A composition comprising plant growth promoting microbes and methods for treating a plant seed and f

专利摘要:
abstract microbial strains, compositions, and methods of use thereof to enhance growth and / or yield of a plant are provided. also provided are materials and methods for presenting, inhibiting, or treating the development of plant pathogens or phytopathogenic diseases. the disclosure also provides non-naturally occurring plant and derivatives thereof such as artificially infected plants with a microbial strain of the invention. plant growth promoting microbes and uses thereof are presented microbial strains, compositions and their methods of use to improve the growth and / or yield of a plant. Materials and methods for presenting, inhibiting or treating the development of plant pathogens or phytopathogenic diseases are also provided. The disclosure also provides non-naturally occurring plants and their derivatives, such as plants artificially infected with a microbial strain of the invention.
公开号:BR112014014000B1
申请号:R112014014000-6
申请日:2012-12-13
公开日:2019-05-28
发明作者:David T. Bullis；Christopher J. Grandlic；Ryan McCann；Janne S. Kerovuo
申请人:Monsanto Technology Llc；
IPC主号:

专利说明:

DESCRIPTION REPORT OF THE PATENT FOR COMPOSITION UNDERSTANDING MICROBES THAT PROMOTES PLANT GROWTH AND METHODS FOR TREATING A PLANT SEED AND TO IMPROVE GROWTH AND / OR YIELD OF A PLANT.
[001] This patent application claims the benefit of U.S. Provisional Application No. 61 / 570,237 filed on December 13, 2011 and which is incorporated by reference in its entirety, including all tables, figures and claims.
FIELD OF THE INVENTION [002] The present invention relates to the field of sustainable agriculture. Specifically, the description provides microbial compositions and methods useful for the production of plant species. In particular, the compositions and methods disclosed in this document are useful for improving plant growth and / or suppressing the development of plant pathogens and pathogenic diseases.
SEQUENCE LISTING INCORPORATION [003] The material in the accompanying sequence listings is incorporated herein by reference in this patent. The accompanying file, called SGI1540_1WO_CRF_OF_SL_ST25.txt was created on December 13, 2012 and is 20 KB. The files can be accessed using Microsoft Word on a computer using the Windows operating system.
BACKGROUND OF THE INVENTION [004] The microflora around plants is very diverse, including bacteria, fungi, yeasts, algae. Some of these microorganisms can be harmful to plants and are often referred to as pathogens, while others can be beneficial to plants, promoting plant growth and harvest productivity. Recent advances in soil microbiology and
Following is sheet 1a / 83
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1a / 83 plant biotechnology resulted in increased interest in the use
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2/83 environmental management. In particular, a number of microorganisms known to be present in the ecological niche of soil, generally known as rhizosphere and rhizoplane have received considerable attention with regard to their ability to promote plant growth. In fact, the rhizosphere soil represents a good reservoir of microbes for the potential isolation of beneficial microbes. The plant's rhizosphere can contain billions of microorganisms in one gram of soil. In theory, microbial inoculants, without human intervention, have a low rate of survival and effectiveness in their natural soil environment because of insufficient colony formation units per gram of soil. Therefore, since the 1960s, a number of biofertilizers that have an increased concentration of potential colony inoculum have been developed and marketed in an attempt to reduce the need for chemical fertilizers.
[005] In addition, research carried out in recent years has shown that microorganisms can be used as biological control agents to increase efficiency and agricultural productivity. These studies have shown that several microorganisms are capable of suppressing plant pathogens and / or complementing plant growth, thus offering an attractive alternative to chemical pesticides that are less favored because of their potentially negative impact on the quality of human health and the environment. environment.
[006] Microorganisms that can colonize plant roots and stimulate plant growth are generally known as plant growth promoting microbes (PGPM). In the past two decades, many species of PGPM having a positive influence on the growth of a wide variety of plant cultures have been reported. PGPM are often
3/83 universal symbionts of higher plants and are capable of increasing the adaptive potential of their hosts through a series of mechanisms, such as the fixation of molecular nitrogen, the mobilization of recalcitrant nutrients from the soil (for example, iron, phosphorus, sulfur etc.), the synthesis of phytohormones and vitamins and the decomposition of plant materials in soils that often increases the organic matter of the soil. Also, certain microbes can facilitate plant growth through microbial control of species pathogenic to the plant (ie, phytopathogens). For example, some beneficial microbes can control root rot in plants competing with fungi for space on the root surface of the plant. In other cases, competition between various microbial strains in the plant's native microflora can stimulate root growth and increase the absorption of mineral nutrients and water to increase plant yield. Therefore, biofertilizers can be developed as products based on microorganisms that live naturally in the soil. By increasing the population of beneficial microorganisms in the soil through artificial inoculation, these soil microorganisms can increase their biological activity and thus provide plants with important nutrients and beneficial factors that improve their growth.
[007] The inoculation of plants grown with PGPM is generally seen as a promising agricultural approach, as this allows pests to be controlled without the use of pesticides in large quantities. As environmental concerns about groundwater quality, with excessive exposure to fertilizers and pesticides in food cultivation, biological alternatives are becoming necessary. Thus, developing biological treatment compatible with fertilizers and pesticides, or even
4/83 reducing the amount of these chemical compounds could be a significant advance in the agricultural industry. It has been established that stimulation of plant growth by PGPM is often closely related to the ability of PGPM to colonize plant roots. However, relatively little attention has been paid to the development of efficient selection procedures for obtaining microbial strains with a high capacity for root colonization. The lack of such selection procedures delays the study of plant bacterial symbiosis and the implementation of PGPM in agriculture.
[008] Therefore, there is a permanent need for the identification of new PGPM and / or testing its compatibility with commercially available plantation management products. In addition, further research is also needed to compare strains of pure culture versus complementary mixed strains of microorganisms that form synergistic consortia. Such mixed consortia may have greater potential for consistent performance with better competitive capacity under different environmental and growth conditions.
SUMMARY OF THE INVENTION [009] Microbial cultures and strains are provided in this document. Microbial compositions and methods of use to improve the growth and / or yield of a plant are also provided. Methods for treating plant seeds using the microbial compositions disclosed in this document are also provided. Methods for preventing, inhibiting or treating the development of plant pathogens or the development of phytopathogenic diseases are still provided. The description also provides varieties of non-naturally occurring plants that are artificially infected with a microbial endophyte of the invention.
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Seed, reproductive tissue, vegetative tissue, regenerative tissue, plant parts or the offspring of unnatural plant varieties are also provided. The description further provides a method for preparing agricultural compositions.
[0010] In one aspect, the present description provides isolated microbial strains, respective isolated cultures, respective biologically pure cultures and respective enriched cultures. In certain preferred embodiments of this aspect, the microbial strain can be SGI-003-H11 (deposited as NRRL B-50483); SGI-020-A01 (deposited as NRRL B-50484); SGI-026-G06 (deposited as NRRL B-50485); SGI-026-G07 (deposited as NRRL B-50486), or a strain derived from any of the strains mentioned. In some other preferred embodiments, the microbial strain may comprise a nucleotide or amino acid sequence that exhibits at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or at least one identity sequence 99.5% of any of the 16S ribosomal / recA nucleotide sequences or amino acids in the Sequence Listing. In some modalities of the microbial strain there is also an activity to promote plant growth as described in this document.
[0011] Microbial compositions that include a microbial strain of the invention or a respective culture are also provided. Such microbial compositions according to some preferred embodiments may include an agronomically effective amount of an additional compound or composition, in which the additional compound or composition may be a fertilizer, an acaricide, a bactericide, fungicide, insecticide, a microbicide,
6/83 a nematicide or a pesticide. In some other preferred embodiments, the microbial compositions may further include a carrier. In still other preferred embodiments, the carrier can be a plant seed. In certain embodiments of this aspect, the microbial composition is prepared as a formulation which can be an emulsion, a colloid, a powder, a granule, a pellet, a powder, a spray, an emulsion or a solution. In some other preferred embodiments, the microbial compositions can be seed coating formulations. In another aspect, plant seeds that are coated with a microbial composition in accordance with the present invention are also provided.
[0012] In another aspect, methods are provided for the treatment of plant seeds. Such methods include exposing or contacting the seeds of the plant with a microbial strain according to the present invention or a respective culture.
[0013] In another aspect of the invention, methods are provided here to improve the growth and / or yield of a plant. In some embodiments, such a method involves the application of an effective amount of a microbial strain in accordance with the present invention or a respective culture for the plant, or the surroundings of the plant. In some other embodiments, the method involves the growth of a microbial strain in accordance with the present invention or even a culture, in a growth medium or the soil of a host plant before or simultaneously with the growth of host plants in said culture medium or soil. In preferred embodiments, the plant can be a corn plant or a wheat plant. In some other modalities, the microbial strain or respective culture can be
7/83 established as an endophyte in the plant.
[0014] In another aspect of the present invention, methods are provided for preventing, inhibiting or treating the development of a plant pathogen. Such methods include the cultivation of a microbial strain according to the invention or a culture thereof, in a culture medium or the soil of a host plant before or concomitant with the growth of host plants in said culture medium or soil. In some preferred embodiments, the plant pathogen may be a microorganism of the genus Colletotrichum, Fusarium, Gibberella, Monographella, Penicillium, or Stagnospora. In some particularly preferred embodiments, the plant pathogen can be Colletotrichum gramnicola, Fusarium graminearum, Gibberella zeae, Monographella nivalis, Penicillium sp., Or Stagnospora nodurum.
[0015] Yet another aspect of the invention provides methods for preventing, inhibiting or treating the development of plant pathogenic plant diseases. Such methods include applying to the plant, or to the surroundings of the plant, an effective amount of a microbial strain according to the invention or a respective culture. In some preferred embodiments, the microbial strain or a respective crop can be applied to the soil, a seed, a root, a flower, a leaf, a part of the plant, or the entire plant.
[0016] Another 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 respective culture. They are also provided in some modalities of this aspect: seed, reproductive tissue, vegetative tissue, regenerative tissues, parts of the plant and the offspring of non-naturally occurring plants.
[0017] Another aspect of the invention provides methods for
8/83 preparation of an agricultural composition. Such methods involve inoculating the microbial strain according to the present invention or a respective culture inside or to a substrate and allowing it to grow.
[0018] In another aspect of the invention it provides an isolated strain, a respective isolated culture, a respective biologically pure culture and an enriched culture of a microorganism of the genus Pantoea. In one embodiment of the microorganism it comprises a DNA sequence or amino acid sequence that codes for a 16S rRNA gene or a recA protein, having at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or at least 99.5% of sequence identity for an encoding of sequence of the 16S rRNA gene or recA protein disclosed in the Sequence Listing. In another embodiment, the invention provides a genus of microorganisms that comprises any DNA sequence or amino acid sequence described above and that increases the growth and / or yield of a plant, as described in this document.
[0019] These and other objects and resources of the invention will become more fully apparent from the following detailed description of the invention and the claims.
DETAILED DESCRIPTION OF THE INVENTION [0020] Unless otherwise defined, all terms of the technique, notations and other scientific terms or terminology used in this document 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 in this document for purposes of
9/83 clarification and / or for immediate reference, and the inclusion of such definitions here should not necessarily be construed to represent a substantial difference in what is generally understood in the art. Many of the techniques and procedures described or mentioned in this document are well understood and commonly used using conventional methodology by those skilled in the art.
[0021] The singular form one, one and a include plural references, unless the context clearly expresses the opposite. For example, the term a cell includes one or more cells, including mixtures thereof.
[0022] Bactericidal: the term bactericidal, as used here, refers to the ability of a substance to increase mortality or inhibit the growth of bacteria or composition.
[0023] Biological control: the term biological control and its abbreviated form biocontrol, in this document, is defined as the control of a pathogen or insect or any other undesirable organism by using at least one second organism other than man. An example of known mechanisms of biological control is the use of microorganisms that control root rot by fungi competing with fungi for space on the root surface, or microorganisms that inhibit growth 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 isolating an organism, such as a bacterium or fungus species, from its natural environment, the host plant is a plant that supports the growth of the bacterium or fungus, for example, a plant of a species of bacterium or fungus is a endophyte.
[0024] An effective amount, as used here, is an amount sufficient for beneficial or desired effect results.
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An effective amount can be administered in one or more administrations. In terms of treatment, inhibition or protection, an effective amount is that amount sufficient to improve, stabilize, reverse, delay or delay the progression of the target infection from disease states. The term effective microorganism used here in reference to a microorganism is intended to mean that the strain of the object has a degree of promoting plant growth and / or yield or a degree of inhibition of pathogenic diseases that exceeds, in a statistically significant level, or an untreated control. In some cases, the term an effective amount is used in this document in reference to that amount of microbial treatment that is necessary to obtain a beneficial or desired result over what occurs in an untreated control under appropriate treatment conditions as described in this document. . For the purposes of the present description, the actual application rate of a liquid formulation will generally range from a minimum of approximately 1 and 10 ³ to approximately 1 and 10 ¹⁰ viable cells / mL and, preferably, from approximately 1 and 10 ⁶ to approximately 5 and 10 ⁹ viable cells / mL. In most conditions, the strains of the invention described in the examples below would be optimally effective at application rates in the range of approximately 1 and 10 ⁶ to 1 and 10 ⁹ viable cells / mL, assuming a mode of application that would achieve substantially uniform contact of at least 50% of the plant's tissues. If the microorganisms are applied as a solid formulation, the application rate must be controlled to result in a comparable number of viable cells per unit surface area of the plant tissue as obtained by the liquid treatment rates mentioned above. In general, the microbial compositions of the present invention are biologically effective when delivered in
11/83 concentrations greater than 10 ⁶ CFU / g (colony forming units per gram), preferably greater than 10 ⁷ CFU / g, more preferably 10 ⁸ CFU / g, and more, preferably 10 ¹⁰ CFU / g .
[0025] Composition: A composition is intended to mean a combination of active agent and at least one other compound, carrier or composition, which can be inert (for example, a detectable or labeling agent or liquid carrier) or active, such as a fertilizer.
[0026] The control plant, as used in this description, provides a reference point for measuring changes in the phenotype of the plant object, it can be any suitable plant cell, seed, plant component, plant tissue, plant organ or all plant. A control plant may include, for example, (a) a wild-type plant or a cell, that is, with the same genotype as the raw material for the genetic alteration that resulted in the plant or cell object; (b) a plant or cell of the genotype as a primary material, but which has been transformed with a null construct (i.e., a construct that has no known effect on the characteristic of interest, such as a construct comprising a reporter gene); (c) a plant or cell that is an unprocessed segregant between the descendants of a plant or cell object; (d) a plant or cell that is genetically identical to the plant or cell object, but which is not exposed to the same regime (ie, fertilizer treatment) as the plant or cell object; (e) the plant or cell object itself, under conditions where the gene of interest is not expressed; or (f) the plant object or the cell itself, under conditions where it has not been exposed to a specific treatment, such as a fertilizer or combination of fertilizers and / or other chemicals.
