MX358751B

Plant growth-promoting microbes and uses therefor.

Abstract

Microbial strains, compositions, and methods of use thereof to enhance the growth and/or yield of a plant are provided. Also provided are materials and methods for presenting, inhibiting, or treating the development of plant pathogens or phytopathogenic diseases. The disclosure also provides non-naturally occurring plant and derivatives thereof such as plants artificially infected with a microbial strain of the invention.

MX358751B, drawing sheet 1 — Sheet 1 of 111

Term

6.2 yearsleft in the term

Expires 13 December 2032.

13 claims: 2 independent, 11 dependent

1
NOVEDAD DE LA INVENCIÓN NOVELTY OF THE INVENTION CLAIMS REIVINDICACIONES 1 - A seed of coated plant having a coating comprising a carrier and an endophyte, characterized in that the endophyte comprises the microbial strain SGI-003-H11, deposited as NRRL B50483. 1,- Una semilla de planta recubierta que tiene un recubrimiento que comprende un portador y un endófito, caracterizada porque el endófito comprende la cepa microbiana SGI-003-H11, depositada como NRRL B50483.
10
10 - A method to inhibit or treat the development of a pathogenic disease of a plant caused by a plant pathogen, said 10, - Un método para inhibir o tratar el desarrollo de una enfermedad patogénica de una planta causada por un patógeno vegetal, dicho 20 método caracterizado porque comprende crecer una planta a partir de la semilla recubierta como la que se reclama en la reivindicación 1. twenty method characterized in that it comprises growing a plant from the coated seed as claimed in claim 1.

Independent claims2

563 paragraphs in 112 sections, as filed

(54) Title: MICROBES THAT PROMOTE PLANT GROWTH AND USE OF THESE. (54) Title: PLANT GROWTH-PROMOTING MICROBES AND USES THEREFOR.

(57) Summary

The present invention relates to microbial strains, compositions, and methods of using these to enhance the growth and / or yield of a plant; Materials and methods are also provided to present, inhibit, or treat the development of plant pathogens or plant pathogenic diseases; The disclosure also provides plants of non-natural origin and derivatives thereof such as plants artificially infected with microbial strains of the invention.

(57) Abstract

Microbial strains, compositions, and methods of use thereof to enhance the growth and / or yield of a plant are provided. Also provided are materials and methods for presenting, inhibiting, or treating the development of plant pathogens or phytopathogenic diseases. The disclosure also provides non-naturally occurring plant and derivatives thereof such as plants artlficially infected with a microbial strain of the invention.

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PATENT TITLE No. 358751

Owner (s): MONSANTO TECHNOLOGY LLC

Address: 800 North Lindbergh Blvd., St. Louis, Missouri, 63167, USA

Denomination: MICROBES THAT PROMOTE PLANT GROWTH AND USE OF THESE.

Classification: CIP: C12R1 / 07; A01N6j3 / Q2; CQ5G3 / 02; C12N1 / 20

CPC: C12R1 / 07; A01N63 / 02; C0SG3 / 02; C12N1 / 20

Inventor (s): DAVID T, BULLIS; CHRISTOPHER J. GRÁNDLJC; RYAN MCCANN; JANNE S.

KEROVUO

Number:

MX / a / 2014/007070

Ffeha de »PfosimMwión Internacional:

de, p, eterníce 2012

PRIORITY

Country: PmM;

ΊUS December 13, 2011

Validity: Twenty years

Expiration Date: December 13, 2Q32

Number:

61/570,237

Issue Date: August 31, 2018

The reference patent is granted on the basis of artreufos 1 °, F fraesiph V, 6? ftaccÍpay.lll. and 59 of the Law «on Industrial Property.

Pursuant to article 23 of the Industrial Law, the patent shall be valid for twenty years. .

Whoever signs this title does so based on the provisions of the | adiqptqs 5th sections III and 7th bis 2 of the Industrial Property Law (Official Gazette of the Federation (D.OF.) 06/27/1991, reformed the 08/02/19 ^ 4 ¡0 / 1ΟΠ9β6. · - # »12 / 19ΒΤ / 17/06/1999. 01/26/2004, 06/16/2005, 01/25/2006, 05/06/2009, 06/01 / 2010,18 / 06/2010, 28/06 / 201¾ 27 / ^ 1/2012 ^ 0 ^ 06/2012) 08/01/2046 and 03/13/2018), articles 1. 3rd section V Section a), 4 "and 12th sections I and III of the Reglameoto = of the Mexican Instinct of the IndustiaMELQ Property /. 14ij12 / il989í'Tafonado on 01/07/2002, 07/15/2004, 07/28/2004 and 09/07/2007): articles 1, 3, 6, 5 * section V subsection e0,) 6 fractions! and III and 30 of the Organic Statute of the Mexican Institute of Industrial Property (DOF 12/27/1999, amended on 10/10/2002, 07/29/2004 04/08/2004 and 13 (59/2007), 1st, 3rd and 5th paragraph a) of the Agreement that delegates powers to the Deputy Directors General, ^ üordiiiad & r, EBrtolPles Qiviskmatep, THulaníS dpMs Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Instituto Meditarás de I »Industrial Property (DOF 12/15/1999, amended on 02/04/2000, 07/29 / 2004.04 / 08/2004 and 13/09/2007). ·.

This letter is signed with an advanced electronic signature (FIEL), based on articles 7 BIS 2 of the Industrial Property Law; 3 of its Regulations, and 1 fraction III, 2 fraction V, 26 BIS and 26 TER of the Agreement establishing the guidelines for the use of the Electronic Payment and Services Portal (PASE) of the Mexican Institute of Industrial Property, in the procedures indicated.

THE DIVISIONAL DIRECTOR OF PATENTS

NAHANNY CANAL REYES

Original string:

NAHANNY MARISOL CANAL REYES | 00001000000403252793 | Tax Administration Service | 1695 || MX / 2018/76595 | MX / a / 2014/007070 | Patent title PCT | 1223 | GAGV | Pág (s) | t2llzxSmCk8EQ3U2TyQZLWf

Digital stamp:

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MX / 2018/76595

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11 * 3 II MU MUCAMO

MICROBES THAT PROMOTE PLANT GROWTH AND tBtSSBS

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This application claims the benefit of provisional application 5 61 / 570,237, filed on December 13, 2011, which is incorporated herein in its entirety by this reference, including all tables, figures and claims.

FIELD OF THE INVENTION 10

The present invention relates to the field of sustainable agriculture. Specifically, the disclosure provides useful microbial compositions and methods for the production of crop plants. In particular, the compositions and methods described 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 attached sequence listing is incorporated into this application in its entirety by this reference. The attached file, named “SGI1540_1WO_CRF_OF_SL_ST25.txt, was created on December 13, 2012 and is 20 KB. Files can be accessed using Microsoft

Word on a computer with Windows operating system.

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INDUSTRIAL

BACKGROUND OF THE INVENTION

The microflora that surrounds plants is very diverse, including bacteria, fungi, yeast, algae. Some of these microorganisms can be harmful to plants, and are sometimes called pathogens, while others are beneficial to plants because they promote plant growth and crop productivity. Recent advances in soil microbiology and plant biotechnology have resulted in an interesting increase in the use of microbial agents in agriculture, horticulture, forestry, and environmental management. In particular, a number of microorganisms known to be present in the ecological niche of the soil, generally known as the rhizosphere or rzoplane, have received considerable attention regarding their ability to promote plant growth. In fact, the rhizosphere soil represents an adequate deposit of microbes for the potential isolation of beneficial microbes. The plant rhizosphere can contain billions 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, due to insufficient colony-forming units per gram of soil. Therefore, since 1960, a number of biofertilizers have been developed that have an increased colony inoculum potential concentration, and have been commercialized in an attempt to reduce the need for chemical fertilizers.

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In addition, research carried out on its lus úlíiusus diius, Ira demonstrated that microorganisms can be used as biological control agents to increase agricultural productivity and efficiency. These studies have shown that various microorganisms are capable of eliminating plant pathogens and / or complement plant growth, thus offering an attractive alternative to chemical pesticides that are less favorable due to their potentially negative impact on human health and environmental quality.

Microorganisms that can colonize plant roots and stimulate plant growth are generally known as plant growth promoting microorganisms (PGPMs). Many species of PGPM have been described in the past two decades as having a positive influence on the growth of a variety of crop plants. PGPMs are often symbiotes of older plants, and are able to enhance the adaptive potential of their hosts through a number of mechanisms, such as molecular nitrogen fixation, mobilization of nutrients from decay-resistant soils (eg, iron, phosphorus, sulfur, etc.), the synthesis of phytohormones and vitamins, and the decomposition of plant materials in soils that often increase soil organic matter. Certain microbes can also facilitate plant growth by controlling microbial species pathogenic for plants (ie, plant pathogens). For example, some beneficial microbes can control decay

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MEXICAN INSTITUTE

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INDUSTWal of the root in plants, by competition with the ntlliyus μυι · aspüüiu on the surface of the root of the plant. In other cases, competition between various microbial strains in the plant's natural microflora can stimulate root growth and increase absorption of mineral nutrients and water to improve plant performance. Therefore, biofertilizers can be developed as products based on microorganisms that naturally live in the soil. By increasing the population of beneficial microorganisms in the soil through artificial inoculation, these soil microorganisms can enhance their biological activity and thus provide plants with important nutrients and beneficial factors that enhance their growth.

Inoculating cultivated plants with PGPM is generally seen as a promising agricultural approach, because it allows pests to be controlled without the use of high amounts of pesticides. Given the growing concern about groundwater quality and food exposure to pesticides, biological alternatives are necessary. Therefore, the development of a biological treatment compatible with fertilizers and pesticides or even the reduction of the amount of these chemical compounds could be a significant advance in the agricultural industry. It has been established that stimulation of plant growth by means of PGPM is often closely related to the ability of PGPM to colonize plant roots. However, relatively little attention has been paid to the development of

effective selection to obtain microbial strains with caponad. I have to control the colonization of the roots. The lack of such selection procedures slows down the study of plant bacterial symbiosis, and the implementation of PGPM in agriculture.

Therefore, there is a continuing need to identify new PGPMs and / or assess their compatibility with existing commercially available crop management products. Furthermore, additional research is needed to compare pure culture strains with complementary mixed strains of microorganisms that form synergistic consortia. Such mixed consortia may have greater potential for consistent performance with competitive capacity under different growth and environmental conditions.

BRIEF DESCRIPTION OF THE INVENTION

Microbial strains and cultures are provided herein. Microbial compositions and methods of use are also provided to enhance the growth and / or yield of a plant. Plant seed treatment methods are also provided through the use of microbial compositions described herein. In addition, methods are provided to prevent, inhibit, or treat the development of plant pathogens or the development of plant pathogenic diseases. The description also provides non-natural plant varieties that are

varieties artificially infected with an endophyte ^^ íTflOfebieeeu-de.Ja ™ »^ Invention. Seeds, reproductive tissue, vegetative tissue, regenerative tissue, plant parts, or plant progeny of non-natural origin are also provided. The description additionally 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 cultures thereof. In certain preferred embodiments of this aspect, the microbial strain may be SGI-003-H11 (deposited as NRRL B-50483); SGI-020-A01 (filed as NRRL B50484); SGI-026-G06 (filed as NRRL B-50485); SGI-026-G07 (deposited as NRRL B-50486), or a strain derived from one of said strains. In some other preferred embodiments, the microbial strain may comprise a nucleotide or amino acid sequence that exhibits at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or at least 99.5% sequence identity with any of the nucleotide sequences and / or amino acid sequences of 16S rlbosomlco and / or recA in the sequence listing. In some embodiments, the microbial strain also has an activity that promotes plant growth as described herein.

Microbial compositions including a microbial strain of the invention or a culture thereof are also provided. Such

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Microbial compositions in accordance with some popular trends 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 additionally include a carrier. In still other preferred embodiments, the carrier may be a vegetable seed. In certain embodiments of this aspect, the microbial composition is prepared as a formulation that can be an emulsion, a colloid, a powder, a granule, a sediment, a powder, an aerosol, an emulsion, or a solution. In some other preferred embodiments, the microbial compositions can be seed coating formulations. In yet another aspect, vegetable seeds are also provided which are coated with a microbial composition in accordance with the present invention.

In another aspect, methods of treating vegetable seeds are provided. Such methods include exposing or contacting the plant seeds with a microbial strain according to the present invention or a culture thereof.

In another aspect of the invention, methods for improving the growth and / or yield of a plant are provided herein. In some embodiments, said method involves applying an effective amount of a microbial strain according to the present invention or a culture thereof to

IMPI the plant, or around the plant. In some other modalities ·, the · method involves cultivating a microbial strain according to the present invention or a cultivation thereof in a culture medium or the soil of a host plant before or at the time of growth of the host plant in said culture medium or soil. In preferred embodiments, the plant can be a corn plant or a wheat plant. In some other modalities, the microbial strain or culture of this can be established as an endophyte in the plant.

In another aspect of the present invention, methods are provided to prevent, inhibit, or treat the development of a plant pathogen. Such methods include cultivating a microbial strain according to the invention or a culture thereof in a culture medium or the soil of a host plant before or at the same time as the growth of the host plant occurs in said culture medium or soil. In some preferred embodiments, the plant pathogen may be a microorganism of the genus Colletotrichum, Fusarium, Gibberella, Monographella, Penicillium, or Stagnospora. In some particularly preferred embodiments, the plant pathogen may be Colletotrichum graminicola, Fusarium graminearum, Gibberella zeae, Monographella nivalis, Penicillium sp. or Stagnospora nodurum.

Yet another aspect of the invention provides methods of preventing, inhibiting, or treating the development of a pathogenic disease in a plant. Such methods involve application to the plant, or to the surroundings

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of the plant, an effective amount of a microbial strain LAccording to the invention or a culture thereof. In some preferred embodiments, the microbial strain or culture thereof can be applied to the soil, a seed, a root, a flower, a leaf, a part of the plant, or the entire plant.

Yet another aspect of the invention provides plants of non-natural origin. Plants of non-natural origin are artificially infected with a microbial strain of the invention or a culture thereof. In additional modalities of this aspect, seeds, reproductive tissue, vegetative tissue, regenerative tissue, parts of plants and progeny of plants of non-natural origin are provided.

Another aspect of the invention provides methods for preparing an agricultural composition. Such methods involve inoculating the microbial strain according to the present invention or a culture of it in or on a substrate and allowing its growth.

In another aspect the invention provides an isolated strain, an isolated culture thereof, a biologically pure culture thereof or an enriched culture of a microorganism of the genus Pantoea. In one embodiment the microorganism comprises a DNA sequence or an amino acid sequence encoding 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 with a sequence encoding the gene of 16S rRNA or the recA protein

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described in the sequence listing. In another way, it provides a genus of microorganisms comprising any of the DNA sequences or amino acid sequences described above and which enhance the growth and / or performance of a plant, as described herein.

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

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical terms, notations, and other scientific terms or terminology employed herein have the meanings commonly understood by those skilled in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for the purpose of clarity and / or to make specific reference, and the inclusion of such definitions herein is not necessarily to be construed as representing a substantial difference from what which is generally understood in technique. Many of the techniques and procedures described or mentioned herein are commonly understood and employed using conventional methodology by those skilled in the art.

