Streptomyces endophyte compositions and methods for improved agronomic traits in plants

This application is the National Stage of International Application No. PCT/US2016/036504, filed 8 Jun. 2016, which claims the benefit of and priority to U.S. Provisional Application No. 62/172,748 filed 8 Jun. 2015, and of U.S. Provisional No. 62/172,750 filed on 8 Jun. 2015, and of U.S. Provisional Application No. 62/172,755 filed on 8 Jun. 2015, and of U.S. Provisional Application No. 62/316,386 filed on 31 Mar. 2016, all of which are hereby incorporated by reference in their entireties.

The instant application includes a Sequence Listing with 4779 sequences which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 8, 2016, is named 33897PCT_sequencelisting.txt, and is 8.83 MB in size.

This invention relates to compositions and methods for improving the cultivation of plants, particularly agricultural plants, such as soybeans and maize. For example, this invention describes bacteria, such as strains of the genus Streptomyces , that are capable of living in or otherwise associated with a plant, which may be used to impart improved agronomic traits to plants. The disclosed invention also describes methods of improving plant characteristics by introducing bacteria to those plants. Further, this invention also provides methods of treating seeds and other plant elements with Streptomyces bacteria that are capable of living within or otherwise associated with a plant, to impart improved agronomic characteristics to plants, particularly agricultural plants, for example soybeans or maize.

According the United Nations Food and Agricultural Organization (UN FAO), the world's population will exceed 9.6 billion people by the year 2050, which will require significant improvements in agricultural to meet growing food demands. At the same time, conservation of resources (such as water, land), reduction of inputs (such as fertilizer, pesticides, herbicides), environmental sustainability, and climate change are increasingly important factors in how food is grown. There is a need for improved agricultural plants and farming practices that will enable the need for a nearly doubled food production with fewer resources, more environmentally sustainable inputs, and with plants with improved responses to various biotic and abiotic stresses (such as pests, drought, disease).

Today, crop performance is optimized primarily via technologies directed towards the interplay between crop genotype (e.g., plant breeding, genetically-modified (GM) crops) and its surrounding environment (e.g., fertilizer, synthetic herbicides, pesticides). While these paradigms have assisted in doubling global food production in the past fifty years, yield growth rates have stalled in many major crops and shifts in the climate have been linked to production instability and declines in important crops, driving an urgent need for novel solutions to crop yield improvement. In addition to their long development and regulatory timelines, public fears of GM-crops and synthetic chemicals have challenged their use in many key crops and countries, resulting in a lack of acceptance for many GM traits and the exclusion of GM crops and many synthetic chemistries from some global markets. Thus, there is a significant need for innovative, effective, environmentally-sustainable, and publically-acceptable approaches to improving the yield and resilience of crops to stresses.

Improvement of crop resilience to biotic and abiotic stresses has proven challenging for conventional genetic and chemical paradigms for crop improvement. This challenge is in part due to the complex, network-level changes that arise during exposure to these stresses.

Like humans, who utilize a complement of beneficial microbial symbionts, plants have been purported to derive a benefit from the vast array of bacteria and fungi that live both within and around their tissues in order to support the plant's health and growth. Endophytes are symbiotic organisms (typically bacteria or fungi) that live within plants, and inhabit various plant tissues, often colonizing the intercellular spaces of host leaves, stems, flowers, fruits, seeds, or roots. To date, a small number of symbiotic endophyte-host relationships have been analyzed in limited studies to provide fitness benefits to model host plants within controlled laboratory settings, such as enhancement of biomass production (i.e., yield) and nutrition, increased tolerance to stress such as drought and pests. There is still a need to develop better plant-endophyte systems to confer benefits to a variety of agriculturally-important plants such as soybean and maize, for example to provide improved yield and tolerance to the environmental stresses present in many agricultural situations for such agricultural plants.

Thus, there is a need for compositions and methods of providing agricultural plants with improved yield and tolerance to various biotic and abiotic stresses. Provided herein are novel compositions including bacteria that are capable of living within a plant, formulations comprising these compositions for treatment of plants and plant elements, and methods of use for the same, created based on the analysis of the key properties that enhance the utility and commercialization of an endophyte composition.

In an aspect, the invention provides a method for preparing a plant reproductive element composition, comprising contacting the surface of a plant reproductive element of a plant with a formulation comprising a purified microbial population that comprises a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO: 2 through SEQ ID NO:18. In another aspect, the invention provides a method for preparing a plant reproductive element composition, comprising contacting the surface of a plant reproductive element of a plant with a formulation comprising a purified microbial population that comprises a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a strain deposit IDAC Deposit ID 081111-06. In yet another aspect, the invention provides a method for preparing a plant reproductive element composition, comprising contacting the surface of a plant reproductive element of a plant with a formulation comprising a purified microbial population that comprises a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a Streptomyces species selected from the group consisting of: albidoflavus, albus, aureofaciens, ginsengisoli, griseus, lydicus, mutabilis, neyagawaensis, praecox , and SMCD2215; wherein in any of the preceding methods the endophyte is present in the formulation in an amount capable of modulating at least one of: trait of agronomic importance, transcription of a gene, level of a transcript, the expression of a protein, level of a hormone, level of a metabolite, and population of endogenous microbes; in plants grown from the plant reproductive elements, as compared to isoline plants grown from plant reproductive elements not contacted with the formulation.

In certain aspects the invention provides for any of the preceding methods, wherein the Streptomyces endophyte is optionally present in the plant reproductive element in an amount capable of providing a benefit to a plant derived from the plant reproductive element, as compared to a plant derived from a plant reproductive element not treated with said Streptomyces endophyte.

An embodiment of the invention is a plant derived from the composition of any of the preceding methods, wherein the plant comprises in at least one of its plant elements said Streptomyces endophyte. Another embodiment of the invention comprises the progeny of the plant derived from the composition of any of the preceding methods, wherein the progeny comprises in at least one of its plant elements the Streptomyces endophyte.

Another embodiment of the invention is a plurality of plant reproductive element compositions prepared according to any of the proceeding methods, wherein the compositions are confined within an object selected from the group consisting of: bottle, jar, ampule, package, vessel, bag, box, bin, envelope, carton, container, silo, shipping container, truck bed, and case.

In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element, comprising treating the plant reproductive element with a formulation comprising a Streptomyces endophyte that comprises at least 2× higher acetoin production as compared to the strain represented by SEQ ID NO: 1. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element, comprising treating the plant reproductive element with a formulation comprising a Streptomyces endophyte that comprises at least 1.5× higher siderophore production as compared to the strain represented by SEQ ID NO: 1. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element, comprising treating the plant reproductive element with a formulation comprising a Streptomyces endophyte that comprises greater utilization of a primary carbon source selected from the group consisting of: D-Galactose, Glycerol, alpha-D-Glucose, Sucrose, beta-methyl-D-glucoside, D-cellobiose, L-alanine, L-alanyl-glycine, mono methyl succinate, glycyl-L-proline, L-lyxose, as compared to the strain represented by SEQ ID NO: 1. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element, comprising treating the plant reproductive element with a formulation comprising a Streptomyces endophyte that secretes at least one protein listed in Table 5C with at least a 0.4× higher rate, as compared to the strain represented by SEQ ID NO: 2. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element, comprising treating the plant reproductive element with a formulation comprising a Streptomyces endophyte that secretes at least one protein selected listed in Table 5D with at least a 0.7× lower rate, as compared to the strain represented by SEQ ID NO: 2. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element, comprising treating the plant reproductive element with a formulation comprising a Streptomyces endophyte that comprises at least 3 arabinose transporter genes in its genome. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element, comprising treating the plant reproductive element with a formulation comprising a Streptomyces endophyte that secretes at least one protein selected from Table 5A. In yet another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element, comprising treating the plant reproductive element with a formulation comprising a Streptomyces endophyte that does not secrete a protein selected from the proteins listed in Table 5B.

In an aspect, the invention also provides a method of using a beneficial Streptomyces endophyte that confers a trait of agronomic importance to a plant, wherein the endophyte comprises at least 97% identity to at least 600 nucleotides of SEQ ID NO: 3. In another aspect, the invention also provides a method of using a beneficial Streptomyces endophyte that confers a trait of agronomic importance to a plant, wherein said endophyte comprises at least 2× higher acetoin production as compared to the strain represented by SEQ ID NO: 1. In an aspect, the invention provides a method of using a beneficial Streptomyces endophyte that confers a trait of agronomic importance to a plant, wherein the endophyte comprises at least 1.5× higher siderophore production as compared to the strain represented by SEQ ID NO: 1. In another aspect, the invention provides a method of using a beneficial Streptomyces endophyte that confers a trait of agronomic importance to a plant, wherein the endophyte comprises greater utilization of a primary carbon source selected from the group consisting of: D-Galactose, Glycerol, alpha-D-Glucose, Sucrose, beta-methyl-D-glucoside, D-cellobiose, L-alanine, L-alanyl-glycine, mono methyl succinate, glycyl-L-proline, L-lyxose, as compared to the strain represented by SEQ ID NO: 1. In an aspect, the invention provides a method of using a beneficial Streptomyces endophyte that confers a trait of agronomic importance to a plant, wherein the endophyte secretes at least one protein listed in Table 5C with at least a 0.4× higher rate, as compared to the strain represented by SEQ ID NO: 2. In an aspect, the invention provides a method of using a beneficial Streptomyces endophyte that confers a trait of agronomic importance to a plant, wherein the endophyte secretes at least one protein listed in Table 5D with at least a 0.7× lower rate, as compared to the strain represented by SEQ ID NO: 2. In an aspect, the invention provides a method of using a beneficial Streptomyces endophyte that confers a trait of agronomic importance to a plant, wherein the endophyte comprises at least 3 arabinose transporter genes in its genome. In another aspect, the invention provides a method of using a beneficial Streptomyces endophyte that confers a trait of agronomic importance to a plant, wherein the endophyte secretes at least one protein selected from Table 5A. In yet another aspect, the invention provides a method of using a beneficial Streptomyces endophyte that confers a trait of agronomic importance to a plant, wherein the endophyte does not secrete a protein selected from the proteins listed in Table 5B.

In a certain aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO: 2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises modulation of transcription of at least one gene involved in a pathway selected from the group consisting of: symbiosis enhancement, resistance to biotic stress, resistance to abiotic stress, growth promotion, cell wall composition, and developmental regulation. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO: 2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises modulation of at least one hormone involved in a pathway selected from the group consisting of: developmental regulation, seed maturation, dormancy, response to environmental stresses, stomatal closure, expression of stress-related genes, drought tolerance, defense responses, infection response, pathogen response, disease resistance, systemic acquired resistance, transcriptional reprogramming, mechanical support, protection against biotic stress, protection against abiotic stress, signaling, nodulation inhibition, endophyte colonization, fatty acid deoxygenation, wound healing, antimicrobial substance production, metabolite catabolism, cell proliferation, and abscission. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO: 2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises modulation of least one metabolite in at least one of the following plant metabolic pathways: alkaloid metabolism, phenylpropanoid metabolism, flavonoid biosynthesis, isoflavonoid biosynthesis, lipid metabolism, nitrogen metabolism, and carbohydrate metabolism. In yet another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO: 2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises modulation of at least one transcript involved in at least one of the following pathways: symbiosis enhancement, resistance to biotic stress, resistance to abiotic stress, growth promotion, cell wall composition, and developmental regulation.

In an embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises upregulation of at least one gene in root tissue, selected from the upregulated genes listed in Table 8A. In an embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises upregulation of at least one gene in leaf tissue, selected from the upregulated genes listed in Table 8A. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises upregulation of at least one gene in stem tissue, selected from the upregulated genes listed in Table 8A. In an embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises downregulation of at least one gene in root tissue, selected from the downregulated genes listed in Table 8A. In an embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises downregulation of at least one gene in leaf tissue, selected from the downregulated genes listed in Table 8A. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises downregulation of at least one gene in stem tissue, selected from the downregulated genes listed in Table 8A. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in hormone level in root tissue, selected from the group consisting of: abscisic acid, salicylic acid, cinnaminic acid, traumatic acid. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in hormone level in root tissue, selected from the group consisting of: jasmonic acid, jasmonic acid-isoleucine, 12-oxo-phytodienoic acid, 10-oxo-11 phytoenoic acid. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in hormone level in stem tissue, selected from the group consisting of: jasmonic acid, jasmonic acid-isoleucine, traumatic acid, 12-oxo-phytodienoic acid. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in hormone level in stem tissue, selected from the group consisting of: abscisic acid, salicylic acid, cinnaminic acid, 10-oxo-11 phytoenoic acid. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in hormone level in leaf tissue, selected from the group consisting of: abscisic acid, salicylic acid, cinnaminic acid, jasmonic acid, jasmonic acid-isoleucine, traumatic acid. In an embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in hormone level in leaf tissue, selected from the group consisting of: 12-oxo-phytodienoic acid, 10-oxo-11 phytoenoic acid. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in metabolite level in root tissue, selected from the group consisting of: pipecolic acid, octadecanoic acid. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in metabolite level in root tissue, selected from the group consisting of: aspartic acid, glutamic acid, histidine, serine, D-glucopyranose, galactose, galacturonic acid. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in metabolite level in stem tissue, selected from the group consisting of: galactose, tryptophan, caffeic acid, daidzein, allantoin, glutamine, isoleucine, leucine, proline, tryptophan, trehalose. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in metabolite level in stem tissue, selected from the group consisting of: ethanolaminephosphate, hexadecanoic acid, asparagine. In an embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in metabolite level in leaf tissue, selected from the group consisting of: ethanolaminephosphate, hexadecanoic acid, asparagine, galactose, tryptophan, daidzein, allantoin, glutamine, isoleucine, leucine, proline, tryptophan, trehalose, histidine, D-glucopyranose, octadecanoic acid, phenylalanine, tyrosine, benzoic acid, nicotinic acid, phenylalanine, shikimic acid, quinic acid, sinapic acid, quinic acid, shikimic acid, hesperetin, ethanolamine, sphingosine, glycerol, octadecadienoic acid, dodecanol, campesterol, alanine, phenylalanine, threonine, tyrosine, valine, lyxose, threose, xylose, salicylic acid, vanillic acid, beta tocopherol. In an embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in metabolite level in leaf tissue, selected from the group consisting of: sucrose, pyrogallol, lumichrome. In another embodiment, the invention provides for a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under normal watering conditions, comprising treating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from reproductive elements treated with said Streptomyces endophyte, wherein the characteristic comprises an enrichment of at least one gene described in Table 15A, 15B, 15C, or 15D.

In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO: 2 through SEQ ID NO: 18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises modulation of transcription of at least one gene involved in at least one of the following pathways: symbiosis enhancement, resistance to biotic stress, resistance to abiotic stress, growth promotion, cell wall composition, and developmental regulation. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises modulation of transcription of at least one transcript involved in at least one of the following pathways: symbiosis enhancement, resistance to biotic stress, resistance to abiotic stress, growth promotion, cell wall composition, and developmental regulation. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises modulation of levels of at least one hormone involved in a pathway selected from the group consisting of: developmental regulation, seed maturation, dormancy, response to environmental stresses, stomatal closure, expression of stress-related genes, drought tolerance, defense responses, infection response, pathogen response, disease resistance, systemic acquired resistance, transcriptional reprogramming, mechanical support, protection against biotic stress, protection against abiotic stress, signaling, nodulation inhibition, endophyte colonization, fatty acid deoxygenation, wound healing, antimicrobial substance production, metabolite catabolism, cell proliferation, and abscission. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises modulation of at least one metabolite in at least one of the following plant metabolic pathways: alkaloid metabolism, phenylpropanoid metabolism, flavonoid biosynthesis, isoflavonoid biosynthesis, lipid metabolism, nitrogen metabolism, and carbohydrate metabolism. In yet another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises modulation of microbiome community profile.