[0027] Culture, isolated culture, biologically pure culture and
12/83 enriched culture: As used here, a strain isolated from a microbe is a strain that has been removed from its natural environment. As such, the term isolated does not necessarily reflect the extent to which the microbe has been purified. But in different modalities an isolated culture has been purified at least 2x or 5x or 10x or 50x or 100x from the raw material from which it is isolated. As an example of non-limitation, if a crop is isolated from the soil as a raw material, the organism can be isolated so that its concentration in a certain amount of purified or partially purified material (eg soil) is at least 2x or 5x or 10x or 50x or 100x that, in original raw material. A substantially pure culture of the microbe strain refers to a culture that contains substantially no other microbes than that of the desired strain or strain of the microbes. In other words, a substantially pure culture of a strain of the microbe is substantially free of other contaminants, which may include microbial contaminants, as well as unwanted chemical contaminants. Furthermore, as used here, a biologically pure strain is intended to mean the separate stress of 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. In different modalities a biologically pure culture has been purified at least 2x or 5x or 10x or 50x or 100x from the material with which it is normally associated in nature. As a non-limiting example, if a crop is normally associated with soil in nature, the organism can be biologically pure so that its concentration in a certain amount of purified or partially purified material
Purified 13/83 with which it is normally associated in nature (for example, soil) is at least 2x or 5x or 10x or 50x or 100x than in the original impure material. As used here, the term enriched culture of an isolated microbial strain refers to a microbial culture in which the total microbial population of the culture contains more than 50%, 60%, 70%, 80%, 90% or 95% strain isolated.
[0028] Cultivation: The term 'cultivation,' as used here, refers to the spread of organisms on or in media of various types.
[0029] As used in this document, an endophyte is an endosymbiont that lives within a plant for at least part of its life without causing apparent disease. Endophytes can be transmitted either vertically (directly from parent to child) or horizontally (from individual to independent individual). Vertically transmissible fungal endophytes are normally asexual and transmit from the maternal plant to the offspring through fungal hyphae penetrating the host's seeds. Endophyte bacteria can also be transferred vertically from seeds to seedlings (Ferreira et al., FEMS Microbiol. Lett. 287: 8-14, 2008). On the other hand, horizontally transmitted endophytes are typically sexual and transmitted through spores that can be spread by the wind and / or insect vectors. Endophyte microbes from crop plants have received considerable attention with regard to their ability to control disease and insect infestation, as well as their potential to promote plant growth.
[0030] Fungal pathogen: For the purposes of the present invention it is understood that the use of the term fungal pathogen or fungus is intended to include both the sexual (teleomorphic) stage of this organism and also the asexual (anamorphic) stage, also known as the fungal stages perfect and imperfect, respectively. For example, the anamorphic phase of Fusarium graminearum is Gibberella zeae.
14/83 [0031] Fungicide: As used here, fungicide refers to the ability of a composition or substance to decrease the growth rate of fungi or increase the death rate of fungi.
[0032] 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 Burkholderia the parental strain is defined here as the original Burkholderia strain before mutagenesis. Mutants or variants can occur in nature without human intervention. They are also obtained 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-N-nitrosoguanidine, ethylmethanesulfone, or by irradiation using gamma irradiation, x-ray or UV irradiation or by other means known to those skilled in the art.
[0033] Nematicide: the term nematicide, as used here, refers to the ability of a substance to increase mortality or inhibit the growth of bacteria or nematodes.
[0034] Pathogen: The term pathogen in this document refers to an organism such as algae, arachnid, bacteria, fungus, insect, nematode, parasitic plant, protozoan, yeast or virus capable of producing a disease in a plant or animal. The term phytopathogen in this document refers to a pathogen that infects a plant.
[0035] Percentage of sequence identity: the percentage of sequence identity, as used here, is determined by comparing two sequences aligned locally ideally over a comparison window defined by the length of the alignment
15/83 location between the two strings. The amino acid sequence in the comparison window can comprise additions or deletions (eg, gaps or bumps) in comparison to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The local alignment between two sequences includes only the segments of each sequence that are considered sufficiently similar according to a criterion that varies according to the algorithm used to perform the alignment (eg BLAST). The percentage of sequence identity is calculated by determining the number of positions in which the identical nucleic acid base or amino acid residue occurs in both sequences to produce the number of corresponding positions, dividing the number of corresponding positions by the total number of positions in the comparison window and multiply the result by 100. The ideal sequence alignment for comparison can be achieved by the local homology algorithm of Smith and Waterman (1981) Add.APL.Math.2: 482, by the global homology alignment algorithm Needleman and Wunsch (J Mol. Biol. 48: 443, 1970), for the search for Pearson and Lipman's similarity method (Proc. Natl. Acad. Sci. USA 85: 2444, 1988), for heuristic implementations of these algorithms ( NCBI BLAST, WU-BLAST, BLAT, YES, BLASTZ), or by inspection. Since two sequences have been identified by comparison, GAP and BESTFIT are preferably employed to determine the optimal alignment. Typically, default values of 5.00 for gap weight and 0.30 for gap weight length are used. The term substantial sequence identity between polynucleotides or polynucleotide sequences refers to the polynucleotide or polypeptide comprising a sequence of at least 50% sequence identity, preferably at least 70%, preferably at least 80%, more preferably at least
16/83 minus 85%, more preferably at least 90%, even more, preferably at least 95%, more preferably at least 96%, 97%, 98% or 99% of the identity sequence and compared to a sequence of reference using the programs. In addition, paired sequence homology or sequence similarity, as used, refers to the percentage of residues that are similar between two aligned sequences. Amino acid residue families that have side chains similar to amino acid residues have been well defined in the art. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine , threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and side chains aromatic (eg tyrosine, phenylalanine, tryptophan, histidine).
[0036] Query amino acid and nucleic acid sequences can be searched in the nucleic acid or amino acid sequences of the object that is residing in the public or exclusive databases. Such searches can be done using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST v 2.18). The NCBI BLAST program is available on the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov/Blast.cgi). Typically, the following NCBI BLAST parameters can be used: Filter options set to default, the Comparison Matrix set to BLOSUM62, the Gap Costs (gap) set to Existence: 11, extent: 1, the word size set to 3, the
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Expected (threshold E) set to 1e-3 and the minimum length of the local alignment set to 50% of the query string length. Sequence of identity and similarity can also be determined using the GenomeQuest ^TM software (Gene-IT, Worcester Massachusetts USA).
[0037] The term pests as used in this document refers to an undesirable organism that may include, but is not limited to, bacteria, fungi, plants (for example, weeds), nematodes, insects and other pathogenic animals. Pesticide, as used here, refers to the ability of a substance or composition to slow the growth rate of a pest, that is, an undesirable organism, or to increase the mortality rate of a pest.
[0038] Offspring: As used here, offspring includes descendants of a particular plant or plant lineage. The progeny of a current plant includes seeds formed in F ₁ , F ₂ , F ₃ , F ₄ , F ₅ , F ₆ and subsequent generations of plants, or seeds formed in BC1, BC2, BC3, and subsequent generations of plants, or seeds formed in F ₁ BC ₁ , F ₁ BC ₂ , F ₁ BC ₃ , and subsequent generations of plants. The designation F1 refers to the offspring of a cross between two parents who are genetically distinct. The designations F2, F3, F4, F5 and F6 refer to the subsequent generations of offspring by auto- or sibpolinization of an F1 plant.
[0039] Variant: as used here in reference to a nucleic acid and polypeptide, the term variant is used in this document to denote a polypeptide, protein molecule or polynucleotide with some differences, generated synthetically or naturally, in its amino acids or sequences of nucleic acids compared to a reference polypeptide or
18/83 polynucleotide, respectively. For example, these differences include substitutions, insertions, deletions, or any desired combination of such changes in a reference polypeptide or polypeptide. Variants of polypeptides and proteins may also consist of changes in charge and / or post-institutional changes (such as glycosylation, methylation, phosphorylation, etc.). [0040] The term variant, when used in this document in reference to a microorganism, is a microbial strain having characteristics of identification of the species to which it belongs, having at least one variation of nucleotide sequence or identifiably different trait with respect to the parental strain, where the trait is genetically based (hereditary). For example, for a Bacillus thuringiensis 020_A01 strain with a plant growth promoting activity, identifiable traits include 1) the ability to suppress the development of phytopathogenic fungi, including Fusarium graminearum Gibberella zeae, Stagnospora nodurum, Colletotrichum gramnicola; 2) the ability to increase the seed yield in wheat; and 3) with a 16S rRNA gene with nucleotide sequence greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% sequence identity for the 16S rRNA gene of Bacillus thuringiensis ; can be used to confirm a variant like Bacillus thuringiensis 020_A01 020_A01.
[0041] Yield: As used here, the term yield refers to the amount of cultivable plant material or product of plant origin and is usually defined as the measurable product of the economic value of a crop. For crop plants yield also means the amount of material harvested per acre or unit of production. Yield can be defined in terms of quantity or quality. The harvest material can
19/83 vary from crop to crop, for example, they may be seeds, above soil biomass, roots, fruits, cotton fibers, any other part of the plant, or any product of plant origin, which is of economic value. The term income also includes the income potential, which is the maximum income obtained. Yield can be dependent on a number of yield components, which can be monitored by certain parameters. These parameters are well known to people skilled in the art and vary from culture to culture. The term yield also includes the harvest index, which is the relationship between the biomass harvested and the total amount of biomass.
[0042] All publications and patent applications mentioned in this specification are incorporated into this document by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[0043] No admission is made in such a way that any reference constitutes state of the art. The discussion of references states that their authors declare, and candidates reserve the right to contest the accuracy and relevance of the documents cited. It will be clearly understood that, although a number of prior art publications are referred to in this document, such reference does not constitute an admission that any of these documents is part of the general common knowledge in the art.
[0044] The discussion of the general methods given in this document is for illustrative purposes only. Other alternative methods and modalities will be evident to those skilled in the art after reviewing the description.
Plant Growth Promoting Microorganisms [0045] Several microorganisms associated with the plant can
20/83 positively affect the health and physiology of the plant in several ways. These beneficial microbes are generally referred to as plant growth promoting microorganisms (PGPMs). The term plant growth promoting activity, as used here, encompasses a wide range of improved plant properties, including, for example, without limitation, improved nitrogen fixation, improved root development, increased leaf area, increased plant yield, increased seed germination, increased photosynthesis or an increase in the accumulated biomass of the plant. In several modalities, the improvement is at least 10% increase or at least 25% increase or at least 50% increase or at least 75% increase or at least a 100% increase on the property being measured. Thus, as a non-limitation of examples, microbes can produce an increase above the percentage indicated in nitrogen fixation, or an increase above that indicated, either in the total weight of the root, or in the leaf area or in the yield of plant product (for example , an increase above the indicated percentage in the weight of the plant product, or a percentage increase in seeds that germinate within 10 days or 14 days or 30 days, or the rate of photosynthesis (for example: determined by CO2 consumption) or accumulated of the plant's biomass (for example, determined by the weight of the plant). The plant product is the item generally but not necessarily - a food produced by the plant. Yield can be determined using any convenient method, for example, bushels or pounds of product strains produced per hectare of planting. To date, isolated strains from two dozen genera of microorganisms have been reported for the activity of promoting plant growth and / or activity. of biocontrol, and new genera and species with similar activities are still being discovered. Furthermore, within some genres of
21/83 bacteria, various species and subspecies of biocontrol agents have been identified and can be found across multiple spatial scales, from the global to the farm level and even on individual plants. In addition, it has been reported that some isolated individual microbials may exhibit biocontrol and / or growth-promoting activity not only in the plants or crops from which they were obtained, but also in other plant cultures. This indicates the generalist nature of some genotypes, especially those with a wide geographical distribution. As discussed above, if introduced in sufficient numbers and active for a sufficient period, a single microbial population can have a significant impact on the health of the plant.
[0046] Several mechanisms have been postulated to provide an explanation for the positive impact of PGPMs on improving plant growth. The beneficial effects of microorganisms on plant growth can be direct or indirect.
[0047] The term direct plant growth promoting microorganism, for purposes of description, refers to a microorganism that can improve plant growth in the absence of pathogens. As discussed in more detail below, examples of promoting direct plant growth include (a) biofertilization, (b) stimulation of root growth, (c) rhizoremediation and (d) control of plant stress. In addition, several PGPMs have been reported to promote plant growth indirectly through biological control mechanisms, that is, reducing the level of the disease, for example, antibiosis, induction of systemic resistance and competition with pathogens for nutrients and niches.
[0048] Biofertilizers: Microbial fertilizers feed the plant with nutrients and, thus, can promote plant growth in the absence of pressure from the pathogen. Examples of no
22/83 limitation of isolated microbials that can directly promote plant growth and yield include species of N ₂ fixing bacteria Rizobio and Bradirizobio that, through the symbiotic nitrogen fixation, can form nodules in the roots of leguminous plants, in which they convert Atmospheric N2 in ammonia which, in contrast, atmospheric N2, can be used by the plant as a nitrogen source. Other examples include species of Azospiril species, which are free-living N ₂ fixers that can fertilize and increase the yield of cereal crops such as wheat, sorghum and corn. Despite the ability to fix N ₂ - Azospiril -, the increase in yield caused by inoculation by Azospiril is often attributed to the increase in root development and, therefore, to the increase in water and mineral absorption rates. In this regard, several rhizobacteria such as Azotobacter spp. have been reported to be capable of producing a wide variety of phytohormones (eg, auxins, cytokinins) and enzymes (eg, pectinase). Many of these phytohormones and enzymes have been shown to be closely involved in the infection process of symbiotic associations of plant-bacteria that have a regulatory influence on Rizobium nodulation.
[0049] In many cases, PGPMs can also affect plant growth and development by modifying nutrient absorption. They can alter nutrient absorption rates, for example, by the direct effects on the roots, by the effects on the environment, which in turn modify the behavior of the root and compete directly for nutrients (Gaskin et al., Agricult. Ecosyst Environ 12: 99-116, 1985). Some factors by which PGPM can play a role in modifying nutrient use efficiency in soils include, for example, root geometry, nutrient solubility, availability of nutrient, producing
