NZ625835B2 - Plant growth-promoting microbes and uses therefor - Google Patents

Abstract

Disclosed is an isolated microbial strain SGI-003-H11 (Pantoea agglomerans) deposited as NRRL B-50483. Further, disclosed is an isolated microbial strain comprising a DNA sequence exhibiting at least 99% sequence identity to the nucleotide sequences of SEQ ID NO:1 in the Sequence Listing; and further wherein said microbial strain has a plant growth-promoting activity. r wherein said microbial strain has a plant growth-promoting activity.

Description

PLANT GROWTH-PROMOTING MICROBES AND USES THEREFOR This application claims the benefit of US. provisional application 61/570,237, filed December 13, 2011 and which is incorporated by reference herein in its entirely, including all , , and claims.

FIELD OF THE INVENTION The present invention relates to the field of sustainable agriculture. Specifically, the disclosure provides microbial itions and methods useful for the production of crop plants. In particular, the compositions and methods disclosed herein are useful for enhancing plant growth and/or suppressing the development of plant pathogens and pathogenic diseases.

INCORPORATION OF SEQUENCE LISTING The material in the accompanying Sequence Listings is hereby incorporated by reference into this application. The accompanying file, named “SGIl540_1WO_CRF_OF_SL_ST25.txt” was created on December 13, 2012 and is 20 KB.

The files can be ed using Microsoft Word on a computer that uses Windows OS.

BACKGROUND OF THE INVENTION The microflora surrounding plants is very diverse, ing bacteria, fungi, yeast, algae. Some of these microorganisms may be deleterious to plants, and are often referred to as pathogens, while others may be beneficial to plants by promoting plant growth and crop productivity. Recent advances in soil iology and plant biotechnology have resulted in an increased interest in the use of microbial agents in agriculture, horticulture, forestry and nmental management. In particular, a number of microorganisms known to be present in soil ecological niche, generally known as rhizosphere and rhizoplane, have received considerable attention with respect to their ability to promote plant . Indeed, the rhizosphere soil ents a good reservoir of microbes for the potential ion of beneficial microbes. Plant rhizosphere can n ns of microorganisms in one gram of soil. In theory, microbial inoculants, without human intervention, have a low survival rate and efficacy in their natural soil environment because of the insufficient colony forming units per gram of soil. Therefore, since the 1960’s, a number of biofertilizers that have an increased 2012/069579 colony um potential concentration have been developed and commercialized in an attempt to reduce the need for chemical fertilizers.

In addition, research ted in recent years has shown that microorganisms can be used as biological l agents to increase agricultural tivity and efficiency.

These studies have shown that various rganisms are able to suppress plant pathogens and/or supplement plant growth, thus offering an attractive alternative to chemical pesticides with are less favored because of their ially negative impact on human health and environment quality.

Microorganisms which can colonize plant roots and stimulate plant growth are generally known as plant growth-promoting microbes (PGPM). In the past two decades, many PGPM species having positive influence on the grth of a wide variety of crop plants have been reported. PGPM are often universal symbionts of higher plants, and are able to enhance the adaptive potential of their hosts through a number of mechanisms, such as the fixation of molecular nitrogen, the mobilization of recalcitrant soil nutrients (e.g., iron, phosphorous, sulfur etc), the synthesis of phytohormones and vitamins, and the decomposition of plant materials in soils which often increases soil organic matter. Also, certain microbes can tate plant growth by controlling microbial species pathogenic to the plant (i.e., phytopathogens). For example, some beneficial microbes can control root rot in plants by competing with filngi for space on the e of the plant root. In other instances, competition among various microbial strains in a s native microflora can stimulate root growth and increase the uptake of mineral nutrients and water to enhance plant yield.

Therefore, biofertilizers can be developed as products based on microorganisms that naturally live in the soil. By increasing the population of ial microorganisms in the soil through artificial inoculation, these soil rganisms can boost their biological activity and, thus, supply the plants with important nutrients and beneficial factors that enhance their growth.

The inoculation of cultivated plants with PGPM is generally seen as a promising agricultural approach, for it allows pests to be controlled without using ides in large amounts. As environmental concerns about groundwater quality with excess fertilizer and pesticide exposure in foods grow, biological alternatives are becoming necessary. Thus, developing biological treatment compatible with fertilizers and ides or even reducing the amount of these chemical compounds could be a significant advancement in the agricultural industry. It has been ished that stimulation of plant growth by PGPM is 2012/069579 often closely related to the ability of the PGPM to colonize plant roots. However, vely little attention has been given to the development of efficient selection procedures for obtaining microbial s with high root-colonizing ability. The lack of such selection procedures slows down the study of plant-bacterial symbioses, and the ment of PGPM in agriculture.

Therefore, there is a continuing need for the identification of new PGPM and/or testing of their compatibility with existing commercially available crop management products. Moreover, additional investigation is also needed to compare pure culture strains versus complementary mixed strains of microorganisms that form synergistic consortia. Such mixed consortia might have greater potential for consistent performance with better itive ability under different environmental and growth conditions.

SUMMARY OF THE INVENTION Microbial s and cultures are provided herein. Microbial itions and methods of use thereof to enhance the growth and/or yield of a plant are also ed. Also provided are methods for the treatment of plant seeds by using the microbial compositions disclosed herein. Further provided are methods for preventing, inhibiting, or treating the development of plant pathogens or the development of phytopathogenic diseases. The disclosure also provides non-naturally occurring plant varieties that are varieties artificially infected with a microbial endophyte of the invention. Seed, uctive tissue, vegetative tissue, regenerative tissues, plant parts, or progeny of the non-naturally occurring plant varieties are also provided. The disclosure further provides a method for preparing agricultural compositions.

In one aspect, the present disclosure provides isolated microbial strains, isolated cultures thereof, biologically pure cultures thereof, and enriched es thereof. In n preferred embodiments of this aspect, the microbial strain can be 3-Hll ited as NRRL B-50483); SGI-OZO-AOl ited as NRRL B-50484); SGIG06 (deposited as NRRL B-50485); 6-G07 (deposited as NRRL B-50486), or a strain derived from any one of said strains. In some other preferred embodiments, the microbial strain can comprise a nucleotide or amino acidsequence that exhibits 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% sequence identity to any one of the 168 ribosomal and/or recA nucleotide sequences and/or amino acid sequences in the ce Listing. In some embodiment the microbial strain also has a plant growth-promoting activity as described herein.

Also provided are microbial itions that include a microbial strain of the invention or a culture thereof. Such microbial compositions according to some red embodiments may comprise an agriculturally effective amount of an additional compound or composition, in which the additional compound or composition may be a fertilizer, an acaricide, a bactericide, a fungicide, an insecticide, a microbicide, a nematicide, or a pesticide. In some other preferred embodiments, the microbial compositions may fiarther include a carrier. In yet other preferred embodiments, the carrier may be a plant seed. In certain embodiments of this aspect, the microbial ition is prepared as a formulation that can be an emulsion, a colloid, a dust, a granule, a pellet, a powder, a spray, an on, or a solution. In some other preferred embodiments, the microbial compositions may be seed coating formulations. In yet another aspect, plant seeds that are coated with a microbial composition in accordance with the present invention are also ed.

In another aspect, there are provided methods for ng plant seeds. Such methods include exposing or contacting the plant seeds with a microbial strain according to the present invention or a culture f.

In another aspect of the invention, provided herein are methods for enhancing the growth and/or yield of a plant. In some embodiments, such method involves applying an ive amount of a microbial strain in accordance with the present invention or a culture f to the plant, or to the plant's surroundings. In some other embodiments, the method involves growing a microbial strain in accordance with the t invention or a e f in a grth medium or soil of a host plant prior to or concurrent with host plant growth in said grth medium or soil. In preferred embodiments, the plant may be a corn plant or a wheat plant. In some other embodiments, the microbial strain or culture thereof may be established as an endophyte on the plant.

In another aspect of the present invention, there are provided s for preventing, inhibiting or treating the development of a plant pathogen. Such methods include growing a microbial strain according to the invention or a culture thereof in a growth medium or soil of a host plant prior to or concurrent with host plant grth in said grth medium or soil. In some preferred embodiments, the plant pathogen may be a microorganism of the genus Colletotrz'chum, Fusarz'um, Gibberella, Monographella, Penicillium, or Stagnospora.

In some particularly preferred embodiments, the plant pathogen may be Colletotrz'chum graml'nz'cola, Fusarz'um graml'nearum, Gibberella zeae, Monographella nivall's, Penicillium sp., or Stagnospora nodurum.

Another fiarther aspect of the invention provides methods for preventing, inhibiting or treating the development of plant pathogenic e of a plant. Such s e applying to the plant, or to the plant's surroundings, an effective amount of a microbial strain according to the invention or a culture thereof. In some preferred embodiments, the microbial strain or a culture f may be applied to soil, a seed, a root, a flower, a leaf, a portion of the plant, or the whole plant.

Another further aspect of the invention provides non-naturally occurring plants.

The turally occurring plants are artificially infected with a microbial strain of the invention or a culture thereof Further provided in some embodiments of this aspect are seed, reproductive , tive , regenerative tissues, plant parts, and progeny of the non-naturally occurring plants.

Another aspect of the invention provides methods for preparing an agricultural composition. Such methods involve ating the microbial strain according to the present ion or a culture f into or onto a substratum and allowing it to grow.

In another aspect the invention provides an isolated strain, an isolated culture thereof, a biologically pure culture thereof, and an enriched culture of a microorganism of the genus Pantoea. In one embodiment the rganism comprises a DNA sequence or amino acid sequence coding 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% sequence identity to a sequence coding for 16S rRNA gene or recA n disclosed in the Sequence Listing. In another embodiment the invention provides a genus of microorganisms comprising any of the DNA sequences or amino acid sequences described above and which enhances the grth and/or yield of a plant, as described herein.

These and other objects and features of the invention will become more fully apparent from the following detailed description of the ion and the claims.

DETAILED DESCRIPTION OF THE INVENTION Unless otherwise , all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such ions herein should not necessarily be ued to ent a substantial difference over what is generally understood in the art. Many of the techniques and ures described or referenced herein are well understood and commonly employed using conventional methodology by those d in the art.

The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates ise. For example, the term “a cell” includes one or more cells, including mixtures thereof Bactericidal: the term “bactericidal”, as used herein, refers to the ability of a ition or substance to increase mortality or inhibit the growth rate of bacteria.

Biological control: the term “biological control” and its iated form “biocontrol”, as used herein, is defined as control of a pathogen or insect or any other undesirable organism by the use of at least a second organism other than man. An example of known mechanisms of biological control is the use of microorganisms that control root rot by mpeting filngi for space on the surface of the root, or microorganisms that either inhibit the growth of or kill the pathogen. The “host plant” in the context of biological control is the plant that is susceptible to disease caused by the pathogen. In the context of isolation of an organism, such as a bacterium or fungal species, from its natural environment, the “host plant” is a plant that supports the growth of the bacterium or filngus, for example, a plant of a species the bacterium or fiangus is an endophyte of.

An "effective amount", as used herein, is an amount sufficient to effect beneficial or desired results. 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 rate, stabilize, reverse, slow or delay progression of the target infection or disease . The expression "effective microorganism" used herein in nce to a microorganism is intended to mean that the subject strain exhibits a degree of promotion of plant grth and/or yield or a degree of inhibition of a pathogenic disease that exceeds, at a statistically significant level, that of an untreated control. In some instances, the expression "an effective amount" is used herein in reference to that ty of microbial treatment which is necessary to obtain a beneficial or desired result relative to that ing in an untreated control under suitable conditions of treatment as described herein. For the purpose of the t disclosure, the actual rate of application of a liquid formulation will usually vary from a minimum of about l X 103 to about l X 1010 viable cells/mL and preferably from about l X 106 to about X 109 viable cells/mL. Under most conditions, the strains of the invention described in the es below would be optimally effective at application rates in the range of about l X 106 to l X 109 viable cells/mL, assuming a mode of application which would achieve substantially uniform t of at least about 50% of the plant tissues. If the microorganisms are applied as a solid formulation, the rate of application should be controlled to result in a comparable number of viable cells per unit area of plant tissue surface as obtained by the entioned rates of liquid treatment. Typically, the microbial compositions of the present invention are biologically effective when delivered at a concentration in excess of 106 CFU/g (colony g units per gram), preferably in excess of 107 CFU/g, more preferably 108 CFU/g, and most preferably at 109 CFU/g.

Composition: A “composition” is intended to mean a ation of active agent and at least another compound, carrier, or composition, which can be inert (for example, a detectable agent or label or liquid carrier) or active, such as a fertilizer.

A "control plant", as used in the present disclosure, provides a nce point for measuring changes in phenotype of the subject plant, may be any suitable plant cell, seed, plant component, plant tissue, plant organ or whole plant. A control plant may comprise, for example, (a) a wild-type plant or cell, z'.e., of the same genotype as the starting material for the c alteration which resulted in the subject plant or cell; (b) a plant or cell of the genotype as the starting material but which has been transformed with a null construct (i.e., a construct which has no known effect on the trait of st, such as a construct comprising a reporter gene); (c) a plant or cell which is a non-transformed segregant among progeny of a subject plant or cell; (d) a plant or cell which is genetically identical to the t plant or cell but which is not exposed to the same treatment (e.g., fertilizer treatment) as the t plant or cell; (e) the subject plant or cell itself, under conditions in which the gene of interest is not sed; or (f) the subject plant or cell itself, under conditions in which it has not WO 90628 been exposed to a particular treatment such as, for example, a fertilizer or combination of fertilizers and/or other chemicals.

Culture, isolated e, biologically pure culture, and enriched e: As used herein, an isolated strain of a microbe is a strain that has been removed from its natural milieu. As such, the term ted" does not necessarily reflect the extent to which the microbe has been purified. But in different embodiments 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 a non-limiting example, if a culture is isolated from soil as raw material, the organism can be isolated to an extent that its concentration in a given quantity of purified or partially purified material (e.g., soil) is at least 2x or 5x or 10x or 50x or 100x that in the original raw material. A "substantially pure culture" of the strain of e refers to a culture which contains substantially no other microbes than the d strain or s of microbe. In other words, a substantially pure e of a strain of microbe is substantially free of other inants, which can include microbial contaminants as well as undesirable al contaminants. Further, as used herein, a "biologically pure" strain is intended to mean the strain separated from materials with which it is normally associated in nature. Note that a strain associated with other s, or with compounds or materials that it is not normally found with in nature, is still defined as "biologically pure." A monoculture of a particular strain is, of course, "biologically pure." In different embodiments 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 culture is normally associated with soil in nature, the organism can be biologically pure to an extent that its concentration in a given quantity of purified or partially purified material with which it is normally associated in nature (e.g. soil) is at least 2x or 5x or 10x or 50x or 100x that in the al unpurified material. As used herein, the term "enriched culture" of an isolated ial strain refers to a microbial culture wherein the total microbial population of the culture contains more than 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain .

Culturing: The term ‘culturing’, as used herein, refers to the propagation of organisms on or in media of various kinds.

As used herein, an hyte" is an endosymbiont that lives within a plant for at least part of its life without causing apparent disease. Endophytes may be transmitted either ally (directly from parent to offspring) or horizontally (from individual to unrelated 2012/069579 individual). Vertically-transmitted fiangal endophytes are typically asexual and transmit from the maternal plant to offspring Via fiangal hyphae penetrating the host's seeds. Bacterial endophytes can also be transferred vertically from seeds to seedlings (Ferreira et al., FEMS Microbiol. Lett. 287:8-14, 2008). Conversely, horizontally-transmitted endophytes are typically sexual, and transmit Via spores that can be spread by wind and/or insect vectors.

Microbial endophytes of crop plants have received erable attention with respect to their ability to control disease and insect infestation, as well as their potential to promoting plant growth.

Fungal en: For purposes of this invention it is understood that the use of term fungal pathogen or fungus is intended to include both the sexual (teleomorphic) stage of this organism and also the asexual (anamorphic) stage, also referred to as the perfect and imperfect fungal stages, respectively. For example, the anamorphic stage of Fusarium gramz'nearum is ella zeae.

Fungicidal: As used herein, “fiangicidal” refers to the ability of a ition or nce to decrease the rate of grth of fiangi or to increase the mortality of fungi.

Mutant: As used herein, the term “mutant” or “variant” in nce 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 lderia the “parental strain” is defined herein as the original Burkholderz’a strain before mutagenesis. s or variants may occur in nature without the intervention of man. They also are obtainable by treatment with or by a variety of methods and compositions known to those of skill in the art. For example, a parental strain may be treated with a chemical such as N—methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfone, or by irradiation using gamma, x-ray, or adiation, or by other means well known to those practiced in the art.

Nematicidal: The term icidal”, as used herein, refers to the ability of a substance or composition to increase mortality or inhibit the growth rate of nematodes.

Pathogen: The term "pathogen" as used herein refers to an organism such as an alga, an arachnid, a bacterium, a fungus, an insect, a nematode, a parasitic plant, a protozoan, a yeast, or a virus capable of producing a disease in a plant or animal. The term "phytopathogen" as used herein refers to a enic organism that infects a plant.

Percentage of sequence identity: “percentage of sequence identity”, as used , is determined by comparing two optimally locally aligned ces over a comparison window defined by the length of the local alignment between the two sequences.