In a certain aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises upregulation of at least one gene in root tissue, selected from the upregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. In certain other aspects, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO: 2 through SEQ ID NO: 18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises upregulation of at least one gene in leaf tissue, selected from the upregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises upregulation of at least one gene in stem tissue, selected from the upregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises downregulation of at least one gene in root tissue, selected from the downregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises downregulation of at least one gene in leaf tissue, selected from the downregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises downregulation of at least one gene in stem tissue, selected from the downregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises expression of at least one sugar transporter gene selected from Table 9. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises upregulation of at least one transcript in root tissue, selected from the upregulated transcripts listed in Table 8F. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises upregulation of at least one transcript in leaf tissue, selected from the upregulated transcripts listed in Table 8F. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises upregulation of at least one transcript in stem tissue, selected from the upregulated transcripts listed in Table 8F. In another aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises downregulation of at least one transcript in root tissue, selected from the downregulated transcripts listed in Table 8F. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises downregulation of at least one transcript in leaf tissue, selected from the downregulated transcripts listed in Table 8F. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises downregulation of at least one transcript in stem tissue, selected from the downregulated transcripts listed in Table 8F. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises upregulation of a sugar transporter transcript in leaf tissue or root tissue. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in hormone level in root tissue, selected from the group consisting of: abscisic acid, salicylic acid, cinnaminic acid jasmonic acid, jasmonic acid-isoleucine, traumatic acid, 12-oxo-phytodienoic acid, 10-oxo-11 phytoenoic acid. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in hormone level in stem tissue, selected from the group consisting of: 12-oxo-phytodienoic acid, 10-oxo-11 phytoenoic acid. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in hormone level in stem tissue, selected from the group consisting of: abscisic acid, salicylic acid, cinnaminic acid jasmonic acid, jasmonic acid-isoleucine, traumatic acid. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in hormone level in leaf tissue, selected from the group consisting of: salicylic acid, cinnaminic acid, 12-oxo-phytodienoic acid, 10-oxo-11 phytoenoic acid. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in hormone level in leaf tissue, selected from the group consisting of: abscisic acid, jasmonic acid, jasmonic acid-isoleucine, traumatic acid. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in metabolite level in root tissue, selected from the group consisting of: pipecolic acid, hexadecanoic acid, octadecanoic acid. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in metabolite level in root tissue, selected from the group consisting of: tryptophan, tyrosine, benzoic acid, nicotinic acid, tyrosine, quinic acid, sinapic acid, ferulic acid, caffeic acid, quinic acid, daidzein, dodecanol, alanine, allantoin, asparagine, aspartic acid, glutamic acid, glutamine, histidine, leucine, methionine, proline, threonine, tryptophan, tyrosine, valine, D-glucopyranose, salicylic acid, pyrogallol, beta tocopherol, galacturonic acid. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in metabolite level in stem tissue, selected from the group consisting of: tryptophan, ferulic acid, allantoin, glutamine, histidine, leucine, tryptophan, valine, D-glucopyranose, salicylic acid, hexadecanoic acid, octadecanoic acid, hesperetin, ethanolamine, glycerol, vanillic acid. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in metabolite level in stem tissue, selected from the group consisting of: sphingosine. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in metabolite level in leaf tissue, selected from the group consisting of: lumichrome. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises a decrease in metabolite level in leaf tissue, selected from the group consisting of: sphingosine, tryptophan, ferulic acid, allantoin, glutamine, histidine, leucine, tryptophan, valine, salicylic acid, octadecanoic acid, hesperetin, ethanolamine, vanillic acid, tyrosine, benzoic acid, nicotinic acid, tyrosine, quinic acid, sinapic acid, caffeic acid, quinic acid, daidzein, dodecanol, alanine, glutamic acid, methionine, proline, threonine, tyrosine, phenylalanine, tryptamine, phenylalanine, shikimic acid, shikimic acid, ethanolaminephosphate, octadecadienoic acid, campesterol, β-alanine, isoleucine, phenylalanine, serine, galactose, lyxose, threose, trehalose, gallic acid. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises a reduced abundance of organisms of the Escherica - Shigella genera in the plant's leaf microbiome community. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in abundance of organisms of the Rhizophagus genera in the plant's root microbiome community. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in abundance of organisms of the Glomus genera in the plant's root microbiome community. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises a reduced abundance of organisms of the Enterobacteriaceae family in the plant's leaf microbiome community. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in abundance of organisms of the Nectriaceae family in the plant's root microbiome community. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in abundance of organisms of the Glomeraceae family in the plant's root microbiome community. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises the presence of at least one OTU described in Table 13A or Table 13B. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an increase in the presence of at least one OTU selected from Table 13C. In an aspect, the invention provides a method of modulating a trait of agronomic importance in a plant derived from a plant reproductive element under water-limited conditions, comprising associating said plant reproductive element with a formulation comprising a Streptomyces endophyte that is heterologous to the plant reproductive element and comprises a 16S nucleic acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical over at least 600 nucleotides to a nucleic acid sequence selected from SEQ ID NO:2 through SEQ ID NO:18, and modulating at least one characteristic of said plant as compared to an isoline plant not grown from a reproductive element treated with said Streptomyces endophyte, wherein the characteristic comprises an enrichment of at least gene described in Table 15A, 15B, 15C, or 15D.

In certain embodiments, the invention provides for methods of altering the native microbiome community of a plant, comprising deriving the plant from a plant reproductive element treated with a formulation comprising a beneficial Streptomyces endophyte. In an aspect, the invention provides for methods of altering the native microbiome community of a plant, comprising deriving said plant from a plant reproductive element treated with a formulation comprising a beneficial Streptomyces endophyte, wherein the microbiome community alteration comprises a reduction in abundance of organisms of the Escherica - Shigella genera in the plant's leaf microbiome community. In an aspect, the invention provides for methods of altering the native microbiome community of a plant, comprising deriving said plant from a plant reproductive element treated with a formulation comprising a beneficial Streptomyces endophyte, wherein the microbiome community alteration comprises an increase in abundance of organisms of the Rhizophagus genera in the plant's root microbiome community. In an aspect, the invention provides for methods of altering the native microbiome community of a plant, comprising deriving said plant from a plant reproductive element treated with a formulation comprising a beneficial Streptomyces endophyte, wherein the microbiome community alteration comprises an increase in abundance of organisms of the Glomus genera in the plant's root microbiome community. In an aspect, the invention provides for methods of altering the native microbiome community of a plant, comprising deriving said plant from a plant reproductive element treated with a formulation comprising a beneficial Streptomyces endophyte, wherein the microbiome community alteration comprises a reduction in abundance of organisms of the Enterobacteriaceae family in the plant's leaf microbiome community. In an aspect, the invention provides for methods of altering the native microbiome community of a plant, comprising deriving said plant from a plant reproductive element treated with a formulation comprising a beneficial Streptomyces endophyte, wherein the microbiome community alteration comprises an increase in abundance of organisms of the Nectriaceae family in the plant's root microbiome community. In an aspect, the invention provides for methods of altering the native microbiome community of a plant, comprising deriving said plant from a plant reproductive element treated with a formulation comprising a beneficial Streptomyces endophyte, wherein the microbiome community alteration comprises an increase in abundance of organisms of the Glomeraceae family in the plant's root microbiome community. In an aspect, the invention provides for methods of altering the native microbiome community of a plant, comprising deriving said plant from a plant reproductive element treated with a formulation comprising a beneficial Streptomyces endophyte, wherein the microbiome community alteration comprises a presence of at least one OTU described in Table 13A or Table 13B. In an aspect, the invention provides for methods of altering the native microbiome community of a plant, comprising deriving said plant from a plant reproductive element treated with a formulation comprising a beneficial Streptomyces endophyte, wherein the microbiome community alteration comprises an increase in presence of at least one OTU selected from Table 13C.

The invention also provides any of the preceding methods; wherein the plant is optionally soybean or maize; wherein the formulation of any of the preceding methods optionally comprises a purified population of the Streptomyces endophyte at a concentration of at least about 10 {circumflex over ( )}2 CFU/ml in a liquid formulation or about 10 {circumflex over ( )}2 CFU/gm in a non-liquid formulation; wherein the Streptomyces endophyte is optionally capable auxin production, nitrogen fixation, production of an antimicrobial compound, mineral phosphate solubilization, siderophore production, cellulase production, chitinase production, xylanase production, or acetoin production; wherein the trait of agronomic importance is optionally selected from the group consisting of: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, increase in yield, increase in yield under water-limited conditions, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increase in biomass, increase in shoot length, increase in root length, improved root architecture, increase in seed weight, altered seed carbohydrate composition, altered seed oil composition, increase in radical length, number of pods, delayed senescence, stay-green, altered seed protein composition, increase in dry weight of mature plant reproductive elements, increase in fresh weight of mature plant reproductive elements, increase in number of mature plant reproductive elements per plant, increase in chlorophyll content, increase in number of pods per plant, increase in length of pods per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, increase in number of non-wilted leaves per plant, improved plant visual appearance; wherein the Streptomyces endophyte is optionally capable of localizing in a plant element of said plant, said plant element selected from the group consisting of: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, keikis, and bud; wherein the plant reproductive element is optionally a seed or a transgenic seed; wherein the plant reproductive element is optionally placed into a substrate that promotes plant growth and/or wherein the substrate promotes plant growth in soil; wherein the formulation optionally further comprises one or more of the following: a stabilizer, or a preservative, or a carrier, or a surfactant, or an anticomplex agent, or any combination thereof; and wherein the formulation optionally further comprises at least one additional bacterial endophyte.

Certain embodiments of the invention are any of the preceding methods; wherein the Streptomyces endophyte is optionally capable of localizing in a plant element of the plant, the plant element selected from the group consisting of: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, keikis, and bud; and wherein the plant element is optionally a seed; wherein the Streptomyces endophyte is optionally present in at least two compartments of the seed, selected from the group consisting of: embryo, seed coat, endosperm, cotyledon, hypocotyl, and radicle.

In certain aspects, the invention provides a synthetic composition comprising a plant reproductive element treated with a formulation comprising a purified Streptomyces endophyte population, wherein said Streptomyces endophyte is heterologous to the plant reproductive element, and comprises at least 600 nucleotides at least 95% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NO:2 through SEQ ID NO:18, wherein the endophyte is present in the synthetic combination in an amount capable of modulating at least one of: a trait of agronomic importance, the expression of a gene, the level of a transcript, the expression of a protein, the level of a hormone, the level of a metabolite, the population of endogenous microbes in plants grown from said plant reproductive element, as compared to an isoline plant grown from a plant reproductive element not contacted with the bacterial endophyte. In certain other aspects, the invention provides a synthetic composition comprising a plant reproductive element treated with a formulation comprising a purified Streptomyces endophyte population, wherein said Streptomyces endophyte is heterologous to the plant reproductive element, and comprises a strain deposit IDAC Deposit ID 081111-06, wherein the endophyte is present in the synthetic combination in an amount capable of modulating at least one of: a trait of agronomic importance, the expression of a gene, the level of a transcript, the expression of a protein, the level of a hormone, the level of a metabolite, the population of endogenous microbes in plants grown from said plant reproductive element, as compared to an isoline plant grown from a plant reproductive element not contacted with the bacterial endophyte. In certain aspects, the invention provides a synthetic composition comprising a plant reproductive element treated with a formulation comprising a purified Streptomyces endophyte population, wherein said Streptomyces endophyte is heterologous to the plant reproductive element, and comprises a Streptomyces species selected from the group consisting of: albidoflavus, albus, aureofaciens, ginsengisoli, griseus, lydicus, mutabilis, neyagawaensis, praecox , and SMCD2215, wherein the endophyte is present in the synthetic combination in an amount capable of modulating at least one of: a trait of agronomic importance, the expression of a gene, the level of a transcript, the expression of a protein, the level of a hormone, the level of a metabolite, the population of endogenous microbes in plants grown from said plant reproductive element, as compared to an isoline plant grown from a plant reproductive element not contacted with the bacterial endophyte.

The invention also provides the synthetic composition of any of the preceding claims, wherein the plant is optionally soybean or maize; wherein the formulation optionally comprises a purified population of the Streptomyces endophyte at a concentration of at least about 10 {circumflex over ( )}2 CFU/ml in a liquid formulation or about 10 {circumflex over ( )}2 CFU/gm in a non-liquid formulation; wherein the Streptomyces endophyte is optionally capable of auxin production, nitrogen fixation, production of an antimicrobial compound, mineral phosphate solubilization, siderophore production, cellulase production, chitinase production, xylanase production, or acetoin production; wherein the trait of agronomic importance is selected from the group consisting of: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, increase in yield, increase in yield under water-limited conditions, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increase in biomass, increase in shoot length, increase in root length, improved root architecture, increase in seed weight, altered seed carbohydrate composition, altered seed oil composition, increase in radical length, number of pods, delayed senescence, stay-green, altered seed protein composition, increase in dry weight of mature plant reproductive elements, increase in fresh weight of mature plant reproductive elements, increase in number of mature plant reproductive elements per plant, increase in chlorophyll content, increase in number of pods per plant, increase in length of pods per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, increase in number of non-wilted leaves per plant, improved plant visual appearance; wherein the Streptomyces endophyte is optionally capable of localizing in a plant element of a plant grown from said seed, said plant element selected from the group consisting of: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, keikis, and bud; wherein the plant reproductive element is optionally a seed or a transgenic seed; wherein the plant reproductive element is optionally placed into a substrate that promotes plant growth; wherein the substrate that promotes plant growth is optionally soil; and wherein the substrate that promotes plant growth is soil and wherein a plurality of the plant reproductive elements are optionally placed in the soil in rows, with substantially equal spacing between each seed within each row.

The invention also provides the synthetic composition of any of the preceding claims, wherein the formulation optionally further comprises one or more of the following: a stabilizer, or a preservative, or a carrier, or a surfactant, or an anticomplex agent, or any combination thereof; wherein the formulation optionally further comprises one or more of the following: fungicide, nematicide, bactericide, insecticide, and herbicide; and wherein the formulation optionally further comprises at least one additional bacterial endophyte; wherein the plant reproductive element is optionally a seed or a transgenic seed.

An embodiment of the invention is a plant derived from the synthetic composition of any of the preceding claims, wherein the plant optionally comprises in at least one of its plant elements the bacterial endophyte; and wherein the progeny of the plant optionally comprises in at least one of its plant elements bacterial endophyte.

Another embodiment of the invention is a plurality of synthetic compositions of any of the preceding claims, wherein the compositions are optionally confined within an object selected from the group consisting of: bottle, jar, ampule, package, vessel, bag, box, bin, envelope, carton, container, silo, shipping container, truck bed, and case.

In certain other aspects, the invention provides the synthetic compositions of any of the preceding claims, wherein the Streptomyces endophyte is optionally present in the plant reproductive element in an amount capable of providing a benefit to the plant reproductive element or to a plant derived therefrom;

wherein the bacterial endophyte is optionally present in at least two compartments of the seed, selected from the group consisting of: embryo, seed coat, endosperm, cotyledon, hypocotyl, and radicle.

Included with the invention, is a plurality of synthetic combinations of any of the synthetic compositions of the preceding claims, wherein the synthetic combinations are shelf-stable.

An aspect of the invention provides a plant grown from the synthetic combination of any of the synthetic compositions of the preceding claims, wherein the plant comprises modulation of the transcription of at least one gene involved in at least one of the following pathways: symbiosis enhancement, resistance to biotic stress, resistance to abiotic stress, growth promotion, cell wall composition, and developmental regulation. Another aspect of the invention provides a plant grown from the synthetic combination of any of the synthetic compositions of the preceding claims, wherein the plant comprises modulation of the transcription of at least one transcript involved in at least one of the following pathways: symbiosis enhancement, resistance to biotic stress, resistance to abiotic stress, growth promotion, cell wall composition, and developmental regulation. Another aspect of the invention provides a plant grown from the synthetic combination of any of the synthetic compositions of the preceding claims, wherein the plant comprises modulating the level of at least one hormone involved in a pathway selected from the group consisting of: developmental regulation, seed maturation, dormancy, response to environmental stresses, stomatal closure, expression of stress-related genes, drought tolerance, defense responses, infection response, pathogen response, disease resistance, systemic acquired resistance, transcriptional reprogramming, mechanical support, protection against biotic stress, protection against abiotic stress, signaling, nodulation inhibition, endophyte colonization, fatty acid deoxygenation, wound healing, antimicrobial substance production, metabolite catabolism, cell proliferation, and abscission. Yet another aspect of the invention provides a plant grown from the synthetic combination of any of the synthetic compositions of the preceding claims, wherein the plant comprises modulating at least one metabolite in at least one of the following plant metabolic pathways: alkaloid metabolism, phenylpropanoid metabolism, flavonoid biosynthesis, isoflavonoid biosynthesis, lipid metabolism, nitrogen metabolism, and carbohydrate metabolism.

An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises at most 18% total microbes from the Escherica - Shigella genera in the total microbiome of the plant's root microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises at least 5% total microbes from the Glomus genera in the total microbiome of the plant's root microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises at least 8% total microbes from the Rhixophagus genera in the total microbiome of the plant's root microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises at most 18% total microbes from the Enterobacteriaceae family of the total microbiome in the plant's leaf microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises at least 25% total microbes from the Nectriaceae family in the total microbiome of the plant's root microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises at least 5% total microbes from the Glomeraceae family in the total microbiome of the plant's root microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises a bacterial or fungal OTU selected from Table 13A. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises a bacterial or fungal OTU selected from Table 13B. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises upregulation of at least one gene in root tissue, selected from the upregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises upregulation of at least one gene in leaf tissue, selected from the upregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises upregulation of at least one gene in stem tissue, selected from the upregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises downregulation of at least one gene in root tissue, selected from the downregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises downregulation of at least one gene in leaf tissue, selected from the downregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises downregulation of at least one gene in stem tissue, selected from the downregulated genes listed in Tables 8A, 8B, 8C, 8D, and 8E. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises expression of at least one sugar transporter gene selected from Table 9. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises upregulation of at least one transcript in root tissue, selected from the upregulated transcripts listed in Table 8F. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises upregulation of at least one transcript in leaf tissue, selected from the upregulated transcripts listed in Table 8F. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises upregulation of at least one transcript in stem tissue, selected from the upregulated transcripts listed in Table 8F. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises downregulation of at least one transcript in root tissue, selected from the downregulated transcripts listed in Table 8F. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises downregulation of at least one transcript in leaf tissue, selected from the downregulated transcripts listed in Table 8F. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises downregulation of at least one transcript in stem tissue, selected from the downregulated transcripts listed in Table 8F. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises upregulation of a sugar transporter transcript in leaf tissue or root tissue. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises decrease in hormone level in root tissue, selected from the group consisting of: abscisic acid, salicylic acid, cinnaminic acid jasmonic acid, jasmonic acid-isoleucine, traumatic acid, 12-oxo-phytodienoic acid, 10-oxo-11 phytoenoic acid. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an increase in hormone level in stem tissue, selected from the group consisting of: 12-oxo-phytodienoic acid, 10-oxo-11 phytoenoic acid. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises a decrease in hormone level in stem tissue, selected from the group consisting of: abscisic acid, salicylic acid, cinnaminic acid jasmonic acid, jasmonic acid-isoleucine, traumatic acid. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an increase in hormone level in leaf tissue, selected from the group consisting of: salicylic acid, cinnaminic acid, 12-oxo-phytodienoic acid, 10-oxo-11 phytoenoic acid. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises a decrease in hormone level in leaf tissue, selected from the group consisting of: abscisic acid, jasmonic acid, jasmonic acid-isoleucine, traumatic acid. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an increase in metabolite level in root tissue, selected from the group consisting of: pipecolic acid, hexadecanoic acid, octadecanoic acid. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises a decrease in metabolite level in root tissue, selected from the group consisting of: tryptophan, tyrosine, benzoic acid, nicotinic acid, tyrosine, quinic acid, sinapic acid, ferulic acid, caffeic acid, quinic acid, daidzein, dodecanol, alanine, allantoin, asparagine, aspartic acid, glutamic acid, glutamine, histidine, leucine, methionine, proline, threonine, tryptophan, tyrosine, valine, D-glucopyranose, salicylic acid, pyrogallol, beta tocopherol, galacturonic acid. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an increase in metabolite level in stem tissue, selected from the group consisting of: tryptophan, ferulic acid, allantoin, glutamine, histidine, leucine, tryptophan, valine, D-glucopyranose, salicylic acid, hexadecanoic acid, octadecanoic acid, hesperetin, ethanolamine, glycerol, vanillic acid. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises a decrease in metabolite level in stem tissue, selected from the group consisting of: sphingosine. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an increase in metabolite level in leaf tissue, selected from the group consisting of: lumichrome. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises a decrease in metabolite level in leaf tissue, selected from the group consisting of: sphingosine, tryptophan, ferulic acid, allantoin, glutamine, histidine, leucine, tryptophan, valine, salicylic acid, octadecanoic acid, hesperetin, ethanolamine, vanillic acid, tyrosine, benzoic acid, nicotinic acid, tyrosine, quinic acid, sinapic acid, caffeic acid, quinic acid, daidzein, dodecanol, alanine, glutamic acid, methionine, proline, threonine, tyrosine, phenylalanine, tryptamine, phenylalanine, shikimic acid, shikimic acid, ethanolaminephosphate, octadecadienoic acid, campesterol, β-alanine, isoleucine, phenylalanine, serine, galactose, lyxose, threose, trehalose, gallic acid. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises a reduced abundance of organisms of the Escherica - Shigella genera in the plant's leaf microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an increase in abundance of organisms of the Rhizophagus genera in the plant's root microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an increase in abundance of organisms of the Glomus genera in the plant's root microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises a reduced abundance of organisms of the Enterobacteriaceae family in the plant's leaf microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an increase in abundance of organisms of the Nectriaceae family in the plant's root microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an increase in abundance of organisms of the Glomeraceae family in the plant's root microbiome community. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises the presence of at least one OTU described in Table 13A or Table 13B. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an increase in presence of at least one OTU selected from Table 13C. An embodiment of the invention is a plant grown from the synthetic combination of any of the preceding synthetic compositions, wherein the plant comprises an enrichment of at least one gene described in Table 15A, 15B, 15C, or 15D.

