COMPOSITIONS AND METHODS TO PREVENT, INHIBIT OR TREAT THE CEREAL FUSARIOSIS DISEASE, COATED SEED AND

专利摘要:
abstract compositions and methods for controlling head blight disease compositions comprising microbiological strains and cultures and methods of use thereof are provided herein. certain strains, cultures, and compositions thereof are useful for the control of head blight disease, for example, of various crop plants. biological control compositions, and methods of use thereof to prevent, inhibit or treat the development of plant pathogens or disease and for preserving plant yield, are also provided. "Compositions and Method for Controlling Cereal Fusarium Disease". Compositions comprising microbiological strains and cultures and methods for using them are provided herein. Certain strains, cultures and compositions thereof are useful for the control of cereal fusarium disease, for example of various cereals. Biological control compositions, and methods of using them to prevent, inhibit or treat the development of plant pathogens or disease and to preserve plant yield are also provided.
公开号:BR112014001815B1
申请号:R112014001815-4
申请日:2012-07-24
公开日:2019-09-24
发明作者:Christopher J. Grandlic；Wayne A. Green；Janne S. Kerovuo；Ryan T. McCann
申请人:Monsanto Technology Llc；
IPC主号:

专利说明:

[001] The material in the Accompanying Sequence Listing is incorporated herewith by reference in its entirety. The accompanying file, named SGI1520-1 WO_ST25.txt, was created on July 24, 2012 and is 55 Kb. The file can be accessed using Microsoft Word on a computer using Windows OS.
FIELD OF THE INVENTION [002] The present invention relates to the biological control of phytopathogenic diseases. Specifically, it relates to compositions and methods useful for controlling the disease of cereal fusariosis in cereal plants, such as wheat and barley.
BACKGROUND OF THE INVENTION [003] Grain Fusarium, also known as head scab, pink mold, white heads (whiteheads) and tombstone scab, is a devastating disease that afflicts wheat, barley, and various other cereal crops worldwide, particularly in the USA, Europe and China. This disease can reach epidemic levels and cause extensive damage to grains, especially wheat and barley in humid and semi-humid cereal growing areas of the world, including India, Russia, France, Germany and the United Kingdom. In particular, wheat scab or cereal fusariosis is one of the most damaging wheat diseases in the United States. Across the country, this disease has caused the wheat industry millions of dollars in lost earnings. In the Midwest and the Highlands, wheat scab is the main obstacle to wheat production in recent years. CePetition Fusariosis 870190046231, of 17/05/2019, p. 6/90
2/75 reais in addition to attacking wheat also attacks and reproduces in barley, oats, corn and many other cereals.
[004] This serious plant disease can be caused by several phytopathogens, but primarily by several species in the fungal genus Fusarium. For example, causal agents of cereal fusarium disease in wheat include a number of different species of Fusarium species, for example F. culmorum, F. graminearum (teleomorph, Gibberella zeae), F. avenaceum (teleomorph, G. avenacea), F. poae, as well as non-Fusarium pathogens such as Microdochium nivale (teleomorph, Monographella nivalis) and Microdochium majus. In the United States, Europe and the most agronomically important areas of the world, the predominant causative agent of cereal fusariosis is Fusarium graminearum (teleomorph, Gibberella zeae strict sense).
[005] These pathogens normally survive on plant remains. They invade and damage the spikelets of the grain head during flowering, consequently preventing or partially preventing the development of the grain in the grain head. As a result, the invading mange pathogen can either kill part of the grain head or the whole grain head. Some infected seeds are inferior in force and often fail to germinate. Infected seeds that germinate often die early in the seedling stage due to crown rot or root rot, causing poor stands on the next crop plant. Healthy seedlings can also become infected in an emergency. In addition to the weak and poor stand, loss of income due to pathogen infestation can be very high if conditions are favorable for the development of the disease.
[006] The fungal pathogens of the Fusarium genus spread through grain cultivation areas worldwide, and are known as cau
Petition 870190046231, of 5/17/2019, p. 7/90
3/75 for serious damage, particularly in areas with high rainfall between the flowering and the grain supplement. When Fusarium graminearum is the causative agent, this disease is of primary concern because it not only reduces commercial values of the contaminated grains, in addition to yield losses, but because Fusarium infection can also lead to the accumulation of trichothecene mycotoxins in the grains consequently threatening health human and livestock. Trichothecenes are major mycotoxin contaminants in cereals worldwide, causing food refusal, vomiting, diarrhea and weight loss in non-ruminant animals and posing a threat to the health of other animals and humans when exposure levels are high. This threat has been exacerbated by the recent shift in strains of F. graminearum in the United States towards the production and vigor of the larger toxin. Mycotoxins most often found are deoxynivalenol (DON, also known as vomitoxin) and zearalenone (ZEA). Deoxynivalenol in particular is a very dangerous toxin, causing gastrointestinal disorders accompanied by hemorrhagic and other conditions in humans and animals that eat infected grains, leading to death in some cases. Since deoxynivalenol is generally stable against changes in pH and high temperature, detoxification can be very difficult. Therefore, contaminated grains beyond a certain level cannot be used in any form of preparation, processing or livestock feed, and consequently often need to be discarded.
[007] So far, several strategies have been employed to control fusariosis from cereals to cereals. Promising options include chemical measurements, the development of resistant crop crops, and traditional crop rotation and cropping practices. Among these options, chemical pesticides can be somewhat effective in reducing infestation by cereal fusariosis, but
Petition 870190046231, of 5/17/2019, p. 8/90
4/75 residue problems in relation to the use of late fungicides in the development of the crop, usually in the flowering stages just a few weeks before harvest, decrease its attractiveness. Advances in the development of resistant crops using traditional reproduction and genetic manipulation show that other alternatives for disease control are also occurring. Reported examples of advances in genetic manipulation include altering the production of a plant hormone or manipulating the plant hormone signaling pathway. In recent years, considerable advances in the area of traditional breeding have been made in understanding the genetic basis of resistance to cereal fusarium disease and a number of genes and quantitative traits (QTL) conferring resistance have been reported. However, progress in improving breeding resistance to cereal fusariosis disease has been slow, largely because of the difficulty of studying this disease. In fact, relatively little is currently understood about the mechanisms involved in resistance or susceptibility. In addition, the genetic diversity of Fusarium species, which are the predominant causative agents of the disease, often raises concerns about how long the effectiveness of chemical fungicides and resistant crops can be. As a result, virtually all wheat crops currently in large-scale production remain vulnerable to infection.
[008] Furthermore, although some success in controlling the disease of cereals fusariosis can be expected by traditional practices such as plowing fields to bury crop residues infested with a causative agent, for example, of F. graminearum, after harvesting, crop conventional harvesting of fields is not compatible with the practice of conserving the minimum tillage soil. Considering the potential for spreading inoculation over long distances and cultivating
Petition 870190046231, of 5/17/2019, p. 9/90
5/75 diverse species that can act as alternative hosts for pathogens, crop rotation is often an unsustainable solution. In addition to the problem of pesticide residues in the environment, reports of pesticide resistance and cases of increased content of DON in the grain can also be problems with its use. In addition, rising costs and problems in the public and private sectors about pesticide residues in the environment and food product safety yield this less attractive disease control alternative, and have led to requests for crop cultivation using as few pesticides as possible.
[009] In summary, despite considerable advances in the development of techniques to control cereal fusariosis, reducing the impact of this devastating disease on grain production and quality remains an unsolvable problem. Therefore, the identification and development of new techniques for controlling cereals fusariosis is essential in improving the production and quality of many cereal crops. These problems require an urgent solution not only in the United States, but also across the globe, including Asia and Europe.
[0010] Biological control of cereal fusarium disease has attracted considerable interest since the mid-1990s. Biological control agents (BCAs), although currently in very limited numbers, could be an environmentally acceptable method to substantially decrease the level of disease caused by pathogens. such as Fusarium. Public acceptance, compatibility with other measures of disease management, and durability are among favorable factors in supporting the development of strategies to biologically control cereal fusarium disease. Biological control agents can play an important role in the production of organic cereal. In conventional production, such agents can extend
Petition 870190046231, of 5/17/2019, p. 10/90
6/75 spike protection after the flowering stage after chemical fungicides can no longer be applied. So far, significant advances in the area of biological control have been achieved. For example, certain strains of spore-producing bacteria (such as Bacillus and Pseudomonas species) and yeasts (such as Cryptococcus species) show some promise for controlling cereal fusarium disease and reducing mycotoxin contamination. However, despite these and other advances, the need remains for improved microorganisms for use in the biological control of cereal fusarium disease. Although BCAs have become a more acceptable solution for plant pathogens and BCA products have been advertised to a greater extent than before, there have been few attempts to develop antagonistic strategies and microorganisms to biologically control cereal fusariosis disease. In addition, the life cycle of Fusarium spp. and other agents that cause cereal fusariosis disease suggest that pathogens can potentially be susceptible to biological control techniques using antagonistic microorganisms at different stages of development. Consequently, there is a need to identify new biological control agents, preferably with different modes of action, as well as biocontrol methods that can help to effectively prevent or suppress the development of cereal fusarium disease.
SUMMARY OF THE INVENTION [0011] Compositions comprising microbiological strains and cultures are provided here. Certain strains, cultures and compositions thereof are useful for controlling the disease of cereals fusariosis, for example, of various cereals including wheat and other cereal plants. Biological control compositions, and methods of using them to prevent, inhibit or treat the development of pathogens from
Petition 870190046231, of 5/17/2019, p. 11/90
7/75 plant or disease and to preserve plant yield, are also provided. Also provided are methods for using such compositions as biological control agents in combination with other agriculturally effective compounds or compositions for controlling dangerous plant pathogens.
[0012] In one aspect, the present invention provides isolated microbial strains having suppressive activity against cereal fusarium disease. The microbial strains according to this aspect of the present invention are selected from the group consisting of the genera Microbacterium, Bacillus, Mycosphaerella and Variovorax. In some preferred modalities, microbial strains are selected from the group consisting of Mycosphaerella sp. SGI-010- HI 1 (deposited as NRRL 50471), strain of Microbacterium sp. SGI-014C06 (deposited as NRRL B-50470), strain of Microbacterium sp. SGI-005-G08 (deposited as NRRL -), strain of Variovorax sp. SGI014-G01 (deposited as NRRL B-50469), strain of Bacillus sp. SGI015-F03 (deposited as NRRL B-50760), strain of Bacillus sp. SGI015-H06 (deposited as NRRL B-50761), and variants of pesticide assets of any of them. The microbial strain according to some other preferred embodiments may comprise a DNA sequence that exhibits at least 85% sequence identity for any of the nucleotide sequences in the Sequence Listing. Biologically pure cultures and enriched cultures of the microbial strains disclosed here are also provided.
[0013] 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, are also provided in another aspect of the present invention. an insecticide, a mi
Petition 870190046231, of 5/17/2019, p. 12/90
8/75 crobicide, nematicide, pesticide and fertilizer. Compositions in some modalities of this aspect can be prepared as a formulation selected from the group consisting of an emulsion, a colloid, a dust, a granule, a lozenge, a powder, a spray, an emulsion and a solution. In some other embodiments, the compositions can be provided with a vehicle. In some preferred embodiment, the vehicle is an agriculturally acceptable vehicle. In some particularly preferred embodiment, the vehicle is a plant seed. In other preferred embodiments, the composition is a seed coating formulation. In addition, in the present disclosure, seeds are coated with a composition according to the present invention.
[0014] In another aspect of the present invention, methods are provided to prevent, inhibit or treat the development of a plant pathogen. The methods involve cultivating a microbial strain of the invention or a culture thereof in a cultivation medium or soil of a host plant prior to or competing with growth of the host plant in the cultivation medium or soil. In some preferred embodiments of this aspect, the plant pathogen causes disease of cereals fusariosis. In some particularly preferred embodiments, the plant pathogen is Fusarium graminearum.
[0015] In yet another aspect of the present invention, methods are provided to prevent, inhibit or treat the development of a plant's cereal fusariosis disease. The methods involve applying to the plant, or the surroundings of the plant, an effective amount of a microbial strain of the invention or a culture thereof. In one embodiment, such a disease of cereals fusariosis is caused by the fungus Fusarium graminearum. In some embodiments of this aspect, the microbial strain or a culture thereof is applied to the soil, a seed, a root, a flower, a leaf, a portion of the plant or the entire plant
Petition 870190046231, of 5/17/2019, p. 13/90
9/75 ra. In a preferred embodiment, the plant is susceptible to Fusarium graminearum. In some other preferred embodiments, the plant is a wheat plant, a corn plant, a barley plant, or an oat plant. In another preferred embodiment, the microbial strain of the present invention or a culture thereof is established as an endophyte on the plant.
[0016] Another additional aspect of the invention provides non-naturally occurring plants. Non-naturally occurring plants are artificially infected with a microbial strain of the invention or a culture thereof. In addition, in some preferred embodiments of this aspect, seed, reproductive tissue, vegetative tissue, plant parts and offspring of non-naturally occurring plants are provided.
[0017] Yet another aspect of the invention provides a method for preparing an agricultural composition. The method involves inoculating the microbial strain according to the present invention or a culture thereof within or on a substrate and allowing it to grow at a temperature of 1-37 ° C until obtaining a number of cells or spores of at least 102- 10 ³ per millimeter or per gram.
[0018] These and other objectives and resources of the invention will become fully evident from the following detailed description of the invention and the claims.
DETAILED DESCRIPTION OF THE INVENTION
Some definitions [0019] Unless otherwise defined, all terms of the technique, notations and other scientific terms or terminology used here are intended to have the meanings commonly understood by those versed in the technique to which this invention belongs. In some cases, terms with commonly understood meanings are defined here for clarity and / or for immediate reference, and the inclusion of such defi
Petition 870190046231, of 5/17/2019, p. 14/90
10/75 here should not necessarily be interpreted to represent a substantial difference about which is generally understood in the art. Many of the techniques and procedures described or referenced here are well understood and commonly used using conventional methodology by those skilled in the art.
[0020] The singular form one, one, and o / a include plural references unless the context clearly indicates otherwise. For example, the term a cell includes one or more cells, including mixtures of them.
[0021] The terms antagonistic microorganism and microbial antagonist are used here interchangeably in reference to a microorganism that is intended to mean that the object strain exhibits a degree of inhibition of excess cereal fusarium disease, at a statistical level significant, than from an untreated control.
[0022] Antibiotic: the term antibiotic, as used here, refers to any substance that is capable of killing or inhibiting the growth of a microorganism. Antibiotics can be produced by any one or more of the following: 1) a microorganism, 2) a synthetic process, or 3) a semi-synthetic process. An antibiotic can be a microorganism that secretes a volatile organic compound. In addition, an antibiotic can be a volatile organic compound secreted by a microorganism.
[0023] Bactericidal: the term bactericidal, as used here, refers to the ability of a composition or substance to increase mortality or inhibit the rate of growth of the bacterium. Inhibition of the growth rate of the bacteria can be commonly quantified as the reduction of viable bacterial cells over time.
[0024] Biological control: the term biological control and its abbreviated form biocontrol, as used here, is defined as a con
Petition 870190046231, of 5/17/2019, p. 15/90
11/75 trolley of a pathogen or insect or any other undesirable organism by using at least one following non-human organism. An example of a known biological control mechanism is the use of microorganisms that control root rot competing outside the fungus for space on the root surface, or microorganisms that either inhibit the growth of or kill the pathogen. The host plant in the context of biological control is the plant that is susceptible to disease caused by the pathogen. In the context of the isolation of an organism, such as a fungus species, from its natural environment, the host plant is a plant that supports the growth of fungi, for example, a plant of a species the fungus is an endophyte.
[0025] The term cereal as used here is intended to refer to any species of cereal that may be susceptible to the disease of cereal fusariosis. Cereals reported to be susceptible include wheat, barley, oats, and tritical, although wheat and barley are the two crops in which this disease presents a significant economic problem.
[0026] An effective amount refers to an amount sufficient to affect beneficial or desired results. In terms of disease management, treatment, inhibition or protection, an effective amount is that amount sufficient to suppress, stabilize, reverse, slow or slow the progression of the target infection or disease states. As such, the term an effective amount is used here in reference to that amount of antagonist treatment that is required to achieve a reduction in the level of pathogen development and / or in the level of pathogenic disease compared to that which occurs in an untreated control. . Usually, an effective amount of a given antagonist treatment provides a reduction of at least 20%; or more typically, between 30 to 40%; more normally, between 50-60%; even more normally, between 70 to 80%; and ain
Petition 870190046231, of 5/17/2019, p. 16/90
12/75 of the most commonly, between 90 to 95%, in relation to the level of disease and / or the level of development of pathogen occurring in an untreated control under appropriate treatment conditions. An effective amount can be administered in one or more administrations. The actual rate of application of a liquid formulation will normally range from a minimum of about 1 X 10 3 to about 1 X 10 ¹⁰ viable cells / ml and preferably from about 1 X 10 ⁶ to about 5 x 10 ⁹ cells / ml viable. Under most conditions, the antagonistic microbial strains of the invention described in the Examples below would be optimally effective at application rates in the range of about 1 X 10 ⁶ to 1 x 10 ⁹ viable cells / mL, assuming an application mode that would reach substantially uniform contact of at least about 50% of plant tissues. If the antagonists are applied as a solid formulation, the application rate must be controlled to result in a comparable number of viable cells per plant tissue surface unit area as obtained by the aforementioned liquid treatment rates. Typically, the biological control agents of the present invention are biologically effective when delivered in a concentration in excess of 10 ⁶ CFU / g (colony forming units per gram), preferably in excess of 10 ⁷ CFU / g, more preferably 108 CFU / g, and more preferably at 10 ⁹ CFU / g.