The singular form "a", "a" and "the" includes references in

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plural unless context clearly indicates TTé ÓlIU llianeia. For example, the term a cell includes one or more cells, and includes mixtures of these.

Bactericidal: The term "bactericidal", as used herein, refers to the ability of a composition or substance to increase mortality or inhibit the growth rate of bacteria.

Biological control: The term "biological control" and its short form "biocontrol", as used herein, is defined as the control of a pathogen or insect or any unwanted organism by using at least one second organism that is not be a human being. An example of a known biological control mechanism is the use of microorganisms that control root decay by occupying the space that would be occupied by fungi on the root surface, or microorganisms that inhibit growth or kill the pathogen. The host plant in the context of biological control is the plant that is susceptible to disease caused by the pathogen. In the context of isolating an organism, such as a bacterial or fungal species, from its natural environment, the host plant is a plant that supports the growth of the bacterium or fungus, for example, a plant of a species from which the bacteria or fungus is an endophyte.

An effective amount, as used herein, is an amount sufficient to produce 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 the

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enough to improve, stabilize, reverse, slow down, or delay the progression of the target infection or disease. The term "effective microorganism" used herein with reference to a microorganism, is intended to mean that the strain herein exhibits a degree of promotion of plant growth and / or yield or a degree of inhibition of a pathogenic disease that exceeds, in a statistically significant level, that of an untreated control. In some instances, the term "effective amount" is used herein in reference to an amount of a microbial treatment that is necessary to obtain a beneficial or desired result relative to that occurring in an untreated control under suitable treatment conditions, as described herein. For the purposes of this description, the actual application rate of a liquid formulation will usually range from a minimum of about 1 x 103 to about 1 x 101° viable cells / mL and preferably around 1 x 106 to about 5 x 109 viable cells / mL. Under most conditions, the strains of the invention described in the examples below will be effectively optimal with application rates in the range of about 1 X 106 a 1 X 109 viable cells / mL, assuming an application mode that would achieve a substantially uniform contact of at least around 50% of the plant tissues. If microorganisms are applied as a solid formulation, the application rate should be controlled to achieve a similar number of viable cells per unit area of plant tissue area, as obtained

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through the aforementioned treatment rates irqmVTiT'Τίμΐ ..... jL"Microbial compositions of the present invention are biologically effective when administered at a concentration in excess of 106 CFU / g (colony forming units per gram), preferably in excess of 107 CFU / g, more preferably 108 CFU / g and more preferably at 109 CFU / g.

Composition: Composition is intended to mean a combination of active agent and at least one other compound, carrier, or composition, which may be inert (eg, a detectable agent or label or liquid carrier) or active, such as a fertilizer.

A control plant, as used in the present description, provides a reference point to measure changes in the plant phenotype of the present, it can be any plant cell, seed, plant component, plant tissue, plant organ or plant integer suitable. A control plant may comprise, for example, (a) a wild-type plant or cell, that is, the same genotype as the starting material for the genetic alteration that resulted in the plant or cell herein; (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 that has no known effect on the trait of interest, such as a construct comprising a reporter gene ); (c) a plant or cell that is an untransformed segregator between the progeny of a plant or cell herein; (d) a plant or cell that is genetically identical to

IMPI the plant or cell of the present but which is not Assumed to the same treatment (for example, treatment with fertilizer) as the plant or cell of the present; (e) the plant or cell of the present one itself, under conditions in which the gene of Interest is not expressed; or (f) the plant or cell itself herein, under conditions in which it has not been exposed to a particular treatment such as, for example, a fertilizer or a combination of fertilizers and / or other chemicals.

Culture, isolated culture, biologically pure culture, and enriched culture: As used herein, an isolated strain of a microbe is a strain that has been removed from its natural environment. As such, the term "isolated" does not necessarily reflect the extent to which the microbe has been purified. But in different modalities an isolated culture has been purified at least 2x or 5x or 10x or 50x or 100x from the raw material from which it was isolated. As a non-exhaustive example, if a crop is isolated from the soil as raw material, the organism can be isolated to an extent where its concentration in a given quantity of purified or partially purified material (eg soil) is at least 2x or 5x or 10x or 50x or 10Ox than in the original raw material. A substantially pure culture of the microbe strain refers to a culture that substantially does not contain any other microbes than the desired strain or strains of microbes. In other words, a substantially pure culture of a strain of microbes is substantially free of other contaminants, which may include microbial contaminants, as well as unwanted chemical contaminants. Also, as used in

IMPI Dt In this industry, a biologically pure strain is intended to mean the separate strain of materials with which it is normally associated in nature. Note that a strain associated with other strains, or with compounds or materials with which it is not normally found in nature, is still referred to 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-exhaustive example, if a culture is normally associated with the soil in nature, the organism can be biologically pure to an extent in which its concentration in a given quantity of purified or partially purified material with which it is normally associated in the Nature (eg earth) is at least 2x or 5x or 10x or 50x or 100x than in the original unpurified material. As used herein, the term "enriched culture of an isolated microbial strain" refers to a microbial culture where the total microbial population of the culture contains more than 50%, 60%, 70%, 80%, 90%, or 95%. of the isolated strain.

Cultivate: The term cultivar, as used herein, refers to the spread of organisms in media of various types.

As used herein, an endophyte is an endosymbiont that lives within a plant for at least part of its life without causing obvious disease. Endophytes can be transmitted

IMPI either vertically (directly from parent to offspring) or horizontally (from Individual to unrelated individual). Vertically transmitted fungal endophytes are typically asexual and are transmitted from the parent plant to the progeny by penetration of the fungal hypha into the seeds of the host plant. Bacterial endophytes can also be transferred vertically from seeds to seedlings (Ferreira et al „FEMS Microbio !. Lett. 287: 8-14, 2008). In contrast, horizontally transmitted endophytes are typically sexual, and are transmitted by spores, and can spread by wind and / or insect vectors. Microbial endophytes of crop plants have received considerable attention regarding their ability to control both disease and insect infestation, as well as their potential to promote plant growth.

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

Fungicide: As used herein, fungicide refers to the ability of a composition or substance to decrease the growth rate of fungi or to increase fungal mortality.

Mutant: As used herein, the term mutant or

Variant in reference to a microorganism refers to a modification of the parent strain in which the desired biological activity is similar to that expressed by the parent strain. For example, in the case of Burkholderia, the parent strain is defined herein as the original Burkholderia strain prior to mutagenesis. Mutants or variants can occur in nature without human intervention. They can also be obtained by a variety of methods and compositions known to those skilled in the art. For example, a parent strain can be treated with a chemical such as N-methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfone, or by radiation using gamma, X, or ultraviolet radiation, or by other means known to those of skill in the art. The technique.

Nematicide: The term "nematicide" 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 algae, an arachnid, a bacterium, a fungus, an insect, a nematode, a parasitic plant, a protozoan, a yeast, or a virus capable of of producing a disease in a plant or animal. The term plant pathogen as used herein refers to a pathogenic organism that infects the plant.

Sequence Identity Percentage: The sequence identity percentage, as used herein, is determined by comparing two aligned sequences located

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optimal, in a comparison window defined by the length of the local alignment between the two sequences. The amino acid sequence in the comparison window may comprise additions or deletions (eg, gaps or surpluses) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The local alignment of two sequences only includes segments of each sequence that are considered sufficiently similar, according to a criterion that depends on a logarithm used to carry out the alignment (for example BLAST). The sequence identity percentage is calculated by determining the number of positions in which the nucleic acid base or amino acid residue appears in both sequences to obtain the number of coincident positions, dividing the number of coincident positions by the total number of positions in the comparison window and multiplying the result by 100. Optimal sequence alignment for comparison can be accomplished using the Smith and Waterman (1981) Add local homology algorithm. APL. Math. 2: 482, using the Needleman and Wunsch homology alignment algorithm (J Mol. Biol. 48: 443, 1970); by searching for the Pearson and Lipman similarity method (Proc. Nati. Acad.

Sci. USA 85: 2444, 1988), by heuristic implementations of these algorithms (NCBI BLAST, WU-BLAST, BLAT, SIM, BLASTZ), or by inspection. Since two sequences have been identified for comparison, GAP and BESTFIT are preferably used to determine

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its optimal alignment. Typically, the default poi values are used for the gap weight of 5.00 and 0.30 for the gap weight length. The term "substantial sequence identity between polynucleotide or polypeptide sequences" refers to polynucleotides or polypeptides that comprise a sequence identity that is at least 50%, 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 more preferably at least 96%, 97%, 98 % or 99% substance identity compared to a reference sequence using the programs. Furthermore, sequence homology or pairwise sequence similarity, as used, refers to the percentage of residues that are similar between two aligned sequences. 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 (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).

The query of nucleic acids and amino acid sequences is

IMPI ικητηπο ΜίικΑΝο MLAFaOnilMD moumiAi can search against amino acid sequences o-éeMee- ^ weleicos-deda invention found in public or private databases. Such searches can be performed using the Basic Local Alignment Search tool program of the National Center for Biotechnology Information (NCBI BLAST v 2.18). The NCBI BLAST program is available online through the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov/Blast.cgi). Typically the following parameters can be used for NCBI BLAST: Filter options are set to default, Comparison Matrix is set to BLOSUM62, Gap Cost is set to Existence: 11,

Extension: 1 ”, the word length is set to 3, the Expectation (threshold E) is set to 1e-3, and the minimum length for local alignment is set to 50% of the query sequence length. The identity and sequence similarity can be determined using the GenomeQuest ™ software (Gene-IT, Worcester Mass., USA).

The term pest as used herein refers to an unwanted organism that can include, but is not limited to, bacteria, fungi, plants (eg, weeds), nematodes, insects, and other pathogenic animals. Pesticide, as used herein, refers to the ability of a substance or composition to decrease the growth rate of a pest, i.e., an unwanted organism, or to increase the mortality of a pest.

Progeny: As used herein, progeny includes

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descendants of a particular plant or line of plants. The progeny of a plant of the invention include seeds formed in Ft, F2, F3, F4, F5i F6 and subsequent generations of plants, or seeds formed in BC ^ BC2, BC3, and subsequent generations of plants, or seeds formed in F1BC1,

FiBC2, F1BC3, and subsequent generations of plants. The designation F1 refers to the offspring of a cross between two parents that are genetically different. The F designations2, F3, F4, F5 and F6 refer to subsequent generations of progeny self pollinated or pollinated by a plant of the same hybrid of a Fv plant

Variant: As used herein in reference to a nucleic acid and polypeptide, the term variant is used herein to designate a polypeptide, protein or polynucleotide molecule with some differences, synthetically or naturally generated, in its amino acid sequences or nucleic acid, compared 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 may additionally consist of charge changes 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 that has identifying characteristics of the species to which it belongs, while having the

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minus a nucleotide sequence variation or a different identifiable trait with respect to the parent strain, where the trait is genetic (hereditary). For example, for a Bacillus thuringiensis strain 020_A01 that has plant growth promoting activity, identifiable traits include 1) the ability to inhibit the development of fungal phytopathogens, including Fusarium graminearum, Gibberella zeae, Stagnospora nodurum, Colletotrichum graminicola-, 2) the ability to improve wheat yield; and 3) have a 16S rRNA gene with a nucleotide sequence with a Sequence Identity greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% to the Bacillus 16S rRNA gene thuringiensis 020_A01; can be used to confirm a variant such as Bacillus thuringiensis 020_A01.

Yield: As used herein, the term yield refers to the amount of harvestable plant material or product derived from plants, and is normally defined as the measurable product of economic value of a crop. For crop plants, yield also means the amount of harvestable material per acre or unit of production. Yield can be defined in terms of quantity or quality. The harvested material can vary from crop to crop, for example, it can be seeds, above-ground biomass, roots, fruits, cotton fibers and other parts of plants, or any plant-derived product that is of economic value. The term yield also includes yield potential, which is the maximum yield obtainable. The

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Performance may depend on a quantity of -G component gives performance, which can be monitored by certain parameters. These parameters are known to those of skill in the art and vary from crop to crop. The term yield also includes the harvest index, which is the ratio of the biomass harvested to the total amount of biomass.

All publications and patent applications cited herein are incorporated herein by this reference to the same extent as if each individual publication or patent application is specifically and individually indicated as incorporated herein by this reference.

It is not admitted that any of the references constitutes prior art. The discussion of the references establishes what their authors affirm, and the applicants reserve the right to question the precision and relevance of the cited documents. It will be clearly understood that, although there are a number of prior art publications mentioned herein, these references do not constitute an admission that any of these documents form part of the common general knowledge of the art.

The discussion of general methods provided herein is intended for illustrative purposes only. Other alternative methods and modalities will be apparent to those skilled in the art in light of the present disclosure.

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Plant Growth-Promoting Microorganisms Various 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 (PGPM). The term plant growth promoting activity, as used herein, encompasses a wide variety of improved plant properties, including, for example, non-limitingly, improved nitrogen fixation, improved root development, increased leaf area , increased plant yield, increased seed germination, increased photosynthesis or an increase in the accumulated biomass of the plant. In various modalities the improvement is an increase of at least 10% or an increase of at least 25% or an increase of at least 50% or an increase of at least 75% or an increase of at least 100% in the property being is measuring. Therefore, as non-exhaustive examples, microbes can produce an aforementioned percentage increase in nitrogen fixation, or an aforementioned percentage increase in total root weight, or in leaf area or in plant product yield. (for example, an aforementioned percentage increase in the weight of plant product), or an aforementioned percentage increase in seeds that germinate within 10 days or 14 days or 30 days, or the photosynthesis rate (for example, determined by CO2 consumption) or the accumulated biomass of the plant (for example, determined by the weight of the plant). The product

Vegetable IMPI is e, product - usual but not necessarily - a food product produced by a plant. Yield can be determined using any convenient method, for example bushels or pounds of plant product produced per acre of plantation. To date, isolates of more than two dozen genera of microorganisms have been described that have activity that promotes plant growth 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 on many spatial scales, from the global to the farm level, and even in individual plants. Furthermore, it has been described that some individual microbial isolates can exhibit biocontrol and / or plant growth promoting activity, not only in the plants or crops from which they were obtained but also in other crops. This indicates the general nature of some genotypes, especially those with a wide geographic distribution. As discussed above, if introduced in sufficient quantities and if they are active for a sufficient time, an individual microbial population can have a significant impact on plant health.

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

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The term "direct plant ui eiiii iltiitu promoter microorganism", for the purposes of this description, refers to a microorganism that can enhance plant growth in the absence of pathogens. As discussed in more detail below, examples of direct plant growth promotion include (a) blofertilization, (b) stimulation of root growth, (c) rzorremedlaclón, and (d) control of plant stress. Additionally, several PGPMs have been shown to promote plant growth indirectly through biological control mechanisms, that is, by reducing the level of disease, for example antlblosls, induction of systemic resistance, and competition with pathogens for nutrients and niches.

