. agents capable of improving germination and product quality.

[0091] Known methods and compositions for coating seeds can be especially useful when they are modified by adding one of the embodiments of this invention. Such suitable coating methods and apparatuses are described, for example, in (U.S. Pat Nos. 5918413; 5554445; 5389399; 4759945; 4465017). Всевозможные композиции для создания покрытий на семенах описаны, например, в (U.S. Pat. Appl. Nos. US20110033432, US20100154299, U.S. Pat. Nos. 5939356; 5876739, 5849320; 5791084, 5661103; 5580544, 5328942; 4735015; 4634587; 4372080, 4339456 ; and 4245432).

[0092] A wide variety of additives can be incorporated into seed treatment formulations containing the compositions of the invention. Such additives may be, for example, binders, preferably consisting of an adhesive polymer, which may be natural or artificial and should not have a phytotoxic effect on the seeds to be coated. The binder may be selected from the following materials: polyvinyl acetates; polyvinyl acetate copolymers; copolymers of ethylene vinyl acetate (EVA); polyvinyl alcohol; polyvinyl alcohol copolymers; celluloses, including ethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose and carboxymethylcellulose; polyvinyl pyrolidones; polysaccharides, including starch, modified starch, dextrins, maltodextrins, alginate, and chitosans; fats; vegetable oils; proteins, including gelatin and zeins; gum arabic; shellacs; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers; polyvinyl acrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.

[0093] Any dye can be used, including organic chromophores belonging to the nitroso class; nitro; azo, including monazo, bisazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. Other acceptable additives include trace nutritional supplements such as iron, manganese, boron, copper, cobalt, molybdenum and zinc salts. To hold these substances on the surface of the seeds, polymeric or other dust-like agents can be added.

[0094] In some specific embodiments of the invention, the applied coatings, in addition to microbial cells or spores, may also contain an adhesive layer. The adhesive must be non-toxic, biodegradable and tacky. Suitable materials are, for example: polyvinyl acetates; polyvinyl acetate copolymers; polyvinyl alcohols; copolymers of polyvinyl alcohols; celluloses such as methylcellulose, hydroxymethylcellulose and hydroxymethylpropylcellulose; dextrins; alginates; Sahara; molasses; polyvinylpyrrolidones; polysaccharides; proteins; fats; vegetable oils; gum arabic; gelatins; syrups; and starches. The materials described in (U.S. Pat. No. 7213367 and U.S. Pat. Appln. No. US20100189693) may also be suitable.

[0095] Various additives such as adhesives, dispersants, surfactants, nutritional and buffer ingredients may also be included in the seed treatment preparation. Common additives in a seed treatment formulation are, for example, coating agents, wetting agents, buffering agents and polysaccharides, as well as at least one agriculturally suitable carrier such as water, solids or dry powders. Dry powders can be prepared from a wide variety of materials such as calcium carbonate, gypsum, vermiculite, talc, humus, activated carbon and various phosphorus compounds.

[0096] In one embodiment, the seed coating composition may comprise at least one organic or inorganic, natural or artificial excipient with which the active ingredients are combined to facilitate their application to the seed. Desirably, the filler is an inert, solid material such as clay, natural or artificial silicate, silica, resin, wax, solid fertilizer (e.g. ammonium salt), natural soil mineral such as kaolin, clay, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earth, or an artificial mineral such as silica, alumina or silicate, of which aluminum or magnesium silicates are particularly suitable.

[0097] The seed treatment may additionally contain one or more of the following ingredients: other pesticides, including compounds that act only underground; fungicides such as captan, thiram, metaxyl, fludioxonil, oxadilyl and isomers of each of these materials, and the like; herbicides, including compounds selected from glyphosphate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol esters, pyridazinones, uracils, phenoxy, ureas and benzoic acids; herbicidal protective agents such as benzoxazine, benzhydryl derivatives, N,N-diallyldichloroacetamide, various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone, naphthalic anhydride compounds, and oxime derivatives; fertilizers; and biocontrol agents such as other natural or recombinant bacteria and fungi of the genera Rhizobium , Bacillus , Pseudomonas , Serratia , Trichoderma , Glomus , Gliocladium and mycorrhizal fungi. These ingredients may be added in the form of a separate layer on the seed or, alternatively, as part of a seed coating composition according to the invention.

[0098] Preferably, the amount of new composition or other ingredients used in the seed treatment should not inhibit seed germination or cause phytotoxic damage to the seed.

[0099] Microorganism-treated seeds can be further coated with a film on top of the coating applied to them to protect the latter. Such surface coatings are well known and can be applied by fluidized bed and rotary drum techniques.

[0100] In principle, any plant seed capable of germinating and producing a plant susceptible to attack by nematodes and/or pathogenic fungi can be treated in accordance with the invention. Seeds of cereals, coffee, cabbage crops, fiber crops, flowers, fruits, legumes, oilseeds, trees, tubers, vegetables, and other plants of monocot and dicotyledonous species are suitable for this. Preferred seeds for coating are, for example, legumes, carrots, corn, cotton, grasses, lettuce, peanuts, peppers, potatoes, rapeseed, rice, rye, sorghum, soybeans, sugar beets, sunflowers, tobacco and tomatoes. In the most preferred coating compositions according to the invention are barley or wheat (spring wheat or winter wheat) seeds.

[0101] In another aspect of the present invention, there is provided a novel cereal plant created by artificially introducing a microbial endophyte of the invention into a cereal plant free of endophytic microorganisms. In some embodiments of this aspect, the microbial endophyte introduced into the cereal plant may be an endophytic antagonist having suppressive activity against Fusarium or Fusarium blight. In addition, the endophytic antagonist introduced into the cereal plant may be the fungal strain SGI-010-H11. There are many methods that have demonstrated their effectiveness for the introduction of microbial endophyte in cereal grasses. Examples of such methods can be found in (U.S. Pat. Appl. No. 20030195117A1, U.S. Pat. Appl. No. 20010032343A1, U.S. Pat. No. 7084331). One skilled in the art would appreciate that many of the above methods can be used in the cultivation of the new cereal plant according to the invention.

[0102] After artificial infection, it is desirable to amplify the DNA of the isolated endophytic antagonist by a PCR reaction, and confirm its antagonism by performing homology studies on the amplified DNA. Further, it is desirable to introduce a foreign gene expressing an identifiable agent into the aforementioned endophytic antagonist, and confirm that the aforementioned identification agent has colonized the plant-infecting endophytic antagonist with the aforementioned foreign gene.

Preparation of a biocontrol composition according to the present invention

[0103] Cultures of microbial antagonists can be prepared for use in the biocontrol compositions of the invention using known standard static drying and liquid fermentation techniques. Cultivation, as usual, is carried out in a bioreactor.

[0104] A bioreactor is any device that provides the conditions for a biologically active environment. In the context of this description, a bioreactor is a vessel in which microorganisms, including microbial antagonists, according to the invention can be grown. The bioreactor may be of any shape and size suitable for culturing microorganisms. The bioreactor can range in size from 10 ml to liters and cubic meters. It can be made of stainless steel or any other suitable material. The bioreactor may be batch-acting or continuous (eg, continuously agitated). By its type, a bioreactor can refer to chemostats, which are used in microbiology for growing and collecting bacteria. Suitable commercially available bioreactors include, for example, those manufactured by Bioreactor System Design, Asenjo & Merchuk, CRC Press, 1995.

[0105] For small scale needs, such as testing and developing new processes and processes that cannot be converted to continuous, a batch bioreactor can be used.

[0106] Microorganisms cultured in the bioreactor may be suspended or immobilized. Cultivation in a bioreactor is generally carried out under aerobic conditions at appropriate temperatures and pH. To obtain organisms according to the invention, cell culture is carried out at temperatures in the range of 5-37°C, in which temperatures in the ranges of 15-30°C, 15-28°C, 20-30°C and 15-25°C are preferred. The pH value of the nutrient medium can vary from 4.0 to 9.0, but the preferred operating range is from slightly sour to neutral with a pH of 4.0-7.0, or 4.5-6.5, or pH 5.0- 6.0. As a rule, the maximum cell output is obtained in the period of 20-72 hours after inoculation.

[0107] It is clear that the optimal conditions for the cultivation of antagonists according to the invention depend on the particular strain. However, the main ingredients and nutritional conditions can be determined based on the conditions used in the selection process and the general requirements for most microorganisms. Microbial antagonists should generally be grown in aerobic liquid cultures on a medium containing sources of carbon, nitrogen, and inorganic salts that can be assimilated by the microorganism and promote efficient cell growth. Preferred carbon sources are hexoses such as glucose. However, they can be replaced by other, easily assimilated sources such as amino acids. Numerous inorganic and protein materials can serve as sources of nitrogen during the growing process. Preferred sources of nitrogen are amino acids and ureas, but gaseous ammonia, inorganic nitrate and ammonium salts, vitamins, pure nutrients, pyrimidines, yeast extract, meat extract, peptone proteose, soy flour, casein hydrolysates, vinasse filtrate, etc. can also be used. . Among the inorganic minerals that can be introduced into the nutrient medium, one can name common salts that can supply calcium, zinc, iron, manganese, magnesium, copper, cobalt, potassium, sodium, molybdate, phosphate, sulfate, chloride, borate, etc. ions. Preferred the nutrient medium for fungal antagonists is liquid potato dextrose, and for bacterial strains, R2A premix broth.

[0108] In the present description, various sources of information are identified and incorporated by reference, including, for example, articles from scientific journals, patent documents, tutorials, and Internet page addresses. References to these sources are for the sole purpose of showing the general state of the art at the time of filing. While the content and ideas set forth in each and every one of these sources can serve as a basis and be used to implement and apply embodiments of the invention in practice, none of the discussions and none of the comments in a particular source of information can be considered an admission that that such a comment was broadly accepted as the general opinion of the industry.

[0109] The description of general methods provided herein is for illustrative purposes only. Other alternative methods and embodiments of the invention may follow from this description and are also within the spirit and scope of the present application.

[0110] The following are examples that also serve solely to illustrate the invention and are not intended to be limiting in any way.

EXAMPLES

EXAMPLE 1.Description of antagonistic microorganisms capable of suppressing the development Fusarium graminaerum and Gibberella zeae , which are the causative agents of fusarium

[0111] This example describes a high-throughput process for the collection and screening of candidate microorganisms developed by Synthetic Genomics, Inc. for the isolation of strains of microorganisms with suppressive activity against Fusarium pathogens and, in particular, against Fusarium graminearum . New microbial antagonistic strains have been isolated from plant tissue samples collected from several locations throughout the United States. Bacterial strains SGI-014-C06, SGI-014-G01, SGI-015-F03, and SGI-015-H06 were isolated from plant root tissue collected at the Eagle Peak Preserve near Julian, CA. The fungal strain SGI-010-H11 and bacterial strain SGI-005-G08 were isolated from stem tissue from two different plant samples collected at San Elijo Lagoon, Encinitas, CA.

[0112] The isolation of these microorganisms was performed as follows. To isolate the bacterial strain, plant root tissues were irradiated with ultrasound and serially diluted on plates with 2xYT (yeast extract with tryptone) and nitrogen-free agar medium. After that, according to morphological characteristics, individual colonies were selected, which were individually cultivated in a liquid broth medium. To isolate the fungi, the plant tissue was first subjected to surface sterilization by immersion in 70% ethanol and a brief flame treatment. The tissue was then dissected and placed on potato dextrose agar (PDA), after which it was incubated at room temperature. When mycelial growth began to be observed, the mycelial culture segment was transferred to another PDA plate. Microbial strains were isolated, purified and stored at -80°C in 15% glycerol for bacterial strains and on dried barley seeds for fungal strains.

[0113] The isolated strains of microorganisms were determined for their ability to inhibit the growth of F. graminearum mycelium in in vitro antagonism tests, which were carried out on agar plates with potato dextrose agar (PDA) nutrient medium in accordance with the high throughput screening method with slight modifications described in the application (US Pat. Appl. No. US20120107915A1), incorporated here by reference. Briefly, the isolated strains of microorganisms were grown on trypsin-soy broth agar (TSBA/5) at one-fifth normal concentration for 24 hours before consumption. A conidial inoculum of F. graminearum NRRL-5883 was produced by hyphal pinching of an actively growing colony of the fungus and transferring the hyphal filaments to PDA agar medium. After incubating the plates for 7 days at 25° C. with a 12 h/day photoperiod, the conidia were washed from the PDA plates with a weak phosphate buffer (0.004% phosphate buffer, pH 7.2 with 0.019% MgCl ₂ ). Then, a suspension of F. graminearum conidia in weak phosphate buffer (1×10 ⁵ conidia/ml) was immediately spread over the surface of the agar, and then the plates were incubated at 25°C for 48-72 hours. To initiate the antagonism test, cells of the isolated microbial strains were pointwise inoculated at equal distances within the perimeter of the plate. Five days later, the strains were marked as antibiosis-positive, when a light area was visually observed along the perimeter of the microbial colonies, on which there was no mycelial growth.

[0114] More than 4000 microbial strains were isolated and analyzed according to the above method. Among them, six strains showed the ability to significantly suppress the mycelial growth of F. graminearum NRRL-5883 on PDA medium. These new microbial antagonists have been named SGI-005-G08, SGI-010-H11, SGI-014-C06, SGI-014-G01, SGI-015-F03 and SGI-015-H06.

[0115] The above novel microbial antagonists were also tested for antagonism for their ability to inhibit the growth of the fungal pathogen Gibberella zeae , which is a teleomorphic species of the fungus Fusarium graminearum. All methods used were identical to those described above, except that the pathogenic strain tested in this way was a pathogenic strain of the fungus Gibberella zeae. Among these six microbial antagonists, four strains SGI-010-H11, SGI-014-C06, SGI-015-F03 and SGI-015-H06 showed the ability to significantly inhibit the mycelial growth of the fungus Gibberella zeae.