[0027] In addition, the term effective microbial antagonist used here in reference to a microorganism is intended to mean that the subject's microbial strain exhibits a degree of inhibition of excess fusarium disease, on a statistically significant level, that of an untreated control. Typically, an effective microbial antagonist has the ability to effect a reduction of at least 20%; or more normally, between 30 to 40%; more normally, between 50-60%; even more normally, between 70 to
Petition 870190046231, of 5/17/2019, p. 17/90
13/75
80%; and even more normally, between 90 to 95%, in relation to the level of disease and / or the level of development of pathogen occurring in an untreated control under adequate treatment conditions.
[0028] Composition: The composition is intended to mean a combination of active agent and at least one other compound, vehicle or composition, inert (for example, a detectable agent or label or liquid vehicle) or active, such as a pesticide.
[0029] Isolated microbial strain, isolated culture, biologically pure culture, and enriched culture: as used here, the term isolated as applied to a microorganism (for example, bacteria or microfungus) refers to a microorganism that has been removed and / or purified from an environment in which it occurs naturally. As such, a strain isolated from a microbe as used here is a strain that has been removed and / or purified from its natural environment. Consequently, an isolated microorganism does not include a resident in an environment where it occurs naturally. In addition, the term isolated does not necessarily reflect the extent to which the microbe has been purified. A substantially pure culture of the microbe strain refers to a culture that contains substantially no other microbes than the desired strain or strains of microbes. In other words, a substantially pure culture of a microbe strain is substantially free of other contaminants, which can include microbial contaminants as well as unwanted chemical contaminants. Additionally, as used here, a biologically pure strain is intended to mean the strain separate from the materials with which it is normally associated in nature. Note that a strain associated with other strains or with compounds or materials that are not normally found in nature, is still defined as biologically pure. A monoculture of a particular strain is, of course, biologically pure. As used here, the term
Petition 870190046231, of 5/17/2019, p. 18/90
14/75 enriched culture of an isolated microbial strain refers to a microbial culture that contains more than 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain.
[0030] As used here, an endophyte is an endosymbiont that lives with a plant for at least part of its life without causing apparent disease. Endophytes can be transmitted either vertically (directly from father to children) or horizontally (from individual to unrelated individual). Vertically transmitted fungal endophytes are normally asexual and transmit from the maternal plant to their children by means of fungal hyphae penetrating the host seeds. Bacterial endophytes can also be transferred vertically from seeds to sowing (Ferreira et al., 2008). In contrast, endophytes transmitted horizontally normally sexed, and transmit through spores that can be spread by the wind and / or insect vectors. Cereal endophytes have received considerable attention regarding their ability to control both disease and insect infestation, as well as to promote plant growth.
[0031] Functionally comparable protein: the phrase functionally comparable protein as used here describes those proteins that have at least one characteristic in common. Such characteristics include sequence similarity, biochemical activity, similarity of transcriptional pattern and phenotypic activity. Typically, functionally comparable proteins share some sequence similarity or at least one biochemist. Within this definition, counterparts, orthologs, analogues and analogues are considered to be functionally comparable. In addition, functionally comparable proteins generally share at least one biochemical and / or phenotypic activity. Functionally comparable proteins will give rise to the same characteristics as a similar one, but
Petition 870190046231, of 5/17/2019, p. 19/90
15/75 not necessarily the same, grade. Normally, functionally comparable proteins give the same characteristics where the quantitative measurement due to one of the comparable is at least 20% of the other; more typically, between 30 to 40%; more normally, between 50-60%; even more normally, between 70 to 80%; even more normally, between 90 to 95% or; even more normally, between 98 to 100% of the other.
[0032] Fungicide: As used here, fungicide refers to the ability of a composition or substance to slow the growth rate of the fungus or increase the mortality of the fungus.
[0033] Fusarium fungus: for the purposes of this invention it is understood that the use of the term Fusarium fungus is intended to include both the sexual (teleomorphic) stage of this organism and the asexual (anamorphic) stage, also referred to as the perfect and imperfect stages , respectively. For example, the anamorphic stage of Fusarium graminearum is Gibberella zeae, a causative agent of cereal fusariosis disease. This disease usually occurs when the flower or seed head begins to inoculate with conidia produced by the imperfect shape or aspaspores produced by the perfect shape of this fungus.
[0034] Mutant: As used here, the term mutant or variant in reference to a microorganism refers to a modification of the parental strain in which the desired biological activity is similar to that expressed by the parental strain. For example, in the case of Microbacterium, the parental strain is defined here as the original Microbacterium strain before mutagenesis. Mutants or variants can occur in nature without human intervention. They are also obtainable by treatment with or by a variety of methods and compositions known to those skilled in the art. For example, a parent strain can be treated with a chemical such as N-methyl-N'-nitro
Petition 870190046231, of 5/17/2019, p. 20/90
16/75
N-nitrosoguanidine, ethylmethanesulfone, or by irradiation using X-ray gamma or UV irradiation, or by other means well known to those practiced in the art.
[0035] Nematicide: The term nematicide, as used here, refers to the ability of a substance or composition to increase mortality or inhibit the rate of growth of nematodes.
[0036] Pathogen: The term pathogen as used here refers to an organism such as an algae, an arachnid, a bacterium, a fungus, an insect, a nematode, a parasitic plant, yeast, a protozoan, or a virus capable of producing a disease in a plant or animal. The term phytopathogen as used here refers to a pathogenic organism that infects a plant.
[0037] Percentage of percentage identity: percentage of sequence identity, as used here, is determined by comparing two locally aligned strings optimally over a comparison window defined by the length of the local alignment between the two sequences. The amino acid sequence in the comparison window may comprise additions or deletions (for example, gaps or bumps) when compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of two sequences. Local alignment between two sequences includes only segments of each sequence that are considered sufficiently similar according to a criterion that depends on the algorithm used to perform the alignment (for example BLAST). The percentage identity is calculated by determining the number of positions where the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of equivalent positions, dividing the number of equivalent positions by the total number of positions in the window comparison and multiplying the result by 100. Optimal sequence alignment for comparison
Petition 870190046231, of 5/17/2019, p. 21/90
17/75 to be conducted by Smith and Waterman's local homology algorithm (1981) Add. APL. Math. 2: 482, by Needleman and Wunsch's global homology alignment algorithm (J. Mol. Biol. 48: 443, 1970), by Pearson and Lipman's similarity method search (Proc. Natl. Acad. Sci. USA 85 : 2444, 1988), by heuristic implementations of these algorithms (NCBI BLAST, WU-BLAST, BLAT, SIM, BLASTZ), or by inspection. Since two sequences have been identified for comparison, GAP and BESTFIT are preferably used to determine their optimal alignment. Normally, the default values of 5.00 for span weight and 0.30 for span weight length are used. The term substantial sequence identity between polynucleotides or polypeptide sequences refers to the polynucleotide or polypeptide comprising a sequence that has at least 50% sequence identity, preferably at least 70%, preferably at least 80%, more preferably at least 85 % or more preferably at least 90%, even more preferably at least 95%, and most preferably at least 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using the programs.
[0038] Amino acid and nucleic acid sequences can be searched against amino acid or nucleic acid sequences residing in public or private databases. Such searches can be done using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST v 2.18) program. The NCBI BLAST program is available on the Internet from the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov/Blast.cgi). Usually the following parameters for NCBI BLAST can be used:
Petition 870190046231, of 5/17/2019, p. 22/90
18/75 filter options adjusted for presets, the Comparison Matrix adjusted for BLOSUM62, so Gap Costs adjusted for Existence: 11, Extension: 1, the Word Size adjusted for 3, the Expect (limit E) adjusted for 3, and the minimum local alignment length adjusted to 50% of the query string length. Sequence identity and similarity can also be determined using GenomeQuest ^TM software (Gene-IT, Worcester Mass. USA).
[0039] The term pesticide, as used here, refers to the ability of a substance or composition to slow the growth rate of a plague, that is, an unwanted organism, [0040] By suppressing activity of a biological control agent against a phytopathogen means the ability of the agent to suppress, inhibit, stabilize, reverse, decrease, or delay or increase the mortality of a plague. Development of the pathogen itself, or the progression of infection or disease states caused by the pathogen.
[0041] Variant: a variant, as used here in reference to a microorganism, is a strain having characteristics identifying the species to which it belongs, although having at least one variation of nucleotide sequence or identifiably different trait with respect to the parental strain , where the trait is genetically based (transmissible). For example, for a strain of Microbacterium sp. SGI-SGI-014-C06 having fungicidal activity, identifiable traits include 1) the ability to suppress the growth of Fusarium graminearum and its teleomorphic Gibberella zeae; 2) the ability to suppress the development of cereal fusariosis disease; 3) having maintenance genes with sequence identity greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% to the maintenance genes of Microbacterium sp. SGI-014-C06 can be used
Petition 870190046231, of 5/17/2019, p. 23/90
19/75 to confirm a variant like Microbacterium sp. SGI-014-C06. [0042] For nucleic acids and polypeptides, the term variant is used here to denote a polypeptide, protein or polynucleotide molecule with some differences, generated synthetically or naturally, in its amino acid or nucleic acid sequences when compared to a polypeptide or polynucleotide of reference, respectively. For example, these differences include substitutions, insertions, deletions or any desired combinations of such changes in a reference polypeptide or polypeptide. Polypeptide and protein variants may additionally consist of changes in charge and / or post-translational changes (such as glycosylation, methylation, phosphorylation, etc.).
[0043] All publications and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication or patent application had specifically and individually indicated to be incorporated by reference.
[0044] No admission is made that any reference constitutes state of the art. The discussion of the reference states that their authors claim, and the applicants reserve the right to challenge the accuracy and pertinence of the documents cited. It will be clearly understood that, although a number of prior art publications are referred to here, this reference does not constitute an admission that any such documents form part of general common knowledge in the prior art.
Methods for Taxonomic Identification [0045] Microorganisms can often be distinguished based on direct microscopic analysis (making all of the cells in a sample look the same on examination), staining characteristics, simple molecular analysis (such as a poly determination
Petition 870190046231, of 5/17/2019, p. 24/90
20/75 simple restriction fragment length morphism (RFLP), and so on. In addition to illustrative examples of such taxonomic analysis techniques as described in Examples 2-3 of the present disclosure, taxonomic identification of a microorganism can involve up to several different levels of analysis, and each analysis can be based on a different characteristic of the organism. Such taxonomic analyzes may include analysis based on nucleic acid (for example, analysis of specific individual genes, or as for their presence or their exact sequence, or expression of a particular gene or family of genes), protein-based analysis ( for example, at a functional level using direct or indirect enzyme assays, or at a structural level using immunological detection techniques), and so on.
[0046] Analysis based on nucleic acid: someone skilled in the art will appreciate that a wide variety of techniques based on nucleic acid are known and can be useful in obtaining taxonomic identification of a given microorganism. These techniques can be used to identify cells by gene sequence or to identify cells that have particular genes or gene families. Common gene families useful for taxonomic studies include the 16S gene family, the actin gene family, and the recombinase A (recA) gene family. These methods typically include amplifying and sequencing genes from very small numbers of cells, and therefore often overcome the problems of contracting cells and their DNA from diluted suspensions. The term nucleic acid amplification generally refers to techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen. Useful techniques for nucleic acid amplification are well known in the art. An example of nucleic acid amplification is the polymerase chain reaction
Petition 870190046231, of 5/17/2019, p. 25/90
21/75 (PCR), in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow the hybridization of the primers to the nucleic acid model in the sample. The primers are extended under suitable conditions, dissociated from the model, and then re-hybridized, extended and dissociated to increase the nucleic acid copy number. Other examples of in vitro magnification techniques include filament displacement magnification; transcription-free isothermal amplification; expansion of the repair chain reaction; ligase chain reaction; amplification of gap filling ligase chain reaction; detection of coupled ligase and PCR; and RNA transcription-free amplification.
[0047] In addition to the illustrative example primers provided here, see, for example Examples 2-3 and the Sequence Listing, primers have also been routinely designed, and new ones are continually being designed, for individual species or phylogenetic groups of microorganisms. Such strictly targeted primers can be used with the methods described here to hide and / or specifically identify only the microorganisms of interest. [0048] Methods for preparing and using 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). Magnification primer pairs can be derived from a known sequence, for example, using computer programs designed for that purpose such as Primer (Whitehead Institute for Biomedical Research, Cambridge, Mass.). Someone skilled in the art will appreciate that the specificity of a particular probe or primer increases with its length. Consequently, for example, a primer comprising 30 nucleotides
Petition 870190046231, of 5/17/2019, p. 26/90
22/75 consecutive nucleotide encoding rRNA or flanking regions thereof will pellet into a target sequence with a higher specificity than a corresponding primer of only 15 nucleotides. Consequently, for greater specificity, probes and primers can be selected that comprise at least 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides from a target nucleotide sequence such as the 16S rRNA.
[0049] Common techniques for preparing nucleic acids useful for nucleic acid applications (eg, PCR) include extraction of phenol / chloroform or use of one of the many DNA extraction kits that are available on the market. Another way that DNA can be amplified is by adding cells directly to the nucleic acid amplification reaction mixture and based on the stage of denaturation of the amplification for cell lysis and DNA release.
The product of the nucleic acid amplification reactions can be further characterized by one or more standard techniques that are well known in the art, including electrophoresis, restriction endonuclease cleavage patterns, hybridization or oligonucleotide binding, and / or sequencing nucleic acid. When hybridization techniques are used for cell identification purposes, a variety of probe labeling methods can be useful, including fluorescent labeling, radioactive labeling and non-radioactive labeling. When nucleic acid sequencing techniques are used, homology search for the nucleotide sequence of the extended nucleic acid molecules can be conducted using several databases of known sequences, including but not limited to the DDBJ / GenBank / EMBL databases.
[0051] b. Protein-based analysis: in addition to nucleic acid analysis, microorganisms can be characterized taxonomi
Petition 870190046231, of 5/17/2019, p. 27/90
23/75 and identified based on the presence (or absence) of specific proteins directly. Such an analysis can be based on specific protein activity, for example, through an enzyme assay or by the response of co-cultured organisms, or by the mere presence of specified protein (which can, for example, be determined using immunological methods, such as as immunofluorescent antibody staining in situ).
[0052] Enzyme assays: by way of example, fluorescent or chromogenic substrate analogs can be included within the growth medium (for example, microtiter plate cultures), followed by the incubation and screening of the reaction products, thus identifying cultures on a basis of their enzyme activities.
[0053] Co-culture response: In some embodiments of the present invention, the activity of an enzyme performed by an isolated microbe can be assayed based on the response (or degree of response) of a co-cultured organism (such as an organism news reporter).
[0054] A variety of methods can also be used to identify microorganisms selected and isolated from a source environment by binding at least one antibody-derived molecule to a molecule, or more particularly an epitope of a molecule, of the micro- body.
[0055] Anti-microorganism protein antibodies can be produced using standard procedures described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The determination that a particular agent binds substantially only to a protein of the desired microorganism can be made readily using or adapting routine procedures. A suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988)).
Petition 870190046231, of 5/17/2019, p. 28/90
24/75 [0056] Smaller antibody fragments (antibody-derived molecules, for example, FAbs, Fvs, and single chain Fvs (SCFvs)) can also serve as specific binding agents. Methods of making these fragments are routine.
[0057] Detection of antibodies that bind to cells in an arrangement of this invention can be performed using standard techniques, for example ELISA assays that provide a detectable signal, for example a fluorescent or luminescent signal.
Cultures isolated from the invention [0058] As described in more detail in the Examples section of the present disclosure, applicants have found a number of new agriculturally beneficial microorganisms, for example, effective suppressors of cereal fusarium disease. In particular, these new antagonistic microorganisms are effective in reducing the severity of cereal fusariosis and in inhibiting the growth of Fusarium graminearum, a primary causative agent of cereal fusarium disease in wheat. Microbial antagonists have been identified from a set of approximately 5,000 microbial strains obtained from wild plant samples collected from various locations in the United States. Initial selection of the antagonistic microorganism was based on the ability of the microorganisms to suppress the development of the pathogen of F. graminearum and that of its Gibberella zeae teleomorph in an in vitro antagonism assay. Selected microbial antagonists were then bioassayed in a greenhouse study on wheat seedings, which involved inoculating the seedings with the microbial strains, followed by repeated inoculations of F. graminearum spores, for the ability of the microbial strains to reduce gravity fungus infestation and its ability to preserve seed yield. The antagonistic microorganisms selected in this way were found
Petition 870190046231, of 5/17/2019, p. 29/90