The amino acid sequence in the comparison window may comprise ons or deletions (e. g., gaps or overhangs) as compared to the reference ce (which does not comprise additions or deletions) for l alignment of the two ces. Local alignment n two sequences only includes segments of each ce that are deemed to be ently similar according to a criterion that depends on the algorithm used to perform the alignment (e. g. BLAST). The percentage of sequence identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and an (1981) Add. APL. Math. 2:482, by the global homology alignment algorithm of Needleman and Wunsch (J M01. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85: 2444, 1988), by heuristic implementations of these algorithms (NCBI BLAST, WU-BLAST, BLAT, SIM, BLASTZ), or by inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. The term “substantial sequence identity” between polynucleotide or polypeptide ces refers to polynucleotide or polypeptide comprising a sequence that has at least 50% ce identity, preferably at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 96%, 97%, 98% or 99% sequence identity compared to a reference ce using the programs. In addition, pairwise sequence homology or sequence similarity, as used refers to the percentage of residues that are similar between two sequences aligned. Families of amino acid residues having similar side chains have been well defined in the art. These families include amino acids with basic side chains (e.g., , arginine, histidine), acidic side chains (e.g., aspartic acid, ic acid), uncharged polar side chains (e.g., glycine, gine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ranched side chains (e.g., ine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Query nucleic acid and amino acid sequences can be searched against subject nucleic acid or amino acid sequences residing in public or proprietary databases. Such searches can be done using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST V 2.18) program. The NCBI BLAST program is available on the intemet from the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov/Blast.cgi). Typically the ing parameters for NCBI BLAST can be used: Filter options set to "default”, the Comparison Matrix set to M62”, the Gap Costs set to “Existence: 11, Extension: 1”, the Word Size set to 3, the Expect (E threshold) set to le-3, and the minimum length of the local alignment set to 50% of the query ce length. Sequence identity and similarity may also be determined using GenomeQuestTM re (Gene-IT, Worcester Mass. USA).

The term “pest” as used herein refers to an undesired organism that may include, but not limited to, bacteria, fungi, plants (e.g., weeds), nematodes, insects, and other pathogenic animals. “Pesticidal”, as used , refers to the ability of a substance or composition to decrease the rate of growth of a pest, z'.e., an undesired organism, or to increase the mortality of a pest. y: As used herein, "progeny" includes descendants of a particular plant or plant line. Progeny of an instant plant include seeds formed on F1, F2, F3, F4, F5, F6 and uent generation plants, or seeds formed on BC1, BC2, BC3, and subsequent generation plants, or seeds formed on F1BC1, F1BC2, F1BC3, and subsequent generation plants. The designation F1 refers to the y of a cross between two parents that are genetically distinct. The designations F2, F3, F4, F5 and F6 refer to subsequent generations of self— or sib- pollinated progeny of an F1 plant.

Variant: as used herein in reference to a c acid and ptide, the term “variant” is used herein to denote a polypeptide, protein or polynucleotide molecule with some differences, generated synthetically or naturally, in their amino acid or nucleic acid sequences as ed to a reference polypeptide or polynucleotide, respectively. For example, these differences include substitutions, insertions, deletions or any desired combinations of such changes in a reference polypeptide or polypeptide. Polypeptide and protein variants can further consist of changes in charge and/or post-translational modifications (such as glycosylation, methylation. phosphorylation, etc.) The term “variant”, when used herein in reference to a microorganism, is a microbial strain having fying characteristics of the species to which it belongs, while having at least one nucleotide sequence variation or identifiably different trait with respect to the parental strain, where the trait is genetically based able). For example, for a Bacillus thuringiensis 020_AOl strain having a plant growth-promoting activity, identifiable traits include 1) the ability to suppress the development of fungal phytopathogens, including Fusarium earum, Gibberella zeae, spora nodurum, Colletotrichum graminicola; 2) the ability to enhance seed yield in wheat; and 3) having a 168 rRNA gene with nucleotide ce with greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% sequence identity to the 168 rRNA gene of Bacillus thuringiensis 020_A01; can be used to confirm a variant as Bacillus thuringiensis 020_A01.

Yield: As used herein, the term "yield" refers to the amount of harvestable plant material or plant-derived product, and is normally defined as the measurable produce of economic value of a crop. For crop plants, "yield" also means the amount of harvested material per acre or unit of production. Yield may be defined in terms of quantity or quality.

The harvested material may vary from crop to crop, for example, it may be seeds, above ground biomass, roots, fruits, cotton fibers, any other part of the plant, or any plant-derived product which is of economic value. The term "yield" also encompasses yield potential, which is the m obtainable yield. Yield may be dependent on a number of yield components, which may be red by certain ters. These parameters are well known to persons skilled in the art and vary from crop to crop. The term "yield" also encompasses harvest index, which is the ratio between the harvested biomass over the total amount of s.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent ation was cally and individually indicated to be incorporated by reference.

No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants e the right to challenge the accuracy and ence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

The sion of the general methods given herein is ed for illustrative purposes only. Other alternative methods and embodiments will be apparent to those of skill in the art upon review of this disclosure.

Plant Growth-Promoting Microorganisms Diverse plant-associated microorganisms can positively impact plant health and physiology in a variety of ways. These beneficial microbes are generally referred to as plant growth-promoting microorganisms (PGPMs). The term “plant growth-promoting activity”, as used herein, asses a wide range of improved plant properties, including, for example without limitation, improved en fixation, improved root development, increased leaf area, increased plant yield, increased seed germination, increased photosynthesis, or an increased in accumulated biomass of the plant. In various embodiments the improvement is an at least 10% increase or at least 25% increase or at least 50% se or at least 75% increase or at least a 100% increase in the property being measured. Thus, as non-limiting examples, the microbes may produce an above stated percentage increase in nitrogen fixation, or an above stated increase in total root , or in leaf area or in plant product yield (e. g., an above stated percentage increase in plant product weight), or an increased percentage of seeds that germinate within 10 days or 14 days or 30 days, or rate of photosynthesis (e.g., determined by C02 consumption) or accumulated biomass of the plant (e.g., determined by weight of the plant). The plant product is the item — usually but not arily — a food item produced by the plant. The yield can be determined using any convenient method, for example, bushels or pounds of plant product ed per acre of planting. To date, isolated strains of over two dozen genera of microorganisms have been reported to have plant growth- promoting ty and/or biocontrol activity, and new genera and species with similar activities are still being discovered. Additionally, within some bacterial genera, multiple species and subspecies of biocontrol agents have been identified and can be found across multiple spatial , from the global level to farm level, and even on single . rmore, it has been reported that some individual microbial isolates may display trol and/or plant growth-promoting ty not only on the plants or crops from which they were obtained but also on other crops. This indicates the generalist nature of some WO 90628 genotypes, especially those with a wide geographic distribution. As discussed above, if introduced in sufficient numbers and active for a sufficient duration, a single microbial population can have a significant impact on plant health.

Several mechanisms have been postulated to provide an explanation for the positive impact of PGPMs on plant growth enhancement. The beneficial effects of the microorganisms on plant growth can be direct or indirect.

The term “direct plant -promoting microorganism”, for the purpose of this disclosure, refers to a microorganism that can enhance plant grth in the absence of pathogens. As discussed in more detail below, examples of direct plant grth promotion include (a) biofertilization, (b) stimulation of root growth, (c) rhizoremediation, and (d) plant stress control. In addition, several PGPMs have been reported to promote plant growth indirectly via isms of biological control, z'.e., by reducing the level of disease, for example antibiosis, induction of systemic resistance, and competition with pathogens for nutrients and niches.

Biofertilizers: Microbial fertilizers supply the plant with nutrients and thereby can promote plant growth in the absence of pathogen re. Non-limiting es of microbial isolates that can directly promote plant grth and/yield include Ng-fixing bacteria Rhizobz'um and hl'zobium species that, through symbiotic nitrogen fixation, can form s on roots of leguminous plants, in which they convert atmospheric N2 into a which, in contrast to heric N2, can be used by the plant as a nitrogen source. Other examples include Azospz'rz'llum species, which are free-living N2-fixers that can ize and increase yield of cereal crops such as wheat, sorghum, and maize. Despite 'rz'llum ’s N2- fixing capacity, the yield increase caused by inoculation by Azospz'rillum is often attributed to increased root development and thus to increased rates of water and mineral . In this respect, several rhizobacteria like Azotobacter spp. have been reported to be capable of producing a wide array of ormones (e.g., auxins, cytokinins) and enzymes (e.g., pectinase). Many of these phytohormones and enzymes have been shown to be intimately involved in the infection process of symbiotic bacteria-plant associations which have a regulatory influence on nodulation by Rhizobz'um.

In many ces, PGPMs also can affect the plant growth and development by modifying nutrient uptake. They may alter nt uptake rates, for example, by direct effects on roots, by effects on the environment which in turn modify root behavior, and by competing directly for nutrients (Gaskin et al., Agricult. Ecosyst. n. 12: 99-116, 1985).

Some factors by which PGPM may play a role in modifying the nutrient use efficiency in soils include, for example, root geometry, nutrient solubility, nutrient availability by producing plant ial ion form, ioning of the nutrients in plant and ation efficiency. For example, a low level of e phosphate can limit the growth of plants.

Some plant growth-promoting microbes are capable of solubilizing phosphate from either organic or inorganic bound phosphates, thereby facilitating plant growth. Several enzymes of microbial origin, such as nonspecific phosphatases, phytases, phosphonatases, and C-P lyases, release soluble phosphorus from organic compounds in soil. For example, an increased solubilization of inorganic phosphorous in soil has been found to enhance phosphorus uptake in canola seedling using Pseudomonas puticla as well as increased sulfur- oxidation and sulfur uptake ton and Germida, Can. J. Microbiol. 37: 521-529, 1991; Banerjee, Phytochemicals and Health, vol. 15, May 18, 1995).

Phytostimulators: Some microorganisms can produce substances that ate the growth of plant in the e of pathogens. For example, the production of plant hormones is a characteristic of many plant-associated rganisms. For all five classical phytohormones, i. e., auxin, ethylene, abscisic acid, nin, and gibberellin, synthesis as a secondary metabolite has been demonstrated for at least one bacterial and/or fungal species (for review, see, e.g., Kim et al., Appl. n. Microbiol, Vol. 77, 5:1548—1555, 2011).

Some microorganisms can also produce secondary lites that affect phytohormone production in plants. ly, the best-known example is hormone auxin, which can promote root growth. Other examples include pseudomonads which have been reported to produce indole acetic acid (IAA) and to enhance the amounts of IAA in plants, thus having a profound impact on plant biomass production (Brown, Annual Rev. Phytopathology, 68: 181- 197, 1974). For example, Tien et al. (Applied Environmental Microbiol, 37:1016-1024, 1979) ed that inoculation of nutrient solutions around roots of pearl millet with Azospirillum brasiliense resulted in increased shoot and root weight, an increased number of lateral roots, and all lateral roots were densely covered with root hairs. Plants supplied with combinations of IAA, gibberellins and kinetin showed an increase in the production of lateral roots similar to that caused by Azospirilla. Although the biological cance of these ormones and plant-hormone-like materials are not fillly understood, the growth 2012/069579 stimulating activity of these microorganisms is commonly attributed to their production of these materials.

In addition, other hormones as well as certain volatile c compounds (VOCs) and the cofactor pyrrolquinoline quinone (PQQ) also stimulate plant growth. For example, some rhizobacteria, such as strains of the bacterial species B. sabtilis, B. amyloliqaefaciens, and Enterobacter cloacae, promote plant growth by ing VOCs. The highest level of growth promotion has been observed with 2,3-butanediol and 3-hydroxybutanone (also referred to as n) as elicitors of induced systemic resistance. The cofactor PQQ has been described as a plant growth promoter, which acts as an antioxidant in plants. Some reports suggests that effect may be indirect because PQQ is a cofactor of several enzymes, e. g., involved in ngal activity and induction of systemic resistance.

Stress controllers: Plant -promoting microorganisms that contain the enzyme l-aminocyclopropane-l-carboxylic acid (ACC) deaminase facilitate plant growth and development by decreasing plant ethylene . Such microorganisms take up the ethylene precursor ACC and convert it into 2-oxobutanoate and NH3. Several types of stress have been reported to be relieved by ACC deaminase producers, such as, for example, stress from the effects of athogenic bacteria, stress from polyaromatic hydrocarbons, stress from heavy metal such as Ca2+ and Ni2+, and stress from salt and drought.

In addition, several PGPM strains that induced yield increases of potato have been ed to produce extracellular siderophores that bind Fe“, making it less available to certain member of natural microflora (Kloepper et al., Nature 286: 885-886, 1980). These rhizobacteria excrete low molecular weight, high affinity -chelating microbial cofactors that specifically enhance their acquisition of iron by binding to membrane bound siderophore receptors. One of the siderophores produced by some pseudomonad PGPMs is known as pseudobactin that inhibits the growth of Erwinia cartovora (causal organism for soft-rot of ) (see, e.g., Kloepper et al., Current Microbiol. 4: 317-320, 1980). Additions of pseudobactin to the grth medium inhibited soft-rot infection and also d the number of pathogenic fiangi in the potato plant along with a significant increase in potato yield. Most evidence to support the siderophore theory of biological control by PGPM comes from work with the dines, one class of sideophores that comprises the fluorescent pigments of fluorescent pseudomonads [Demange et al., in Iron Transport in es, Plants and Animals eman et al., eds.), pp 167-187, 1987]. According to the siderophore theory, pyoverdines demonstrate certain onal strain specificity which is due to selective recognition of outer membrane phore receptors r et al., Soil Biology and Biochemistry 19: 443-450, 1989).

Isolated cultures of the invention As described in more detail in the Examples section of the present disclosure, Applicants have discovered several novel microorganisms that are effective promoters of plant growth and plant yield. In many cases, the isolated rganisms are also effective in suppressing the development of several plant enic diseases. The microbial es were selected from a pool of approximately 5,000 microbial strains obtained from environmental samples collected from several ons throughout the United States. Initial selection of the microorganisms was based on the ability of the microorganisms to colonize plant roots and to produce chemical compounds and s that are considered to be important for their ction with plants. The microorganisms were also bio-assayed for their y to suppress the development of various fungal phytopathogens in an in vitro antagonism assay. Selected microbial microorganisms were then bio-assayed in greenhouse studies on commercial wheat and corn ies for the ability of the microbial strains to promote plant growth and for their ability to preserve seed yield potential.

Taxonomic analysis further determined that representative microorganisms bed in the present disclosure are closely related to the ial genera Bacillus, lderia, Herbaspirillum, Pantoea, and Pedobacter.

Deposit of Biological Material Purified cultures of microbial strains described in the present disclosure were deposited in the Agricultural Research Service Culture Collection located at 1815 N. sity Street, Peoria, IL 61604, USA (NRRL) in accordance with the Budapest Treaty for the purpose of patent procedure and the regulations thereunder (Budapest Treaty).

Accession numbers for these deposits are as follows: Table 1: Microbial isolates and corresponding accession numbers Strain ID Provisional Taxonomy SGIH11 NRRL B-50483 Pantoea agglomerans 003_H11 WO 90628 SGI-OZO-AOl NRRL B-50484 Bacillus thuringiensis 020_AOl SGTG06 NRRL B-50485 Burkholderz'a metallica 026_G06 SGTG07 NRRL B-50486 Burkholderz'a Vietnamz'ensz's 026_G07 The microbial strains have been deposited under conditions that ensure that access to the culture will be available during the cy of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §l.l4 and 35 U.S.C. §122. The deposits represent ntially pure cultures of the deposited strains. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application or its progeny are filed. r, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in tion of patent rights granted by mental action.

Preferred microorganisms of the present invention have all of the fying characteristics of the deposited strains and, in particular, the fying characteristics of being able to e plant growth and/or yield as described herein, and the identifying characteristics as being able to suppress the development of fungal phytopathogen as described herein. In particular, the preferred microorganisms of the present invention refer to the deposited microorganisms as bed above, and strains derived therefrom.

The microbiological compositions of the present invention that comprise isolated microbial strains or cultures f can be in a variety of forms, including, but not d to, still cultures, whole cultures, stored stocks of cells, mycelium and/or hyphae (particularly glycerol stocks), agar strips, stored agar plugs in glycerol/water, freeze dried stocks, and dried stocks such as lyophilisate or mycelia dried onto filter paper or grain seeds. As defined elsewhere , “isolated culture” or grammatical equivalents as used in this disclosure and in the art is understood to mean that the referred to culture is a culture fluid, pellet, scraping, dried sample, lyophilisate, or section (for example, hyphae or mycelia); or a support, ner, or medium such as a plate, paper, filter, matrix, straw, pipette or pipette tip, fiber, needle, gel, swab, tube, vial, particle, etc. that contains a single type of organism. In the present invention, an isolated culture of a microbial antagonist is a culture fluid or a scraping, pellet, dried preparation, lyophilisate, or section of the microorganism, or a support, container, or medium that contains the microorganism, in the absence of other organisms.

The present disclosure further provides compositions that contain at least one ed ial strains or cultures thereof of the present invention and a r. The carrier may be any one or more of a number of carriers that confer a variety of ties, such as sed stability, wettability, dispersability, etc. g agents such as natural or synthetic tants, which can be nonionic or ionic surfactants, or a combination thereof can be included in a composition of the invention. Water-in-oil emulsions can also be used to formulate a composition that includes at least one isolated microorganism of the present invention (see, for example, US. Patent No. 7,485,451, incorporated by reference herein).