Included with the invention is a plant grown from any of the preceding synthetic combinations, wherein the plant optionally exhibits a trait of agronomic interest, selected from the group consisting of: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, increase in yield, increase in yield under water-limited conditions, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increase in biomass, increase in shoot length, increase in root length, improved root architecture, increase in seed weight, altered seed carbohydrate composition, altered seed oil composition, increase in radical length, number of pods, delayed senescence, stay-green, altered seed protein composition, increase in dry weight of mature plant reproductive elements, increase in fresh weight of mature plant reproductive elements, increase in number of mature plant reproductive elements per plant, increase in chlorophyll content, increase in number of pods per plant, increase in length of pods per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, increase in number of non-wilted leaves per plant, improved plant visual appearance; wherein the plant is optionally soybean or maize; and wherein the plant or progeny of the plant, optionally comprises in at least one of its plant elements the Streptomyces endophyte.

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying figure, where:

FIG. 1 : Culture plate of Strain A

FIG. 2 : Culture plate of Strain B

FIG. 3 : Culture plate of Strain C

FIG. 4 : Greenhouse phenotypes of plants grown from seeds treated with Strain C, under normal watering conditions. Soybean plants grown from Strain C-treated seeds confer improved phenotypes, including a 3.2% increase in seed count, a 0.8% increase in fresh seed weight, a 1.0% increase in dry seed weight, a 9.5% increase in pod conts (>1 cm), and a 51.4% reduction in small pods (<1 cm) count.

FIG. 5 : Greenhouse phenotypes of plants grown from seeds treated with Strain C, under water-limited conditions. Soybean plants grown from Strain C-treated seeds exhibit increased tolerance to extreme drought (1st drought cycle: 18 days post watering). NT=plant grown from non-Strain C treated/non-formulation treated seed, Formulation Control=plant grown from seed treated with formulation without Strain C, Strain C treated plant=treated with Strain C microbial endophyte in formulation.

FIG. 6 : Greenhouse phenotypes of plants grown from seeds treated with different Streptomyces strains, under water-limited conditions. Soybean plants grown from seeds treated with any Streptomyces strain demonstrate improved visual phenotype tolerance to water-limited growth conditions, and those treated with Strain C exhibit the most improvement.

FIG. 7 : Community sequencing graphs showing average abundance of bacterial genera, as a proportion of the community, in leaf tissue of water stressed soybean plants grown from seeds treated with Strain B (left), Strain C (middle), and untreated controls (right). The average abundance of organisms in the Eschericia - Shigella genera are reduced from approximately 21% of the bacterial community of untreated soybean leaves to approximately 13% of the bacterial community in Strain C treated soybean leaves and 16% of the bacterial community in Strain B treated leaves. Treatment with Strain C reduces the abundance of bacteria in the Eschericia - Shigella genera on soybean leaves by 37% relative to untreated controls.

FIG. 8 : Community sequencing graphs showing the average abundance of fungal genera, as a proportion of the community, in root tissue of water stressed soybean plants grown from seeds treated with Strain B (left), Strain C (middle), and untreated controls (right). The average abundance of fungi in the Rhizophagus genera are increased from approximately 4.7% of the fungal community of untreated soybean roots and 4.3% of the fungal community in Strain B treated soybean roots to approximately 8.9% of the fungal community of Strain C treated soybean roots. Treatment with Strain C resulted in a 87.7% increase in the abundance of fungi in the Rhizophagus genera in soybean roots relative to untreated controls. Fungi of the genus Glomus are also increased in the roots of soybeans treated with Strain C and Strain B treatments relative to untreated controls.

FIG. 9 : Community sequencing graphs showing the average abundance of bacterial families, as a proportion of the community, in leaf tissue of water stressed soybean plants grown from seeds treated with Strain B (left), Strain C (middle), and untreated controls (right).

FIG. 10 : Community sequencing graphs showing the average abundance of bacterial families, as a proportion of the community, in root tissue of water stressed soybean plants grown from seeds treated with Strain B (left), Strain C (middle), and untreated controls (right).

Terms used in the claims and specification are defined as set forth below unless otherwise specified.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

An “endophyte” is an organism capable of living within a plant or otherwise associated therewith, and does not cause disease or harm the plant otherwise. Endophytes can occupy the intracellular or extracellular spaces of plant tissue, including the leaves, stems, flowers, fruits, seeds, or roots. An endophyte can be for example a bacterial or fungal organism, and can confer a beneficial property to the host plant such as an increase in yield, biomass, resistance, or fitness. An endophyte can be a fungus or a bacterium.

A “plurality of endophytes” means two or more types of endophyte entities, e.g., of simple bacteria or simple fungi, complex fungi, or combinations thereof. In some embodiments, the two or more types of endophyte entities are two or more strains of endophytes. In other embodiments, the two or more types of endophyte entities are two or more species of endophytes. In yet other embodiments, the two or more types of endophyte entities are two or more genera of endophytes. In yet other embodiments, the two or more types of endophyte entities are two or more families of endophytes. In yet other embodiments, the two or more types of endophyte entities are two or more orders of endophytes.

As used herein, the term “microbe” or “microorganism” refers to any species or taxon of microorganism, including, but not limited to, archaea, bacteria, microalgae, fungi (including mold and yeast species), mycoplasmas, microspores, nanobacteria, oomycetes, and protozoa. In some embodiments, a microbe or microorganism is an endophyte. In some embodiments, a microbe is an endophyte, for example a bacterial or fungal endophyte, which is capable of living within a plant. In some embodiments, a microbe or microorganism encompasses individual cells (e.g., unicellular microorganisms) or more than one cell (e.g., multi-cellular microorganism). A “population of microorganisms” may thus refer to a multiple cells of a single microorganism, in which the cells share common genetic derivation.

As used herein, the term “bacterium” or “bacteria” refers in general to any prokaryotic organism, and may reference an organism from either Kingdom Eubacteria (Bacteria), Kingdom Archaebacteria (Archae), or both. In some cases, bacterial genera or other taxonomic classifications have been reassigned due to various reasons (such as but not limited to the evolving field of whole genome sequencing), and it is understood that such nomenclature reassignments are within the scope of any claimed taxonomy. For example, certain species of the genus Erwinia have been described in the literature as belonging to genus Pantoea (Zhang and Qiu, 2015).

The term 16S refers to the DNA sequence of the 16S ribosomal RNA (rRNA) sequence of a bacterium. 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of bacteria.

As used herein, the term “fungus” or “fungi” refers in general to any organism from Kingdom Fungi. Historical taxonomic classification of fungi has been according to morphological presentation. Beginning in the mid-1800's, it was became recognized that some fungi have a pleomorphic life cycle, and that different nomenclature designations were being used for different forms of the same fungus. In 1981, the Sydney Congress of the International Mycological Association laid out rules for the naming of fungi according to their status as anamorph, teleomorph, or holomorph (Taylor, 2011). With the development of genomic sequencing, it became evident that taxonomic classification based on molecular phylogenetics did not align with morphological-based nomenclature (Shenoy, 2007). As a result, in 2011 the International Botanical Congress adopted a resolution approving the International Code of Nomenclature for Algae, Fungi, and Plants (Melbourne Code) (2012), with the stated outcome of designating “One Fungus=One Name” (Hawksworth, 2012). However, systematics experts have not aligned on common nomenclature for all fungi, nor are all existing databases and information resources inclusive of updated taxonomies. As such, many fungi referenced herein may be described by their anamorph form but it is understood that based on identical genomic sequencing, any pleomorphic state of that fungus may be considered to be the same organism. For example, the genus Alternaria is the anamorph form of the teleomorph genus Lewia (Kwasna 2003), ergo both would be understood to be the same organism with the same DNA sequence. For example, it is understood that the genus Acremonium is also reported in the literature as genus Sarocladium as well as genus Tilachilidium (Summerbell, 2011). For example, the genus Cladosporium is an anamorph of the teleomorph genus Davidiella (Bensch, 2012), and is understood to describe the same organism. In some cases, fungal genera have been reassigned due to various reasons, and it is understood that such nomenclature reassignments are within the scope of any claimed genus. For example, certain species of the genus Microdiplodia have been described in the literature as belonging to genus Paraconiothyrium (Crous and Groenveld, 2006).

“Internal Transcribed Spacer” (ITS) refers to the spacer DNA (non-coding DNA) situated between the small-subunit ribosomal RNA (rRNA) and large-subunit (LSU) rRNA genes in the chromosome or the corresponding transcribed region in the polycistronic rRNA precursor transcript. ITS gene sequencing is a well-established method for studying phylogeny and taxonomy of fungi. In some cases, the “Large SubUnit” (LSU) sequence is used to identify fungi. LSU gene sequencing is a well-established method for studying phylogeny and taxonomy of fungi. Some fungal endophytes of the present invention may be described by an ITS sequence and some may be described by an LSU sequence. Both are understood to be equally descriptive and accurate for determining taxonomy.

As used herein with respect to fungi and bacteria, the term “marker gene” refers to an organism's 16S (for bacteria) or ITS (for fungi) polynucleotide sequence, by which a microbe may be specifically identified and assigned taxonomic nomenclature.

The terms “pathogen” and “pathogenic” in reference to a bacterium or fungus includes any such organism that is capable of causing or affecting a disease, disorder or condition of a host comprising the organism.

A “spore” or a population of “spores” refers to bacteria or fungi that are generally viable, more resistant to environmental influences such as heat and bactericidal or fungicidal agents than other forms of the same bacteria or fungi, and typically capable of germination and out-growth. Bacteria and fungi that are “capable of forming spores” are those bacteria and fungi comprising the genes and other necessary abilities to produce spores under suitable environmental conditions.

“Biomass” means the total mass or weight (fresh or dry), at a given time, of a plant tissue, plant tissues, an entire plant, or population of plants. Biomass is usually given as weight per unit area. The term may also refer to all the plants or species in the community (community biomass).

The term “isolated” is intended to specifically reference an organism, cell, tissue, polynucleotide, or polypeptide that is removed from its original source and purified from additional components with which it was originally associated. For example, an endophyte may be considered isolated from a seed if it is removed from that seed source and purified so that it is isolated from any additional components with which it was originally associated. Similarly, an endophyte may be removed and purified from a plant or plant element so that it is isolated and no longer associated with its source plant or plant element.

As used herein, an isolated strain of a microbe is a strain that has been removed from its natural milieu. “Pure cultures” or “isolated cultures” are cultures in which the organisms present are only of one strain of a particular genus and species. This is in contrast to “mixed cultures,” which are cultures in which more than one genus and/or species of microorganism are present. As such, the term “isolated” does not necessarily reflect the extent to which the microbe has been purified. A “substantially pure culture” of the strain of microbe refers to a culture which contains substantially no other microbes than the desired strain or strains of microbe. In other words, a substantially pure culture of a strain of microbe is substantially free of other contaminants, which can include microbial contaminants. Further, as used herein, a “biologically pure” strain is intended to mean the strain separated from materials with which it is normally associated in nature. A strain associated with other strains, or with compounds or materials that it is not normally found with in nature, is still defined as “biologically pure.” A monoculture of a particular strain is, of course, “biologically pure.” As used herein, the term “enriched culture” of an isolated microbial strain refers to a microbial culture that contains more that 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain.

A “host plant” includes any plant, particularly a plant of agronomic importance, which an endophytic entity such as an endophyte can colonize. As used herein, an endophyte is said to “colonize” a plant or plant element when it can be stably detected within the plant or plant element over a period time, such as one or more days, weeks, months or years, in other words, a colonizing entity is not transiently associated with the plant or plant element. Such host plants are preferably plants of agronomic importance.

A “non-host target” means an organism or chemical compound that is altered in some way after contacting a host plant that comprises an endophyte, as a result of a property conferred to the host plant by the endophyte.

As used herein, a nucleic acid has “homology” or is “homologous” to a second nucleic acid if the nucleic acid sequence has a similar sequence to the second nucleic acid sequence. The terms “identity,” “percent sequence identity” or “identical” in the context of nucleic acid sequences refer to the residues in the two sequences that are the same when aligned for maximum correspondence. There are a number of different algorithms known in the art that can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990). In some embodiments, sequences can be compared using Geneious (Biomatters, Ltd., Auckland, New Zealand). In other embodiments, polynucleotide sequences can be compared using the multiple sequence alignment algorithm MUSCLE (Edgar R C, 2004).

The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 76%, 80%, 85%, or at least about 90%, or at least about 95%, 96%, 97%, 98% 99%, 99.5% or 100% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, Gap, MUSCLE, or any other method known in the art.

As used herein, the terms “operational taxonomic unit,” “OTU,” “taxon,” “hierarchical cluster,” and “cluster” are used interchangeably. An operational taxon unit (OTU) refers to a group of one or more organisms that comprises a node in a clustering tree. The level of a cluster is determined by its hierarchical order. In one embodiment, an OTU is a group tentatively assumed to be a valid taxon for purposes of phylogenetic analysis. In another embodiment, an OTU is any of the extant taxonomic units under study. In yet another embodiment, an OTU is given a name and a rank. For example, an OTU can represent a domain, a sub-domain, a kingdom, a sub-kingdom, a phylum, a sub-phylum, a class, a subclass, an order, a sub-order, a family, a subfamily, a genus, a subgenus, or a species. In some embodiments, OTUs can represent one or more organisms from the kingdoms eubacteria, protista, or fungi at any level of a hierarchal order. In some embodiments, an OTU represents a prokaryotic or fungal order.

In some embodiments, the present invention contemplates the synthetic compositions comprising the combination of a plant element, seedling, or whole plants and an endophyte population, in which the endophyte population is “heterologously disposed.” In some embodiments, “heterologously disposed” means that the native plant element, seedling, or plant does not contain detectable levels of the microbe in that same plant element, seedling, or plant. For example if said plant element or seedling or plant does not naturally have the endophyte associated with it and the endophyte is applied, the endophyte would be considered to be heterologously disposed. In some embodiments, “heterologously disposed” means that the endophyte is being applied to a different plant element than that with which the endophyte is naturally associated. For example, if said plant element or seedling or plant has the endophyte normally found in the root tissue but not in the leaf tissue, and the endophyte is applied to the leaf, the endophyte would be considered to be heterologously disposed. In some embodiments, “heterologously disposed” means that the endophyte being applied to a different tissue or cell layer of the plant element than that in which the microbe is naturally found. For example, if endophyte is naturally found in the mesophyll layer of leaf tissue but is being applied to the epithelial layer, the endophyte would be considered to be heterologously disposed. In some embodiments, “heterologously disposed” means that the endophyte being applied is at a greater concentration, number, or amount of the plant element, seedling, or plant, than that which is naturally found in said plant element, seedling, or plant. For example, an endophyte concentration that is being applied is at least 1.5 times greater, between 1.5 and 2 times greater, 2 times greater, between 2 and 3 times greater, 3 times greater, between 3 and 5 times greater, 5 times greater, between 5 and 7 times greater, 7 times greater, between 7 and 10 times greater, 10 times greater, or even greater than 10 times higher number, amount, or concentration than that which is naturally present, the endophyte would be considered to be heterologously disposed. In some embodiments, “heterologously disposed” means that the endophyte is applied to a developmental stage of the plant element, seedling, or plant in which said endophyte is not naturally associated, but may be associated at other stages. For example, if an endophyte is normally found at the flowering stage of a plant and no other stage, an endophyte applied at the seedling stage may be considered to be heterologously disposed. For example, an endophyte that is normally associated with leaf tissue of a cupressaceous tree sample would be considered heterologous to leaf tissue of a maize plant. In another example, an endophyte that is normally associated with leaf tissue of a maize plant is considered heterologous to a leaf tissue of another maize plant that naturally lacks said endophyte. In another example, an endophyte that is normally associated at low levels in a plant is considered heterologous to that plant if a higher concentration of that endophyte is introduced into the plant. In yet another example, an endophyte that is associated with a tropical grass species would be considered heterologous to a wheat plant.

The term “isoline” is a comparative term, and references organisms that are genetically identical, but may differ in treatment. In one example, two genetically identical maize plant embryos may be separated into two different groups, one receiving a treatment (such as transformation with a heterologous polynucleotide, to create a genetically modified plant) and one control that does not receive such treatment. Any phenotypic differences between the two groups may thus be attributed solely to the treatment and not to any inherency of the plant's genetic makeup. In another example, two genetically identical soybean seeds may be treated with a formulation that introduces an endophyte composition. Any phenotypic differences between the plants derived from (e.g., grown from or obtained from) those seeds may be attributed to the treatment, thus forming an isoline comparison.

Similarly, by the term “reference agricultural plant,” it is meant an agricultural plant of the same species, strain, or cultivar to which a treatment, formulation, composition or endophyte preparation as described herein is not administered/contacted. A reference agricultural plant, therefore, is identical to the treated plant with the exception of the presence of the endophyte and can serve as a control for detecting the effects of the endophyte that is conferred to the plant.