Biofertilizers: Microbial fertilizers supply nutrients to the plant and therefore can promote plant growth in the absence of pathogen pressure. Non-exhaustive examples of microbial isolates that can directly promote plant growth and yield Include N-fixing bacteria2, as Bradyrbizobium and Rhizobium species that by symbiotic nitrogen fixation can form nodules in the roots of leguminous plants, in which they convert N2 atmospheric ammonia which unlike N2 Atmospheric, it can be used by the plant as a nitrogen source. Other examples include Azospiríllum species, which are fixatives of N2 Autonomous who can fertilize and increase the yield of cereal crops such as wheat, sorghum, and corn. Despite the fixation capacity of N2 of Azospiríllum, the yield increase caused by Azospiríllum inoculation

27 IMPI

OE THE PROPERTY

INDUSTRIAL generally attributes this to high root development and »therefore to high rates of absorption of water and minerals. In this regard, several rhizobacteria such as Azotobacter spp. have been shown to be capable of producing a broad set of phytohormones (eg, auxins, cytokinins) and enzymes (eg, pectinase). Many of these phytohormones and enzymes have been shown to be closely involved in the infection process of symbiotic associations between plants and bacteria that have a regulatory influence on Rhizobium nodulation.

In many cases, PGPMs can also affect plant growth and development by modifying nutrient absorption. They can alter nutrient uptake rates, for example, by direct effects on roots, by effects on the environment that in turn modify root behavior, and by competing directly for nutrients (Gaskin et al., Farming. Ecosyst. Environ. 12: 99-116, 1985). Some of the factors by which PGPMs can play an important role in modifying the efficiency of nutrient use in the soil include, for example, root geometry, nutrient solubility, nutrient availability by producing the form ionic compatible with the plant, division of nutrients in the plant and efficiency in their use. For example, a low level of soluble phosphate can limit the growth of plants. Some microbes that promote plant growth are able to solubilize phosphate from inorganic or organic bound phosphates, thereby facilitating plant growth. Various enzymes of microbial origin, such

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OC INJTTTUTO MEXICANO

ZO DI LA rmniOAD

INDUSTRIAL as non-specific phytases, phosphonatases, CP liases and phosphatases, release soluble phosphorus from organic compounds in the soil. For example, it has been found that a high solubilization of inorganic phosphorus in the soil improves the absorption of phosphorus in cañala seedlings using

Pseudomonas putida as well as high sulfur absorption and sulfur oxidation (Grayston and Germida, Can. J. Microbiol. 37: 521-529, 1991; Baneijee, Phytochemicals and Health, vol. 15, May 18, 1995).

Phytostimulators: Some microorganisms can produce substances that stimulate plant growth in the absence of pathogens.

For example, the production of plant hormones is a characteristic of many microorganisms associated with plants. For all five classical phytohormones, i.e. auxin, ethylene, abscisic acid, cytokinin, and gibberellin, synthesis has been shown to be a secondary metabolite for at least one species of fungus and / or bacterial (for a review, see, for example, Kim et al., Appl. Environ. Microbiol., Vol. 77, 5: 1548-1555, 2011). Some microorganisms can also produce secondary metabolites that affect the production of phytohormones in plants. Probably the best known example is the hormone auxin, which can promote root growth. Other examples include pseudomonas that have been shown to produce indole acetic acid (AAI) and to improve AAI amounts in plants, causing 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,

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1979) demonstrated that inoculation of STreaedbí nutrtéfltés solutions from the roots of pearl millet with Azospiríllum brasiliense caused an increase in the weight of the root and stem, a high amount of lateral roots, and all the lateral roots were densely covered with radical hairs . Plants supplied with combinations of AAI, giberilins, and kinetins showed an increase in the production of lateral roots similar to those caused by Azospirílla. Although the biological importance of these plant hormones and plant hormone-like materials is not fully understood, the growth-stimulating activity of these microorganisms is commonly attributed to the production of these materials.

In addition, other hormones as well as certain volatile organic compounds (VOCs) and the cofactor pyrrolquinoline quinone (PQQ) also stimulate plant growth. For example, some rhizobacteria, such as strains of bacterial species B. subtilis, B. amyloliquefaciens, and Enterobacter cloacae promote plant growth by releasing VOCs. The highest level of growth promotion has been observed with 2,3-butanediol and 3-hydroxy-2-butanone (also called acetoin) as triggers of induced systemic resistance. The PQQ cofactor has been described as a plant growth promoter that acts as an antioxidant in plants. Some reports suggest that the effect may be indirect because PQQ is a cofactor of various enzymes, for example involved in antifungal activity and resistance induction

Systemic IMPI. Stress controllers: Plant growth promoting microorganisms that contain the enzyme 1-aminocyclopropane-1carboxylic acid (ACC) deaminase facilitate plant growth and development by lowering plant ethylene levels. These microorganisms absorb the ethylene precursor ACC and convert it to 2-oxobutanoate and NH3. Different types of stress have been shown to be attenuated by ACC deaminase producers, such as, for example, stress caused by effects of phytopathogenic bacteria, stress caused by polyaromatic hydrocarbons, stress caused by heavy metals such as Ca2+ and not2*, and stress caused by salt and drought.

In addition, several PGPM strains that caused increases in potato yields have been shown to produce extracellular Fe-binding siderophores.3+, making it less available for a certain member of the natural microflora (Kloepper et al., Nature 286: 885-886, 1980). These rhizobacteria excrete high affinity, low molecular weight iron chelating microbial cofactors that specifically enhance their iron acquisition by binding to membrane bound siderophore receptors. One of the siderophores produced by some pseudomonas PGPM is known as pseudobactin that inhibits the growth of Erwinia cartovora (organism that causes potato soft rot) (see, for example, Kloepper et al., Current Microbiol. 4: 317-320 , 1980). Additions of pseudobactin to

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Growth media inhibited infection of Blánda rot and also reduced the number of pathogenic fungi in the potato plant along with a significant increase in potato yield. Most of the evidence supporting the siderophore theory of biological control by PGPM comes from work with pyoverines, a class of sideophores comprising the fluorescent pigments of fluorescent pseudomonas [Demange et al., In Iron Transport in Microbes, Plañís and Animáis (Winkleman et al., Eds.), Pp 167-187, 1987], According to the siderophore theory, pyoverines show certain functional strain specificity that is due to selective recognition of outer membrane siderophore receptors (Bakker et al., Soil Biology and Biochemistry 19: 443-450, 1989).

Isolated cultures of the invention

As described in more detail in the section on

Examples of the present disclosure, Applicants have discovered a number of novel microorganisms that are effective promoters of plant growth and plant yield. In many cases, isolated microorganisms are also effective in suppressing the development of various pathogenic plant diseases. Microbial isolates were selected from a group of approximately 5,000 microbial strains obtained from environmental samples collected from various locations throughout the United States. The initial selection of the microorganisms was based on the ability of the microorganisms to colonize the roots of the plants.

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and to produce chemical compounds and enzymes that are considered important for their interaction with plants. The microorganisms were also subjected to a biological assay to analyze their ability to suppress the development of various fungal phytopathogens in an in vitro antagonism assay. The selected microbial microorganisms were then biologically assayed in greenhouse studies on commercial wheat and corn varieties to analyze the ability of microbial strains to promote plant growth and to analyze their ability to preserve seed yield potential.

Taxonomic analyzes further determined that the representative microorganisms described in the present disclosure are closely related to bacteria of the genus Bacillus, Burkholderia, Herbaspirillum, Pantoea, and Pedobacter.

Deposit of biological material

The purified cultures of microbial strains described in the present description were deposited in the Culture Collection of the Agricultural Research Service located at 1815 N. University Street, Peoría, IL 61604, USA (NRRL) in accordance with the Budapest Treaty for the purposes of the patent procedure and its regulations (Budapest Treaty). The access numbers of these deposits are as follows:

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TABLE 1 - Microbial isolates and corresponding access numbers

<td>Strain identification</td><td>Access number</td><td>Provisional taxonomy</td>

<td>SGI-003-H11</td><td>NRRL B-50483</td><td>Pantoea aggtomerans 003_H11</td>

<td>SGI-020-A01</td><td>NRRL B-50484</td><td>Bacillus thuríngiensis 020 A01</td>

<td>SGI-026-G06</td><td>NRRL B-50485</td><td>Burkholderia metallica 026 G06</td>

<td>SGI-026-G07</td><td>NRRL B-50486</td><td>Vietnamese burkholderia 026 G07</td>

The microbial strains have been deposited under conditions that ensure that access to the culture will be available, while this patent application is pending, for whoever designates the Patent and Trademark Manager in accordance with article 1.14, title 37 of the CFR [Code of Federal Regulations] and Article 122, Title 35 of the USC [United States Code]. The deposits basically represent pure cultures of the deposited strains. Deposits are available as required by foreign patent law in the countries where the equivalents of this application or their progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the object of the invention to the detriment of patent rights granted by government actions.

The preferred microorganisms of the present invention have all the identifying characteristics of the deposited strains and, in particular, the identifying characteristics that they are capable of

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promote plant growth and / or yield as described herein, and the identifying characteristics of which are capable of suppressing the development of fungal phytopathogens as described herein. In particular, the preferred microorganisms of the present invention refer to deposited microorganisms as described above, and strains derived therefrom.

Microbiological compositions

The microbiological compositions of the present invention comprising the isolated microbial strains or cultures thereof can be found in a variety of forms, including, but not limited to, immobile cultures, whole cultures, cell solution, mycelium, and / or stored hyphae (in (glycerol solutions), agar strips, agar plugs stored in glycerol / water, lyophilized solutions and dry solutions such as lyophilisate or mycelium dried on filter paper or grain seeds. As defined herein, isolated culture or grammatical equivalents, as used in this description and in the art, is understood to mean that the culture referred to is a fluid, sediment, scraped, sample culture. dry, lyophilized or section (eg hypha or mycelium); or a support, container, or medium such as a plate, paper, filter, matrix, straw, pipette, or pipet tip, fiber, needle, gel, swab, tube, container, particle, etc. it contains a single type of organism. In the present invention, an isolated culture of an antagonist

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Microbial is a culture fluid or a scraping, sediment, dry preparation, lyophilized, or section of the microorganism, or a support, container, or medium containing the microorganism, in the absence of other organisms.

The present disclosure further provides compositions containing at least one isolated microbial strain or cultures of the present invention and a carrier. The carrier can be any one or more of a number of carriers that confer a variety of properties such as increased stability, wettability, dispersibility, etc. Wetting agents such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination of these 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, which is incorporated herein by this reference) . Suitable formulations that can be prepared include wetting powders, granules, gels, agar strips or sediments, thickeners and the like, microencapsulated particles, and the like, liquids such as aqueous fluids, aqueous suspensions, water-in-oil emulsions, etc. The formulation may include grain or legume products (eg, ground grain or beans, broth or flour derived from the 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

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composition can be applied or used as a coatingfexjMabffiJa-seaaillaja. let it saturate the seed.

In some embodiments, the agricultural carrier may be soil or a plant growth medium. Other agricultural carriers that can be used include water, fertilizers, vegetable-based oils, moisturizers, or combinations of these. Alternatively, the agricultural carrier may be a solid, such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, pods, other animal and plant products, or combinations, including granules, sediments, or suspensions. Mixtures of any of the above ingredients are also considered carriers, such as, but not limited to, pesta (kaolin flour and clay), agar-based sediments or loam, sand or clay flour, etc. The formulations may include food sources for cultured organisms, such as barley, rice, or other biological materials such as seed, plant parts, sugarcane bagasse, husks or reeds from grain processing, plant material on the Soil (for example, garden waste) or wood from construction site waste, sawdust, or small fibers from recycling paper, cloth, or wood. Other suitable formulations will be known to those skilled in the art.

In the liquid form, for example, the solutions or suspensions, the microorganisms of the present invention can be mixed or suspended in water or in aqueous solutions. Liquid diluents or carriers

Suitable include water, aqueous solutions, i Ir _ IililHt, Ul pLtióloc n »trns liquid carriers.

Solid compositions can be prepared by dispersing microorganisms of the invention in a suitably divided solid carrier, such as peat, wheat, bran, vermlcullta, clay, talc, bentonlta, earth earth, fuller's earth, pasteurized soil, and the like. When such formulations are used as wettable powders, biologically compatible dispersing agents such as nonionic, anlonyl, amphoteric or cationic dispersing and emulsifying agents can be employed.

In a preferred embodiment, the compositions contemplated herein improve the growth and performance of crop plants, such as wheat, barley, oats, and corn, and, when used in sufficient quantities, act as a microbial fertilizer. These compositions, in a similar way to other blofertllizing agents, have a high margin of safety since they normally do not burn or damage the plant.

As described in greater detail in the present disclosure, improving plant growth and plant yield can be accomplished by applying one or more ml-microblock compositions of the present invention to a host plant or parts of a host plant. The compositions can be applied in an effective amount to improve plant growth or performance related to an untreated control. Active constituents are used in a concentration

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Enough to enhance the growth of the target plant when applied to the plant. As will be apparent to one skilled in the art, effective concentrations can vary depending on factors such as: (a) the type of the plant or agricultural raw material; (b) the physiological state of the plant or agricultural raw material; (c) the concentration of pathogens that affect the plant or agricultural raw material; (d) the type of disease damage to the plant or agricultural raw material; (e) weather conditions (eg, temperature, humidity); and (f) the stage of plant disease. In accordance with the present invention, normal concentrations are those greater than 1 X 102 CFU / mL of carrier. Preferred concentrations are in the range of about 1 x 104 to about 1 x 109 CFU / mL, such as concentrations within the 1 x 10 range6 at 1 x 10® CFU / mL. The most preferred concentrations with those from about 37.5 to about 150 mg of dry bacterial mass per milliliter of carrier (liquid composition) or per gram of carrier (dry formulation).

In some embodiments, the amount of one or more of the microorganisms in the compositions of the present invention may vary depending on the final formulation as well as the size or type of plant or seed used. Preferably, the one or more microorganism in the compositions is present in about 2% w / w to about 80% w / w of the total formulation. More preferably, the one or more microorganism used in the compositions is

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about 5% w / w about ~ ~5 -% - w / w — and preferably about 10% w / w about 60% w / w by weight of the total formulation.

As will be understood by those skilled in the art, the microbiological compositions of the invention can be applied to the target plants using a variety of conventional methods such as dusting, coating, injection, rubbing, rolling, dipping, spraying or brushing, or any another suitable technique that does not significantly damage the target plants to be treated. Particularly preferred methods include inoculating the growth medium or soil with microbial cell suspensions and coating the seeds of the plants with microbial cells and / or spores.

Typically, the compositions of the invention are chemically inert; therefore they are basically compatible with any constituent of the application program. They can also be used in combination with substances that affect plant growth, 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 active pesticidal agents such as, for example, herbicides, nematicides, fungicides, insecticides, and the like.

When used as biofertilizers in their commercially available formulations and in their use forms, prepared from

IMPI these formulations, the active microbial layers and Jas-proximal pas-da. according to the present invention they may additionally be present in the form of a mixture with synergists. Synergists are compounds by which the activity of the active compositions is increased without the need for the added synergist to be active in itself.