23/83 the appropriate ion form of the plants, partitioning of nutrients in the plant and the efficiency of use. For example, a low level of soluble phosphate can limit plant growth. Some plant growth promoting microbes are able to solubilize phosphate from any organic or inorganic bound phosphate, facilitating plant growth. Various enzymes of microbial origin, such as nonspecific phosphatases, phytases, phosphonatases and C-P lyases, release soluble phosphorus from organic compounds in the soil. For example, greater solubilization of inorganic phosphorus in the soil has been found to increase phosphorus absorption in canola seedlings using Pseudomonas putida, as well as increased absorption of sulfur oxidation and sulfur (Grayston and Germida, Can. J. Microbiol. 37 : 521-529, 1991; Banerjee,
Phytochemicals and Health, vol. 15, May 18, 1995).
[0050] Phyto-stimulators: some microorganisms can produce substances that stimulate plant growth in the absence of pathogens. For example, the production of plant hormones is a characteristic of many microorganisms associated with the plant. For all five classic phytohormones, ie auxin, ethylene, abscisic acid, cytokinin and gibberellin, synthesis as a secondary metabolite has been demonstrated by at least one bacterial or fungal species (for review, see, for example, Kim et al ., Appl. Environ. Microbiol., Vol. 77, 5: 1548-1555, 2011). Some microorganisms can produce secondary metabolites that affect the production of phytohormone in plants. Probably the best known example is auxin, a hormone that can promote root growth. Other examples include pseudomonas that have been reported for the production of indole acetic acid (IAA) and increased amounts of IAA in plants, thus having a profound impact on the production of plant biomass Brown, Annual Rev. Phytopathology, 68: 181-197,
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1974). For example, Tien et al. (Applied Environmental Microbiol., 37: 1016-1024, 1979) reported that inoculation of nutrient solutions around millet roots with Azospirillum brasiliense resulted in an increase in shoot and root weight, an increase in the number of side roots, and all the lateral roots were densely covered with root hair. Plants fed with combinations of IAA, gibberellins and kinetin showed an increase in the production of lateral roots similar to that caused by Azospiril. Although the biological importance of these phytohormones and materials similar to plant hormones are not fully understood, the growth-stimulating activity of these microorganisms is commonly attributed to their production of these materials.
[0051] In addition, other hormones, as well as certain volatile organic compounds (VOCs) and cofactor quinone pyrrolquinoline (PQQ) also stimulate plant growth. For example, some rhizobacteria, such as strains of the bacterial species B. subtilis, b. amiloliquefaciense Enterobacter cloacae, promote plant growth by releasing volatile organic compounds. The highest level of growth promotion has been observed with 2,3-butanediol and 3-hydroxy-2-butanone (also referred to as acetoin) as elicitors of induced systemic resistance. The PQQ cofactor has been described as a plant growth promoter, which acts as an antioxidant in plants. Some reports suggest that the effect may be indirect because PQQ is a cofactor of several enzymes, for example, involved in antifungal activity and induction of systemic resistance.
[0052] Stress controllers: plant growth promoting microorganisms that contain the enzyme 1 aminocyclopropane-1-carboxylic acid (ACC) deaminase facilitate the growth and development of plants, decreasing the levels of
25/83 ethylene from plants. Such microorganisms occupy the precursor of ethylene ACC and convert it into 2-oxobutanoate and NH ₃ . Various types of stress have been reported to be relieved by ACC deaminase producers, such as, for example, stress from the effects of phytopathogenic bacteria, stress from polycyclic aromatic hydrocarbons, heavy metal stress such as Ca ^{2 +} and Ni ² + and salt stress and dry. [0053] In addition, several PGPM strains that induce increased potato yield have been reported for extracellular production of siderophores that bind Fe ^{3 +} , making it less available to certain members of the natural microflora (Kloepper et al., Nature 286 : 885-886, 1980). These rhizobacteria excrete low molecular weight, high-affinity ferric-chelating microbial cofactors that specifically improve your iron acquisition by binding siderophore receptors attached to the membrane. One of the siderophores produced by some pseudomona PGPMs is known as pseudobactin which inhibits the growth of Erwinia cartovora (causal organism for soft rot in potatoes) (See, for example, Kloepper et al., Current Microbiol. 4: 317-320, 1980 ). Additions of pseudobactin to the growth medium inhibited soft rot infection and also reduced the number of pathogenic fungi in the potato plant, along with a significant increase in yield. Most of the evidence to support the siderophore theory of biological control by PGPM comes from work with pioverdines, a class of sideophores that comprise the fluorescent pigments of fluorescent pseudomonas [Demange et al., In Iron Transport in Microbes, Plants and Animals ( Winkleman et al., Eds.), Pp 167-187, 1987]. According to the siderophore theory, pioverdines demonstrate some functional specificity of the strain that is due to the selective recognition of the siderophore outer membrane receptors (Bakker et al., Soil Biology and Biochemistry 19: 443-450,
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1989).
Isolated cultures of the invention [0054] As described in more detail in the examples section of the present description, depositors have discovered several new microorganisms that are effective promoters of plant growth and plant yield. In many cases, isolated microorganisms are also effective in suppressing the development of various plant pathogenic diseases. The isolated microbials were selected from a total of approximately 5,000 microbial strains obtained from environmental samples collected at various locations in the United States. The initial selection of microorganisms was based on the ability of microorganisms to colonize plant roots and the production of chemical compounds and enzymes that are considered important for their interaction with plants. Microorganisms were also bioanalyzed for their ability to suppress the development of various phytopathogenic fungi in an in vitro antagonism assay. Selected microbial microorganisms were then bioassayed in greenhouse studies on commercial wheat and corn varieties for the ability of microbial strains to promote plant growth and for their ability to preserve potential seed yield.
[0055] Taxonomic analysis determined even though representative microorganisms described in the present description are closely related to the bacterial genera Bacillus, Burkholderia, Herbaspirillum, Pantoea, and Pedobacter.
Biological Material Deposit [0056] Purified cultures of microbial strains described in this description were deposited at Agricultural Research Service Culture Collection located at 1815 N. University Street,
27/83
Peoria, IL 61604, USA (NRRL) in accordance with the Budapest Treaty for the purposes of patenting and application regulations (Budapest Treaty). Membership numbers for these deposits are as follows:
Table 1: Isolated microbials and corresponding membership numbers
Cepe ID Membership Number Provisional Taxonomy SGI-003-H11 NRRL B-50483 Pantoea agglomerans 003_H11 SGI-020-A01 NRRL B-50484 Bacillus thuringiensis 020_A01 SGI-026-G06 NRRL B-50485 Burkholderia metallica 026_G06 SGI-026-G07 NRRL B-50486 Burkholderia vietnamiensis 026_G07
[0057] The microbial strains were deposited under conditions that guarantee that access to the culture will be available pending the present patent application for one determined by the Commissioner of Patents and Trademarks to be entitled to them below 37 C.F.R. §1.14 and 35 §122 U.S.C. The deposits represent substantially pure cultures of the deposited strains. Deposits are available as required by foreign patent laws in countries where counterparts of the requested object or their descendants are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice inventing the subject in derogation from the patent rights granted by government action.
[0058] Preferred microorganisms of the present invention have all the identification characteristics of the deposited strains and, in particular, the identification characteristics of being able to promote plant growth and / or yield as described in this document and the identification characteristics as being able to suppress the development of phytopathogenic fungus as described in this document. In particular, micro
Preferred organisms of the present invention refer to deposited microorganisms as described above, and strains derived from them.
Microbiological compositions [0059] The microbiological compositions of the present invention that comprise isolated microbial strains or respective cultures can be in a variety of forms, including, but not limited to resting culture, complete cultures, stored stocks of cells, mycelium and / or hyphae (particularly glycerol stocks), agar strips, agar plugs stored in glycerol / water, dry frozen stocks and lyophilized stocks or dried mycelia on filter paper or cereal seeds. As defined elsewhere in this document, isolated culture or grammatical equivalents as used in the description and in the art it is understood that the culture referred to is a culture fluid, pellet, scraping, dry sample, lyophilisate or section (for example, hyphae or mycelia); or a support, container or medium such as a plate, paper, filter, matrix, straw, pipette or pipette tip, fiber, needle, gel, cotton swab, tube, flask, particle, etc. that contains a single type of organism. In the present invention, a culture isolated from a microbial antagonist is a fluid culture or a scraping, pellet, dry preparation, lyophilisate or section of the microorganism, or a support, container or medium containing the microorganisms, in the absence of other organisms.
[0060] The present additional description provides compositions that contain at least one isolate of microbial strains or fraction cultures of the present invention and an aircraft carrier. The carrier can be any one or more of a number of carriers that impart a variety of properties, such as increased stability, wettability, dispersibility, etc. wetting agents such as natural or synthetic surfactants, which can be
Non-ionic 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 in this document). Suitable formulations that can be prepared include wettable powders, granules, gels, agar strips or pellets, thickeners and the like, microencapsulated particles and the like, liquids such as aqueous dispersible, aqueous suspensions, water-in-oil emulsions, etc. The formulation can include grain or vegetable products (for example, crushed grain or beans, broth or flour derived from grain or beans), starch, sugar, or oil. The carrier can be an agricultural carrier. In certain preferred embodiments, the carrier is a seed, and the composition can be applied or coated on the seed or left to saturate the seed.
[0061] In some modalities, the agricultural carrier can be soil or plant growth medium. Other agricultural carriers that can be used include water, fertilizers, herbal oils, humectants or their combinations. Alternatively, the agricultural carrier can be a solid, such as diatomaceous earth, clay, silica, alginate, clay, bentonite, vermiculite, seed cases, other plant and animal products or combinations, including granules, pellets or suspensions. Mixtures of any of the above mentioned ingredients are also provided as carriers, such as, but not limited to, pesta (flour clay and kaolin), agar agar or clay pellets based on flour, sand or clay, etc. formulations can include food sources for cultivated organisms, such as barley, rice or other biological materials, such as seeds, plant parts, cane bagasse,
30/83 grain processing hulls or stalks, land made of vegetable material (cultural remains) or construction site waste wood, sawdust or small fibers of recycled paper, fabric or wood. Other suitable formulations will be known to those skilled in the art.
[0062] In liquid form, for example, solutions or suspensions, the microorganisms of the present invention can be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates or other carrier liquids.
[0063] Solid compositions can be prepared by dispersing the microorganisms of the invention in an appropriately divided solid carrier, such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, Fuller's earth, pasteurized soil and the like. When such formulations are used as wettable powders, biologically compatible dispersing agents, such as non-ionic, anionic, amphoteric or dispersing and emulsifying cationic agents can be used.
[0064] In a preferred embodiment, the compositions contemplated in this document reinforce the growth and yield of vegetable species, such as wheat, barley, oats and corn and, when used in sufficient quantities, to act as a microbial fertilizer. These compositions, similar to other biofertilizer agents, can have a high safety margin because they do not normally burn or injure the plant.
[0065] As described in detail throughout the present description, the improvement of plant growth and plant yield can be effected by applying one or more of the microbiological compositions of the present invention to a host plant or parts of the host plant. The compositions can be applied in
31/83 an effective amount to increase plant growth or yield in relation to an untreated control. The active constituents are used in a concentration sufficient to improve the growth of the target plant when applied to the plant. As will be evident to those skilled in the art, effective concentrations may vary depending on several factors, such as, for example, (a) the type of plant or agricultural commodities; (b) the physiological state of the plant or agricultural commodities; (c) the concentration of pathogens that affect the plant or agricultural commodities; (d) the type of disease injury on the plant or agricultural commodities; (e) climate (for example, temperature, humidity); and the disease (f) the plant phase. According to the present invention, typical concentrations are those greater than 1 and 10 ² CFU / ml of the carrier. A preferred concentration range from approximately 1 and 10 ⁴ to approximately 1 and 10 ⁹ CFU / ml, such as concentrations ranging from 1 and 10 ⁶ to 1 and 10 ⁸ CFU / ml. Most preferred concentrations are those from approximately 37.5 to approximately 150 mg of dry bacterial mass per milliliter of the carrier (liquid composition) or per gram of the carrier (dry formulation).
[0066] In some embodiments, the amount of one or more of the microorganisms in the 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 microorganisms in the compositions are present in about 2% w / w / to approximately 80% w / w of the entire formulation. More preferably, the one or more microorganisms employed in the compositions is about 5% w / w to approximately 65% w / w and more, preferably about 10% w / w, to approximately 60% w / w by weight of the entire formulation.
[0067] As will be appreciated by those skilled in the art, the
32/83 microbiological compositions of the invention can be applied to the target plant using a variety of conventional methods, such as sprinkling, coating, injecting, rubbing, rolling, dipping, spraying, or brushing or any other appropriate technique, which will not significantly hurt the target plant to be treated. Particularly preferred methods include inoculating the growth medium or soil with suspensions of microbial cells and coating seeds with microbial cells and / or spores.
[0068] Usually, the compositions of the invention are chemically inert; therefore, they are compatible with substantially any other constituents of the order schedule. They can also be used in combination with substances that affect plant growth, such as fertilizers, plant growth regulators and the like, as long as those compounds or substances are biologically compatible. They can also be used in combination with biologically compatible active pesticide agents such as herbicides, nematocides, fungicides, insecticides and the like.
[0069] When used as biofertilizers in their commercially available formulations and in the forms of use, prepared from these formulations, the active microbial strains and compositions according to the present invention may, in addition, be present in the form of a mixture with synergistic agents. Synergistic agents are compounds by which the activity of the active compositions is increased without it being necessary for the added synergist to be active in itself.
[0070] When used as biofertilizers in their commercially available formulations and in the forms of use, prepared from these formulations, the active microbial strains and compositions according to the invention, in addition, may be
33/83 present in the form of a mixture with inhibitors that reduce the degradation of the active compositions after application in the plant's habitat, on the surface of plant parts or in plant tissues. [0071] The active microbial strains and compositions according to the invention, such as, or in their formulations, may also be used as a mixture with known fertilizers, acaricides, bactericides, fungicides, insecticides, microbicides, nematicides, pesticides or combinations of anyone, for example in order to broaden the spectrum of action or prevent the development of resistance to pesticides in this way. In many cases, the result of synergistic effects, that is, the activity of the mixture may exceed the activity of the individual components. A mixture with other known active compounds, such as growth regulators, protective agents and / or semi-chemicals is also contemplated.
[0072] In a preferred embodiment of the present invention, the compositions can also include at least one chemical or biological fertilizer. The amount of at least one chemical or biological fertilizer 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, at least one chemical or biological fertilizer employed is about 0.1% w / w, to about 80% w / w based on the entire formulation. More preferably, at least one chemical or biological fertilizer is present in an amount of approximately 1% w / w to approximately 60% w / w and more, preferably approximately 10% w / w, to approximately 50% w / w.
[0073] The microbiological compositions of the present invention preferably include at least one biological fertilizer. Exemplary biological fertilizers that are suitable for use in this document and can be included in a microbiological composition according to the present invention to promote plant growth and