A “reference environment” refers to the environment, treatment or condition of the plant in which a measurement is made. For example, production of a compound in a plant associated with an endophyte can be measured in a reference environment of drought stress, and compared with the levels of the compound in a reference agricultural plant under the same conditions of drought stress. Alternatively, the levels of a compound in plant associated with an endophyte and reference agricultural plant can be measured under identical conditions of no stress.

A “plant element” is intended to generically reference either a whole plant or a plant component, including but not limited to plant tissues, parts, and cell types. A plant element is preferably one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, kelkis, shoot, bud. As used herein, a “plant element” is synonymous to a “portion” of a plant, and refers to any part of the plant, and can include distinct tissues and/or organs, and may be used interchangeably with the term “tissue” throughout.

Similarly, a “plant reproductive element” is intended to generically reference any part of a plant that is able to initiate other plants via either sexual or asexual reproduction of that plant, for example but not limited to: seed, seedling, root, shoot, cutting, scion, graft, stolon, bulb, tuber, corm, keikis, or bud.

A “population” of plants refers to more than one plant, that are of the same taxonomic category, typically be of the same species, and will also typically share a common genetic derivation.

As used herein, an “agricultural seed” is a seed used to grow a plant typically used in agriculture (an “agricultural plant”). The seed may be of a monocot or dicot plant, and may be planted for the production of an agricultural product, for example feed, food, fiber, fuel, industrial uses, etc. As used herein, an agricultural seed is a seed that is prepared for planting, for example, in farms for growing.

“Agricultural plants,” or “plants of agronomic importance,” include plants that are cultivated by humans for food, feed, fiber, fuel, and/or industrial purposes. Agricultural plants include monocotyledonous species such as: maize ( Zea mays ), common wheat ( Triticum aestivum ), spelt ( Triticum spelta ), einkorn wheat ( Triticum monococcum ), emmer wheat ( Triticum dicoccum ), durum wheat ( Triticum durum ), Asian rice ( Oryza sativa ), African rice ( Oryza glabaerreima ), wild rice ( Zizania aquatica, Zizania latifolia, Zizania palustris, Zizania texana ), barley ( Hordeum vulgare ), Sorghum ( Sorghum bicolor ), Finger millet ( Eleusine coracana ), Proso millet ( Panicum miliaceum ), Pearl millet ( Pennisetum glaucum ), Foxtail millet ( Setaria italica ), Oat ( Avena sativa ), Triticale ( Triticosecale ), rye ( Secale cereal ), Russian wild rye ( Psathyrostachys juncea ), bamboo ( Bambuseae ), or sugarcane (e.g., Saccharum arundinaceum, Saccharum barberi, Saccharum bengalense, Saccharum edule, Saccharum munja, Saccharum officinarum, Saccharum procerum, Saccharum ravennae, Saccharum robustum, Saccharum sinense , or Saccharum spontaneum ); as well as dicotyledonous species such as: soybean ( Glycine max ), canola and rapeseed cultivars ( Brassica napus ), cotton (genus Gossypium ), alfalfa ( Medicago sativa ), cassava (genus Manihot ), potato ( Solanum tuberosum ), tomato ( Solanum lycopersicum ), pea ( Pisum sativum ), chick pea ( Cicer arietinum ), lentil ( Lens culinaris ), flax ( Linum usitatissimum ) and many varieties of vegetables.

The term “synthetic composition” means one or more plant elements associated by human endeavor with an isolated, purified endophyte composition, said association which is not found in nature. In some embodiments of the present invention, “synthetic composition” is used to refer to a treatment formulation comprising an isolated, purified population of endophytes associated with a plant element. In some embodiments of the present invention, “synthetic composition” refers to a purified population of endophytes in a treatment formulation comprising additional compositions with which said endophytes are not found associated in nature.

A “treatment formulation” refers to a mixture of chemicals that facilitate the stability, storage, and/or application of the endophyte composition(s). Treatment formulations may comprise any one or more agents such as: surfactant, a buffer, a tackifier, a microbial stabilizer, a fungicide, an anticomplex agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, a desiccant, a nutrient, an excipient, a wetting agent, a salt.

In some embodiments, an “agriculturally compatible carrier” can be used to formulate an agricultural formulation or other composition that includes a purified endophyte preparation. As used herein an “agriculturally compatible carrier” refers to any material, other than water, that can be added to a plant element without causing or having an adverse effect on the plant element (e.g., reducing seed germination) or the plant that grows from the plant element, or the like.

The compositions and methods herein may provide for an improved “agronomic trait” or “trait of agronomic importance” to a host plant, which may include, but not be limited to, the following: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, yield improvement, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, modulation of a metabolite, modulation of the proteome, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition, compared to an isoline plant derived from a seed without said seed treatment formulation.

As used herein, the terms “water-limited condition” and “drought condition,” or “water-limited” and “drought,” may be used interchangeably. For example, a method or composition for improving a plant's ability to grow under drought conditions means the same as the ability to grow under water-limited conditions. In such cases, the plant can be further said to display improved tolerance to drought stress.

As used herein, the terms “normal watering” and “well-watered” are used interchangeably, to describe a plant grown under typical growth conditions with no water restriction.

Additionally, “altered metabolic function” or “altered enzymatic function” may include, but not be limited to, the following: altered production of an auxin, altered nitrogen fixation, altered production of an antimicrobial compound, altered production of a siderophore, altered mineral phosphate solubilization, altered production of a cellulase, altered production of a chitinase, altered production of a xylanase, altered production of acetoin, altered utilization of a carbon source.

An “increased yield” can refer to any increase in biomass or seed or fruit weight, seed size, seed number per plant, seed number per unit area, bushels per acre, tons per acre, kilo per hectare, or carbohydrate yield. Typically, the particular characteristic is designated when referring to increased yield, e.g., increased grain yield or increased seed size.

“Nutrient” or “seed nutrient” refers to any composition of the associated plant element, most particularly compositions providing benefit to other organisms that consume or utilize said plant element.

“Agronomic trait potential” is intended to mean a capability of a plant element for exhibiting a phenotype, preferably an improved agronomic trait, at some point during its life cycle, or conveying said phenotype to another plant element with which it is associated in the same plant. For example, a plant element may comprise an endophyte that will provide benefit to leaf tissue of a plant from which the plant element is grown; in such case, the plant element comprising such endophyte has the agronomic trait potential for a particular phenotype (for example, increased biomass in the plant) even if the plant element itself does not display said phenotype.

In some cases, the present invention contemplates the use of compositions that are “compatible” with agricultural chemicals, including but not limited to, a fungicide, an anticomplex compound, a bactericide, a virucide, an herbicide, a nematicide, a parasiticide, a pesticide, or any other agent widely used in agricultural which has the effect of killing or otherwise interfering with optimal growth of another organism. As used herein, a composition is “compatible” with an agricultural chemical when the organism is modified, such as by genetic modification, e.g., contains a transgene that confers resistance to an herbicide, or is adapted to grow in, or otherwise survive, the concentration of the agricultural chemical used in agriculture. For example, an endophyte disposed on the surface of a plant element is compatible with the fungicide metalaxyl if it is able to survive the concentrations that are applied on the plant element surface.

As used herein, a “colony-forming unit” (“CFU”) is used as a measure of viable microorganisms in a sample. A CFU is an individual viable cell capable of forming on a solid medium a visible colony whose individual cells are derived by cell division from one parental cell.

The terms “decreased,” “fewer,” “slower” and “increased” “faster” “enhanced” “greater” as used herein refers to a decrease or increase in a characteristic of the endophyte treated plant element or resulting plant compared to an untreated plant element or resulting plant. For example, a decrease in a characteristic may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%, at least 15%, at least 20%, between 20% and 30%, at least 25%, at least 30%, between 30% and 40%, at least 35%, at least 40%, between 40% and 50%, at least 45%, at least 50%, between 50% and 60%, at least about 60%, between 60% and 70%, between 70% and 80%, at least 75%, at least about 80%, between 80% and 90%, at least about 90%, between 90% and 100%, at least 100%, between 100% and 200%, at least 200%, at least about 300%, at least about 400% or more lower than the untreated control and an increase may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%, at least 15%, at least 20%, between 20% and 30%, at least 25%, at least 30%, between 30% and 40%, at least 35%, at least 40%, between 40% and 50%, at least 45%, at least 50%, between 50% and 60%, at least about 60%, between 60% and 70%, between 70% and 80%, at least 75%, at least about 80%, between 80% and 90%, at least about 90%, between 90% and 100%, at least 100%, between 100% and 200%, at least 200%, at least about 300%, at least about 400% or more higher than the untreated control.

As demonstrated herein, agricultural plants may be associated with symbiotic microorganisms, termed endophytes, particularly bacteria and fungi, which may contribute to plant survival, performance, and characteristics. However, modern agricultural processes may have perturbed this relationship, resulting in increased crop losses, diminished stress resilience, biodiversity losses, and increasing dependence on external chemicals, fertilizers, and other unsustainable agricultural practices. There is a need for novel compositions and methods for generating plants with novel microbiome properties that can sustainably increase yield, improve stress resilience, and decrease fertilizer and chemical use.

Currently, the generally accepted view of plant endophytic communities focuses on their homologous derivation, predominantly from the soil communities in which the plants are grown (Hallman, et al., (1997) Canadian Journal of Microbiology. 43(10): 895-914). Upon observing taxonomic overlap between the endophytic and soil microbiota in A. thaliana , it was stated, “Our rigorous definition of an endophytic compartment microbiome should facilitate controlled dissection of plant-microbe interactions derived from complex soil communities” (Lundberg et al., (2012) Nature. 488, 86-90). There is strong support in the art for soil representing the repository from which plant endophytes are derived. New Phytologist (2010) 185: 554-567. Notable plant-microbe interactions such as mycorrhyzal fungi and complex rhizobia fit the paradigm of soil-based colonization of plant hosts and appear to primarily establish themselves independently of seed. As a result of focusing attention on the derivation of endophytes from the soil in which the target agricultural plant is currently growing, there has been an inability to achieve commercially significant improvements in plant yields and other plant characteristics such as increased root biomass, increased root length, increased height, increased shoot length, increased leaf number, increased water use efficiency, increased overall biomass, increase grain yield, increased photosynthesis rate, increased tolerance to drought, increased heat tolerance, increased salt tolerance, increased resistance to insect and nematode stresses, increased resistance to a fungal pathogen, increased resistance to a complex pathogen, increased resistance to a viral pathogen, a detectable modulation in the level of a metabolite, and a detectable modulation in the proteome relative to a reference plant.

The inventors herein have conceived of using endophytes that are capable of living within or otherwise associated with plants to improve plant characteristics, as well as methods of using endophytes that are capable of being associated with plants, to impart novel characteristics to a host plant, as well as to distinct plant elements of the host plant. In an embodiment of this invention, endophyte compositions are isolated and purified from plant or fungal sources, and synthetically combined with a plant element, to impart improved agronomic potential and/or improved agronomic traits to the host plant. In another embodiment of the invention, endophytes that are capable of living within plants are isolated and purified from their native source(s) and synthetically combined with a plant element, to impart improved agronomic potential and/or improved agronomic traits to the host plant or the host plant's elements. Such endophytes that are capable of living within plants may be further manipulated or combined with additional elements prior to combining with the plant element(s).

As described herein, beneficial organisms can be robustly obtained from heterologous, homologous, or engineered sources, optionally cultured, administered heterologously to plant elements, and, as a result of the administration, confer multiple beneficial properties. This is surprising given the variability observed in the art in endophytic microbe isolation and the previous observations of inefficient plant element pathogen colonization of plant host's tissues.

In part, the present invention provides preparations of endophytes that are capable of living within plants, and the creation of synthetic compositions of plant elements and/or seedlings with heterologous endophytes, and formulations comprising the synthetic compositions, as well as the recognition that such synthetic compositions display a diversity of beneficial properties present in the agricultural plants and the associated endophyte populations newly created by the present inventors. Such beneficial properties include metabolism, transcript expression, proteome alterations, morphology, and the resilience to a variety of environmental stresses, and any combination of such properties. The present invention also provides methods of using such endophytes to benefit the host plant with which it is associated.

Endophyte Compositions and Methods of Isolation

The endophytes of the present invention provide several unexpected and significant advantages over other plant-associated microbes. Different environments can comprise significantly different populations of endophytes and thus may provide reservoirs for desired endophytes. Once a choice environment is selected, plant elements of choice plants to be sampled can be identified by their healthy and/or robust growth, or other desired phenotypic characteristics.

In some embodiments of the present invention, endophytes may be bacteria identified from a plant source. In some embodiments of the present invention, endophytes are bacteria identified from a non-plant source, yet be capable of living within a plant, to create a new endophyte entity.

Endophyte Selection: Sourcing

In some embodiments of the present invention, endophytes may be isolated from plants or plant elements. In an embodiment of the present invention, endophytes described herein can also be isolated from plants, plant elements, or endophytic fungi of plants or plant elements adapted to a particular environment, including, but not limited to, an environment with water deficiency, salinity, acute and/or chronic heat stress, acute and/or chronic cold stress, nutrient deprived soils including, but not limited to, micronutrient deprived soils, macronutrient (e.g., potassium, phosphate, nitrogen) deprived soils, pathogen stress, including fungal, nematode, insect, viral, and complex pathogen stress.

In one embodiment, a plant is harvested from a soil type different than that in which the plant is normally grown. In another embodiment, the plant is harvested from an ecosystem where the agricultural plant is not normally found. In another embodiment, the plant is harvested from a soil with an average pH range that is different from the optimal soil pH range of the agricultural plant. In one embodiment, the plant is harvested from an environment with average air temperatures lower than the normal growing temperature of the agricultural plant. In one embodiment, the plant is harvested from an environment with average air temperatures higher than the normal growing temperature of the agricultural plant. In another embodiment, the plant is harvested from an environment with average rainfall lower than the optimal average rainfall received by the agricultural plant. In one embodiment, the plant is harvested from an environment with average rainfall higher than the optimal average rainfall of the agricultural plant. In another embodiment, the plant is harvested from a soil type with different soil moisture classification than the normal soil type that the agricultural plant is grown on. In one embodiment, the plant is harvested from an environment with average rainfall lower than the optimal average rainfall of the agricultural plant. In one embodiment, the plant is harvested from an environment with average rainfall higher than the optimal average rainfall of the agricultural plant. In another embodiment, the plant is harvested from an agricultural environment with a yield lower than the average yield expected from the agricultural plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an agricultural environment with a yield lower than the average yield expected from the agricultural plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment with average yield higher than the optimal average yield of the agricultural plant. In another embodiment, the plant is harvested from an environment with average yield higher than the optimal average yield of the agricultural plant. In another embodiment, the plant is harvested from an environment where soil contains lower total nitrogen than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment where soil contains higher total nitrogen than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment where soil contains lower total phosphorus than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment where soil contains higher total phosphorus than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment where soil contains lower total potassium than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment where soil contains higher total potassium than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment where soil contains lower total sulfur than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment where soil contains higher total sulfur than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment where soil contains lower total calcium than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment where soil contains lower total magnesium than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land. In another embodiment, the plant is harvested from an environment where soil contains higher total sodium chloride (salt) than the optimum levels recommended in order to achieve average yields for a plant grown under average cultivation practices on normal agricultural land.

Endophytes can be obtained from a host plant or a plant element of many distinct plants. In an embodiment, the endophyte can be obtained a plant element of the same or different crop, and can be from the same or different cultivar or variety as the plant element to which the composition is heterologously associated.

In another embodiment, endophytes used in a composition or used to make a synthetic composition can be obtained from the same cultivar or species of agricultural plant to which the composition is intended for heterologous association, or can be obtained from a different cultivar or species of agricultural plant. For example, endophytes from a particular corn variety can be isolated and coated onto the surface of a corn plant element of the same variety.

In another embodiment, endophytes used in a composition or used to make a synthetic composition can be obtained from a plant element of a plant that is related to the plant element to which the composition is intended to be association. For example, an endophyte isolated from Triticum monococcum (einkorn wheat) can be coated onto the surface of a T. aestivum (common wheat) plant element; or, an endophyte from Hordeum vulgare (barley) can be isolated and coated onto the plant element of a member of the Triticeae family, for example, plant elements of the rye plant, Secale cereale ).

In still another embodiment, endophytes used in a composition or used to make a synthetic composition can be obtained from a plant element of a plant that is distantly related to the plant element onto which the endophyte is to be coated. For example, a tomato-derived endophyte can be isolated and coated onto a soybean plant element.

In some embodiments, a purified endophytes population is used that includes two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or greater than 25) different endophytes, e.g., obtained from different families of plant or fungus, or different genera of plant or fungus, or from the same genera but different species of plant or fungus.

In yet another embodiment, endophytes used in a composition or used to make a synthetic composition can be obtained from different individual plants of the same variety, each of which has been subjected to different growth conditions. For example, an endophyte obtained from a drought-affected plant of one variety can be isolated and coated onto the plant element that was derived from a plant of the same variety not subjected to drought. In such cases, the endophyte is considered to be heterologously associated with the plant element onto which it is applied.

The heterologous relationship between the endophyte and the host plant element may result from an endophyte obtained from any different plant or plant element than that which with it becomes associated. In some cases, the endophyte is obtained from a different cultivar of the same species. In some cases, the endophyte is obtained from a different plant species. In some cases, the endophyte is obtained from the same plant species but from two different plants, each exposed to some different environmental condition (for example, differences in heat units or water stress). In some cases, the endophyte is obtained from the same plant individual but from different plant elements or tissues (for example, a root endophyte applied to a leaf).

In some embodiments, compositions described herein comprise a purified endophyte population is used that includes at least two or more, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or greater than 25) different endophytes, e.g., obtained from different families of plants, or different genera of plant or fungus, or from the same genera but different species of plants.

The different endophytes can be obtained from the same cultivar of agricultural plant (e.g., the same maize, wheat, rice, or barley plant), different cultivars of the same agricultural plant (e.g., two or more cultivars of maize, two or more cultivars of wheat, two or more cultivars of rice, or two or more cultivars of barley), or different species of the same type of agricultural plant (e.g., two or more different species of maize, two or more different species of wheat, two or more different species of rice, or two or more different species of barley). In embodiments in which two or more endophytes are used, each of the endophytes can have different properties or activities, e.g., produce different metabolites, produce different enzymes such as different hydrolytic enzymes, confer different beneficial traits, or colonize different elements of a plant (e.g., leaves, stems, flowers, fruits, seeds, or roots). For example, one endophyte can colonize a first and a second endophyte can colonize a tissue that differs from the first tissue. Combinations of endophytes are disclosed in detail below.