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

The active microbial strains and compositions according to the invention, as such or in their formulations, can also be used as a mixture with known fertilizers, acaricides, bactericides, fungicides, insecticides, microbicides, nematicides, pesticides or combinations of any of these. for example, in order to broaden the spectrum of action or prevent the development of resistance to pesticides in this way. In many cases, they result in synergistic effects, that is, the activity of the mixture can exceed the activity of the individual components. Also contemplated is a mixture with other known active compounds, such as growth regulators, protectors and / or semiochemicals.

In a preferred embodiment of the present invention,

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Compositions may additionally include at least one chemical or biological strength. The amount of at least one chemical or biological fertilizer used in the compositions can vary depending on the final formulation as well as the size of the plant and the seed to be treated. Preferably, the at least one chemical or biological fertilizer employed is about 0.1% w / w to about 80% w / w based on the total 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 more preferably about 10% w / w to about 50% w / w.

The microbiological compositions of the present invention preferably include at least one biological fertilizer. Example biological fertilizers that are suitable for use herein and that can be included in a microbiological composition according to the present invention to promote plant growth and performance 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 before formulation with an additional organism. For example, microbes such as, but not limited to, Achromobacter, Ampelomyces, Aureobasidium, Azospirillum, Azotobacter, Bacillus, Beauvería, Bradyrhizobium, Candida, Chaetomium, Cordyceps, Cryptococcus, Dabaryomyces, Delftia, Erftiaophys , Herbaspirillum, Lactobacillus, Mariannaea, Microccocus, Paecilomyces, Paenibacillus, Pantoea, Pichia, Pseudomonas,

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

Rhizobium, Saccharomyces, Sporoboiomyces, Stenotrophomonas, Streptomyces, Talaromyces, and Trichoderma can be provided in a composition with the microorganisms of the present invention. The use of microbiological compositions in accordance with the present invention in combination with the microbial microorganisms disclosed in US Patent Application No. US20030172588A1, US20030211119A1; US Patent No. 7,084,331; 7,097,830; 7,842,494; PCT Application No. W02010109436A1 is also particularly preferred.

In a preferred embodiment of the present invention, the 10 compositions may additionally include at least one chemical or biological pesticide. The amount of at least one chemical or biological pesticide used in the compositions can vary depending on the final formulation as well as the size of the plant and the 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 total formulation. More preferably, the at least one chemical or biological pesticide is present in an amount of about 1% w / w to about 60% w / w and more preferably about 10% w / w to about 50% w / w.

A variety of chemical pesticides are apparent to one skilled in the art and can be used. Example chemical pesticides include those found in the carbamate, organophosphate, organochlorine, and pyrethroid classes. It also includes chemical control agents such as, non-exhaustively, benomyl, borax, captafol, captan, chorotalonil,

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copper-containing formulations; formulations containing dictate / dichloran, iodine, zinc; fungicides that inhibit ergosterol biosynthesis such as non-exhaustively blasticidin, cymoxanil, fenarimol, flusilazole, folpet, imazalil, iprodione, maneb, mancozeb, metalaxyl, oxycarboxine, miclobutanil, oxytetracycline, PCNB, pentachlorophenol, sodium, sodium DNOC, sodium hypochlorite, sodium phenylphenate, streptomycin, sulfur, tebuconazole, terbutrazol, thiabendazole, methyl thiophanate, triadimefon, tricyclazole, triforine, validamicin, vinclozolin, zineb and ziram.

The microbiological compositions of the present invention preferably include at least one biological pesticide. Example biological pesticides that are suitable for use herein and that can be included in a microbiological composition according to the present invention to prevent a pathogenic plant disease that includes microbes, animals, bacteria, fungi, genetic material, plant and natural products of living organisms. In these compositions, the microorganism of the present invention is isolated before formulation with an additional organism. For example, microbes such as, but not limited to, Ampelomyces, Aureobasidium, Bacillus, Beauveria, Candida, Chaetomium, Cordyceps, Cryptococcus, Dabaryomyces, Erwinia, Exophilia, Gliocladium, Mariannaea, Paeciiomyces, Paenibacillus, Paenibacillus, Paenibacillus, , Spotobolomyces, Talaromyces and Tríchoderma, can be provided in a composition, in particular the

aa IMPI mrmvnjMUiCANo

DELAMORIDAC

INDUSTRIAL microorganisms of the present invention, with fungal strains of the Muscodor genus. The use of the microbiological compositions according to the present invention in combination with the microbial antagonists described in US Patent No. 7,518,040; US Patent No. 7,601,346; US Patent No. 6,312,940 is also particularly preferred.

Examples of fungi that can be combined with compositions and microbial strains of the present invention in one composition include, but are not limited to, Muscodor species, Aschersonia aleyrodis, Beauveria bassiana (white muscarina), Beauveria brongniartii, Chladosporíum herbarum, Cordyceps clavulata, Cordyceps entomorrhiza, Cordyceps facis, Cordyceps gracilis, Cordyceps melolanthae, Cordyceps militaris, Cordyceps myrmecophila, Cordyceps ravenelii, Cordyceps sinensis, Cordyceps sphecocephala, Cordyceps subsessilis, Cordyceps unilateralis,

Cordyceps variabilis, Cordyceps washingtonensis, Culicinomyces clavosporus, Entomophaga grylli, Entomophaga maimaiga, Entomophaga muscae, Entomophaga praxibulli, Entomophthora plutellae, Fusarium lateritium, Hirsutella citriformis, Hirsutella thompsoni, Metarhizium anisopliae (green muscarine), Metarhizium flaviride, Muscodor albus, Neozygitesfloridana,

Nomuraea rileyi, Paecilomyces farinosus, Paecilomyces fumosoroseus, Pandora neoaphidis, Tolypocladium cylindrosporum, Verticiilium lecanii, Zoophthora radicaos, and mycorrhizal species such as Lacearía bicolor. Other mycopesticide species will be apparent to experts in the

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technique.

The present invention also provides methods for treating a plant by applying any of a variety of common formulations in an effective amount either to the soil (i.e., in the furrow), to a part of the plant (i.e. wetting) or seed before planting (i.e. seed coating or compost). Common formulations include solutions, emulsifiable concentrate, wettable powders, suspension concentrate, soluble powders, granules, suspension-emulsion concentrate, natural and synthetic materials impregnated with the active compound, and very fine controlled release capsules in polymeric substances. In certain embodiments of the present invention, the microbial compositions are formulated into powders that are available in either a ready-to-use formulation or are mixed with each other at the time of use. In any of these modalities, the powder can be mixed with the soil before or at the time of planting. In an alternative embodiment, one or both of either the plant growth promoting agent or the biocontrol agent is a liquid formulation that is mixed at the time of treatment. A person skilled in the art understands that an effective amount of the compositions of the invention 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 the method of application, one or more suitable additives may also be introduced into the

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compositions of the present invention. Adhesives, such as carboxymethyl cellulose, and natural and synthetic polymers in the form of powders, granules, or latex, such as acacia, chitin, polyvinyl alcohol, and polyvinyl acetate, may be added, as well as natural phospholipids, such as cephalins and lecithins, and synthetic phospholipids. , to the compositions herein.

In a preferred embodiment, the microbiological compositions are formulated in a single, stable solution, emulsion, or suspension. For solutions, the active chemical compounds are typically dissolved in solvents before the biological agent is added. Suitable liquid solvents include petroleum-based aromatics, such as xylene, toluene, or alkylnaphthalenes, aliphatic hydrocarbons, such as cyclohexane, or paraffins, eg, 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 centone or cyclohexanone, highly polar solvents, such as dimethylformamide and dimethyl sulfoxide. For emulsions and suspensions, 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 suspension embodiment, the chemical and biological agent are combined in a ready-to-use formulation that has a reasonably long shelf life. In use, the liquid can be sprayed or applied foliarly as a spray or in the furrow at the time of planting the crop. The liquid composition can be introduced in a quantity

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effective in the seed (i.e. seed coat or compost) or in the soil (i.e. in the furrow) prior to seed generation or directly in the soil in contact with the roots by using a variety of techniques known in the art, including, but not limited to, drip irrigation, sprinklers, injection into the soil or by soaking the soil.

Optionally, stabilizers and buffers can be added, including alkali and alkaline earth metal salts and organic acids, such as nitric acid and ascorbic acid, inorganic acids, such as hydrochloric acid or sulfuric acid. Biocides may also be added and may include formaldehyde or formaldehyde releasing agents, and benzoic acid derivatives, such as phidroxybenzoic acid.

Pathogens

Those skilled in the art will recognize that the methods and compositions according to the present invention can in principle be applied to inhibit the development of any plant pathogen or any plant pathogenic disease. The invention is not intended to be limited to 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.

IMPI οαππηοΜΚΑΜο

4fi DI IA F * 6TOMD n <J INDUSTMAL

Examples of plant pathogenic diseases that are suitable for applications of the methods and materials of the present invention include, but are not limited to, diseases caused by a wide range of pathogenic fungi. The methods of the present invention are preferably applied against pathogenic fungi that are important or interesting for agriculture, horticulture, plant biomass for the production of biofuel molecules and other chemicals, and / or forestry. The pathogenic Pseudomonas species (eg Pseudomonas solanacearum), Xylella fastidiosa; Ralstonia solanacearum, Xanthomonas campestris, Erwinia amylovora, Fusarium species, Phytophthora species (e.g. P. infestaos), Botrytis species, Leptosphaeria species, pepper lines (Ascomycota), and rusts (Basidiomycota), etc., have a particular interest.

Non-exhaustive examples of plant pathogens of interest include, for example, Acremonium strictum, Agrobacteríum tumefaciens, Alternaría altemata, Alternaría solani, Aphanomyces euteiches, Aspergillus fumigatus, Athelia rolfsii, Aureobasidium pullulans, Bipolarís zeicolaé, Botanical zeicolay Cercospora medicaginis, Cercospora sojina, Colletotríchum coccodes, Colletotríchum fragaríae, Colletotríchum graminicola, Coniella diplodiella, Coprynopsis psychromorbida, Corynespora cassiicola, Curvularía pallescens, Cylindrocladium crotalaríae, Diplocarpon earíianum, Diplodia gossyina, Diplodia spp., Epicoccum nigrum, Erysiphe cichoracearum, Fusarium

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graminearum, Fusarium oxysporum, Fusarium oxysporum f.sp. tuberosi, Fusarium proliferatum var. Proliferatum, Fusarium solani, Fusarium verticillioides, Ganoderma boninense, Geotrichum candidum, Glomerella tucumanensis, Guignardia bidwellii, Kabatiella zeae, Leptosphaerulina briosiana, Monoptera, Mygains, Macrophomina, fragariae, Nigrospora oryzae, Ophiostoma ulmi, Pectobacterium carotovorum, Pellicularia sasakii (Rhizoctonia solani), Peronospora manshurica, Phakopsora pachyrhizi, Phoma foveata, Phoma medicaginis, Phomopsis longicolla, Phytophthora cinnamomi, erythroseptica Phytophthora, Phytophthora fragariae, Phytophthora infestans, Phytophthora medicaginis, Phytophthora megasperma, Phytophthora paimivora, Podosphaera leucotricha, Pseudopeziza medicaginis, Puccinia graminis subsp. Tritici (UG99), Puccinia sorghi, Pyricularia grísea, Pyricularia oryzae, Pythium ultimum, Rhizoctonia solani, Rhizoctonia zeae, Rosellinia sp., Sclerotinia sclerotoriaum, Setleriacapica, Splerotinia, Septoria , Stemphylium sp, Synchytrium endobioticum, Thecaphora (Angiosorus), Thielaviopsis, Tilletia indica, Trichoderma viride, Ustilago maydis, Verticillium albo-atrum, Verticillium dahliae, Verticillium dahliae, Xanthomonas axonopodis, Xanthomonas oryzae pv. oryzae.

In a preferred embodiment of the present invention, the

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Methods and materials of the invention are useful pathogens Aspergillus fumigatus, Botrytis cinérea, Cerpospora betae, Colletotrichum sp., Curvularia spp., Fusaríum sp., Ganoderma boninense, Geotrichum candidum, Gibberella sp., Monographella sp., Mycosphaerella fi. Phytophthora ramorum, Penicillium sp., Pythium ultimum, Rhizoctonia solani, Rhizopus spp., Schizophyllum spp., Sclerotinia sclerotiorum, Stagnospora sp., Verticillium dahliae, or Xanthomonas axonopodis. In a particularly preferred embodiment, the methods and materials of the invention can be used to inhibit the development of various plant pathogens of Commercial Importance, including Fusaríum graminearum NRRL-5883, Monographella nivalis ATCC MYA-3968, Gibberella zeae ATCC-16106, Stagnospora nodurum ATCC-26369, Colletotrichum graminicola ATCC-34167, and pathogens Penicillium sp.

Seed coating formulation

MEXICAN INSTITUTE Ot LA KOREDAD INDUSTRIAL

In a particularly preferred embodiment, the microbial compositions of the present invention are formulated as a seed treatment. It is contemplated that the seeds may be substantially uniformly covered with one or more layers of the microbial compositions disclosed herein using conventional mixing, spraying, or a combination thereof through the use of treatment application equipment that is designed and specifically manufactured to accurately, safely and efficiently apply

IMPI

seeds to seeds. Such equipment uses various types of coating technology such as rotary coaters, drum coaters, fluidized bed, jetted bed techniques, rotary sprinklers, or a combination of these. Liquid seed treatments such as those of the present invention can be applied by means of either a rotating atomizer disc or a spray nozzle that evenly distributes the seed treatment to the seeds while moving along a spray pattern . Preferably, the seed is then mixed or precipitated for an additional period of time to achieve further distribution of treatment and drying. The seeds may or may not be primed prior to coating with the compositions of the invention to increase uniformity of germination and emergence. In an alternative modality, a dry powder formulation can be placed in measured size on the moving seeds and allowed to mix until fully distributed.

Another aspect of the invention provides seeds treated with the microbial compositions herein. One embodiment provides seeds that have at least part of the surface area coated with a microbiological composition in accordance with the present invention. In a specific embodiment, seeds treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 106 to around 109 per seed. Seeds can also have more spores or microbial cells per seed, such as

IMPI

like for example 101θ, 1011 or 1012 spores per seed> microbial sputum and / or cells can freely coat the seeds or, preferably, can be formulated in a liquid or solid composition prior to coating the seeds. For example, a solid composition comprising microorganisms can be prepared by mixing a solid carrier with a spore suspension until the solid carriers are impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.

In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the present invention additionally contain functional agents capable of protecting the seeds from the damaging effects of selected herbicides, such as activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the products or a combination of these.