EXAMPLE 2 Extraction of DNA. Sequencing and taxonomy

Lysis of fungal cells and obtaining information on the ITS-5.8S rDNA sequence

[0116] Fungal biomass was transferred to a 96-well PCR microplate containing 50 μl of 2x lysis buffer (100 mM Tris HCL, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS, 400 μg/ml proteinase K). The lysis was carried out under the following conditions: 55°C for 60 minutes, 94°C for 4 minutes. An aliquot of the lysis product was used as a source of template DNA for PCR amplification. The ITS-5.8S rDNA sequence was amplified by PCR reaction with two primers M13-ITS1 (SEQ ID NO: 15) and ITS4 M13-tail (SEQ ID NO: 16).

[0117] For amplification of the ITS-5.8S rDNA region, each PCR mixture was prepared in a 20 μl final volume reaction mixture containing 4 μl from the fungal lysis reaction mixture, 0.2 μM of each primer (ITS1/ITS4), 6% Tween-20 and 10 µl 2x ImmoMix (Bioline USA Inc, Taunton, MA). PCR was performed under the following conditions: 94°C for 10 minutes; 94°C for 30 seconds, 52°C for 30 seconds, 72°C for 75 seconds for 30 cycles; 72°C for 10 minutes. A 2 µl aliquot of the PCR product was placed on a 1.0% agarose gel to confirm a single band of the expected size. Positive bands were sent for forward and reverse Sanger sequencing using M13 primers. The sequence of the 5.8S intergenic region (ITS) of fungal strain SGI-010-H11 is shown in the Sequence Listing under SEQ ID NO: 11. Homology studies for a specific nucleotide sequence of the 5.8S intergenic region (ITS) were performed using the DDBJ/GenBank/EMBL database. . After that, the phylogenetic relationship of the nucleotide sequence of the 5.8S intergenetic region (ITS) was analyzed among the isolated fungal antagonist SGI-010-H11 described here, microorganisms of genera and species showing high sequence homologies with the isolated fungal antagonist SGI-010-H11, and other diverse genera. and microbial species using the ClustalW program for building a phylogenetic tree. The fungal strain SGI-010-H11 is considered to be related to the family Mycosphaerellaceae , based on ~97% similarity of its ITS-5.8S rDNA sequence with that of Mycosphaerellapunctiformis (GenBank EU343182) and Ramulariapratensis (GenBank EUO19284), whose 5.8S ITS sequences show a clear relationship with Mycosphaerellaceae.

Lysis of bacterial cells and obtaining information about the 16S rRNA sequence

[0118] A 20 µl aliquot of the cell suspension was transferred to a 96-well PCR plate containing 20 µl of 2x lysis buffer (100 mM Tris HCL, pH 8.0.2 mM EDTA, pH 8.0.1% SDS, 400 µg/ ml Proteinase K). The lysis was carried out under the following conditions: 55°C for 30 minutes, 94°C for 4 minutes. An aliquot of the lysis product was used as a source of template DNA for PCR amplification. The 16S rRNA sequence was amplified by PCR reaction with primers M13-27F (SEQ ID NO: 17) and 1492R M13 tail (SEQ ID NO: 18).

[0119] For amplification of the 16S rRNA region, each PCR mixture was prepared in a reaction mix with a final volume of 20 µl containing 4 µl of the bacterial lysis reaction mixture, 2 µl of each primer (27F/1492R), 6% Tween-20 and 10 µl of 2x ImmoMix (Bioline USA Inc, Taunton, MA). PCR reaction was carried out under the following conditions: 94°C for 10 minutes; 94°C for 30 seconds, 52°C for 30 seconds, 72°C for 75 seconds for 30 cycles; 72°C for 10 minutes. A 2 µl aliquot of the PCR product was placed on a 1.0% agarose gel to confirm a single band of the expected size. Positive bands were sent for forward and reverse Sanger sequencing with Ml3 primers. The 16S rRNA sequences of bacterial strains SGI-014-C06, SG1-005-G08, SGI-014-G01, SGI-015-F03, and SGI-015-H06 were approximately 1.4 Kb in length and are listed in the Sequence Listing under SEQ ID numbers NO: 1, 10, 12, 13 and 14, respectively. Homology studies for certain nucleotide sequences were performed using the DDBJ/GenBank/EMBL database. After that, the phylogenetic relationship of the nucleotide sequence of the 16 rRNA genes among the isolated bacterial antagonists described here, bacteria genera and species showing high sequence homology with the isolated bacterial antagonists, and other diverse bacterial genera and species was analyzed using the ClustalW phylogenetic tree construction program. Sequence identity and similarity were also determined using the GenomeQuest™ program (Gene-IT, Worcester Mass. USA). Sequence analysis results indicated that the bacterial isolate SGI-014-G01 could be considered related to the genus Variovorax based on ~99% similarity of its 16S rRNA sequence to those of several Variovorax species, including Variovorax . R-21938 (GenBank AJ786799) and Variovoraxparadoxus (GenBank GUI 86109), whose 16S rRNA sequences show a clear relationship to the Variovorax species. In addition, sequence analysis results indicated that the two bacterial isolates SGI-015-F03 and SGI-015-H06 can be considered related to the Bacillaceae family based on >99% similarity of their respective 16S sequences to those of several Bacillus species.

[0120] The results of sequence analysis showed that the two bacterial isolates SGI-014-C06 and SGI-055-G08 can be considered related to the family Microbacteriaceae based on >99% similarity of their respective 16S sequences to those of several Microbacterium species. Namely, the 1430 nt 16S rRNA sequence of the SGI-014-C06 gene (SEQ ID NO: 1) is identical to the 16S rRNA gene of Microbacterium oxydans strain DSM 20578 and several other strains of Microbacterium (e.g. sequences Y17227.1, FJ200406, EU086800 and EU714335 from GenBank) over the entire 1430 nt length.

[0121] In particular, among the thousands of microbial strains that were isolated and tested for their ability to inhibit the development of Fusarium in the antagonism assay as described in Example 1, eighty-eight strains were thereafter identified as a species of Microbacterium based on the similarity of their 16S sequences to those in known Microbacterium species. However, the authors found that only two strains, SGI-014-C06 and SGI-055-G08 Microbacterium , had suppressive activity against Fusarium graminearum . As mentioned above, several microbes of natural origin have been reported to date with antagonistic activity against Fusarium. However, prior to the present invention, there have been no reports of a microorganism of the genus Mycobacterium having antagonistic activity. In addition, prior to the present invention, no method or process was known to the present inventors that utilizes a bacterial strain of the genus Mycobacterium as a biocontrol agent to prevent, inhibit, or treat the development of a Fusarium pathogen.

EXAMPLE 3 Sequence Analysis of Mandatory Genes from Isolate SGI-014-C06

[0122] Recently, (Richert et al. Syst. Appl. Microbiol. 30:102-108, 2007) reported phylogenetic studies of several mandatory genes from 27 species of the genus Microbacterium . These studies concluded that although the merits of 16S rRNA sequence analysis for systematic taxonomy are unsurpassed, focusing only on a single taxonomic parameter will not lead to systematic conclusions. As stated above, the nucleotide sequence of the 16S rRNA gene of strain SGI-014-C06 (SEQ ID NO: 1) is identical to the 16S rRNA gene of several Microbacterium strains, including the nucleotide sequence of the 16S rRNA gene of Microbacterium oxydans strain DSM 20578, which was included in the study (Richert et al (2007) (GenBank Accession Y17227.1)). The inventors further extended the phylogenetic analysis to the four mandatory genes of the SGI-014-C06 isolate, which are DNA gyrase B subunit ( gyrB) , RNA polymerase B subunit ( rpoE) , recombinase A ( recA) , and polyphosphate kinase ( ppk) . Towards this end, the entire genome of isolate SGI-014-C06 was shotgun sequenced, assembled and annotated according to the methods described in (PCT Patent Application No. WO2010115156A2). Genomic DNA was prepared from a fresh culture of SGI-014-C06. High molecular weight DNA was extracted using a cell bead and the UltraClean® Mega Soil DNA Isolation Kit (Cat. No. 12900-10) available from MO BIO Laboratories, Inc. according to the protocol recommended by the manufacturer. After that, genomic DNA was prepared from SGI-014-C06 for shotgun 454 pyrosequencing. Genomic DNA (7.5 µg) was used to construct the library according to the recommended protocol (454 Life Sciences) for single long reads. Sequences were generated by two consecutive GS FLX Titanium sequencing passes.

[0123] The sequences of the four mandatory genes gyrB , rpoB , recA , andppk of the SGI-014-C06 isolate, shown in the Sequence Listing, have been obtained. Homology studies for certain nucleotide sequences were performed using the DDBJ/GenBank/EMBL database. Sequence identity and similarity were also determined using the GenomeQuest™ program (Gene-IT, Worcester Mass. USA). It is shown below in more detail that the result of binding gene sequencing revealed the novelty of the SGI-014-C06 isolate described here, which is a novel bacterial strain and can be considered related to the Microbacteriaceae family.

[0124] The polynucleotide sequence of the gyrB gene of strain SGI-014-C06 has the highest identity with the sequence of the gyrB gene of strain Microbacterium testaceum with accession number AP012052 in GenBank (82.69% by alignment of 936/2172 nucleotides). Compared to the gyrB gene of Microbacterium oxydans strain DSM 20578 (GenBank AMI 81493; Richert et al. , 2007), the sequence homologies between the two genes were ~62% identical at the nucleotide level and ~37% identical at the amino acid level.

[0125] The polynucleotide sequence of the rpoB gene of strain SGI-014-C06 has the highest identity with the rpoB gene sequence of the Microbacterium maritypicum strain accession number AM181582 in GeneBank (96.98% by alignment of 1093/3504 nucleotides). Compared to the rpoB gene of Microbacterium oxydans strain DSM 20578 (GenBank AM181583; Richert et al. , 2007), the sequence homologies between the two genes were ~96% identical at the nucleotide level and 99% identical at the amino acid level.

[0126] The polynucleotide sequence of the recA gene of strain SGI-014-C06 has the highest identity with the sequence of the recA gene of strain Microbacterium testaceum accession number AP012052 in GeneBank (85.45% in alignment of 962/1188 nucleotides). Compared to the recA gene of Microbacterium oxydans strain DSM 20578 (GenBank AMI 81527; Richert et al. , 2007), the sequence homologies between the two genes were ~92% identical at the nucleotide level and 100% identical at the amino acid level.

[0127] The polynucleotide sequence of the ppk gene of strain SGI-014-C06 has the highest identity with the sequence of the ppk gene of strain Microbacterium luteolum with accession number AM181554 in GenBank (91.38% by alignment of 1380/2175 nucleotides). Compared to the ppk gene of Microbacterium oxydans strain DSM 20578 (GenBank AMI 81556; Richert et al. , 2007), the sequence homologies between the two genes were ~91% identical at the nucleotide level and ~98% identical at the amino acid level.

EXAMPLE 4 Protection of Wheat from Fusarium graminearum Infection with Microbacterium.beta . Microbial Antagonists . (NRRL B-50470) Mycophaerella . fNRRL 50471) and Variovorax . (NRRL B-50469)

[0128] The microbial strains described in Example 1 that showed a positive antibiosis test in an antagonism screen were further subjected to plant bioassays in which cells of the microbial strains were applied directly to the seeds of a susceptible cereal cultivar followed by inoculation with conidial spores of the fungus F. graminearum . Microbial strains were grown to sufficient turbidity and diluted in water. Two grams of susceptible wheat seeds (Hank; WestBred LLC, Bozeman, MT) were sown in one liter pots of pasteurized soil medium. After sowing, 20 ml of microbial culture solution was spread over the seeds. Wheat seeds were kept under greenhouse conditions for germination under fluorescent lighting for a 14 h photoperiod. At the beginning of flowering, the ear of wheat was infected by spraying conidial spores NRRL 5883 F. graminearum. Conidial spores were harvested from five day old PDA plates by pouring 0.01% Tween 20 water onto the plates and scraping the spores into the suspension. After spraying the spores, the wheat plants were transferred to a fog chamber with 100% humidity and kept there for three days for infection. Thus, the following samples were prepared:

[0129] 1. Untreated samples: no microbial or chemical fungicide treatment + Fusarium infestation.

[0130] 2. Uninfected specimens: no microbial or chemical fungicide treatment, no Fusarium infestation.

[0131] 3. SGI-010-H11: mushroom treatment + Fusarium challenge.

[0132] 4. SGI-014-GO1: bacterial treatment + Fusarium challenge.

[0133] 5. SGI-014-C06: bacterial treatment + Fusarium challenge.

[0134] Twenty days after infection, irrigation was stopped and the wheat plants were allowed to dry for three weeks before harvest. Each ear of wheat was then cut and harvested individually. The severity of the disease was determined for each ear that had symptoms of Fusarium. The severity of the disease was numerically determined by dividing the number of affected spikelets by the total number of spikelets per spike. The results (Table 2) showed that wheat treated with any of the three microbial antagonists SGI-014-C06 (NRRL B-50470), SGI-010-H11 (NRRL 50471), and SGI-014-G01 (NRRL B-50469) , had significantly lower disease severity and Fusarium graminearum infestations than untreated controls grown under the same conditions (P<0.05).