34/83 yield includes microbes, animals, bacteria, fungi, genetic material, plants 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, the species of Achromobacter, Ampelomyces, Aureobasidium, Azospirillum, Azotobacter, Bacillus, Beauveria, Bradyrhizobium, Candida, Chaetomium, Cordyceps, Cryptococcus, Dabaryomyces, Delftia, Erwiniocilll, Exophilia, , Mariannaea, Microccocus, Paecilomyces, Paenibacillus, Pantoea, Pichia, Pseudomonas, Rhizobium, Saccharomyces, Sporobolomyces, Stenotrophomonas, Streptomyces, Talaromyces, and Trichoderma can be supplied in a composition with the microorganisms of the present invention. Using microbiological compositions according to the present invention in combination with microbial microorganisms, disclosed in US Patent Applications ^Nos US20030172588A1, US20030211119A1; US Patent ^Nos 7,084,331; 7,097,830; 7,842,494; PCT application No. WO2010109436A1 are also particularly preferred.
[0074] 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, at least one chemical or biological pesticide employed is approximately 0.1% w / w, approximately 80% w / w based on the entire formulation. More preferably, at least one chemical or biological pesticide is present in an amount of approximately 1% w / w to approximately 60% w / w and more, preferably approximately 10% w / w, approximately 50% w / w.
35/83 [0075] A variety of chemical pesticides are evident to those skilled in the art and can be used. Exemplary chemical pesticides include those in carbamate, organophosphates, organochlorines and pretroid grades. Also included are chemical agents such as, but not limited to, benomyl, borax, captafol, captan, chorotalonil, copper-containing formulations; formulations containing diclone, dichloran, iodine, zinc; fungicides that inhibit the ergosterol biosynthesis, such as, but not limited to, blastididin, cymoxanil, fenarimol, flusilazole, folpete, imazalil, ipordione, manebe, manocozeb, metalaxyl, oxycarboxin, miclobutanil, oxytetracycline, procnol, pentathinolone, PCN quinometionate, sodium aresenite, sodium DNOC, sodium hypochlorite, sodium phenylphenate, streptomycin, sulfur, tebuconazole, terbutrazole, thiabendazolel, thiophanate-methyl, triadimefon, tricyclazole, triforine, validimicin, vinclozolin, zineb and zineb.
[0076] The microbiological compositions of the present invention preferably include at least one biological pesticide. Exemplary biological pesticides that are suitable for use in this document and can be included in a microbiological composition according to the present invention to prevent a plant pathogenic disease include microbes, animals, bacteria, fungi, genetic material, plants 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, the species of Ampelomyces, Aureobasidium, Bacillus, Beauveria, Candida, Chaetomium, Cordyceps, Cryptococcus, Dabaryomyces, Erwinia, Exophilia, Gliocladium, Mariannaea, Paecilomyces, Paenibacillus, Pantoea, Pantoea, Pantoea Sporobolomyces, Talaromyces, and Trichoderma can be supplied in a composition with,
36/83 microorganisms of the present invention, with fungal strains of Muscodor being particularly preferred. The use of microbiological compositions according to the present invention in combination with microbial antagonists, disclosed in U.S. Patent No. 7,518,040; U.S. Patent No. 7,601,346; U.S. Patent No. 6,312,940 is also particularly preferred.
[0077] Examples of fungi that can be combined with the microbial strains and compositions of the present invention in one composition include, without limitation, Muscodor species, Aschersonia aleyrodis, Beauveria bassiana (white Muscarina), Beauveria brongniartii, Chladosporium herbarum, Cordyceps clavulata, Cordyceps entomorrhiza, FACIS Cordyceps, gracilis Cordyceps, Cordyceps melolanthae, Cordyceps militaris, myrmecophila Cordyceps, Cordyceps ravenelii, Cordyceps sinensis, Sphecocephala Cordyceps, subsessilis Cordyceps, unilateral Cordyceps, variabilis Cordyceps, Cordyceps washingtonensis, Culicinomyces clavosporus, Entomophaga grylli, Entomophaga maimaiga, Entomophaga muscae , Entomophaga praxibulli, Entomophthora plutellae, Fusarium lateritium, Hirsutella citriformis, Hirsutella thompsoni, Metarhizium anisopliae (Muscarina verde), Metarhizium flaviride, Muscodor albus, Neozygitesfloridana, Nomuracesororaisomy, Pauraeaomyomy, paura lypocladium cylindrosporum, Verticillium lecanii, Zoophthora radicans, and mycorrhizal species such as Laccaria bicolor. Other species of mycopesticide will be evident to those skilled in the art.
[0078] The present invention also provides methods of treating a plant by applying any of a variety of usual formulations in an amount effective for any soil (ie, in the furrow), or a part of the plant (i.e., growth regulator) or on
37/83 seed before planting (ie seed coating or dressing). Usual formulations include solutions, emulsion concentrate, wettable powders, suspension concentrate, soluble powders, granules, suspension-emulsion concentrate, natural and synthetic materials, impregnated with active compound and control release capsules in very fine polymeric substances. In certain embodiments of the present invention, microbial compositions are formulated into powders that are available in either formulation ready for use, or are mixed at the time of use. In each modality, the powder can be mixed with the soil before or at the time of planting. In an alternative embodiment, one or both of any plant growth promoting agent, or biocontrol agent is a liquid formulation that is mixed at the time of treatment. Those skilled in the art understand that an effective amount of the inventive compositions depends on the final formulation of the composition, as well as the size of the plant or the size of the seed to be treated.
[0079] Depending on the final formulation and the 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 powder, granules or latex, 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 for the present compositions.
[0080] In a preferred embodiment, microbiological compositions are formulated in a single, stable solution, or emulsion or suspension. For solutions, the active chemical compounds are usually dissolved in solvents before the biological agent is added. Suitable liquid solvents include
38/83 petroleum based aromatic compounds, such as xylene, toluene or alkylnaphthalenes, aliphatic hydrocarbons, such as cyclohexane or paraffins, for example, petroleum fractions, minerals and vegetable oils, alcohols such as butanol or glycol, as well such 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 or suspension, the liquid medium is water. In one embodiment, the chemical agent and the biological agent are suspended in separate liquids and mixed at the time of application. In a preferred embodiment of the suspension, the chemical agent and the biological agent are combined in a ready-to-use formulation that exhibits a reasonably long service life. In use, the liquid can be sprayed or can be applied via the leaf as an atomized spray or in the furrow when planting the crop. The composition of the liquid can be introduced in an effective amount into the seed (ie, seed coating or compress) or into the soil (ie, in the furrow) before the seed germinates 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, sprayers, soil injection or soil soaking.
[0081] Optionally, stabilizers and buffers can be added, including 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 derivatives of benzoic acid, such as p-hydroxybenzoic acid.
Pathogenic Agents [0082] One skilled in the art will recognize that the methods and
39/83 the compositions according to the present invention in principle can be applied to suppress the development of any plant pathogen or any phytopathogenic diseases. The invention is not intended to be limited to a particular type of culture or cell types. For example, microbial cells that undergo complex forms of differentiation, filamentation, sporulation, etc. they can also be used for the methods and compositions of the present invention.
[0083] Examples of phytopathogenic diseases that are suitable for applications of the methods and materials of the present inventions include, but are not limited to, diseases caused by a wide range of pathogenic fungi. The methods of the present invention are preferably applied against pathogenic fungi that are important or interesting for agriculture, horticulture, biomass plants for the production of biofuel molecules and other chemicals and / or forest products. Of particular interest are pathogenic species Pseudomonas (for example, species Pseudomonas solanacearum), Xylella fastidiosa; Ralstonia solanacearum, Xanthomonascampestris, Erwinia amylovora, Fusarium species, Phytophthora species (for example, P. infestans), Botrytis species, Leptosphaeria species, powdery mildews (Ascomycota) and rusts (Basidiomycota), etc.
[0084] Non-limiting examples of plant pathogens of interest include, for example, Acremonium strictum, Agrobacterium tumefaciens, Alternaria alternata, Alternaria solani, Aphanomyces euteiches, Aspergillus fumigatus, Athelia rolfsii, Aureobasidium pullulans, Bipolaris zeicola, Botryisporia cysticis, Cyrilisporia, Cisterneidae maydis, Cercospora medicaginis, Cercospora sojina, Colletotrichum coccodes, Colletotrichum fragariae, Colletotrichum graminicola, Coniella diplodiella, Coprinopsis psychromorbida,
40/83
Corynespora cassiicola, Curvularia pallescens, Cylindrocladium crotalariae, Diplocarpon earlianum, Diplodia gossyina, Diplodia spp., Epicoccum nigrum, Erysiphe cichoracearum, Fusarium graminearum, Fusarium oxysporum, Fusarium oxysporum f. sp. tuberosi, Fusarium proliferatum var. proliferatum, Fusarium solani, Fusarium verticillioides, Ganoderma boninense, Geotrichum candidum, Glomerella tucumanensis, Guignardia bidwellii, Kabatiella zeae, Leptosphaerulina briosiana, Leptotr Backpack medicaginis, Macrophomina, Macrophomina, Magnaportheia, Magnaportheia, Magnaportheia, Magnaporthe fragariae, Nigrospora oryzae, Ophiostoma ulmi, Pectobacterium carotovorum, Pellicularia sasakii (Rhizoctonia solani), Peronospora manshurica, Phakopsora pachyrhizi, foveata Phoma, Phoma medicaginis, longicolla Phomopsis, Phytophthora cinnamomi, erythroseptica Phytophthora fragariae Phytophthora infestans, Phytophthora medicaginis Phytophthora Phytophthora megasperma , Phytophthora palmivora, Podosphaera leucotricha, Pseudopeziza medicaginis, Puccinia graminis subsp. Tritici (UG99), Puccinia sorghi, Pyricularia grisea, Pyricularia oryzae, Pythium ultimum, Rhizoctonia solani, Rhizoctonia zeae, Rosellinia sp. , Stemphylium sp, Synchytrium endobioticum, Thecaphora (Angiosorus), Thielaviopsis, Tilletia indica, Trichoderma viride, Ustilago maydis, Verticillium albo-atrum, Verticillium dahliae, Verticillium dahliae, Xanthomonas xonthomye axonopodisis. oryzae.
[0085] In a preferred embodiment of the present invention, the methods and materials of the invention are useful in suppressing the development of the pathogens Aspergillus fumigatus, Botrytis
41/83 cinerea, Cerpospora betae, Colletotrichum sp., Curvularia spp., Fusarium sp., Ganoderma boninense, Geotrichum candidum, Gibberella sp., Monographella sp., Mycosphaerella fijiensis, Phytophthora palmivora, Phytophthora ramorum, Penicillium, Pyicillium. Rhizoctonia solani, Rhizopus spp., Schizophyllum spp., Sclerotinia sclerotiorum, Stagnospora sp., Verticillium dahliae, or Xanthomonas axonopodis. In a particularly preferred embodiment, the inventive methods and materials can be used to suppress the development of several commercially important plant pathogens, including Fusarium graminearum NRRL-5883, Monographella nivalis ATCC MYA-3968, Gibberella zeae ATCC-16106, Stagnospora nodurum ATCC-26369, Colletotrichum graminicola ATCC-34167, and Penicillium sp.
Seed coating formulation [0086] In a particularly preferred embodiment, the microbial compositions of the present invention are formulated as a seed treatment. It is envisaged that the seeds can be substantially uniformly coated with one or more layers of the microbial compositions disclosed in this document using conventional methods of mixing, spraying or a combination of them using treatment application equipment that is specifically designed and manufactured to apply accurately, safely and effectively seed treatment products in the seeds. This equipment uses various types of coating technology such as rotary coaters, drum coaters, fluidized bed techniques, spouted beds, rotary mists or a combination thereof. Liquid seed treatments such as those of the present invention can be applied through a rotating atomizing disk or a spray nozzle that evenly distributes the seed treatment in the
42/83 seed, as it moves, despite the spray pattern. Preferably, the seed is then mixed or agitated for an additional period of time to achieve the distribution of additional treatment 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.
[0087] Another aspect of the invention provides seeds treated with the microbial compositions in question. 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 with microorganisms have a concentration of microbial spores or a concentration of microbial cells of about 10 ⁶ to about 10 ⁹ per seed. The seeds may also have more spores or microbial cells per seed, such as, for example, 10 ¹⁰ , 10 ¹¹ or 10 ¹² spores per seed. Spores and / or microbial cells can be coated freely on the seeds or, preferably, they can be formulated in a solid or liquid composition before being coated on the seeds. For example, a solid composition comprising microorganisms can be prepared by mixing a solid carrier with a spore suspension until the solid carriers are impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.
[0088] In some other embodiments, it is envisaged that the solid or liquid microbial compositions of the present invention additionally contain functional agents capable of protecting the
43/83 seeds of the 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.
[0089] 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 coating methods and apparatus for their application are disclosed in , for example, in US Patents ^Nos 5,918,413; 5,554,445; 5,389,399; 4,759,945; and 4,465,017.
Seed coating compositions are disclosed, for example, in US Patent Application No. US20100154299, US Patent ^Nos 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.
[0090] A variety of additives can be added to seed treatment formulations comprising the inventive compositions. Binders can be added and include those compounds preferably of an adhesive polymer that can be natural or synthetic without a phytotoxic effect on the seed to be coated. The binder can be selected from polyvinyl acetates; polyvinyl acetate copolymers; 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; arabic gums; shellacs; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers;
44/83 polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.
[0091] 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 oligonutrients such as iron, manganese, boron, copper, cobalt, molybdenum and zinc salts. A polymer or other dust control agent can be applied to maintain treatment on the seed surface.
[0092] In some specific embodiments, in addition to microbial cells or spores, the coating may additionally comprise a layer of adherents. The adhesive must be non-toxic, biodegradable, and adhesive. Examples of such materials include, but are not limited to, polyvinyl acetates; polyvinyl acetate copolymers; 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; arabic gums; gelatines; syrups; and starches. More examples can be found, for example, in U.S. Patent No. 7,213,367 and U.S. Patent Application No. US20100189693.
[0093] 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, coating agents, wetting agents, buffering agents, and polysaccharides. At least one carrier
45/83 agronomically acceptable 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, plaster, vermiculite, talc, humus, activated carbon, and various phosphorus compounds.
[0094] In some embodiment, the seed coating composition may comprise at least one filler that is an organic or inorganic component, natural or synthetic, with which the active components are combined to facilitate its application in the seed. Preferably, the filler is an inert solid such as clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (eg ammonium 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.
[0095] The seed treatment formulation may additionally include one or more of the following ingredients: other pesticides, including compounds that act just below the ground; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixil, and isomers of each of these materials, and the like; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxis, ureas, and benzoic acids; herbicide protectors such as benzoxazine, benzhydryl derivatives, N, Ndialyl dichloroacetamide, various dihaloacil compounds, oxazolidinyl and thiazolidinyl, ethanone, naphthalic anhydride compounds, and oxime derivatives; chemical fertilizers; biological fertilizers; and biocontrol agents, such as other recombinant or naturally occurring bacteria and fungi of the genera Rhizobium, Bacillus,
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Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. These ingredients can be added as a separate layer on top of the seed or, alternatively, can be added as part of the seed coating composition of the invention.
[0096] Preferably, the amount of the new composition or other ingredients used in seed treatment should not inhibit seed germination, or cause phytotoxic damage to the seed.
[0097] The formulation that is used to treat the seed in the present invention can be in the form of a suspension; emulsion; particulate slurry in the aqueous medium (for example, water); wettable powder; wettable granules (dry fluid); and dry granules. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation is preferably from about 0.5% to about 99% by weight (w / w), preferably from 5 to 40% or as otherwise formulated by those skilled in the art.
[0098] As mentioned above, other inactive or conventional inert ingredients can be incorporated into the formulation. Such inert ingredients include, but are not limited to: conventional tacking agents; dispersing agents such as methylcellulose, for example, serve as combined dispersing / tacking agents for use in seed treatments; polyvinyl alcohol; lecithin, polymeric dispersants (for example, polyvinylpyrrolidone / vinyl acetate); thickeners (eg clay thickeners to improve viscosity and reduce sedimentation of particle suspensions); emulsion stabilizers; surfactants; antifreeze compounds (eg, urea), paints, dyes, and the like. In addition, inert ingredients useful in the present invention can be found in McCutcheon's, vol. 1,
47/83
Emulsifiers and Detergents, MC Publishing Company, Glen Rock,
N.J., U.S.A., 1996. Additional inert ingredients useful in the present invention can be found in McCutcheon's, vol. 2, Functional Materials, MC Publishing Company, Glen Rock, N.J., U.S.A., 1996.