Seed coating methods and compositions known in the art can be particularly useful when modified by adding one of the embodiments of the present invention. Such coating methods and apparatus for application are described in, for example, US Patent Nos. 5,918,413; 5,554,445; 5,389,399; 4,759,945; and 4,465,017. Seed coating compositions are described, for example, in US Patent Applications No. US20100154299, US Patent No. 5,939,356;

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4,634,587; 4,372,080, 4,339,456; and 4,245,432, among othersi

A variety of additives can be added to the seed treatment formulations comprising the compositions of the invention. Binders can be added, and include those compounds preferably of an adhesive polymer that can be natural or synthetic without phytotoxic effect on the seed to be coated. The binder can be selected from polyvinyl acetates; copolymers of polyvinyl acetate; copolymers of ethylene vinyl acetate (EVA); polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, including ethyl celluloses, methyl celluloses, hydroxymethyl celluloses, hydroxypropyl celluloses and carboxymethyl cellulose; polyvinylpyrrolidones; polysaccharides, including starch, modified starch, dextrins, maltodextrins, alginate, and chitosans; fats; oils; proteins, including gelatin and zeins; gum arabic; shellac; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.

Any of a variety of colorants can be employed, including organic chromophores classified as nitrous, 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, including oligonutrients such as iron, manganese, boron, copper,

IMPI _. nmnVfDMUKANO

DEMnSÜMD

INDUSTRIAL cobalt, molybdenum and zinc. A polymer or other dust control agent can be applied to preserve the treatment on the seed surface.

In some specific embodiments, in addition to microbial cells and spores, the coatings may additionally comprise an adherent layer. The adherent should be non-toxic, biodegradable and adhesive. Examples of such materials include, but are not limited to, polyvinyl acetates; copolymers of polyvinyl acetates; polyvinyl alcohols; copolymers of polyvinyl alcohols; celluloses, such as methylcelluloses, hydroxymethyl celluloses and hydroxymethylpropyl celluloses;

dextrins; alginates; sugars; molasses; polyvinylpyrrolicfones; polysaccharides; proteins; fats; oils; gum arabic; jellies; syrups; and starches. More examples can be found in, for example, US Patent No. 7,213,367 and in US Patent Application No. US20100189693.

Various additives, such as tackifiers, dispersants, surfactants and nutrients, and buffering ingredients, may also be included in the seed treatment formulation. Other conventional seed treatment additives include, but are not limited to, coating agents, wetting agents, buffering agents, and polysaccharides. At least one agriculturally acceptable carrier can be added to the seed treatment formulation such as water, solids, or dry powders. Dry powders can be derived from a variety of materials, such as calcium carbonate, gypsum, vermiculite, talc, humus, activated carbon, and various phosphorous compounds.

In some modalities, the composition flSléLUUIIIIIItílllU iftp

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mnipuru Μ MUCAMO μ uragniDAD seeds can comprise at least one filler that is a natural or synthetic component, organic or inorganic, with which the active components are combined to facilitate its application on the seed.

Preferably, the filler is an inert solid such as clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (eg, ammonium salts), natural soil minerals, such as kaolin, clay, talc, lime, quartz , attapulgite, montmorillonite, bentonite or diatomaceous earth, or synthetic minerals, such as silica, alumina or silicates, in particular aluminum or magnesium silicates.

The seed treatment formulation may additionally include one or more of the following ingredients: other pesticides, including compounds that act only below ground; fungicides, such as captan, tiram, metalaxyl, fludioxonllo, oxadlxilo, and isomers of each of these materials, and the like; herbicides, including selected compounds of glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxies, ureas, and benzoic acids; Herbicidal protectors such as benzoxazine, benzhydryl derivatives, Ν, Ν-diallyl dichloroacetamide, various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone, naphthalic anhydride compounds, and oxime derivatives; Chemical fertilizers; biological fertilizers; and biocontrol agents such as naturally occurring or recombinant bacteria and fungi of the genera Rhizobium, Bacillus, Pseudomonas, Serratia,

IMPI

Tríchoderma, Glomus, Gliocíadium and mycorrhizal fungi. These ingredients can be added as a separate layer on the seed or, alternatively, can be added as part of the seed coating composition of the invention.

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

The formulation that is used to treat the seed in the present invention may be in the form of a suspension; emulsion; suspension of particles in an aqueous medium (eg, water); wettable powder; wettable granules (dry fluid); and dry granules. If formulated in a suspension, 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 otherwise formulated by those skilled in the art .

As mentioned above, other conventional inactive or inert ingredients can be incorporated into the formulation. Such ingredients include, but are not limited to: bonding agents; dispersing agents, such as methylcellulose, for example, serve as a combined dispersant / bonding agent for use in seed treatments; polyvinyl alcohol; lecithin, polymeric dispersants (eg, polyvinylpyrrolidone / vinyl acetate); thickeners (for example, thickeners in clay to improve viscosity and reduce sedimentation of suspensions

IMPI

MEXICAN INSTITUTE of industrial philosophy

particle); emulsion stabilizers; surfactants; antifreeze compounds (eg urea), dyes, colorants, and the like. More inert ingredients useful for the present invention can be found in McCutcheon's, vol. 1, Emulsifiers and Detergents, MC Publishlng Company,

Glen Rock, NJ, USA, 1996. Additional inert ingredients useful for the present invention can be found in McCutcheon's, vol. 2, Functional Materials, MC Publishing Company, Glen Rock, NJ, USA, 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 (eg, bottle or bag), mechanical application, precipitation, spraying, and dipping. . A variety of active or inert materials can be used to contact the seeds with microbial compositions in accordance with the present invention, such as conventional film coating materials, including but not limited to water-based film coating materials. , such as SEPIRET ™ (Seppic, Inc., NJ) and OPACOAT ™ (Berwind Pharm. Services, PA).

The amount of composition according to the present invention that is used for seed treatment will vary depending on the type of seed and the type of active ingredients, but the treatment will comprise contacting the seeds with an agriculturally effective amount of the inventive composition. . As discussed above, an effective amount means the amount of inventive composition that is sufficient

IMPI '«mnOMWCANO

WUJMBUUD

INDUSTRIAL

to produce beneficial or desired results. An effective amount can be administered in one or more administrations.

In addition to the topcoat, the seed can be treated with one or more of the following ingredients: other pesticides, including fungicides and herbicides: herbicide protectors, fertilizers, and / or biocontrol agents. These ingredients can be added as a separate layer or alternatively they can be added in the coating layer.

The seed coating formulations of the present invention can be applied to seeds using a variety of techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic seed treaters, and drum coaters. Other methods, such as jetted beds, may also be useful. Seeds can be separated by size before coating. After coating, the seeds are commonly dried and transferred to a machine to sort them by size. Such procedures are known in the art.

Seeds treated with microorganisms can also be coated with a second coating film to protect the coating. Such second coatings are known in the art and can be applied using drum film and fluidized bed coating techniques.

In another embodiment of the present invention, the compositions according to the present invention can be introduced into a seed

IMPI

M ESCAÑO INSTITUTE OF NON IDAD

INDUSTRIAL

by using solid metric conditioning. For example, the amount of a composition of the invention can be mixed with a solid matrix material and then the seed can be contacted with the solid matrix material for a period to allow the composition to enter the seed. The seed can then be optimally separated from the solid matrix material and stored or used, or the mixture of solid matrix material plus the seed can be directly stored or planted. Solid matrix materials that are useful for the present invention include polyacrylamide, starch, clay, silica, alumina, earth, sand, polyurea, chicklaclate or any other material capable of absorbing or adsorbing the composition of the invention for a time and releasing that composition in the seed. It is useful to ensure that the composition of the invention and the solid matrix material are compatible with each other. For example, the solid matrix material should be chosen so that it can release the composition at a reasonable rate, for example, over a period of minutes, hours, or days.

In principle, any vegetable seed capable of germinating to form a plant can be treated according to the invention. Suitable seeds include those of cereals, coffee, cabbage crops, fiber crops, flowers, fruits, legumes, oil crops, trees, tuber crops, vegetables, as well as other plants of the monocot and dicot species. Preferably crop seeds are covered, including, but not limited to, beans, carrots, corn,

IMPI

cotton, grasses, lettuce, peanuts, bell peppers, potatoes, rape, arró ^^ C & ñtebó ^ 'gOTgü;

soy, sugar beet, sunflower, tobacco and tomato. Most preferably, barley or wheat seeds (especially winter wheat or wheat) are coated with the compositions of the present invention.

Preparation of the microbial compositions according to the present invention

Microorganism cultures can be prepared for use in the microbial compositions of the invention using standard static drying and liquid fermentation techniques known in the art. Growth is commonly carried out in a bioreactor.

A bioreactor refers to any device or system that supports a biologically active environment. As described herein, a bioreactor is a container in which microorganisms, including microorganisms of the present invention, are grown. A bioreactor can be of any shape or size suitable for the cultivation of microorganisms. A bioreactor can be in the range of 10 mL to liters per cubic meter and can be made of stainless steel or any other suitable material as is known and used in the art. The bioreactor may be a batch type reactor, a batch feed type, or a continuous type bioreactor (eg, a continuous stirring reactor). For example, a bioreactor can be a known chemostat and used in the microbiology technique for cultivation and harvesting.

IMPI imrnW »M« tlCAI «) ntlAfBOFMMC

INnUCTMM microorganisms. A bioreactor can be obtained from any commercial proreectur (see also Bioreactor System Design, Asenjo and Merchuk, CRC

Press, 1995).

For small-scale operations, a batch bioreactor 5 can be used, for example, to evaluate and develop new processes, and for processes that cannot be converted to continuous operations.

Microorganisms grown in a bioreactor can be suspended or immobilized. Culture in the bioreactor is generally under aerobic conditions at temperatures and pH suitable for growth.

For the organisms of the invention, cell growth can be achieved at temperatures between 5 and 37 ° C, with the preferred temperature in the range of 15 to 30 ° C, 15 to 28 ° C, 20 to 30 ° C, or 15 to 25 ° C. The pH of the nutrient medium can vary between 4.0 and 9.0, but the preferred operating range is generally slightly acidic or neutral at pH 4.0 to 7.0, or 4.5 to 6.5, or pH 5.0 to 6.0.

Typically, maximum cell yield is obtained 20-72 hours after inoculation.

Optimal conditions for cultivation of microorganisms of this invention will, of course, depend on the particular strain. However, by virtue of the conditions applied in the selection process and general requirements of most microorganisms, a person skilled in the art will be able to determine essential nutrients and conditions. The microorganisms would typically be grown in liquid aerobic cultures in media containing carbon, nitrogen, and inorganic salt sources that

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they can be assimilated by microorganisms and be efficient cellular support-4e * 4M-ccecÍBÜeüta. Preferred carbon sources are hexoses, such as glucose, but can be substituted with other sources that are easily assimilated, such as amino acids. Many inorganic and prototelnaceous materials can be used as nitrogen sources in the culture processes. Preferred nitrogen sources are amino acids and urea, but others include ammonia gas, inorganic nitrate and ammonium salts, vitamins, purines, pyrimidines, yeast extract, calf extract, protease peptone, milled soy, casein hydrolysate, soluble of distillates and the like. Among the Inorganic minerals that can be incorporated into the nutrient medium are the common salts that are capable of producing calcium, zinc, iron, manganese, magnesium, copper, cobalt, potassium, sodium, molybdate, phosphate, sulfate, chloride, borate and similar ions. . Without limiting it, the use of liquid dextrose and potato medium is preferred for fungal strains and R2A broth premix for bacterial strains.

Novel plant varieties

Also provided, in another aspect of the present invention, is a novel plant by artificially introducing a microbial endophyte of the invention into a plant that is free of endophytic microorganisms. In some modalities of this aspect, the microbial endophyte introduced into the plant can be an endophytic microorganism that has an activity that promotes plant growth, a

IMPI

Biological control activity or a combination of a variety of methods previously known to be effective in the art for introducing a microbial endophyte into a species of cereal grass. Examples of such methods include the 5 described in US Patent Application No. 20030195117A1, US Patent Application No. 20010032343A1, and US Patent No. 7,084,331, among others. It will be apparent to those skilled in the art that many of the aforementioned methods can be useful in producing 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 microogranism is confirmed by conducting a homology search for the amplified DNA. It is preferably preferred that the foreign gene expressing an identifiable medium is introduced into the aforementioned endophytic microorganism, and the presence of colonization of the aforementioned endophytic microorganism infecting the plant is confirmed by the aforementioned identifiable medium, using the foreign gene.

MEXICAN INSTITUTE OF LA MOCEDAD

INDUSTRIAL

Plants suitable for the methods of the invention

In principle, the methods and compositions according to the present invention can be implemented with any plant species. Monocotyledonous plant species, as well as dicotyledons, are particularly suitable. The methods and compositions are used

IMPI preferably with plants that are Important or int ^ S§antSS * TJara,^ lsr agriculture, horticulture, for the production of biomass used in the production of liquid fuel molecules and other chemicals, and / or in forestry.

Therefore, the invention can be used on a wide range of plants, preferably higher plants of the Angiospermae and Gymnospermae classes. Plants of the Dicotylodenae and Monocotyledonae subclasses are particularly suitable. Dicotyledonous plants belong to the orders of Aristochiales, Asterales, Bátales, Campanulales, Capparales, Caryophyllales, Casuarínales, Celastrales, Comales, Diapensales, Dilleniales, Dipsacales, Ebenales, Encales, Eucomiales, Euphorbiales, Fabales, Fágales, Gentianales, Geraniales, Hamamelidales, initials, Juglandales, Lamíales, Laureles, Lecythidales, Leitneríales, Magniolales, Málvales, Myrícales, Myrtales, Nymphaeales, Papeverales, Piperales, Plantaginales, Plumbaginales, Podostemales, Polemoniales, Polygalales, Polygonales, Prímulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales y Viólales. Monocotyledonous plants belong to the orders of Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eríocaulales, Hydrocharítales, Juncales, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Tríuridales, Typhales and Zingiberales. Plants that belong to the classes of Gymnospermae

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They are Cycadales, Ginkgoales, Gnetales and Piñales. —— _

Suitable species may include members of the genera Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberís, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum , Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbit, Cynodon, Datura, Dianthus, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucathroxylum Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Pan Parthenium, Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticose Triticum, Unióla, 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 example, Panicum virgatum (rod grass), Sorghum bicolor (sorghum, sudan grass), Miscanthus giganteus (miscanthus), Saccharum sp.