[0135] Seeds from each ear were collected by hand and weighed. The average weight of seeds per pot and in each treatment mode was determined. As shown in Table 3, all microbial antagonists produced a significant reduction in crop losses due to Fusarium infection.

table 2
Efficiency of microbial antagonists in reducing the incidence of Fusarium disease in wheat Treatment Disease severity, % Standard deviation P value uninfected 1.30 0.0025 <0.0001 raw 15.46 0.0364 Not determined SGI-010-H11 7.42 0.0219 0.0005 SGI-014-G01 6.59 0.0315 0.0001 SGI-014-C06 7.21 0.0225 0.0004

Table 3
Efficacy of microbial antagonists in maintaining seed yield in Fusarium-infected wheat Treatment Seed weight, g P value uninfected 0.8438±0.1633 0.0191 raw 0.4669±0.1209 Not determined SGI-010-H11 0.6200±0.1 844 0.7025 SGI-014-G01 0.7963±0.2032 0.5277 SGI-014-C06 0.6744±0.0948 0.0551

EXAMPLE 5.growth inhibition Fusarium graminearum microbial antagonists on wheat seedlings

[0136] Microbial strains that demonstrated suppressor activity against F. graminearum in a laboratory antagonism test were also investigated for their ability to reduce the incidence of Fusarium seed blight in wheat. Seeds of a susceptible wheat variety (Hank; WestBred LLC, Bozeman, MT) were sown in 1 liter 20 cm diameter plastic pots with pasteurized soil medium. Microbial cell suspensions were prepared as follows. 2YT or other similar nutrient medium was inoculated with broth cultures from glycerol matrix solutions of isolates or streak plates. Typically, cultures were initiated 48-72 hours prior to placing them in a culture chamber, greenhouse, or field, where they reached the late exponential growth phase. Isolates with longer doubling times were also initiated in advance. Cultures were incubated at 30°C on a rotary shaker at 200 rpm. After culturing, the cells were formed into 10,000×g granules for 15 min. at 4°C and resuspended in 10 mm MgSO ₄ buffer (pH 7.0). Cell density was normalized for each isolate in units of CFU/mL (cell-forming units per milliliter). As a rule, ~10 ⁹ cfu/ml suspension was prepared for each isolate, which was delivered to the inoculation site on ice. Inoculation was carried out by diluting these cell suspensions 1/20 in irrigation water to a final density of 5×10 ⁷ cfu/ml. For experiments in 1 liter pots, 20 ml of this diluted cell suspension was evenly distributed over the cultivation surface of each pot. For the micropot experiments, the cell suspensions were not diluted and 1 ml of each 10 ⁹ cfu/ml suspension was pipetted onto the cultivation surface of each pot like a spray over the top of submerged seeds. Seeds and plants were kept in a greenhouse at room temperature with a 14-hour light photoperiod.

[0137] Fusarium graminearum strain NRRL-5883 mycelium was grown on PDA plates for 5 days under continuous illumination. Hyphae and conidia were harvested by pouring a few milliliters of sterile water (0.01% Tween 20) onto the plates and scraping the surface of the agar with a sterile spatula. The spore concentration in the inoculum was approximately 2×10 ⁵ spores/ml, however, the concentration of the hyphal fragment was not determined.

[0138] Inoculation with the F. graminearum species was started after the emergence of short shoots from the seeds and was repeated every other evening for 10 days. The F. graminearum inoculum was applied by spraying in an amount of approximately 30 ml per pot. Immediately after inoculation, each time the plants were sprayed from above with a fogger. When using a chemical fungicide, it was prepared by diluting 2 ml of a fungicide stock solution (Banner Maxx, Syngenta) in 1 L of distilled water and applied with a sprayer in an amount of approximately 30 ml per pot.

[0139] Fusarium damage was assessed by the degree of Fusarium infection, determining it by the number of Fusarium colony forming units per gram (CFU/g) of plant tissues. All processing operations were performed in four blocks of four pots each. The following types of processing were carried out:

[0140] 1. Untreated: no microbial or chemical fungicide treatment + Fusarium infestation.

[0141] 2. Uninfected: no microbial or chemical fungicide treatment and no Fusarium infestation.

[0142] 3. Fungicide: chemical fungicide treatment + Fusarium infection.

[0143] 4. SGI-010-H11: fungal treatment + Fusarium challenge.

[0144] 5. SGI-014-G01: bacterial treatment + Fusarium challenge.

[0145] 6. SGI-014-C06: bacterial treatment + Fusarium challenge.

[0146] the Results, which are shown in table 4, showed that all three microbial treatments gave a significant reduction in the severity of fusarium compared with uninfected control. In particular, two microbial antagonists, SGI-014-C06 and SGI-010-H11, significantly reduced Fusarium graminearum infection in wheat compared to an untreated control grown under the same conditions. Protection of wheat against Fusarium infection by either of the two microorganisms, SGI-014-C06 or SGI-010-H11, was statistically comparable to that of a commercial chemical fungicide. When using the microorganism SGI-014-G01, the observed protection of wheat against Fusarium infection was much less noticeable, and its effectiveness varied significantly from one cultivation pot to another.

Table 4
Effect of microbial antagonists on Fusarium infection Treatment cfu/g P value raw 1487±600 Not determined uninfected 0±0 <0.0001 Chemical fungicide 290±146 <0.0001 SGI-010-H11 671±450 0.0077 SGI-014-G01 1246±465 0.8387 SGI-014-C06 367±234 0.0001

EXAMPLE 6 Cultivation and storage of microbial antagonists

[0147] Species Mycosphaerella . Several methods were used to store the isolated fungus as a pure culture, one of which used filter paper. In another case, after cultivation of the fungus on PDA, it was cut into small squares, which were placed in vials with 15% glycerol solution and stored at -70°C. In a similar experiment, this fungus was stored at 4°C, but not in glycerol, but in distilled water. However, one of the preferred methods was storage on infected sterile barley seeds at -80°C.

[0148] Bacillus , Microbacterium and Variovorax species . The isolated bacteria were kept as a pure culture. The bacterial colony was transferred into a vial with R2A liquid broth medium (Tecknova) and cultured there at 30°C on a shaker at 250 rpm. during two days. After that, the culture was transferred into vials with 15% glycerol solution and kept in them at -80°C.

EXAMPLE 7 Treatment for Spore Production and Seed Coating

[0149] Spore production . Following a typical spore production protocol, one liter of 2xYT culture medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) was inoculated with 5 ml of starter culture or scraping from a Petri dish and incubated overnight at 30°C in a rotary shaker at 225 rpm. Bacterial cells were pelleted by centrifugation and washed with 1x PBS buffer (8 g/l NaCl; 0.2 g/l KC1; 1.44 g/l Na ₂ HPO ₄ ; 0.24 g KH ₂ PO ₄ ; pH 7.4) . Cells were resuspended in CDSM medium (Hageman et al , J. Bacterial. , 438-441, 1984) and cultured for an additional four nights at 30° C. in a rotary shaker. Sporulation in the bacterial culture was monitored daily using a phase contrast microscope until the entire culture was formed from free floating spores. The duration of such incubation usually does not exceed four days or lasts more than six days, depending on the cultivated species. Under a phase-contrast microscope, endospores are visible inside cells as bright white, flattened spheroids. Bacterial spores were granulated by centrifugation at 10,000 X g for 15 minutes, washed twice with sterile dH ₂ O and, if necessary, concentrated to 50 ml, and could either be used immediately or refrigerated for future use. Spore concentration was measured by the OD ₆₀₀ method. This technique typically produced at least 2000 OD of bacterial spores.

[0150] In particular, several bacterial strains in accordance with the invention, for example, SGI-015-F03 of the Bacillus species, can produce large numbers of spores after 4 days of incubation with a simple inoculation of a large overnight growing starter culture (~15 ml) in 1 liter of 2xSG culture medium followed by 4 days of incubation at the appropriate temperature in a rotary shaker at 225 rpm. The nutrient medium 2xSG of the following composition was used: 16 g/l nutrient broth; 0.25 g/l MgSO ₄ ; 2.0 g/l KCl; 0.15 g/l CaCl ₂ .2H ₂ O; 0.025 g/l MnCl ₂ .2H ₂ O; 0.28 mg/l FeSO ₄ -7H ₂ O; 1.0 g/l dextrose.

[0151] Coating of wheat and corn seeds. A small scale seed treatment experiment was carried out according to the minimally modified procedure described in (Sudisha et al. , 2009). Typically, a biopolymer stock solution was prepared by adding 6 g of gum arabic powder (MP Biomedical) to 36 ml of water in a 50 ml Falcon tube and then mixing until homogeneous using a wheeled mixer. A mixing plate was used to mix large quantities of coated seeds where necessary.

[0152] If plant cells were used, then cloudy cultures of actively growing microbial cells were washed with PBS (phosphate buffered saline) and adjusted to OD ₆₀₀ ~5.0. Alternatively, microbial spore suspensions were prepared as described above. Bacterial spores and/or plant cells were thoroughly resuspended in ~32 ml of gum arabic-biopolymer stock solution prepared as described above, and the resulting suspension was thoroughly mixed in a 1 liter bottle. Approximately 400 g of seeds (wheat or corn) were added to the bottle and mixed vigorously on a shaker or vortex shaker until a uniform distribution of the gum/cell suspension was achieved. The coated seeds were then spread over plastic trays for laminar flow drying until detackified, generally for 3-6 hours with occasional agitation. In some cases, spore-covered seeds can be dried overnight. However, crop-covered seeds were generally stored before they were completely dehydrated. A viability test periodically subjected to microbes used in seed coating preparations showed that these microbes remained viable for at least three months. The germination rate of coated seeds was almost identical to that of uncoated control seeds.

EXAMPLE 8. Effect of microbial seed treatment on the development of Fusarium in wheat

[0153] Seeds of Fusarium-susceptible variety (RB07) wheat were coated with microorganisms SGI-014-C06 or SGI-015-F03 or SGI-015-H06 in accordance with the procedure described in Example 8. Coated seeds were sown in 1 liter pots with pasteurized soil media [(Metromix-Mix 200; Scotts-Sierra Horticultural Products, Marysville, OH; and 3 grams of 14-14-14 (NPK) fertilizer]. Pots were seeded with 7-8 plants each, after which, in the growth phase 3rd leaf, their seeding was reduced to 5 plants per pot.Pots were combined into randomized blocks of 5 pots per treatment.Further, the coated seeds were placed to germinate in greenhouse conditions with fluorescent lighting for 16 hours per day.When the wheat began to bloom, it was infected by spraying on the ear with macroconidial spores of strain NRRL 5883 of the species F. graminearum at a concentration of 100,000 spores/ml After spraying the spores, the wheat was placed in a climatic chamber with 100% humidity and dew formation, and kept in these favorable conditions. conditions suitable for infection within three days. The following types of sample processing were carried out:

[0154] 1. Untreated: no microbial or chemical fungicidal treatment + Fusarium infestation.

[0155] 2. SGI-014-C06: bacterial seed treatment + Fusarium challenge.

[0156] 3. SGI-015-F03: bacterial seed treatment + Fusarium challenge.

[0157] 4. SGI-015-H06: bacterial seed treatment + Fusarium challenge.

[0158] The severity of Fusarium was assessed visually on the tenth day and twenty-first day after infection. The degree of infection was determined for each spikelet showing Fusarium symptoms, and the percentage of symptomatic spikelets was calculated, that is, the number of spikelets showing symptoms of Fusarium multiplied by 100 was divided by the total number of spikelets. The results obtained (Table 5) showed that wheat seeds coated with any of the three tested microbial antagonists - SGI-014-C06, SGI-015-F03, SGI-015-H06 - had a significantly reduced infection rate with the fungus Fusarium graminearum compared to " untreated control grown under the same conditions (P<0.05).

Table 5
Development of Fusarium symptoms in wheat after seed treatment with antagonistic microorganisms Treatment Degree of infection, % 10 days after infection 21 days after infection Raw control 49.31 66.10 SGI-014-C06 2.08 7.74 SGI-015-F03 23.12 31.53 SGI-015-H06 9.42 19.81

EXAMPLE 9 Biocontrol of Wheat Fusarium in Greenhouse Trials

[0159] Each of the microorganisms SGI-014-C06, SGI-015-F03, and SGI-015-H06 was tested in a larger greenhouse trial. The trials were conducted in a "plant growth containment room" (PGCR: plant growth containment room) of Synthetic Genomics, Inc., and included the following treatments: infected control, uninfected control, and treatment with industrial fungicides of four brands. These included chemical fungicides Banner MAXX® (Syngenta) at 2 ml/L and Prosaro® (Bayer CropScience) at 3 ml/L, which were sprayed on the leaves according to the manufacturer's recommendations, and biological fungicides of the Actinovate brands. ® (Natural Industries) and RhizoVital® (ABiTEP GmbH), which were applied by spraying the soil also in accordance with the manufacturer's recommendations.

[0160] The microbial treatment preparation was applied either by spraying the soil or by coating the seed. When the microbial treatment was carried out by spraying the soil, cell suspensions of each microorganism were individually applied to the seeds before covering them with a large amount of nutrient medium. For this, freshly prepared cultures were granulated to remove nutrients and resuspended in 10 mM magnesium sulfate (MgSO ₄ ) buffer. Thereafter, cell suspensions were added by pipetting and spreading 20 ml of the suspension evenly over the seeds for a total of ~10 ⁹ cells per pot. In the case of microbial seed coating treatment, microorganisms (cells/spores) were prepared essentially as described above in Example 8. Coated seeds were obtained by incorporating a solution of gum arabic into the cell suspension and thus creating a sticky mixture for subsequent application. her for seeds. After that, the microbial coating of the seeds was dried, resulting in the formation of a solid, but water-soluble, microorganism-capturing biopolymer (cells/spores). The aim of these manipulations was to provide a cell titer of at least 10 ⁶ -10 ⁷ viable cells/seed. In this greenhouse experiment, the seeds were coated the day before sowing.