In an embodiment, the endophyte is an endophytic microbe isolated from a different plant than the inoculated plant. For example, in an embodiment, the endophyte is an endophyte isolated from a different plant of the same species as the inoculated plant. In some cases, the endophyte is isolated from a species related to the inoculated plant.

Endophyte Selection: Compatibility with Agrichemicals

In certain embodiments, the endophyte is selected on the basis of its compatibility with commonly used agrichemicals. As mentioned earlier, plants, particularly agricultural plants, can be treated with a vast array of agrichemicals, including fungicides, biocides (anticomplex agents), herbicides, insecticides, nematicides, rodenticides, bactericides, virucides, fertilizers, and other agents.

In some embodiments, the endophytes of the present invention display tolerance to an agrichemical selected from the group consisting of: Aeris®, Avicta® DuoCot 202, Cruiser®, Syntenta CCB® (A), Clariva®, Albaugh, Dynasty®, Apron®, Maxim®, Gaucho®, Provoke® ST, Syngenta CCB®, Trilex®, WG Purple, WG Silver, Azoxystrobin, Carboxin, Difenoconazole, Fludioxonil, fluxapyroxad, Ipconazole, Mefenoxam, Metalaxyl, Myclobutanil, Penflufen, pyraclostrobin, Sedaxane, TCMTB, Tebuconazole, Thiram, Triadimenol (Baytan®), Trifloxystrobin, Triticonazole, Tolclofos-methyl, PCNB, Abamectin, Chlorpyrifos, Clothianidin, Imidacloprid, Thiamethoxam, Thiodicarb.

In some cases, it can be important for the endophyte to be compatible with agrichemicals, particularly those with anticomplex properties, in order to persist in the plant although, as mentioned earlier, there are many such anticomplex agents that do not penetrate the plant, at least at a concentration sufficient to interfere with the endophyte. Therefore, where a systemic anticomplex agent is used in the plant, compatibility of the endophyte to be inoculated with such agents will be an important criterion.

In an embodiment, natural isolates of endophytes that are compatible with agrichemicals can be used to inoculate the plants according to the methods described herein. For example, endophytes that are compatible with agriculturally employed anticomplex agents can be isolated by plating a culture of endophytes on a petri dish comprising an effective concentration of the anticomplex agent, and isolating colonies of endophytes that are compatible with the anticomplex agent. In another embodiment, an endophyte that is compatible with an anticomplex agent is used for the methods described herein.

Bactericide-compatible endophyte can also be isolated by selection on liquid medium. The culture of endophytes can be plated on petri dishes without any forms of mutagenesis; alternatively, endophytes can be mutagenized using any means known in the art. For example, endophyte cultures can be exposed to UV light, gamma-irradiation, or chemical mutagens such as ethylmethanesulfonate (EMS), ethidium bromide (EtBr) dichlovos (DDVP, methyl methane sulphonale (MMS), triethylphosphate (TEP), trimethylphosphate (TMP), nitrous acid, or DNA base analogs, prior to selection on fungicide comprising media. Finally, where the mechanism of action of a particular bactericide is known, the target gene can be specifically mutated (either by gene deletion, gene replacement, site-directed mutagenesis, etc.) to generate an endophyte that is resilient against that particular chemical. It is noted that the above-described methods can be used to isolate endophytes that are compatible with both bacteriostatic and bactericidal compounds.

It will also be appreciated by one skilled in the art that a plant may be exposed to multiple types of anticomplex compounds, either simultaneously or in succession, for example at different stages of plant growth. Where the target plant is likely to be exposed to multiple anticomplex agents, an endophyte that is compatible with many or all of these agrichemicals can be used to inoculate the plant. An endophyte that is compatible with several agents can be isolated, for example, by serial selection. An endophyte that is compatible with the first agent can be isolated as described above (with or without prior mutagenesis). A culture of the resulting endophyte can then be selected for the ability to grow on liquid or solid media comprising the second agent (again, with or without prior mutagenesis). Colonies isolated from the second selection are then tested to confirm its compatibility to both agents.

Likewise, endophytes that are compatible to biocides (including herbicides such as glyphosate or anticomplex compounds, whether bacteriostatic or bactericidal) that are agriculturally employed can be isolated using methods similar to those described for isolating compatible endophytes. In one embodiment, mutagenesis of the endophyte population can be performed prior to selection with an anticomplex agent. In another embodiment, selection is performed on the endophyte population without prior mutagenesis. In still another embodiment, serial selection is performed on an endophyte: the endophyte is first selected for compatibility to a first anticomplex agent. The isolated compatible endophyte is then cultured and selected for compatibility to the second anticomplex agent. Any colony thus isolated is tested for compatibility to each, or both anticomplex agents to confirm compatibility with these two agents.

Compatibility with an antimicrobial agent can be determined by a number of means known in the art, including the comparison of the minimal inhibitory concentration (MIC) of the unmodified and modified endophytes. Therefore, in one embodiment, the present invention discloses an isolated modified endophyte, wherein the endophyte is modified such that it exhibits at least 3 fold greater, for example, at least 5 fold greater, between 5 and 10 fold greater, at least 10 fold greater, between 10 and 20 fold greater, at least 20 fold greater, between 20 and 30 fold greater, at least 30 fold greater or more MIC to an antimicrobial agent when compared with the unmodified endophyte.

In one embodiment, disclosed herein are endophytes with enhanced compatibility to the herbicide glyphosate. In one embodiment, the endophyte has a doubling time in growth medium comprising at least 1 mM glyphosate, for example, between 1 mM and 2 mM glyphosate, at least 2 mM glyphosate, between 2 mM and 5 mM glyphosate, at least 5 mM glyphosate, between 5 mM and 10 mM glyphosate, at least 10 mM glyphosate, between 10 mM and 15 mM glyphosate, at least 15 mM glyphosate or more, that is no more than 250%, between 250% and 100%, for example, no more than 200%, between 200% and 175%, no more than 175%, between 175% and 150%, no more than 150%, between 150% and 125%, or no more than 125%, of the doubling time of the endophyte in the same growth medium comprising no glyphosate. In one particular embodiment, the endophyte has a doubling time in growth medium comprising 5 mM glyphosate that is no more than 150% the doubling time of the endophyte in the same growth medium comprising no glyphosate.

In another embodiment, the endophyte has a doubling time in a plant tissue comprising at least 10 ppm glyphosate, between 10 and 15 ppm, for example, at least 15 ppm glyphosate, between 15 and 10 ppm, at least 20 ppm glyphosate, between 20 and 30 ppm, at least 30 ppm glyphosate, between 30 and 40 ppm, at least 40 ppm glyphosate or more, that is no more than 250%, between 250% and 200%, for example, no more than 200%, between 200% and 175%, no more than 175%, between 175% and 150%, no more than 150%, between 150% and 125%, or no more than 125%, of the doubling time of the endophyte in a reference plant tissue comprising no glyphosate. In one particular embodiment, the endophyte has a doubling time in a plant tissue comprising 40 ppm glyphosate that is no more than 150% the doubling time of the endophyte in a reference plant tissue comprising no glyphosate.

The selection process described above can be repeated to identify isolates of endophytes that are compatible with a multitude of agents.

Candidate isolates can be tested to ensure that the selection for agrichemical compatibility did not result in loss of a desired bioactivity. Isolates of endophytes that are compatible with commonly employed agents can be selected as described above. The resulting compatible endophyte can be compared with the parental endophyte on plants in its ability to promote germination.

The agrichemical compatible endophytes generated as described above can be detected in samples. For example, where a transgene was introduced to render the endophyte compatible with the agrichemical(s), the transgene can be used as a target gene for amplification and detection by PCR. In addition, where point mutations or deletions to a portion of a specific gene or a number of genes results in compatibility with the agrichemical(s), the unique point mutations can likewise be detected by PCR or other means known in the art. Such methods allow the detection of the endophyte even if it is no longer viable. Thus, commodity plant products produced using the agrichemical compatible endophytes described herein can readily be identified by employing these and related methods of nucleic acid detection.

Endophyte Selection: Combinations

Combinations of endophytes can be selected by any one or more of several criteria. In one embodiment, compatible endophytes are selected. As used herein, “compatibility” refers to endophyte populations that do not significantly interfere with the growth, propagation, and/or production of beneficial substances of the other. Incompatible endophyte populations can arise, for example, where one of the populations produces or secrets a compound that is toxic or deleterious to the growth of the other population(s). Incompatibility arising from production of deleterious compounds/agents can be detected using methods known in the art, and as described herein elsewhere. Similarly, the distinct populations can compete for limited resources in a way that makes co-existence difficult.

In another embodiment, combinations are selected on the basis of compounds produced by each population of endophytes. For example, the first population is capable of producing siderophores, and another population is capable of producing anti-fungal compounds. In an embodiment, the first population of endophytes or endophytic components is capable of a function selected from the group consisting of auxin production, nitrogen fixation, and production of an antimicrobial compound, siderophore production, mineral phosphate solubilization, cellulase production, chitinase production, xylanase production, and acetoin production, carbon source utilization, and combinations thereof. In another embodiment, the second population of endophytes or endophytic component is capable of a function selected from the group consisting of auxin production, nitrogen fixation, production of an antimicrobial compound, siderophore production, mineral phosphate solubilization, cellulase production, chitinase production, xylanase production, and acetoin production, and combinations thereof. In still another embodiment, the first and second populations are capable of at least one different function.

In still another embodiment, the combinations of endophytes are selected for their distinct localization in the plant after colonization. For example, the first population of endophytes or endophytic components can colonize, and in some cases preferentially colonize, the root tissue, while a second population can be selected on the basis of its preferential colonization of the aerial parts of the agricultural plant. Therefore, in an embodiment, the first population is capable of colonizing one or more of the tissues selected from the group consisting of a root, shoot, leaf, flower, and seed. In another embodiment, the second population is capable of colonizing one or more tissues selected from the group consisting of root, shoot, leaf, flower, and seed. In still another embodiment, the first and second populations are capable of colonizing a different tissue within the agricultural plant.

In some embodiments, combinations of endophytes are selected for their ability to confer a benefit to the host plant at different points in the life cycle of said host plant. In one example, one endophyte can be selected to impart improved seedling vigor, and a second endophyte can be selected to improve soil nutrient acquisition by roots of the mature plant.

In still another embodiment, combinations of endophytes are selected for their ability to confer one or more distinct fitness traits on the inoculated agricultural plant, either individually or in synergistic association with other endophytes. In another embodiment, one endophyte may induce the colonization of a second endophyte. Alternatively, two or more endophytes may induce the colonization of a third endophyte. For example, the first population of endophytes or endophytic components is selected on the basis that it confers significant increase in biomass, while the second population promotes increased drought tolerance on the inoculated agricultural plant. Therefore, in one embodiment, the first population is capable of conferring at least one trait selected from the group consisting of thermal tolerance, herbicide tolerance, drought resistance, insect resistance, fungus resistance, virus resistance, bacteria resistance, male sterility, cold tolerance, salt tolerance, increased yield, enhanced nutrient use efficiency, increased nitrogen use efficiency, increased fermentable carbohydrate content, reduced lignin content, increased antioxidant content, enhanced water use efficiency, increased vigor, increased germination efficiency, earlier or increased flowering, increased biomass, altered root-to-shoot biomass ratio, enhanced soil water retention, or a combination thereof. In another embodiment, the second population is capable of conferring a trait selected from the group consisting of thermal tolerance, herbicide tolerance, drought resistance, insect resistance, fungus resistance, virus resistance, bacteria resistance, male sterility, cold tolerance, salt tolerance, increased yield, enhanced nutrient use efficiency, increased nitrogen use efficiency, increased fermentable carbohydrate content, reduced lignin content, increased antioxidant content, enhanced water use efficiency, increased vigor, increased germination efficiency, earlier or increased flowering, increased biomass, altered root-to-shoot biomass ratio, and enhanced soil water retention. In still another embodiment, each of the first and second population is capable of conferring a different trait selected from the group consisting of thermal tolerance, herbicide tolerance, drought resistance, insect resistance, fungus resistance, virus resistance, bacteria resistance, male sterility, cold tolerance, salt tolerance, increased yield, enhanced nutrient use efficiency, increased nitrogen use efficiency, increased fermentable carbohydrate content, reduced lignin content, increased antioxidant content, enhanced water use efficiency, increased vigor, increased germination efficiency, earlier or increased flowering, increased biomass, altered root-to-shoot biomass ratio, and enhanced soil water retention.

The combinations of endophytes can also be selected based on combinations of the above criteria. For example, the first population of endophytes can be selected on the basis of the compound it produces (e.g., its ability to fix nitrogen, thus providing a potential nitrogen source to the plant), while the second population can be selected on the basis of its ability to confer increased resistance of the plant to a pathogen (e.g., a fungal pathogen).

In some embodiments of the present invention, it is contemplated that combinations of endophytes can provide an increased benefit to the host plant, as compared to that conferred by a single endophyte, by virtue of additive effects. For example, one endophyte strain that induces a benefit in the host plant may induce such benefit equally well in a plant that is also colonized with a different endophyte strain that also induces the same benefit in the host plant. The host plant thus exhibits the same total benefit from the combination of different endophyte strains as the additive benefit to individual plants colonized with each individual endophyte of the combination. In one example, a plant is colonized with two different endophyte strains: one provides a 1× increase in biomass when associated with the plant, and the other provides a 2× increase in biomass when associated with a different plant. When both endophyte strains are associated with the same plant, that plant would experience a 3× (additive of 1×+2× single effects) increase in auxin biomass. Additive effects are a surprising embodiment of the present invention, as non-compatibility of endophytes may result in a cancellation of the beneficial effects of both endophytes.

In some embodiments of the present invention, it is contemplated that a combination of endophytes can provide an increased benefit to the host plant, as compared to that conferred by a single endophyte, by virtue of synergistic effects. For example, one endophyte strain that induces a benefit in the host plant may induce such benefit beyond additive effects in a plant that is also colonized with a different endophyte strain that also induces that benefit in the host plant. The host plant thus exhibits the greater total benefit from the combination of different endophyte strains than could be seen from the additive benefit of individual plants colonized with each individual endophyte of the combination. In one example, a plant is colonized with two different endophyte strains: one provides a 1× increase in biomass when associated with a plant, and the other provides a 2× increase in biomass when associated with a different plant. When both endophyte strains are associated with the same plant, that plant would experience a 5× (greater than an additive of 1×+2× single effects) increase in biomass. Synergistic effects are a surprising embodiment of the present invention.

Endophyte Selection: Compositions of the Invention

In some embodiments, the endophyte is selected from the genus Streptomyces . In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 1. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 2. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 3. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 4. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 5. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 6. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 7. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 8. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 9. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 10. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 11. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 12. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 13. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 14. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 15. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 16. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 17. In some embodiments, the endophyte comprises a nucleotide sequence that is at least 97% identical to SEQ ID NO: 18.

In some embodiments, the endophyte is at least 97% identical to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:18. In some embodiments, the endophyte is between 97% and 98% identical, at least 98% identical, between 98% identical and 99% identical, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:18.

In some cases, the endophyte, or one or more components thereof, is of monoclonal origin, providing high genetic uniformity of the endophyte population in an agricultural formulation or within a synthetic plant element or plant combination with the endophyte.

In some embodiments, the endophyte can be cultured on a culture medium or can be adapted to culture on a culture medium.

The compositions provided herein are preferably stable. The endophyte may be shelf-stable, where at least 0.01%, of the CFUs are viable after storage in desiccated form (i.e., moisture content of 30% or less) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10 weeks at 4° C. or at room temperature. Optionally, a shelf-stable formulation is in a dry formulation, a powder formulation, or a lyophilized formulation. In some embodiments, the formulation is formulated to provide stability for the population of endophytes. In an embodiment, the formulation is substantially stable at temperatures between about −20° C. and about 50° C. for at least about 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3 or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or one or more years. In another embodiment, the formulation is substantially stable at temperatures between about 4° C. and about 37° C. for at least about 5, 10, 15, 20, 25, 30 or greater than 30 days.

Endophytes and Synthetic Compositions with Plants and Plant Elements

It is contemplated that the methods and compositions of the present invention may be used to improve any characteristic of any agricultural plant. The methods described herein can also be used with transgenic plants comprising one or more exogenous transgenes, for example, to yield additional trait benefits conferred by the newly introduced endophytic microbes. Therefore, in one embodiment, a plant element of a transgenic soybean plant is contacted with an endophytic microbe. In one embodiment, a plant element of a transgenic maize plant is contacted with an endophytic microbe.

For example, the endophyte may provide an improved benefit or tolerance to a plant that is of at least 3%, between 3% and 5%, at least 5%, between 5% and 10%, least 10%, between 10% and 15%, for example at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared with uninoculated plants grown under the same conditions.

In one embodiment, it is contemplated that the plant of the present invention is soybean ( Glycine max ).

The primary uses for harvested soybean crops include: soybean oil, soybean meal, livestock feed, and uses for human consumption. All parts of a soy plant are utilized, including the starch, flours, oils, and proteins.

The primary uses for harvested maize crops include: livestock feed, food for human consumption, biofuels, high fructose corn syrup, sweeteners, dry distiller grains, plastics, cosmetics, and textiles. All parts of a corn plant are utilized, including the starch, fiber, proteins, and oils.

The endophyte compositions and methods of the present invention are capable of providing improvements of agronomic interest agricultural plants, for example soybeans and maize.

In some embodiments, the present invention contemplates the use of endophytes that can confer a beneficial agronomic trait upon the plant element or resulting plant with which it is associated.