[0099] The coating formulations of the present invention can be applied to seeds by a variety of methods, including, but not limited to, mixing in a container (e.g., a bottle or bag), mechanical application, stirring, spraying, and immersion. A variety of active or inert material can be used to contact seeds with microbial compositions according to the present invention, such as conventional film coating materials, including, but not limited to, water-based film coating materials such as SEPIRET ™ (Seppic, Inc., NJ) and OPACOAT ™ (Berwind Pharm. Services, PA).
[00100] The amount of a composition according to the present invention that is used for seed treatment will vary depending on the type of seed and the type of active ingredients, but the treatment will comprise bringing the seeds into contact with an agronomically effective amount of seed. inventive composition. As discussed above, an effective amount means that the amount of the inventive composition is sufficient to affect beneficial or desired results. An effective amount can be administered in one or more administrations.
[00101] In addition to the coating layer, seeds can be treated with one or more of the following ingredients: other pesticides including fungicides and herbicides; herbicide protectors; fertilizers and / or biocontrol agents. These ingredients can be added as a separate layer or alternatively they can be added to the coating layer.
[00102] The seed coating formulations of this
48/83 invention can be applied to seeds using a variety of techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic seed disinfectants, and drum coaters. Other methods, such as gushing beds, may also be useful. The seeds can be pre-sized before coating. After coating, the seeds are normally dried and then transferred to a sizing machine for sizing. Such procedures are known in the art.
[00103] Seeds treated by microorganisms can also be wrapped with a film overcoat to protect the coating. Such overcoats are known in the art and can be applied using fluidized bed and drum film coating techniques.
[00104] In another embodiment of the present invention, the compositions according to the present invention can be introduced into a seed by using solid matrix initiation. For example, an amount of an inventive composition can be mixed with a solid matrix material and then the seed can be brought into contact with the solid matrix material for a period to allow the composition to be introduced into the seed. The seed can then optionally be separated from the solid matrix material and stored or used, or the mixture of solid matrix material plus the seeds can be stored or planted directly. Solid matrix materials that are useful in the present invention include polyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea, polyacrylate, or any other material capable of absorbing or adsorbing the inventive composition for a time and releasing that composition into the or in the seed. It is useful to make sure that the inventive composition and solid matrix material are
49/83 compatible with each other. For example, the solid matrix material must be chosen so that it can release the composition at a reasonable rate, for example, over a period of minutes, hours, or days.
[00105] In principle, any plant seed capable of germinating to form a plant can be treated in accordance with the invention. Suitable seeds include those from cereals, coffee, Brassica crops, fiber crops, flowers, fruits, legumes, oilseeds, trees, tuber crops, vegetables, as well as other plants of monocot and dicot species. Preferably, harvest seeds that are coated include, but are not limited to, beans, carrots, corn, cotton, grasses, lettuce, peanuts, pepper, potatoes, canola, rice, rye, sorghum, soy, beets, sunflower , tobacco and tomatoes. Preferably, barley or wheat seeds (spring wheat or winter wheat) are coated with the present compositions.
Preparation of the microbial compositions according to the present invention [00106] Cultures of the microorganisms can be prepared for use in the microbial compositions of the invention using standard liquid fermentation and static drying techniques known in the art. Growth is commonly performed in a bioreactor.
[00107] A bioreactor refers to any device or system that supports a biologically active environment. As described in this document, a bioreactor is a receptacle in which microorganisms including the microorganism of the invention can be grown. A bioreactor can be of any shape or size appropriate for the growth of microorganisms. A bioreactor can vary in size and scale from 10 mL per cubic meter of liter
50/83 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, a powered batch type bioreactor or a continuous type (for example, a continuous agitated reactor). For example, a bioreactor can be a chemostat as known and used in the microbiology technique for the growth and harvesting of microorganisms. A bioreactor can be obtained from any commercial supplier (See also Bioreactor System Design, Asenjo & Merchuk, CRC Press, 1995).
[00108] 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 into continuous operations.
[00109] Microorganisms grown in a bioreactor can be suspended or immobilized. Growth in the bioreactor is generally under aerobic conditions at temperatures and pH suitable for growth. For the organisms of the invention, cell growth can be obtained 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 at 25 ° C. The pH of the medium can vary between 4.0 and 9.0, but the preferred operating range is generally 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 to 72 hours after inoculation.
[00110] Ideal conditions for the cultivation of the microorganisms of this invention will certainly depend on the specific strain. However, due to the conditions applied in the selection process and the general requirements of most microorganisms, a person skilled in the art would be able to determine the essential conditions and nutrients. Microorganisms would normally be grown in
51/83 aerobic liquid cultures in media containing sources of carbon, nitrogen, and inorganic salts that can be assimilated by microorganisms and favorable to effective cell growth. Preferred carbon sources are hexoses, such as glucose, but other sources that are easily assimilated such as amino acids, can be substituted. Many inorganic and 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, meat extract, peptone proteose, soy flour, casein hydrolysates, soluble in distiller , and the like. Among the inorganic minerals that can be incorporated into the nutrient medium are usual salts capable of producing calcium, zinc, iron, manganese, magnesium, copper, cobalt, potassium, sodium, molybdate, phosphate, sulfate, chloride, borate, and similar ions. Without limiting them, the use of potato dextrose liquid medium for fungal strains and R2A broth premix for bacterial strains is preferred.
New plant varieties [00111] In addition, in another aspect of the present invention, a new plant created artificially by introducing a microbial endophyte of the invention into a plant that is free of endophytic microorganisms is provided. In some embodiments of this aspect, the microbial endophyte introduced into the plant may be an endophytic microorganism having an activity to promote plant growth, a biological control activity, or a combination of both activities. A variety of methods previously considered effective for introducing a microbial endophyte into cereal grass species are known in the art. Examples of such methods include those described in
52/83
U.S. Patent Application No. 20030195117A1, U.S. Patent Application No. 20010032343A1, and U.S. Patent No. 7,084,331, among others. It will become apparent to those skilled in the art that many of the methods mentioned above may be useful for the production of a new plant of the invention.
[00112] After artificial infection, it is preferable that a DNA sequence of the isolated endophytic microorganism is amplified by PCR and the endophyte is confirmed by performing a homology search for the amplified DNA sequence. In addition, it is preferable that a foreign gene expressing identifiable media is introduced into the aforementioned endophytic microorganism, and the presence of the colonization of the aforementioned endophytic microorganism that infects the plant is confirmed by the above identifiable means using the foreign gene.
Plants suitable for the methods of the invention [00113] In principle, the methods and compositions according to the present invention can be implanted for any species of plant. Monocotyledonous plants as well as dicotyledonous plants are particularly suitable. The methods and compositions are preferably used with plants that are important or interesting for agriculture, horticulture, for the production of biomass used in the production of liquid fuel molecules and other chemicals, and / or forest products.
[00114] Thus, the invention has use over a wide range of plants, preferably superior plants belonging to the classes of Angiospermae and Gymnospermae. Plants from the Dicotylodenaee subclasses of Monocotyledonae are particularly suitable. Dicotyledonous plants belong to the orders of Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, Diapensales,
53/83
Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Illiciales, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Papalales, Miales, Malaleses, Miales, Males Plantaginales, Plumbaginales, Podostemales, Polemoniales, Polygalales, Polygonales, Primulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendales, Umbrellas, Trochodendales, Umbrellas, Trochodendales, Urales, Trochodendes. Monocotyledonous plants belong to the orders of Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingibes. The plants belonging to the Gymnospermae class are Cycadales, Ginkgoales, Gnetales, and Pinales.
[00115] Suitable species may include members of the genus Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythus, Erythus, Erythy Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum, Papaia, Papaver, Parumum, Papaia Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Pink, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,
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Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.
[00116] The methods and compositions of the present invention are preferably used in plants that are important or interesting for agriculture, horticulture, biomass for the production of biofuel molecules and other chemicals, and / or forestry. Non-limiting examples include, for example, Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, Capim Sudan), Miscanthus giganteus (miscanto), Saccharum sp. (energy cane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soy), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus ( sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugar beet), Pennisetum glaucum (millet), Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (bluestem large), Pennisetum purpureum (elephant grass), Phalaris arundinacea (yellow grass), Cynodon dactylon (grass-shorts), Festuca arundinacea (tall fescue), Spartina pectinata (prairie spartan), Arundo donax (giant cane), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale spp. (triticum - wheat X rye), Bamboo, Carthamus tinctorius (saffron), Jatropha curcas (Jatropha), Ricinus communis (castor), Elaeis guineensis (oil palm), Phoenix dactylifera (date palm), Archontophoenix cunninghamiana (royal palm), Syagrusus (jerivá), Linum usitatissimum (flax), Brassica juncea, Manihot esculenta (cassava), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cabbage) flower, brussels sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (pepper and pepper) ,
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Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (pumpkin), Cucurbita moschata (pumpkin), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanum melongena (eggplant), Papaver somniferum (opium poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis, Coichicum autumnale, Veratrumumornata Digitalis purpurea, Dioscorea spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp. ., Rauwolfia serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guaiúl e), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosa spp. (pink), Dianthus caryophyllus (carnation), Petunia spp. (Petunia), Poinsettia pulcherrima (poinsettia) Nicotiana tabacum (tobacco), Lupinus albus (lupine), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (spruce), Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis (grasses), Lolium spp. (ryegrass), Phleum pratense (red grass), and conifers. Of interest are plants grown for energy production, so called energy crops, such as cellulose-based energy crops such as Panicum virgatum (switchgrass) Sorghum bicolor (sorghum, Sudan grass), Miscanthus giganteus (miscanto), Saccharum sp. (energy cane), Populus balsamifera (poplar), Andropogon gerardii (bluestem grande), Pennisetum purpureum (elephant grass), Phalaris arundinacea (grass
56/83 yellow), Cynodon dactylon (grass-shorts), Festuca arundinacea (tall fescue), Spartina pectinata (prairie spartan), Medicago sativa (alfalfa), Arundo donax (giant cane), Secale cereale (rye), Salix spp . (willow), Eucalyptus spp. (eucalyptus), Triticosecale spp. (triticum - wheat X rye) and Bamboo; and starch-based energy crops such as Zea mays (corn) and Manihot esculenta (cassava); and energy-based sugar crops like Saccharum sp. (sugar cane), Beta vulgaris (sugar beet), and Sorghum bicolor (L.) Moench (sweet sorghum); and energy crops for biofuel production such as Glycine max (soy), Brassica napus (canola), Helianthus annuus (sunflower), Carthamus tinctorius (saffron), Jatropha curcas (Jatropha), Ricinus communis (castor), Elaeis guineensis (oil palm) African), Elaeis oleifera (American oil palm), Cocos nucifera (coconut), Camelina sativa (wild flax), Pongamia pinnata (Pongam), Olea europaea (olive), Linum usitatissimum (flax), Crambe abyssinica (Abyssinian-kale) , and Brassica juncea.
[00117] The discussion of the general methods given here is for illustrative purposes only. Other alternative methods and modalities will be apparent to those skilled in the art after reviewing this description, and should be included within the spirit and scope of this application.
[00118] It should also be understood that the following examples are offered to illustrate, but not limit, the invention.
EXAMPLES
EXAMPLE 1: Isolation of the microorganism from environmental samples [00119] Identification of spore-forming rhizobacteria using a serial dilution method and sonicated roots. The following microorganisms were isolated using a sonicated root method, serial dilutions, as described below:
57/83 isolates SGI-026-G06 and SGI-026-G07, which were isolated from a needle-type grass sample; isolate SGI-041-B03, which was isolated from a sample of wild rye; and isolate SGI020-A01, which was isolated from wheat root tissues grown in a composite soil sample.
[00120] An enrichment procedure was developed to specifically identify spore-forming rhizobacteria. Soon, sonicated root extracts were treated with heat to kill vegetative cells and then bathed in a rich medium. Microorganisms that survived heat treatment and formed colonies were considered spore-forming. This method was considered to be particularly effective for the selection of Gram-positive bacteria. Freshly sampled roots were used as starting material for these advances. Thin cuts found at the tip of the roots are the youngest, can have a high density of root hair, and generally have high densities of rhizobacteria. A sterile blade was used to cut these root areas into 5 to 10 cm segments, which were then washed under sterile milliQ water to remove large soil particles. When necessary, a more rigorous wash was performed by placing the roots in a 50 mL Falcon tube with 25 mL 1 and sterile phosphate buffered saline (ie, PBS buffer) and vortexing for 1 minute. Each root sample was subsequently suspended in 20 mL of sterile PBS buffer and sonicated on ice for two 1-minute intervals at 8 watts using a Fisher Scientific Sonic Dismembrator. For heat treatment, normally 1 ml of sonicated root cell suspension was transferred to a sterile Eppendorf tube incubated in a water bath at 80 ° C for 20 minutes. The heat-treated cell suspensions were allowed to cool to room temperature before being
58/83 serially diluted to concentrations of 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , and 10 ^-7 . 100 μL of each 10-fold dilution was spread on culture plates containing a microbiological medium solidified with agar and 100 mg / L of cycloheximide to inhibit the growth of fungi. In some cases, it was necessary to perform a 1/10 or 1/100 dilution before plating in order to obtain the CFU density suitable for colony harvesting. Isolated colonies were chosen using sterile pipette tips, placed in 96-well microtiter plates each containing 150 μL 2 and YT liquid medium per well. The microtiter plates were incubated for 1 to 2 days at 30 ° C in order to obtain a high density cell for further characterization and archiving.
[00121] Isolation of bacteria forming biopellicle. The following microbial isolates were isolated using a bio-film-forming method as described below: isolate SGI-003H11, which was isolated from a Yucca plant root sample; isolate SGI-034-C09 isolated, which was isolated from a grass root sample; and isolate SGI-034-E10, which was from a sample of Queen Anne's Lace plant.
[00122] Bio-film-forming method: In this procedure, the bio-film-forming bacteria were isolated from sonicated root segments, as described by Fall et al. (Syst. Appl. Microbiol. 27.372-379, 2004). As described above, bacteria that form bio-films on the surface of a root are usually very good root colonizing bacteria. In general, when such bacteria are present at high densities, they can have a significant influence on the health of the plant and can competitively exclude invasive pathogens. Briefly, sonication was used to remove bacterial and fungal cells slightly associated with the root, leaving behind only microbes that were strongly
59/83 adhered to the root surface. Both Gram-positive and Gram-negative biopellicle-forming bacteria were selected using this method.