Suitable formulations that may be prepared include wettable powders, granules, gels, agar strips or pellets, thickeners, and the like, microencapsulated particles, and the like, liquids such as aqueous flowables, aqueous suspensions, water-in-oil emulsions, etc. The formulation may include grain or legume products (e.g., ground grain or beans, broth or flour derived from grain or beans), starch, sugar, or oil. The carrier may be an agricultural carrier. In certain preferred embodiments, the carrier is a seed, and the composition may be applied or coated onto the seed or allowed to saturate the seed.

In some embodiments, the agricultural carrier may be soil or plant growth medium. Other agricultural carriers that may be used include water, izers, plant-based oils, humectants, or combinations thereof Alternatively, the agricultural carrier may be a solid, such as diatomaceous earth, loam, silica, alginate, clay, bentonite, ulite, seed cases, other plant and animal products, or combinations, including granules, pellets, or suspensions. Mixtures of any of the aforementioned ingredients are also contemplated as carriers, such as but not limited to, pesta (flour and kaolin clay), agar or flour-based pellets in loam, sand, or clay, etc. Formulations may include food sources for the ed organisms, such as barley, rice, or other biological materials such as seed, plant parts, sugar cane bagasse, hulls or stalks from grain processing, ground plant al (“yard ) or wood from ng site refuse, t or small fibers from recycling of paper, fabric, or wood.

Other suitable formulations will be known to those skilled in the art.

In the liquid form, e.g., ons or suspensions, the microorganisms of the present invention may be mixed or suspended in water or in aqueous solutions. Suitable WO 90628 liquid diluents or carriers include water, aqueous solutions, petroleum distillates, or other liquid carriers.

Solid compositions can be prepared by dispersing the microorganisms of the invention in and on an appropriately divided solid carrier, such as peat, wheat, bran, vermiculite, clay, talc, ite, diatomaceous earth, fuller's earth, pasteurized soil, and the like. When such ations are used as wettable powders, biologically compatible dispersing agents such as non-ionic, anionic, amphoteric, or cationic dispersing and emulsifying agents can be used.

In a preferred embodiment, the compositions contemplated herein enhance the growth and yield of crop plants, such as wheat, barley, oat, and corn and, when used in sufficient amounts, to act as microbial izer. These compositions, similarly to other biofertilizer agents, can have a high margin of safety because they typically do not burn or injury the plant.

As described in great detail throughout the present disclosure, ing plant growth and plant yield may be ed by application of 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 an amount ive to enhance plant growth or yield relative to that in an untreated l. The active constituents are used in a concentration sufficient to enhance the grth of the target plant when applied to the plant. As will be apparent to a skilled person in the art, ive concentrations may vary depending upon various factors such as, for example, (a) the type of the plant or agricultural commodity; (b) the physiological condition of the plant or agricultural commodity; (c) the concentration of pathogens affecting the plant or agricultural commodity; (d) the type of e injury on the plant or agricultural commodity; (e) weather conditions (e.g., temperature, humidity); and (f) the stage of plant disease. According to the t invention, typical concentrations are those higher than l X 102 CFU/mL of carrier. Preferred concentrations range from about l X 104 to about 1 X 109 CFU/mL, such as the concentrations g from l X 106 to l X 108 CFU/mL. More preferred concentrations are those of from about 37.5 to about 150 mg dry bacterial mass per milliliter of carrier (liquid composition) or per gram of carrier (dry formulation).

In some ments, the amount of one or more of the microorganisms in the compositions of the present invention can vary depending on the final formulation as well as size or type of the plant or seed utilized. ably, the one or more microorganisms in the compositions are present in about 2% w/w/ to about 80% w/w of the entire formulation. More preferable, the one or more microorganisms employed in the compositions is about 5% w/w to about 65% w/w and most ably about 10% w/w to about 60% w/w by weight of the entire formulation.

As it will be appreciated by those skilled in the art, the microbiological compositions of the invention may be applied to the target plant using a variety of conventional methods such as dusting, coating, injecting, rubbing, rolling, dipping, spraying, or brushing, or any other appropriate technique which does not significantly injure the target plant to be treated. Particularly preferred methods include the inoculation of growth medium or soil with suspensions of microbial cells and the coating of plant seeds with ial cells and/or spores.

Typically, the compositions of the invention are chemically inert; hence they are compatible with substantially any other constituents of the application schedule. They may also be used in ation with plant growth affecting substances, such as fertilizers, plant growth regulators, and the like, provided that such compounds or substances are biologically compatible. They can also be used in combination with biologically compatible pesticidal active agents as for example, ides, nematocides, fiangicides, insecticides, and the like.

When used as biofertilizers in their commercially available formulations and in the use forms, prepared from these formulations, the active microbial strains and compositions according to the present invention can furthermore be present in the form of a e with synergists. Synergists are compounds by which the ty of the active compositions is increased without it being necessary for the synergist added to be active itself.

When used as biofertilizers in their commercially available formulations and in the use forms, prepared from these formulations, the active microbial strains and itions according to the invention can furthermore be present in the form of a mixture with inhibitors which reduce the degradation of the active compositions after application in the t of the plant, on the surface of parts of plants or in plant tissues.

The active microbial strains and compositions according to the invention, as such or in their ations, can also be used as a mixture with known izers, acaricides, icides, fiangicides, insecticides, microbicides, nematicides, pesticides, or combinations of any thereof, for example in order to widen the spectrum of action or to t the development of resistances to pesticides in this way. In many cases, synergistic effects result, z'.e., the activity of the mixture can exceed the activity of the individual components. A e with other known active compounds, such as growth tors, safeners and/or semiochemicals is also contemplated.

In a preferred embodiment of the present invention, the compositions may further include at least one chemical or biological fertilizer. The amount of at least one chemical or ical fertilizer employed in the compositions can vary depending on the final formulation as well as the size of the plant and seed to be treated. Preferably, the at least one al or biological fertilizer employed is about 0.1% w/w to about 80% w/w based on the entire formulation. More preferably, the at least one chemical or biological fertilizer is present in an amount of about 1% w/w to about 60% w/w and most preferably about 10% w/w to about 50% w/w.

The microbiological compositions of the present invention preferably include at least one biological fertilizer. ary biological fertilizers that are suitable for use herein and can be included in a microbiological ition according to the present invention for promoting plant growth and/yield include microbes, s, bacteria, fiangi, genetic material, plant, and natural products of living organisms. In these compositions, the microorganism of the present invention is isolated prior to formulation with an additional sm. For example, microbes such as but not limited to species of Achromobacter, Ampelomyces, Aareobasidiam, Azospl'rillam, Azotobacter, Bacillus, Beaaveria, Bradyrhizobiam, Candida, Chaetomz'am, Cordyceps, Cryptococcas, Dabaryomyces, 'a, Erwinia, Exophz'lz'a, Gliocladz'am, Herbaspz'rz'llam, Lactobacz'llas, Mariannaea, Microecocas, Paecz'lomyces, Paenibacillas, Pantoea, , Pseudomonas, Rhizobz'am, Saccharomyces, Sporobolomyces, Stenotrophomonas, Streptomyces, Talaromyces, and Trichoderma can be provided in a composition with the microorganisms of the present invention. Use of the microbiological compositions according to the present invention in ation with the microbial microorganisms disclosed in US. Patent Appl. Nos. USZOO30172588A1, US20030211119A1; US. Pat. Nos. 7,084,331; 7,097,830; 7,842,494; PCT Appl. No.

WO2010109436A1 is also particularly preferred.

In a preferred embodiment of the present ion, the compositions may further include at least one chemical or biological pesticide. The amount of at least one chemical or ical pesticide employed in the compositions can vary depending on the final formulation as well as the size of the plant and seed to be treated. Preferably, the at least one chemical or biological pesticide employed is about 0.1% w/w to about 80% w/w based on the entire formulation. More preferably, the at least one chemical or biological pesticide is present in an amount of about 1% w/w to about 60% w/w and most preferably about 10% w/w to about 50% w/w.

A y of chemical pesticides is apparent to one of skill in the art and may be used. Exemplary chemical pesticides include those in the carbamate, organophosphate, organochlorine, and prethroid classes. Also included are chemical control agents such as, but not limited to, benomyl, borax, captafol, captan, chorothalonil, formulations containing copper; ations containing dichlone, an, iodine, zinc; fiangicides that inhibit ergosterol biosynthesis such as but not limited to blastididin, cymoxanil, fenarimol, flusilazole, folpet, imazalil, one, maneb, manocozeb, metalaxyl, oxycarboxin, myclobutanil, oxytetracycline, PCNB, pentachlorophenol, prochloraz, onazole, quinomethionate, sodium ite, sodium DNOC, sodium lorite, sodium phenylphenate, streptomycin, sulfur, tebuconazole, terbutrazole, thiabendazolel, thiophanate- methyl, triadimefon, tricyclazole, triforine, validimycin, vinclozolin, zineb, and ziram.

The microbiological compositions of the t invention preferably include at least one biological pesticide. Exemplary biological pesticides that are suitable for use herein and can be included in a microbiological ition according to the present ion for preventing a plant pathogenic disease include microbes, animals, bacteria, fungi, genetic material, plant, and natural products of living organisms. In these compositions, the microorganism of the present invention is isolated prior to formulation with an additional organism. For example, es such as but not limited to species of Ampelomyces, Aareobasidiam, Bacillus, Beaaveria, a, Chaetoml'am, Cordyceps, Cryptococcas, Dabaryomyces, Erwinia, Exophz'lz'a, Gliocladz'am, Mariannaea, Paecilomyces, Paenibacillas, Pantoea, Pichia, Pseudomonas, Sporobolomyces, Talaromyces, and Trichoderma can be ed in a composition with the rganisms of the present invention, with fungal strains of the Muscodor genus being particularly preferred. Use of the microbiological compositions according to the present invention in combination with the microbial nists disclosed in US Patent No. 7,518,040; US Patent No. 346; US Patent No. 6,312,940 is also ularly preferred.

Examples of fiangi that can be ed with microbial s and compositions of the t invention in a composition include, without limitation, Muscodor species, Aschersom'a aleyrodz's, Beauverl'a bassiana ("white muscarine"), Beauveria brongm'artz'z', Chladosporl'um herbarum, Cordyceps clavulata, Cordyceps entomorrhl'za, Cordyceps facis, eps gracilz's, Cordyceps melolanthae, Cordyceps militaris, Cordyceps myrmecophila, Cordyceps ravenell'l', Cordyceps sinensis, Cordyceps sphecocephala, Cordyceps subsessz'lz's, Cordyceps unilateralz's, Cordyceps variabilis, Cordyceps washingtonensis, Culicinomyces clavosporus, phaga gryllz', Entomophaga maimaiga, Entomophaga muscae, phaga bullz', Entomophthora plutellae, Fusarz'um laterz'tz'um, Hirsutella citrz'formz's, Hirsutella thompsom', Metarhz'zz'um anisoplz'ae ("green muscarine"), Metarhz'zz'um flaviride, Muscodor albus, Neozygz'tesflorz'dana, Nomuraea rileyi, Paecilomyces farinosus, Paecz'lomyces roseus, Pandora neoaphidis, Tolypocladz'um cylindrosporum, Verticz'llz'um lecam’z’, Zoophthora radicans, and mycorrhizal species such as Laccarz’a bicolor.

Other mycopesticidal species will be apparent to those skilled in the art.

The present invention also provides methods of treating a plant by application of any of a variety of customary formulations in an effective amount to either the soil (i.e., in- furrow), a n of the plant (i.e., drench) or on the seed before planting (126., seed coating or dressing). Customary formulations include solutions, emulsifiable concentrate, wettable powders, sion concentrate, soluble powders, granules, suspension-emulsion concentrate, natural and synthetic materials nated with active compound, and very fine control release capsules in polymeric substances. In certain embodiments of the present invention, the microbial itions are formulated in s that are available in either a ready-to-use formulation or are mixed er at the time of use. In either embodiment, the powder may be admixed with the soil prior to or at the time of planting. In an alternative embodiment, one or both of either the plant growth-promoting agent or biocontrol agent is a liquid formulation that is mixed together at the time of treating. One of ordinary skill in the art tands 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.

Depending on the final formulation and method of application, one or more suitable additives can also be introduced to the compositions of the present ion. ves such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or latexes, such as gum arabic, chitin, polyvinyl alcohol and polyvinyl e, as well as natural phospholipids, such as cephalins and lecithins, and synthetic olipids, can be added to the present compositions.

In a preferred embodiment, the microbiological compositions are formulated in a single, stable solution, or emulsion, or suspension. For solutions, the active chemical compounds are typically dissolved in solvents before the ical agent is added. Suitable liquid solvents include petroleum based aromatics, such as xylene, toluene or alkylnaphthalenes, aliphatic arbons, such as cyclohexane or ns, for example petroleum fractions, mineral and vegetable oils, alcohols, such as butanol or glycol as well as their ethers and esters, ketones, such as methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, strongly polar ts, such as dimethylformamide and dimethyl sulphoxide.

For on or suspension, the liquid medium is water. In one embodiment, the chemical agent and biological agent are suspended in separate liquids and mixed at the time of application. In a preferred embodiment of suspension, the chemical agent and biological agent are combined in a ready-to-use formulation that exhibits a reasonably long shelf-life. In use, the liquid can be d or can be applied foliarly as an ed spray or in-fiarrow at the time of planting the crop. The liquid composition can be introduced in an effective amount on the seed (i.e., seed coating or ng) or to the soil (226., III-filI'I'OW) before germination of the seed or directly to the soil in contact with the roots by utilizing a variety of techniques known in the art including, but not limited to, drip irrigation, sprinklers, soil injection or soil drenching.

Optionally, stabilizers and buffers can be added, ing alkaline and alkaline earth metal salts and organic acids, such as citric acid and ic acid, inorganic acids, such as hydrochloric acid or sulfuric acid. Biocides can also be added and can include formaldehydes or formaldehyde- releasing agents and derivatives of benzoic acid, such as phydroxybenzoic acid.

Pathogens The skilled artisan in the art will recognize that the methods and compositions according to the t invention in principle can be applied to suppress the development of any plant pathogens or any phytopathogenic diseases. It is not intended that the invention be limited to a particular culture types or cell types. For example, microbial cells that undergo complex forms of differentiation, filamentation, sporulation, etc. can also be used for the methods and compositions of the present invention.

Examples of phytopathogenic diseases that are suitable for applications of the s and materials of the present ions include, but are not limited to, diseases caused by a broad range of pathogenic fungi. The methods of the present ion are preferably applied against pathogenic fungi that are important or interesting for agriculture, horticulture, plant biomass for the production of biofuel molecules and other chemicals, and/or ry. Of particular interest are pathogenic Pseudomonas species (e.g., Pseudomonas solanacearum), Xylella fastidiosa; Ralstonia solanacearum, monas campestris, Erwim'a ora, Fusarz'um species, Phytophthora species (e.g., P. infestans), Botrytz's species, Leptosphaerz’a species, y mildews (Ascomycota) and rusts iomycota), etc. miting examples of plant pathogens of interest include, for instance, Acremonium strictum, Agrobacterium tumefaciens, Alternarz'a ata, Alternarz'a solanz', Aphanomyces euteiches, Aspergillus fumigatus, Athelz'a 'z', Aureobasidium pullulans, Bz'polarz's zeicola, Botrytis cinerea, Calonectrl'a kyotensz's, Cephalosporz'um maydz's, Cercospora medicagz'nz's, Cercospora sojina, Colletotrichum coccodes, Colletotrichum fragarl'ae, Colletotrichum m'cola, Coniella dl'plodz'ella, opsz's psychromorbida, Corynespora cassz'z'cola, Curvularz'a pallescens, Cylindrocladl'um crotalarz'ae, Dz'plocarpon earlianum, Diplodl'a gossyz'na, Dz'plodl'a spp., Epicoccum , Erysz'phe cichoracearum, Fusarz'um gramz'nearum, Fusarz'um oxysporum, Fusarz'um oxysporumfsp. tuberosz', Fusarz'um prolz'feratum var. prolz'feratum, Fusarz'um solanz', Fusarz'um verticillz'oides, Ganoderma boninense, 'chum candidum, Glomerella tucumanensis, Guignardz'a bidwellz'z', Kabatz'ella zeae, Leptosphaerulina briosz'ana, Leptotrochila medicagz'nz's, Macrophomina, Macrophomina lina, Magnaporthe , Magnaporthe oryzae, Microsphaera manshurica, Monilz'm'a fructz’cola, haerella fijiensis, Mycosphaerella fragarz'ae , Nigrospora oryzae, Ophz'ostoma ulmz', Pectobacterz'um carotovorum, Pellz'cularz'a sasakl'z' (Rhizoctom'a salami), Peronospora manshurica, Phakopsora pachyrhizz', Phoma faveata, Phoma medicagz'nz's, Phomopsz's longicolla, Phytophthora cinnamomi, Phytophthora erythroseptz'ca, Phytophthora l'ae, Phytophthora infestans, Phytophthora medicagz'nz's, Phytophthora megasperma, Phytophthora palmz'vora, haera leucotrz'cha, Pseudopezz'za medicagz'm's, Puccinia graml'm's subsp. Triticz' (UG99), Puccinia sorghz', Pyricularz'a grisea, Pyricularz'a oryzae, Pythz'um ultimum, Rhizoctom'a ', Rhizoctom'a zeae, Rosellz'nz'a sp., Sclerotz'm'a tiorum, Sclerotinina trl'folz'orum, Sclerotl'um 'z', Septorz'a glycines, Septorz'a lycopersz'cz', Setomelanomma turcica, Sphaerotheca macularz's, Spongospora subterranea, Stemphylz'um Sp, Synchytrl'um 'otl'cum, Thecaphora (Angiosorus), Thielavz'opsz's, Tilletz'a indica, Trichoderma viride, Ustz'lago maydz's, Verticz'llz'um albo-atrum, Verticz'llz'um ae, Verticz'llz'um dahlz'ae, Xanthomonas axonopodz's, Xanthomonas oryzae pv. oryzae.