(energy cane), Populus balsamifera (poplar), Zea mays (corn), Glycine

IMPI

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max (soybean), Brassica napus (canela), Triticum aestiCuiil (Lllyu), Uujjppm / w · hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugar beet) , Pennisetum glaucum (pearl millet), Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Eríanthus spp., Populus spp., Andropogon gerardii (scrub blue stem), Pennisetum purpureum (elephant grass), Phalaris arundina ( yellow lupine), Cynodon dactylon (common grass), Festuca arundinacea (tall fescue), Spartina pectinata (cable prairie grass), Arundo donax (giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Tríticosecale spp. (trít¡cum - tr¡go X rye), Bamboo, Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricinus communis (castor bean), Elaeis guineensis (oil palm), Phoenix dactylifera (date palm), Archontophoenix cunninghamiana (king palm), Syagrus romanzoffíana (queen palm), Linum usitatissimum (flax), Brassica júncea, Manihot esculenta (manioc), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato) , Brassica olerácea (broccoli, cauliflower, Brussels sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobtoma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot and sweet pepper) ), AHium cepa (onion), Cucumis meló (melon), Cucumis sativus (cucumber), Cucúrbita maxima (squash), Cucúrbita moschata (squash), Spinacea oleomaceous (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (quimbombó) Solanum melongena (aubergine), Papaver somniferum (poppy), Papaver

impi

ΙΕβΝΙυτ® MUCAMO

Orient industrial MUMomSMD, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva; Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis, Coichicum autumnale, Veratrum califomica, Digitalis ¡anata, Digitalis purpurea, Dioscorea spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberís spp., Cephalotaxus spp., Cephalotaxus spp., Cephalotaxus spp., Cephalotaxus spp., Cephalotaxus spp., Cephalotax spp. spp., Erythroxylum coca, Galanthus womorii, Scopolia spp., Lycopodium serratum (Huperzia serrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium,

Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosa spp. (pink), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (white lupine), Unióla paniculata (oats), bentgrass (Agrostis spp.), Populus tremuloides (trembling), Pinus spp. (pine), Abies spp. (spruce), Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis (barley), Lolium spp. (ryegrass), Phleum pratense (fleo) and conifers. Plants of interest that are grown for energy production, called energy crops, such as cellulose-based energy crops, such as Panicum virgatum (rod grass),

Sorghum bicolor (sorghum, sudan grass), Miscanthus giganteus (miscanthus), Saccharum sp. (energy cane), Populus balsamifera (poplar), Andropogon gerardii (scrub blue stem), Pennisetum purpureum (elephant grass), Phalaris arundinacea (yellow lupine), Cynodon dactylon (common grass),

Festuca arundinacea (tall fescue), Spartina pectindid (llltílbá grassland cable), Medicago sativa (alfalfa), Arundo donax (giant cane), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale spp. (triticum-wheat X rye), and Bamboo; and starch-based energy crops, such as Zea mays (corn) and Manihot esculenta (cassava); and sugar-based energy crops, such as Saccharum sp. (sugar cane), Beta vulgarís (sugar beet), and Sorghum bicolor (L.) Moench (sweet sorghum); and energy crops that produce biofuels, such as Glycine max (soy), Brassica napus (cane), Helianthus annuus (sunflower),

Carthamus tinctoríus (safflower), Jatropha curcas (Jatropha), Ricinus communis (castor bean), Elaeis guineensis (African oil palm), Elaeis oleifera (American oil palm), Cocos nucífera (coconut), Camelina sativa (wild flax), Pongamia pinnata (Pongam), Olea europaea (olive), Linum usitatissimum (flax), Crambe abyssinica (Abyssinian cabbage) and Brassica júncea.

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

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IWTWWO MGUCANO

RILA KOREDAD INDUSTRIAL

EXAMPLES

EXAMPLE 1

Isolation of microorganisms from environmental samples

Identification of spore-forming rhizobacteria using a sonicated root method and serial dilutions.

The following microorganisms were isolated using a sonicated root method, serial dilutions as described below: Isolates SGI-026-G06 and SGI-026-G07 were isolated from a needle grass sample; isolate SGI-041-B03 was isolated from a sample of wild rye; and isolate SGI-020-A01 was isolated from root tissue grown in a composite soil sample.

An enrichment procedure was developed to specifically identify the rhizobacteria that form spores. Broadly, the sonicated root extracts were heat treated to remove vegetative cells and then plated with enrichment medium. Microorganisms that survived heat treatment and formed colonies were considered spore-forming. This method was found to be particularly effective in selecting gram-positive bacteria. The new root samples were used as starting material for these enrichments. The thin sections at the tip of the roots are the newest, they may have a

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high root filament density and typically have Uní i JJjiLj altao of rhizobacteria. A sterile blade was used to cut these root areas into 5 -10 cm segments, which were washed in sterile milliQ water to remove large soil particles. When necessary, stricter washing was achieved by placing the roots in a 50 mL Falcon tube with 25 mL of 1x phosphate buffered saline (i.e. PBS buffer) and mixed vigorously for 1 minute. Each root sample was subsequently suspended in 20 mL of sterile PBS buffer and sonicated on ice for 1 minute intervals at 8 watts, using a Fisher Scientific sonic dismembrator. For heat treatment, typically 1 mL of the sonicated root cell suspension is transferred to a sterile Eppendorf tube, incubated in a 80 ° C water bath for 20 minutes. The heat treated cell suspensions were allowed to cool to room temperature before serially diluting them to concentrations of 10 '1, 10'2, 10’3, 104, 10'5, 10'6 and 10 '7. 100 pL of each dilution multiple of 10 were plated in culture plates containing agar-solidified microbiological medium and 100 mg / L cycloheximide to inhibit fungal growth. In some cases it was necessary to carry out a 1/10 or 1/100 dilution before plating to obtain the proper CFU density to collect colonies. Isolated colonies were collected using sterile pipet tips, placed in 96-well microtiter plates, each containing 150 pL of 2 x liquid YT medium per well. The microtiter plates were incubated for 1-2 days.

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at 30 ° C to obtain a high cell density for ^ fta-eafactecizaciÓD.

additional file.

ΙΝίΙΤΠΠΌ MEXICAN

DELAraomoAD.

industrial

Isolation of bacteria that form biofilms.

The following microbial isolates were isolated using a biofilm-forming method as described below: SGI-003-H11 isolate was isolated from a Yucca plant root sample; isolate SGI-034-C09 was isolated from a grass root sample; and SGI-034-E10 isolate was isolated from a sample of green chervil plant.

Biofilm-forming method: In this procedure, biofilm-forming bacteria were isolated from sonicated root segments, as described in Fall et al. (Syst. Appl. Microbiol. 27,372-379,

2004). As described above, bacteria in biofilms on the surface of a root are typically highly effective 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 invasive pathogens. Broadly speaking, sonication was used to remove bacterial and fungal cells that lightly attach to the root, leaving behind only those microbes that strongly adhered to the root surface. Both gram-positive and gram-negative biofilm-forming bacteria are

IMPI ικππυτο ΜΒΧΚΑΝΟ

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INDUSTXlAi.

selected using this method. ..... »··· ^ ·. ·

The new root samples were used as starting material for these enrichments. The thin sections at the root tip were the newest tissues, had a high root filament density, and typically had high densities of rhizobacteria. A sterile sheet was used to cut these root areas into 5-10 cm segments, which were washed by placing them in a 50 mL Falcon tube with 25 mL 1 x PBS and mixed vigorously for 1 minute. Wash debris was allowed to settle, and then sterile forceps were used to transfer the washed root segments to 50 mL Falcon tubes filled with 25 mL 1 x PBS, and sonicated on ice using a Fisher Scientific sonic dismembrator for two 30-second intervals with a 30-second pause between pulses. Sonicated root samples were transferred to sterile plastic Petri dishes and allowed to dry completely capless in a biosafety cabinet. Each root segment was then placed on a separate CMA plate containing 1% agar (10 g / L casein digestion, 10 g / L mannitol, 10 g / L agar). Sometimes, sterile forceps were used to place the root segment in the agar medium. The plates were subsequently incubated at

37 ° C and were monitored to verify microbial growth. Typically, after 1-2 days, multiple microbial growths emerged from the roots and passed to the CMA medium. A sterile pipet tip was used to collect growths with unique morphologies throughout the segment and

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each of these growths was transferred to the center of the face of the CMA which contained 0.3% agarose. The CMA plates were subsequently incubated for 1-2 days at 37 ° C and monitored for growth. Commonly, isolates that form biofilms exhibited dendritic growth in this medium.

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

EXAMPLE 2

Growth and storage of microbial isolates

The isolated bacteria were stored as a pure culture. A bacterial colony was transferred to a container containing R2A broth liquid medium (Tecknova) and allowed to cultivate at 30 ° C with stirring at 250 rpm for two days. The culture was then transferred to containers containing 15% glycerol and stored at -80 ° C.

EXAMPLE 3

DNA extraction, sequencing and taxonomy

An aliquot of 20 pl of cell suspension was transferred

IMPI nwTrtyrcMEWCANo

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INDUSTRIAL bacterial to a 96-well PCR plate containing 20 pl of?v lysis buffer (100 mM Tris HCI, pH 8.0, 2 mM EDTA, pH 8.0, 1% SDS, 400 pg / mL Proteinase K). The lysis conditions were as follows: incubation at 55 ° C for 30 minutes, followed by incubation at

94 ° C for 4 minutes. An aliquot of lysis product was used as the source of the DNA template for PCR amplification.

For amplification of the 16S rDNA region, each PCR mix was prepared in a 20 µ final volume reaction containing 4 µl of the bacterial lysis reaction, 2 pM of each PCR primer, Tween-20 al

6 % and 10 pl of 2x ImmoMix (Bioline USA Inc, Taunton, MA). The primers used for the PCR amplification were M13-27F (5'TGTAAAACGACGGCCAGTTAGAGTTTGATCCTGGCTCAG-3 'SEQ ID NO:

8) and 1492R M13 with tail (5'CAGGAAACAGCTATGACCGGTTACCTTGTTACGACTT-3 '; SEQ ID NO: 9).

PCR was carried out in a PCT-200 personal thermocycler (MJ-Research, MA, USA) as follows: 94 ° C for 10 minutes; 94 ° C for 30 seconds, 52 ° C for 30 seconds, 72 ° C for 75 seconds for 30 cycles; 72 ° C for 10 minutes. A 2 µl aliquot of each PCR product was added to 1.0% agarose gel to confirm the presence of a single band of the expected size. Positive bands were isolated, purified, and submitted for PCR sequencing. Sequencing was performed in the forward and reverse direction by the J. Craig Venter Institute in San Diego, California, using 454 technologies.

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The homology search for the determined nucleotide sequence was carried out in the DDBJ / GenBank / EMBL database. Later, the phylogenic relationship of the nucleotide sequence of the 16 rRNA genes was analyzed between the isolated bacterial strains described herein, bacteria of the genus and species that show high sequence homologies with respect to the isolated bacterial strains, and others. broad varieties of genera and species of microorganisms, using the ClustalW program for creating phylogenic trees. Sequence identity and similarity was also determined using the GenomeQuest ™ software (Gene-IT, Worcester Mass. USA). The result of the sequence analysis revealed that the bacterial isolates SGI-003_H11, SGI-020_A01, SGI026 G06, SGI-026_G07, SGI-034_C09, SGI-034_E10, SGI-041_B03 could be considered related to the species of Pantoea agglomerans, Bacillis th Burkholderia metallica, Burkholderia vietnamiensis, Bacillus pumilus, Herbaspirillum sp., Pedobactersp., Respectively, based on> 98% of the sequence homologies of each of the 16 rRNA sequences with the respective microorganisms.

EXAMPLE 4

Biochemical characteristics of bacterial isolates

Isolated bacteria were further studied to verify important properties in their interaction with plants. The

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Properties studied include nitrogen fixation, siderophore secretion, inorganic phosphorus solubilization, production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, production of 2,3-butanediol, and production of plant growth hormone auxin. The results of the in vitro biochemical tests are shown in Table 2.

Fixation of nitrogen:

The bacterial cell suspensions were streaked on a solid medium of the following composition that did not include a nitrogen source: 4.0 g / L KOH; 0.5 g / L K2HPO4; 0.2 g / L MgSO4-7H2OR; 0.1 g / L of NaCI; 0.02 g / L of CaCI2; 0.005g / L FeSO4-7H2OR; 0.002 g / L NaMoO4-2H2OR; 0.01 g / L MnSO4 7H2OR; 5.0 g / L malic acid; 0.1 - 1.0 g / L gellan gum; and optionally 0.5% v / v bromothymol blue, pH 7.0. Gellan gum and agar concentrations may vary as necessary to achieve the desired average thickness; typically 0.5 g / L was used. Stretch marks were incubated at 30 ° C for 2-5 days. These plates were monitored daily and colonies were selected when they appeared. In some cases, longer growth periods (up to two weeks or more) were allowed to capture slow growing isolates. These streak plates were used to collect colonies using 20 or 200 pL aerosol barrier pipet tips in 96-well cell culture plates, filled with 150 pL / well of 2YT medium. Alternatively, isolate colonies were collected directly in N-free medium to

IMPI nwmuro MBUCANO '' OF THE NAME

INDUSTRIAL confirm its N-free growth capacity. The iBsultatlDS, as 55 'summarized in Table 2, indicated that only the SGI-026-G07 isolate showed nitrogen binding activity at detectable levels.

Siderophore discharge:

This assay was used to identify bacterial isolates that produced siderophores, which are Fe chelating compounds.3+ with high affinity, in vitro. Typically, the bacterial isolates were cultured in minimal medium that was essentially Fe-free. All glassware used during this assay was acid washed and rinsed three times with mineral water to remove residual Fe that could alter the assay results. . The composition of the MM9 medium was as follows: O.5g / L of K2HPO4; 1.0g / L NH4CI; 0.2g / L MgSO4 H2OR; 0.5g / L of NaCI; 7.55g / L of PIPES Buffer; 10.0g / L glucose; 2.5g / L gluconic acid; 2.5g / L of melic acid; 0.5 g / L of casamino acids. The medium was adjusted to pH 7.0 with 5N KOH and sterilized using a 0.2 pM filter (Corning).

This assay was typically performed in a high throughput format using a Beckman FX liquid handling station and 96-well cell culture plates with 150 pL of MM9 culture medium per well. Cultures and media were aseptically distributed and transferred using an autoclave needle tool in a laminar flow shroud. After transfer, they were incubated at 30 ° C for 5 days. After incubation, the culture supernatants were cultured by

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centrifugation using a 0.22 µΜΤΚΓ90 pULIIlUb filter plate. Transfer 10 ml of filtered supernatant from each well to a Falcon assay plate. A standard curve was prepared using deferrloxamine (DFO) diluted in MM9 medium. Two hundred mlcroliters of CAS Assay Solution [10mM HDTMA, Fe (lll) Solution: 1mM FeCh.6H2O, HC110mM, 2mM CAS] were added to each of the supernatants and standard wells, followed by Incubation at temperature environment for 20-30 minutes. The absorbance of the blue 630 nm CAS Test Solution (SpectroMax M2) is inversely proportional to the concentration of slderophores in each well (i.e. the test solution should change to a deep orange with higher amounts of slderophores).

Solubillza of inorganic phosphorus:

The ability of bacterial isolates to solubilize mineral phosphate in vitro was evaluated as follows. The bacteria to be tested were streaked on a phosphate agar culture medium [5.0g / L hldroxllapatlta-Ca10(PO4)5(OH)2; 1.0g / L NH4CI; 0.2g / L MgSO4H2OR; 0.5g / L of NaCI; 0.01 g / L FeSO4-7H2OR; 0.01 g / L of Na2MoO4-7H2OR; 0.01g / L of MnSO4 7H2OR; 5.0g / L glucose; 2.5g / L gluconic acid; 2.5 g / L of melic acid; 0.5g / L casaminoacids; 20.0g / L gellan gum; pH 7.2)], and its growth was monitored daily. The culture medium had an opaque appearance due to the presence of calcium phosphate. Bacterial growth and discoloration of the medium could be observed if the

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bacteria has the ability to dissolve from luIcíü phosphate. ' your isolated1 that they have the capacity to solubilize the phosphate in the mineral phase, they would produce a transparent halo in the opaque medium that surrounds the colony. As summarized in Table 2, the ability to solubilize the mineral phosphate could not be detected in any of the microorganisms tested as determined by the in vitro assay described herein.