25/75 of the effective number in reducing the severity of cereals fusariosis in attempts of greenhouse effect.
[0059] Taxonomic analysis further determined that each of the antagonistic microorganisms described in the present disclosure 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, the type genus of the Microbacteriaceae family, is generally considered to accommodate a Gram-positive spore forming rod-shaped bacteria that were originally isolated during previous studies on lactic acid-producing bacteria. Members of the Microbacterium genus are originally largely characterized by their noted heat resistance, presence in journals, and production of small amounts of L (+) lactic acid from glucose. Unlike other genera of the Microbacteriaceae family in which species are characterized by a coherent type of peptidoglycan, Microbacterium species have ornithine or lysine or in inter-peptide bridge or in position 3 of the type B peptidoglycan. In other chemotaxonomic properties, such isoprenoid quinones (MK-11, MK-12, MK-13), polar lipids, fatty acids and DNA-based composition, members of the genus exhibit the usual range of diversity found in other genera of Microbacteriaceae. Two bacterial genera Microbacterium and Aureobacterium can be joined according to some taxonomy studies. So far, members of the Microbacterium genus comprise at least 33 species, which have been isolated from a wide range of habitats, including soil, dairy products, plant galls, insects or clinical specimens. Various aspects of its ecology, phylogeny, taxonomy, culture methods, and long-term preservation conditions have recently been summarized by Evtushenko and Takeuchi
Petition 870190046231, of 5/17/2019, p. 30/90
26/75 (2006). Someone skilled in the art will readily appreciate that the microorganism of the genus Microbacterium can be taxonomically identified widely by any of the taxonomic identification techniques described above, including taxonomic chemoanalysis of cell wall peptidoglycan and comparative 16S rDNA sequence analysis as described in Evtushenko and Takeuchi (2006) and references cited therein, as well as those described in Examples 2-3 of the present disclosure. As discussed in detail below, several naturally occurring microorganisms have so far been reported to have antagonistic activity against cereal fusarium disease. However, there are no reports prior to the present invention that describe a microorganism of the genus Mycobacterium having such antagonistic activity. In addition, prior to the present invention, the present inventors were not aware of any methods or processes of using a bacterial strain of the genus Mycobacterium as a biocontrol agent in preventing, inhibiting or treating the development of a pathogen causing the disease of cereals fusariosis.
[0061] The genus Bacillus is a genus of rod-shaped, spore-forming, Gram-positive / variable bacteria. Similar to other genera associated with the previous history of microbiology, such as Pseudomonas or Vibrio, approximately 266 members of species of the genus Bacillus are found ubiquitous, and this is widely considered to be one of the genera with the greatest diversity of 16S and environmental diversity. Bacillus species can be mandatory aerobic or facultative anaerobes, and positive for catalase enzyme. Ubiquitous in nature, Bacillus includes both species living free and pathogenic. Under stressful environmental conditions, cells produce endo-spores that can remain dormant for long periods. These characteristics originally defined
Petition 870190046231, of 5/17/2019, p. 31/90
27/75 define the genus, but not all of these species are closely related, and many have been removed for other genera. In fact, several studies have attempted to reconstruct the phylogeny of the genus. The specific study of Bacillus with the greatest diversity covered is by Xu and Cote [Intl. J. of Syst. Evol. Microbiol. 53 (3): 695-704; 2003], using 16S and the ITS region, where they divide 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 large number of nested taxa and especially in both 16S and 23S it is considered as paraphyletic to Lactobacillales (Lactobacillus, Streptococcus, Staphylococcus, Listeria, etc.), due to Bacillus coahuilensis and others (see, for example, Yarda et al., Syst. Appl. Microbiol. 31 (4): 241-250, 2008; Yarda et al., Syst. Appl. Microbiol. 33 (6): 291-299, 2010]. A particular Glade, formed by B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis and B. weihenstephanensis under current classification standards, must be a unique species (within 97% of identity 16S), but due to medical reasons, they are considered separate species. In addition to the taxonomy analysis methods described in Examples 2-3 of this disclosure, phylogenetic and taxonomic analyzes of Bacillus species can be performed by a variety of techniques , including those discussed details in Xu and Cote, 2003; Yarda et al., 2008; Yarda et al., 2010.
[0062] The genus Variovorax was originally created by reclassifying Alcaligenes paradoxus as Variovorax paradoxus (Willems et al., 1991), which is widely considered to be a species of this genus. V paradoxus has been extensively studied as a model for new biodegradation agents, as well as microbe / microbe and microbe / plant interactions. Other species include V. dokdonen
Petition 870190046231, of 5/17/2019, p. 32/90
28/75 sis, V. soli (Kim et al., 2006), and V. boronicumulans (Miwa et al., 2008). Variovorax species are catabolically very diverse and engage in mutually beneficial interactions with other bacterial species in many biodegradations, and therefore have ecological importance and high application potential. For example, a soil methanotrophic, just as co-cultivated together with a V. paradoxus strain, exhibits a high affinity for methane (a potent greenhouse gas), and this trait is not normally seen in laboratory cultures. Similarly, a relative close to Variovorax has been reported to be the central pair, not photosynthetic within the phototrophic consortium Chlorochromatium aggregatum. Some species of the genus Variovorax also have the ability to interfere with the communication of other bacteria. Yet some other species of the genus Variovorax can interact closely with another biota (for example, plants) in various ecosystems. In addition, some species of Variovorax, residing in the area just outside the plant's roots and / or inside a plant, have been reported to be able to promote plant growth by reducing ethylene levels, the repression of pathogenesis controls by quorum sensitivity, and the increased resistance to heavy metals that largely benefits phytoremediation. In addition to the taxonomy analysis methods described in Examples 2-3 of the present disclosure, phylogenetic and taxonomic analyzes of Variovorax species can be performed by a variety of techniques including those discussed in detail elsewhere elsewhere here.
[0063] Mycosphaerella is a very large genus of fungus, with more than 2,000 species names and at least 500 species associated with more than 40 anamorphic genera (especially Cercospora, Pseudocercospora, Septoria, Rarnularia, etc.). In addition, several thousand anamorphs lack telomorphs. The Mycosphaerella genus
Petition 870190046231, of 5/17/2019, p. 33/90
29/75 includes species that are pathogens, saprobes, endophytes, or mutualistic associations. Various aspects of its ecology, phylogeny, taxonomy, culture methods, and long-term preservation conditions have recently been reported (see, for example, Crous et al., Persoonia, 23: 119-146,2009). Someone skilled in the art will readily appreciate that the microorganism of the genus Mycosphaerella can be identified taxonomically by any of the taxonomic techniques described above, or a combination thereof. Most common techniques include comparative sequence analyzes using 16S rDNA sequences and the internal transcribed spacer regions as described in, for example, Crous et al., Studies in Mycology, 55: 235-253,2006; Crous et al., 2009, supra; Goodwin et al., Phytopathology 91: 648-658, 2001; and those described in Examples 2-3 of the present disclosure.
Biological Material Deposit [0064] Purified cultures of microbial strains identified as having suppressive activity against cereal fusariosis disease were deposited at the Agricultural Research Service Culture Collection located at 1815 N. University Street, Peoria , IL 61604, USA (NRRL) according to the Budapest Treaty for the purpose of the patent procedure and the regulations under it (Budapest Treaty). Access numbers for these deposits are as follows:
SGI Cepa ID Access number Provisional Taxonomy SGI-005-G08 NRRL __-___ Microbacterium sp. SGI-010-H11 NRRL 50471 Mycosphaerella sp. SGI-014-C06 NRRL B-50470 Microbacterium sp. SGI-014-G01 NRRL B-50469 Variovorax sp. SGI-015-F03 NRRL B-50760 Bacillus sp. SGI-015-H06 NRRL B-50761 Bacillus sp.
Petition 870190046231, of 5/17/2019, p. 34/90
30/75 [0065] The microbial strains were deposited under conditions that guarantee the crop will be available pending this patent application to someone identified by the Patent and Trademark Commissioner to be entitled there under 37 G.F.R. §1.14 and 35 U.S.C. § 122. The deposits represent substantially pure cultures of the deposited strains. Deposits are available as required by foreign patent laws in countries where counterparties to the object application or their progeny are deposited. However, it should be understood that the availability of a deposit does not constitute a license to practice object invention in derogation from patent rights granted by government action.
[0066] Preferred microorganisms of the present invention have all the characteristics of identification of deposit strains and, in particular, the characteristics of identification of being able to suppress the development of cereal fusarium disease as described here, and as being capable to suppress the pathogen development of Fusarium graminearum and its teleomorphic Gibberella zeae as described here. In particular, the preferred microorganisms of the present invention refer to deposited microorganisms as described above, and mutants thereof.
Microbiological Compositions [0067] The microbiological compositions of the present invention comprising isolated microbial strains or cultures thereof can be in a variety of forms, including, but not limited to, yet cultures, total cultures, stored stocks of cells, mycelium and / or hifae (particularly glycerol stocks), agar strips, agar plugs stored in glycerol / water, dry frozen stocks, and dry stocks such as lyophilisates or dried mycelia on filter paper or grain seeds. As defined anywhere here, isolated culture or grammar equivalents as used
Petition 870190046231, of 5/17/2019, p. 35/90
31/75 in this disclosure and in the technique is understood to mean that said culture is a culture fluid, pellet, scraping, dry sample, lyophilate, or section (for example, hyphae or mycelia); or a support, vessel or medium such as a plate, paper, filter, matrix, straw, pipette or pipette tip, fiber, needle, gel, swab, tube, vial, particle, etc. that contains a unique type of organism. In the present invention, a culture isolated from a microbial antagonist is a culture fluid or a scrape, pellet, dry preparation, lyophilate, or section of the microorganism, or a support, vessel, or medium containing the microorganism, in the absence of other organisms .
[0068] The present disclosure further provides compositions that contain at least one isolated microbial strain or cultures of the same invention and a vehicle. The vehicle can be any or more than a number of vehicles that impart a variety of properties, such as high stability, wettability, dispersibility, etc. Wetting agents such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination thereof, can be included in a composition of the invention. Water-in-oil emulsions can also be used to formulate a composition that includes at least one isolated microorganism of the present invention (see, for example, U.S. Patent No. 7,485,451, incorporated by reference here). Suitable formulations that can be prepared include wettable powders, granules, gels, agar strips or pellets, thickeners, and others, microencapsulated patulas and others, liquids such as aqueous fluids, aqueous suspensions, water-in-oil emulsions, etc. The formulation can include grain or vegetable products (for example, ground grain or beans, broth or flour derived from grain or beans), starch, sugar or oil. The vehicle can be an agricultural vehicle. In certain preferred embodiments, the vehicle is a seed, and the composition can be
Petition 870190046231, of 5/17/2019, p. 36/90
32/75 applied or coated on the seed or allowed to saturate the seed. [0069] In some modalities, the agricultural vehicle can be soil or plant growth medium. Other agricultural vehicles that can be used include water, fertilizers, plant-based oils, humectants, or combinations thereof. Alternatively, the agricultural vehicle can be a solid, such as a diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, seed cases, other plant and animal products, or combinations, including granules, pellets, or suspensions. Mixtures of any of the aforementioned ingredients are also contemplated as carriers, such as but not limited to, pesta (flour and kaolin clay), agar or pellets based on marl, sand, or clay flour, etc. Formulations may include food sources for cultured organisms, such as barley, rice or other biological materials such as seed, plant parts, sugar cane bagasse, husks or stalks from grain processing, creeping material (eg garden waste) or construction waste, sawdust from small fibers of paper, fabric or wood recycling. Other suitable formulations will be known to those skilled in the art.
[0070] In liquid form, for example solutions or suspensions, microorganisms can be mixed or resuspended in water or in aqueous solutions. Suitable liquid thinners or vehicles include water, aqueous solutions, petroleum distillates or other liquid vehicles.
[0071] Solid compositions can be prepared by dispersing anatgonist microorganisms in and over a properly divided solid vehicle, such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, fuller soil, pasteurized soil, and others. When such formulations are used as wettable powders,
Petition 870190046231, of 5/17/2019, p. 37/90
33/75 biologically compatible dispersion agents such as nonionic, anionic, amphoteric, or cationic dispersion and emulsifiers can be used.
[0072] In a preferred embodiment, the compositions contemplated here can prevent attack by disease of the fusariosis of cereals in plants, particularly cereal plants, such as wheat, barley, oats, and corn and, when used in sufficient amounts, to act as microbial antagonists. These compositions, similar to other biocontrol agents, have a high safety margin because they do not normally burn or injure the plant.
[0073] As described in more detail throughout the present disclosure, control of cereal fusariosis disease can be effected by applying one or more microbiological compositions of the present invention to a host plant or parts of the host plant. The compositions can be applied in an effective amount to reduce the level of cereal fusariosis compared to that in an untreated control. The active constituents are used in a concentration sufficient to inhibit plant pathogen development from the targeted plant pathogen when applied to a cereal plant. As will be apparent to a person skilled in the art, effective concentrations can vary depending on factors such as: (a) the type of plant or agricultural product; (b) the physiological condition of the plant or agricultural product; (c) the concentration of pathogens affecting the plant or agricultural product; (d) the type of disease injury on the plant or agricultural product; (e) weather conditions (for example temperature, humidity); and (f) the stage of plant disease. According to the invention, typical concentrations are those higher than 1 X 10 ² CFU / ml of the vehicle. Preferred concentration ranges from about 1 X 104 to about 1 x 109 CFU / ml, as well as concentrations ranging from 1 X 106 to 1 X 108 CFU / ml. Most preferred concentrations Petition 870190046231, of 17/05/2019, p. 38/90
34/75 more are those of about 35 to about 150 mg of dry microbial mass per gram of vehicle (dry formulation) or per millimeter of vehicle (liquid composition). In solid formulations, the application rate must be controlled to result in a comparable number of viable cells per unit area of the plant's tissue surface as obtained by the previously mentioned liquid treatment rates. Normally, the biological control agents of the present invention are biologically effective when delivered in a concentration in excess of 106 CFU / g (colony forming units per gram), preferably in excess of 10 ⁷ CFU / g, more preferably 108 CFU / g, and most preferably at 109 CFU / g.
[0074] In some embodiments, the amount and one or more of the biological control agents in the microbial compositions of the present invention may vary depending on the final formulation as well as the size or type of plant or seed used. Preferably, the one or more biological control agents in the microbial compositions are present in about 2% w / w / to about 80% w / w of the entire formulation. More preferably, the one or more biological control agents employed in the compositions is about 5% w / w about 65% w / w and more preferably about 10% w / w and about 60% w / w by weight of the whole formulation.
[0075] As will be appreciated by those skilled in the art, the microbiological compositions of the invention can be applied to wheat or other cereal plants using a variety of conventional methods such as dusting, coating, injection, friction, rolling, dipping, spraying, or brushing, or any other appropriate technique that does not significantly harm the wheat plant or other cereals to be treated. The method of spraying is particularly preferred.
[0076] Usually, the compositions of the invention are chemical
Petition 870190046231, of 5/17/2019, p. 39/90
35/75 inert; consequently they are compatible with substantially any other constituents of the spray schedule. They can also be used in combination with biologically compatible active pesticidal agents such as herbicides, nematicides, fungicides, insecticides, and others. It can also be used in combination with substances related to plant growth, such as fertilizers, plant growth regulators and others, provided that such compounds or substances are biologically compatible.
[0077] When used as pesticides or fungicides in their commercially available formulations and in the forms of use, prepared from these formulations, the active microbial antagonists and compositions according to the present invention may still be present in the form of a mixture with synergists. Synergists are compounds by which the activity of active compositions is increased without it being necessary for the added synergist to be active himself.
[0078] When used as pesticides in their commercially available formulations and in the forms of use, prepared from these formulations, the active microbial antagonists and compositions according to the invention may additionally be present in the form of a mixture with inhibitors that reduce degradation of compositions active after application in the plant habitat, on the surface of plant parts or on plant tissues.
[0079] The active microbial antagonists and compositions according to the invention, as such or in their formulations, can also be used as a mixture with known acaricides, bactericides, fungicides, insecticides, microbicides, nematicides, pesticides, or combinations thereof, for example to broaden the spectrum of action or to avoid the development of resistance in this
Petition 870190046231, of 5/17/2019, p. 40/90
36/75 sense. In many cases, synergistic effects result, i.e. the activity of the mixture may exceed the activity of the individual components. A mixture with other known active compounds, such as fertilizers, growth regulators, protectors and / or semi-chemicals is also contemplated.
[0080] In a preferred embodiment of the present invention, the compositions can further include at least one chemical or biological pesticide. The amount of at least one chemical or biological pesticide used in the compositions can vary depending on the final formulation as well as the size of the plant and seed to be treated. Preferably, the at least one chemical or biological pesticide employed is about 0.1% w / w to about 80% w / w based on the entire formulation. More preferably, the at least one chemical or biological pesticide is present in an amount of about 1% w / w to about 60% w / w and more preferably about 10% w / w to about 50% w / w.
[0081] A variety of pesticides are apparent to someone skilled in the art and can be used. Exemplary chemical pesticides include those in the carbamate, organophosphate, organochlorine, and pretroid class. Also included are chemical control agents such as, but not limited to, benomyl, borax, captafol, captane, chorotalonil, copper-containing formulations; formulations containing diclone, dichloran, iodine, zinc; fungicides that inhibit ergosterol biosynthesis such as, but not limited to, blastididine, cymoxanil, phenarimol, flusilazole, folpet, imazalil, ipordione, maneb, manocozeb, metalaxyl, oxycarboxine, miclobutanil, oxytetracycline, PCNB, pentachlorol, propachlorolen, pentachlorolen sodium, sodium DNOC, sodium hypochlorite, sodium phenylphenate, streptomycin, sulfur, tebuconazole, terbutrazol, thiabendazolel, thiophanate-methyl, triadimefon, tricyclazole, triforine, validimycin, vinclozolin, zineb, and ziram. A variety
Petition 870190046231, of 5/17/2019, p. 41/90