[0161] For each type of treatment, four identical sets of samples were provided, placed according to a randomized full block design of the experiment (RCBD: Randomized Complete Block Design) on three-level shelves located in two rows along the sides of the greenhouse. The shelves were equipped with a fluorescent lighting system (Agrosun, 5850K) that provided an average illumination of 800 µmol measured on the surface of the pot. Each set of samples consisted of four fixed 1 L pots. The pots were filled with an artificial nutrient medium consisting of Arabidopsis Growth Medium AIS (from Lehle Seeds) and washed coarse sand in a 3:1 volume ratio. In all pots, two grams of spring wheat seeds (Hank, WestBred) were spread over the surface of the nutrient medium. The pots had a bottom water supply with maintenance of ~2 cm water level and were visually controlled for germination and growth.

[0162] In the flowering phase (Feekes 10.5.1 cereal growth and development identification system), a wheat ear was infected by spraying a Fusarium graminearum conidial spore suspension. For this greenhouse experiment, fungal spores were prepared by planting homogenized mycelium of F. graminearum strain NRRL 5883 on large PDA plates and then incubating for five days under continuous illumination at room temperature. Thereafter, fungal spores were collected by flooding the plates with magnesium sulfate buffer (10 mM MgSO ₄ ; 0.01% Tween 20) and scraping the surface of the agar with a sterile spatula. After adjusting the spore concentration, approximately 50,000 conidial spores per spike were applied to the plants using a hand sprayer. The relative humidity in the greenhouse was raised to 90% and kept for three days for infection, and then returned to normal levels. After the wheat seeds had set, the water supply was stopped and the spikelets were allowed to dry completely. Then the ears of wheat were collected individually. The severity of the disease was determined for each spike that had Fusarium symptoms by counting the number of affected spikelets and dividing it by the total number of spikelets per spike. The results (Table 6) showed that wheat treated with any of the three microbial antagonists tested, SGI-014-C06, SGI-015-F03, SGI-015-H06, had a significantly lower severity and number of Fusarium graminearum infections compared to " untreated control grown under the same conditions (P<0.05).

Table 6
The effectiveness of microbial antagonists in reducing the incidence of Fusarium in wheat Treatment Degree of infection, % Protection against infection, % uninfected 2.1±2.9 Not determined Infected 37.2±9.8 0.00 Prosario® 4.6±2.8 93.00 Banner-MAXX® 12.5±5.8 70.00 Actinovate® 38.9±12.1 -5.00 Rhizo Vital® 28.0±5.4 26.00 SGI-014-C06 18.4±9.1 30.00 SGI-015-F03 25.4±6.9 54.00 SGI-015-H06 26.6±7.4 34.00

[0163] To determine the yield of seeds, each ear of wheat was cut by hand, the seeds were collected and cleaned of husks and other detritus, counted and weighed. The total seed yield was defined as the total weight of seeds obtained from plants in 16 pots for each treatment. As shown in Table 7, all three microbial antagonists tested showed significant retention of seed yield, ie seed losses due to Fusarium infection were significantly reduced.

Table 7
The effectiveness of microbial antagonists in maintaining the yield of wheat seeds affected by Fusarium Treatment Total seed yield, g Seed yield protection, % uninfected 14.36 Undefined raw 11.05 0.00 Prosario® 13.44 72.00 Banner-MAXX® 14.41 102.00 Actinovate® 11.99 28.00 RhizoVital® 12.65 48.00 SGI-014-C06 14.70 110.00 SGI-015-F03 15.03 120.00 SGI-015-H06 14.14 93.00

[0164] Thus, the treatment of wheat with any of the considered microorganisms made it possible to significantly protect the yield of seeds from losses caused by Fusarium infection. In particular, wheat treated with either of the SGI-014-C06 or SGI-015-F03 strains showed a significant improvement in overall seed yield, ie 110% and 120%, respectively, compared to the uninfected control. In contrast, commercial fungicides of the Actinovate®, RhizoVital® and Prosario® brands have not shown significant yield protection in their treated wheat against losses caused by Fusarium infection.

EXAMPLE 10. Biocontrol with fusarium wheat in the field

[0165] Antagonistic microorganisms that have shown their suppressive activity against the pathogen F. graminearum and Fusarium in the in vitro antagonism assay and in the greenhouse studies described in Examples 4-9 continue to be investigated further, in a series of field trials in various geographical areas throughout the United States. States. Some experiments are carried out on agricultural areas that are used for microbiological control of Fusarium wheat, where natural infection with this disease occurs. These tests use wheat varieties that are susceptible to F. graminearum infection. In these trials, wheat treated with different methods is sown in small plots of 12 rows, about 3 meters long. Row spacing is approximately 20 cm. The treated areas in each experiment are entered into a randomized block design. The efficacy of various wettable powder compositions containing microbial antagonists according to the invention in reducing the severity and incidence of Fusarium infections is also being evaluated. In these experiments, microbial antagonists are evaluated both individually and in combinations.

[0166] In these tests, antagonistic microorganisms are evaluated in various coatings on seeds and/or in the field, where microbial suspensions are applied directly to flowering wheat ears. In each of these experiments, microorganisms are evaluated at identical sites. The experiments can use antagonists, pathogens and agricultural production methods described in Examples 4-9.

[0167] Coating the seeds. In addition to the seed coating methods described above in Example 7, a wide variety of known technologies and formulations for treating wheat seeds with microbial antagonists, such as those described in (Fernando et al , 2002; Bello et al. , 2002; Kim et al. al , 1997). In general, wheat seeds are first moistened and kept at room temperature to initiate germination. Before sowing, germinated seeds can be immersed in microbial suspensions. Pathogenic spores can be inoculated either before or after bacterial inoculation. Methods for preparing antagonists, pathogens and plants for this seed treatment may be as described in Examples 4 and 5.

[0168] Assessment of disease severity and seed yield.

Antagonists are amenable to further evaluation in greenhouse conditions for their suppressive activity against Fusarium on flowering wheat and barley using a variety of methods and techniques, including those described, for example, in (Schisler, Plant Disease, Vol 86(12), 1350-1356, 2002; Schisler et al Biological Control, 39:497-506, 2006; and Khan and Doohan, Biological Control, 48:42-47, 2009). In one such experiment, G. zeae strain inoculums are prepared on sterile, yellow grain corn as described in (Khan et al. Biological Control , 29:245-255, 2004). Fully colonized lipid cultures (48 hour culture) of microbial strains are diluted to one-fourth the concentration with phosphate buffer. The final number of CFU per ml for antagonistic treatment ranges from 1×10 ⁹ CFU/ml to 6×10 ⁹ CFU/ml. After that, suspension treatment is applied to the flowering wheat ears using a CO ₂ hand sprayer. The treatment is usually carried out after sunset to minimize possible UV degradation of the antagonistic cells. Prepare 5 replicates (groups of sample copies) per treatment, introduced into a randomized block diagram. The first treatment control are the plants treated with the buffer solution. The second treatment control is the untreated plants. Infection severity and incidence of Fusarium in the field are assessed by counting 60 ears per replicate (i.e. 300 ears/treatment) where the plant develops from a medium milky phase to a soft waxy phase. When the grains reach full maturity, the wheat ears are harvested by hand and threshed using an Almaco brand single plant and ear thresher (Almaco, IA). Samples of grains obtained from each row of replicates are evaluated by weight of 100 grains. Data on disease severity, incidence and weight of 100 caryopses are analyzed by one-way analysis of variance (ANOVA).

[0169] Compositions containing microbial antagonists according to the invention, alone or in combination, demonstrate a significant reduction in morbidity. These results indicate that biological control with these active microbial agents can play an important role in the control of scab in cereal plants such as wheat.

EXAMPLE 11 Development of non-natural cultivars and their breeding programs

[0170] Endophytic microorganisms in accordance with the invention are introduced into cultivated plants, including crops, of various genotypes and geographical origin. However, similar endophytic microorganisms with which it would be possible to create plant-endophyte combinations with improved agronomic characteristics using methods similar to those known in the industry, including, among others, those described in (U.S. Pat Appl. No. 20030195117A1; U.S. Pat. Appl. No. 20010032343A1; and U.S. Pat. No. 7084331) are missing. Thus, desired artificial plant-endophyte combinations can be created and selected by a breeding and cultivar development program based on their ability to form and maintain a mutualistic combination resulting in an agronomic effect. Such a breeding program may also use a ranking of the agronomic characteristics of a given combination. These characteristics may include, for example, drought tolerance, biomass accumulation, resistance to insect infestation, edibility by livestock (e.g., herbivores), ease of reproduction, seed yield, etc. Such combinations may differ in the level of accumulation of microbial metabolites that are toxic to agricultural pests. and weeds, including ergot alkaloid levels, lolin levels, peramine levels, or lolitrem levels, while demonstrating the required agronomic characteristics of cultivated plants, including resistance to infection or eating by insects, resistance to abiotic stress, livestock palatability, biomass accumulation, ease of propagation and seed output, etc.

[0171] Here is a description of many embodiments of the invention. However, it is understood that the elements of the embodiments of the invention described herein may be combined in combination to form new embodiments of the invention and various modifications thereof, which do not go beyond the spirit and scope of the present invention. Therefore, all other embodiments of the invention, alternatives and equivalents are covered by the scope of the invention in accordance with this description of the invention and its claims.

[0172] The title in this application is given solely for ease of understanding by the reader and in no way limits the scope of the invention or its embodiments.

[0173] All publications and patent applications mentioned in the present description are incorporated here by reference to the same extent as if each publication or patent application is individually indicated as incorporated by reference.

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SEQUENCE LIST

<110> SYNTHETIC GENOMICS, INC.

GRANDLIC, CHRISTOPHER J.

GREEN, WAYNE A.

KEROVUO, JANNE S.

MCCANN, Ryan T.

<120> COMPOSITIONS AND METHODS FOR FIGHTING FUSARIA

<130> SGI1520-1WO

<150> US 61/511,467

<151> 2011-07-25

<160> 18

<170>PatentIn version 3.5

<210> 1

<211> 1430

<212> DNA

<213> Microbacterium sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-014-C06

<220>

<221> various signs

<223> encodes 16S ribosomal RNA

<400> 1

acgctggcgg cgtgcttaac acatgcaagt cgaacggtga acacggagct tgctctgtgg60

gatcagtggc gaacgggtga gtaacacgtg agcaacctgc ccctgactct gggataagcg 120

ctggaaacgg cgtctaatac tggatatgtg acgtgaccgc atggtctgcg tctggaaaga 180

atttcggttg gggatgggct cgcggcctat cagcttgttg gtgaggtaat ggctcaccaa 240

ggcgtcgacg ggtagccggc ctgagagggt gaccggccac actgggactg agacacggcc 300

cagactccta cgggaggcag cagtggggaa tattgcacaa tgggcgcaag cctgatgcag 360

caacgccgcg tgagggacga cggccttcgg gttgtaaacc tcttttagca gggaagaagc 420

gaaagtgacg gtacctgcag aaaaagcgcc ggctaactac gtgccagcag ccgcggtaat 480

acgtagggcg caagcgttat ccggaattat tgggcgtaaa gagctcgtag gcggtttgtc 540

gcgtctgctg tgaaatccgg aggctcaacc tccggcctgc agtgggtacg ggcagactag 600

agtgcggtag gggagattgg aattcctggt gtagcggtgg aatgcgcaga tatcaggagg 660

aacaccgatg gcgaaggcag atctctgggc cgtaactgac gctgaggagc gaaagggtgg 720

ggagcaaaca ggcttagata ccctggtagt ccaccccgta aacgttggga actagttgtg 780

gggtccattc cacggattcc gtgacgcagc taacgcatta agttccccgc ctggggagta 840

cggccgcaag gctaaaactc aaaggaattg acggggaccc gcacaagcgg cggagcatgc 900

ggattaattc gatgcaacgc gaagaacctt accaaggctt gacatatacg agaacgggcc 960

agaaatggtc aactctttgg acactcgtaa acaggtggtg catggttgtc gtcagctcgt 1020

gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc ctcgttctat gttgccagca 1080

cgtaatggtg ggaactcatg ggatactgcc ggggtcaact cggaggaagg tggggatgac 1140

gtcaaatcat catgcccctt atgtcttggg cttcacgcat gctacaatgg ccggtacaaa 1200

gggctgcaat accgcgaggt ggagcgaatc ccaaaaagcc ggtcccagtt cggattgagg 1260

tctgcaactc gacctcatga agtcggagtc gctagtaatc gcagatcagc aacgctgcgg 1320

1380

1430

<210> 2

<211> 2172

<212> DNA

<213> Microbacterium sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-014-C06