[00123] Freshly sampled roots were used as starting material for these advances. Thin cuts found at the tip of the roots were the youngest tissues, had a high density of root hair, and generally had high densities of rhizobacteria. A sterile blade was used to cut these root areas into 5 to 10 cm segments, which were then washed, placing them in a 50 mL Falcon tube with 25 mL 1 and PBS and vortexing for 1 minute. The washing debris was allowed to rest, and then a sterile forceps was used to transfer the washed root segments into 50 ml Falcon tubes filled with 25 ml 1e PBS, and sonicated on ice using a Fisher Scientific Sonic Dismembrator for two intervals of 30 seconds with a 30-second pause between pulses. The sonicated root samples were transferred to sterile plastic Petri dishes and allowed to dry completely without lids inside a biosafety cabin. Each root segment was then placed on a separate CMA plate containing 1% agar (10 g / L of casein digest, 10 g / L of mannitol, 10 g / L of agar). From time to time, sterile forceps were used to push the root segment into the agar media. The plates were then incubated at 37 ° C and monitored for microbial growth. Usually after 1 to 2 days, multiple microbial growths have emerged from the root and into the CMA media. A sterile pipette tip was used to choose growths with unique morphologies along the segment and each growth was transferred to the center of a CMA plate containing 0.3% agarose. The CMA plates were then incubated for 1 to 2 days at 37 ° C and
60/83 monitored for growth. Normally, biopellet forming isolates exhibited dendritic growth in this medium.
[00124] A sterile loop was used to transfer the biomass and purify each isolate from the CMA plates on CMKA plates (2% agar, 1.2 g / L K2HPO4) by strip. The CMKA medium restricts the growth of biopellicle and allows the choice of individual colonies for archiving.
EXAMPLE 2: Growth and storage of microbial isolates [00125] 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 stirring at 250 rpm for two days. The culture was then transferred to flasks containing 15% glycerol and stored at 80 ° C.
EXAMPLE 3: DNA Extraction, Sequencing and Taxonomy [00126] An aliquot of 20 pl of bacterial cell suspension was transferred to a 96-well PCR plate containing 20 pl of a 2x lysis buffer (100 mM Tris HCL, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS, 400 pg / ml Proteinase K). The Lysis conditions were as follows: Incubation at 55 ° C for 30 minutes, followed by incubation at 94 ° C for 4 minutes. An aliquot of the lysis product was used as the source of the DNA model for PCR amplification.
[00127] For the amplification of the 16S rRNA region, each PCR mixture was prepared in a final 20 µl volume reaction containing 4 µl of the bacterial lysis reaction, 2 μΜ of each PCR primer, 6% Tween-20 , and 10 μl of 2 x ImmoMix (Bioline USA Inc, Taunton, MA). The primers used for PCR amplification were M13-27F
5-TGTAAAACGACGGCCAGTTAGAGTTTGATCCTGGCTCAG-3 ’
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SEQ ID NO: 8) and M13 tail 1492R (5'CAGGAAACAGCTATGACCGGTTACCTTGTTACGACTT-3 '; SEQ ID NO: 9). PCR was performed on a PTC-200 personal thermocycler (MJ-Research, MA, USA) 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 each PCR product was run on a 1.0% agarose gel to confirm a single band of the expected size. Positive bands were isolated, purified, and subjected to PCR sequencing. The sequencing was performed in advanced and reverse initiation directions by J. Craig Venter Institute in San Diego, California, using 454 technologies.
[00128] The search for homology 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 strains described here, bacteria of the genera and species that have high sequence homologies for the isolated bacterial strains, and other large varieties of bacterial genera and species, using the ClustalW phylogenetic tree building program. Sequence identity and similarity were also determined using the GenomeQuest ™ software (Gene-IT, Worcester Mass. USA). The result of the sequence analysis revealed that the isolates of bacteria SGI-003_H11, SGI-020_A01, SGI026_G06, SGI-026_G07, SGI-034_C09, SGI-034_E10, SGI-041_B03 can be considered to be related to the species of Pantoea agglomerans, Bacillus thuringiens , Burkholderia metallica, Burkholderia vietnamiensis, Bacillus pumilus, Herbaspirillum sp., Pedobacter sp., Respectively, based on sequence homologies of> 98% of each of the 16 rRNA sequences for the
62/83 respective microorganisms.
EXAMPLE 4: Biochemical characteristics of Bacterial Isolates [00129] The isolated bacteria were further studied for important properties in their interaction with plants. The studied properties include nitrogen fixation, siderophore secretion, inorganic phosphorus solubilization, production of 1-aminocyclopropane-1- carboxylic acid (ACC) deaminase, production of 2,3-butanediol, and production of plant growth hormone auxin. The results of in vitro biochemical tests are shown in Table 2.
Nitrogen fixation:
[00130] Bacterial cell suspensions were drawn in a solid medium, with the following composition that did not include a nitrogen source: KOH 4.0 g / L; K ₂ HPO ₄ 0.5 g / L; MgSO <7H ₂ O 0.2 g / L; 0.1 g / L NaCl; 0.02 g / L CaCl ₂ ; FeSO <7H ₂ O 0.005 g / L; NaMoO4 ^ 2H ₂ O 0.002 g / L; MnSO4YH ₂ O 0.01 g / L; Malic acid 5.0 g / L; Gellan gum 0.1 to 1.0 g / L; and optionally, 0.5% v / v Bromothymol blue, pH 7.0. Concentrations of Gellan gum or agar may vary as needed to achieve the desired average thickness; normally 0.5 g / L was used. The layers were incubated at 30 ^o C for 2 to 5 days. These plates were monitored daily and colonies were selected as they appeared. In some cases, long growth periods (up to two weeks or more) allowed the capture of the slowest growing isolates. These layer plates were normally chosen by colony using 20 or 200 µl aerosol barrier pipette tips in 96-well cell culture plates filled with 150 µl / well of 2YT medium. Alternatively, the isolates were chosen by plaque colony directly in free N medium to confirm their free N growth abilities. The results, as summarized in Table 2,
63/83 indicate that only the SGI-026-G07 isolate showed nitrogen fixation activity at a detectable level.
Siderophore secretion:
[00131] This assay was used to identify bacterial isolates that were producing siderophores, which are high-affinity Fe ³⁺ chelating compounds, in vitro. Typically, microbial isolates were grown in a minimal medium, which was essentially Fe-free. All glass products used throughout this assay were washed with acid and rinsed three times with milliQ water to remove residual Fe which can alter the results of the test. The composition of the MM9 medium was as follows: K ₂ HPO ₄ 0.5 g / L; NH ₄ Cl 1.0 g / L; MgSO <H ₂ O 0.2 g / L; 0.5 g / L NaCl; TUBE Buffer 7.55 g / L; Glucose 10.0 g / L; Gluconic Acid 2.5 g / L; Malic Acid 2.5 g / L; Casamino acids 0.5 g / L. The medium was adjusted to pH 7.0 with 5N KOH, and sterilized using a 0.2 μΜ filter (Corning).
[00132] This assay was typically performed in a high throughput format using a Beckman FX liquid handling station and 96-well cell culture plates with 150 μL of MM9 growth medium per well. Cultures and media were distributed and transferred aseptically using an autoclavable precision tool under a laminar flow hood. After transfer, cultures were incubated at 30 ^o C for 5 days. After incubation, culture supernatants were harvested by centrifugation using a 0.22 μΜ 96-well filter plate. Ten microliters of filtered supernatant was transferred from each well to a Falcon assay plate. A standard curve was prepared using desferrioxamine (DFO) diluted in the MM9 medium. Two hundred microliters of the CAS assay solution [10 mM HDTMA, Fe (III) Solution: 1 mM FeCl ₃ .6H2O, 10 mM HCl, 2 mM CAS] were added to each of the supernatants and standard wells, followed by
64/83 incubation at room temperature for 20 to 30 minutes. The absorbance of the blue CAS test solution at 630 nm (SpectroMax M2) is inversely proportional to the siderophore concentration in each well (that is, the test solution should change to an intense orange, with greater amounts of siderophores). Solubilization of inorganic phosphorus:
[00133] The ability of microbial isolates to solubilize mineral phosphate in vitro was evaluated as follows. Bacteria to be tested were strained in the agar phosphate growth medium [Hydroxylapatite - Ca ₁₀ (PO ₄ ) ₅ (OH) ₂ 5.0 g / L; NH ₄ Cl 1.0 g / L; MgSO ₄ -H ₂ O 0.2 g / L; 0.5 g / L NaCl; FeSO ₄ -7H ₂ O 0.01 g / L; Na ₂ MoO ₄ -7H ₂ O 0.01 g / L; MnSO ₄ -7H ₂ O 0.01 g / L; Glucose 5.0 g / L; Gluconic Acid 2.5 g / L; Malic Acid 2.5 g / L; Casamino Acids 0.5 g / L; Gellan gum 20.0 g / L; pH 7.2)], and its growth was monitored daily. The culture medium had an opaque appearance, due to the presence of calcium phosphate. Bacterial growth and loss of color in the medium would be observed if the bacteria were capable of dissolving calcium phosphate. Isolates having the ability to solubilize the mineral phase phosphate would produce a clear halo in the opaque medium around the colony. As summarized in Table 2, the ability to solubilize mineral phosphate was not detectable in any of the microorganisms tested, as determined by the in vitro assay described here. Production of ACC deaminase:
[00134] One of the main mechanisms used by plant growth-promoting rhizobacteria (PGPM) to facilitate plant growth and development is the reduction of ethylene levels by deaminating 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in plants. ACC deaminase catalyzes the hydrolysis of 1-aminocyclopropane-1-carboxylic acid (ACC) to α-ketobutyrate and ammonia. The presence of the α-ketobutyrate product
65/83 can then be determined indirectly through a reaction with 2,4-dinitrophenylhydrazine in HCl to form a phenylhydrazone derivative. After adding NaOH, the amount of phenihidrazone in the solution can be determined spectrophotometrically by measuring the absorbance at 540 nm (Penrose and Glick, Physiol Plant. May; 118: 10-1, 2003). This assay was performed normally in a high throughput format using 96-well cell culture plates. Each well contained 150 pL of DF salt growth medium supplemented with 2.0 g / L of (NH ₄ ) ₂ SO ₄ . Cultures and media were distributed and transferred aseptically using an autoclavable precision tool under a laminar flow hood. After transfer, cultures were incubated at 30 ^o C for 2 days. After reaching turbidity, the cultures were transferred a second time using a sterile precision tool under a laminar flow hood in 96-well plates, containing 150 µl per well of DF salt growth medium supplemented with 5 mM ACC as single source of nitrogen, followed by a 4-day incubation at 30 ^o C. The absorbance of each culture at 600 nm was measured using a spectrophotometer. The isolates that exhibited robust growth under these conditions (OD> 0.2) were carried out for further testing for ACC deaminase activity as described in Penrose and Glick, 2003, supra.
[00135] The test results, as summarized in Table 2, indicated that the following isolates produced significant amounts of ACC deaminase: SGI-003-H11, SGI-026-G06, SGI026-G07, and SGI-041-B03.
2,3-Butanediol production:
[00136] The ability of bacterial isolates to synthesize 2,3-butanediol in vitro was evaluated as follows using capillary gas chromatography mass spectroscopy, as
66/83 described by Ryu et al. (Proc. Natl. Acad. Sci. USA 100: 4927-4932, 2003). This assay was normally performed in a high throughput format using 96-well cell culture plates with 150 µl DF salt growth medium per well. A titer-tek can also be used when preparing a large number of plates for primary fabrics from large collections of isolates. Cultures and media were distributed and transferred aseptically using an autoclavable precision tool under a laminar flow hood. After transfer, cultures were incubated at 30 ^o C for 5 days. After incubation, culture supernatants were harvested by centrifugation using a 0.22 μΜ 96-well filter plate. 50 microliters of filtered supernatant from each well were transferred to the corresponding wells of a 96-well deep plate containing 450 μL 50% methanol per well, using an L200 multichannel pipette and sealed with an adhesive plate seal, followed by quantification of 2,3-butanediol using the protocol described by Ryu et al. (2003, supra). The test results, as summarized in Table 2, indicated that the following isolates produced significant amounts of 2,3-butanediol: SGI003-H11, SGI-034-C09, and SGI-041-B03.
Auxine production:
[00137] Auxins are hormones that can directly affect plant growth. This assay was performed to determine whether bacterial isolates produced auxins, since many endophytic and rhizosphere bacterial isolates are known to have biochemical pathways that synthesize auxin indole-3-acetic acid (IAA) and its derivatives. Tryptophan is often a precursor to this synthesis; and therefore, this assay quantified the production of IAA (auxin) from bacterial isolates grown in a medium supplemented with a low concentration of the amino acid tryptophan.
67/83 [00138] This assay was normally performed in a high throughput format using 96-well cell culture plates with 150 µl of YT growth medium per well. When preparing a large number of plates for primary screens from large collections of isolates, a titer-tek was used. Cultures and media were distributed and transferred aseptically using an autoclavable precision tool under a laminar flow hood. After transfer, cultures were incubated at 30 ^o C for 5 days. After incubation, culture supernatants were harvested by centrifugation using a 0.22 μΜ 96-well filter plate. Ten microliters of filtered supernatant from each well was transferred to a Falcon assay plate. Two hundred microliters of the Salkowsky assay solution (Gordon and Weber, Plant Physiol. 26: 192-195, 1951) were added to each of the supernatants and standard wells, followed by incubation at room temperature for 15 to 20 minutes. The reaction was monitored by plate absorbance over SpectroMax ax2 at 535 nm as the color change from yellow to purple / pink in the Salkowsky test solution was proportional to the concentration of auxin (IAA) in each well. The test results, as summarized in Table 2, indicated that the following isolates produced significant amounts of phytohormone auxin: SGI003-H11, SGI-020-A01, SGI-034-C09, SGI-034-C09, and SGI-041- B03.
Table 2: Biochemical characteristics of bacterial isolates (ND: not detectable).
Bacterial isolates Biochemical activity Isolated ID TaxonomyProvisional Auxine production ACCdeaminas and 2,3butanedio l Nfixation Phosphorus solubilization 003_H11 Pantoea agglomerans Yes Yes Yes ND ND
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020_A01 Bacillus thuringiensis Yes ND ND ND ND 026_G06 Burkholderiametallica ND Yes ND ND ND 026_G07 Burkholderia vietnamiensis ND Yes ND Yes ND 034_C09 Bacilluspumilus Yes ND Yes ND ND 034_E10 Herbaspirillumsp. ND ND ND ND ND 041_B03 Pedobactersp. Yes Yes Yes ND ND
[00139] EXAMPLE 5: Biocontrol activity of bacterial isolates against fungal phytopathogens [00140] An in vitro antagonism assay was used to assess the ability of isolated bacterial strains to suppress the development of various fungal pathogens, including Fusarium graminearum NRRL-5883, Monographella nivalis ATCC MYA-3968, Gibberella zeae ATCC-16106, Stagnospora nodurum ATCC-26369, Colletotrichum gramnicola ATCC-34167, and a pathogen Penicillium sp. The test was performed on potato dextrose agar (PDA). Strains isolated from bacteria were grown on Triptych soy broth agar with a strength of one fifth (TSBA / 5) for 24 h before use.
[00141] For each fungal pathogen, a conidial inoculum was produced by hyphae, actively dropping a growing colony of the fungus and transferring the hyphae strains to PDA agar medium. After incubating the plates for 7 days at 25 ^o C using a 12 h / day photoperiod, fungal conidia were washed from PDA plates using a weak phosphate buffer (0.004% phosphate buffer, pH 7.2, with 0.019% MgCl ₂ ). A suspension of
69/83 fungal conidia in the weak phosphate buffer (approximately 1 χ 10 ⁵ conidia / mL) was then immediately sprayed onto the agar surface, and the sprayed plates were then incubated at 25 ^o C for 48 to 72 h before use in antagonism tests.
[00142] To start the antagonism tests, cells of isolated microbial strains were inoculated by spot at equal distances within the perimeter of the plate. After five days, the bacterial strains were marked as positive antibiosis when there was a visibly clear area (ie, growth inhibition zone) with no mycelial growth around the perimeter of the microbial colonies. The results of the antagonism assays, as summarized in Table 3, demonstrated that each of the microorganisms disclosed in this document inhibited the development of several fungal phytopathogens, including Fusarium graminearum, Monographella nivalis, Gibberella zeae, Stagnospora nodurum, Colletotrichum gramnicola, Penicillium spnicola .
Table 3: Biocontrol activity of bacterial isolates against fungal phytopathogens
Bacterial isolates Growth suppression of fungal pathogens (marked inhibition zone after 5 days of incubation) Isolated ID TaxonomyProvisional Fusariu m gramine arum Monogr aphella nivalis Gibber ella zeae Stagnos pora nodurum Colletotric um gramnicol a Penicilli sp. 003_H11 Pantoea agglomerans No Yes No No No No 020_A01 Bacillus thuringiensis Yes No Yes Yes Yes No
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026_G06 Burkholderiametallica No Yes Yes Yes Yes Yes 026_G07 Burkholderia vietnamiensis No Yes No No No No 034_C09 Bacilluspumilus No No No Yes No No 034_E10 Herbaspirillu m sp. No No No Yes No No 041_B03 Pedobactersp. No Yes No Yes Yes Yes
EXAMPLE 6: Intensification of wheat yield potential [00143] Effects of bacterial inoculation on plant growth and yield were studied in a greenhouse with isolate SGI-020-A01. Microbial cell suspensions were prepared as follows. Broth cultures, 2YT media, or similar growth media, were inoculated from glycerol stocks of the isolate or streak plates. Normally, prior to use in the growth chamber, greenhouse, or field, bacterial cultures were started 48 to 72 hours to allow the cultures to reach the final exponential phase. Isolates that have longer doubling times have already started early. The cultures were incubated at 30 ° C on a rotary shaker at 200 rpm. After growth, the cells were pelleted at 10,000 χ g for 15 min at 4 ^o C and resuspended in 10 mM MgSO ₄ buffer (pH7.0). Cell densities were normalized for each isolate on a CFU / mL basis. Normally, suspensions of ~ 10 ⁹ CFU / mL were prepared for each isolate and transported on ice to the inoculation site. Inoculations were performed by diluting these cell suspensions 1/20 in the irrigation water to a final density of 5 and 10 ⁷ CFU / mL. For 1 liter vessel tests, 20 mL of the suspension of
71/83 diluted cells were evenly distributed over the surface of each replication vessel.