In a preferred embodiment of the present invention, the methods and materials of the invention are useful in suppressing the development the pathogens Aspergillusfumz’gatus, Botrytis cinerea, Cerpospora betae, Colletotrz'chum sp., Curvularl'a spp., Fusarz'um sp., Ganoderma boninense, Geotrz'chum candidum, Gibberella sp., Monographella sp., haerella fijiensis, Phytophthora palmz'vora, hthora ramorum, Penicillium sp., Pythz'um ultimum, Rhizoctom'a solam', Rhizopus spp., phyllum spp., Sclerotz'm'a sclerotiorum, Stagnospora sp., Verticz'llz'um dahlz'ae, or Xanthomonas axonopodz's. In a particularly red ment, the inventive methods and materials may be used to suppress the development of several plant pathogens of cial importance, including 'um graminearum NRRL-5883, Monographella nivalz's ATCC MYA-3968, Gibberella zeae ATCC-l6lO6, Stagnospora nodurum ATCC-26369, Colletotrz'chum graminz'cola ATCC-34l67, and Penicillium sp. pathogens.

Seed coating formulation In a particularly preferred embodiment, the microbial itions of the present invention are formulated as a seed treatment. It is contemplated that the seeds can be substantially mly coated with one or more layers of the microbial compositions disclosed herein using tional methods of mixing, spraying or a combination thereof through the use of treatment application equipment that is specifically ed and manufactured to accurately, safely, and efficiently apply seed treatment products to seeds.

Such equipment uses various types of coating technology such as rotary coaters, drum coaters, fluidized bed ques, spouted beds, rotary mists or a combination f. Liquid seed treatments such as those of the present invention can be d via either a spinning "atomizer" disk or a spray nozzle which evenly distributes the seed treatment onto the seed as it moves though the spray pattern. Preferably, the seed is then mixed or tumbled for an additional period of time to achieve additional treatment distribution and drying. The seeds can be primed or ed before coating with the inventive compositions to increase the uniformity of germination and emergence. In an ative embodiment, a dry powder ation can be metered onto the moving seed and allowed to mix until completely distributed.

Another aspect of the invention provides seeds treated with the subject microbial compositions. One embodiment provides seeds having at least part of the surface area coated with a microbiological composition according to the present invention. In a specific embodiment, the microorganism-treated seeds have a microbial spore concentration or microbial cell concentration from about 106 to about 109 per seed. The seeds may also have more spores or microbial cells per seed, such as, for example 1010, 1011 or 1012 spores per seed. The microbial spores and/or cells can be coated freely onto the seeds or, preferably, they can be formulated in a liquid or solid composition before being coated onto the seeds.

For example, a solid ition comprising the microorganisms can be prepared by mixing a solid carrier with a sion of the spores until the solid carriers are impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.

In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the t invention further contain functional agents capable of protecting seeds from the harmful effects of selective herbicides such as ted carbon, nutrients (fertilizers), and other agents capable of ing the germination and quality of the products or a combination thereof.

Seed g 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, US. Pat. 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. Pat. Appl. No. US20100154299, US. Pat. 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; 587; 080, 4,339,456; and 4,245,432, among others.

WO 90628 2012/069579 A variety of additives can be added to the seed treatment formulations comprising the inventive compositions. Binders can be added and include those composed preferably of an adhesive polymer that can be natural or synthetic Without phytotoxic effect on the seed to be . The binder may be selected from polyvinyl es; polyvinyl acetate copolymers; ethylene vinyl acetate (EVA) copolymers; polyvinyl alcohols; nyl alcohol copolymers; celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrolidones; ccharides, including starch, modified starch, dextrins, maltodextrins, alginate and chitosans; fats; oils; proteins, including gelatin and zeins; gum arabics; shellacs; vinylidene chloride and vinylidene chloride copolymers; m lignosulfonates; acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.

Any of a variety of colorants may be employed, including organic chromophores classified as nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. Other additives that can be added include trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc. A polymer or other dust control agent can be applied to retain the ent on the seed surface.

In some specific ments, in on to the microbial cells or spores, the coating can further comprise a layer of adherent. The adherent should be non-toxic, biodegradable, and adhesive. Examples of such materials include, but are not limited to, nyl acetates; polyvinyl acetate mers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, such as methyl celluloses, hydroxymethyl celluloses, and hydroxymethyl propyl celluloses; dextrins; alginates; sugars; molasses; polyvinyl pyrrolidones; polysaccharides; proteins; fats; oils; gum arabics; gelatins; ; and starches.

More examples can be found in, for example, US. Pat. No. 7,213,367 and US. Pat. Appln.

OlOOl$9693.

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 d to, coating agents, wetting agents, buffering agents, and polysaccharides. At least one agriculturally acceptable carrier can be added to the seed treatment formulation such as water, solids or dry powders. The dry powders can be derived from a variety of materials such as calcium carbonate, gypsum, vermiculite, talc, humus, activated charcoal, and various phosphorous compounds.

In some embodiment, the seed coating composition can comprise at least one filler which is an organic or inorganic, natural or synthetic component with which the active components are combined to facilitate its application onto the seed. Preferably, the filler is an inert solid such as clays, natural or synthetic silicates, silica, , waxes, solid fertilizers (for example ammonium salts), natural soil minerals, such as kaolins, clays, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous , or synthetic minerals, such as silica, alumina or silicates, in particular ium or magnesium silicates.

The seed treatment formulation may fiarther include one or more of the following ingredients: other pesticides, including compounds that act only below the ; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and s of each of those materials, and the like; herbicides, including compounds ed from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, ol ethers, pyridazinones, uracils, phenoxys, ureas, and benzoic acids; herbicidal safeners such as benzoxazine, benzhydryl derivatives, N,N-diallyl dichloroacetamide, various dihaloacyl, idinyl and thiazolidinyl compounds, ethanone, naphthalic anhydride compounds, and oxime derivatives; chemical fertilizers; ical fertilizers; and trol agents such as other naturally-occurring or recombinant bacteria and fungi from the genera Rhizobz'um, Bacillus, monas, Serratz'a, Trichoderma, Glomus, Gliocladz'um and mycorrhizal fungi.

These ingredients may be added as a separate layer on the seed or alternatively may be added as part of the seed g composition of the invention.

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

The ation that is used to treat the seed in the present invention can be in the form of a suspension; emulsion; slurry of particles in an aqueous medium (e.g., ; wettable powder; wettable granules (dry flowable); and dry granules. If ated as a suspension or slurry, the concentration of the active ingredient in the formulation is preferably about 0.5% to about 99% by weight (w/w), preferably 5-40% or as otherwise formulated by those skilled in the art.

As mentioned above, other conventional inactive or inert ingredients can be orated into the formulation. Such inert ingredients include but are not limited to: conventional sticking agents; dispersing agents such as methylcellulose, for example, serve as combined dispersant/sticking agents for use in seed treatments; polyvinyl alcohol; lecithin, polymeric dispersants (e. g., polyvinylpyrrolidone/vinyl acetate); thickeners (e.g, clay thickeners to improve viscosity and reduce settling of particle sions); emulsion stabilizers; surfactants; antifreeze compounds (e.g, urea), dyes, colorants, and the like.

Further inert ingredients useful in the present invention can be found in McCutcheon's, vol. 1, "Emulsifiers and Detergents," MC hing 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., , 1996.

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, tumbling, ng, and immersion. A variety of active or inert material can be used for contacting 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 SEPIRETTM (Seppic, Inc., NJ.) and OPACOATTM (Berwind Pharm. Services, PA.) The amount of a composition according to the present invention that is used for the treatment of the seed will vary depending upon the type of seed and the type of active ingredients, but the treatment will comprise contacting the seeds with an agriculturally ive amount of the inventive composition. As discussed above, an ive amount means that amount of the ive ition that is sufficient to affect beneficial or desired results. An effective amount can be stered in one or more administrations.

] In addition to the coating layer, the seed may be treated with one or more of the following ingredients: other pesticides including fungicides and herbicides; herbicidal safeners; fertilizers and/or biocontrol agents. These ients may be added as a separate layer or alternatively may be added in the coating layer.

] The seed coating formulations of the present invention may be applied to the seeds using a variety of ques and machines, such as fluidized bed techniques, the roller mill method, rotostatic seed treaters, and drum coaters. Other methods, such as spouted beds may also be useful. The seeds may be pre-sized before coating. After coating, the seeds are typically dried and then transferred to a sizing machine for sizing. Such procedures are known in the art.

The microorganism-treated seeds may also be enveloped with a film overcoating to t the coating. Such overcoatings are known in the art and may be applied using fluidized bed and drum film coating techniques.

In another embodiment of the present invention, compositions according to the present ion can be uced onto a seed by use of solid matrix priming. For example, a ty of an inventive composition can be mixed with a solid matrix material and then the seed can be placed into contact with the solid matrix material for a period to allow the composition to be introduced to 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 seed can be stored or planted directly. Solid matrix materials which are useful in the present invention include polyacrylamide, starch, clay, silica, alumina, soil, sand, ea, rylate, or any other material capable of absorbing or adsorbing the inventive composition for a time and releasing that composition into or onto the seed. It is useful to make sure that the inventive composition and the solid matrix material are compatible with each other. For example, the solid matrix material should be chosen so that it can e the composition at a reasonable rate, for example over a period of minutes, hours, or days.

In principle, any plant seed capable of germinating to form a plant can be treated in accordance with the invention. Suitable seeds include those of cereals, coffee, cole crops, f1ber crops, flowers, , legume, oil crops, trees, tuber crops, vegetables, as well as other plants of the monocotyledonous, and ledonous s. Preferably, crop seeds are coated include, but are not limited to, bean, carrot, corn, cotton, grasses, lettuce, peanut, pepper, potato, rapeseed, rice, rye, sorghum, soybean, sugarbeet, sunflower, tobacco, and tomato seeds. Most preferably, barley or wheat (spring wheat or winter wheat) seeds are coated with the t compositions.

Preparing the microbial compositions according to the present invention Cultures of the microorganisms may be prepared for use in the microbial compositions of the invention using rd static drying and liquid fermentation techniques known in the art. Growth is commonly ed in a bioreactor.

A bioreactor refers to any device or system that supports a biologically active environment. As described herein a bioreactor is a vessel in which microorganisms including the microorganism of the invention can be grown. A bioreactor may be any riate shape or size for growing the microorganisms. A bioreactor may range in size and scale from 10 mL to liter’s to cubic meters and may be made of stainless steel or any other appropriate material as known and used in the art. The bioreactor may be a batch type bioreactor, a fed batch type or a continuous-type bioreactor (e.g., a continuous stirred r). For e, a bioreactor may be a chemostat as known and used in the art of iology for growing and harvesting microorganisms. A ctor may be obtained from any cial supplier (See also Bioreactor System Design, Asenjo & Merchuk, CRC Press, 1995).

For small scale operations, a batch bioreactor may be used, for example, to test and develop new processes, and for ses that cannot be converted to continuous operations. rganisms grown in a ctor may be suspended or immobilized. Growth in the bioreactor is generally under aerobic conditions at suitable temperatures and pH for growth. For the organisms of the invention, cell growth can be achieved at temperatures between 5 and 37°C, with the preferred temperature being in the range of 15 to 30°C, 15 to 28°C, 20 to 30°C, or 15 to 25°C. The pH of the nutrient medium can vary n 4.0 and 9.0, but the preferred operating range is usually slightly acidic to neutral at pH 4.0 to 7.0, or 4.5 to 6.5, or pH 5.0 to 6.0. Typically, maximal cell yield is obtained in 20-72 hours after inoculation.

Optimal conditions for the cultivation of the microorganisms of this invention will, of course, depend upon the particular strain. However, by virtue of the conditions applied in the selection process and general requirements of most microorganisms, a person of ry skill in the art would be able to determine essential nutrients and conditions. The microorganisms would typically be grown in aerobic liquid cultures on media which contain sources of carbon, nitrogen, and inorganic salts that can be assimilated by the microorganism and supportive of efficient cell growth. Preferred carbon sources are hexoses such as glucose, but other sources that are readily lated such as amino acids, may be substituted. Many inorganic and proteinaceous als may be used as nitrogen sources in the growth s.

Preferred en sources are amino acids and urea but others include gaseous ammonia, inorganic salts of nitrate and ammonium, vitamins, purines, pyrimidines, yeast extract, beef extract, proteose peptone, soybean meal, hydrolysates of casein, distiller's solubles, and the like. Among the inorganic ls that can be incorporated into the nutrient medium are the customary salts e of yielding calcium, zinc, iron, ese, ium, copper, cobalt, potassium, sodium, molybdate, phosphate, e, chloride, borate, and like ions.

Without being limited thereto, use of potato dextrose liquid medium for fungal strains and R2A broth premix for bacterial strains is preferred.

Novel p_lant varieties ] Also ed, in another aspect of the present invention, is a novel plant created by artificially introducing a microbial endophyte of the invention into a plant that is fiee of endophytic microorganisms. In some ments of this aspect, the microbial endophyte introduced into the plant may be an endophytic microorganism having a plant growth- promoting activity, a biological control activity, or a combination of both activities. A variety of methods previously found effective for the introduction of a ial yte into cereal grass species are known in the art. Examples of such methods include those described in US. Pat. Appl. No. 20030195117Al, US. Pat. Appl. No. 20010032343Al, and US. Pat.

No. 7,084,331, among others. It will become nt to those skilled in the art that many of the aforementioned methods can be useful for the making of a novel plant of the invention.

After artificial infection, it is preferred that a DNA sequence of the isolated endophytic microorganism is amplified by PCR and the endophyte is confirmed by carrying out a homology search for the DNA sequence amplified. Further, it is preferred that a foreign gene that expresses an identifiable means is introduced into the above-mentioned endophytic microorganism, and the presence of the colonization of the above-mentioned endophytic microorganism infecting the plant is confirmed by the above-identifiable means using the foreign gene.

Plants suitable for the methods of the invention In principle, the methods and compositions according to the t invention can be ed for any plant species. Monocotyledonous as well as dicotyledonous plant s 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 producing liquid fuel molecules and other als, and/or forestry.

Thus, the invention has use over a broad range of plants, preferably higher plants pertaining to the classes ofAngiospermae and Gymnospermae. Plants of the subclasses of the Dicotylodenae and the Monocotyledonae are particularly suitable. Dicotyledonous plants belong to the orders of the Aristochiales, Asterales, s, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, sales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, biales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, lidales, Illiciales, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales, Piperales, Plantaginales, Plumbaginales, Podostemales, Polemoniales, Polygalales, Polygonales, ales, les, Rafilesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, es, Sapindales, Sarraceniaceae, ulariales, s, Trochodendrales, Umbellales, Urticales, and Violales. Monocotyledonous plants belong to the orders of the Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, es, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plants belonging to the class of the Gymnospermae are Cycadales, Ginkgoales, Gnetales, and Pinales.

Suitable species may include members of the genus Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, ca, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coflea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, thus, Musa, Nicotiana, Oryza, m, Papaver, Parthenium, Pennisetum, Petunia, Phalaris, , Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum, Salix, naria, Scopolia, Secale, Solanum, Sorghum, Spartina, ea, tum, Taxus, Theobroma, Triticosecale, Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

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 instance, Panicum virgatum (switchgrass), Sorghum r (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. ycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), ca napus (canola), Triticum aestivum (wheat), Gossypium hirsutum n), Oryza sativa , Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), Pennisetum glaucum (pearl millet), Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., hus spp., Populus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon clactylon (bermudagrass), a arundinacea (tall fescue), Spartina pectinata (prairie cord-grass), Arundo donax (giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), osecale spp. (triticum--wheat X rye), Bamboo, Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricinus communis (castor), Elaeis guineensis (oil palm), Phoenix clactylifera (date palm), Archontophoenix cunninghamiana (king palm), Syagrus romanzofiana (queen palm), Linum usitatissimum (flax), Brassica juncea, Manihot esculenta (cassaya), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato), ca ea (broccoli, cauliflower, brusselsprouts), ia sinensis (tea), Fragaria ananassa berry), Theobroma cacao (cocoa), Cofi’ea arabica e), Vitis vinifera ), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion), s melo (melon), Cucumis sativus (cucumber), Cucurbita maxima h), Cucurbita moschata (squash), 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 ofiicinalis, Coichicum autumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperzia serrata), Lycopodium spp., Rauwolfia serpentina, fia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula ofiicinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosa spp.

, Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (f1r), Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp. (ryegrass), Phleum pratense (timothy), and conifers. Of interest are plants grown for energy production, so called energy crops, such as cellulose-based energy crops like Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant , Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata (prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp. w), Eucalyptus spp. (eucalyptus), osecale spp. (triticum— wheat X rye), and ; and starch-based energy crops like Zea mays (corn) and Manihot esculenta (cassava); and sugar-based energy crops like Saccharum sp. (sugarcane), Beta vulgaris beet), and Sorghum bicolor (L) Moench (sweet sorghum); and biofiJel- producing energy crops like Glycine max (soybean), Brassica napus (canola), Helianthus annuus wer), Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricinus communis (castor), Elaeis guineensis an oil palm), Elaeis oleifera (American oil palm), Cocos nucifera (coconut), Camelina sativa (wild flax), Pongamia pinnata (Pongam), Olea europaea ), Linum usitatissimum (flax), Crambe abyssinica (Abyssinian-kale), and Brassicajuncea.

The discussion of the general methods given herein is intended for rative purposes only. Other alternative methods and embodiments will be apparent to those of skill in the art upon review of this sure, and are to be included within the spirit and w of this ation.

It should also be tood that the following examples are offered to illustrate, but not limit, the invention.

EXAMPLES EXAMPLE 1: Microorganism isolation from environmental samples Identification of spore-forming rhizobacteria using a sonicated roots and serial dilutions method. The ing microorganisms were isolated using a “sonicated roots, serial dilutions” method as described below: the 6-G06 and SGIG07 isolates, which were isolated from a needle-like grass sample; the SGI-04l-B03 isolate, which was isolated from a wild rye sample; and the O-AOl isolate, which was isolated from a wheat root tissues grown in a composite soil sample.

An enrichment procedure was developed to specifically fy spore-forming rhizobacteria. Briefly, sonicated root extracts were heat treated to kill vegetative cells and then plated onto a rich medium. Microorganisms that survived the heat treatment and formed colonies were considered to be spore-formers. This method was found to be particularly effective for selection of Gram-positive bacteria. Freshly sampled roots were used as starting material for these enrichments. Fine sections found at the tip of roots are the youngest, can have a high root hair density, and typically have high densities of rhizobacteria. A sterile blade was used to section these areas of the roots into 5 - 10 cm ts, which were then washed under sterile milliQ water to remove large soil particles. When needed, a more rigorous wash was accomplished by g the roots into a 50 mL Falcon tube with 25 mL 1 X sterile phosphate buffered saline (1.6. PBS buffer) and vortexing for 1 minute. Each root sample was subsequently suspended in 20 mL sterile PBS buffer and sonicated on ice for two l-minute intervals at 8 watts using a Fisher Scientific Sonic Dismembrator. For heat treatment, typically 1 mL of the sonicated root cell sion was transferred into a e Eppendorf tube incubated in an 80°C water bath for 20 minutes. The heat treated cell suspensions were allowed to cool to room temperature before ly diluted to concentrations of 101, 102, 10'3, 10“, 10's, 106, and 107. 100 uL of each 10-fold dilution was spread onto culture plates containing a microbiological medium fied with agar and 100 mg/L cycloheximide to inhibit fungal growth. In some cases, it was necessary to perform a 1/10 or 1/100 dilution prior to plating in order to obtain the proper CFU density for colony picking. Isolated es were picked using sterile pipette tips, arrayed into 96-well microtiter plates each containing 150 uL 2 X YT liquid medium per well. The microtiter plates were incubated for 1-2 days at 30°C in order to obtain a high cell y for r characterization and archiving. 2012/069579 ion of biofilm-forming bacteria. The following microbial isolates were isolated using a “biofilm former” method as described below: the 3-Hll isolate, which was ed from a Yucca plant root sample; the SGIC09 isolate, which was isolated from a grass root sample; and the 4-ElO isolate, which was from a Queen Anne’s Lace plant sample.

Biofilm former method: In this procedure, biofilm-forming bacteria were ed from sonicated root segments, as described by Fall et al. (Syst. Appl. Microbiol. 27,372-379, 2004). As described above, bacteria that form biofilms the e of a root are typically very good root colonizing bacteria, In general, when such bacteria are present at high densities, they can have a significant influence on plant health and can competitively exclude invading pathogens. Briefly, sonication was used to remove bacterial and fungal cells that are loosely attached to the root, leaving behind only those microbes that were strongly adhered to the root surface. Both Gram-positive and Gram-negative biofilm-forming ia were selected using this method. y sampled roots were used as starting material for these enrichments. Fine sections found at the tip of roots were the youngest tissues, had a high root hair density and typically had high densities of rhizobacteria. A sterile blade was used to section these areas of the roots into 5 - 10 cm segments, which were then washed by placing them into a 50 mL Falcon tube with 25 mL l X PBS and vortexed for 1 minute. The debris from the wash was allowed to settle, and then a sterile forceps was used to transfer the washed root segments to 50 mL Falcon tubes filled with 25 mL l X PBS, and sonicated on ice using a Fisher Scientific Sonic Dismembrator for two 30 second intervals with a 30 second pause between . The sonicated root samples were transferred to sterile plastic Petri dishes and allowed to dry completely without lids inside a biosafety cabinet. Each root segment was then placed onto a separate CMA plate containing 1% agar (10 g/L Casein digest, 10 g/L mannitol, 10 g/L agar).

Sometimes, a sterile forceps was used to push the root t into the agar media. The plates were subsequently ted at 37°C and monitored for microbial growth. Typically after 1-2 days, multiple microbial growths d from the root and onto the CMA media.

A sterile pipette tip was used to pick growths with unique morphologies along the segment and each of these growths was transferred to the center of a CMA plate containing 0.3% agarose. The CMA plates were uently incubated for 1-2 days at 37°C and monitored for growth. Typically, biofilm-forming isolates displayed dendritic growth on this medium.

] A sterile loop was used to transfer biomass and streak-purify each isolate from the CMA plates onto CMKA plates (2% agar, 1.2 g/L K2HPO4). The CMKA medium restricts biofilm growth and allows for the picking of individual colonies for archiving.

EXAMPLE 2: Growth and storage of the ial isolates The isolated bacteria were stored as a pure e. A bacterial colony was transferred to a vial containing R2A broth liquid medium (Tecknova) and d to grow at °C with shaking at 250 rpm for two days. The e was then transferred into vials containing 15% glycerol and stored at -80°C.

EXAMPLE 3: DNA extraction, Sequencing and Taxonomy A 20 ul aliquot of bacterial cell suspension was transferred to a 96-well PCR plate containing 20 ul of a 2x lysis buffer (100 mM Tris HCL, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS, 400 ug/mL Proteinase K). Lysis conditions were as follows: 55°C incubation for 30 minutes, followed by 94°C incubation for 4 minutes. An aliquot of the lysis product was used as the source of template DNA for PCR amplification.

For amplification of 16S rRNA region, each PCR mixture was prepared in a 20 ul final volume reaction containing 4 ul of the bacterial lysis reaction, 2 uM of each PCR primer, 6% Tween-20, and 10 ul of 2x ImmoMix (Bioline USA Inc, Taunton, MA). The primers used for PCR amplification were M13-27F (5 ’ - TGTAAAACGACGGCCAGTTAGAGTTTGATCCTGGCTCAG-3’ SEQ ID NO: 8) and 1492R M13-tailed (5’-CAGGAAACAGCTATGACCGGTTACCTTGTTACGACTT-3’; SEQ ID NO: 9). The PCR was carried out in 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 s for 30 cycles; 72°C for 10 minutes. A 2 ul t of each PCR product was run on a 1.0% e gel to confirm a single band of the expected size. Positive bands were isolated, purified, and submitted for PCR cing. Sequencing was performed in the forward and reverse priming directions by the J. Craig Venter Institute in San Diego, Calif. using 454 technologies.

Homology search for the determined nucleotide sequence was conducted using the DDBJ/GenBank/EMBL database. uently, the phylogenetic relationship of the nucleotide sequence of the 16 rRNA genes was analyzed among the isolated ial strains described herein, bacteria of the genera and species that exhibit high sequence homologies to 2012/069579 the isolated ial strains, and other wide varieties of bacterial genera and species, using the ClustalW phylogenetic tree building program. Sequence identity and similarity were also determined using GenomeQuestTM software (Gene-IT, Worcester Mass. USA). The sequence analysis result revealed that the ial isolates SGI-003_Hl l, SGI-020_A01, SGI- 026_G06, SGI—026_G07, SGI—034_C09, SGI-034_E10, SGI—04l_B03 can be considered to be related to the species of a agglomerans, Bacillus thuringiensis, lderz’a metallica, Burkholderia vietnamz'ensz's, Bacillus pumilus, Herbaspirillum sp., Pedobacter sp., respectively, based upon >98% sequence homologies of each of the 16 rRNA sequences to the respective microorganisms.

EXAMPLE 4: Biochemical Characteristics of the Bacterial Isolates The isolated bacteria were further studied for properties important in their interaction with plants. The studied properties included nitrogen fixation, siderophore secretion, lization of inorganic phosphorus, production of ocyclopropane-l- carboxylic acid (ACC) deaminase, tion of 2,3 diol, and the production of plant growth hormone auxin. The s of in vitro mical assays are shown in Table 2.

Nitrogen fixation: Bacterial cell suspensions were streaked on a solid medium of the following composition which did not include a nitrogen source: KOH 4.0 g/L; K2HPO4 0.5 g/L; MgSO4'7H20 0.2 g/L; NaCl 0.1 g/L; CaClz 0.02 g/L; FeSO4'7H20 0.005g/L; NaMoO4'2H20 0.002 g/L; MnSO4'7H20 0.01 g/L; Malic Acid 5.0 g/L; Gellan Gum 0.l — 1.0 g/L; and optionally 0.5% v/v Bromothymol blue, pH 7.0. Gellan gum or agar concentrations may be varied as necessary to achieve desired medium thickness; typically 0.5 g/L was used. Streaks were incubated at 30°C for 2 — 5 days. These plates were monitored daily and colonies were ed as they appeared. In some cases, longer growth periods (up to two weeks or greater) allowed for the capture of slower growing es. These streak plates were typically colony-picked using 20 or 200 uL aerosol barrier pipette tips into 96- well cell e plates filled with 150 uL/well of 2YT medium. Alternatively, isolates were colony-picked from plates directly into N—free medium to confirm their N—free growth abilities. The results, as summarized in Table 2, indicated that only the isolate SGIG07 showed nitrogen fixing activity at a detectable level.

Siderophore secretion: This assay was used to identify bacterial isolates that were producing siderophores, which are high-affinity Fe3+-chelating compounds, in vitro. Typically, the microbial isolates were cultured on a minimal medium which was essentially free of Fe. All glassware used throughout this assay was acid-washed and rinsed three times with milliQ water to remove residual Fe which may alter assay results. The composition of the MM9 medium was as s: K2HPO4 0.5g/L; NH4Cl l.0g/L; MgSO4'H20 ; NaCl 05ng PIPES Buffer 7.55g/L; Glucose 10.0g/L; Gluconic Acid 2.5g/L; Malic Acid 2.5g/L; Casamino Acids 0.5 g/L. The medium was ed to pH 7.0 with 5N KOH, and sterilized using a 0.2 uM filter (Corning).

This assay was typically run in a high-throughput format using a Beckman FX liquid handling station and l cell culture plates with 150 uL MM9 growth medium per well. Cultures and media were distributed and transferred aseptically using an autoclavable pin-tool under a laminar flow hood. Following transfer, cultures were incubated at 30°C for 5 days. After tion, the culture supematants were ted via centrifiJgation using a 96- well 0.22 uM 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 MM9 medium. Two-hundred microliters of the CAS assay solution [10 mM HDTMA, Fe(III)-Solution: 1 mM F6C13.6H20, 10 mM HCl, 2 mM CAS] was added to each of the supematants and standard wells, followed by incubation at room temperature for 20 — minutes. The absorbance of the blue CAS assay on at 630 nm (SpectroMax M2) is ely proportional to the siderophore concentration in each well (i.e., the assay solution should change to an intense orange with greater quantities of siderophores).

Solubilization of inorganic phosphorus: The ability of the ial isolates to lize l phosphate in vitro was assessed as follows. Bacteria to be tested were streaked on an agar phosphate growth medium [Hydroxylapatite — Ca10(PO4)5(OH)2 5.0g/L; NH4Cl l.0g/L; MgSO4'H20 0.2g/L; NaCl 05ng 7H20 0.01g/L; NazMoO4'7H20 0.01g/L; MnSO4'7H20 0.01g/L; Glucose ; Gluconic Acid 2.5g/L; Malic Acid 2.5 g/L; Casamino Acids 0.5g/L; Gellan Gum .0g/L; pH 7.2)], and their growth was monitored daily. The culture medium had an opaque appearance due the present of calcium phosphate. Bacterial growth and loss of the color of the medium would be observed if the bacteria have dissolving ability of calcium phosphate.

Isolates having the y to solubilize the mineral phase phosphate would produce a clear halo on the opaque medium surrounding the colony. As summarized in Table 2, the ability to solubilize mineral phosphate was not able in any of the tested microorganisms as determined by the in vitro assay described herein.

ACC deaminase p_roduction: One of the major isms utilized by plant growth-promoting acteria (PGPM) to facilitate plant grth and development is the lowering of ethylene levels by deamination of l-aminocyclopropane-l-carboxylic acid (ACC), the immediate sor of ethylene in plants. ACC deaminase catalyzes the hydrolysis of l-aminocyclopropane-l- carboxylic acid (ACC) into (x-ketobutyrate and ammonia. The presence of the obutyrate product can then be determined indirectly via a reaction with 2, 4-dinitrophenylhydrazine in HCl to form a phenylhydrazone derivative. After an addition of NaOH, the amount of ydrazone in solution can be determined spectrophotometrically by measuring its absorbance at 540 nm (Penrose and Glick, Physiol Plant. May;ll8:lO-l, 2003). This assay was typically run in a high-throughput format using 96-well cell culture plates. Each well contained 150 uL DF salts growth medium supplemented with 2.0 g/L (NH4)2SO4. es and media were distributed and transferred aseptically using an autoclavable pin-tool under a laminar-flow hood. Following transfer, cultures were ted at 30°C for 2 days. After ng turbidity, the cultures were transferred a second time using a sterile pin-tool under a laminar-flow hood into 96-well plates containing 150 uL per well of DF salts grth medium supplemented with 5 mM ACC as the sole nitrogen source, followed by a 4 day incubation at °C. Absorbance of each culture at 600 nm was measured using a spectrophotometer. es that displayed robust growth under these conditions (OD >0.2) were taken forward for fiarther assay for ACC deaminase activity as described in Penrose and Glick, 2003, supra.

The test results, as summarized in Table 2, indicated that the following isolates produced significant amounts of ACC deaminase: SGIHll, SGIG06, SGI G07, and SGI-04l-B03. 2,3-butanediol production: The ability of the bacterial isolates to synthesize tanediol in vitro was assessed as follows using capillary gas chromatography mass oscopy as described by Ryu et al. (Proc. Natl. Acad. Sci. USA. 100:4927-4932, 2003). This assay was typically run in a hroughput format using 96-well cell culture plates with 150 uL DF salts growth medium per well. A titer-tek may also be used when preparing a large number of plates for primary screens of large isolate collections. Cultures and media were distributed and erred aseptically using an autoclavable pin-tool under a laminar-flow hood. Following transfer, cultures were incubated at 30°C for 5 days. After incubation, the culture supematants were harvested via fiagation using a 96-well 0.22 uM filter plate. Fifty microliters of filtered atant from each well was transferred to corresponding wells of a deep 96-well plate containing 450 uL 50% methanol per well using a L200 multichannel pipette and sealed with an adhesive plate seal, ed by 2,3-butanediol quantification assay using the protocol described by Ryu et al. (2003, supra). The test results, as summarized in Table 2, indicated that the following isolates ed significant s of 2, 3-butanediol: SGI- 003-Hl l, SGIC09, and SGI-04l-B03.

Production of auxin: Auxins are es that can directly affect plant growth. This assay was performed to determine if the bacterial isolates produced auxins, since many rhizosphere and endophytic bacterial isolates are known to possess biochemical pathways that synthesize the auxin indoleacetic acid (IAA) and its derivatives. Tryptophan is often a precursor in this synthesis; and therefore, this assay quantified 1AA (auxin) production from bacterial es grown on a medium supplemented with a low concentration of the amino acid phan.

This assay was typically run in a high-throughput format using 96-well cell culture plates with 150 uL YT growth medium per well. When preparing a large number of plates for primary screens of large isolate collections, a titer-tek was used. Cultures and media were distributed and transferred aseptically using an autoclavable pin-tool under a laminar-flow hood. Following transfer, cultures were incubated at 30°C for 5 days. After incubation, the culture supematants were ted via filgation using a l 0.22 uM filter plate. Ten microliters of filtered supernatant from each well was transferred to a Falcon assay plate. Two hundred microliters of the sky’s assay solution (Gordon and Weber, Plant Physiol. 26:192-195, 1951) was added to each of the supernatant and standard wells, followed by incubation at room temperature for 15 — 20 minutes. The reaction was monitored by absorbance of the plate on the SpectroMax M2 at 535 nm as color change from yellow to purple/pink of the Salkowsky’s assay solution was proportional to the concentration of auxin (1AA) in each well. The test results, as summarized in Table 2, indicated that the WO 90628 following isolates produced significant amounts of the phytohormone auxin: SGI—003-Hl l, SGIA01, SGIC09, SGI—034-C09, and SGI—04l-B03.