<220>

<221> various signs

<223> SGI Gene ID SG1MICG850682

<220>

<221> various signs

<223> encodes the peptide sequence SED ID NO 3

<400> 2

gtgtctgcgg ccggagagcg gctgtcgaga atagactcgg agattgtgaa tgccgagtat60

tccgcccatc atctccaggt gcttgagggg ctcgaagctg tccgcaagcg ccggggcatg 120

tacatcgggt cgaacggctc gccgggcctc atgcactgcc tttgggagat catcgacaac 180

tctgtcgacg aggcggtggc aggcaacggc acgaagatcg acatcatcct gcactccgac 240

ggcagcgtcg aggtgcacga ccgcggtcgc ggcatccctg tcgacgtcga gccgcgcacc 300

ggtctcaccg gtgtcgaggt cgtctacacc aagctgcacg ccggaggaaa gttcggcggc 360

ggctcgtacg cggcatccgg tggactgcac ggtgtcggcg cctccgtcgt gaacgcgctc 420

tccgagcgcc tcgacgtcga ggtcgaccgc gggggcaaga cctacgcgat gtcgttccat 480

cgcggtgagc ccggcatctt cacggactcc ggcgagaagc ggccggacgc cccgttcacg 540

ccgttcgagg agaacagcga gctgcgcgtc atcggcaagg cgccgcgtgg cgtgaccggc 600

acccgggtgc gctactgggc cgaccggcag atcttcacca aggatgcggc gttccagctc 660

tcggagctcg agacccgcgc acgccagacg gcgttcctcg ttcccggtct cgagatcgtc 720

gtcaaggacg cacgggcggc cggtcaggtc atccccgtcc cggactccga cggcgagacg 780

accgtcgtcg ggaccgatga gacctcgtac ctctacgagg gcggcatctc cgagttcgtc 840

gagtacctcg ccatcgaccc tcccgtgacc gacacctggc gtatccaggg cgagggcatg 900

ttcaaggaga ccgtgcccgt cctgcaggca gacggccaca tggtcgccac cgaggtggag 960

cgtgtgtgcg ccgtcgacat cgcgctgcgc tgggggaccg gctacgacac ccgtgtgcgc 1020

tccttcgtga acatcatcgc gacgcccaag ggcggaaccc accagcaggg cttcgagcag 1080

gagctcctga aggtgctgcg ctcccaggtc gagcagaacg cccgccgtct gaaggtcggc 1140

aacgacaagc tggagaagga cgacgtcctc gccggcctca ccgccgtgct cacggtcaac 1200

gtgccggagc cgcagttcga gggccagacc aaggaagtgc tcggcacccc cgcggtgcgg 1260

1320

1380

1440

ctgccgacca agctcgtcga ctgccgcacg aacgaggtcg agcgcagcga gctcttcatc 1500

gtggagggcg actcggctct cggcaccgcc aagaacgcgc gcaacagcga gttccaggcg 1560

ctgctcccga tccgagggaa gatcctcaac gtgcagaagg cctctgtcgg cgacatgctg 1620

tcgaacaccg agtgcgcgtc gatcatccag gtgatcggcg ccggatccgg acgcaccttc 1680

gacatcgatg cggcgcgcta cggcaaggtg atcctgatga gcgacgccga tgtcgacggc 1740

gcgcacatcc gtaccctgct gctcacgctg ttcttccgct acatgcgacc gctgatcgag 1800

cacgggcgtg tgttcgccgc ggtgccgccg ttgcaccgggg tgatcgtgat gaacccgggg 1860

tccaagccga acgagacgat ctacacctac agcgagcagg agatgcacgc gctgctggcg 1920

aagctccgca aggccggcaa gcgctggcac gagccgatcc agcgctacaa gggtctcggt 1980

gagatggacg cggaacagct cgcgaacacc accatggacc gctccggccg tctgctgcgc 2040

cgtgtgcgca tggaagacgc cgaggccgcc ggtcgcgtgt tcgagctgct gatgggcaac 2100

gaggtcgcgc cgcgccgcga gttcatcatc gactcctccg accggttgtc gcgcgagtcc 2160

atcgacgcct ga 2172

<210> 3

<211> 723

<212> PROTEIN

<213> Microbacterium sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-014-C06

<220>

<221> various signs

<223> SGI peptide ID SG1MICT850682

<220>

<221> various signs

<223> gyrase B subunit

<400> 3

Met Ser Ala Ala Gly Glu Arg Leu Ser Arg Ile Asp Ser Glu Ile Val

1 5 10 15

Asn Ala Glu Tyr Ser Ala His His Leu Gln Val Leu Glu Gly Leu Glu

20 25 30

Ala Val Arg Lys Arg Pro Gly Met Tyr Ile Gly Ser Asn Gly Ser Pro

35 40 45

Gly Leu Met His Cys Leu Trp Glu Ile Ile Asp Asn Ser Val Asp Glu

50 55 60

Ala Val Ala Gly Asn Gly Thr Lys Ile Asp Ile Ile Leu His Ser Asp

65 70 75 80

Gly Ser Val Glu Val His Asp Arg Gly Arg Gly Ile Pro Val Asp Val

85 90 95

Glu Pro Arg Thr Gly Leu Thr Gly Val Glu Val Val Tyr Thr Lys Leu

100 105 110

His Ala Gly Gly Lys Phe Gly Gly Gly Ser Tyr Ala Ala Ser Gly Gly

115 120 125

Leu His Gly Val Gly Ala Ser Val Val Asn Ala Leu Ser Glu Arg Leu

130 135 140

Asp Val Glu Val Asp Arg Gly Gly Lys Thr Tyr Ala Met Ser Phe His

145 150 155 160

Arg Gly Glu Pro Gly Ile Phe Thr Asp Ser Gly Glu Lys Arg Pro Asp

165 170 175

Ala Pro Phe Thr Pro Phe Glu Glu Asn Ser Glu Leu Arg Val Ile Gly

180 185 190

Lys Ala Pro Arg Gly Val Thr Gly Thr Arg Val Arg Tyr Trp Ala Asp

195 200 205

Arg Gln Ile Phe Thr Lys Asp Ala Ala Phe Gln Leu Ser Glu Leu Glu

210 215 220

Thr Arg Ala Arg Gln Thr Ala Phe Leu Val Pro Gly Leu Glu Ile Val

225 230 235 240

Val Lys Asp Ala Arg Ala Ala Gly Gln Val Ile Pro Val Pro Asp Ser

245 250 255

Asp Gly Glu Thr Thr Val Val Gly Thr Asp Glu Thr Ser Tyr Leu Tyr

260 265 270

Glu Gly Gly Ile Ser Glu Phe Val Glu Tyr Leu Ala Ile Asp Pro Pro

275 280 285

Val Thr Asp Thr Trp Arg Ile Gln Gly Glu Gly Met Phe Lys Glu Thr

290 295 300

Val Pro Val Leu Gln Ala Asp Gly His Met Val Ala Thr Glu Val Glu

305 310 315 320

Arg Val Cys Ala Val Asp Ile Ala Leu Arg Trp Gly Thr Gly Tyr Asp

325 330 335

Thr Arg Val Arg Ser Phe Val Asn Ile Ile Ala Thr Pro Lys Gly Gly

340 345 350

Thr His Gln Gln Gly Phe Glu Gln Glu Leu Leu Lys Val Leu Arg Ser

355 360 365

Gln Val Glu Gln Asn Ala Arg Arg Leu Lys Val Gly Asn Asp Lys Leu

370 375 380

Glu Lys Asp Asp Val Leu Ala Gly Leu Thr Ala Val Leu Thr Val Asn

385 390 395 400

Val Pro Glu Pro Gln Phe Glu Gly Gln Thr Lys Glu Val Leu Gly Thr

405 410 415

Pro Ala Val Arg Gln Ile Val Ala Gln Val Ile Arg Lys Asp Leu Ala

420 425 430

Gln Arg Phe Ser Ser Thr Lys Arg Asp Asp Lys Asn Gln Ala Thr Gln

435 440 445

Leu Leu Asp Lys Ile Val Ser Glu Met Lys Ala Arg Val Ser Ala Arg

450 455 460

Ala His Lys Glu Thr Gln Arg Arg Lys Asn Ala Leu Glu Ser Ser Thr

465 470 475 480

Leu Pro Thr Lys Leu Val Asp Cys Arg Thr Asn Glu Val Glu Arg Ser

485 490 495

Glu Leu Phe Ile Val Glu Gly Asp Ser Ala Leu Gly Thr Ala Lys Asn

500 505 510

Ala Arg Asn Ser Glu Phe Gln Ala Leu Leu Pro Ile Arg Gly Lys Ile

515 520 525

Leu Asn Val Gln Lys Ala Ser Val Gly Asp Met Leu Ser Asn Thr Glu

530 535 540

Cys Ala Ser Ile Ile Gln Val Ile Gly Ala Gly Ser Gly Arg Thr Phe

545 550 555 560

Asp Ile Asp Ala Ala Arg Tyr Gly Lys Val Ile Leu Met Ser Asp Ala

565 570 575

Asp Val Asp Gly Ala His Ile Arg Thr Leu Leu Leu Thr Leu Phe Phe

580 585 590

Arg Tyr Met Arg Pro Leu Ile Glu His Gly Arg Val Phe Ala Ala Val

595 600 605

Pro Pro Leu His Arg Val Ile Val Met Asn Pro Gly Ser Lys Pro Asn

610 615 620

Glu Thr Ile Tyr Thr Tyr Ser Glu Gln Glu Met His Ala Leu Leu Ala

625 630 635 640

Lys Leu Arg Lys Ala Gly Lys Arg Trp His Glu Pro Ile Gln Arg Tyr

645 650 655

Lys Gly Leu Gly Glu Met Asp Ala Glu Gln Leu Ala Asn Thr Thr Met

660 665 670

Asp Arg Ser Gly Arg Leu Leu Arg Arg Val Arg Met Glu Asp Ala Glu

675 680 685

Ala Ala Gly Arg Val Phe Glu Leu Leu Met Gly Asn Glu Val Ala Pro

690 695 700

Arg Arg Glu Phe Ile Ile Asp Ser Ser Asp Arg Leu Ser Arg Glu Ser

705 710 715 720

Ile Asp Ala

<210> 4

<211> 3504

<212> DNA

<213> Microbacterium sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-014-C06