37/75 of insecticidal compounds may be useful for compositions of the invention, including but not limited to those cited in Ped. Pat. US No. 20110033432A1.
[0082] The microbiological compositions of the present invention preferably include at least one biological pesticide. Exemplary biological pesticides that are suitable for use here and can be included in a microbiological composition according to the present invention to prevent pathogenic plant disease include microbes, animals, bacteria, fungi, genetic material, plant, and natural products from living organisms. In these compositions, the microorganism of the present invention is isolated before formulation with an additional organism. For example, microbes such as but not limited to Ampelomyces, Aureobasidium, Bacillus, Beauveria, Candida, Chaetomium, Cordyceps, Cryptococcus, Dabaryomyces, Erwinia, Exophilia, Gliocladium, Mariannaea, Paecilomyces, Paenibacillus, Pantoea, Pantoea, Pantoea, Pantoea, Pantoea, Pantoea, Pantoea Talaromyces, and Trichoderma can be provided in a composition with the antagonistic microorganisms of the present invention, with fungal strains of the genus Muscodor being particularly preferred. Use of the microbiological compositions according to the present invention in combination with the microbial antagonists disclosed in US Patent No. 7,518,040; US Patent No. 7,601,346; US Patent No. 6,312, .940 is also particularly preferred.
[0083] Examples of fungi that can be combined with microbial antagonists and compositions of the present invention in a composition include, without limitation, Muscodor species, Aschersonia aleyrodis, Beauveria bassiana (white muscarine), Beauveria brongniartii, Chladosporium herbarum, Cordyceps clavulata, Cordycepsh , Cordyceps facts, Cordyceps gracilis, Cordyceps melolanthae, Cordyceps militaris, Cordyceps myrmecophila, Color
Petition 870190046231, of 5/17/2019, p. 42/90
38/75 dyceps ravenelii, Cordyceps sinensis, Cordyceps sphecocephala, Cordyceps subsessilis, unilateralis Cordyceps, Cordyceps variabilis, Cordyceps washingtonensis, Culicinomyces clavosporus, Entomophaga grylli, Entomophaga maimaiga, Entomophaga muscae, Entomophaga praxibulli, Entomophthora plutellae, Fusarium lateritium, citriformis Hirsutella, Hirsutella thompsoni , Metarhizium anisopliae (green muscarina), Metarhizium flaviride, Muscodor albus, Neozygitesfloridana, Nomuraea rileyi, Paecilomyces farinosus, Paecilomyces fumosoroseus, Pandora neoaphidis, Tolypocladium cylindrosporum, Verticillophora lichani, and Verticillophium lecani. Other micropesticide species will be apparent from those skilled in the art.
[0084] The present invention also provides methods of treating a plant by applying any variety of customary formulations in an effective amount or to the soil (ie, in soil), a portion of the plant (ie, watering) or over the seed before planting (ie, seed coating or dressing). Usual formulations include solutions, emulsifiable concentrate, wettable powders, suspension concentrate, soluble powders, granules, suspension emulsion concentrate, natural and synthetic materials impregnated with active compound, and very fine control release capsules in polymeric substances. In certain embodiments of the present invention, biological control compositions are formulated into powders that are available either a ready-to-use formulation or are mixed together at the time of use. In another embodiment, the powder can be mixed with the soil before or at the time of planting. In an alternative embodiment, one or both of the biological control agent or insect control agent is a liquid formulation that is mixed together at the time of treatment. Someone ordinarily skilled in the art understands that an effective amount of
Petition 870190046231, of 5/17/2019, p. 43/90
39/75 inventive positions depends on the final formulation of a composition as well as the size of the plant or the size of the seed to be treated.
[0085] Depending on the final formulation and method of application, one or more suitable additives can also be introduced for the compositions of the present invention. Adhesives such as carboxymethylcellulose and natural and synthetic 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 lecithins, and synthetic phospholipids can be added to present compositions.
[0086] In a preferred embodiment, microbiological compositions are formulated in a single stable solution, or emulsion or suspension. For solutions, the active chemical compounds (ie, pest control agents) are normally dissolved in solvents before the biological control agent is added. Suitable liquid solvents include petroleum based aromatics, such as xylene, toluene or alkynaphthalenes, 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, strongly polar solvents, such as dimethylformamide and dimethyl sulfoxide. For emulsion and suspension, the liquid medium is water. In one embodiment, the chemical control agent and biological control agent are suspended in separate liquids and mixed at the time of application. In a preferred suspension mode, the insect and biological control agent are combined in a ready-to-use formulation that exhibits a shelf life of at least two years. In use, the liquid can be sprayed or applied foliarly as an atomized spray or in
Petition 870190046231, of 5/17/2019, p. 44/90
40/75 furrow when planting the crop. The liquid composition can be introduced into the soil prior to seed germination or directly into the soil in contact with the roots using a variety of techniques known in the art including, but not limited to, drip irrigation, sprinkler, soil injection or water flooding. solador.
[0087] Optionally, stabilizers and buffers can be added, 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 can also be added and may include formaldehydes or formaldehyde releasing agents and benzoic acid derivatives, such as phydroxybenzoic acid.
Seed coating formulations [0088] In some particularly preferred embodiments, the biocontrol compositions of the present invention are formulated as a seed treatment. The seed treatment preferably comprises at least one insect control agent and at least one biological control agent. It is contemplated that the seeds can be substantially uniformly coated with one or more layers of the biocontrol compositions disclosed here using conventional methods of mixing, spraying or a combination of them using treatment application equipment that is specifically designed and manufactured for use. apply precisely, safely and efficiently from seed to seed treatment products. Such equipment uses several types of coating technology such as rotary coaters, cylindrical coaters, fluidized core techniques, spouted cores, mixed rotors or a combination thereof. Liquid seed treatments such as those of the present invention can be applied
Petition 870190046231, of 5/17/2019, p. 45/90
41/75 by means of either a spinning “atomizer” disk or a spray nozzle that evenly distributes the seed treatment over the seed as it moves through the spray model. Preferably, the seed is then mixed or tumbled for an additional period of time to achieve additional treatment distribution and drying. The seeds can be prepared or not prepared before coating with the inventive compositions to increase uniformity of germination and emergence. In an alternative embodiment, a dry powder formulation can be measured on the moving seed and allowed to mix until completely distributed.
[0089] Another aspect of the invention provides seeds treated with the microbiological object compositions. One embodiment provides seeds having at least part of the surface area coated with a microbiological composition according to the present invention. In a specific embodiment, seeds treated by microorganisms have a spore concentration or microbial cell concentration of about 106 to about 10 ⁹ per seed. The seeds may also have more spores or microbial cells per seed, such as, for example, 10 ¹⁰ , 1 0 ¹¹ or 10 ¹² spores per seed. The microbial spores and / or cells can be freely coated on the seeds or, preferably, they can be formulated in a liquid or solid composition before being coated on the seeds. For example, a solid composition comprising microorganisms can be prepared by mixing a solid vehicle with a spore suspension until the solid vehicles are impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.
[0090] In some other embodiments, it is contemplated that the solid or liquid biocontrol compositions of the present invention additionally contain functional agents capable of protecting themselves
Petition 870190046231, of 5/17/2019, p. 46/90
42/75 minds of harmful effects of selective herbicides such as activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the products or a combination of them.
[0091] Seed coating methods and compositions that are known in the art can be particularly useful when they are modified by the addition of one of the embodiments of the present invention. Such methods of coatings and apparatus for their application are disclosed in, for example, Pat. We. US 5,918,413; 5,554,445; 5,389,399; 4,759,945; and 4,465,017. Various seed coating compositions are disclosed, for example, in Ped. Pat. Appl. We. US20110033432, US20100154299, U.S. Pat. We. 5,939,356; 5,876,739, 5,849,320; 5,791,084, 5,661,103; 5,580,544,
5,328,942; 4,735,015; 4,634,587; 4,372,080, 4,339,456; and 4,245,432, among others.
[0092] A variety of additives can be added to seed treatment formulations comprising the inventive compositions. Binders can be added and include those preferably composed of an adhesive polymer that can be natural or synthetic with no phytotoxic effect on the seed to be coated. The binder can be selected from polyvinyl acetates; copolymers of polyvinyl acetates; copolymers of ethylene vinyl acetate (EVA); polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrrolidones; polysaccharides, including starch, modified starch, dextrins, maltodextrins, alginate and chitosans; fats; oils; proteins, including gelatin and zeins; gum arabic; shellac; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers;
Petition 870190046231, of 5/17/2019, p. 47/90
43/75 polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene. [0093] Any of a variety of dyes can be used, including organic chromophores classified as nitrous; nitro; azo, including monoazo, bisazo and polyaz; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methyl, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. Other additives that can be added include trace nutrients such as iron, manganese, boron, copper, cobalt, molybdenum and zinc salts. A polymer or other dust control agent can be applied to retain the treatment on the seed surface.
[0094] In some specific embodiments, in addition to microbial cells or spores, the coating may additionally comprise an adherent layer. The adhesive must be non-toxic, biodegradable and adhesive. Examples of such materials include, but are not limited to, polyvinyl acetates; copolymers of polyvinyl acetate; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, such as methyl celluloses, hydroxymethyl celluloses, and hydroxymethyl propyl celluloses; dextrins; alginates; sugars; molasses; polyvinyl pyrrolidones; polysaccharides; proteins; fats; oils; gum arabic; gelatines; syrups and starches. More examples can be found in, for example, Pat. No. U.S, 7,213,367 and Ped. Pat. No. US20100189693.
[0095] Various additives, such as adherents, dispersants, surfactants, and nutrient and buffer ingredients, can also be included in the seed treatment formulation. Other conventional seed treatment additives include, but are not limited to, wetting agents, buffering agents and polysaccharides. At least one agriculturally acceptable carrier can be added to the seed treatment formulation such as water, solids or dry powders. Dry powders can be derived from a variety of materials such as calcium carbonate, gypsum, vermiculite, talc, humus,
Petition 870190046231, of 5/17/2019, p. 48/90
44/75 activated carbon and various phosphorous compounds.
[0096] In some embodiment, the seed coating composition may comprise at least one filler that is a natural or synthetic, organic or inorganic component with which the active components are combined to facilitate its application on the seed. Preferably, the filler is an inert solid such as rings, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (eg ammonia salts), natural soil minerals, such as kaolins, clays, talc, lime, quartz, atapulgite, montmorillonite, bentonite or diatomaceous earth, or synthetic minerals, such as silica, alumina or silicates, in particular aluminum or magnesium silicates.
[0097] The seed treatment formulation can also include one or more of the following ingredients: other pesticides, including compounds that act just below the ground; fungicides, such as captan, extract, metalaxyl, fludioxonil, oxadixil, and isomers of each of those materials, and others; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxis, ureas, and benzoic acids; herbicidal protectors such as benzoxazine, benzhydryl derivatives, N, N-diallyl dichloroacetamide, various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone, naphthyl anhydride compounds, and oxime derivatives; fertilizers; and biocontrol agents such as other recombinant or naturally occurring bacteria and fungi of the genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium and mycorrhizal fungus. These ingredients can be added as a separate layer on top of the seed or alternatively they can be added as part of the composition of the seed coating invention.
[0098] Preferably, the amount of the new composition or
Petition 870190046231, of 5/17/2019, p. 49/90
45/75 other ingredients used in seed treatment should not inhibit seed germination; or cause phytotoxic damage to the seed.
[0099] Microorganism-treated seeds can also be additionally wrapped with a film overcoat to protect the coating. Such overlays are known in the art and can be applied using fluidized core and cylindrical film coating techniques.
[00100] In principle, any plant seed capable of germinating to form a plant that is susceptible to attack by nematodes and / or pathogenic fungus can be treated according to the invention. Suitable seeds include those from cereals, coffee, cole crops, fiber crops, flowers, fruits, vegetables, oil crops, trees, tuber crops, vegetables, as well as other monocot plants, and dicot species. Preferably, crop seeds are coated which includes, but is not limited to, beans, carrots, corn, cotton, grasses, lettuce, peanuts, pepper, potatoes, rapeseed, rice, rye, sorghum, soya beans, sugar beet, sunflower, tobacco, and tomato seeds. Most preferably, barley or wheat seeds (spring wheat or winter wheat) are coated with the present compositions.
[00101] Also provided, in another aspect of the present invention, is a new cereal plant created by artificially introducing a microbial endophyte of the invention into a cereal plant that is 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 cereal fusariosis disease or an agent causing cereal fusariosis disease. In addition, the endophytic antagonist introduced into the cereal plant may be a strain of fungus SGI-010-H11. A variety of methods previously found effective for
Petition 870190046231, of 5/17/2019, p. 50/90
46/75 introduction of a species of cereal grass is known in the art. Examples of such methods include those described in Ped. Pat. US No. 20030195117A1, Ped. Pat. No. 20010032343A1, and Pat. No. U.S, 7,084,331, among others. It will become apparent to those skilled in the art that many of the aforementioned methods may be useful in making a new cereal plant of the invention.
[00102] After artificial infection, it is preferred that the DNA of the isolated endophytic antagonist is amplified by PCR and the antagonist is confirmed by performing homology research for the amplified DNA. Additionally, it is preferable that a foreign gene expressing an identifiable medium is introduced into the endophytic antagonist mentioned above, and the presence of colonization of the above endophytic antagonist infecting the plant is confirmed by the means identified above using the foreign gene.
Preparing the biocontrol compositions according to the present invention [00103] Cultures of the microbial antagonists can be prepared for use in the biocontrol compositions of the invention using standard liquid fermentation and static drying techniques known in the art. Growth is commonly carried out in a bioreactor. [00104] A bioreactor refers to any device or system that supports a biologically active environment. As described here, a bioreactor is a flask in which microorganisms including the microbial antagonists of the invention can be grown. A bioreactor can be any suitable shape or size for cultivating microorganisms. A bioreactor can vary in size and scale from 10 mL to liters to cubic meters and can be made of stainless steel or any other suitable material as known and used in the art. The bioreactor can be a batch type bioreactor, the batch type of
Petition 870190046231, of 5/17/2019, p. 51/90
47/75 feed or a continuous type bioreactor (for example, a continuous agitated reactor). For example, a bioreactor can be a chemostat as known and used in the microbiology technique for cultivating and harvesting the bacteria. A bioreactor can be obtained from any commercial supplier (see also Bioreactor System Design, Asenjo & Merchuk, CRC Press, 1995).
[00105] For small scale operations, a batch bioreactor can be used, for example, to test and develop new processes, and for processes that cannot be converted to continuous operations.
[00106] Microorganisms grown in a bioreactor can be suspended or immobilized. Cultivation in the bioreactor is usually under aerobic conditions at appropriate temperatures and pH for growth. For the organisms of the invention, cell growth can be achieved at temperatures between 5 and 37 ° C, with the preferred temperature being in the range of 15 to 30 ° C, 15 to 28 ° C, 20 to 30 ° C, or 15 to 25 ° C. The pH of the nutrient medium can vary between 4.0 and 9.0, but the preferred operating range is usually slightly acidic to neutral at pH 4.0 to 7.0, or 4.5 to 6.5, or pH 5.0 to 6.0. Typically, maximum cell yield is obtained 20-72 hours after inoculation.
[00107] Optimal conditions for the cultivation of antagonists of this invention will, of course, depend on the particular strain. However, due to the conditions applied in the selection process and the general requirements of most microorganisms, a person ordinarily skilled in the art would be able to determine essential nutrients and conditions. Microbial antagonists would normally be cultured in aerobic liquid cultures in medium containing sources of carbon, nitrogen and inorganic salts that can be assimilated by the microorganism and efficient cell growth support. Preferred carbon sources are hexoses such as glucose, but
Petition 870190046231, of 5/17/2019, p. 52/90
48/75 other sources that are readily assimilated such as amino acids, can be substituted. Many inorganic or proteinaceous materials can be used as sources of nitrogen in the growth process. Preferred nitrogen sources are amino acids and urea, but others include gaseous ammonia, inorganic nitrate and ammonium salts, vitamins, purines, pyrimidines, yeast extract, steak extract, peptone proteose, soy bran, casein hydrolysates, soluble distillers , and others. Among the inorganic materials that can be incorporated into the nutrient medium are the usual salts capable of yielding calcium, zinc, iron, manganese, magnesium, copper, cobalt, potassium, sodium, molybdate, phosphate, sulfate, chloride, borate, and ions similar. Without being limited to them, the use of liquid medium of potato dextrose for fungal antagonists and premix of R2A broth for bacterial strains is preferred.
[00108] Throughout the disclosure, several sources of information are referred to and incorporated by reference. Sources of information include, for example, scientific articles from newspapers, patent documents, textbooks and addresses of World Wide Web browser-inactive page addresses. The reference to such sources of information is only for the purpose of providing an indication of the general state of the art at the time of deposit. Although the contents and teachings of each of all sources of information can be supported and used by someone skilled in the art to make and use modalities of the invention, any discussion and comment on a specific source of information should in no way be considered as a admission that such a comment was widely accepted as a general opinion in the field.
[00109] The discussion of the general methods given here is intended for illustrative purposes only. Other alternative methods and modalities will be evident for those skilled in the art through
Petition 870190046231, of 5/17/2019, p. 53/90
49/75 are of that disclosure, and are to be included within the spirit and scope of that request.
[00110] It should be understood that the following examples are offered to illustrate, but not to limit the invention.
EXAMPLES
EXAMPLE 1: discovery of antagonistic microorganisms capable of suppressing the development of Fusarium graminaerum and Gibberella zeae, agents that cause the Fusarium disease of cereals.
[00111] This example describes a high-throughput process for the collection and screening of candidate microorganisms that have been developed internally at Synthetic Genomics, Inc. to isolate strain of microorganisms having suppressive activity against agents causing the disease of cereal fusariosis, particularly against Fusarium graminearum. New strains of microbial antagonists have been isolated from plant tissue samples collected from various locations across the United States. The bacterial strains SGI-014C06, SGI-014-G01, SGI-015-F03, and SGI-015-H06 were isolated from the root tissues of the plant collected from Eagle Peak Preserve near Julian, CA. The fungal strain SGI-010-H11 and the bacterial strain SGI-005-G08 were isolated from the stem tissues of two different plants collected from San Elijo Lagoon, Encinitas, CA.