In some cases, the endophytes described herein are capable of moving from one tissue type to another. For example, the present invention's detection and isolation of endophytes within the mature tissues of plants after coating on the exterior of a plant element demonstrates their ability to move from the plant element into the vegetative tissues of a maturing plant. Therefore, in one embodiment, the population of endophytes is capable of moving from the plant element exterior into the vegetative tissues of a plant. In one embodiment, the endophyte that is coated onto the plant element of a plant is capable, upon germination of the plant element into a vegetative state, of localizing to a different tissue of the plant. For example, endophytes can be capable of localizing to any one of the tissues in the plant, including: the root, adventitious root, seminal root, root hair, shoot, leaf, flower, bud, tassel, meristem, pollen, pistil, ovaries, stamen, fruit, stolon, rhizome, nodule, tuber, trichome, guard cells, hydathode, petal, sepal, glume, rachis, vascular cambium, phloem, and xylem. In an embodiment, the endophyte is capable of localizing to the root and/or the root hair of the plant. In another embodiment, the endophyte is capable of localizing to the photosynthetic tissues, for example, leaves and shoots of the plant. In other cases, the endophyte is localized to the vascular tissues of the plant, for example, in the xylem and phloem. In still another embodiment, the endophyte is capable of localizing to the reproductive tissues (flower, pollen, pistil, ovaries, stamen, fruit) of the plant. In another embodiment, the endophyte is capable of localizing to the root, shoots, leaves and reproductive tissues of the plant. In still another embodiment, the endophyte colonizes a fruit or plant element tissue of the plant. In still another embodiment, the endophyte is able to colonize the plant such that it is present in the surface of the plant (i.e., its presence is detectably present on the plant exterior, or the episphere of the plant). In still other embodiments, the endophyte is capable of localizing to substantially all, or all, tissues of the plant. In certain embodiments, the endophyte is not localized to the root of a plant. In other cases, the endophyte is not localized to the photosynthetic tissues of the plant.

In some cases, endophytes are capable of replicating within the host plant and colonizing the plant.

As shown in the Examples section below, the endophyte populations described herein are capable of colonizing a host plant. Successful colonization can be confirmed by detecting the presence of the endophyte population within the plant. For example, after applying the fungi to the plant elements, high titers of the fungi can be detected in the roots and shoots of the plants that germinate from the plant elements. Detecting the presence of the endophyte inside the plant can be accomplished by measuring the viability of the endophyte after surface sterilization of the plant element or the plant: endophyte colonization results in an internal localization of the endophyte, rendering it resistant to conditions of surface sterilization. The presence and quantity of endophyte can also be established using other means known in the art, for example, immunofluorescence microscopy using microbe-specific antibodies, or fluorescence in situ hybridization (see, for example, Amann et al. (2001) Current Opinion in Biotechnology 12:231-236, incorporated herein by reference in its entirety). Alternatively, specific nucleic acid probes recognizing conserved sequences from an endophyte can be employed to amplify a region, for example by quantitative PCR, and correlated to CFUs by means of a standard curve.

In some cases, plants are inoculated with endophytes that are isolated from the same species of plant as the plant element of the inoculated plant. For example, an endophyte that is normally found in one variety of a plant is associated with a plant element of a plant of another variety of that plant that in its natural state lacks said endophyte. For example, an endophyte that is normally found in one variety of Glycine max (soybean) is associated with a plant element of a plant of another variety of Glycine max that in its natural state lacks said endophyte. In an embodiment, the endophyte is obtained from a plant of a related species of plant as the plant element of the inoculated plant. For example, an endophyte that is normally found in one species of a plant is applied to another species of the same genus, or vice versa. In some cases, plants are inoculated with endophytes that are heterologous to the plant element of the inoculated plant. In an embodiment, the endophyte is obtained from a plant of another species. For example, an endophyte that is normally found in dicots is applied to a monocot plant, or vice versa. In other cases, the endophyte to be inoculated onto a plant is obtained from a related species of the plant that is being inoculated. In one embodiment, the endophyte is obtained from a related taxon, for example, from a related species. The plant of another species can be an agricultural plant. In another embodiment, the endophyte is part of a designed composition inoculated into any host plant element.

In another embodiment, the endophyte is disposed, for example, on the surface of a reproductive element of an agricultural plant, in an amount effective to be detectable in the mature agricultural plant. In one embodiment, the endophyte is disposed in an amount effective to be detectable in an amount of at least about 100 CFU between 100 and 200 CFU, at least about 200 CFU, between 200 and 300 CFU, at least about 300 CFU, between 300 and 400 CFU, at least about 500 CFU, between 500 and 1,000 CFU, at least about 1,000 CFU, between 1,000 and 3,000 CFU, at least about 3,000 CFU, between 3,000 and 10,000 CFU, at least about 10,000 CFU, between 10,000 and 30,000 CFU, at least about 30,000 CFU, between 30,000 and 100,000 CFU, at least about 100,000 CFU or more in the mature agricultural plant.

In some cases, the endophyte is capable of colonizing particular plant elements or tissue types of the plant. In an embodiment, the endophyte is disposed on the plant element or seedling in an amount effective to be detectable within a target tissue of the mature agricultural plant selected from a fruit, a seed, a leaf, or a root, or portion thereof. For example, the endophyte can be detected in an amount of at least about 100 CFU, at least about 200 CFU, at least about 300 CFU, at least about 500 CFU, at least about 1,000 CFU, at least about 3,000 CFU, at least about 10,000 CFU, at least about 30,000 CFU, at least about 100,000 CFU or more, in the target tissue of the mature agricultural plant.

Beneficial Attributes of Synthetic Compositions of Plant Elements and Endophytes

Improved Attributes Conferred by Endophytes

The present invention contemplates the establishment of a relationship between a symbiont and a plant element. In one embodiment, endophyte association results in a detectable change to the plant element, or the whole plant. The detectable change can be an improvement in a number of agronomic traits (e.g., improved general health, increased response to biotic or abiotic stresses, or enhanced properties of the plant or a plant element, including fruits and grains). Alternatively, the detectable change can be a physiological or biological change that can be measured by methods known in the art. The detectable changes are described in more detail in the sections below. As used herein, an endophyte is considered to have conferred an improved agricultural trait whether or not the improved trait arose from the plant, the endophyte, or the concerted action between the plant and endophyte. Therefore, for example, whether a beneficial hormone or chemical is produced by the plant or the endophyte, for purposes of the present invention, the endophyte will be considered to have conferred an improved agronomic trait upon the host plant, as compared to an isoline plant that has not been associated with said endophyte.

In some embodiments, provided herein, are methods for producing a plant element of a plant with a heritably altered trait. The trait of the plant can be altered without known genetic modification of the plant genome, and comprises the following steps. First, a preparation of an isolated endophyte that is heterologous to the plant element of the plant is provided, and optionally processed to produce an endophyte formulation. The endophyte formulation is then contacted with the plant. The plants are then allowed to go to seed, and the seeds are collected.

Improved General Health

Also described herein are plants, and fields of plants, that are associated with beneficial endophytes, such that the overall fitness, productivity or health of the plant or a portion thereof, is maintained, increased and/or improved over a period of time. Improvement in overall plant health can be assessed using numerous physiological parameters including, but not limited to, height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof. Improved plant health, or improved field health, can also be demonstrated through improved resistance or response to a given stress, either biotic or abiotic stress, or a combination of one or more abiotic stresses, as provided herein.

Other Abiotic Stresses

Disclosed herein are endophyte-associated plants with increased resistance to an abiotic stress. Exemplary abiotic stresses include, but are not limited to: drought, heat, salt content, metal content, low nutrient conditions, cold, excess water conditions.

Drought and Heat Tolerance.

In some cases, a plant resulting from seeds or other plant elements treated with an endophyte can exhibit a physiological change, such as a compensation of the stress-induced reduction in photosynthetic activity. Fv/Fm tests whether or not plant stress affects photosystem II in a dark adapted state. Fv/Fm is one of the most commonly used chlorophyll fluorescence measuring parameter. The Fv/Fm test is designed to allow the maximum amount of the light energy to take the fluorescence pathway. It compares the dark-adapted leaf pre-photosynthetic fluorescent state, called minimum fluorescence, or Fo, to maximum fluorescence called Fm. In maximum fluorescence, the maximum number of reaction centers have been reduced or closed by a saturating light source. In general, the greater the plant stress, the fewer open reaction centers available, and the Fv/Fm ratio is lowered. Fv/Fm is a measuring protocol that works for many types of plant stress. For example, there would be a difference in the Fv/Fm after exposure of an endophyte treated plant that had been subjected to heat shock or drought conditions, as compared to a corresponding control, a genetically identical plant that does not contain the endophytes grown in the same conditions. In some cases, the endophyte-associated plant as disclosed herein can exhibit an increased change in photosynthetic activity ΔFv(ΔFv/Fm) after heat-shock or drought stress treatment, for example 1, 2, 3, 4, 5, 6, 7 days or more after the heat-shock or drought stress treatment, or until photosynthesis ceases, as compared with corresponding control plant of similar developmental stage but not comprising endophytes. For example, a plant having an endophyte able to confer heat and/or drought-tolerance can exhibit a ΔFv/Fm of from about 0.1 to about 0.8 after exposure to heat-shock or drought stress or a ΔFv/Fm range of from about 0.03 to about 0.8 under one day, or 1, 2, 3, 4, 5, 6, 7, or over 7 days post heat-shock or drought stress treatment, or until photosynthesis ceases. In some embodiments, stress-induced reductions in photosynthetic activity can be compensated by at least about 0.25% (for example, at least about 0.5%, between 0.5% and 1%, at least about 1%, between 1% and 2%, at least about 2%, between 2% and 3%, at least about 3%, between 3% and 5%, at least about 5%, between 5% and 10%, at least about 8%, at least about 10%, between 10% and 15%, at least about 15%, between 15% and 20%, at least about 20%, between 20$ and 25%, at least about 25%, between 25% and 30%, at least about 30%, between 30% and 40%, at least about 40%, between 40% and 50%, at least about 50%, between 50% and 60%, at least about 60%, between 60% and 75%, at least about 75%, between 75% and 80%, at least about 80%, between 80% and 85%, at least about 85%, between 85% and 90%, at least about 90%, between 90% and 95%, at least about 95%, between 95% and 99%, at least about 99% or at least 100%) as compared to the photosynthetic activity decrease in a corresponding reference agricultural plant following heat shock conditions. Significance of the difference between endophyte-associated and reference agricultural plants can be established upon demonstrating statistical significance, for example at p<0.05 with an appropriate parametric or non-parametric statistic, e.g., Chi-square test, Student's t-test, Mann-Whitney test, or F-test based on the assumption or known facts that the endophyte-associated plant and reference agricultural plant have identical or near identical genomes (isoline comparison).

In some embodiments, the plants comprise endophytes able to increase heat and/or drought-tolerance in sufficient quantity, such that increased growth or improved recovery from wilting under conditions of heat or drought stress is observed. For example, an endophyte population described herein can be present in sufficient quantity in a plant, resulting in increased growth as compared to a plant that does not contain endophytes, when grown under drought conditions or heat shock conditions, or following such conditions. Increased heat and/or drought tolerance can be assessed with physiological parameters including, but not limited to, increased height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, wilt recovery, turgor pressure, or any combination thereof, as compared to a reference agricultural plant grown under similar conditions. For example, the endophyte may provide an improved benefit or tolerance to a plant that is of at least 3%, between 3% and 5%, at least 5%, between 5% and 10%, least 10%, between 10% and 15%, for example at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared with uninoculated plants grown under the same conditions.

In various embodiments, endophytes introduced into the plant can confer in the resulting plant thermal tolerance, herbicide tolerance, drought resistance, insect resistance, fungus resistance, virus resistance, bacteria resistance, male sterility, cold tolerance, salt tolerance, increased yield, enhanced nutrient use efficiency, increased nitrogen use efficiency, increased protein content, increased fermentable carbohydrate content, reduced lignin content, increased antioxidant content, enhanced water use efficiency, increased vigor, increased germination efficiency, earlier or increased flowering, increased biomass, altered root-to-shoot biomass ratio, enhanced soil water retention, or a combination thereof. A difference between the endophyte-associated plant and a reference agricultural plant can also be measured using other methods known in the art.

Salt Stress.

In other embodiments, endophytes able to confer increased tolerance to salinity stress can be introduced into plants. The resulting plants comprising endophytes can exhibit increased resistance to salt stress, whether measured in terms of survival under saline conditions, or overall growth during, or following salt stress. The physiological parameters of plant health recited above, including height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared with the growth rate of reference agricultural plants (e.g., isogenic plants without the endophytes) grown under identical conditions. For example, the endophyte may provide an improved benefit or tolerance to a plant that is of at least 10%, between 10% and 15%, for example at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared with uninoculated plants grown under the same conditions. In other instances, endophyte-associated plants and reference agricultural plants can be grown in soil or growth media comprising different concentration of sodium to establish the inhibitory concentration of sodium (expressed, for example, as the concentration in which growth of the plant is inhibited by 50% when compared with plants grown under no sodium stress). Therefore, in another embodiment, a plant resulting from plant elements comprising an endophyte able to confer salt tolerance described herein exhibits an increase in the inhibitory sodium concentration by at least 10 mM, between 10 mM and 15 mM, for example at least 15 mM, between 15 mM and 20 mM, at least 20 mM, between 20 mM and 30 mM, at least 30 mM, between 30 mM and 40 mM, at least 40 mM, between 40 mM and 50 mM, at least 50 mM, between 50 mM and 60 mM, at least 60 mM, between 60 mM and 70 mM, at least 70 mM, between 70 mM and 80 mM, at least 80 mM, between 80 mM and 90 mM, at least 90 mM, between 90 mM and 100 mM, at least 100 mM or more, when compared with the reference agricultural plants.

High Metal Content.

Plants are sessile organisms and therefore must contend with the environment in which they are placed. Plants have adapted many mechanisms to deal with chemicals and substances that may be deleterious to their health. Heavy metals in particular represent a class of toxins that are highly relevant for plant growth and agriculture, because many of them are associated with fertilizers and sewage sludge used to amend soils and can accumulate to toxic levels in agricultural fields. Therefore, for agricultural purposes, it is important to have plants that are able to tolerate soils comprising elevated levels of toxic heavy metals. Plants cope with toxic levels of heavy metals (for example, nickel, cadmium, lead, mercury, arsenic, or aluminum) in the soil by excretion and internal sequestration. Endophytes that are able to confer increased heavy metal tolerance may do so by enhancing sequestration of the metal in certain compartments away from the seed or fruit and/or by supplementing other nutrients necessary to remediate the stress. Use of such endophytes in a plant would allow the development of novel plant-endophyte combinations for purposes of environmental remediation (also known as phytoremediation). Therefore, in one embodiment, the plant comprising endophytes shows increased metal tolerance as compared to a reference agricultural plant grown under the same heavy metal concentration in the soil.

Alternatively, the inhibitory concentration of the heavy metal can be determined for endophyte-associated plant and compared with a reference agricultural plant under the same conditions. Therefore, in one embodiment, the plants resulting from plant elements comprising an endophyte able to confer heavy metal tolerance described herein exhibit an increase in the inhibitory metal concentration by at least 0.1 mM, between 0.1 mM and 0.3 mM, for example at least 0.3 mM, between 0.3 mM and 0.5 mM, at least 0.5 mM, between 0.5 mM and 1 mM, at least 1 mM, between 1 mM and 2 mM, at least 2 mM, between 2 mM and 5 mM, at least 5 mM, between 5 mM and 10 mM, at least 10 mM, between 10 mM and 15 mM, at least 15 mM, between 15 mM and 20 mM, at least 20 mM, between 20 mM and 30 mM, at least 30 mM, between 30 mM and 50 mM, at least 50 mM or more, when compared with the reference agricultural plants.

Finally, plants inoculated with endophytes that are able to confer increased metal tolerance exhibit an increase in overall metal excretion by at least 10%, between 10% and 15%, for example at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared with uninoculated plants grown under the same conditions.

Low Nutrient Stress.

Endophytes described herein may also confer to the plant an increased ability to grow in nutrient limiting conditions, for example by solubilizing or otherwise making available to the plants macronutrients or micronutrients that are complexed, insoluble, or otherwise in an unavailable form. In one embodiment, a plant is inoculated with an endophyte that confers increased ability to liberate and/or otherwise provide to the plant with nutrients selected from the group consisting of phosphate, nitrogen, potassium, iron, manganese, calcium, molybdenum, vitamins, or other micronutrients. Such a plant can exhibit increased growth in soil comprising limiting amounts of such nutrients when compared with reference agricultural plant. Differences between the endophyte-associated plant and reference agricultural plant can be measured by comparing the biomass of the two plant types grown under limiting conditions, or by measuring the physical parameters described above. Therefore, in one embodiment, the plant comprising endophyte shows increased tolerance to nutrient limiting conditions as compared to a reference agricultural plant grown under the same nutrient limited concentration in the soil, as measured for example by increased biomass or seed yield of at least 10%, between 10% and 15%, for example at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared with uninoculated plants grown under the same conditions.

Cold Stress.

In some cases, endophytes can confer to the plant the ability to tolerate cold stress. As used herein, cold stress refers to both the stress induced by chilling (0° C.-15° C.) and freezing (<0° C.). Some cultivars of agricultural plants can be particularly sensitive to cold stress, but cold tolerance traits may be multigenic, making the breeding process difficult. Endophytes able to confer cold tolerance can reduce the damage suffered by farmers on an annual basis. Improved response to cold stress can be measured by survival of plants, production of protectant substances such as anthocyanin, the amount of necrosis of parts of the plant, or a change in crop yield loss, as well as the physiological parameters used in other examples. Therefore, in an embodiment, the plant comprising endophytes shows increased cold tolerance exhibits as compared to a reference agricultural plant grown under the same conditions of cold stress. For example, the endophyte may provide an improved benefit or tolerance to a plant that is of at least 3%, between 3% and 5%, at least 5%, between 5% and 10%, least 10%, between 10% and 15%, for example at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared with uninoculated plants grown under the same conditions.

Biotic Stress.

In other embodiments, the endophyte protects the plant from a biotic stress, for example, insect infestation, nematode infestation, complex infection, fungal infection, bacterial infection, oomycete infection, protozoal infection, viral infection, and herbivore grazing, or a combination thereof. For example, the endophyte may provide an improved benefit or tolerance to a plant that is of at least 3%, between 3% and 5%, at least 5%, between 5% and 10%, least 10%, between 10% and 15%, for example at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared with uninoculated plants grown under the same conditions.

Insect Herbivory.

There are an abundance of insect pest species that can infect or infest a wide variety of plants. Pest infestation can lead to significant damage. Insect pests that infest plant species are particularly problematic in agriculture as they can cause serious damage to crops and significantly reduce plant yields. A wide variety of different types of plant are susceptible to pest infestation including commercial crops such as cotton, soybean, wheat, barley, and corn (maize).