<220>

<221> various signs

<223> SGI Gene ID SG1MICG850290

<220>

<221> various signs

<223> encodes the peptide sequence of SEQ ID NO 5

<400> 4

ttggctgctg ctcgcaacgc atccacatcc accaccacca agaacggacg cggagcttcc60

cgtctttcgt tcgccaagat ctccgacacg ctgacggtcc ctgaccttct cgccctgcag 120

accgaatcct tcggttggct ggtcggcaac gacgcctgga aggcgcgcgt ggccgaggcc 180

aagaagcagg ggcgcaccga cgtcaacgag aacagcggtc tgggcgagat cttcgaggag 240

atctctccga tcgaggacct cggcgagacg atgcagctgt cgttcacgaa cccctacctc 300

gagccggaga agtactccat cgaggagtgc aaggagcgtg gcaagaccta cgccgctccg 360

ctgtacgtcg aggccgagtt catgaaccac ctcacgggtg agatcaagac ccagacggtc 420

ttcatgggcg acttcccgct gcagaccgac aagggaacgt tcatcatcaa cggctccgag 480

cgcgtcgtcg tctcgcagct ggtgcgttcg cccggtgtct acttcgacaa gacccccgac 540

aagacgtccg acaaggacat cgtctcggca cgcgtcatcc cgagccgtgg tgcctggctc 600

gagttcgaga tcgacaagcg cgaccaggtc ggcgtgcgcg tcgaccgcaa gcgcaagcag 660

tcggtcacgg tcttcctcaa ggcgctgggc atgaccagcg aggagatcct cgccgagttc 720

gccggctaca cctcgatcga ggagacgctc gcgaaggaca cgatcgtcac gaaggaagat 780

gcgctccgcg acatctaccg caagctccgt ccgggcgagc aggtcgccgc cgaggccgcc 840

cgcgcgctcc tggacaactt ctacttcaac ccgaagcgct acgacctggc caaggtcggt 900

cgctacaaga tcaaccacaa gctgggcctg gaccagccgc tgaactcgtc ggtcctgacc 960

gtcgaagaca tcgtggccac gatcaagtac ctggtgcgtc tgcacgccgg caccgaggag 1020

accttcacgg gcatccgcgg tggtaagaag gccgagatcc gtctcgcgac cgacgacatc 1080

gacaacttcg gcaaccgtcg catccgcgcg gtcggcgagc tgatccagaa ccaggtccgc 1140

accggtctct cccgcatgga gcgcgtcgtc cgcgagcgca tgaccacgca ggacatcgag 1200

gcgatcacgc cgcagaccct gatcaacgtg cgccccgtcg tcgccgcgat caaggagttc 1260

ttcggaacgt cgcagctgtc gcagttcatg gaccagaaca acccgctcgc gggtctgacg 1320

aacaagcgtc gtctgtctgc gctcggcccc ggtggtctct cgcgagaccg cgccggcgtc 1380

gaggtccgtg acgtccaccc ctcgcactac ggccgcatgt gcccgatcga gactccggaa 1440

ggcccgaaca tcggtctgat cggtgctctc gcgaccttcg cgcgcatcaa ctcgttcgga 1500

ttcatcgaga ccccgtaccg caaggtcgtc gacggtgtcg tgaccgacca gatcgactac 1560

ctgacggctt ccgaagaggt cgacttcaac atcgcgcagg ccaacgcccc gctcgatgcc 1620

1680

gacctgttcg tccccgagga gatcggctac atcgacgtct ccccgcgcca gatggtgtcg 1740

gtcgcgacct cgctcgtgcc cttcctcgag cacgacgacg cacagcgcgc cctcatgggt 1800

gccaacatgc agcgtcaggc tgtgccgctg ctgcgcagcg actcgccgct cgtcggaacc 1860

ggtatggagg gctacacggc catcgacgcc ggtgacgtgc tcaccgccga gaaggccggt 1920

gtcgtctccg aggtctccgc agaccgcgtc gtcgtcatgc tcgacgaggg cggaacgcag 1980

gagtaccacc tgcgcaagtt cgaccgctcc aaccagggca cgtcgtacaa ccagaaggtc 2040

gtcgtcaccg ccggtgagcg cgtcgaggtc ggagaggtca tcgccgatgg ccccgccacc 2100

gagaacggcg agctggccct cggaaagaac ctcctcgtcg cgttcatgac gtgggaggggc 2160

tacaacttcg aggacgcgat catcctgagc caggacctgg tgaaggacga caccctctcc 2220

tcgatccaca tcgaggagta cgaggtcgat gctcgcgaca ccaagctcgg caaggaggag 2280

atcacgcgtg acctccccaa cgtcagcccg gagctgctga aggacctcga cgagcgcggc 2340

atcatccgca tcggtgccga ggtccgccct ggcgacatcc tcgtcggcaa ggtcacgccg 2400

aagggtgaga ccgagctgtc ggccgaggag cgcctgctcc gcgcgatctt caacgagaag 2460

agccgcgaag tccgtgacac ctcgctgaag gtgccccacg gtgagcaggg cacgatcatc 2520

gccgtcaagg agttcaacgc tgaggacggc gacgacgagc tcggctccgg cgtcaaccgc 2580

cgcgtcgtgg tctacatcgc ccagaagcgc aagatcaccg agggtgacaa gctcgccggc 2640

cgtcacggca acaagggtgt catcgcgaag atcctcccga tcgaggacat gccgttcctt 2700

tcggacggta ccccggtcga catcgtgctg aacccgctcg gtatccccgg tcgaatgaac 2760

ttcggtcagg tcctggagac ccacctcggg tggatcgcga agcagggctg gaaggtcgag 2820

ggcaacccgg agtgggctgt gaagctcccg aaggacgcat tcgaggccgc ccccggcacg 2880

aaggtcgcca ccccggtgtt cgacggtgcg agcgaggagg agatcgctgg tctcctcgac 2940

gcgaccaccc cgacccgtga cggcgtccgc ctgatcgact cgagcggcaa gacgcagctg 3000

ttcgacggtc gttcgggtga gccgttcccg gcgccgatct ccgtgggcta catgtacatc 3060

ctgaagctgc accacctggt cgacgacaag atccacgcac gttccacggg tccgtactcg 3120

atgatcaccc agcagccgct cggtggtaag gcgcagttcg gtggacagcg cttcggtgag 3180

atggaggtgt gggccctcga ggcctacggc gccgcatacg cgctccagga gctcctcacg 3240

atcaagtccg acgacatcct cggccgcgtc aaggtgtacg aggcgatcgt caagggcgag 3300

aacatccagg agccggcat ccccgagtcg ttcaaggtgc tcatgaagga gatgcagtcg 3360

ctctgcctga acgtcgaggt cctctcggcc gacggcacgc tggtcaacct ccgcgacacc 3420

gacgacgagg cgttccgcgc cgcggaagag ctcggtatca acatctccag ccgcttcgag 3480

gccgcctcga tcgacgagat ctaa 3504

<210> 5

<211> 1167

<212> PROTEIN

<213> Microbacterium sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-014-C06

<220>

<221> various signs

<223> SGI peptide ID SG1MICT850290

<220>

<221> various signs

<223> Subunit B of RNA polymerase

<400> 5

Met Ala Ala Ala Arg Asn Ala Ser Thr Ser Thr Thr Thr Lys Asn Gly

1 5 10 15

Arg Gly Ala Ser Arg Leu Ser Phe Ala Lys Ile Ser Asp Thr Leu Thr

20 25 30

Val Pro Asp Leu Leu Ala Leu Gln Thr Glu Ser Phe Gly Trp Leu Val

35 40 45

Gly Asn Asp Ala Trp Lys Ala Arg Val Ala Glu Ala Lys Lys Gln Gly

50 55 60

Arg Thr Asp Val Asn Glu Asn Ser Gly Leu Gly Glu Ile Phe Glu Glu

65 70 75 80

Ile Ser Pro Ile Glu Asp Leu Gly Glu Thr Met Gln Leu Ser Phe Thr

85 90 95

Asn Pro Tyr Leu Glu Pro Glu Lys Tyr Ser Ile Glu Glu Cys Lys Glu

100 105 110

Arg Gly Lys Thr Tyr Ala Ala Pro Leu Tyr Val Glu Ala Glu Phe Met

115 120 125

Asn His Leu Thr Gly Glu Ile Lys Thr Gln Thr Val Phe Met Gly Asp

130 135 140

Phe Pro Leu Gln Thr Asp Lys Gly Thr Phe Ile Ile Asn Gly Ser Glu

145 150 155 160

Arg Val Val Val Ser Gln Leu Val Arg Ser Pro Gly Val Tyr Phe Asp

165 170 175

Lys Thr Pro Asp Lys Thr Ser Asp Lys Asp Ile Val Ser Ala Arg Val

180 185 190

Ile Pro Ser Arg Gly Ala Trp Leu Glu Phe Glu Ile Asp Lys Arg Asp

195 200 205

Gln Val Gly Val Arg Val Asp Arg Lys Arg Lys Gln Ser Val Thr Val

210 215 220

Phe Leu Lys Ala Leu Gly Met Thr Ser Glu Glu Ile Leu Ala Glu Phe

225 230 235 240

Ala Gly Tyr Thr Ser Ile Glu Glu Thr Leu Ala Lys Asp Thr Ile Val

245 250 255

Thr Lys Glu Asp Ala Leu Arg Asp Ile Tyr Arg Lys Leu Arg Pro Gly

260 265 270

Glu Gln Val Ala Ala Glu Ala Ala Arg Ala Leu Leu Asp Asn Phe Tyr

275 280 285

Phe Asn Pro Lys Arg Tyr Asp Leu Ala Lys Val Gly Arg Tyr Lys Ile

290 295 300

Asn His Lys Leu Gly Leu Asp Gln Pro Leu Asn Ser Ser Val Leu Thr

305 310 315 320

Val Glu Asp Ile Val Ala Thr Ile Lys Tyr Leu Val Arg Leu His Ala

325 330 335

Gly Thr Glu Glu Thr Phe Thr Gly Ile Arg Gly Gly Lys Lys Ala Glu

340 345 350

Ile Arg Leu Ala Thr Asp Asp Ile Asp Asn Phe Gly Asn Arg Arg Ile

355 360 365

Arg Ala Val Gly Glu Leu Ile Gln Asn Gln Val Arg Thr Gly Leu Ser

370 375 380

Arg Met Glu Arg Val Val Arg Glu Arg Met Thr Thr Gln Asp Ile Glu

385 390 395 400

Ala Ile Thr Pro Gln Thr Leu Ile Asn Val Arg Pro Val Val Ala Ala

405 410 415

Ile Lys Glu Phe Phe Gly Thr Ser Gln Leu Ser Gln Phe Met Asp Gln

420 425 430

Asn Asn Pro Leu Ala Gly Leu Thr Asn Lys Arg Arg Leu Ser Ala Leu

435 440 445

Gly Pro Gly Gly Leu Ser Arg Asp Arg Ala Gly Val Glu Val Arg Asp

450 455 460

Val His Pro Ser His Tyr Gly Arg Met Cys Pro Ile Glu Thr Pro Glu

465 470 475 480

Gly Pro Asn Ile Gly Leu Ile Gly Ala Leu Ala Thr Phe Ala Arg Ile

485 490 495

Asn Ser Phe Gly Phe Ile Glu Thr Pro Tyr Arg Lys Val Val Asp Gly

500 505 510

Val Val Thr Asp Gln Ile Asp Tyr Leu Thr Ala Ser Glu Glu Val Asp

515 520 525

Phe Asn Ile Ala Gln Ala Asn Ala Pro Leu Asp Ala Lys Gly Arg Phe

530 535 540

Arg Glu Ser His Val Leu Ala Arg Pro Lys Gly Gly Ser Gly Glu Val

545 550 555 560

Asp Leu Phe Val Pro Glu Glu Ile Gly Tyr Ile Asp Val Ser Pro Arg

565 570 575

Gln Met Val Ser Val Ala Thr Ser Leu Val Pro Phe Leu Glu His Asp

580 585 590

Asp Ala Gln Arg Ala Leu Met Gly Ala Asn Met Gln Arg Gln Ala Val

595 600 605

Pro Leu Leu Arg Ser Asp Ser Pro Leu Val Gly Thr Gly Met Glu Gly

610 615 620

Tyr Thr Ala Ile Asp Ala Gly Asp Val Leu Thr Ala Glu Lys Ala Gly

625 630 635 640

Val Val Ser Glu Val Ser Ala Asp Arg Val Val Val Met Leu Asp Glu

645 650 655

Gly Gly Thr Gln Glu Tyr His Leu Arg Lys Phe Asp Arg Ser Asn Gln

660 665 670

Gly Thr Ser Tyr Asn Gln Lys Val Val Val Thr Ala Gly Glu Arg Val

675 680 685

Glu Val Gly Glu Val Ile Ala Asp Gly Pro Ala Thr Glu Asn Gly Glu

690 695 700

Leu Ala Leu Gly Lys Asn Leu Leu Val Ala Phe Met Thr Trp Glu Gly

705 710 715 720

Tyr Asn Phe Glu Asp Ala Ile Ile Leu Ser Gln Asp Leu Val Lys Asp

725 730 735

Asp Thr Leu Ser Ser Ile His Ile Glu Glu Tyr Glu Val Asp Ala Arg

740 745 750

Asp Thr Lys Leu Gly Lys Glu Glu Ile Thr Arg Asp Leu Pro Asn Val

755 760 765

Ser Pro Glu Leu Leu Lys Asp Leu Asp Glu Arg Gly Ile Ile Arg Ile

770 775 780

Gly Ala Glu Val Arg Pro Gly Asp Ile Leu Val Gly Lys Val Thr Pro

785 790 795 800

Lys Gly Glu Thr Glu Leu Ser Ala Glu Glu Arg Leu Leu Arg Ala Ile

805 810 815

Phe Asn Glu Lys Ser Arg Glu Val Arg Asp Thr Ser Leu Lys Val Pro

820 825 830

His Gly Glu Gln Gly Thr Ile Ile Ala Val Lys Glu Phe Asn Ala Glu

835 840 845

Asp Gly Asp Asp Glu Leu Gly Ser Gly Val Asn Arg Arg Val Val Val

850 855 860

Tyr Ile Ala Gln Lys Arg Lys Ile Thr Glu Gly Asp Lys Leu Ala Gly

865 870 875 880

Arg His Gly Asn Lys Gly Val Ile Ala Lys Ile Leu Pro Ile Glu Asp

885 890 895

Met Pro Phe Leu Ser Asp Gly Thr Pro Val Asp Ile Val Leu Asn Pro

900 905 910

Leu Gly Ile Pro Gly Arg Met Asn Phe Gly Gln Val Leu Glu Thr His

915 920 925

Leu Gly Trp Ile Ala Lys Gln Gly Trp Lys Val Glu Gly Asn Pro Glu

930 935 940

Trp Ala Val Lys Leu Pro Lys Asp Ala Phe Glu Ala Ala Pro Gly Thr

945 950 955 960

Lys Val Ala Thr Pro Val Phe Asp Gly Ala Ser Glu Glu Glu Ile Ala

965 970 975

Gly Leu Leu Asp Ala Thr Thr Pro Thr Arg Asp Gly Val Arg Leu Ile

980 985 990

Asp Ser Ser Gly Lys Thr Gln Leu Phe Asp Gly Arg Ser Gly Glu Pro

995 1000 1005

Phe Pro Ala Pro Ile Ser Val Gly Tyr Met Tyr Ile Leu Lys Leu

1010 1015 1020

His His Leu Val Asp Asp Lys Ile His Ala Arg Ser Thr Gly Pro

1025 1030 1035

Tyr Ser Met Ile Thr Gln Gln Pro Leu Gly Gly Lys Ala Gln Phe

1040 1045 1050

Gly Gly Gln Arg Phe Gly Glu Met Glu Val Trp Ala Leu Glu Ala

1055 1060 1065

Tyr Gly Ala Ala Tyr Ala Leu Gln Glu Leu Leu Thr Ile Lys Ser

1070 1075 1080

Asp Asp Ile Leu Gly Arg Val Lys Val Tyr Glu Ala Ile Val Lys

1085 1090 1095

Gly Glu Asn Ile Gln Glu Pro Gly Ile Pro Glu Ser Phe Lys Val

1100 1105 1110

Leu Met Lys Glu Met Gln Ser Leu Cys Leu Asn Val Glu Val Leu

1115 1120 1125

Ser Ala Asp Gly Thr Leu Val Asn Leu Arg Asp Thr Asp Asp Glu

1130 1135 1140

Ala Phe Arg Ala Ala Glu Glu Leu Gly Ile Asn Ile Ser Ser Arg

1145 1150 1155

Phe Glu Ala Ala Ser Ile Asp Glu Ile

1160 1165

<210> 6

<211> 1188

<212> DNA

<213> Microbacterium sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-014-C06