[00112] The microorganisms were isolated as follows. For bacterial strain isolation, root plant tissues were sonicated and subjected to serial dilutions over 2xYT (yeast and tryptone extract) and plates of N-free Agar medium. Individual colonies were then selected on morphological characteristics, and individually grown on liquid broth medium. For fungal isolation, plant tissue was sterilized on the first surface by dipping in 70% ethanol and passing briefly through a flame. The tissue was then dissected and placed in
Petition 870190046231, of 5/17/2019, p. 54/90
50/75 potato dextrose (PDA), followed by incubation at room temperature. When mycelial growth was observed, a segment of mycelial growth was then transferred to another PDA plate. Microorganism strains were isolated, purified and preserved at -80 ° C in 15% glycerol in for bacterial strains and on dry barley seed for fungal strains.
[00113] Strains of isolated microorganisms as described above were therefore tested for their ability to suppress mycelial growth of F. graminearum in an in vitro antagonism test, which was performed on agar plates containing dextrose agar growth medium. and potato (PDA) according to a high yield screening procedure described in Ped. From US Pat No. US20120107915A1, which is incorporated by reference here, with minor modifications. Briefly, strains isolated from microorganisms were grown on one-fifth strength Triptico soy broth agar (TSBA / 5) for 24 hours before use. Conidial inoculum of F. graminearum NRRL-5883 was produced by hyphal typification of a colony of culture actively on fungi and transferring the hyphal filaments to the Agar PDA medium. After incubating the plates for 7 days at 25 ^° C using a 12 h / day photoperiod, conidia were washed from PDA plates using a weak phosphate buffer (0.004% phosphate buffer, pH 7.2, with 0.019% MgCb). A suspension of F. graminearum conidia in weak phosphate buffer (1 ^x 10 ⁵ conidia / ml) was then spread immediately on an agar surface, and the plates were then incubated at 25 ^° C for 4872 hours. To start the antagonism test, cells from isolated microbial strains were inoculated by spot at equal distances within the perimeter of the plate. After five days, the strains were scored as positive antibiosis when a visibly clear area that lacked mycelial growth existed around the perimeter of microbial colonies.
Petition 870190046231, of 5/17/2019, p. 55/90
51/75 [00114] More than 4,000 microbial strains were isolated and tested by the procedure described above. Of those, six strains were found to significantly suppress the growth of mycelium of F. graminearum NRRL-5883 in PDA medium. These new antagonists are identified as SGI-005-G08, SGI-010-H11, SGI-014C06, SGI-014-G01, SGI-015-F03, and SGI-015-H06.
[00115] Antagonism testing was also carried out for new microbial antagonists for their ability to suppress the growth of fungal pathogen Gibberella zeae, which is a teleomorphic species of Fusarium graminearum. All procedures were identical to those described above, except for the fact that the pathogen strain tested in this assay was a pathogenic strain of the fungus Gibberella zeae. Of these six microbial antagonists, four strains SGI-010-H11, SGI-014-C06, SGI-015-F03, and SGI-015-H06 were found to significantly suppress the mycelial growth of Gibberella zeae.
EXAMPLE 2: DNA Extraction, Sequencing and Taxonomy [00116] Fungal cell lysis and Acquisition of ITS-5.8S rDNA sequence information [00117] The fungal biomass was transferred to a 96-well PCR microplate containing 50 pl of a 2 x lysis buffer (100 mM Tris HCL, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS, 400 pg / ml Proteinase K). lysis conditions were as follows: 55 ° C for 60 minutes, 94 ° C for 4 minutes. An aliquot of the lysis product was used as the model DNA source for PCR amplification. The ITS-5.8S rDNA sequence was amplified by PCR using two M13-ITS1 primers (SEQ ID NO: 15) and with ITS4 M13 tail (SEQ ID NO: 16).
[00118] For the amplification of the ITS-5.8S rDNA region, each PCR mixture was prepared in a final volume reaction of 20-pl containing
Petition 870190046231, of 5/17/2019, p. 56/90
52/75 μΙ from the fungal lysis reaction, 0.2 μΜ of each primer (ITS1 / ITS4), 6% Tween-20, and 10 μl of 2x ImmoMix (Bioline USA Inc, Taunton, MA). PCR conditions were as follows: 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-μΙ aliquot of the PCR product was run on a 1.0% agarose gel to confirm a single band of the expected size. Positive bands were sent for Sanger sequencing in the forward and reverse directions using M13 primers. Sequence of the 5.8S intergenic region (ITS) of the fungal strain SGI-010-H11 is provided in the Sequence Listing as SEQ ID NO: 11. Homology search for the determined nucleotide sequence of the 5.8S intergenic region (ITS) was conducted using the DDBJ / GenBank / EMBL database. Subsequently, the phylogenetic relationship of the nucleotide sequence of the 5.8S intergenic region (ITS) was analyzed between the isolated fungal antagonist SGI-010-H11 described here, microorganisms of the genera and species that present high sequence homologies with the fungal antagonist isolate SGI-010H11, and other wide varieties of genera and species of microorganism, using the ClustalW phylogenetic tree building program. The fungal strain SGI-010-H11 is considered to be related to the Mycosphaerellaceae family based on —97% similarity of its ITS-5.8S rDNA sequence to those of Mycosphaerella punctiformis (GenBank EU343182) and Ramularia pratensis (GenBank EU019284) , whose sequences of 5.8S ITS show clear kinship to Mycosphaerellaceae.
[00119] Bacterial cell lysis and Acquisition of 16S rRNA Sequence information [00120] A 20 µl aliquot of cell suspension was transferred to a 96-well PCR plate containing 20 µl of a 2x buffer
Petition 870190046231, of 5/17/2019, p. 57/90
53/75 lysis (100 mM Tris HCL, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS, 400 pg / ml Proteinase K). Lysis conditions were as follows: 55 ° C for 30 minutes, 94 ° C for 4 minutes. An aliquot of the lysis product was used as the source of model DNA for PCR amplification. The 16S rRNA sequence was amplified by PCR using M13-27F (SEQ ID NO: 17) and 1492R M13 tail primers (SEQ ID NO: 18).
[00121] For amplification of 16S rRNA region, each PCR mixture was prepared in a final 20-pl volume reaction containing 4 of the bacterial lysis reaction, 2 pM of each primer (27F / 1492R), 6% Tween-20 , and 10 pl of 2x ImmoMix (Bioline USA Inc, Taunton, MA). PCR conditions were as follows: 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-pl aliquot of the PCR product was run on a 1.0% agarose gel to confirm a single band of the expected size. Positive bands were sent for Sanger sequencing in the forward and reverse directions using M13 primers. Sequences of the rRNA16S of the bacterial strains SGI-014-C06, SGI-005-G08, SGI-014-G01, SGI-015-F03, and SGI-015H06 are approximately 1.4-Kb in length and are provided in the Sequence Listing as SEQ ID NOs: 1, 10, 12, 13, and 14 respectively. Homology research for the determined nucleotide sequence was conducted using the DDBJ / GenBank / EMBL database. Subsequently, the phylogenetic relationship of the nucleotide sequence of the 16 rRNA genes was analyzed among the isolated bacterial antagonists described here, bacteria of the genera and species that show high sequence homologies with the isolated bacterial antagonists, and other wide varieties of the bacterial genera and species , using the ClustalW phylogenetic tree building program. Sequence identity and similarity were also determined using the GenomeQuest software (Gene
Petition 870190046231, of 5/17/2019, p. 58/90
54/75
IT, Worcester Mass. USA). The result of the sequence analysis revealed that the bacterial isolate SGI-014-G01 can be considered related to the Variovorax genus based on —99% similarity of its 16S rRNA sequence to those of several Variovorax species, including Variovorax sp. R-21938 (GenBank AJ786799) and Variovorax paradoxes (GenBank GU186109), whose 16S rRNA sequences show clear kinship to Variovorax sp. In addition, the result of the sequence analysis also revealed 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 spp.
[00122] The result of the sequence analysis revealed that the two bacterial isolates SGI-014-C06 and SGI-055-G08 can be considered related to the Microbacteriaceae family based on> 99% similarity of their respective 16S sequences to those of several Microbacterium spp. Notably, the 1430-nt sequence of the 16S rRNA deSGI-014-C06 gene (SEQ ID NO: 1) is identical to the 16S rRNA gene of a Microbacterium oxydans DSM 20578 strain and several other strains of Microbacterium sp. (for example, GenBank strings Y17227.1, FJ200406, EU086800, and EU714335) along their length 1430-nt.
[00123] In particular, among the hundreds of microbial strains that have been isolated and tested for the ability to suppress Fusarium development in antagonism assays as described in Example 1, a total of eighty-eight strains have subsequently been identified as Microbacterium species with based on the sequence similarity of its 16S sequences to those of known Microbacterium spp. However, Applicants found that SGI-014-C06 and SGI-055-G08 were the only two Microbacterium strains that had
Petition 870190046231, of 5/17/2019, p. 59/90
55/75 am suppression activity against Fusarium graminearum. To date, as discussed above, several naturally occurring microbes have been reported to have antagonistic activity against cereal fusariosis disease. However, there are no reports prior to the present invention that describe a microorganism of the genus Mycobacterium having such antagonistic activity. In addition, prior to the present invention, the present inventors were not aware of any methods or processes of using a bacterial strain of the genus Mycobacterium as a biocontrol agent in preventing, inhibiting or treating the development of a pathogen causing the disease of cereals fusariosis.
EXAMPLE 3: Sequence Analysis of Maintenance Genes from Isolate SGI-014-C06 [00124] A phylogenetic study of several maintenance genes from 27 species of the genus Microbacterium was recently reported by Richert et al. (Syst. Appl. Microbiol. 30: 102-108, 2007). The study concluded that, while the merits of systematic taxonomy 16S rRNA sequence analysis are insurmountable, an exclusive emphasis on a single taxonomic parameter should not guide systematic conclusions. As disclosed above, the nucleotide sequence of the SGS-014-C06 16S rRNA gene (SEQ p NO: 1) is identical to the 16S rRNA gene of several Microbacterium sp. Strains, including the nucleotide sequence of a 16S rRNA gene of a Microbacterium oxydans DSM 20578 strain that was included in the study by Richert et al. (2007) (GenBank Access Y17227.1). Applicants began to perform a phylogenetic analysis on four maintenance genes of the SGI-014-C06 isolate, which are B-subunit DNA gyrase (gyrB), B-subunit RNA polymerase (rpoB), recombinase A (recA), and polyphosphate kinase ( ppk). For this purpose, the entire genome of the SGI014-C06 isolate was shotgun sequenced,
Petition 870190046231, of 5/17/2019, p. 60/90
56/75 used procedures described in PCT Patent Application No. W02010115156A2. Genomic DNA was prepared from a fresh culture of SGI-014-C06. Cellular pellet was used to extract high molecular weight DNA using the UltraClean® Mega Soil DNA Isolation Kit (Cat. No 12900-10) from MO BIO Laboratories, Inc. according to the recommended manufacturing protocol. The genomic DNA of SGI-014-C06 was then prepared by 454-pyrosis shotgun. Genomic DNA (7.5pg) was used to build the library according to the recommended protocol (454 Life Sciences) for single long readings. The sequences were generated by two GS FLX Titanium series sequencing drives (series sequencing runs).
[00125] The sequences of four maintenance genes gyrB, rpoB, recA, and ppk of isolate SGI-014-C06 are provided in the Sequence Listing. Homology search for the determined nucleotide sequence was conducted using the DDBJ / GenBank / EMBL database. Sequence identity and similarity were also determined using GenomeQuest ^TM software (Gene-IT, Worcester Mass. USA). As discussed in detail below, the result of the sequence analysis of the maintenance genes revealed that the SGI-014-C06 isolate described in the present description is a new bacterial strain and can be considered related to the Microbacteriaceae family.
[00126] The polynucleotide sequence of the SGI-014C06 gyrB gene has the greatest sequence identity with a Microbacterium testaceum gyrB gene having GenBank accession number AP012052 (82.69% over a 936/2172 nucleotide alignment). When compared to the gyrB gene of the Microbacterium oxydans DSM 20578 strain (GenBank AM181493; Richert et al., 2007), the sequence homologies between the two genes were —62% identical
Petition 870190046231, of 5/17/2019, p. 61/90
57/75 at the nucleotide level and —37% identical at the amino acid level.
[00127] The polynucleotide sequence of the SGI-014C06 rpoB gene has the greatest sequence identity with a Microbacterium maritypicum rpoB gene having GeneBank accession number AM181582 (96.98% over a nucleotide alignment
1093/3504). When compared to the rpoB gene of the Microbacterium oxydans DSM 20578 strain (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.
[00128] The polynucleotide sequence of the recA gene of SGI-014C06 has the greatest sequence identity with a recA gene of Microbacterium testaceum having GeneBank accession number AP012052 (85.45% over a 962/1188 nucleotide alignment). When compared to the recA gene of the Microbacterium oxydans DSM 20578 strain (GenBank AM181527; 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.
[00129] The polynucleotide sequence of the ppk gene of SGI-014C06 has the greatest sequence identity with a ppk gene of Microbacterium luteolum having GenBank accession number AM181554 (91.38% over a 1380/2175 nucleotide alignment). When compared to the ppk gene of the Microbacterium oxydans DSM 20578 strain (GenBank AM181556; 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 using microbial antagonists Microbacterium sp. (NRRL B50470) Mycophaerella sp. (NRRL 50471), and Variovorax sp. (NRRL B50469)
Petition 870190046231, of 5/17/2019, p. 62/90
58/75 [00130] Microbial strains that were found positive for antibiosis in the antagonism screening described in Example 1 were further evaluated using a plant-based bioassay in which cells from the microbial strains were applied directly to seeds of a susceptible cereal crop , followed by inoculation with F. graminearum conidial spores. The microbial strains were grown at sufficient turbidity and diluted with water. Two grams of wheat seeds from a susceptible wheat crop (Hank; WestBred LLC, Bozeman, MT) were sown in one-liter pots containing pasteurized soil. After sowing, 20 mL of the microbial culture dilution was transferred on top of the sown seeds. The wheat seeds were left to germinate in greenhouse conditions under fluorescent lighting with a photoperiod of 14 hours. At the beginning of wheat flowering, the wheat heads were challenged by spraying with F. graminearum NRRL 5883 conidial spores. Conidial spores were harvested from 5-day-old PDA plates by pouring water with 0.01% Tween20 on the plates and by scraping the suspended spore. After spraying spores, wheat plants were transferred to a nebulization chamber with 100% humidity for three days to allow infection. The treatments were:
1. Untreated: challenge of any microbial or chemical fungicidal treatment + Fusarium.
2. Not infected: challenge of any microbial or chemical fungicidal treatment, without Fusarium.
3. SGI-010-H11: fungal treatment challenge + Fusarium.
4. SGI-014-G01: bacterial treatment challenge + Fusarium.
5. SGI-014-C06: bacterial treatment challenge + Fusarium.
[00131] Twenty days after infection, watering was stopped and wheat plants were left to dry for three weeks before harvesting
Petition 870190046231, of 5/17/2019, p. 63/90
59/75 ta. Individual wheat heads were harvested and collected. The severity of the disease was determined for each head showing symptoms of cereal fusarium disease. The severity of the disease was calculated with the number of sick spikelet divided by the total number of spikelet per head. The results (TABLE 2) revealed that wheat plants treated with each of the three microbial antagonists tested, SGI-014-C06 (NRRL B-50470), SGI-010-H11 (NRRL 50471), and SGI-014-G01 ( NRRL B-50469), had significantly lower severity and incidence of Fusarium graminearum infestation when compared to untreated control growth under the same conditions (P <0.05).
[00132] The seeds of each head were harvested by hand and the seed mass was weighed. The average seed weight per pot in each treatment was calculated. As reported in TABLE 3, each of the microbial antagonists has been found to significantly reduce the loss caused by cereal fusariosis infestation.
TABLE 2. Effectiveness of microbial antagonists in reducing the incidence of cereal fusariosis in wheat.
Treatment Percent Severity (%) STDEV P value Not infected 1.30 0.0025 <, 0001 Not treated 15.46 0.0364 AT 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. Effectiveness of microbial antagonists in preserving seed yield in wheat plants with cereal fusariosis disease.
Treatment Seed mass (g) P value Not infected 0.8438 ± 0.1633 0.0191
Petition 870190046231, of 5/17/2019, p. 64/90
60/75
Not treated 0.4669 ± 0.1209 AT SGI-010-H11 0.6200 ± 0.1844 0.7025 SGI-014-G01 0.7963 ± 0.2032 0.5277 SGI-014-C06 0.6744 ± 0.0948 0.0551
EXAMPLE 5: Fusarium graminearum growth inhibition by microbial antagonists in wheat seeding [00133] The microbial strains found to have suppressive activity against F. graminearum in the antagonism assay were further investigated for their ability to reduce the incidence of cereal fusariosis in the wheat. Seeds from a susceptible wheat cultivar (Hank; WestBred LLC, Bozeman, MT) were sown in 1 liter pots each containing medium of pasteurized soil in plastic pots with a diameter of 20 cm. Microbial cell suspensions were prepared as follows: 2YT, or similar growth medium, broth cultures were inoculated from isolated glycerol stocks or streak plates. Normally, cultures were started 48 - 72 hours before use in a growth chamber, greenhouse or field allowing the culture to reach a later exponential phase. Isolates that had longer doubling moments were additionally started in advance. Cultures were incubated at 30 ^° C on a 200 rpm rotary shaker. After growth, the cells were pelleted at 10,000 xg for 15 min at 4 ° C and resuspended in 10 mM MgSO4 buffer (pH 7.0). Cell densities were normalized for each isolate on a CFU / mL basis (units of cell performance per millimeter). Normally, 109 CFU / mL suspensions were prepared for each isolate and transported to the ice inoculation site. Inoculations were performed by diluting these cell suspensions 1/20 in the irrigation water to a final density 5 X 10 ⁷ CFU / mL. For 1-L pot attempts, 20 mL of this diluted cell suspension was evenly distributed over
Petition 870190046231, of 5/17/2019, p. 65/90
61/75 the surface of each replicated pot. For micro-trial pots, cell suspensions were not diluted and 1 mL of each 109 CFU / mL suspension was pipetted directly over the surface of each replica pot as a soil drench on top of submerged seeds. The seeds and plants were kept in a greenhouse effect at room temperature with a photoperiod of 14 h of light.
[00134] Mycelium strain of Fusarium graminearum NRRL-5883 was grown on PDA plates for 5 days under constant light. Hifae and conidia were collected by pouring a few mL of sterile water (0.01% Tween 20) over the plates and scraping the agar surface with a sterile spatula. The spore concentration in the inoculation was approximately 2x10 ⁵ spores / mL, but the concentration of the hyphal fragment has not been determined.
[00135] Inoculation with F. graminearum started after the shoot emerged from the seeds and continued every other afternoon for 10 days. The F. graminearum inoculum was applied with a sprayer in about 30 ml per pot. Immediately after each inoculation, the plants were sprayed with overload. When chemical fungicide was used, the fungicide (Banner Maxx, Syngenta) was prepared by diluting 2 mL of fungicide stock in 1L of distilled water and was applied with a sprayer in about 30 mL per pot.
[00136] Fusariosis of cereals was assessed by the severity of Fusarium infestation, as determined by the number of Fusarium colony forming units per gram (CFU / g) of plant tissues. All treatments were carried out in four blocks with four replicated pots. The treatments were:
[00137] 1. Untreated: no microbial or chemical fungicidal treatment, no Fusarium challenge [00138] 2. Uninfected: no microbial or chemical fungicidal treatment, no Fusarium challenge.
Petition 870190046231, of 5/17/2019, p. 66/90
62/75 [00139] 3. Fungicide: chemical fungicide + Fusarium treatment challenge.
[00140] 4. SGI-010-H11: fungal treatment challenge + Fusarium.
[00141] 5. SGI-014-G01: bacterial treatment challenge + Fusarium.
[00142] 6. SGI-014-C06: challenge of bacterial treatment + Fusarium.