Table 2: mical characteristics of the bacterial isolates (ND: not detectable).

Bacterial Isolates Biochemical Activity Isolate ID Provisional Auxin ACC- 2,3— N-fixation Phosphoms- Taxonomy production deaminase butanediol solubilization 003_Hll Pantoea Yes Yes Yes ND ND agglomerans 020_AOl Bacillus Yes ND ND ND ND thuringiensis 026_G06 Burklzolderz'a ND Yes ND ND ND metallica 026_G07 Burklzolderz'a ND Yes ND Yes ND vietnamiensis 034_C09 Bacillus pumilus ND Yes ND ND 034_ElO Herbaspz'rz'llum ND ND ND ND ND 04 l_B03 Pedobacter Sp. Yes Yes Yes ND ND EXAMPLE 5: Biocontrol ty of the bacterial isolates t fungal phytopathogens An in vitro antagonism assay was used to assess the ability of the ed bacterial s to suppress the pment of several plant fiangal pathogens, including Fusarz'um graminearum NRRL-5883, aphella nivalz's ATCC MYA-3968, Gibberella zeae ATCC-l6lO6, Stagnospora nodurum 6369, Colletotrichum graminz'cola ATCC-34l67, and a Penicillium sp. pathogen. The assay was performed on potato dextrose agar (PDA) medium. Isolated strains of bacteria were grown on one-fifth th Tryptic soy broth agar (TSBA/S) for 24 h prior to use.

For each fungal pathogen, a conidial inoculum was ed by hyphal tipping an actively growing colony of the fungus and transferring the hyphal strands to FDA agar medium. After incubating the plates for 7 days at 25°C using a 12 h/day photoperiod, fiangal conidia were washed from FDA plates using a weak phosphate buffer (0.004% phosphate buffer, pH 7.2, with 0.019% MgClz). A suspension of fungal conidia in the weak phosphate buffer ximately l X 105 conidia/mL) was then immediately sprayed over the agar surface, and the sprayed plates were then ted at 25°C for 48-72 h prior to use in antagonism tests.

To initiate the antagonism tests, cells of isolated ial strains were pointinoculated at equal distances inside the perimeter of the plate. After five days, the bacterial strains were scored as antibiosis positive when a visibly clear area (z'.e., growth inhibition zone) that lacked mycelial grth existed around the perimeter of the ial colonies. The results of antagonism assays, as summarized in Table 3, trated that each of the microorganisms disclosed herein inhibited the development of several fungal phytopathogens, including Fusarium graminearum, Monographella nivalis, Gibberella zeae, Stagnospora m, Colletotrichum graminicola, Penicillium sp.

Table 3: Biocontrol actiVity of the bacterial isolates t fitngal phytopathogens.

Bacterial Isolates Growth ssion of fungal pathogen (inhibition zone scored after 5 days of incubation) Isolate Provisional Fusarium Monographella Gibberella Stagnospora Colletom'chum Penicillium ID Taxonomy gramz'nearum nivalz's zeae nodurum gramz'm'cola sp. 003_H1 1 Pantoea No Yes No No No No agglomerans 020_A01 Bacillus Yes No Yes Yes Yes No thuringz'ensz's 026_G06 lderia Yes Yes Yes Yes Yes metallica 026_G07 lderia Yes No No No No viemamiensis 034_C09 Bacillus No No Yes No No pumilus 034_E10 Herbaspz'rillum No No Yes No No 041_B03 Pedobacter sp. \0 Yes No Yes Yes Yes EXAMPLE 6: Enhancement ofwheat yield potential Effects of bacterial inoculation on plant growth and yield were studied in a greenhouse with the isolate 0-A01. Microbial cell suspensions were prepared as follows. 2YT medium, or similar growth media, broth cultures were inoculated from the isolate’s glycerol stocks or streak plates. Typically, prior to use in the growth chamber, greenhouse, or field, bacterial cultures were initiated 48 — 72 hours to allow the cultures to reach late exponential phase. Isolates that have longer doubling times were initiated fiarther in advance. Cultures were incubated at 30°C on a rotary shaker at 200 rpm. After growth, the cells were pelleted at 10,000 X g for 15 min at 4°C and resuspended in 10 mM MgSO4 buffer (pH 7.0). Cell densities were normalized for each isolate on a CFU/mL basis. Typically, ~109 CFU/mL suspensions were prepared for each isolate and transported on ice to the inoculation site. Inoculations were performed by diluting these cell suspensions 1/20 in irrigation water to a final density 5 X 107 CFU/mL. For 1 liter pot trials, 20 mL of this dilute cell suspension was distributed evenly over the surface of each replicate pot.

] Greenhouse trial was conducted with a nutrient deficient field soil. After ng large rocks and debris, f1eld soil was mixed thoroughly to ensure homogeneity. After filling, soil in each of the pots was pressed down ~2 cm for a firm sowing layer. Seeds of a commercial wheat cultivar (hard red spring wheat; Howe Seeds, Inc.) were sown in 1 liter pots containing field soil medium (10.5 cm X 12.5 cm d er plastic pots). Two grams of spring wheat seeds (approximately 70 seeds) were distributed evenly in each pot and 50 mL of field soil were applied and spread evenly over seed layer. ing uniform emergence of wheat coleoptile and uent emergence of first leaf, the plant population was inoculated with 20 mL of 109 CFU/mL of SGI—020-A01. Plants of negative ls received 20 mL of inoculum buffer only. Each ion was performed in 8 replicate flats, each containing four 1 liter pots (n=4 per flat). The flats were randomly distributed over four experimental blocks. The seeds and plants were then maintained in a greenhouse for 60 days at ambient temperature (ranging from about 8°C to about 22°C) with l light cycles of approximately 11.5 hours sunlight/ 12 hours dark throughout the trial. Plants were mly bottom watered to appropriate hydration level depending on the temperature and stage of growth. At approximately 30 days post , approximately 70 individuals per pot were staked and loosely tied together to prevent cross contamination and to minimize positional effects due to variation in plants g into other pots. At approximately 60 days post sowing, plants were allowed to dry out in preparation for harvest. Wheat heads were harvested at approximately 80 days post . Each wheat head was removed by g just below the head. Wheat heads within each pot replicate were pooled, weighted, and subsequently used as an estimate of yield potential. All plants in the population were harvested on the same day and ents were harvested in a randomized order to eliminate large differences in time in between harvesting between ents. As a result, wheat plants treated with the isolate SGIA01 showed a 40% increase in yield potential compared to control eated plants (2.95gram/pot vs. 2.10gram/pot). Averages and standard deviations were documented across all 8 replicates and an ANOVA (Analysis of Variance) was med. Efficacy of the microbial e SGIA01 in enhancing wheat yield potential was fied by analyzing the wheat head yield in weight for each pot replicate. P-values of <05 were considered significant.

E 7: Enhancement ofbiomass production in maize ] Effects of bacterial inoculation on plant grth and yield were studied in greenhouse experiments with each of the following bacterial isolates: SGI—034-C09, SGI- 034-E10, SGIH11, SGIB03, SGIG06, and SGIG07. The greenhouse trials were conducted with a nutrient deficient field soil. After removing large rocks and debris, field soil was mixed thoroughly with potting soil (70:30) to ensure homogeneity.

After filling, soil in each of the pots was pressed down ~2 cm for a firm sowing layer. Seeds of a commercial maize cultivar (Dow AgroSciences) were sown in 1 liter pots (10.5 cm X 12.5 cm tapered pots) each containing soil medium. Two maize s were distributed evenly in each pot in embryo-up orientation, followed by application of 50 mL of field soil, which was spread evenly over the seed layer. After germination, culling of one ng per pot was performed if necessary so that each pot contained only one plant.

Following uniform emergence of maize coleoptile and subsequent emergence of first leaf, the plant population was inoculated with ~20 mL of 109 CFU/ml of a microbial isolate selected from the group of SGIC09, SGIE10, 3-H11, SGIB03, SGIG06, and SGIG07. Microbial cell suspensions were prepared as described in Example 6 above. Plants of negative controls received 20 mL of inoculum buffer only.

Each condition was performed in 8 replicate flats, each containing two 1 liter pots (n=2 per flat). The flats were ly distributed over four experimental blocks. The seeds and plants were then maintained in a greenhouse for 60 days at ambient temperature (ranging from about 8°C to about 22°C) with diurnal light cycles of approximately 11.5 hours sunlight/ 12 hours dark throughout the trial. Plants were uniformly bottom d to appropriate hydration level depending on the temperature and stage of growth. Maize ground biomass was harvested at approximately 60 days post sowing.

] All plants in the population were harvested on the same day and ents were harvested in a randomized order to eliminate large differences in time in between harvesting n treatments. Maize plants were analyzed for difference in total biomass. As documented in Table 4, maize plants treated with each of the microbial isolates showed a significant increase in total biomass as compared to control non-treated plants. Averages and standard deviations were documented across all 8 replicates and an ANOVA (Analysis of Variance) was performed.

Table 4: Efficacy of the microbial isolates in enhancing total plant biomass.

Treatment Plant biomass (g) p-Value Biomass Increase (%) Non-treated 58.6 N/A N/A SGIC09 106.3 <.0001 181% SGIE10 103.6 <.0001 177% SGIH11 100.7 <.0001 172% 1-B03 99.5 0.0001 170% SGIG06 98.3 0.0002 168% SGIG07 97.3 0.0003 166% EXAMPLE 8: Seed coating treatment of wheat seeds and corn seeds ] Small scale seed treatment experiments were conducted by ing a procedure described in Sudisha et al. (Phytoparasitz’ca, 37:161-169, 2009) with minor modifications.

Typically, a biopolymer stock solution was made by adding 1 gram of gum arabic powder (MP Biomedical) to 9 mL water and mixing to homogeneity. Turbid cultures of actively g ial cells or microbial spore preparations were washed with PBS and adjusted to an OD600 of ~5.0. Three mL of the adjusted cell suspension was pelleted via filgation in a 50 mL Falcon tube. The resulting supernatant was decanted, replaced with 3 mL biopolymer stock solution and the resulting suspension was mixed thoroughly.

Typically, approximately 25 g of seeds were added to the Falcon tube and usly shaken or vortexed to ensure a uniform distribution of the gum/cell suspension. Coated seeds were spread across plastic weigh boats to dry in a laminar flow hood until no longer tacky, generally 3 hours with periodic mixing. The coated seeds were then stored at 4°C and periodically tested for stability. A variety of wheat seeds and corn seeds were coated and tested in the manner described above, including common hard red spring wheat varieties Briggs, Faller, Glenn, Hank, RB07, ; hard red winter wheat varieties Jerry, McGill, Overland; and maize seed variety DKC62-6l as well as a cial maize cultivar (Dow AgroSciences).

Viability testing on the microbes used in seed coating formulation was performed using a standard plate count method. Typically, a pre-determined amount of coated seeds was tested for the presence of viable microbes by washing the seeds in an aliquot of appropriate buffer and plating equivalent amounts of buffer on nutrient agar media. Viable colony- forming-units were ined after 1-4 days incubation at 30°C. Viability test showed that between l X 104 and 4 X 107 viable colony-forming-units per seed were present after approximately five weeks of storage at 4°C. When seeds were coated with microbial spores, the viability of the ty of tested microbes remained stable for at least four months, including multiple interstate shipments across the United States, in and out of refrigerated containers. When stored under refrigeration (4°C) the microbes survived on the seed coat with little loss in viability over the test s. The results indicated that seeds coated with compositions disclosed herein could be stored for extended periods under refrigeration and ted that microbes would survive during periods of higher temperatures for distribution.

In on, germination rate of the coated seeds was tested and determined to be essentially identical to l seeds, which were either seeds coated with gum arabic only or uncoated seeds.

EXAMPLE 9: Solid State Formulation of the Microbial Comp_ositions This section describes an ary ation of a microbial fertilizer where the bacteria in accordance with the present invention are encapsulated and the fertilizer is in solid form. Alginate beads are prepared as follows: One milliliter of 30% glycerol is added to l, 1.5 or 2% sodium alginate solution, depending on the alginate properties (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 from a combination of two or more isolates is pelleted via centrifuged, then washed with a saline solution (0.85% NaCl, w/v), suspended in 25 mL of alginate mixture, and mixed thoroughly. This cell suspension is then added drop wise into a pre-cooled sterile 1.5 or 2% (w/v) aqueous on of CaClz under mild agitation to obtain the bacterial-alginate beads.

These beads are allowed to harden for 2-4 h at room ature. Beads are collected by g and are washed several times with sterile water and stored at 4°C. In order to preserve the formulation, the fresh wet beads can be frozen at about -80°C prior to lyophilization at about -45°C for 15 h. The lyophilized dry beads can be stored in appropriate ners, such as sterile glass bottles.

To estimate the viable counts, the encapsulated bacteria can be ed from the beads by resuspending 100 mg of beads in ate buffered saline (pH 7.0) for 30 min followed by homogenization. The total number of released bacteria is determined by standard plate count method after incubating at 30°C for 48 h. At one month intervals the cell densities in the beads are enumerated using similar method.

E 10: Compatibility of the ial compositions with commercial filngicides As environmental concerns are increasing about using pesticides in agriculture, biological alternatives are increasingly perceived as inevitable. However, new biological formulations must also allow organisms to survive and express their c beneficial . Chemical ides are lly toxic not only towards deleterious microorganisms but also to the beneficial ones. However, chance of survivability of these microbial agents might have been enhanced when applied at reduced rates.

In the present study, peat-based carrier material is used for inoculation of both the fungicide treated as well as bare crop seed. ial tolerability of filngicide is generally evaluated in the following manner: a) bacteria inoculated bare seeds grown on an appropriate bacterial growth medium such as trypticase soy agar (TSA; Tryptone 15g/L; Soytone 5 g/L, sodium chloride 5g/L, and agar l5g/L) plates, b) fungicide-treated bacteria inoculated seeds grown on common TSA plates, and, c) fungicide-treated bacteria inoculated seeds grown in the sterile growth pouches. Typically, three trations of fiangicide are used in each of the experiments: manufacturer’s recommended dose and two lower doses (at 75% and 50% of the ended dose). Fungicide-treated bacteria inoculated seeds are stored after inoculation and used at different time intervals (2 hrs, 4 hrs and 6 hrs) to examine the impact on seed germination. Both seed germination and ial presence are monitored in petri- plates. For the grth pouch study, fungicide-treated (recommended dose) seeds are used, and root and hypocotyl lengths were measured at 7 days of seedling growth.

Some rhizobacterial isolates of the invention are compatible with several commonly used fiangicides as ined by ial growth on fungicide-enriched TSA plates. In general, both bare and fungicide-treated seeds, coated with inoculated peat show no cant variation in germination compared to non-inoculated control. Moreover, growth- promoting effects on root and total seedling lengths are observed in all rhizobacterial treatments compared to non-inoculated control.

EXAMPLE 11: Development of non-naturally occurring cultivars and breeding program Endophytic bacteria of the present ion are introduced into crop plants, including cereals, of varying genotypes and geographic origin, lacking such endophytic fungi, to create plant-endophyte combinations with improved agronomic characteristics, using procedures analogous to those known in the art, including those described in US. Pat. Appl.

No. 20030195ll7Al; US. Pat. Appl. No. 20010032343Al; and US. Pat. No. 331, among others. Thus, synthetic plant-endophyte combinations may be created and selected in a breeding/cultivar pment program based on their ability to form and maintain a mutualistic combination that results in an agronomic benefit. Rating of agronomic characteristics of the combination may also be utilized in such a breeding m. These characteristics may include, without limitation, drought tolerance, biomass accumulation, ance to insect infestation, palatability to livestock (e.g., herbivores), ease of reproduction, and seed yield, among others. Such combinations may differ in levels of accumulation of microbial metabolites that are toxic to pests and weeds, including ergot id levels, loline levels, peramine levels, or lolitrem levels, while displaying desired agronomic teristics of crop plants, including resistance to insect feeding or ation, ance to abiotic stress, bility to livestock, biomass accumulation, ease of reproduction, and seed yield, among other traits.

E 12: Yield Study Corn seeds (Zea mays) were coated with different ial treatments and sown in a ed field. Each ent was replicated 5 times in random complete block design.

A single replicate consisted of four 30 feet long beds (rows), 60 seeds were sown (6 inches apart) in each bed. For the observation purpose data was taken from the middle two rows only.

Plant emergence was ed twice as shown in Table 5 below as the percentage of plants in the replicate that had sprouted. Ten plants in the middle two rows of each plot were tagged with a plastic ribbon to record the vital statistics such as the plant height, chlorophyll measurement, plant weight etc. of the plants.

The plant heights (measuring the tip of the tallest/longest leaf) recorded 31 and 56 days after planting indicated that the plant height among the treatments was not significantly different. Most of the plants were dry and leaves had shrunk by day 110 post planting, therefore, in some of the cases, plants looked (measured) shorter than in previous measurement, but overall, the plant height did not differ among the treatments. On the 5th week of planting, chlorophyll content was measured (in SPAD units) from the lower leaves (ca. 60 cm above the ground) and upper leaves (second fiJlly expanded leaf from the top) of ten tagged plants of each plot. The chlorophyll content among the treatments did not significantly differ. On the 110th and 111th day of ng, the crop was harvested. Ten tagged plants from each plot were cut at the soil level and above the ground parts of the plants were weighed (whole plant weight or WPtWt), corn ears were removed and the length of the cob was measured (ear length with kernels) (region filled with marketable kernels only), then the kernels were removed from the cob and their weight was measured for the kernel weight per ear ear).