Production of ACC deamlnasa:

One of the main mechanisms used by the plant growth promoting rhizobacterium (PGPM) to facilitate plant growth and development is to decrease ethylene levels through the deamlination of 1-aminocylpropropane-1-carboxylic acid (ACC ), the immediate precursor to ethylene in plants. ACC deaminase catalyzes the hydrolysis of 1-amlnoclclopropane-1-carboxylic acid (ACC) into α-ketobutyrate and ammonia. The presence of the α-ketobutlrate product can be determined Indirectly by reaction with 2,4-dlnltrofenllhldrazlna in HCI to form a phenylhydrazone derivative. After addition of NaOH, the amount of phenllhydrazone in solution can be determined spectrophotometrically by measuring its absorbance at 540 nm (Penrose and Glick, Physiol Plant. Mayo;

118: 10-1, 2003). This assay was typically performed in a high throughput format using 96-well cell culture plates. Each well contained 150 pL of DF salt culture medium supplemented with 2.0 g / l (NH4)2SO4. Cultures and media were distributed and transferred in a manner

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ΠΒΤΠυΤΟ MEXICAN MIAMMEDAD

Aseptic using an autoclave needle tool on a laminar flow shroud. After transfer, they were incubated at 30 ° C for 2 days. After turbidity is reached, cultures are transferred a second time using a sterile needle tool under a laminar flow hood in 96-well plates containing 150 pL per well of DF salt culture medium supplemented with 5mM ACC such as single nitrogen source, followed by a 4 day incubation at 30 ° C. The absorbance of each culture at 600nm was measured using a spectrophotometer. Isolates that exhibited strong growth under these conditions (OD> 0.2) were taken to the next step for further testing to analyze 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: SGI-003-H11, SGI-026-G06, SGI-026-G07, and SGI-041-B03. .

2,3-Butanediol production:

The ability of bacterial isolates to synthesize 2,3-butanediol in vitro was evaluated as follows using mass spectroscopy and capillary gas chromatography as described in

Ryu et al. (Proc. Nati. Acad. Sci. USA 100: 4927-4932, 2003). This assay was typically performed in a high throughput format using 96-well cell culture plates with 150 pL of DF salt culture medium per well. A titer-tek can also be used when preparing a large quantity of

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plates for primary reaming of large sets of isolates. Cultures and media were aseptically distributed and transferred using an autoclave needle tool in a laminar flow shroud. After transfer, they were incubated at 30 ° C for 5 days. Following incubation, culture supernatants were cultured by centrifugation using a 0.22 µΜ 96-well filter plate. Fifty microliters of filtered supernatant from each well was transferred to the corresponding wells of a 96-well deep plate containing 450 pL 50% methanol per well using an L200 multi-channel pipette and sealed with an adhesive plate closure, followed by 2,3-butanedlol 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 produced significant amounts of 2,3-butanediol: SGI-003-H11, SGI-034-C09, and SGI-041-B03.

Auxin production:

Auxins are hormones that can directly affect plant growth. This assay was performed to determine if bacterial isolates produced auxins, as many endophytic bacterial rhizospheres and isolates are known to have biochemical pathways that synthesize Indole-3-acetic acid (IAA) auxin and its derivatives. Generally tryptophan is a precursor in this synthesis; and therefore, this assay quantified AAI (auxin) production from bacterial isolates.

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grown in a medium supplemented with a low concentration of amino acid tryptophan.

This assay was typically performed in a high throughput format using 96-well cell culture plates with 150 pL of YT salt culture medium per well. When preparing a large number of plates for primary scans of large sets of isolates, a titer-tek was used. Cultures and media were aseptically distributed and transferred using an autoclave needle tool in a laminar flow shroud. After transfer, they were incubated at 30 ° C for 5 days.

Following incubation, culture supernatants were cultured by centrifugation using a 0.22 µΜ 96-well filter plate. Ten microliters of filtered supernatant from each well was transferred to a Falcon assay plate. Two hundred microliters of Salkowsky test solution (Gordon and Weber, Plant Physiol. 26: 192-195, 1951) was added to each of the supernatants and standard wells, followed by incubation at room temperature for 15-20 minutes. The reaction was monitored by absorbance of the plate in the SpectroMax m2 at 535 nm while the color change of the Salkowsky test solution from yellow to purple / pink was proportional to the auxin concentration (AAI) in each well. Test results, as summarized in Table 2, indicated that the following isolates produced significant amounts of auxin phytohormones: SGI-003-H11, SGI-020-A01, SGI-034-C09, SGI-034C09, and SGI -041-B03.

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Biochemical characteristics of bacterial isolates (DN: not detectable)

TABLE 2

<td colspan="2">Bacterial isolates</td><td colspan="5">Biochemical activity</td>

<td>ID of the isolated</td><td>Taxonomy provisional</td><td>Auxin production</td><td>ACC deaminase</td><td>2.3- butanediol</td><td>fixing N</td><td>phosphorus solubilization</td>

<td>003 H11</td><td>Pantoea agglomerans</td><td>Yes</td><td>Yes</td><td>Yes</td><td>ND</td><td>ND</td>

<td>020_A01</td><td>Bacillus thuríngiensis</td><td>Yes</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td>

<td>026_G06</td><td>Burkholderia metallica</td><td>ND</td><td>Yes</td><td>ND</td><td>ND</td><td>ND</td>

<td>026_G07</td><td>Burkholderia vietnamiensls</td><td>ND</td><td>Yes</td><td>ND</td><td>Yes</td><td>ND</td>

<td>034_C09</td><td>Bacillus pumilus</td><td>Yes</td><td>ND</td><td>Yes</td><td>ND</td><td>ND</td>

<td>034_E10</td><td>Herbaspirillum sp.</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td>

<td>041_B03</td><td>Pedobacter sp.</td><td>Yes</td><td>Yes</td><td>Yes</td><td>ND</td><td>ND</td>

EXAMPLE 5

Biological control activity of bacterial isolates against fungal phytopathogens

An in vitro antagonism assay was used to assess the ability of isolated bacterial strains to suppress the development of various plant fungal pathogens, including Fusarium graminearum NRRL-5883, Monographella nivalis ATCC MYA-3968, Gibberella zeae ATCC-

Colletotríchum graminicola The test was performed in a

16106, Stagnospora nodurum ATCC-26369,

ATCC-34167, and a pathogen of Penicillium sp.

potato dextrose agar medium (APD). Isolated strains of bacteria were grown on tryptic soy broth agar (TSBA / 5) for 24 h before use.

For each fungal pathogen, a conidial inoculum was produced by exposing the hita to an actively growing colony of the fungus and transferring the hypha threads to the PDA agar medium. After incubating the plates for 7 days at 25 ° C using a 12 h / day photoperiod, the PDA plates were washed from the conidia using weak phosphate buffer (0.004% phosphate buffer, pH 7.2, with 0.019% MgCI2). Immediately a suspension of fungal conidia was sprayed into the weak phosphate buffer (approximately 1 * 105 conidia / mL) on the agar surface, and the sprayed plates were then incubated at 25 ° C for 48-72 h before using in antagonism tests.

To initiate antagonism tests, cells from isolated microbial strains were inoculated punctually at equal distances within the perimeter of the plate. After five days, the bacterial strains were evaluated as positive for antibiosis when there was a visible transparent area (that is, a growth inhibition zone) that lacked mycelial growth around the perimeter of the microbial colonies. The results of the antagonism tests, as summarized in Table 3, demonstrated that each of the microorganisms disclosed herein

IMPI nr. «ΣπτνιοΜΚΚΑΝο« WMWIBBAD íÑoumuAi inhibited the development of various fungal phytopathogens, including Pusarium graminearum, Monographella nivalis, Gibberella zeae, Stagnospora nodurum,

Colletotrichum graminicola, Penicillium sp.

TABLE 3

Biological control activity of bacterial isolates against fungal phytopathogens.

<td colspan="2">Bacterial isolates</td><td colspan="2">Qualified growth suppression 5 days after</td><td colspan="3">of fungal pathogens (the incubation area)</td><td>incubation</td>

<td>Identification of the isolate</td><td>Taxonomy provisional</td><td>Fusarium gramine arum</td><td>Monographella nivalis</td><td>Gibberella zeae</td><td>Stagnosp pray nodurum</td><td>Colletotri chum graminic wave</td><td>Penicilliu m sp.</td>

<td>003_H11</td><td>Pantoea agglomerans</td><td>No</td><td>Sf</td><td>No</td><td>No</td><td>No</td><td>No</td>

<td>020_A01</td><td>Bacillus thuringiensis</td><td>Yes</td><td>No</td><td>Yes</td><td>Sl</td><td>Yes</td><td>No</td>

<td>026_G06</td><td>Burkholderia metallica</td><td>No</td><td>Sf</td><td>Yes</td><td>YES</td><td>Sl</td><td>Yes</td>

<td>026_G07</td><td>Burkholderia vietnamese</td><td>No</td><td>Sl</td><td>No</td><td>No</td><td>No</td><td>No</td>

<td>034_C09</td><td>Bacillus pumilus</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td>

<td>034_E10</td><td>Herbaspirillum sp.</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td>

<td>041J303</td><td>Pedobacter sp-</td><td>No</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td><td>Sl</td>

EXAMPLE 6

Improving the yield potential of wheat

The effects of bacterial inoculation on plant yield and growth were studied in a greenhouse with isolate SGI-020-

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IWJinUTO MEXICANO

OC DI LA FRORiDAD

OO INDfSTWIAl

A01. Microbial cell suspensions were prepared as follows. 2YT medium, or similar culture medium, broth cultures were inoculated from isolated glycerol stock solutions or striatum plates. Typically, before use in the growth chamber, greenhouse, or field, bacterial cultures were started 48-72 hours to allow the cultures to reach the late exponential phase. The isolates with the highest doubling times were started early. The cultures were incubated at 30 ° C on a rotary shaker at 200 rpm. After growth, cells were pelleted at 10,000 xg for

fifteen min at 4 ° C and resuspended in 10 mM buffer

MgSO4 (pH 7.0). Cell densities were normalized for each isolate on a CFU / mL basis. Typically, suspensions of ~ 109 CFU / mL were prepared for each isolate and transported on ice to the inoculation site. Inoculations were performed by diluting these cell suspensions 1/20 in irrigation water to a final density of 5 x 107 CFU / mL. For assays in 1-liter containers, 20 mL of this diluted cell suspension was distributed uniformly over the surface of each container in duplicate.

The greenhouse test was carried out on field soil with a nutrient deficiency. After removing debris and large rocks, the field soil was mixed well to ensure homogeneity. After filling, the soil in each of the containers was pressed ~ 2 cm for a firm seed coat. The seeds of a variety were sown

IMPI Muicamo Institute

DtunoniBAD υINDUSTRIAL grown from commercial wheat (bread wheat; Howe Seeds, Inc.) in 1-liter containers containing field soil medium (plastics with a narrow diameter of 10.5 cm x 12.5 cm). Two grams of bread wheat seeds (approximately 70 seeds) were evenly distributed in each container and 50 mL of field soil was uniformly applied and sprayed on the seed coat. After the uniform appearance of the wheat coleoptile and the subsequent appearance of the first leaf, the plant population was inoculated with 20 mL of 109 CFI / mL of SGI-020-A01. Negative control plants received only 20 mL of inoculum buffer.

Each condition was performed on 8 duplication surfaces, each containing four 1-liter containers (n = 4 per surface). The surfaces were randomly distributed in four experimental blocks. The seeds and plants were kept in a greenhouse for 60 days at room temperature (in the range of 8 ° C to approximately 22 ° C) with daylight cycles of approximately 11.5 h sunlight / 12 hours of darkness throughout the proof. Plants were evenly watered at the bottom for an adequate level of hydration depending on temperature and stage of growth. Approximately 30 days after planting, approximately 70 individuals per container were stacked and tied to avoid cross contamination and to minimize location effects due to variation in plants falling into other containers. Approximately 60 days after planting, the plants were allowed to dry to prepare them for harvest. Wheat heads were harvested

IMPI „ Λ MEXICAN INSTITUTE OF THE noTKDA »

INDUSTRIAL · approximately 80 days after sowing. Each head of wheat was removed by cutting just below the head. Wheat heads were pooled within each container doubling, and weighed, and then used to estimate yield potential. On the same day, all plants in the population were harvested and treatments were harvested in random order to eliminate large differences in time between harvest between treatments. As a result, wheat plants treated with isolate SGI-020-A01 showed a 40% increase in yield potential compared to untreated control plants (2.95 grams / container compared to 2.10 grams / container). Standard and average deviations were documented in all 8 duplicates and an ANOVA (Analysis of Variance) was performed. The efficacy of SGI-020-A01 microbial isolate to improve wheat yield potential was quantified by analyzing the weight yield of wheat heads for each container duplicate. P values <.05 were considered significant.

EXAMPLE 7

Improvement of biomass production in corn

The effects of bacterial inoculation on plant yield and growth were studied in a greenhouse experiment with each of the following bacterial isolates: SGI-034-C09, SGI-034-E10, SGI003-H11, SGI-041-B03, SGI-026-G06, and SGI-026-G07. The test of

greenhouse were carried out with field soil with nail,ncia 'of nutrients. After removing debris and large rocks, the soil from the field was mixed well with soil from the container (70:30) to ensure homogeneity. After filling, the soil in each of the containers was pressed ~ 2 cm for a firm seed coat. The seeds of a commercial cultivated variety (Dow AgroSciences) were sown in 1-liter containers (narrow 10.5 cm x 12.5 cm containers) each containing soil medium. Two corn kernels were evenly distributed in each container with the embryo up, followed by the application of 50 mL of field soil, which was evenly dispersed over the seed coat. After germination, one seedling per container was discarded when necessary so that each container had only one plant.

After the uniform appearance of the corn coleoptile and the subsequent appearance of the first leaf, the population of the plant was inoculated with ~ 20 mL of 109 CFU / ml of a microbial isolate selected from the group of

SGI-034-C09, SGI-034-E10, SGI-003-H11, SGI-041-B03, SGI-026-G06, and SGI-026-G07. Microbial cell suspensions were prepared as described in Example 6 above. Negative control plants received only 20 mL of inoculum buffer.

Each condition was performed on 8 duplication surfaces, each containing two 1-liter containers (n = 2 per surface). The surfaces were randomly distributed in four experimental blocks. The seeds and plants were kept in a greenhouse for 60 days at

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ambient temperature (in the range of 8 ° C to approximately 22 ° C) with daylight cycles of approximately 11.5 h sunlight / 12 hours dark throughout the test. Plants were evenly watered at the bottom for an adequate level of hydration depending on temperature and stage of growth. Above-ground corn biomass was harvested approximately 60 days after planting.