[00143] The results, as reported in TABLE 4, revealed that all three microbial treatments provided a significant decrease in the severity of FHB when compared to the infected control. In particular, two microbial antagonists, SGI014-C06 and SGI-010-H11, significantly reduced the infestation of wheat plants by Fusarium graminearum when compared to the growth of the untreated control under the same conditions. The protection of wheat plants from Fusarium infestation by each of the two microorganisms SGI-014-C06 and SGI-010-H11 was statistically comparable with the protection provided by the commercial chemical fungicide. When the SGI-014-G01 microorganism was applied, the observed protection of wheat plants from Fusarium infestation was much less conspicuous and its effectiveness varied greatly among replicated pots.
TABLE 4: Effect of microbial antagonists on Fusarium infestation.
Treatment CFU / g P-value Not treated 1487 ± 600 AT Not infected 0 ± 0 <, 0001 Chemical fungicide 290 ± 146 <, 0001 SGI-010-H11 671 ± 450 0.0077 SGI-014-G01 1246 ± 465 0.8387 SGI-014-C06 367 ± 234 0.0001
EXAMPLE 6: Growth and storage of microPetition antagonists 870190046231, of 5/17/2019, p. 67/90
63/75 bianos [00144] Mycosphaerella sp .: several methods were used to store the isolated fungi as a pure culture, one of which was the filter paper technique. The fungus was also allowed to grow in PDA, and then it was cut into small squares that were placed in flasks containing 15% glycerol and stored at -70 ° C. the fungus was also stored at 4 ° C by a similar method, using distilled water other than glycerol. However, one of the preferred methods of storage was over sterile barley seed infested at -80 ° C.
[00145] Bacillus sp., Microbacterium sp. and Variovorax sp .: the isolated bacteria were stored as a pure culture. A bacterial colony was transferred to a flask containing R2A broth liquid medium (Tecknova) and allowed to grow at 30 ° C with agitation at 250 rpm for two days. The culture was then transferred into flasks containing 15% glycerol and stored at - 80 ° C.
EXAMPLE 7: Spore production and seed coating treatments [00146] Spore production: in a typical spore production procedure, one liter of 2xYT growth medium (16 g / L of Tryptone, 10 g / L of extract) yeast, 5 g / L NaCl) was inoculated with 5 ml of starter culture or scraping of petri dishes and inoculated overnight at 30 ° C on a rotary shaker that was set at 225 rpm. Bacterial cells were pelleted by centrifugation, and washed 1x with PBS buffer (8 g / L NaCl; 0.2 g / L KCI; 1.44 g / L Na2HPO4; 0.24g KH2PO4; pH 7.4) . Cells were resuspended in CDSM medium (Hageman et al., J. Bacteriol., 438-441, 1984), and were cultured for an additional four nights at 30 ° C on the rotary shaker. Sporulation in the bacterial culture was monitored daily using a phase contrast microscope until the culture interiates
Petition 870190046231, of 5/17/2019, p. 68/90
64/75 had made virtually free floating spores. The incubation time usually takes less than four days or more than six days depending on the species. Under a phase-contrast microscope, endospores were detected inside cells as bright white oblate spheroids. Bacterial spores were pelleted by centrifugation at 10,000 X g for 15 minutes, washed twice with sterile dH20 and, if necessary, concentrated to 50 mL and could either be used immediately or refrigerated for later use. Spore concentration was measured at OD600 · This procedure normally produced at least 2000 OD's of bacterial spores. [00147] In particular, several bacterial strains of the present invention, for example Bacillus sp. SGI-015-F03, could conveniently produce large amounts of spores after 4 days of incubation with a simple inoculation of a large overnight starter culture (-15 mL) in to 1 liter of 2x SG of growth medium, followed by a 4-day incubation at an appropriate temperature on a rotary shaker set at 225 rpm. The 2x SG growth medium recipe was as follows: 16 g / L of nutrient broth; 0.25 g / L MgSO4; 2.0 g / L of KCl; 0.15 g / L CaCl2.2H2O; 0.025 g / L of MnCl2.2H2O; 0.28 mg / L of FeSO4.7H2O; 1.0 g / L of Dextrose.
[00148] Seed coating treatments for wheat and corn seeds: small-scale seed treatment experiments were conducted following a procedure described in Sudisha et al., 2009 with minor modifications. Typically, a biopolymer stock solution was made by adding 6 grams of gum arabic powder (MP Biomedical) to 36 mL of water in a 50 mL Falcon tube, which was subsequently mixed for homogeneity using a wheel mixer. A shaking plate was used to mix when larger amounts of coated seeds were needed.
Petition 870190046231, of 5/17/2019, p. 69/90
65/75 [00149] When vegetative cells were used, cloudy cultures of active growth microbial cells were washed with PBS and adjusted to an OD600 of —5.0. Alternatively, microbial spore suspensions were prepared as described above. Bacterial spores and / or vegetative cells were carefully resuspended in —32 mL of Arabica gum biopolymer stock solution prepared as described above, and the resulting suspension was carefully mixed in a 1 L bottle. Approximately 400 g of seeds (either wheat or corn) were added to the bottle and shaken vigorously or vortexed to ensure uniform distribution of the gum / cell suspension. Coated seeds were then spread through sterile plastic weight boats to dry in a laminar flow hood until no longer sticky, usually 3-6 hours with periodic mixing. In some cases, seeds coated with spores could be dried overnight. However, seeds coated with vegetative cultures were usually stored outside before they were completely dehydrated. Viability test performed periodically on the microbes used in seed coating formulation showed that the microbes remained viable for at least three months. Germination rate of the coated seeds was determined to be essentially identical to the uncoated control seeds.
EXAMPLE 8: Effect of microbial seed treatments on the development of Fusarium fusariose from cereals in wheat [00150] Seeds from a wheat crop susceptible to FHB (RB07) were coated with each of the microorganisms SGI-014-C06; SGI-015-F03, and SGI-015-H06, according to the procedure described in Example 8. Coated seeds were subsequently sown in one-liter pots containing pasteurized soil medium [(Metromix-Mix 200; Scotts-Sierra Horticultural Products , Marysville, OH; and 3
Petition 870190046231, of 5/17/2019, p. 70/90
66/75 grams of fertilizer 14-14-14 (N-P-K)]. Pots were sown in 78 plants per pot and subsequently diluted to 5 plants per pot in the 3-leaf stage. Pots were arranged in randomized blocks with 5 pots per treatment. The coated seeds were then allowed to germinate under greenhouse conditions under fluorescent lighting with 16 hours of light per day. At the beginning of the wheat bloom (beforeis), the wheat heads were challenged by spraying with conidial spores of F. graminearum NRRL 5883 at a concentration of 100,000 spores / mL. After spraying spores, wheat plants were transferred to a dew chamber with 100% humidity for three days to allow infection. The treatments were:
1. Untreated: no challenge of microbial or chemical fungicide treatment + Fusarium.
2. SGI-014-C06: treatment challenge for bacterial seed + Fusarium.
3. SGI-015-F03: treatment challenge for bacterial seed + Fusarium.
4. SGI-015-H06: treatment challenge for bacterial seed + Fusarium.
[00151] Severity of cereal fusariosis disease was assessed visually at ten days and twenty one days after infection. Disease severity was determined for each peak showing symptoms of cereal fusariosis disease, and calculated as the percentage of symptomatic spikelet; that is, the number of spikelet showing symptoms of FHB divided by the total number of spikelet. The results (TABLE 5) revealed that wheat seeds coated with each of the three microbial antagonists tested, SGI-014-C06; SGI015-F03, and SGI-015-H06, had significantly lower severity of infestation by Fusarium graminearum when compared to competition 870190046231, of 17/05/2019, p. 71/90
67/75 untreated trolley growing under the same conditions (P <0.05).
Table 5: Development of symptoms of cereals fusariosis in wheat after seed treatments with antagonistic microorganisms
Treatment Severity of infection (% o)10 days after infection 21 days after infection Untreated Control 49.31 66.10 SGI-014-C06 2.08 7.74 SGI-015-F03 23.12 31.53 SGI-015-1106 9.42 19.81
EXAMPLE 9: Biocontrol of wheat cereal fusariosis disease in greenhouse trials [00152] Each of the microorganisms SGI-014-C06; SGI-015F03, and SGI-015-H06 were additionally tested in larger scale greenhouse trials. Attempts were conducted in a plant cultivation containment room (PGCR) located at Synthetic Genomics, Inc., and included the following control treatments: infected control, uninfected control, as well as four commercial fungicide comparison parameters. The chemical fungicides of comparison parameters were Banner MAXX® (Syngenta) in a concentration of 2 ml / L and ProsaroO (Bayer CropScience) in a concentration of 3 ml / L, each applied as a foliar spray, following manufacturing recommendation. The biological fungicides of evaluation parameters were Actinovate® (Natural Industries) and RhizoVital® (ABiTEP GmbH), each applied as a soil drench as per instruction manuals recommended by manufacturers. [00153] Microbial treatments were prepared and applied either as a soil drench or as a seed coating. When microbial treatments were applied as a soil drench, cell suspensions of each of the microorganisms were applied individually to the seeds before coating the seeds with more cultivation medium. For this purpose, cultures prepared
Petition 870190046231, of 5/17/2019, p. 72/90
68/75 fresh pellets were pelleted to remove the culture medium and resuspended in 10 mM magnesium sulfate buffer (MgSO4). Cell suspensions were then added by pipetting and distributing 20 milliliters evenly across the seeds in a total cell number of —109 cells per pot. When microbial treatments were applied as a seed coat, cells / spores of microorganisms were essentially prepared as described in Example 8 above. Coated seeds were produced by incorporating cell suspensions with a solution of gum arabic to form a sticky coating mixture that was subsequently applied to the seeds. The microbial seed coating was then allowed to dry to form a water-soluble biopolymer except for the hard one trapping the cells / spores of microorganisms. The objective was to achieve cell titration in the range of at least 10 ⁶ - 10 ⁷ viable cells / seeds. In this experiment to attempt the greenhouse effect, the seed coatings were prepared the day before for sowing the seeds.
[00154] Each treatment had four replicates that were positioned in a Randomized Complete Block Design (RCBD) on three level shelves arranged in two rows along the sides of a growing room. The shelves were equipped with fluorescent lights (Agrosun, 5850K) that generated an average of 800 pmole of light intensity as measured on the pot surface. Each replica consisted of four 1-liter jars contained in a flat medium. The pots were filled with a synthetic culture medium consisting of a 3: 1 (v / v) ratio of Arabidopsis AIS Culture Medium (Lehle Seeds) to sand (Washed Coarse). In each pot, two grams of spring wheat seeds (Hank, WestBred) were spread over the surface of the culture medium. The pots were watered at the bottom keeping - 2 cm of water at its base and observed for germination Petition 870190046231, from 17/05/2019, p. 73/90
69/75 tion and growth.
[00155] In the flowering stage (Feekes 10,5,1) the wheat heads were infected by spraying with a suspension of Fusarium graminearum conidial spore. For this experiment to attempt the greenhouse effect, fungal spores were prepared by placing homogenized mycelia of strain F, graminearum NRRL 5883 on large PDA plates, followed by incubation for five days under constant light at room temperature. The fungal spores were then harvested by flooding the plates with magnesium sulfate buffer (10 mM MgSO4, 0.01% Tween 20) and gently scraping the agar surface with a sterile spatula. The spore concentration was adjusted and approximately 50,000 conidial spores were applied per head with a pressurized hand sprayer. The relative humidity of the culture room was increased to 90% for three days allowing the infection to be normalized again. After the wheat seed head was adjusted, watering was stopped and the wheat heads were allowed to dry completely. Individual wheat heads were harvested and collected. Severity of disease was determined for each head of wheat showing symptoms of cereal fusarium disease. Severity of disease was calculated as the number of sick spikelet divided by the total number of spikelet per head. The results (TABLE 6) revealed that wheat treated with each of the three microbial antagonists tested, SGI014-C06; SGI-015-F03, and SGI-015-H06, had significantly lower severity and incidence of infestation by Fusarium graminearum when compared to the untreated control grown under the same conditions (P <0.05).
TABLE 6. Effectiveness of microbial antagonists in reducing the incidence of cereal fusariosis in wheat.
Petition 870190046231, of 5/17/2019, p. 74/90
70/75
Treatment Severity of infection (%) Protection from infection% Not infected 2.1 ± 2.9 AT Infected 37.2 ± 9.8 0.00 Prosario® 4.6 ± 2.8 93.00 Banner- 12.5 ± 5.8 70.00 Actinovate® 38.9 ± 12.1 -5.00 RhizoVital® 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
[00156] To determine the seed yield, wheat heads were individually threshed by hand, the seeds collected and cleaned from tares and other debris, counted and weighed. The total seed yield was determined as the total seed mass produced by 16 pots from each treatment. As reported in TABLE 7, each of the three microbial antagonists tested was found to significantly protect seed yield; that is, the loss of yield caused by cereal fusariosis infestation has been significantly reduced.
TABLE 7. Effectiveness of microbial antagonists in preserving seed yield in wheat plants with cereal fusariosis disease.
Treatment Yieldtotal seed (g) Yield protection (%) Not infected 14.36 AT Not treated 11.05 0.00 Prosario® 13.44 72.00 Banner-MAXX® 14.41 102.00 Actinovate® 11.99 28.00 RhizoVitalO 12.65 48.00
Petition 870190046231, of 5/17/2019, p. 75/90
71/75
SGI-014-C06 14.70 110.00 SGI-015-F03 15.03 120.00 SGI-015-H06 14.14 93.00
[00157] Consequently, treatments of wheat plants with each of the tested microorganisms significantly preserved the wheat plants against loss of yield caused by Fusarium infestation. In particular, wheat plants treated with either SGI-014C06 or SGI-015-F03 showed a significant improvement in total seed yield; that is, 110% and 120% respectively, when compared to the uninfected control. In contrast, the comparative assessment fungicides Actinovatee, RhizoVital® and Prosario® did not appear to significantly protect treated wheat plants from loss of yield caused by Fusarium infestation of Fusarium cereals.
EXAMPLE 10: Biocontrol of wheat mange under field conditions [00158] Microorganism antagonists that have been found to have suppressive activity against pathogen F graminearum and cereal fusariosis disease in in vitro antagonism assays and greenhouse studies, such as described in Examples 4-9, are further evaluated in a series of field experiments in different geographic locations throughout the United States. Some experiments are performed in agricultural areas that are used for microbiological control of wheat mange, where natural disease infection occurs. The wheat variety used in the test is a susceptible variety of F graminearum. Generally, wheat plants with each treatment were sown in 12-row plots, about 3 meters long. The space between rows is about 20 cm. Plots (plots) treated in each experiment are arranged in a randomized block design. The effectiveness of various humidifiable powder compositions containing the microbial antagonists of this
Petition 870190046231, of 5/17/2019, p. 76/90
72/75 invention in reducing the severity and incidence of cereal fusariosis are also evaluated. In these experiments, microbial antagonists are evaluated either individually or in combination.
[00159] In these tests, microorganism antagonists are evaluated in various seed coating treatments and / or field tests where microbial suspensions are applied directly to flowering wheat heads. In each of the experiments, the microorganisms are evaluated in replicated plots. Antagonist, pathogen and plant production methods as described in Examples 4-9 can be used in these experiments.
[00160] Seed coating treatment - In addition to the seed coating method described in Example 7 above, a variety of seed coating techniques and formulations known in the art can be employed for treating wheat seeds with microbial antagonists, such as such as those previously described by Fernando et al., 2002; Bello et al., 2002; and Kim et al., 1997. In general, wheat seeds are pre-moistened and stored at room temperature to promote germination. Germinated seeds can then be immersed inside the microbial suspensions before sowing. Pathogen spores can be inoculated either before or after bacterial inoculation. Antagonist, pathogen and plant production methods as described in examples 4 and 5 can be used for these seed treatments.
[00161] Assessment of disease severity and yield - Antagonists are subject to additional assessment in the assessment of greenhouse and field effects for their suppressive activity against cereals fusarium disease in wheat and barley in the flowering stages, using a variety of methods and procedures, typically those described in, for example, Schisler, Plant Disease, Vol
Petition 870190046231, of 5/17/2019, p. 77/90
73/75
86 (12), 1350-1356, 2002; Schisler et al. Biological control, 39: 497506, 2006; and Khan and Doohan, Biological control, 48: 42-47, 2009. In one such experiment, inoculations of G. zeae are prepared in sterile yellow toothed corn as described by Khan et al. (Biological control, 29: 245-255, 2004). Fully colonized lipid cultures (48-hour culture) of microbial strains are diluted by one-quarter strength with phosphate buffer. Total CFU counts per ml for antagonist treatments are between 1 X 10 ⁹ CFU / mL and 6 X 10 ⁹ CFU / mL. Treatment suspensions are then applied to flower wheat heads using a costal CO2 sprayer. Treatments are usually applied after the sun to minimize potential UV degradation of antagonistic cells. There are 5 replicates per treatment that are arranged in a randomized block design. The primary control treatment consists of plants treated with a buffer solution. A second control consists of untreated plants. Field evaluations of cereal fusariosis disease and incidence are made by evaluating 60 heads per replica (ie 300 heads / treatment) when the development of the plant reaches between the development of half milk and soft milk. Wheat heads are harvested by hand and threshed using a single Almaco single plant and head thresher (Almaco, IA) when grains reach full maturity. Grain samples obtained from each replicated daughter are evaluated for 100-kernel weight. Disease severity, incidence and 100-kernel weight data are analyzed using one-way analysis of variance (ANOVA).
[00162] Compositions containing the microbial antagonists of the invention, either individually or in combination, are found to significantly decrease the incidence of disease. These results show that biological control measurements using these active microbial agents play an important role in
Petition 870190046231, of 5/17/2019, p. 78/90
74/75 mange management in cereal plants such as wheat plants.
EXAMPLE 11: Development of non-naturally occurring cultivars and breeding program [00163] Endophytic microorganisms of the present invention are introduced into crop plants, including cereals, of varying genotypes and geographic origin, lacking such endophytic microorganisms, for create combinations of endophyte plants with improved agronomic characteristics, using procedures analogous to those known in the art, including those described in Ped. of Pat. US No. 20030195117A1; Ped. of Pat. US No. 20010032343A1; and Ped. of Pat. No. 7,084,331, among others. Consequently, given the synthetic plant-endophyte combinations it can be created and selected in a breeding / cultivation development program based on its ability to form and maintain a mutualistic combination that results in an agronomic benefit. Classification of the characteristics of the combination can also be used in such a breeding program. These characteristics may include, without limitation, drought tolerance, biomass accumulation, resistance to insect infestation, palatability to cattle (eg herbivores), ease of reproduction, and seed yield, among others. Such combinations may differ in levels of accumulation of microbial metabolites that are toxic to pests and weeds, including ergot alkali levels, lolina levels, peramine levels, or lolitrem levels, while showing desired agronomic characteristics of cereals, including resistance to feeding or insect infestation, resistance to abiotic tension, palatability to cattle, biomass accumulation, ease of reproduction, and seed yield, among other traits.
[00164] A number of embodiments of the invention have been described.
Petition 870190046231, of 5/17/2019, p. 79/90
75/75
However, it will be understood that elements of the modalities described here can be combined to make additional modalities and various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments, alternatives and equivalents are within the scope of the invention as described and claimed here. [00165] Titles within the application are solely for the convenience of the reader, and do not in any way limit the invention or its modalities.
[00166] All publications and patent applications mentioned in that specification are incorporated herein with reference to the same extent as if each individual publication or patent application had been specifically indicated individually to be incorporated by reference.