Shortly after the manual t of 10 plants per plot was over, a mechanical harvester, Gleaner® K2 (Allis-Chalmers Mfrg, Milwaukee, WI) was brought in. This machine mechanically removed the remaining plants from two middle rows of each plot, removed the kernels from the cobs, and took the measurement of kernel moisture and weight (10 ears + machine harvest). The ted total yield at 15.5 % moisture content (pounds of corn kernel per acre) based on the weight (lb.) of kernels (include kernels from machine harvested cobs+manual harvested cobs) was 10368.14 pounds per acre or 185.15 bushels per acre for SGIH11 (Pantoea erans). This was the highest yield among all organism treatments and cantly different from the treatments for us amyloliquefaciens SGI-015_F03. The other high producer was the treatment with Bacillus thuringiensis SGI-020_A01.

In conclusion, all the plants in the different treatments emerged and grew similarly in the field conditions provided with the same amount of fertilizer, ergent herbicide (with manual weed pulling later in the season), and weekly irrigation during the growing and warm season, and pest control of ally the corn earworm. With all conditions equal, treatment with SGIH11 ea agglomerans) produced the highest yield over the non-microbe, control treatment. Thus, 003_H11 produced a yield of approximately 17% higher than the control group. Thus, in various embodiments of the invention application of an effective amount of the organism according to any of the methods described herein produces at least 10% or at least 12.5% or at least 15% or about 17% higher yield than a control group, which in some embodiments can be ined by pounds of plant product (e. g., corn ears) per acre or bushels of plant product per acre. Organisms listed in Table 5 are SGIH11 (Pantoea agglomerans); SGIF03 lus amyloliquefaciens); and SGI- 020-A01 (Bacillus thuringiensis).

Table 5 Organism Emergence Emergence Height Height Height Date 6/20 6/24 7/11 8/5 9/28 Control 97.00 94.50 103.64 277.22 267.82 SGIH11 89.50 93.34 103.32 273.58 272.04 SGIF03 95.67 96.67 105.02 282.02 276.08 SGIA01 94.83 95.33 102.04 268.94 264.80 Organism Upper Leaf Lower Leaf WPtWt Ear Length w/ knls Date 7/18 7/18 9/28 9/28 Control 43.24 60.85 493.24 14.98 3-H11 44.17 59.43 494.92 14.29 SGIF03 44.90 63.96 473.40 14.45 SGIA01 46.45 63.84 488.30 14.20 Organism Knl wt/ear(g) Knl wt/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 3-H11 171.96 0.38 185.15 10368.14 SGIF03 156.24 0.34 161.17 9025.62 SGIA01 158.68 0.35 163.09 9133.16 A number of embodiments of the invention have been described. Nevertheless, it will be understood that elements of the ments described herein can be combined to make additional embodiments and various modifications may be made t departing from the spirit and scope of the invention. Accordingly, other embodiments, alternatives and lents are Within the scope of the invention as described and claimed herein.

Headings Within the application are solely for the convenience of the reader, and do not limit in any way the scope of the invention or its embodiments.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically can individually ted to be incorporated by reference.

[Annotation] tboyc_piz BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE T OF MICROORGANISMS FOR THE E OF PATENT URES INTERNATIONAL FORM TO RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT Patent Dept. issued nt to Rule 7.1 by the Synthetic Genomics, Inc. INTERNATIONAL DEPOSITARY AUTHORITY 11149 North Torrey Pines Rd. ‘ identified at the bottom of this page La jolla, CA 92037 NANLE AND S OF DEPOSITOR I. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the DEPOSITOR: Accession number given by the Pantoea agglomeram ATIONAL DEPOSITARY AUTHORITY: 003_H11 NRRL 13—50483 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I. above was accompanied by: [I 1a scientific ption SJa proposed mic designation III. RECEIPT AND ACCEPTANCE This ational Depositary Authority accepts the microorganism identified under 1. above, which was received by it on March 28, ate of the original deposit)2 IV. RECEIPT OF RECUEST FOR CONVERSION The microorganism identified under I. above, was received by this International Depositary Authority on ___________________ (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).

V. INTERNATIONAL DEPOSITARY AUTHORITY Name: Agricultural Research Culture Signature(s) of person(s) having the power to represent the Collection (NRRL) International Deposita A thority or of authorized official(s): International Depositary Authority Address: 1815 N. University Street Peoria, Illinois 61604 U.S.A. Date: Hflrfl 1 Mark with a cross the applicable box. 2 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired. 5656 [Annotation] tboyc_piz BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT URES INTERNATIONAL FORM TO RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT Patent Dept. issued pursuant to Rule 7.1 by the ’ Synthetic Genomics, Inc. INTERNATIONAL DEPOSITARY AUTHORITY 11149 North Torrey Pines Rd identified at the bottom of this page La Jolla, CA 92037 NAME AND ADDRESS OF DEPOSITOR I. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the DEPOSITOR: Accession number given by the m tburiflgiemir INTERNATIONAL DEPOSITARY AUTHORITY. 020_A01 NRRL 13-50484 II. SCIENTIFIC PTION AND/OR PROPOSED TAXONOMIC DESIGNATION The rganism identified under I. above was accompanied by: [I 1a scientific description E121 proposed taxonomic designation III. RECEIPT AND ACCEPTANCE This International Depositary Authority accepts the microorganism identified under 1. above, which was ed by it on March 28, 2011(date of the original deposit)2 IV. RECEIPT OF RE I UEST FOR CONVERSION The microorganism identified under I. above, was received by this International Depositary Authority on_______________ (date of the original deposit) and a request to convert the original deposit to a deposit under the BudapestmTreaty was received by it on (date of receipt of request for conversion).

V. INTERNATIONAL DEPOSITARY AUTHORITY Name: Agricultural Research Culture Signature(s) of (s) having the power to represent the Collection (NRRL) International Depositary Authoty or of authorized official(s): International tary ity Address: 1815 N. University Street i.

~ Peoria, Illinois 61604 USA. Date: Ll... p-“ 1 Mark with a cross the applicable box. 2 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired. 5757 [Annotation] tboyc_piz [Annotation] piz ST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT PROCEDURES INTERNATIONAL FORM TO RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT Patent Dept. issued pursuant to Rule 7.1 by the Synthetic cs, Inc. INTERNATIONAL DEPOSITARY AUTHORITY 11149 North Torrey Pines Rd. identified at the bottom of this page La Jolla, CA 92037 NAME AND ADDRESS OF DEPOSITOR I. FICATION OF THE MICROORGANISM Identification reference given by the DEPOSITOR: Accession number given by the Burkba/dcria metal/1m INTERNATIONAL TARY ITY: 026_G06 NRRL B—50485 II. SCIENTIFIC PTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under 1. above was accompanied by: E] 1a scientific description Qa proposed taxonomic designation III. RECEIPT AND ACCEPTANCE This International Depositary Authority s the microorganism identified under I. above, which was received by it on March 28, 201 1 (date of the original deposit)2 IV. RECEIPT OF REQUEST FOR CONVERSION ' The microorganism identified under I. above, was received by this ational Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).

V. INTERNATIONAL DEPOSITARY AUTHORITY Agricultural ch Culture Signature(s) of person(s) having the power to represent the Collection (NRRL) International Depositary Authori or of authorized official(s): International Depositary Authority ¢ Address: 1815 N. University Street Peoria, Illinois 61604 U.S.A. Date: 1,, fl 3"” 1 Mark with a cross the applicable box. 2 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired. 5858 [Annotation] tboyc_piz BUDAPEST TREATY ON THE ATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT PROCEDURES ATIONAL FORM TO RECEIPT IN THE CASE OF AN ORIGINAL T Patent Dept. issued pursuant to Rule 7.1 by the tic Genomics, Inc. INTERNATIONAL DEPOSITARY AUTHORITY 11149 North Torrey Pines Rd. identified at the bottom of this page La Jolla, CA 92037 NAME AND ADDRESS OF DEPOSITOR I. IDENTIFICATION OF THE MICROORGANISM fication reference given by the TOR: Accession number given by the Barbe/dank vietnamz'emzk INTERNATIONAL DEPOSITARY AUTHORITY: 026_G07 NRRL B—50486 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The rganism identified under I. above was accompanied by: D 1a ific description % proposed taxonomic designation III. RECEIPT AND ACCEPTANCE This International Depositary ity accepts the microorganism identified under I. above, which was received by it on March 28, 2011(date of the original deposit)2 IV. RECEIPT OF RE 0 UEST FOR CONVERSION The microorganism identified under 1. above, was received by this International Depositary Authority on ________________ (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).

V. INTERNATIONAL DEPOSITARY ITY Name: Agricultural Research Culture Signature(s) of person(s) having the power to represent the Collection (NRRL) International Depositary Authori or of authorized official(s): International Depositary Authority \ 2 Address: 1815 N. University Street Peoria, Illinois 61604 USA. Date: ' “/4 3/1] 1 Mark with a cross the applicable box. 2 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired. 5959 ation] tboyc_piz GRDS003WO sequence listing SEQUENCE LISTING <110> SYNTHETIC GENOMICS, INC.

BULLIS, DAVID T.

GRANDLIC, CHRISTOPHER J.