<220>

<221> various signs

<223> SGI Gene ID SG1MICG851004

<220>

<221> various signs

<223> encodes the peptide sequence of SEQ ID NO 7

<400> 6

ttgccgactc gatgtcggtg gcctctccta cggtggaaaa caagccgaag agccattacg60

ccttgtcgga gagttgctcc caaccgggag cgccgcgaca gcctacaggc ggcaccacgc 120

gcgcagaagg agcacatcat gccatcaccc gccgaccgcg agaagtccct cgagaccgcc 180

ctcgcccaga tcgaccgcca gttcggaaag ggctcggtca tgcggctggg cagcgatgag 240

300

ggaggactcc cgcgtggtcg catcgtcgag atctacggac cggagtcctc cggtaagacg 360

acgctcaccc tgcacgcgat cgcgaacgca cagcgtgccg gtggcatcgc ggcgttcatc 420

gacgccgagc acgcgctcga ccccgactac gccgccaagc tcggcgtcga catcgatgcg 480

ctcctggtct cgcagcccga cacgggtgag caggcgctcg agatcgccga catgctcgtg 540

cgctccggtg cgatcgacct catcgtcatc gactccgtcg cggccctcgt gccgcgcgcc 600

gagatcgagg gcgagatggg tgactcgcac gtcggtctgc aggctcgcct catgtcgcag 660

gcgctgcgaa agctcaccgg tggtctgaac cagacgaaca ccacgatgat cttcatcaac 720

cagctccgcg agaagatcgg tgtcttcttc ggttcgccgg agaccactgc cggcggtaag 780

gcgctcaagt tctacgcctc ggtccgcatg gacatccgtc gtatcgagac gctcaaggac 840

ggtactgacg ctgtcggtaa ccgcaccagg gtcaaggtcg tcaagaacaa gatggctccg 900

cctttcaagc aggccgagtt cgacatcctc tacggcgtcg gcatctcgcg cgagggaagc 960

ctgatcgact tcggtgtcga gcatgcgatc gtcaagaagt ccggttcctg gtatacgtac 1020

gacggtgacc agctgggtca gggcaaggag aacgcgcgga cgttcctgct caacaacccc 1080

gacatcgcgc tggcgatcga gacgcagatc aagcagaagc tcggcatcgg cggtcccgcc 1140

gcggcgcctg ctgcggcaga cgagctcgct gagcgtcgtc cggcctga 1188

<210> 7

<211> 395

<212> PROTEIN

<213> Microbacterium sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-014-C06

<220>

<221> various signs

<223> SGI peptide ID SG1MICT851004

<220>

<221> various signs

<223> recombinase A

<400> 7

Met Pro Thr Arg Cys Arg Trp Pro Leu Leu Arg Trp Lys Thr Ser Arg

1 5 10 15

Arg Ala Ile Thr Pro Cys Arg Arg Val Ala Pro Asn Arg Glu Arg Arg

20 25 30

Asp Ser Leu Gln Ala Ala Pro Arg Ala Gln Lys Glu His Ile Met Pro

35 40 45

Ser Pro Ala Asp Arg Glu Lys Ser Leu Glu Thr Ala Leu Ala Gln Ile

50 55 60

Asp Arg Gln Phe Gly Lys Gly Ser Val Met Arg Leu Gly Ser Asp Glu

65 70 75 80

Arg Ala Pro Val Ala Val Ile Pro Thr Gly Ser Ile Ala Leu Asp Val

85 90 95

Ala Leu Gly Val Gly Gly Leu Pro Arg Gly Arg Ile Val Glu Ile Tyr

100 105 110

Gly Pro Glu Ser Ser Gly Lys Thr Thr Leu Thr Leu His Ala Ile Ala

115 120 125

Asn Ala Gln Arg Ala Gly Gly Ile Ala Ala Phe Ile Asp Ala Glu His

130 135 140

Ala Leu Asp Pro Asp Tyr Ala Ala Lys Leu Gly Val Asp Ile Asp Ala

145 150 155 160

Leu Leu Val Ser Gln Pro Asp Thr Gly Glu Gln Ala Leu Glu Ile Ala

165 170 175

Asp Met Leu Val Arg Ser Gly Ala Ile Asp Leu Ile Val Ile Asp Ser

180 185 190

Val Ala Ala Leu Val Pro Arg Ala Glu Ile Glu Gly Glu Met Gly Asp

195 200 205

Ser His Val Gly Leu Gln Ala Arg Leu Met Ser Gln Ala Leu Arg Lys

210 215 220

Leu Thr Gly Gly Leu Asn Gln Thr Asn Thr Thr Met Ile Phe Ile Asn

225 230 235 240

Gln Leu Arg Glu Lys Ile Gly Val Phe Phe Gly Ser Pro Glu Thr Thr

245 250 255

Ala Gly Gly Lys Ala Leu Lys Phe Tyr Ala Ser Val Arg Met Asp Ile

260 265 270

Arg Arg Ile Glu Thr Leu Lys Asp Gly Thr Asp Ala Val Gly Asn Arg

275 280 285

Thr Arg Val Lys Val Val Lys Asn Lys Met Ala Pro Pro Phe Lys Gln

290 295 300

Ala Glu Phe Asp Ile Leu Tyr Gly Val Gly Ile Ser Arg Glu Gly Ser

305 310 315 320

Leu Ile Asp Phe Gly Val Glu His Ala Ile Val Lys Lys Ser Gly Ser

325 330 335

Trp Tyr Thr Tyr Asp Gly Asp Gln Leu Gly Gln Gly Lys Glu Asn Ala

340 345 350

Arg Thr Phe Leu Leu Asn Asn Pro Asp Ile Ala Leu Ala Ile Glu Thr

355 360 365

Gln Ile Lys Gln Lys Leu Gly Ile Gly Gly Pro Ala Ala Ala Pro Ala

370 375 380

Ala Ala Asp Glu Leu Ala Glu Arg Arg Pro Ala

385 390 395

<210> 8

<211> 2175

<212> DNA

<213> Microbacterium sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-014-C06

<220>

<221> various signs

<223> SGI Gene ID SG1MICG849768

<220>

<221> various signs

<223> encodes the peptide sequence of SEQ ID NO 9

<400> 8

atgtacccca tgatcgatcc cgcactcgcc gacgccggcc tcggtgacgc cgaagacgac60

gacttcgacg ccatcgagtc tcccgactcc cagctcccgg accaccgcta cctggatcgc 120

gagctgagct ggctcgcctt caaccagcgc gtaatggagc tcgccgagga tccgtcactg 180

cccgaactcg agcgggcgaa cttcctggcg atcttcgcca gcaacctcga cgagttcttc 240

atggtgcgcg tcgccggcct caagcgccgc atcatgaccg gcctggccgt gccgacgaac 300

atcggccgct cccccgtcga cgcgctcgcc gacatctccc gcgaagcgca cgccctgcag 360

ctgcgtcacg ccgaggcctg gacctcgctc gtgcgccccg ccctggccga ctccggcatc 420

gagatcacgg actggtccga gctgaccgac gacgagcgct ccggattgtc cgagtacttc 480

cagctccagg tcttcccggt gctgatgccg ctcgcggtcg acccggcgca tccgttcccc 540

tacatctccg gcctgtcgct gaacctcgcg atccgcatcc gcaatgcccg caccgggcgc 600

caggagttcg cgcgcctcaa ggtgccgccc atgctccccc gcttcgtgga ggtgccgggc 660

ggcggcgaga tcaagcgctt cctgcgcctg gaggaactga tcgcgaacca cctcggcgac 720

ctgttccccg gcatggaggt gctcgaccac cacgcgttcc gcctcacccg caacgaagac 780

gtggagatcg aggaagacga gagcgagaac ctcatccagg cgctcgaggc cgagctgctg 840

cgccgtcgat tcggcccgcc gatccgcctc gagatcacgg acgacatgga cgaggtcacg 900

atggacctgc tcgtccgcga gctcgacatc accgacctgg aggtctaccg cctccccggt 960

ccgctcgacc tgcgcggact gttcgatctg tcccgcatcg accgtcccga cctgcgctac 1020

1080

gacatcttca aagcgatccg caagtcggat gtgctcgtgc accacccgta cgagtcgttc 1140

acgaccagcg tgcaggcgtt cctcgaacag gccgcccgcg acccgcacgt gctcgccatc 1200

1260

gccgaagccg gcaagcaggt gctggccctc gtcgaggtga aggcccgttt cgacgaggcc 1320

aacaacatcg tctgggcacg caagctcgag aaggccggcg tgcacgtggt ctacggtctc 1380

gtcggactca agacccactg caagctcgcc ctcgtcatcc gcgaggaaga ggggatgctg 1440

cgccactact cgcacgtcgg caccggcaac tacaacccca agaccagccg catctacgag 1500

gacttcggtc tgttcaccgc agacgcgcag gtcggcaaag acctgacacg cctgttcaac 1560

gagctcagcg gctacgcgat cgagaagaag ttcaagcgcc tgctggtcgc cccgctgcac 1620

ctgcgcaagg gcctcatccg ccagatcgac gccgagcgca ggaacgccga ggcggggatc 1680

cccgcgcaca tccgcatcaa ggtgaactcg atggtcgatg aggagatcat cgacgcgctc 1740

taccgcgcga gcgcggccgg ggtgaaggtc gacgtgtggg tgcgcggcat ctgcagcctg 1800

cgcaccgacc tcgacggcat cagtgacaac atcacggtgc gcagcatcct cggccgctac 1860

ctcgagcact cccgcatctt cgcgttcgag aacgccggcg acccgcaggt gtacatcggc 1920

agcgccgaca tgatgcaccg caacctcgac cgtcgtgtgg aggcgctggt gcgcgtcacc 1980

gacgccgacc acctcaagga actgcaggcg ttcttcgacc tcgcgatgga cgacggaacc 2040

tcgtcgtggc atctcggcgc cggcggcgtc tgggagcgcc acgccgtgaa cgccgacggc 2100

aagccgctga tcgacctgca ggataagacc atggggttga tccagcggcg ccgccgcgcg 2160

cgggcggttc gatga 2175

<210> 9

<211> 724

<212> PROTEIN

<213> Microbacterium sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-014-C06

<220>

<221> various signs

<223> SGI peptide ID SG1MICT849768

<220>

<221> various signs

<223> polyphosphate kinase

<400> 9

Met Tyr Pro Met Ile Asp Pro Ala Leu Ala Asp Ala Gly Leu Gly Asp

1 5 10 15

Ala Glu Asp Asp Asp Phe Asp Ala Ile Glu Ser Pro Asp Ser Gln Leu

20 25 30

Pro Asp His Arg Tyr Leu Asp Arg Glu Leu Ser Trp Leu Ala Phe Asn

35 40 45

Gln Arg Val Met Glu Leu Ala Glu Asp Pro Ser Leu Pro Glu Leu Glu

50 55 60

Arg Ala Asn Phe Leu Ala Ile Phe Ala Ser Asn Leu Asp Glu Phe Phe

65 70 75 80

Met Val Arg Val Ala Gly Leu Lys Arg Arg Ile Met Thr Gly Leu Ala

85 90 95

Val Pro Thr Asn Ile Gly Arg Ser Pro Val Asp Ala Leu Ala Asp Ile

100 105 110

Ser Arg Glu Ala His Ala Leu Gln Leu Arg His Ala Glu Ala Trp Thr

115 120 125

Ser Leu Val Arg Pro Ala Leu Ala Asp Ser Gly Ile Glu Ile Thr Asp

130 135 140

Trp Ser Glu Leu Thr Asp Asp Glu Arg Ser Gly Leu Ser Glu Tyr Phe

145 150 155 160

Gln Leu Gln Val Phe Pro Val Leu Met Pro Leu Ala Val Asp Pro Ala

165 170 175

His Pro Phe Pro Tyr Ile Ser Gly Leu Ser Leu Asn Leu Ala Ile Arg

180 185 190

Ile Arg Asn Ala Arg Thr Gly Arg Gln Glu Phe Ala Arg Leu Lys Val

195 200 205

Pro Pro Met Leu Pro Arg Phe Val Glu Val Pro Gly Gly Gly Glu Ile

210 215 220

Lys Arg Phe Leu Arg Leu Glu Glu Leu Ile Ala Asn His Leu Gly Asp

225 230 235 240

Leu Phe Pro Gly Met Glu Val Leu Asp His His Ala Phe Arg Leu Thr

245 250 255

Arg Asn Glu Asp Val Glu Ile Glu Glu Asp Glu Ser Glu Asn Leu Ile

260 265 270

Gln Ala Leu Glu Ala Glu Leu Leu Arg Arg Arg Phe Gly Pro Pro Ile

275 280 285

Arg Leu Glu Ile Thr Asp Asp Met Asp Glu Val Thr Met Asp Leu Leu

290 295 300

Val Arg Glu Leu Asp Ile Thr Asp Leu Glu Val Tyr Arg Leu Pro Gly

305 310 315 320

Pro Leu Asp Leu Arg Gly Leu Phe Asp Leu Ser Arg Ile Asp Arg Pro

325 330 335

Asp Leu Arg Tyr Pro Pro His Leu Pro Thr Thr Ala Val Ala Phe Gln

340 345 350

Pro Ala Gly Ser Ser Asn Arg Ala Asp Ile Phe Lys Ala Ile Arg Lys

355 360 365

Ser Asp Val Leu Val His His Pro Tyr Glu Ser Phe Thr Thr Ser Val

370 375 380

Gln Ala Phe Leu Glu Gln Ala Ala Arg Asp Pro His Val Leu Ala Ile

385 390 395 400

Lys Gln Thr Leu Tyr Arg Thr Ser Gly Asp Ser Pro Val Val Gln Ala

405 410 415

Leu Ile Asp Ala Ala Glu Ala Gly Lys Gln Val Leu Ala Leu Val Glu

420 425 430

Val Lys Ala Arg Phe Asp Glu Ala Asn Asn Ile Val Trp Ala Arg Lys

435 440 445

Leu Glu Lys Ala Gly Val His Val Val Tyr Gly Leu Val Gly Leu Lys

450 455 460

Thr His Cys Lys Leu Ala Leu Val Ile Arg Glu Glu Glu Gly Met Leu

465 470 475 480

Arg His Tyr Ser His Val Gly Thr Gly Asn Tyr Asn Pro Lys Thr Ser

485 490 495

Arg Ile Tyr Glu Asp Phe Gly Leu Phe Thr Ala Asp Ala Gln Val Gly

500 505 510

Lys Asp Leu Thr Arg Leu Phe Asn Glu Leu Ser Gly Tyr Ala Ile Glu

515 520 525

Lys Lys Phe Lys Arg Leu Leu Val Ala Pro Leu His Leu Arg Lys Gly

530 535 540

Leu Ile Arg Gln Ile Asp Ala Glu Arg Arg Asn Ala Glu Ala Gly Ile

545 550 555 560

Pro Ala His Ile Arg Ile Lys Val Asn Ser Met Val Asp Glu Glu Ile

565 570 575

Ile Asp Ala Leu Tyr Arg Ala Ser Ala Ala Gly Val Lys Val Asp Val

580 585 590

Trp Val Arg Gly Ile Cys Ser Leu Arg Thr Asp Leu Asp Gly Ile Ser

595 600 605

Asp Asn Ile Thr Val Arg Ser Ile Leu Gly Arg Tyr Leu Glu His Ser

610 615 620

Arg Ile Phe Ala Phe Glu Asn Ala Gly Asp Pro Gln Val Tyr Ile Gly

625 630 635 640

Ser Ala Asp Met Met His Arg Asn Leu Asp Arg Arg Val Glu Ala Leu

645 650 655

Val Arg Val Thr Asp Ala Asp His Leu Lys Glu Leu Gln Ala Phe Phe

660 665 670

Asp Leu Ala Met Asp Asp Gly Thr Ser Ser Trp His Leu Gly Ala Gly

675 680 685

Gly Val Trp Glu Arg His Ala Val Asn Ala Asp Gly Lys Pro Leu Ile

690 695 700

Asp Leu Gln Asp Lys Thr Met Gly Leu Ile Gln Arg Arg Arg Arg Ala

705 710 715 720

Arg Ala Val Arg

<210> 10

<211> 1390

<212> DNA

<213> Microbacterium sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-005-G08