权利要求:
Claims (21)
[1]
1. Composition, characterized by the fact that it comprises a microbial strain or culture of the same, in which the microbial strain is selected from the group consisting of:
a) strain SGI-014-C06 of Microbacterium sp., a representative sample of it deposited as NRRL B-50470;
b) strain SGI-010-H11 of Mycosphaerella sp., a representative sample of the same deposited as NRRL 50471;
c) strain SGI-014-G01 of Variovorax sp., a representative sample of the same deposited as NRRL B-50469;
d) strain SGI-015-F03 of Bacillus sp., a representative sample of it deposited as NRRL B-50760; and
e) strain SGI-015-H06 from Bacillus sp., a representative sample of it deposited as NRRL B-50761;
the composition further comprising 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, a pesticide and a fertilizer.
[2]
2. Composition according to claim 1, characterized by the fact that the microbial strain is the strain SGI-014-C06 of Microbacterium sp., A representative sample of the same deposited as NRRL B-50470.
[3]
3. Composition according to claim 1, characterized by the fact that the microbial strain is the SGI-010-H11 strain of Mycosphaerella sp., A representative sample of it deposited as NRRL 50471.
[4]
4. Composition according to claim 1, characterized by the fact that the microbial strain is strain SGI-014-G01 of Variovorax sp., A representative sample of it deposited as NRRL B-50469.
Petition 870190046231, of 5/17/2019, p. 81/90
2/5
[5]
5. Composition according to claim 1, characterized by the fact that the microbial strain is the strain SGI-015-F03 of Bacillus sp., A representative sample of the same deposited as NRRL B-50760.
[6]
6. Composition according to claim 1, characterized by the fact that the microbial strain is the strain SGI-015-H06 of Bacillus sp., A representative sample of the same deposited as NRRL B-50761.
[7]
7. Composition according to claim 1, characterized by the fact that it comprises a vehicle.
[8]
8. Composition according to claim 1, characterized by the fact that said vehicle is an agriculturally acceptable vehicle.
[9]
9. Composition according to claim 1, characterized by the fact that said vehicle is a plant seed.
[10]
10. Composition according to claim 1, characterized by the fact that said composition is prepared as a formulation selected from the group consisting of an emulsion, a colloid, a dust, a granule, a lozenge, a powder, a spray, an emulsion and a solution.
[11]
11. Composition according to claim 1, characterized by the fact that said composition is a seed coating formulation.
[12]
12. Seed, characterized by the fact that it has a coating that comprises the composition, as defined in claim 1.
[13]
13. Method to prevent the development of cereals fusariosis, characterized by the fact that it comprises growing a microbial strain or culture of it in a cultivation medium or soil of a host plant before or competing with the growth
Petition 870190046231, of 5/17/2019, p. 82/90
3/5 of the host plant in said growth medium or soil, where the microbial strain is selected from the group consisting of:
a) strain SGI-014-C06 of Microbacterium sp., a representative sample of it deposited as NRRL B-50470;
b) strain SGI-010-H11 of Mycosphaerella sp., a representative sample of the same deposited as NRRL 50471;
c) strain SGI-014-G01 of Variovorax sp., a representative sample of the same deposited as NRRL B-50469;
d) strain SGI-015-F03 of Bacillus sp., a representative sample of it deposited as NRRL B-50760; and
e) strain SGI-015-H06 from Bacillus sp., a representative sample of it deposited as NRRL B-50761.
[14]
14. Method, according to claim 13, characterized by the fact that the disease of cereals fusariosis is caused by Fusarium graminearum.
[15]
15. Method to prevent, inhibit or treat the development of the disease of the fusariosis of cereals of a plant, characterized by the fact that it comprises applying to the plant, or the surroundings of the plant, an effective amount of a microbial strain or culture of the same, in that the microbial strain is selected from the group consisting of:
a) strain SGI-014-C06 of Microbacterium sp., a representative sample of it deposited as NRRL B-50470;
b) strain SGI-010-H11 of Mycosphaerella sp., a representative sample of the same deposited as NRRL 50471;
c) strain SGI-014-G01 of Variovorax sp., a representative sample of the same deposited as NRRL B-50469;
d) strain SGI-015-F03 of Bacillus sp., a representative sample of it deposited as NRRL B-50760; and
e) strain SGI-015-H06 from Bacillus sp., a representative sample of it deposited as NRRL B-50761.
Petition 870190046231, of 5/17/2019, p. 83/90
4/5
[16]
16. Method, according to claim 15, characterized by the fact that said microbial strain or culture thereof is applied to the soil, a seed, a root, a flower, a leaf, a portion of the plant or the entire plant.
[17]
17. Method, according to claim 15, characterized by the fact that said plant is susceptible to Fusarium graminearum.
[18]
18. Method according to claim 15, characterized in that said plant is a wheat plant, a corn plant, a barley plant, or an oat plant.
[19]
19. Method, according to claim 15, characterized by the fact that said microbial strain or culture of the same is established as an endophyte on said plant.
[20]
20. Method for preparing an agricultural composition, characterized by the fact that it comprises inoculating a microbial strain or culture of the same inside or on a substrate and allowing said microbial strain or culture to grow at a temperature of 1-37 ° C until obtaining a number of cells or spores of at least 10 ² -10 ³ per millimeter or per gram, where the microbial strain is selected from the group consisting of:
a) strain SGI-014-C06 of Microbacterium sp., a representative sample of it deposited as NRRL B-50470;
b) strain SGI-010-H11 of Mycosphaerella sp., a representative sample of it deposited as NRRL 50471;
c) strain SGI-014-G01 of Variovorax sp., a representative sample of it deposited as NRRL B-50469;
d) strain SGI-015-F03 of Bacillus sp., a representative sample of it deposited as NRRL B-50760; and
e) strain SGI-015-H06 from Bacillus sp., a representative sample of it deposited as NRRL B-50761.
Petition 870190046231, of 5/17/2019, p. 84/90
5/5
[21]
21. Composition, characterized by the fact that it comprises a microbial strain or culture thereof, where the microbial strain comprises a DNA sequence of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO : 13, or SEQ ID NO: 14; the composition further comprising 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, a pesticide and a fertilizer.