In some cases, endophytes described herein may confer upon the host plant the ability to repel insect herbivores. In other cases, endophytes may produce, or induce the production in the plant of, compounds which are insecticidal or insect repellant. The insect may be any one of the common pathogenic insects affecting plants, particularly agricultural plants.

The endophyte-associated plant can be tested for its ability to resist, or otherwise repel, pathogenic insects by measuring, for example, insect load, overall plant biomass, biomass of the fruit or grain, percentage of intact leaves, or other physiological parameters described herein, and comparing with a reference agricultural plant. In an embodiment, the endophyte-associated plant exhibits increased biomass as compared to a reference agricultural plant grown under the same conditions (e.g., grown side-by-side, or adjacent to, endophyte-associated plants). In other embodiments, the endophyte-associated plant exhibits increased fruit or grain yield as compared to a reference agricultural plant grown under the same conditions (e.g., grown side-by-side, or adjacent to, endophyte-associated plants).

Nematodes.

Nematodes are microscopic roundworms that feed on the roots, fluids, leaves and stems of more than 2,000 row crops, vegetables, fruits, and ornamental plants, causing an estimated $100 billion crop loss worldwide and accounting for 13% of global crop losses due to disease. A variety of parasitic nematode species infect crop plants, including root-knot nematodes (RKN), cyst- and lesion-forming nematodes. Root-knot nematodes, which are characterized by causing root gall formation at feeding sites, have a relatively broad host range and are therefore parasitic on a large number of crop species. The cyst- and lesion-forming nematode species have a more limited host range, but still cause considerable losses in susceptible crops.

Signs of nematode damage include stunting and yellowing of leaves, and wilting of the plants during hot periods. Nematode infestation, however, can cause significant yield losses without any obvious above-ground disease symptoms. The primary causes of yield reduction are due to underground root damage. Roots infected by SCN are dwarfed or stunted. Nematode infestation also can decrease the number of nitrogen-fixing nodules on the roots, and may make the roots more susceptible to attacks by other soil-borne plant nematodes.

In an embodiment, the endophyte-associated plant has an increased resistance to a nematode when compared with a reference agricultural plant. As before with insect herbivores, biomass of the plant or a portion of the plant, or any of the other physiological parameters mentioned elsewhere, can be compared with the reference agricultural plant grown under the same conditions. Particularly useful measurements include overall plant biomass, biomass and/or size of the fruit or grain, and root biomass. In one embodiment, the endophyte-associated plant exhibits increased biomass as compared to a reference agricultural plant grown under the same conditions (e.g., grown side-by-side, or adjacent to, the endophyte-associated plants, under conditions of nematode challenge). In another embodiment, the endophyte-associated plant exhibits increased root biomass as compared to a reference agricultural plant grown under the same conditions (e.g., grown side-by-side, or adjacent to, the endophyte-associated plants, under conditions of nematode challenge). In still another embodiment, the endophyte-associated plant exhibits increased fruit or grain yield as compared to a reference agricultural plant grown under the same conditions (e.g., grown side-by-side, or adjacent to, the endophyte-associated plants, under conditions of nematode challenge).

Fungal Pathogens.

Fungal diseases are responsible for yearly losses of over $10 Billion on agricultural crops in the US, represent 42% of global crop losses due to disease, and are caused by a large variety of biologically diverse pathogens. Different strategies have traditionally been used to control them. Resistance traits have been bred into agriculturally important varieties, thus providing various levels of resistance against either a narrow range of pathogen isolates or races, or against a broader range. However, this involves the long and labor intensive process of introducing desirable traits into commercial lines by genetic crosses and, due to the risk of pests evolving to overcome natural plant resistance, a constant effort to breed new resistance traits into commercial lines is required. Alternatively, fungal diseases have been controlled by the application of chemical fungicides. This strategy usually results in efficient control, but is also associated with the possible development of resistant pathogens and can be associated with a negative impact on the environment. Moreover, in certain crops, such as barley and wheat, the control of fungal pathogens by chemical fungicides is difficult or impractical.

The present invention contemplates the use of endophytes that are able to confer resistance to fungal pathogens to the host plant. Increased resistance to fungal inoculation can be measured, for example, using any of the physiological parameters presented above, by comparing with reference agricultural plants. In an embodiment, the endophyte-associated plant exhibits increased biomass and/or less pronounced disease symptoms as compared to a reference agricultural plant grown under the same conditions (e.g., grown side-by-side, or adjacent to, the endophyte-associated plants, infected with the fungal pathogen). In still another embodiment, the endophyte-associated plant exhibits increased fruit or grain yield as compared to a reference agricultural plant grown under the same conditions (e.g., grown side-by-side, or adjacent to, the endophyte-associated plants, infected with the fungal pathogen). In another embodiment, the endophyte-associated plant exhibits decreased hyphal growth as compared to a reference agricultural plant grown under the same conditions (e.g., grown side-by-side, or adjacent to, the endophyte-associated plants, infected with the fungal pathogen). For example, the endophyte may provide an improved benefit to a plant that is of at least 3%, between 3% and 5%, at least 5%, between 5% and 10%, least 10%, between 10% and 15%, for example at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared with uninoculated plants grown under the same conditions.

Viral Pathogens.

Plant viruses are estimated to account for 18% of global crop losses due to disease. There are numerous examples of viral pathogens affecting agricultural productivity. In an embodiment, the endophyte provides protection against viral pathogens such that the plant has increased biomass as compared to a reference agricultural plant grown under the same conditions. In still another embodiment, the endophyte-associated plant exhibits greater fruit or grain yield, when challenged with a virus, as compared to a reference agricultural plant grown under the same conditions. In yet another embodiment, the endophyte-associated plant exhibits lower viral titer, when challenged with a virus, as compared to a reference agricultural plant grown under the same conditions.

Complex Pathogens.

Likewise, bacterial pathogens are a significant problem negatively affecting agricultural productivity and accounting for 27% of global crop losses due to plant disease. In an embodiment, the endophyte described herein provides protection against bacterial pathogens such that the plant has greater biomass as compared to a reference agricultural plant grown under the same conditions. In still another embodiment, the endophyte-associated plant exhibits greater fruit or grain yield, when challenged with a complex pathogen, as compared to a reference agricultural plant grown under the same conditions. In yet another embodiment, the endophyte-associated plant exhibits lower complex count, when challenged with a bacterium, as compared to a reference agricultural plant grown under the same conditions.

Improvement of Other Traits

In other embodiments, the endophyte can confer other beneficial traits to the plant. Improved traits can include an improved nutritional content of the plant or plant element used for human consumption. In one embodiment, the endophyte-associated plant is able to produce a detectable change in the content of at least one nutrient. Examples of such nutrients include amino acid, protein, oil (including any one of Oleic acid, Linoleic acid, Alpha-linoleic acid, Saturated fatty acids, Palmitic acid, Stearic acid and Trans fats), carbohydrate (including sugars such as sucrose, glucose and fructose, starch, or dietary fiber), Vitamin A, Thiamine (vit. B1), Riboflavin (vit. B2), Niacin (vit. B3), Pantothenic acid (B5), Vitamin B6, Folate (vit. B9), Choline, Vitamin C, Vitamin E, Vitamin K, Calcium, Iron, Magnesium, Manganese, Phosphorus, Potassium, Sodium, Zinc. In an embodiment, the endophyte-associated plant or part thereof contains at least one increased nutrient when compared with reference agricultural plants.

In other cases, the improved trait can include reduced content of a harmful or undesirable substance when compared with reference agricultural plants. Such compounds include those which are harmful when ingested in large quantities or are bitter tasting (for example, oxalic acid, amygdalin, certain alkaloids such as solanine, caffeine, nicotine, quinine and morphine, tannins, cyanide). As such, in one embodiment, the endophyte-associated plant or part thereof contains less of the undesirable substance when compared with reference agricultural plant. In a related embodiment, the improved trait can include improved taste of the plant or a part of the plant, including the fruit or plant reproductive element. In a related embodiment, the improved trait can include reduction of undesirable compounds produced by other endophytes in plants, such as degradation of Fusarium -produced deoxynivalenol (also known as vomitoxin and a virulence factor involved in Fusarium head blight of maize and wheat) in a part of the plant, including the fruit or plant reproductive element.

In other cases, the improved trait can be an increase in overall biomass of the plant or a part of the plant, including its fruit or plant reproductive element.

The endophyte-associated plant can also have an altered hormone status or altered levels of hormone production when compared with a reference agricultural plant. An alteration in hormonal status may affect many physiological parameters, including flowering time, water efficiency, apical dominance and/or lateral shoot branching, increase in root hair, and alteration in fruit ripening.

The association between the endophyte and the plant can also be detected using other methods known in the art. For example, the biochemical, metabolomics, proteomic, genomic, epigenomic and/or transcriptomic profiles of endophyte-associated plants can be compared with reference agricultural plants under the same conditions.

Methods of Using Endophytes and Synthetic Compositions Comprising Endophytes

As described herein, purified endophyte populations and compositions comprising the same (e.g., formulations) can be used to confer beneficial traits to the host plant including, for example, one or more of the following: increased root biomass, increased root length, increased height, increased shoot length, increased leaf number, improved water use efficiency (drought tolerance), increased overall biomass, increase grain yield, increased photosynthesis rate, increased tolerance to drought, increased heat tolerance, increased salt tolerance, increased resistance to nematode stress, increased resistance to a fungal pathogen, increased resistance to a complex pathogen, increased resistance to a viral pathogen, a detectable modulation in the level of a metabolite, and a detectable modulation in the proteome relative to a reference plant. For example, in some embodiments, a purified endophyte population can improve two or more such beneficial traits, e.g., water use efficiency and increased tolerance to drought.

In some cases, the endophyte may produce one or more compounds and/or have one or more activities, e.g., one or more of the following: production of a metabolite, production of a phytohormone such as auxin, production of acetoin, production of an antimicrobial compound, production of a siderophore, production of a cellulase, production of a pectinase, production of a chitinase, production of a xylanase, nitrogen fixation, or mineral phosphate solubilization. For example, an endophyte can produce a phytohormone selected from the group consisting of an auxin, a cytokinin, a gibberellin, ethylene, a brassinosteroid, and abscisic acid. In one particular embodiment, the endophyte produces auxin (e.g., indole-3-acetic acid (IAA)). Production of auxin can be assayed as described herein. Many of the microbes described herein are capable of producing the plant hormone auxin indole-3-acetic acid (IAA) when grown in culture. Auxin plays a key role in altering the physiology of the plant, including the extent of root growth. Therefore, in another embodiment, the endophytic population is disposed on the surface or within a tissue of the seed or seedling in an amount effective to detectably increase production of auxin in the agricultural plant when compared with a reference agricultural plant. In one embodiment, the increased auxin production can be detected in a tissue type selected from the group consisting of the root, shoot, leaves, and flowers.

In some embodiments, the endophyte can produce a compound with antimicrobial properties. For example, the compound can have antibacterial properties, as determined by the growth assays provided herein. In one embodiment, the compound with antibacterial properties shows bacteriostatic or bactericidal activity against E. coli and/or Bacillus sp. In another embodiment, the endophyte produces a compound with antifungal properties, for example, fungicidal or fungistatic activity against S. cerevisiae and/or Rhizoctonia.

In some embodiments, the endophyte is a bacterium capable of nitrogen fixation, and is thus capable of producing ammonium from atmospheric nitrogen. The ability of a bacterium to fix nitrogen can be confirmed by testing for growth of the bacterium in nitrogen-free growth media, for example, LGI media, as described herein.

In some embodiments, the endophyte can produce a compound that increases the solubility of mineral phosphate in the medium, i.e., mineral phosphate solubilization, for example, using the growth assays described herein. In one embodiment, the endophyte produces a compound that allows the bacterium to grow in growth media comprising Ca 3 HPO 4 as the sole phosphate source.

In some embodiments, the endophyte can produce a siderophore. Siderophores are small high-affinity iron chelating agents secreted by microorganisms that increase the bioavailability of iron. Siderophore production by the endophyte can be detected, for example, using any known method in the art.

In some embodiments, the endophyte can produce a hydrolytic enzyme. For example, in one embodiment, an endophyte can produce a hydrolytic enzyme selected from the group consisting of a cellulase, a pectinase, a chitinase and a xylanase. Hydrolytic enzymes can be detected using the methods known in the art.

In some embodiments, metabolites in plants can be modulated by making synthetic compositions of purified endophytic populations. For example, an endophyte described herein can cause a detectable modulation (e.g., an increase or decrease) in the level of various metabolites, e.g., indole-3-carboxylic acid, trans-zeatin, abscisic acid, phaseic acid, indole-3-acetic acid, indole-3-butyric acid, indole-3-acrylic acid, jasmonic acid, jasmonic acid methyl ester, dihydrophaseic acid, gibberellin A3, salicylic acid, upon colonization of a plant.

In some embodiments, the endophyte modulates the level of the metabolite directly (e.g., the microbe itself produces the metabolite, resulting in an overall increase in the level of the metabolite found in the plant). In other cases, the agricultural plant, as a result of the association with the endophytic microbe (e.g., an endophyte), exhibits a modulated level of the metabolite (e.g., the plant reduces the expression of a biosynthetic enzyme responsible for production of the metabolite as a result of the microbe inoculation). In still other cases, the modulation in the level of the metabolite is a consequence of the activity of both the microbe and the plant (e.g., the plant produces increased amounts of the metabolite when compared with a reference agricultural plant, and the endophytic microbe also produces the metabolite). Therefore, as used herein, a modulation in the level of a metabolite can be an alteration in the metabolite level through the actions of the microbe and/or the inoculated plant.

The levels of a metabolite can be measured in an agricultural plant, and compared with the levels of the metabolite in a reference agricultural plant, and grown under the same conditions as the inoculated plant. The uninoculated plant that is used as a reference agricultural plant is a plant that has not been applied with a formulation with the endophytic microbe (e.g., a formulation comprising a population of purified endophytes). The uninoculated plant used as the reference agricultural plant is generally the same species and cultivar as, and is isogenic to, the inoculated plant.

The metabolite whose levels are modulated (e.g., increased or decreased) in the endophyte-associated plant may serve as a primary nutrient (i.e., it provides nutrition for the humans and/or animals who consume the plant, plant tissue, or the commodity plant product derived therefrom, including, but not limited to, a sugar, a starch, a carbohydrate, a protein, an oil, a fatty acid, a mineral, or a vitamin). The metabolite can be a compound that is important for plant growth, development or homeostasis (for example, a phytohormone such as an auxin, cytokinin, gibberellin, a brassinosteroid, ethylene, or abscisic acid, a signaling molecule, or an antioxidant). In other embodiments, the metabolite can have other functions. For example, in one embodiment, a metabolite can have bacteriostatic, bactericidal, fungistatic, fungicidal or antiviral properties. In other embodiments, the metabolite can have insect-repelling, insecticidal, nematode-repelling, or nematicidal properties. In still other embodiments, the metabolite can serve a role in protecting the plant from stresses, may help improve plant vigor or the general health of the plant. In yet another embodiment, the metabolite can be a useful compound for industrial production. For example, the metabolite may itself be a useful compound that is extracted for industrial use, or serve as an intermediate for the synthesis of other compounds used in industry. In a particular embodiment, the level of the metabolite is increased within the agricultural plant or a portion thereof such that it is present at a concentration of at least 0.1 ug/g dry weight, between 0.1 ug/g to 0.3 ug/g, for example, at least 0.3 ug/g dry weight, between 0.3 ug/g to 1.0 ug/g, 1.0 ug/g dry weight, between 1 ug/g and 3 ug/g, 3.0 ug/g dry weight, between 3 ug/g and 10 ug/g, 10 ug/g dry weight, between 10 ug/g and 30 ug/g, 30 ug/g dry weight, between 30 ug/g and 100 ug/g, 100 ug/g dry weight, between 100 ug/g and 300 ug/g, 300 ug/g dry weight, between 300 ug/g and 1 mg/g, 1 mg/g dry weight, between 1 mg/g and 3 mg/g, 3 mg/g dry weight, between 3 mg/g and 10 mg/g, 10 mg/g dry weight, between 10 mg/g and 30 mg/g, 30 mg/g dry weight, between 30 mg/g and 100 mg/g, 100 mg/g dry weight or more, of the plant or portion thereof.

Likewise, the modulation can be a decrease in the level of a metabolite. The reduction can be in a metabolite affecting the taste of a plant or a commodity plant product derived from a plant (for example, a bitter tasting compound), or in a metabolite which makes a plant or the resulting commodity plant product otherwise less valuable (for example, reduction of oxalate content in certain plants, or compounds which are deleterious to human and/or animal health). The metabolite whose level is to be reduced can be a compound that affects quality of a commodity plant product (e.g., reduction of lignin levels).

In some embodiments, the endophyte is capable of generating a complex network in the plant or surrounding environment of the plant, which network is capable of causing a detectable modulation in the level of a metabolite in the host plant.

In a particular embodiment, the metabolite can serve as a signaling or regulatory molecule. The signaling pathway can be associated with a response to a stress, for example, one of the stress conditions selected from the group consisting of drought stress, salt stress, heat stress, cold stress, low nutrient stress, nematode stress, insect herbivory stress, fungal pathogen stress, complex pathogen stress, and viral pathogen stress.

The inoculated agricultural plant is grown under conditions such that the level of one or more metabolites is modulated in the plant, wherein the modulation is indicative of increased resistance to a stress selected from the group consisting of drought stress, salt stress, heat stress, cold stress, low nutrient stress, nematode stress, insect herbivory stress, fungal pathogen stress, complex pathogen stress, and viral pathogen stress. The increased resistance can be measured at about 10 minutes after applying the stress, between 10 minutes and 20 minutes, for example about 20 minutes, between 20 and 30 minutes, 30 minutes, between 30 and 45 minutes, about 45 minutes, between 45 minutes and 1 hour, about 1 hour, between 1 and 2 hours, about 2 hours, between 2 and 4 hours, about 4 hours, between 4 and 8 hours, about 8 hours, between 8 and 12 hours, about 12 hours, between 12 and 16 hours, about 16 hours, between 16 and 20 hours, about 20 hours, between 20 and 24 hours, about 24 hours, between 24 and 36 hours, about 36 hours, between 36 and 48 hours, about 48 hours, between 48 and 72 hours, about 72 hours, between 72 and 96 hours, about 96 hours, between 96 and 120 hours, about 120 hours, between 120 hours and one week, or about a week after applying the stress.