[00144] The greenhouse test was conducted with field soil deficient in nutrients. After removing debris and large rocks, the field soil was carefully mixed to ensure homogeneity. After filling, the soil in each pot was pressed down ~ 2 cm to a layer of solid seed. Seeds from a commercial wheat crop (durum red spring wheat; Howe Seeds, Inc.) were sown in 1 liter pots containing field soil medium (10.5 cm x 12.5 cm tapered diameter plastic pots ). Two grams of spring wheat seeds (approximately 70 seeds) were evenly distributed in each pot and 50 mL of field soil was applied and spread evenly over the seed layer. Following the uniform appearance of wheat coleoptile and subsequent appearance of the first leaf, the plant population was inoculated with 20 mL of 10 ⁹ CFU / mL of SGI-020-A01. Negative control plants received 20 mL of inoculum buffer only. Each condition was performed on 8 replication plans, each containing four 1 liter vessels (n = 4 per plan). The plans were randomly distributed in four experimental blocks. The seeds and plants were then kept in a greenhouse for 60 days at room temperature (ranging from about 8 ^o C to about 22 ^o C) with daytime light cycles of approximately 11.5 hours of sunlight / 12 hours dark for the entire rehearsal. The plants were uniformly watered at the bottom to an appropriate level of hydration, depending on temperature and growth stage. In approximately 30 days after sowing, approximately 70 individuals per pot were stacked and loosely tied to avoid cross-contamination and minimize positional effects, due to
72/83 variation in plants falling to other pots. In approximately 60 days after sowing, the plants were allowed to dry in preparation for harvest. Wheat heads were harvested approximately 80 days after sowing. Each head of wheat was removed by cutting just below the head. Wheat heads within each replication of the pot were grouped, weighed, and later used as an estimate of yield potential. All plants in the population were harvested on the same day and treatments were harvested in a random order to eliminate large differences in time between harvest between treatments. As a result, wheat plants treated with SGI-020-A01 isolate showed a 40% increase in potential yield compared to untreated control plants (2.95 g / pot vs. 2.10 g / pot) . The means and standard deviations were documented through all 8 replications and an ANOVA (Analysis of Variance) was performed. The effectiveness of the microbial isolate SGI-020-A01 in enhancing the potential of wheat yield was quantified by analyzing the yield of wheat head by weight for replication of the vessel. P values of <0.05 were considered statistically significant.
EXAMPLE 7: Intensification of biomass production in corn [00145] Effects of bacterial inoculation on plant growth and yield were studied in greenhouse experiments with each of the following bacterial isolates. SGI-034-C09, SGI034-E10, SGI-003-H11, SGI-041-B03, SGI-026-G06, and SGI-026-G07. The greenhouse tests were conducted with field soil deficient in nutrients. After removing debris and large rocks, the field soil was carefully mixed with pot soil (70:30) to ensure homogeneity. After filling, the soil in each pot was pressed to
73/83 low ~ 2 cm for a layer of solid sowing. Seeds from a commercial corn crop (Dow AgroSciences) were sown in 1 liter pots (10.5 cm x 12.5 cm conical pots) each containing soil medium. Two corn grains were distributed evenly in each pot in the orientation of the embryo, followed by the application of 50 mL of field soil, which was spread evenly over the seed layer. After germination, the slaughter of one seedling per pot is performed if necessary so that each pot contains only one plant.
[00146] After the uniform appearance of corn coleoptyl and subsequent appearance of the first leaf, the plant population was inoculated with ~ 20 mL of 10 ⁹ CFU / ml of a microbial isolate selected from the group SGI-034-C09, SGI -034-E10, SGI-003H11, SGI-041-B03, SGI-026-G06, and SGI-026-G07. The microbial cell suspensions were prepared as described in Example 6 above. Negative control plants received 20 mL of inoculum buffer only.
[00147] Each condition was performed on 8 replication plans, each containing two 1 liter vessels (n = 2 per plan). The plans were randomly distributed in four experimental blocks. The seeds and plants were then kept in a greenhouse for 60 days at room temperature (ranging from about 8 ^o C to about 22 ^o C) with daytime light cycles of approximately 11.5 hours of sunlight / 12 hours dark for the entire rehearsal. The plants were uniformly watered at the bottom to an appropriate level of hydration, depending on temperature and growth stage. The crushed biomass above corn was harvested approximately 60 days after sowing.
[00148] All plants in the population were harvested on the same day and treatments were harvested in a random order to
74/83 eliminate large differences in time between harvest between treatments. Corn plants were analyzed for the difference in total biomass. As documented in Table 4, corn plants treated with each of the microbial isolates showed a significant increase in total biomass compared to untreated control plants. The means and standard deviations were documented through all 8 replications and an ANOVA (Analysis of Variance) was performed.
Table 4: Effectiveness of microbial isolates in intensifying the total biomass of the plant.
Treatment Biomass ofplant (g) P value Increase in biomass (%) gives Untreated 58.6 AT AT SGI-034-C09 106.3 <0.0001 181% SGI-034-E10 103.6 <0.0001 177% SGI-003-H11 100.7 <0.0001 172% SGI-041-B03 99.5 0.0001 170% SGI-026-G06 98.3 0.0002 168% SGI-026-G07 97.3 0.0003 166%
EXAMPLE 8: Coating treatment of wheat seeds and corn seeds [00149] Small-scale seed treatment experiments were conducted after a procedure described in Sudisha et al. (Phytoparasitica, 37: 161-169, 2009) with minor modifications. Typically, a biopolymer stock solution was made by adding 1 gram of powdered gum arabic (MP Biomedical) to 9 mL of water and mixing for homogeneity. Cloudy cultures of microbial spore preparations or microbial cells actively growing were washed with PBS and adjusted to OD600 of ~ 5.0. Three mL of adjusted cell suspension was
75/83 pelleted by centrifugation in a 50 mL Falcon tube. The resulting supernatant was decanted, replaced with 3 ml of biopolymer stock solution and the resulting suspension was mixed thoroughly. Typically, about 25 g of seeds were added to the Falcon tube and vigorously shaken or vortexed to ensure uniform distribution of the gum / cell suspension. Coated seeds were spread on plastic weighing boats to dry in a laminar flow hood, until they were no longer sticky, usually 3 hours with periodic mixing. The coated seeds were then stored at 4 ^o C and periodically tested for stability. A variety of wheat and corn seeds were coated and tested in the manner described above, including the common spring red durum wheat varieties Briggs Faller, Glenn, Hank, RB07, Samson; winter durum red wheat varieties Jerry, McGill, Overland; and corn seed variety DKC62-61, as well as a commercial corn crop (Dow AgroSciences).
[00150] The viability test of the microbes used in the seed coating formulation was performed using a standard plate counting method. Normally, a predetermined amount of coated seeds was tested by the presence of viable microbes by washing the seeds in an aliquot of the appropriate buffer and plating equivalent amounts of buffer on nutrient agar media. Viable colony-forming units were determined after incubation for 1 to 4 days at 30 ^o C. The viability test showed that between 1 χ 10 ⁴ and 4 χ 10 ⁷ viable colony-forming units per seed were present after approximately five weeks storage at 4 ^o C. When the seeds were coated with microbial spores, the viability of most of the microbes tested remained stable for at least
76/83 minus four months, including several interstate shipments from the United States, inside and outside refrigerated containers. When stored under refrigeration (4 ^o C), microbes survived in the seed coating with little loss of viability during the test periods. The results indicated that seeds coated with the compositions disclosed in this document could be stored for long periods under refrigeration and pointed out that the microbes would survive during periods of higher temperatures for distribution. In addition, the germination rate of the coated seeds was tested and determined to be essentially identical to that of the control seeds, which were also seeds coated with gum arabic only or uncoated seeds.
EXAMPLE 9: Solid State Formulation of Microbial Compositions [00151] This section describes an exemplary formulation of a microbial fertilizer, where the bacteria in accordance with the present invention are encapsulated and the fertilizer is in solid form. Alginate granules are prepared as follows:
[00152] One milliliter of 30% glycerol is added to 1, 1.5 or 2% sodium alginate solution, depending on the properties of the alginate (M / G ratio) to obtain a final volume of 25 ml. Bacterial cells from a 250 ml culture obtained from one of the bacterial isolates of the invention or a combination of two or more isolates are pelleted via centrifuges, then washed with a saline solution (0.85% NaCl, p / v), suspended in 25 ml of alginate mixture, and mixed thoroughly. This cell suspension is added dropwise in 1.5 or 2% (w / v) of a pre-cooled sterile aqueous solution of CaCl2 with gentle agitation to obtain the bacterial alginate granules. These granules are allowed to harden for 2 to 4 h at room temperature. The granules
77/83 are collected by sieving and washed several times with sterile water and stored at 4 ^o C. In order to preserve the formulation, the fresh wet granules can be frozen at about -80 ^o C before freeze-drying at about 45 ^o C for 15 h. The freeze-dried dry granules can be stored in appropriate containers, such as sterile glass bottles.
[00153] To estimate viable counts, encapsulated bacteria can be released from granules by resuspending 100 mg of granules in phosphate buffered saline (pH 7.0) for 30 min, followed by homogenization. The total number of bacteria released is determined by the standard plaque counting method after incubation at 30 ^o C for 48 h. At one month intervals, the cell densities in the granules are listed using a similar method.
EXAMPLE 10: Compatibility of microbial compositions with commercial fungicides [00154] As environmental concerns are increasing in relation to the use of pesticides in agriculture, biological alternatives are increasingly perceived as inevitable. However, new biological formulations must also allow organisms to survive and express their specific beneficial impact. Chemical fungicides are generally toxic not only to harmful microorganisms, but also to beneficial ones. However, the chance of survival of these microbial agents may have been intensified when applied at reduced rates.
[00155] In the present study, the peat-based carrier material is used to inoculate both the treated fungicide as well as seeds from own cultures. The bacterial tolerability of the fungicide is generally assessed as follows: a) own seeds inoculated by bacteria grown in a growth medium
78/83 appropriate bacterial such as trypticase soy agar plates (TSA; Tryptone 15g / L; Soytone 5g / L, sodium chloride 5g / L and agar 15g / L), b) seeds inoculated by bacteria treated with fungicide grown in common TSA plates and, c) seeds inoculated by fungicide-treated bacteria grown in sterile growth bags. Typically, three concentrations of fungicide are used in each of the experiments: the manufacturer's recommended dose and two lower doses (at 75% and 50% of the recommended dose). Seeds inoculated by fungicide-treated bacteria are stored after inoculation and used at different time intervals (2 h, 4 h and 6 h) to examine the impact of seed germination. Both seed germination and bacterial presence are monitored in petri dishes. For the growth bag study, seeds treated with fungicide (recommended dose) are used, and the lengths of the root and hippocotyl were measured in 7 days of seedling growth.
[00156] Some rhizobacterial isolates of the invention are compatible with various fungicides commonly used as determined by bacterial growth in TSA plates enriched with fungicide. In general, both seeds treated with fungicide and pure, coated with inoculated peat do not show any significant variation in germination in relation to the uninoculated control. In addition, growth promotion effects in total seedling and root lengths are observed in all rhizobacterial treatments in relation to uninoculated control.
EXAMPLE 11: Development of non-naturally occurring crops and breeding program [00157] Endophytic bacteria of the present invention are introduced into crop plants, including cereals, of different genotypes and geographic origin, without such endophytic fungi, to create
79/83 plant-endophyte combinations with improved agronomic characteristics, using procedures analogous to those known in the art, including those described in U.S. Patent Application No. 20030195117A1, U.S. Patent Application No. 20010032343A1; and U.S. Patent No. 7,084,331, among others. In this way, synthetic plant-endophyte combinations can be created and selected in a breeding / cultivation development program based on their ability to form and maintain a mutualistic combination that results in an agronomic benefit. The evaluation of the agronomic 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 for farm animals (eg herbivores), ease of reproduction and seed yield, among others. These combinations may differ in levels of accumulation of microbial metabolites that are toxic to pests and weeds, including levels of ergot alkaloids, levels of lolina, levels of peramine, or levels of lolitrem, exhibiting desired agronomic characteristics of crop plants, including resistance to feeding or insect infestation, resistance to abiotic stress, palatability for farm animals, biomass accumulation, ease of reproduction, and seed yield, among other traits.
EXAMPLE 12: Yield Study [00158] Corn seeds (Zea mays) were coated with different microbial treatments and sown in a prepared field. Each treatment was replicated 5 times in the complete randomized block design. A single replication consisted of four long beds of 30 feet (rows), 60 seeds were sown (6 inches apart) in each bed. For observation purposes, data were
80/83 taken from the two intermediate lines only.
[00159] The plant's appearance was recorded twice, as shown in Table 5 below as the percentage of plants in the replication that had sprouted. Ten plants in the two intermediate lines of each plot were marked with a plastic tape to record vital statistics, such as plant height, chlorophyll measurement, plant weight, etc. of the plants.
[00160] The plant heights (measuring the highest / longest leaf tip) recorded 31 and 56 days after planting indicated that the plant height between treatments was not significantly different. Most of the plants were dry and the leaves shrank on day 110 after planting, therefore, in some cases, plants considered (measured) smaller than in the previous measurement, but in general, the plant height did not differ between treatments. In ^the 5 weeks of planting, chlorophyll content was measured (units in SPAD) of the lower leaves (approximately 60 cm above ground) and upper sheets (second fully expanded leaf from the top) of ten plants of each plotting. The chlorophyll content between treatments did not differ significantly. On day 110 and 111 of planting, the crop was harvested. Ten marked plants from each plot were cut at ground level and above ground, parts of the plants were weighed (weight of the entire plant or WPtWt), ears of corn were removed and the ear length was measured (ear length with grains ) (region filled with negotiable grains only), then the grains were removed from the ear and their weight was measured for the weight of the grain per ear (KnlWt / ear).
[00161] Shortly after the manual harvest of 10 plants per plot was finished, a mechanical harvester, Gleaner® K2 (AllisChalmers Mfrg, Milwaukee, WI) was brought. This machine mechanically removed the remaining plants from two lines
81/83 intermediate of each plot, removed the grains from the ears, and took the measurement of grain moisture and weight (10 ears + harvest in machine). The total projected yield at the moisture content of 15.5% (pounds of corn kernels per acre) based on the weight (lb.) of the kernels (including machine-harvested ears + ears hand-picked) was 10368.14 pounds per acre or 185.15 bushels per acre for SGI-003-H11 (Pantoea agglomerans). This was the highest yield among all treatments of the body and significantly different from the treatments for Bacillus amyloliquefaciens SGI-015_F03. The other top producer was the treatment with Bacillus thuringiensis SGI-020_A01.
[00162] In conclusion, all the plants in the different treatments appeared and grew the same in the field conditions provided with the same amount of fertilizers, preemergent herbicides (with manual weed extraction later in the season) and weekly irrigation during the hot season and growing, and pest control especially of the corn caterpillar. With all conditions equal, treatment with SGI-003-H11 (Pantoea agglomerans) produced the highest yield on non-microbe, control treatment. In this way, 003_H11 produced a yield approximately 17% higher than that of the control group. Thus, in various embodiments of the invention the application of an effective amount of the organism according to any of the methods described in this document produces at least 10% or at least 12.5%, or at least 15% or about 17% of more yield than a control group, which in some modalities can be determined per acre of plant product (for example, ears of corn) per acre or bushels of plant product per acre. The organisms listed in Table 5 are SGI-003-H11 (Pantoea agglomerans); SGI-015-F03 (Bacillus amyloliquefaciens); and SGI-02082/83
A01 (Bacillus thuringiensis).
Table 5
Body Emergence Emergence Height Height Height Date 6/20 6/24 7/11 5/8 9/28 Control 97.00 94.50 103.64 277.22 267.82 SGI-003-H11 89.50 93.34 103.32 273.58 272.04 SGI-015-F03 95.67 96.67 105.02 282.02 276.08 SGI-020-A01 94.83 95.33 102.04 268.94 264.80
Body Top sheet Bottom sheet WPtWt Ear length w / knls Date 7/18 7/18 9/28 9/28 Control 43.24 60.85 493.24 14.98 SGI-003-H11 44.17 59.43 494.92 14.29 SGI-015-F03 44.90 63.96 473.40 14.45 SGI-020-A01 46.45 63.84 488.30 14.20
Body Knl for ear (g) Knl for ear (lb) 10 ears + machine 10 ears + machine Date 9/28 9/28 9/29 9/29 Control 168.62 0.37 157.81 8837.63 SGI-003-H11 171.96 0.38 185.15 10368.14 SGI-015-F03 156.24 0.34 161.17 9025.62 SGI-020-A01 158.68 0.35 163.09 9133.16 [00163] A number of modalities of the invention has been described. At the
However, it will be understood that elements of the modalities described herein can be combined to make additional modalities and various modifications can be made without abandoning the spirit and scope of the invention. In this sense, other modalities, alternatives and equivalents are within the scope of the invention as described and
83/83 claimed here.
[00164] Positions within the application are only for the convenience of the reader and do not in any way limit the scope of the invention or its modalities.
[00165] All publications and patent applications mentioned in this specification are incorporated into this document by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