<220>

<221> various signs

<223> encodes 16S ribosomal RNA

<220>

<221> various signs

<222> (29)..(36)

<223> n is a, c, g, t and others

<220>

<221> various signs

<222> (38)..(41)

<223> n is a, c, g, t and others

<400> 10

aacggtgaac acggagcttg ctctgtggnn nnnnnngnnn ncgggtgagt aacacgtgag60

caacctgccc ctgactctgg gtaagcgct ggaaacggcg tctaatactg gatacgagta 120

gcgatcgcat ggtcagctac tggaaagatt ttttggttgg ggatgggctc gcggcctatc 180

agcttgttgg tgaggtaatg gctcaccaag gcgtcgacgg gtagccggcc tgagagggtg 240

accggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 300

360

ttgtaaacct cttttagcag ggaagaagcg aaagtgacgg tacctgcaga aaaagcgccg 420

gctaactacg tgccagcagc cgcggtaata cgtagggcgc aagcgttatc cggaattatt 480

gggcgtaaag agctcgtagg cggtttgtcg cgtctgctgt gaaatcccga ggctcaacct 540

cgggcctgca gtgggtacgg gcagactaga gtgcggtagg ggagattgga attcctggtg 600

tagcggtgga atgcgcagat atcaggagga acaccgatgg cgaaggcaga tctctgggcc 660

gtaactgacg ctgaggagcg aaaggggtggg gagcaaacag gcttagatac cctggtagtc 720

caccccgtaa acgttgggaa ctagttgtgg ggtccattcc acggattccg tgacgcagct 780

aacgcattaa gttccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga 840

cggggacccg cacaagcggc ggagcatgcg gattaattcg atgcaacgcg aagaacctta 900

ccaaggcttg acatatacga gaacggggcca gaaatggtca actctttggga cactcgtaaa 960

1020

agcgcaaccc tcgttctatg ttgccagcac gtaatggtgg gaactcatgg gatactgccg 1080

gggtcaactc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgtcttgggc 1140

ttcacgcatg ctacaatggc cggtacaaag ggctgcaata ccgtgaggtg gagcgaatcc 1200

caaaaagccg gtcccagttc ggattgaggt ctgcaactcg acctcatgaa gtcggagtcg 1260

ctagtaatcg cagatcagca acgctgcggt gaatacgttc ccgggtcttg tacacaccgc 1320

ccgtcaagtc atgaaagtcg gtaacacctg aagccggtgg cctaaccctt gtggagggag 1380

ccgtcgaagg 1390

<210> 11

<211> 532

<212> DNA

<213> Mycosphaerella sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-010-H11

<220>

<221> various signs

<223> Internal transcribed spacer 1_5.8S ribosomal DNA

<220>

<221> various signs

<222> (43)..(43)

<223> n is a, c, g or t

<220>

<221> various signs

<222> (526)..(526)

<223> n is a, c, g or t

<400> 11

cggagggatc attaatgagt gagggggcca cccccaacct ccnacccttt gtgaacgcat60

catgttgctt cgggggcgac cctgccgttc gcggcattcc ccccgggaggt catcaaaaca 120

ctgcattctt acgtcggagt aaaaagttaa tttaataaaa ctttcaacaa cggatctctt 180

ggttctggca tcgatgaaga acgcagcgaa atgcgataag taatgtgaat tgcagaattc 240

agtgaatcat cgaatctttg aacgcacatt gcgccccctg gtattccggg gggcatgcct 300

gttcgagcgt catttcacca ctcaagcctc gcttggtatt gggcgtcgcg agtctctcgc 360

gcgcctcaaa gtctccggct gttcggttcg tctcccagcg ttgtggcaac tatttcgcag 420

tggagtacga gtcgtggcgg ccgttaaatc tttcaaaggt tgacctcgga tcaggtaggg 480

atacccgctg aacttaagca tatcaataag cggaggaggt catagntgtt tc532

<210> 12

<211> 1431

<212> DNA

<213> Variovorax sp.

<220>

<221> various signs

<223> SGI bacterial isolate SGI-014-G01

<220>

<221> various signs

<223> encodes 16S ribosomal RNA

<400> 12

tgccttacac atgcaagtcg aacggcagcg cgggagcaat cctggcggcg agtggcgaac60

gggtgagtaa tacatcggaa cgtgcccaat cgtgggggat aacgcagcga aagctgtgct 120

aataccgcat acgatctacg gatgaaagca ggggatcgca agaccttgcg cgaatggagc 180

ggccgatggc agattaggta gttggtgagg taaaggctca ccaagccttc gatctgtagc 240

tggtctgaga ggacgaccag ccacactggg actgagacac ggcccagact cctacgggag 300

gcagcagtgg ggaattttgg acaatgggcg aaagcctgat ccagccatgc cgcgtgcagg 360

atgaaggcct tcgggttgta aactgctttt gtacggaacg aaacggcctt ttctaataaa 420

gagggctaat gacggtaccg taagaataag caccggctaa ctacgtgcca gcagccgcgg 480

540

atgtaagaca gttgtgaaat ccccggggctc aacctgggaa ctgcatctgt gactgcatag 600

ctagagtacg gtagaggggg atggaattcc gcgtgtagca gtgaaatgcg tagatatgcg 660

gaggaacacc gatggcgaag gcaatcccct ggacctgtac tgacgctcat gcacgaaagc 720

gtggggagca aacaggatta gataccctgg tagtccacgc cctaaacgat gtcaactggt 780

tgttgggtct tcactgactc agtaacgaag ctaacgcgtg aagttgaccg cctggggagt 840

acggccgcaa ggttgaaact caaaggaatt gacggggacc cgcacaagcg gtggatgatg 900

tggtttaatt cgatgcaacg cgaaaaacct tacccacctt tgacatgtac ggaattcgcc 960

agagatggct tagtgctcga aagagaaccg taacacaggt gctgcatggc tgtcgtcagc 1020

tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgtc attagttgct 1080

acattcagtt gggcactcta atgagactgc cggtgacaaa ccggaggaag gtggggatga 1140

cgtcaagtcc tcatggccct tataggtggg gctacacacg tcatacaatg gctggtacaa 1200

agggttgcca acccgcgagg gggagctaat cccataaaac cagtcgtagt ccggatcgca 1260

gtctgcaact cgactgcgtg aagtcggaat cgctagtaat cgtggatcag aatgtcacgg 1320

tgaatacgtt cccgggtctt gtacacaccg cccgtcacac catgggagcg ggttctgcca 1380

gaagtagtta gcttaaccgc aaggaggggcg attaccacgg cagggttcgt g1431

<210> 13

<211> 1424

<212> DNA

<213> Bacillus amyloliquefaciens

<220>

<221> various signs

<223> SGI bacterial isolate SGI-015-F03

<220>

<221> various signs

<223> encodes 16S ribosomal RNA

<400> 13

ctggcggcgt gcctaataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt60

120

gaaaccgggg ctaataccgg atggttgtct gaaccgcatg gttcagacat aaaaggtggc 180

ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc 240

accaaggcga cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300

cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360

cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa 420

gaacaagtgc cgttcaaata gggcggcacc ttgacggtac ctaaccagaa agccacggct 480

aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattatgggg 540

600

agggtcattg gaaactgggg aacttgagtg cagagagga gagtggaatt ccacgtgtag 660

cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta 720

actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 780

gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg 840

cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900

ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960

gtcttgacat cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag 1020

gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080

gcaacccttg atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac 1140

aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200

acgtgctaca atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa 1260

atctgttctc agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt 1320

aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380

caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac cttt 1424

<210> 14

<211> 1425

<212> DNA

<213> Bacillus subtilis

<220>

<221> various signs

<223> SGI bacterial isolate SGI-015-H06

<220>

<221> various signs

<223> encodes 16S ribosomal RNA

<400> 14

gctggcggcg tgcctaatac atgcaagtcg agcggacaga tgggagcttg ctccctgatg60

ttagcggcgg acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg 120

ggaaaccggg gctaataccg gatggttgtt tgaaccgcat ggttcagaca taaaaggtgg 180

cttcggctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct 240

caccaaggca acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300

acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360

acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420

agaacaagtg ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc 480

taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg 540

gcgtaaaggg ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg 600

gagggtcatt ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta 660

gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt 720

aactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca 780

cgccgtaaac gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac 840

gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900

gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960

ggtcttgaca tcctctgaca atcctagaga taggacgtcc ccttcggggg cagagtgaca 1020

ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080

cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga 1140

caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200

cacgtgctac aatggacaga acaaagggca gcgaaaccgc gaggttaagc caatcccaca 1260

aatctgttct cagttcggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag 1320

taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380

acaccacgag agtttgtaac acccgaagtc ggtgaggtaa ccttt1425

<210> 15

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer M13-ITS1

<400> 15

tgtaaaacga cggccagttt cgtaggtgaa cctgcgg 37

<210> 16

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer ITS4-M13

<400> 16

caggaaacag ctatgacctc ctccgcttat tgatatgc 38

<210> 17

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer M13-27F Bac

<400> 17

tgtaaaacga cggccagtta gagtttgatc ctggctcag 39

<210> 18

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer 1492R-M13 Bac

<400> 18

caggaaacag ctatgaccgg ttaccttgtt acgactt 37

<---

Claims (19)

1. An isolated microbial strain, which is a strain of Bacillus amyloliquefaciens , for which a representative sample is deposited as NRRL B-50760, and the isolated microbial strain has inhibitory activity against Fusarium. 2. An isolated microbial strain according to claim 1, wherein said microbial strain contains a DNA sequence showing at least 99% sequence identity with SEQ ID NO: 13. 3. An enriched culture of the microbial strain according to claim 1 for the suppression of Fusarium, containing at least 50% of the isolated microbial strain. 4. Composition for suppressing fusarium, containing a microbial strain or culture thereof according to any one of claims 1 to 3 and an agriculturally effective amount of a compound or composition selected from the group consisting of acaricide, bactericide, fungicide, insecticide, microbicide, nematicide, pesticide and fertilizers. 5. Composition for the suppression of Fusarium, containing a microbial strain or its culture according to any one of claims 1 to 3 and a carrier. 6. The composition of claim 5 wherein said carrier is an agriculturally acceptable carrier. 7. The composition of claim 5, wherein said carrier is the seed of a plant. 8. The composition of claim 5, wherein said composition is in the form of a composition selected from the group consisting of emulsion, colloid, dust, granule, bead, powder, spray, emulsion and solution. 9. The composition of claim 5, wherein said composition is a seed coating composition. 10. Seed resistant to Fusarium fungal infection, wherein the seed is coated with a seed coating composition comprising the composition of claim 5. 11. A method for preventing the development of a Fusarium plant pathogen, wherein said method comprises growing a microbial strain or culture thereof according to any one of claims 1 to 3 in a growth medium or soil of a host plant prior to, or simultaneously with, growing the host plant in said growth medium. or soil. 12. The method of claim 11 wherein said plant pathogen causes Fusarium. 13. The method of claim 12 wherein said plant pathogen is Fusarium graminearum . 14. A method for preventing the development of Fusarium in a plant, said method comprising applying to the plant or to the environment of the plant an effective amount of a microbial strain or culture thereof according to any one of claims 1 to 3. 15. The method of claim 14, wherein said microbial strain or culture thereof is applied to soil, seed, root, flower, leaf, plant part, or entire plant. 16. The method of claim 14 wherein said plant is susceptible to Fusarium graminearum. 17. The method of claim 14, wherein said plant is a wheat plant, a corn plant, a barley plant, or an oat plant. 18. The method of claim 14 wherein said microbial strain or culture thereof is established as an endophyte to said plant. 19. The method of obtaining an agricultural composition for the suppression of Fusarium, where the specified method includes the inoculation of the microbial strain or its culture according to any one of claims 1-3 in the substrate and ensuring the growth of the specified microbial strain or its culture at a temperature of 1-37°C to obtaining an amount of cells or spores of at least 10 ² -10 ³ per milliliter or per gram.