On the same day, all plants in the population were harvested and treatments were harvested in random order to eliminate large differences in time between harvest between treatments. Corn plants were analyzed for the difference in their total biomass. As documented in Table 4, corn plants treated with each of the microbial isolates showed a significant increase in total biomass as compared to untreated control plants. Standard and average deviations were documented in all 8 duplicates and an ANOVA (Analysis of Variance) was performed.

TABLE 4

Efficacy of microbial isolates in improving the total biomass of the plant

<td>Treatment</td><td>Plant biomass (9)</td><td>P value</td><td>Biomass increase (%)</td>

<td>No treatment</td><td> 58.6</td><td>N / A</td><td>N / A</td>

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EXAMPLE 8

Wheat and corn seed coating treatment

Small-scale seed treatment experiments were performed according to a procedure described in Sudisha et al. (Phytoparasitica, 37.Ί61-169, 2009) with few modifications. In general, a biopolymer stock solution was made by adding 1 grams of gum arabic powder (MP Biomedical) to 9 mL of water and mixing until homogeneous. Turbid cultures of active growing microbial cells or microbial spore preparations were washed with PBS and adjusted to an OD600 of ~ 5.0. Three mL of adjusted cell suspension was pelleted by centrifugation in a 50 mL Falcon tube. The resulting supernatant was decanted, replaced with 3 mL of biopolymer stock, and the resulting suspension was thoroughly mixed. Typically, approximately 25 g of seeds were added to the Falcon tube and vigorously shaken or vortexed to ensure even distribution of the rubber / cell suspension. The coated seeds were spread in plastic weighing containers to dry in a laminar flow cover until they were no longer tacky, usually 3 hours with periodic shaking. The coated seeds were stored at 4 ° C and periodically analyzed to assess their stability. A variety of wheat seeds and corn seeds were coated and analyzed in the manner described above, including varieties

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common bread wheat Briggs, Faller, Glenn, Hank, RÓ07, Samson; winter bread wheat varieties Jerry, McGill, Overland; and the DKC62-61 maize seed variety as well as commercial cultivated maize varieties (Dow AgroSciences).

Feasibility evaluation on the microbes used in the seed coating formulation was performed using a standard plate count method. Typically, a predetermined amount of coated seeds was analyzed to assess the presence of viable microbes by washing the seeds in an aliquot of suitable buffer and planting equivalent amounts of buffer in the nutrient agar medium. Viable colony forming units were determined after 1-4 days of incubation at 30 ° C. The feasibility test showed that between 1 χ 104 and 4 χ 107 of viable colony forming units per seed were present after approximately five weeks of storage at 4 ° C. When the seeds were coated with microbial spores, the viability of most of the analyzed microbes remained stable for at least four months, including in multiple interstate shipments in the United States, entering and leaving refrigerated containers. When stored refrigerated (4 ° C) the microbes survived in the seed coat with little loss of viability during the test periods. The results indicated that the seeds coated with compositions described herein can be stored refrigerated for longer periods and demonstrated that the microbes can survive during periods with higher temperatures for distribution.

Furthermore, the germination rate of the coated seeds was analyzed and determined to be essentially identical to the control seeds, which were seeds coated only with gum arabic or uncoated seeds.

EXAMPLE 9

Solid state formulation of microbial compositions

MEXICAN INSTITUTE OF PROPERTY fWTWSTRIAL

This section describes an exemplary microbial fertilizer formulation where the bacteria according to the present invention is encapsulated and the fertilizer is in solid form. Alginate pearls are prepared as follows:

One milliliter of 30% glycerol is added to a 1, 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 a combination of two or more isolates were pelleted by centrifugation, then washed with a saline solution (0.85% NaCI, w / v) , suspended in 25 mL of alginate mix, and mixed thoroughly. This cell suspension is then added dropwise in a pre-chilled sterile 1.5 or 2% (w / v) aqueous solution of CaCfe with gentle agitation to obtain the alginate bacterial beads. The beads are allowed to harden for 2-4 hours at room temperature. The

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MEXICAN INSTITUTE

INSTTTVTO MEXiCANC DE LA ηΟΠΕΟΑΓ. INDUSTRY!

Beads were collected with sieving and washed several times with sterile water and stored at 4 ° C. To preserve the formulation, fresh wet beads can be frozen at around -80 ° C before lyophilization at around -45 ° C for 15 hours. The freeze dried dried pearls were stored in suitable containers, such as sterile glass bottles.

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

EXAMPLE 10

Compatibility of microbial compositions with commercial fungicides

As environmental concerns in pesticide use in agriculture are on the rise, biological alternatives are perceived as inevitable. However, new biological formulations must also allow organisms to survive and express their specific beneficial impact. Chemical fungicides are generally toxic, not only for harmful microorganisms but also for

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Q 5 MEXICAN INSTITUTE i '*' DE LA MONEDAD

INDUSTRIAL beneficial. However, the survival capacity Ütí tíütóS microbial agents may have improved when applied at lower rates.

In the present study, the peat-based carrier material is used for inoculation of the treated fungicide and the simple culture seed. The tolerance capacity of the fungicide is generally evaluated as follows: a) simple seeds inoculated with bacteria are cultivated in suitable bacterial growth media such as soybean trypticase agar plates (TSA; Trlptone 15 g / L; Soytone 5 g / L, Sodium Chloride 5 g / L, and Agar 15 g / L) b) seeds Inoculated with bacteria treated with fungicides grown in common TSA plates, and c) seeds inoculated with bacteria treated with fungicides in growth compartments . Typically, three concentrations of fungicides are used in each of the experiments: the manufacturer's recommended dose and two lower doses (at 75% and 50% of the recommended dose). Seeds inoculated with fungicide-treated bacteria are stored after inoculation and used at different time intervals (2 hr, 4 hr, and 6 hr) to examine the impact on seed germination. The presence of bacteria and seed germination are monitored in petri dishes. For the study of the growth compartment, the seeds (with recommended doses) treated with fungicides are used, and the root and length of the hlpocótllo were measured 7 days after the growth of the seedling.

Some rlzobacterial isolates of the invention are compatible with various commonly used fungicides as determined by the

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MEXICAN INSTITUTE

PROPERTY Qfi

INDUSTRIAL bacterial growth on TSA plates enriched with fungicide In general, both the fungicide-treated and the simple seeds, coated with inoculated peat do not show a significant variation in germination compared to the non-inoculated control. In addition, growth promoting effects on total seedling and root length are observed in all rhizobacterial treatments compared to non-inoculated controls.

EXAMPLE 11

Development of cultivated varieties of non-natural origin and cultivation program

The endophytic bacteria of the present invention are introduced into crop plants, including cereals, of various genotypes and geographic origin, which lack such endophytic fungi, to create combinations of plants and endophytes with better agronomic characteristics, using procedures analogous to those known in the art, including those described in US Patent Application No. 20030195117A1; US Patent Application No. 20010032343A1; and US Patent No. 7,084,331, among others. Therefore, particular plant and endophyte synthetic combinations can be created and selected in a cultivation / cultivar variety development program based on their ability to form and maintain a combination of

ΙΜΡΙ6 Λ_ INSTITUTO MBÜCANC V \ nE * · * KOREDAD C

INDWSTSIA The mutual benefit that results in an agronomic benefit. The classification of the agronomic characteristics of the combination can also be used in said cultivation program. These characteristics may include, but are not limited to, drought tolerance, blomass accumulation, resistance to insect infection, palatability for livestock (for example, 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 alkaloid levels, loline levels, peramine levels, or lolltrem levels, while exhibiting desired agronomic characteristics of crop plants, including resistance to insect feeding or infection, resistance to abiotic stress, palatability for livestock, accumulation of blomass, ease of reproduction and seed yield, among other traits.

EXAMPLE 12

Performance study

Corn (Zea mays) seeds were coated with different microbial treatments and sown in a prepared field. Each treatment was repeated 5 times in a complete, randomized block design. A single duplicate consisted of four beds (rows) 9.15 meters long, 60 seeds were sown (15.24 centimeters apart) in

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each bed. For the purposes of observation, the data is

the middle two rows.

Plant emergence was recorded twice as shown in Table 5 below as the percentage of plants in duplicate 5 that had sprouted. Ten plants in the middle two rows of each plot were labeled with plastic tape to record plant measurements, such as plant height, chlorophyll measurement, plant weight, etc.

The plant heights (measuring the highest / longest leaf tip) recorded 31 and 56 days after planting indicated that the plant height between treatments was not significantly different. Most of the plants were dry and the leaves had shrunk by day 110 after planting, therefore in some cases the plants appeared (measured) less than in previous measurements, but in general the height of the plant was no different between treatments. Chlorophyll content was measured in the 5to week after planting (in SPAD units) from the lowest leaves (approximately 60 cm above the ground) and the highest leaves (the second leaf fully expanded from the tip) of ten labeled plants from each plot. The chlorophyll content between the treatments was not significantly different. The culture was harvested on day 110 and 111. Ten labeled plants were cut from each plot at ground level and the above ground parts were weighed (total plant weight or WPtWt), ears of corn were removed, and length of corn (length of ear) was measured with grains) (region filled with grains

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QQ MEXICAN INSTITUTE

OF PROHKDAD

INDUSTRIAL (marketable only), then the grains were removed from the corn and weighed to obtain the grain weight per ear (KnlWt / ear).

Shortly after the manual harvesting of 10 plants per plot was completed, a mechanical harvester, Gleaner® K2 (Allis-Chalmers) was brought

Mfrg, Milwaukee, Wl). This machine manually removed the remaining plants from the two middle rows of each plot, the grains were removed from the corn, and the weight and humidity of the grain was measured (10 ears + mechanical harvest). The total projected yield with a moisture content of 15.5% (kilograms of corn grain per hectare) based on the weight (kg.) Of kernels (includes machine-harvested corn kernels + hand-harvested kernels) was 11,618.53 kilograms per hectare or 161.22 hectoliters per hectare for SGI-003-H11 (Pantoea agglomerans).

This was the highest yield among treatments for all organisms and was significantly different from treatments for Bacillus amyloliquefaciens SGI-015_F03. The other with high production was the treatment with Bacillus thuringiensis SGI-020_A01.

In conclusion, all plants in the different treatments emerged and grew similarly under field conditions provided with the same amount of fertilizer, pre-emergence herbicides (with manual weed removal later in the season), and weekly irrigation during temperate and growing season, and pest control, especially of corn worm. All conditions being the same, treatment with SGI-003-H11 (Pantoea

100

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agglomerans) produced the highest yield compared rr> n treatment hwhat control, without microbes. Therefore, 003_H11 produced approximately 17% higher performance than the control group. Therefore, in various embodiments of applying the invention an effective amount of an organism according to any of the methods described herein produces at least a 10% or at least 12.5% or at least 15% yield or about 17% higher than a control group, which in some modalities can be determined by kilograms of plant product (eg, ears of corn) per hectare or hectoliters of plant product per hectare. The organisms listed in Table 5 are SGI-003-H11 (Pantoea agglomerans) -, SGI-015-F03 {Bacillus amyloliquefaciens); and SGI-020A01 (Bacillus thuringiensis).

TABLE 5

<td>Organism</td><td>Appearance</td><td>Appearance</td><td>Height</td><td>Height</td><td>Height</td>

<td>Control</td><td> 97.00</td><td> 94.50</td><td> 103.64</td><td> 277.22</td><td> 267.82</td>

<td>Organism</td><td>Highest blade</td><td colspan="2">Lower sheet WPtVVt</td><td>Length of the grain</td><td>cob with</td>

<td>Control</td><td> 43.24</td><td> 60.85</td><td> 493.24</td><td> 14.98</td><td></td>

101

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INSTITUTO MUICANO DE L * raOnnMD INDUSTRIAL

<td rowspan="2">Organism</td><td rowspan="2">Weight grain / cob</td><td colspan="2">Weight 10</td><td rowspan="2">10 ears + machine</td>

<td>: a (g) grain / ear (lb)</td><td>ears + machine</td>

<td>Control</td><td> 168.62</td><td> 0.37</td><td> 157.81</td><td> 8837.63</td>

A number of modalities of the Invention have been described.

However, it will be understood that the elements of the embodiments described herein can be combined to perform additional embodiments and that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other, alternative, and equivalent embodiments are within the scope of the invention as described and claimed herein.

The headings within the application are only presented for the convenience of the reader and do not limit in any way the scope of the invention or its modalities.

All publications and patent applications cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application is specifically and individually indicated as incorporated herein by this reference.

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102

Contents112

38 members in 17 offices

This recordMember, by publication datePriority claim7 more offices are in the list below

Priority claims9

Priority claims
Document	Office	Kind	Date
201161570237	United States of America	P	2011-12-13
201161570237	United States of America	P	2011-12-13
61570237	United States of America	–	2011-12-13
2012069579	United States of America	W	2012-12-13
2012069579	United States of America	W	2012-12-13
61570237	–	–	–
PCTUS2012069579	–	–	–
US201161570237P	–	–	–
WO2012US69579	–	–	–

Members38

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CA2860848A1	Canada	A1	2013-06-20
CA3105047A1	Canada	A1	2013-06-20
CA3202904A1	Canada	A1	2013-06-20
WO2013090628A1	World Intellectual Property Organization (WIPO)	A1	2013-06-20
AU2012352161A1	Australia	A1	2014-06-26
CO6980622A2	Colombia	A2	2014-06-27
MX2014007070A	Mexico	A	2014-08-08
PH12014501282A1	Philippines	A1	2014-09-08
EP2790513A1	European Patent Office (EPO)	A1	2014-10-22
US2014342905A1	United States of America	A1	2014-11-20
CN104168771A	China	A	2014-11-26
JP2015502751A	Japan	A	2015-01-29
EP2790513A4	European Patent Office (EPO)	A4	2015-10-28
ZA201404047B	South Africa	B	2015-10-28
RU2014128552A	Russian Federation	A	2016-02-10
NZ625835A	New Zealand	A	2016-02-26
AU2012352161B2	Australia	B2	2016-05-19
US9687000B2	United States of America	B2	2017-06-27
CN104168771B	China	B	2017-06-30
CN107418911A	China	A	2017-12-01
JP6243347B2	Japan	B2	2017-12-06
JP2018011600A	Japan	A	2018-01-25
BR112014014000A2	Brazil	A2	2018-01-30
US2018064116A1	United States of America	A1	2018-03-08
UA117340C2	Ukraine	C2	2018-07-25
RU2664863C2	Russian Federation	C2	2018-08-23
MX358751BThis record	Mexico	B	2018-08-31
BR112014014000B1	Brazil	B1	2019-05-28
EP2790513B1	European Patent Office (EPO)	B1	2019-08-21
EP3598899A2	European Patent Office (EPO)	A2	2020-01-29
EP3598899A3	European Patent Office (EPO)	A3	2020-03-04
ES2752451T3	Spain	T3	2020-04-06
PL2790513T3	Poland	T3	2020-04-30
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CA2860848C	Canada	C	2021-02-23
CA3105047C	Canada	C	2023-08-01
EP3598899B1	European Patent Office (EPO)	B1	2023-11-15