权利要求:
Claims (9)
[1]
1. Composition, characterized by the fact that it comprises a strain of Pantoea agglomerans SGI-003-H11, deposited as NRRL B-50483; wherein the composition further comprises a carrier; and an agronomically effective amount of a compound or composition selected from the group consisting of a fertilizer, an acaricide, a bactericide, a fungicide, an insecticide, a microbicide, and a nematicide.
[2]
2. Composition according to claim 1, characterized by the fact that said carrier is a plant seed.
[3]
Composition according to claim 1, characterized by the fact that said composition is a seed coating formulation.
[4]
4. Composition according to claim 1, characterized in that said composition is prepared as a formulation selected from the group consisting of an emulsion, a colloid, a powder, a granule, a pellet, a powder, a spray, an emulsion and a solution.
[5]
5. Method for the treatment of a plant seed, characterized by the fact that it comprises a stage of exposure or contact of said plant seed with a strain or microbial culture as defined in claim 1.
[6]
6. Method for improving the growth and / or yield of a plant, characterized by the fact that it comprises the application of an effective amount of a strain or microbial culture as defined in claim 1, to the plant, or around the plant.
[7]
Method according to claim 6, characterized by the fact that said strain or microbial culture is grown in a growth medium or soil of a previous host plant or
Petition 870190025394, of 03/18/2019, p. 4/9
2/2 simultaneously with the growth of host plants in said growth medium or soil.
[8]
8. Method according to claim 6, characterized in that said plant is a corn plant or a wheat plant.
[9]
9. Method according to claim 6, characterized by the fact that said strain or microbial culture is established as an endophyte in such a plant.

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法律状态:
2018-03-27| B27A| Filing of a green patent (patente verde)|

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

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2018-04-17| B27B| Request for a green patent granted|

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2018-05-02| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2018-05-02| B15K| Others concerning applications: alteration of classification|Ipc: A01N 63/00 (2006.01), C12N 1/20 (2006.01) |

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2018-08-28| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2018-12-18| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2019-03-26| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2019-05-28| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/12/2012, OBSERVADAS AS CONDICOES LEGAIS. (CO) 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/12/2012, OBSERVADAS AS CONDICOES LEGAIS |

优先权:

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申请号 | 申请日 | 专利标题

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US201161570237P| true| 2011-12-13|2011-12-13|

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US61/570,237|2011-12-13|

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PCT/US2012/069579|WO2013090628A1|2011-12-13|2012-12-13|Plant growth-promoting microbes and uses therefor|

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