MCCANN, RYAN KEROVUO, JANNE S. <120> PLANT GROWTH-PROMOTING MICROBES AND USES THEREFOR <130> SGI1540-1WO <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 1436 <212> DNA <213> Pantoea agglomerans <220> <221> misc_feature <223> SGI bacterial isolate SGI-003_H11 <220> <221> misc_feature <223> encodes a 16S ribosomal RNA <220> <221> misc_feature <222> (63) <223> n is a, c, g, or t <400> 1 ggca ggcctaacac gtcg agcggtagca cagagagctt gggt gnngagcggc ggacgggtga gtaatgtctg ggaaactgcc tgatggaggg ggataactac tggaaacggt agctaatacc gcataacgtc gcaagaccaa agtgggggac cttcgggcct atca gatgtgccca gatgggatta gctagtaggt gaggtaatgg ctcacctagg cgacgatccc tagctggtct gagaggatga ccagccacac tggaactgag acacggtcca gactcctacg ggaggcagca gtggggaata aatg ggcgcaagcc tgatgcagcc atgccgcgtg tatgaagaag gccttcgggt tgtaaagtac tttcagcgag gaggaaggcg ataaggttaa taaccttgtc gattgacgtt actcgcagaa gaagcaccgg ctaactccgt gccagcagcc atac ggagggtgca agcgttaatc ggaattactg ggcgtaaagc ation] tboyc_piz GRDS003WO sequence listing gcacgcaggc ggtctgtcaa gtcggatgtg aaatccccgg gctcaacctg ggaactgcat ccgaaactgg caggctagag tcttgtagag gggggtagaa ttccaggtgt agcggtgaaa tgcgtagaga tctggaggaa taccggtggc ggcc ccctggacaa acgc tcaggtgcga gggg agcaaacagg attagatacc ctggtagtcc acgctgtaaa cgatgtcgac ttggaggttg ttcccttgag gagtggcttc cggagctaac gcgttaagtc gaccgcctgg ggagtacggc cgcaaggtta aaactcaaat gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgatg gaag aaccttacct actcttgaca tccagagaac ttagcagaga tgctttggtg ccttcgggaa ctctgagaca ggtgctgcat 1020 ggctgtcgtc agctcgtgtt gtgaaatgtt gggttaagtc ccgcaacgag cgcaaccctt 1080 tgtt gccagcggtt cggccgggaa ctcaaaggag actgccagtg ataaactgga 1140 ggaaggtggg gatgacgtca agtcatcatg gcccttacga gtagggctac acacgtgcta 1200 caatggcata tacaaagaga ctcg cgagagcaag cggacctcat aaagtatgtc 1260 gtagtccgga tcggagtctg caactcgact ccgtgaagtc ggaatcgcta gtaatcgtgg 1320 atcagaatgc cacggtgaat acgttcccgg gccttgtaca ccgt cacaccatgg 1380 gagtgggttg aagt aggtagctta accttcggga gggcgcttac cacttt 1436 <210> 2 <211> 1440 <212> DNA <213> Bacillus thuringiensis <220> <221> misc_feature <223> SGI bacterial isolate 0_A01 <220> <221> misc_feature <223> encodes a 16S ribosomal RNA <400> 2 cgtgcctaat acatgcaagt cgagcgaatg gattaagagc ttgctcttat gaagttagcg [Annotation] piz GRDS003WO sequence listing gggt gagtaacacg tgggtaacct aaga taac tccgggaaac cggggctaat accggataat attttgaact ttcg aaattgaaag gcggcttcgg ctgtcactta gacc cgcgtcgcat tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg cgac ctgagagggt gatcggccac actgggactg ggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga aggctttcgg aaac tctgttgtta gggaagaaca agtgctagtt gaataagctg gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta tccggaatta ttgggcgtaa agcgcgcgca ggtggtttct taagtctgat gtgaaagccc acggctcaac cgtggagggt cattggaaac tgggagactt agaa gaggaaagtg gaattccatg tgtagcggtg aaatgcgtag agatatggag gaacaccagt ggcgaaggcg actttctggt ctgtaactga cactgaggcg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt tagagggttt ccgcccttta gtgctgaagt taacgcatta agcactccgc agta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct ccta gagatagggc ttctccttcg ggagcagagt gacaggtggt 1020 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080 tctt agttgccatc attaagttgg gcactctaag gtgactgccg gtgacaaacc 1140 ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200 ctacaatgga cggtacaaag agctgcaaga ccgcgaggtg gagctaatct cataaaaccg 1260 ttctcagttc ggattgtagg ctgcaactcg cctacatgaa gctggaatcg ctagtaatcg ation] piz GRDS003WO sequence listing 1320 cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380 cgagagtttg ccga agtcggtggg gtaacctttt tggagccagc cgcctaaggt 1440 <210> 3 <211> 1414 <212> DNA <213> lderia metallica <220> <221> misc_feature <223> SGI bacterial isolate SGI-026_G06 <220> <221> misc_feature <223> encodes a 16S ribosomal RNA <400> 3 catgcaagtc cagc acgggtgctt gcacctggtg gcgagtggcg aacgggtgag taatacatcg gaacatgtcc tgtagtgggg gatagcccgg cgaaagccgg attaataccg atct acggatgaaa gcgggggacc ttcgggcctc gcgctatagg gttggccgat ttag ctagttggtg gggtaaaggc ctaccaaggc gacgatcagt agctggtctg agaggacgac cagccacact gggactgaga cacggcccag actcctacgg gaggcagcag tggggaattt tggacaatgg gcgaaagcct gatccagcaa tgccgcgtgt gtgaagaagg ccttcgggtt gtaaagcact tttgtccgga aagaaatcct tgattctaat acagtcgggg gatgacggta ccggaagaat cggc taactacgtg ccagcagccg cggtaatacg gcga gcgttaatcg gaattactgg gcgtaaagcg tgcgcaggcg gtttgctaag accgatgtga cggg ctcaacctgg gaactgcatt ggtgactggc aggctagagt atggcagagg ggggtagaat tccacgtgta gcagtgaaat gcgtagagat gtggaggaat accgatggcg aaggcagccc cctgggccaa tactgacgct catgcacgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccctaaac gatgtcaact agttgttggg [Annotation] tboyc_piz GRDS003WO sequence listing gattcatttc cttagtaacg tagctaacgc gtgaagttga ccgcctgggg agtacggtcg caagattaaa agga attgacgggg acaa gcggtggatg atgtggatta attcgatgca acgcgaaaaa ccttacctac ccttgacatg gtcggaatcc tgctgagagg cgggagtgct cgaaagagaa ccggcgcaca ggtgctgcat ggctgtcgtc agctcgtgtc 1020 gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gtccttagtt gctacgcaag 1080 ctaa ggagactgcc ggtgacaaac cggaggaagg tgac gtcaagtcct 1140 catggccctt atgggtaggg cttcacacgt catacaatgg tcggaacaga gggttgccaa 1200 cccgcgaggg ggagctaatc ccagaaaacc gatcgtagtc gcac tctgcaactc 1260 gagtgcatga agctggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc 1320 ccgggtcttg tacacaccgc ccgtcacacc gtgg gttttaccag aagtggctag 1380 tctaaccgca aggaggacgg cggt agga 1414 <210> 4 <211> 1423 <212> DNA <213> Burkholderia vietnamiensis <220> <221> misc_feature <223> SGI ial isolate SGI-026_G07 <220> <221> misc_feature <223> encodes a 16S ribosomal RNA <400> 4 tgccttacac atgcaagtcg aacggcagca cgggtgcttg cacctggtgg cgagtggcga acgggtgagt aatacatcgg aacatgtcct gtagtggggg atagcccggc gaaagccgga ttaataccgc atacgatcta aggatgaaag cgggggacct tcgggcctcg cgctataggg ttggccgatg gctgattagc tagttggtgg ggcc taccaaggcg acgatcagta gctggtctga gacc agccacactg ggactgagac acggcccaga ctcctacggg [Annotation] tboyc_piz 3WO sequence listing aggcagcagt ggggaatttt ggacaatggg cgaaagcctg atccagcaat gccgcgtgtg tgaagaaggc cttcgggttg taaagcactt ttgtccggaa agaaatcctt ggctctaata cagtcggggg gtac aata agcaccggct aactacgtgc cagcagccgc ggtaatacgt agggtgcaag cgttaatcgg aattactggg cgtaaagcgt gcgcaggcgg tttgctaaga ccgatgtgaa gggc tcaacctggg aactgcattg gtgactggca ggctagagta tggcagaggg gggtagaatt ccacgtgtag cagtgaaatg cgtagagatg tggaggaata ccgatggcga aggcagcccc ctgggccaat actgacgctc atgcacgaaa gcgtggggag caaacaggat tagataccct ggtagtccac gccctaaacg atgtcaacta gttgttgggg attcatttcc ttagtaacgt agctaacgcg tgaagttgac cgcctgggga gtacggtcgc aagattaaaa ctcaaaggaa ttgacgggga cccgcacaag cggtggatga ttaa ttcgatgcaa cgcgaaaaac cttacctacc cttgacatgg tcggaagccc gatgagagtt gggcgtgctc gaaagagaac cggcgcacag gtgctgcatg gctgtcgtca 1020 gctcgtgtcg tgagatgttg ggttaagtcc gagc gcaacccttg tccttagttg 1080 ctacgcaaga gcactctaag gagactgccg gtgacaaacc aggt ggggatgacg 1140 tcaagtcctc atggccctta tgggtagggc ttcacacgtc atacaatggt cggaacagag 1200 ggttgccaac ccgcgagggg gagctaatcc cagaaaaccg atcgtagtcc cact 1260 ctgcaactcg agtgcatgaa gctggaatcg ctagtaatcg agca tgccgcggtg 1320 aatacgttcc cgggtcttgt acacaccgcc cgtcacacca tgggagtggg ttttaccaga 1380 agtggctagt gcaa ggaggacggt caccacggta gga 1423 <210> 5 <211> 1441 <212> DNA [Annotation] tboyc_piz GRDS003WO sequence listing <213> Bacillus pumilus <220> <221> misc_feature <223> SGI bacterial isolate SGI-034_C09 <220> <221> misc_feature <223> encodes a 16S ribosomal RNA <220> <221> misc_feature <222> .(605) <223> n is a, c, g, or t <400> 5 gcggcgtgcc taatacatgc aagtcgagcg gacagaaggg agcttgctcc cggatgttag cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggagcta ataccggata gttccttgaa ccgcatggtt caaggatgaa agacggtttc cact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc acga tgcgtagccg gagg gtgatcggcc acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtgcaag agtaactgct tgcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcatnggaaa ctgggaaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt cgta gagatgtgga ccag tggcgaaggc gactctctgg tctgtaactg agga gcgaaagcgt ggggagcgaa taga taccctggta gtccacgccg taaacgatga gtgctaagtg ttagggggtt tccgcccctt gcag ctaacgcatt tccg cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct [Annotation] tboyc_piz GRDS003WO ce listing tgacatcctc tgacaaccct agagataggg ctttcccttc ggggacagag tgacaggtgg 1020 tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080 cccttgatct tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac 1140 cggaggaagg tgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 1200 gctacaatgg acagaacaaa gggctgcgag accgcaaggt ttagccaatc ccacaaatct 1260 gttctcagtt cggatcgcag tctgcaactc gactgcgtga aatc gctagtaatc 1320 gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1380 acgagagttt gcaacacccg aagtcggtga ggtaaccttt ccag ccgccgaagg 1440 1441 <210> 6 <211> 1410 <212> DNA <213> Herbaspirillum sp. <220> <221> misc_feature <223> SGI ial isolate SGI-034_E10 <220> <221> misc_feature <223> encodes a 16S mal RNA <400> 6 tgcaagtcga aacggcagca taggagcttg ctcctgatgg cgagtggcga acgggtgagt aatatatcgg aacgtgccct agagtggggg ataactagtc gaaagactag ctaataccgc atacgatcta cggatgaaag tgggggatcg caagacctca tgctcctgga gcggccgata tctgattagc tagttggtgg ggtaaaagcc taccaaggca acgatcagta gctggtctga gacc actg ggactgagac acggcccaga ctcctacggg aggcagcagt tttt ggacaatggg ggcaaccctg atccagcaat gccgcgtgag tgaagaaggc [Annotation] tboyc_piz GRDS003WO sequence listing cttcgggttg tctt ttgtcaggga agaaacggta gtagcgaata actattacta atgacggtac ctgaagaata ggct aactacgtgc cagcagccgc ggtaatacgt agggtgcaag tcgg aattactggg gcgt gcgcaggcgg aagt cagatgtgaa atccccgggc tcaacctggg aattgcattt gagactgcac ggctagagtg tgtcagaggg gggtagaatt ccacgtgtag aatg cgtagatatg tggaggaata ccgatggcga aaggcagccc cctgggataa cgct catgcacgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccctaaac gatgtctact cggg tcttaattga cttggtaacg cagctaacgc gtgaagtaga ccgcctgggg agtacggtcg caagattaaa actcaaagga attgacgggg acccgcacaa gatg atgtggatta attcgatgca acgcgaaaaa ccttacctac ccttgacatg gatggaatcc tgaagagatt tgggagtgct cgaaagagaa ccatcacaca ggtgctgcat ggctgtcgtc tgtc 1020 gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gtcattagtt gctacgaaag 1080 ggcactctaa tgagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcct 1140 catggccctt atgggtaggg cttcacacgt catacaatgg tacatacaga ccaa 1200 cccgcgaggg ggagctaatc ccagaaagtg tatcgtagtc cggattggag tctgcaactc 1260 gactccatga agttggaatc gctagtaatc gcggatcagc atgtcgcggt gaatacgttc 1320 ccgggtcttg tacacaccgc ccgtcacacc atgggagcgg gttttaccag aagtgggtag 1380 cctaaccgca aggagggcgc tcaccacggt 1410 <210> 7 <211> 1368 <212> DNA <213> Pedobacter sp. <220> <221> misc_feature [Annotation] piz GRDS003WO sequence listing <223> SGI bacterial isolate SGI-041_B03 <220> <221> misc_feature <223> encodes a 16S ribosomal RNA <220> <221> misc_feature <222> .(529) <223> n is a, c, g, or t <400> 7 gaaagtggcg cacgggtgcg taacgcgtat gcaacctacc tggg ggatagcccg gagaaatccg gattaatacc gcataaaatc acagtactgc atagtgcaat gatcaaacat ttatgggaag aagatgggca tgcgtgtcat tagctagttg gcggggtaac ggcccaccaa ggcgacgatg ggat ggat ggccccccac actggtactg agacacggac cagactccta cgggaggcag cagtaaggaa tattggtcaa tggaggcaac tctgaaccag ccatgccgcg aaga ctgccctatg aaac tgcttttatc cgggaataaa cctgagtacg tgtacttagc tgaatgtacc ggaagaataa ggatcggcta actccgtgcc agcagccgcg gtaatacgga ggatccaagc gttatccgga tttattgggt ttaaagggtg cgtaggcggc ctgttaagtc aggggtgaaa gacggtagct caactatcng cagtgccctt gatactgatg ggcttgaatg gactagaggt aggcggaatg agacaagtag cggtgaaatg catagatatg tctcagaaca ccgattgcga aggcagctta ctatggtctt attgacgctg aggcacgaaa ggat caaacaggat tagataccct ggtagtccac gccctaaacg atgaacactc ggcg atacacagtc agcggctaag cgaaagcgtt aagtgttcca cctggggagt acgctcgcaa gagtgaaact aatt gacgggggcc cgcacaagcg gaggagcatg tggtttaatt cgatgatacg cgaggaacct tacccgggct tgaaagttag tgaatcattt agagataaat gagtgagcaa tcacacgaaa gctg catggctgtc gtcagctcgt gccgtgaggt gttgggttaa gtcccgcaac gagcgcaacc cctatgttta 1020 [Annotation] tboyc_piz GRDS003WO sequence listing gttgccagca cgttatggtg gggactctaa acagactgcc tgtgcaaaca gagaggaagg 1080 aggggacgac gtcaagtcat catggccctt acgtccgggg acgt atgg 1140 atggtacaga ctac atagcaatat gatgcgaatc tcacaaagcc attcacagtt 1200 cggattgggg tctgcaactc gaccccatga agttggattc gctagtaatc cagc 1260 aatgacgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcaagc catggaagtt 1320 gggggtacct aaagtatgta accgcaagga gcgtcctagg gtaaaacc 1368 <210> 8 <211> 39 <212> DNA <213> Artificial sequence <220> <223> PCR primer M13-27F <400> 8 tgtaaaacga cggccagtta gagtttgatc ctggctcag <210> 9 <211> 37 <212> DNA <213> Artificial sequence <220> <223> PCR primer 1492R M13-tailed <400> 9 caggaaacag ctatgaccgg ttaccttgtt acgactt <210> 10 <211> 1059 <212> DNA <213> Pantoea agglomerans <400> 10 atggctatcg acgaaaacaa acagaaagcg ttggcggcag cactgggcca gatcgaaaaa ggta aaggctccat catgcgcctg ggtgaagacc gttccatgga cgtggaaact accg tttc cctggatatc gccctcggcg ctggcggtct gccaatgggc gtcg aaatctacgg gcctgaatct tccggtaaaa cgacgctgac cctgcaggtt [Annotation] tboyc_piz GRDS003WO sequence listing atcgccgccg cgcagcgtga aggtaaaacc ttta tcgatgcaga gcacgcgctg gtat atgcccgcaa gctgggcgtc gatatcgaca acctgctgtg ctctcagccg gacaccggcg aacaggcgct ctgt gacgcgctgg cgcgctccgg tgcggtagac gtgctggtgg tcgactccgt tgcggcgctg acgccgaaag cggaaatcga aggcgagatc tctc acatgggcct cgcggcgcgt atgatgagcc aggcaatgcg ggcc ggtaacctga agcagtccaa cacgctgctg atcttcatca accagatccg tatgaaaatt ggcgtgatgt tcggtaaccc ggaaaccacc accggcggta acgcgctgaa attctacgcc cgtc tggatatccg ccgtatcggt gcggtaaaag atggcgataa cgtcattggt agcgaaaccc gcgtgaaggt cgtgaagaac aaaatcgccg cgccgttcaa gcaggcggag ttccagatcc tctacggcga aggcatcaac ttcttcggcg agctggtcga tctgggcgtg aaagagaagc tgattgaaaa agcgggcgcc tggtatagct acaacggcga caaaattggt cagggtaaag cgaacgctat ctcctggctg aaagagaacc cggctgcggc gaaagagatc gagaagaaag ttcgtgaact gctgctgaac aaccaggatg ccacgccgga cttcgcggtt 1020 gatggtaaaa gcgaagaagc aagcgaacag tga 1059 <210> 11 <211> 352 <212> PRT <213> a agglomerans <400> 11 Met Ala Ile Asp Glu Asn Lys Gln Lys Ala Leu Ala Ala Ala Leu Gly 1 5 10 15 Gln Ile Glu Lys Gln Phe Gly Lys Gly Ser Ile Met Arg Leu Gly Glu 25 30 Asp Arg Ser Met Asp Val Glu Thr Ile Ser Thr Gly Ser Leu Ser Leu 40 45 ation] tboyc_piz GRDS003WO sequence listing Asp Ile Ala Leu Gly Ala Gly Gly Leu Pro Met Gly Arg Ile Val Glu 50 55 60 Ile Tyr Gly Pro Glu Ser Ser Gly Lys Thr Thr Leu Thr Leu Gln Val 65 70 75 80 Ile Ala Ala Ala Gln Arg Glu Gly Lys Thr Cys Ala Phe Ile Asp Ala 85 90 95 Glu His Ala Leu Asp Pro Val Tyr Ala Arg Lys Leu Gly Val Asp Ile 100 105 110 Asp Asn Leu Leu Cys Ser Gln Pro Asp Thr Gly Glu Gln Ala Leu Glu 115 120 125 Ile Cys Asp Ala Leu Ala Arg Ser Gly Ala Val Asp Val Leu Val Val 130 135 140 Asp Ser Val Ala Ala Leu Thr Pro Lys Ala Glu Ile Glu Gly Glu Ile 145 150 155 160 Gly Asp Ser His Met Gly Leu Ala Ala Arg Met Met Ser Gln Ala Met 165 170 175 Arg Lys Leu Ala Gly Asn Leu Lys Gln Ser Asn Thr Leu Leu Ile Phe 180 185 190 Ile Asn Gln Ile Arg Met Lys Ile Gly Val Met Phe Gly Asn Pro Glu 195 200 205 Thr Thr Thr Gly Gly Asn Ala Leu Lys Phe Tyr Ala Ser Val Arg Leu 210 215 220 Asp Ile Arg Arg Ile Gly Ala Val Lys Asp Gly Asp Asn Val Ile Gly 225 230 235 240 Ser Glu Thr Arg Val Lys Val Val Lys Asn Lys Ile Ala Ala Pro Phe 245 250 255 Lys Gln Ala Glu Phe Gln Ile Leu Tyr Gly Glu Gly Ile Asn Phe Phe 260 265 270 Gly Glu Leu Val Asp Leu Gly Val Lys Glu Lys Leu Ile Glu Lys Ala 275 280 285 [Annotation] tboyc_piz GRDS003WO ce listing Gly Ala Trp Tyr Ser Tyr Asn Gly Asp Lys Ile Gly Gln Gly Lys Ala 290 295 300 Asn Ala Ile Ser Trp Leu Lys Glu Asn Pro Ala Ala Ala Lys Glu Ile 305 310 315 320 Glu Lys Lys Val Arg Glu Leu Leu Leu Asn Asn Gln Asp Ala Thr Pro 325 330 335 Asp Phe Ala Val Asp Gly Lys Ser Glu Glu Ala Ser Glu Gln Asp Phe 340 345 350

Claims (24)

CLAIMS What is claimed is

1. An enriched culture of a microbial SGIH11 strain deposited as NRRL B-50483.

2. An isolated culture of a ial SGIH11 strain ted as NRRL B-50483.

3. A biologically pure culture of a microbial SGIH11 strain deposited as NRRL B- 50483.

4. An isolated microbial strain ted as NRRL B-50483 or a strain derived therefrom;

5. An isolated microbial strain comprising a DNA ce exhibiting at least 99% sequence identity to SEQ ID NO:1; and further wherein said microbial strain has a plant growth-promoting activity.

6. A composition comprising a microbial strain or culture according to any one of claims 1-5, and an agriculturally 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, a nematicide, and a pesticide.

7. A composition comprising a microbial strain or culture according to any one of claims 1-5, and a carrier.

8. A composition ing to claim 7, wherein said carrier is a plant seed.

9. A composition according to claim 7, wherein said composition is a seed coating formulation.

10. A ition according to claim 7, wherein said composition is prepared as a formulation selected from the group consisting of an emulsion, a d, a dust, a e, a pellet, a powder, a spray, an emulsion, and a solution.

11. A plant seed having a coating comprising a ition according to claim 9.

12. A method for treating a plant seed, said method comprising a step of exposing or contacting said plant seed with a microbial strain or culture according to any one of claims 1-5.

13. A method for enhancing the growth and/or yield of a plant, said method comprising applying an effective amount of a microbial strain or culture according to any one of claims 1-5 to the plant, or to the s surroundings.

14. A method according to claim 13, wherein said microbial strain or e is grown in a growth medium or soil of a host plant prior to or concurrent with host plant growth in said growth medium or soil.

15. A method according to claim 13, wherein said plant is a corn plant or a wheat plant.

16. A method according to claim 13, wherein said microbial strain or culture is established as an endophyte on said plant.

17. A method for preventing, inhibiting or ng the development of a plant pathogen, said method sing growing a microbial strain or culture according to any one of claims 1-5 in a growth medium or soil of a host plant prior to or concurrent with host plant growth in said growth medium or soil.

18. A method ing to claim 17, wherein said plant pathogen is selected from the group consisting of Colletotrichum, Fusarium, Gibberella, Monographella, Penicillium, and Stagnospora sms.

19. A method according to claim 17, wherein said plant pathogen is selected from the group consisting of Colletotrichum graminicola, um graminearum, Gibberella zeae, Monographella nivalis, Penicillium sp., or Stagnospora nodurum.

20. A method for preventing, inhibiting or treating the pment of a pathogenic disease of a plant, said method comprising applying an effective amount of a microbial strain or culture according to any one of claims 1-5 to the plant, or to the plant's surroundings.

21. A method according to claim 20, wherein said microbial strain or culture is applied to soil, a seed, a root, a flower, a leaf, a portion of the plant, or the whole plant.

22. A non-naturally occurring plant that is a plant artificially infected with a microbial strain or culture according to any one of claims 1-5.

23. Seed, reproductive tissue, vegetative tissue, regenerative tissues, plant parts, or progeny of a turally occurring plant according to claim 22.

24. A method for ing an agricultural composition, said method comprising inoculating a microbial strain or culture according to any one of claims 1-5 into or onto a substratum and allowing said microbial strain or culture to grow at a temperature of 1- 37°C until obtaining a number of cells or spores of at least 102-103 per milliliter or per gram.