类似技术:

---

公开号 | 公开日 | 专利标题

---

AU2018267535B2|2019-10-03|Compositions and methods for controlling head blight disease

---

US11064702B2|2021-07-20|Isolated complex endophyte compositions and methods for improved plant traits

---

US11197457B2|2021-12-14|Designed complex endophyte compositions and methods for improved plant traits

---

EP1761626A2|2007-03-14|Pochonia chlamydosporia strain pcmr and method to use it in biological control of the root-knot-nematode |

---

Brownbridge et al.2006|Field application of biopolymercoated Beauveria bassiana F418 for clover root weevil | control in Waikato and Manawatu

---

Hernández-Hernández2018|Ismael Hernández-Ríos 1 Juan José Almaraz-Suarez 2 Aline López-López 3 Margarita Torres-Aquino 1 § Francisco Javier Morales Flores

---

NZ709801B2|2016-01-06|Compositions and methods for controlling head blight disease

---

NZ712059B2|2016-05-03|Compositions and methods for controlling head blight disease

---

NZ704721B2|2015-11-03|Compositions and methods for controlling head blight disease

---

NZ715042B2|2016-05-27|Compositions and methods for controlling head blight disease

---

NZ620577B2|2015-11-03|Compositions and methods for controlling head blight disease

同族专利:

---

公开号 | 公开日

---

AU2018267535A1|2018-12-06|

---

UA120152C2|2019-10-10|

---

CN103974620A|2014-08-06|

---

AU2012287003A1|2014-02-20|

---

AU2015234389A1|2015-10-29|

---

AU2016259414A1|2016-12-08|

---

AU2017248427B2|2018-09-13|

---

RU2014106863A|2015-08-27|

---

NZ712059A|2016-01-29|

---

US20190364906A1|2019-12-05|

---

WO2013016361A3|2014-02-27|

---

NZ704721A|2015-07-31|

---

UA118331C2|2019-01-10|

---

WO2013016361A2|2013-01-31|

---

NZ715042A|2016-02-26|

---

BR112014001815A2|2017-02-21|

---

AU2016259414B2|2018-05-24|

---

EP2736341B1|2019-01-16|

---

US10405554B2|2019-09-10|

---

AU2017248427A1|2017-11-02|

---

PL2736341T4|2019-08-30|

---

RU2705286C2|2019-11-06|

---

CA2842793A1|2013-01-31|

---

AU2015234389B2|2016-11-17|

---

EP2736341A2|2014-06-04|

---

EP2736341A4|2014-12-24|

---

RU2017129695A|2019-02-04|

---

JP6009564B2|2016-10-19|

---

CO6880050A2|2014-02-28|

---

RU2634386C2|2017-10-26|

---

AU2012287003B2|2015-09-10|

---

MX2014001076A|2014-02-27|

---

MX351864B|2017-10-30|

---

RU2736382C1|2020-11-16|

---

US20170094978A1|2017-04-06|

---

AU2018267535B2|2019-10-03|

---

CN103974620B|2016-11-23|

---

JP2014531893A|2014-12-04|

---

PL2736341T3|2019-08-30|

---

US20130031673A1|2013-01-31|

---

NZ620577A|2015-07-31|

---

ZA201400587B|2014-11-26|

---

US10412971B2|2019-09-17|

---

NZ709801A|2015-09-25|

---

MX368976B|2019-10-23|

---

RU2017129695A3|2019-02-04|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

CA1335364C|1986-12-17|1995-04-25|Ran Lifshitz|Beneficial, direct-acting soil bacteria|

---

US5491077A|1994-07-20|1996-02-13|Merck & Co., Inc.|Microbial method|

---

RU2170511C2|1999-01-10|2001-07-20|Государственный научный центр прикладной микробиологии|Preparation for protection of plants against diseases|

---

US6312940B1|1999-10-07|2001-11-06|The United States Of America As Represented By The Secretary Of Agriculture|Bacillus species for reducing fusarium head blight in cereals|

---

CA2323019C|1999-10-07|2011-03-08|The United States Of America, As Represented By The Secretary Of Agricul Ture|Bacteria and yeasts for reducing fusarium head blight in cereals and selection thereof|

---

BR0009629A|2000-12-08|2002-08-20|Embrapa - Empresa Brasileira De Pesquisa Agropecua|Biocontrol of plant diseases caused by fusarium species with new isolates of bacillus megaterium and pantoea agglomerans|

---

KR100431613B1|2001-05-26|2004-05-17|이기성|THE NOVEL ANTIFUNGAL BACTERIA Bacillus subtilis EBM 31 AND THE BIOPESTICIDES CONTAINING THEREOF|

---

FI114080B|2001-06-21|2004-08-13|Kemira Agro Oy|Surface coated seed and method of coating a seed|

---

JP4313980B2|2002-04-10|2009-08-12|社団法人農林水産先端技術産業振興センター|A method for controlling diseases and pests of gramineous plants using symbiotic fungi, seeds combined with control agents and control agents|

---

RU2235771C2|2002-05-13|2004-09-10|Государственное унитарное предприятие "Государственный научный центр прикладной микробиологии"|Strain of bacterium pseudomonas fluorescens p469 for preparing preparation against plant diseases caused by phytopathogenic fungi and microorganisms|

---

JP3776919B2|2004-02-27|2006-05-24|株式会社イツキ|Plant disease control method and control agent using Bacillus bacteria|

---

RU2284353C1|2005-05-05|2006-09-27|Общество с ограниченной ответственностью "БИО-АГРО"|Strain of microorganism bacillus mycoides var. b. a. for preparing biopreparation used in stimulation of plant growth and protection against diseases|

---

CN100334201C|2005-09-23|2007-08-29|中国农业大学|Bacillus subtilis and its uses|

---

US7601346B1|2005-12-28|2009-10-13|The United States Of America, As Represented By The Secretary Of Agriculture|Choline-utilizing microbial strains for biologically controlling fusarium head blight|

---

JP2010539213A|2007-09-20|2010-12-16|ビーエーエスエフソシエタス・ヨーロピア|Combinations containing bactericidal strains and active ingredients|

---

EA027649B1|2009-01-26|2017-08-31|Зингента Кроп Протекшн Аг|Pasteuria sp. strain for protection of crops against nematodes, composition and method of protection crops against nematodes|

---

JP2011102278A|2009-11-12|2011-05-26|Central Glass Co Ltd|Method for controlling brassicaceous plant disease injury|

---

CN101857848B|2010-05-10|2012-06-20|华中农业大学|Biocontrol bacillus subtilis WJ-1 for rice sheath blight, microbial inoculum and application thereof|

---

CN101993836B|2010-06-13|2012-05-23|河南省农业科学院|Bacillus subtilis strain YB-81, fungicide and preparation method and application thereof|

---

AU2012287003B2|2011-07-25|2015-09-10|Monsanto Technology, Llc|Compositions and methods for controlling head blight disease|AU2012287003B2|2011-07-25|2015-09-10|Monsanto Technology, Llc|Compositions and methods for controlling head blight disease|

---

EP2676536A1|2012-06-22|2013-12-25|AIT Austrian Institute of Technology GmbH|Method for producing plant seed containing endophytic micro-organisms|

---

CN104718289A|2012-10-17|2015-06-17|诺维信公司|Method for production of brewers wort|

---

MX368358B|2013-02-05|2019-09-30|Univ Saskatchewan|Endophytic microbial symbionts in plant prenatal care.|

---

JP2016512043A|2013-03-15|2016-04-25|オハイオ・ステイト・イノベーション・ファウンデーション|Use of Cryptococcus flavesens strains for biological control of head blight|

---

EP3013134B1|2013-06-26|2020-11-25|Indigo AG, Inc.|Seed-origin endophyte populations, compositions, and methods of use|

---

US10136646B2|2013-06-26|2018-11-27|Indigo Ag, Inc.|Agricultural endophyte-plant compositions, and methods of use|

---

WO2015100432A2|2013-12-24|2015-07-02|Symbiota, Inc.|Method for propagating microorganisms within plant bioreactors and stably storing microorganisms within agricultural seeds|

---

EP3659414A1|2013-09-04|2020-06-03|Indigo Ag, Inc.|Agricultural endophyte-plant compositions, and methods of use|

---

EP3161124B1|2014-06-26|2020-06-03|Indigo Ag, Inc.|Endophytes, associated compositions, and methods of use thereof|

---

HUE048814T2|2013-11-06|2020-08-28|Texas A & M Univ Sys|Fungal endophytes for improved crop yields and protection from pests|

---

CN103849617A|2013-12-16|2014-06-11|复旦大学|Connector and method for Permanent preservation of genome DNA |

---

WO2015100431A2|2013-12-24|2015-07-02|Symbiota, Inc.|Plants containing beneficial endophytes|

---

MX367032B|2014-06-20|2019-08-02|The Flinders Univ Of South Australia|Inoculants and methods for use thereof.|

---

EP3240391A4|2014-12-30|2018-07-11|Indigo Agriculture, Inc.|Seed endophytes across cultivars and species, associated compositions, and methods of use thereof|

---

US9364005B2|2014-06-26|2016-06-14|Ait Austrian Institute Of Technology Gmbh|Plant-endophyte combinations and uses therefor|

---

RU2576197C1|2014-12-25|2016-02-27|Общество с ограниченной ответственностью "Научно-производственное объединение АРГО ЭМ-1"|Method for producing composition for growing plants|

---

EP3240403B1|2014-12-29|2019-11-13|FMC Corporation|Microbial compositions and methods of use for benefiting plant growth and treating plant disease|

---

WO2016178086A1|2015-05-01|2016-11-10|Inocucor Technologies, Inc.|Microbial compositions and methods for bioprotection|

---

EP3288361A4|2015-05-01|2018-09-19|Indigo Agriculture, Inc.|Isolated complex endophyte compositions and methods for improved plant traits|

---

AR105315A1|2015-05-01|2017-09-27|Indigo Agriculture Inc|COMPLEX COMPOSITIONS OF DESIGN ENDOPHYTS AND METHODS FOR IMPROVED CHARACTERS IN PLANTS|

---

RU2017146713A|2015-06-08|2019-07-15|Индиго Аг, Инк.|ENDOPHITE COMPOSITIONS WITH STREPTOMYCES AND METHODS FOR IMPROVING AGRONOMIC CHARACTERISTICS IN PLANTS|

---

WO2017019726A1|2015-07-29|2017-02-02|Merck Sharp & Dohme Corp.|Oxy-cyanoquinolinone pde9 inhibitors|

---

WO2017019724A1|2015-07-29|2017-02-02|Merck Sharp & Dohme Corp.|Phenyl-cyanoquinolinone pde9 inhibitors|

---

WO2017019723A1|2015-07-29|2017-02-02|Merck Sharp & Dohme Corp.|Aza-cyanoquinolinone pde9 inhibitors|

---

CN105462890B|2015-12-23|2019-01-01|广西大学|Application of the sophora tonkinensis Gapnep endogenetic bacteria B22 in prevention and treatment notoginseng root rot|

---

CN106116925A|2016-06-29|2016-11-16|李怀峰|A kind of functional type nutrition fertilizer and preparation method thereof|

---

WO2018017106A1|2016-07-21|2018-01-25|Rutgers, The State University Of New Jersey|Endophytic bacterium for application to grasses to increase plant growth|

---

EP3311669B1|2016-10-21|2020-06-10|DCM De Ceuster Meststoffen NV|Novel plant-growth promoting bacteria and the use thereof|

---

US10624351B2|2016-12-01|2020-04-21|Indigo Ag, Inc.|Modulated nutritional quality traits in seeds|

---

MX2019010349A|2017-03-01|2019-11-21|Indigo Ag Inc|Endophyte compositions and methods for improvement of plant traits.|

---

BR112019018232A2|2017-03-01|2020-07-28|Indigo Ag, Inc.|endophytic compositions and methods for improving plant traits|

---

FR3068470B1|2017-07-03|2022-01-28|Inoventeam|SEED MARKING METHOD|

---

US11263707B2|2017-08-08|2022-03-01|Indigo Ag, Inc.|Machine learning in agricultural planting, growing, and harvesting contexts|

---

CN107299069B|2017-08-09|2021-08-03|郑州大学|Agricultural microbial preparation and application thereof in preventing and treating root-knot nematodes of melons and watermelon fusarium wilt|

---

CN108823126B|2018-04-12|2020-01-14|黄山学院|Polygonatum sibiricum endophyte and application thereof|

---

CN108378063A|2018-05-18|2018-08-10|中国农业科学院农业环境与可持续发展研究所|A kind of preparation method of muscardine granule|

---

JP2021530245A|2018-07-25|2021-11-11|リージェンツ オブ ザ ユニバーシティ オブ ミネソタ|Platform for developing a soil-borne phytopathogen-inhibiting microbial consortia|

---

CN109182189B|2018-09-20|2020-10-09|宁国市百立德生物科技有限公司|High-yield microbacterium oxydans and application thereof|

---

US20220073968A1|2019-02-08|2022-03-10|Newleaf Symbiotics, Inc.|Methods for identifying microbial strains having enhanced plant colonization efficiency|

---

WO2020170244A1|2019-02-20|2020-08-27|Taxon Biosciences Inc.|Plant growth-promoting microbes, compositions, and uses thereof|

---

BR112021022596A2|2019-05-24|2022-01-04|Danstar Ferment Ag|Methods for increasing the germination rate of a microbial spore, for controlling a plant pathogen, for increasing the effectiveness of a biological agent, for manufacturing a composition, composition and use|

---

CN110229766B|2019-06-14|2021-06-08|浙江工业大学|Microbacterium oxydans and application thereof in degradation of organic pollutants|

法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2018-08-28| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

---

2018-09-04| B15K| Others concerning applications: alteration of classification|Free format text: AS CLASSIFICACOES ANTERIORES ERAM: A01N 63/00 , C12N 1/20 , C12N 1/21 , C05G 3/02 , A01C 1/06 , A01P 7/02 , A01P 1/00 , A01P 3/00 , A01P 7/04 , A01P 5/00 , A01H 5/00 , A01H 5/10 , C12N 5/04 Ipc: A01N 63/00 (1980.01), C12N 1/20 (1980.01), C05G 3/ |

---

2019-02-19| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

---

2019-08-06| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

优先权:

---

申请号 | 申请日 | 专利标题

---

US201161511467P| true| 2011-07-25|2011-07-25|

---

US61/511,467|2011-07-25|

---

PCT/US2012/048012|WO2013016361A2|2011-07-25|2012-07-24|Compositions and methods for controlling head blight disease|

---