The metabolites or other compounds described herein can be detected using any suitable method including, but not limited to gel electrophoresis, liquid and gas phase chromatography, either alone or coupled to mass spectrometry, NMR, immunoassays (radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA)), chemical assays, spectroscopy and the like. In some embodiments, commercial systems for chromatography and NMR analysis are utilized.

In other embodiments, metabolites or other compounds are detected using optical imaging techniques such as magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), CAT scans, ultra sound, MS-based tissue imaging or X-ray detection methods (e.g., energy dispersive x-ray fluorescence detection).

Any suitable method may be used to analyze the biological sample (e.g., seed or plant tissue) in order to determine the presence, absence or level(s) of the one or more metabolites or other compounds in the sample. Suitable methods include chromatography (e.g., HPLC, gas chromatography, liquid chromatography), mass spectrometry (e.g., MS, MS-MS), LC-MS, enzyme-linked immunosorbent assay (ELISA), antibody linkage, other immunochemical techniques, biochemical or enzymatic reactions or assays, and combinations thereof. The levels of one or more of the recited metabolites or compounds may be determined in the methods of the present invention. For example, the level(s) of one metabolites or compounds, two or more metabolites, three or more metabolites, four or more metabolites, five or more metabolites, six or more metabolites, seven or more metabolites, eight or more metabolites, nine or more metabolites, ten or more metabolites, or compounds etc., including a combination of some or all of the metabolites or compounds including, but not limited to those disclosed herein may be determined and used in such methods.

In some embodiments, a synthetic composition of a plant and a formulation comprising at least one endophytic microbe will cause an increase in the level of a protein in the plant.

In some embodiments, a synthetic composition of a plant and a formulation comprising at least one endophytic microbe will cause a decrease in the level of a protein in the plant.

In some embodiments, a synthetic composition of a plant and a formulation comprising at least one endophytic microbe will cause an increase in the level of expression of a gene in the plant.

In some embodiments, a synthetic composition of a plant and a formulation comprising at least one endophytic microbe will cause a decrease in the level of expression of a gene in the plant.

In some embodiments, a synthetic composition of a plant and a formulation comprising at least one endophytic microbe will cause an increase in the level of a plant hormone.

In some embodiments, a synthetic composition of a plant and a formulation comprising at least one endophytic microbe will cause a modulation in the concentration or amount of a metabolite.

As shown in the Examples and otherwise herein, endophyte-inoculated plants display increased thermal tolerance, herbicide tolerance, drought resistance, insect resistance, fungus resistance, virus resistance, bacteria resistance, male sterility, cold tolerance, salt tolerance, increased yield, enhanced nutrient use efficiency, increased nitrogen use efficiency, increased protein content, increased fermentable carbohydrate content, reduced lignin content, increased antioxidant content, enhanced water use efficiency, increased vigor, increased germination efficiency, earlier or increased flowering, increased biomass, altered root-to-shoot biomass ratio, enhanced soil water retention, or a combination thereof.

Therefore, in an embodiment, the endophytic population is disposed on the surface or on or within a tissue of the seed or seedling in an amount effective to increase the biomass of the plant, or a part or tissue of the plant derived from the seed or seedling. The increased biomass is useful in the production of commodity products derived from the plant. Such commodity products include an animal feed, a fish fodder, a cereal product, a processed human-food product, a sugar or an alcohol. Such products may be a fermentation product or a fermentable product, one such exemplary product is a biofuel. The increase in biomass can occur in a part of the plant (e.g., the root tissue, shoots, leaves, etc.), or can be an increase in overall biomass when compared with a reference agricultural plant. Such increase in overall biomass can be under relatively stress-free conditions. In other cases, the increase in biomass can be in plants grown under any number of abiotic or biotic stresses, including drought stress, salt stress, heat stress, cold stress, low nutrient stress, nematode stress, insect herbivory stress, fungal pathogen stress, complex pathogen stress, and viral pathogen stress.

In another embodiment, the endophytic population is disposed on the surface or within a tissue of the seed or seedling in an amount effective to increase the rate of seed germination when compared with a reference agricultural plant.

In other cases, the microbe is disposed on the seed or seedling in an amount effective to increase the average biomass of the fruit or cob from the resulting plant when compared with a reference agricultural plant.

Plants inoculated with an endophytic population may also show an increase in overall plant height. Therefore, in an embodiment, the present invention provides for a seed comprising an endophytic population that is disposed on the surface or within a tissue of the seed or seedling in an amount effective to increase the height of the plant. For example, the endophytic population is disposed in an amount effective to result in an increase in height of the agricultural plant when compared with a reference agricultural plant. Such an increase in height can be under relatively stress-free conditions. In other cases, the increase in height can be in plants grown under any number of abiotic or biotic stresses, including drought stress, salt stress, heat stress, cold stress, low nutrient stress, nematode stress, insect herbivory stress, fungal pathogen stress, complex pathogen stress, or viral pathogen stress.

In another embodiment, the plant containing the endophyte is able to grown under nutrient stress conditions while exhibiting no difference in the physiological parameter compared to a plant that is grown without nutrient stress. In some embodiments, such a plant will exhibit no difference in the physiological parameter when grown with 2-5% less nitrogen than average cultivation practices on normal agricultural land, for example, at least 5-10% less nitrogen, at least 10-15% less nitrogen, at least 15-20% less nitrogen, at least 20-25% less nitrogen, at least 25-30% less nitrogen, at least 30-35% less nitrogen, at least 35-40% less nitrogen, at least 40-45% less nitrogen, at least 45-50% less nitrogen, at least 50-55% less nitrogen, at least 55-60% less nitrogen, at least 60-65% less nitrogen, at least 65-70% less nitrogen, at least 70-75% less nitrogen, at least 80-85% less nitrogen, at least 85-90% less nitrogen, at least 90-95% less nitrogen, or less, when compared with crop plants grown under normal conditions during an average growing season. In some embodiments, the microbe capable of providing nitrogen-stress tolerance to a plant is diazotrophic. In other embodiments, the microbe capable of providing nitrogen-stress tolerance to a plant is non-diazotrophic.

The host plants inoculated with the endophytic population may also show improvements in their ability to utilize water more efficiently. Water use efficiency is a parameter often correlated with drought tolerance. Water use efficiency (WUE) is a parameter often correlated with drought tolerance, and is the CO 2 assimilation rate per amount of water transpired by the plant. An increase in biomass at low water availability may be due to relatively improved efficiency of growth or reduced water consumption. In selecting traits for improving crops, a decrease in water use, without a change in growth would have particular merit in an irrigated agricultural system where the water input costs were high. An increase in growth without a corresponding jump in water use would have applicability to all agricultural systems. In many agricultural systems where water supply is not limiting, an increase in growth, even if it came at the expense of an increase in water use also increases yield.

When soil water is depleted or if water is not available during periods of drought, crop yields are restricted. Plant water deficit develops if transpiration from leaves exceeds the supply of water from the roots. The available water supply is related to the amount of water held in the soil and the ability of the plant to reach that water with its root system. Transpiration of water from leaves is linked to the fixation of carbon dioxide by photosynthesis through the stomata. The two processes are positively correlated so that high carbon dioxide influx through photosynthesis is closely linked to water loss by transpiration. As water transpires from the leaf, leaf water potential is reduced and the stomata tend to close in a hydraulic process limiting the amount of photosynthesis. Since crop yield is dependent on the fixation of carbon dioxide in photosynthesis, water uptake and transpiration are contributing factors to crop yield. Plants which are able to use less water to fix the same amount of carbon dioxide or which are able to function normally at a low water potential, are more efficient and thereby are able to produce more biomass and economic yield in many agricultural systems. An increased water use efficiency of the plant relates in some cases to an increased fruit/kernel size or number.

Therefore, in one embodiment, the plants described herein exhibit an increased water use efficiency (WUE) when compared with a reference agricultural plant grown under the same conditions. For example, the endophyte may provide an increase in WUE to a plant that is of at least 3%, between 3% and 5%, at least 5%, between 5% and 10%, least 10%, between 10% and 15%, for example at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared with uninoculated plants grown under the same conditions. Such an increase in WUE can occur under conditions without water deficit, or under conditions of water deficit, for example, when the soil water content is less than or equal to 60% of water saturated soil, for example, less than or equal to 50%, less than or equal to 40%, less than or equal to 30%, less than or equal to 20%, less than or equal to 10% of water saturated soil on a weight basis. In some embodiments, the plants inoculated with the endophytic population show increased yield under non-irrigated conditions, as compared to reference agricultural plants grown under the same conditions.

In a related embodiment, the plant comprising endophyte can have a higher relative water content (RWC), than a reference agricultural plant grown under the same conditions.

Formulations for Agricultural Use

The endophyte populations described herein are intended to be useful in the improvement of agricultural plants, and as such, may be formulated with other compositions as part of an agriculturally compatible carrier. It is contemplated that such carriers can include applications such as, but not be limited to: seed treatment, root wash, seedling soak, foliar application, soil inocula, in-furrow application, sidedress application, soil pre-treatment, wound inoculation, drip tape irrigation, vector-mediation via a pollinator, injection, osmopriming, hydroponics, aquaponics, aeroponics. The carrier composition with the endophyte populations, may be prepared for agricultural application as a liquid, a solid, or a gas formulation. Application to the plant may be achieved, for example, as a powder for surface deposition onto plant leaves, as a spray to the whole plant or selected plant element, as part of a drip to the soil or the roots, or as a coating onto the plant element prior to planting. Such examples are meant to be illustrative and not limiting to the scope of the invention.

The formulation useful for these embodiments generally and typically include at least one member selected from the group consisting of a buffer, a tackifier, a microbial stabilizer, a fungicide, an anticomplex agent, an herbicide, a nematicide, an insecticide, a bactericide, a virucide, a plant growth regulator, a rodenticide, a desiccant, and a nutrient.

The carrier can be a solid carrier or liquid carrier, and in various forms including microspheres, powders, emulsions and the like. The carrier may be any one or more of a number of carriers that confer a variety of properties, such as increased stability, wettability, or dispersability. Wetting agents such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination thereof can be included in a composition of the invention. Water-in-oil emulsions can also be used to formulate a composition that includes the purified population (see, for example, U.S. Pat. No. 7,485,451, which is incorporated herein by reference in its entirety). Suitable formulations that may be prepared include wettable powders, granules, gels, agar strips or pellets, thickeners, biopolymers, and the like, microencapsulated particles, and the like, liquids such as aqueous flowables, aqueous suspensions, water-in-oil emulsions, etc. The formulation may include grain or legume products, for example, ground grain or beans, broth or flour derived from grain or beans, starch, sugar, or oil.

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

In an embodiment, the formulation can include a tackifier or adherent. Such agents are useful for combining the complex population of the invention with carriers that can contain other compounds (e.g., control agents that are not biologic), to yield a coating composition. Such compositions help create coatings around the plant or plant element to maintain contact between the endophyte and other agents with the plant or plant element. In one embodiment, adherents are selected from the group consisting of: alginate, gums, starches, lecithins, formononetin, polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinyl acetate, cephalins, Gum Arabic, Xanthan Gum, carragennan, PGA, other biopolymers, Mineral Oil, Polyethylene Glycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, Methyl Cellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate, Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate, Gellan Gum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum Ghatti, and polyoxyethylene-polyoxybutylene block copolymers. Other examples of adherent compositions that can be used in the synthetic preparation include those described in EP 0818135, CA 1229497, WO 2013090628, EP 0192342, WO 2008103422 and CA 1041788, each of which is incorporated herein by reference in its entirety.

It is also contemplated that the formulation may further comprise an anti-caking agent.

The formulation can also contain a surfactant, wetting agent, emulsifier, stabilizer, or anti-foaming agent. Non-limiting examples of surfactants include nitrogen-surfactant blends such as Prefer 28 (Cenex), Surf-N (US), Inhance (Brandt), P-28 (Wilfarm) and Patrol (Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP), Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); and organo-silicone surfactants include Silwet L77 (UAP), Silikin (Terra), Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) and Century (Precision), polysorbate 20, polysorbate 80, Tween 20, Tween 80, Scattics, Alktest TW20, Canarcel, Peogabsorb 80, Triton X-100, Conco NI, Dowfax 9N, Igebapl CO, Makon, Neutronyx 600, Nonipol NO, Plytergent B, Renex 600, Solar NO, Sterox, Serfonic N, T-DET-N, Tergitol NP, Triton N, IGEPAL CA-630, Nonident P-40, Pluronic. In one embodiment, the surfactant is present at a concentration of between 0.01% v/v to 10% v/v. In another embodiment, the surfactant is present at a concentration of between 0.1% v/v to 1% v/v. An example of an anti-foaming agent would be Antifoam-C.

In certain cases, the formulation includes a microbial stabilizer. Such an agent can include a desiccant. As used herein, a “desiccant” can include any compound or mixture of compounds that can be classified as a desiccant regardless of whether the compound or compounds are used in such concentrations that they in fact have a desiccating effect on the liquid inoculant. Such desiccants are ideally compatible with the population used, and should promote the ability of the endophyte population to survive application on the seeds and to survive desiccation. Examples of suitable desiccants include one or more of trehalose, sucrose, glycerol, and methylene glycol. Other suitable desiccants include, but are not limited to, non reducing sugars and sugar alcohols (e.g., mannitol or sorbitol). The amount of desiccant introduced into the formulation can range from about 5% to about 50% by weight/volume, for example, between about 10% to about 40%, between about 15% and about 35%, or between about 20% and about 30%.

In some cases, it is advantageous for the formulation to contain agents such as a fungicide, an anticomplex agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, a bactericide, a virucide, or a nutrient. Such agents are ideally compatible with the agricultural plant element or seedling onto which the formulation is applied (e.g., it should not be deleterious to the growth or health of the plant). Furthermore, the agent is ideally one which does not cause safety concerns for human, animal or industrial use (e.g., no safety issues, or the compound is sufficiently labile that the commodity plant product derived from the plant contains negligible amounts of the compound).

In the liquid form, for example, solutions or suspensions, endophyte populations of the present invention can be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates, or other liquid carriers.

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

The solid carriers used upon formulation include, for example, mineral carriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acid white soil, vermiculite, and pearlite, and inorganic salts such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, ammonium chloride, and calcium carbonate. Also, organic fine powders such as wheat flour, wheat bran, and rice bran may be used. The liquid carriers include vegetable oils (such as soybean oil, maize (corn) oil, and cottonseed oil), glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, etc.

In an embodiment, the formulation is ideally suited for coating of a population of endophytes onto plant elements. The endophytes populations described in the present invention are capable of conferring many fitness benefits to the host plants. The ability to confer such benefits by coating the populations on the surface of plant elements has many potential advantages, particularly when used in a commercial (agricultural) scale.

The endophyte populations herein can be combined with one or more of the agents described above to yield a formulation suitable for combining with an agricultural plant element, seedling, or other plant element. Endophyte populations can be obtained from growth in culture, for example, using a synthetic growth medium. In addition, endophytes can be cultured on solid media, for example on petri dishes, scraped off and suspended into the preparation. Endophytes at different growth phases can be used. For example, endophytes at lag phase, early-log phase, mid-log phase, late-log phase, stationary phase, early death phase, or death phase can be used. Endophytic spores may be used for the present invention, for example but not limited to: arthospores, sporangispores, conidia, chlamadospores, pycnidiospores, endospores, zoospores.

The formulations comprising endophyte populations of the present invention typically contains between about 0.1 to 95% by weight, for example, between about 1% and 90%, between about 3% and 75%, between about 5% and 60%, between about 10% and 50% in wet weight of the population of the present invention. It is preferred that the formulation contains at least about 10 {circumflex over ( )}3 CFU per ml of formulation, for example, at least about 10 {circumflex over ( )}4, at least about 10{circumflex over ( )}5, at least about 10{circumflex over ( )}6, at least about 10{circumflex over ( )}7 CFU, at least about 10{circumflex over ( )}8 CFU per ml of formulation. It is preferred that the formulation be applied to the plant element at about 10{circumflex over ( )}2 CFU/seed, between 10 {circumflex over ( )}2 and 10 {circumflex over ( )}3 CFU, at least about 10 {circumflex over ( )}3 CFU, between 10 {circumflex over ( )}3 and 10 {circumflex over ( )}4 CFU, at least about 10 {circumflex over ( )}4 CFU, between 10 {circumflex over ( )}4 and 10 {circumflex over ( )}5 CFU, at least about 10 {circumflex over ( )}5 CFU, between 10 {circumflex over ( )}5 and 10 {circumflex over ( )}6 CFU, at least about 10 {circumflex over ( )}6 CFU, between 10 {circumflex over ( )}6 and 10{circumflex over ( )}7 CFU, at least about 10{circumflex over ( )}7 CFU, between 10{circumflex over ( )}7 and 10 {circumflex over ( )}8 CFU, or even greater than 10 {circumflex over ( )}8 CFU per seed.

In some embodiments, fungal endophytes may be encapsulated in a fungal host, whether its native host or a heterologous host, before incorporation into a formulation.

Populations of Plant Elements (PEs)

In another embodiment, the invention provides for a substantially uniform population of plant elements (PEs) comprising two or more PEs comprising the endophytic population, as described herein above. Substantial uniformity can be determined in many ways. In some cases, at least 10%, between 10% and 20%, for example, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 90%, at least 90%, between 90% and 95%, at least 95% or more of the PEs in the population, contains the endophytic population in an amount effective to colonize the plant disposed on the surface of the PEs. In other cases, at least 10%, between 10% and 20%, for example, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 90%, at least 90%, between 90% and 95%, at least 95% or more of the plant element s in the population, contains at least 1, between 10 and 10, 10, between 10 and 100, or 100 CFU on the plant element surface or per gram of plant element, for example, between 100 and 200 CFU, at least 200 CFU, between 200 and 300 CFU, at least 300 CFU, between 300 and 1,000 CFU, at least 1,000 CFU, between 1,000 and 3,000 CFU, at least 3,000 CFU, between 3,000 and 10,000 CFU, at least 10,000 CFU, between 10,000 and 30,000 CFU, at least 30,000 CFU, between 30,000 and 100,000 CFU, at least 100,000 CFU, between 100,000 and 300,000 CFU, at least 300,000 CFU, between 300,000 and 1,000,000 CFU, or at least 1,000,000 CFU per plant element or more.

In a particular embodiment, the population of plant elements is packaged in a bag or container suitable for commercial sale. Such a bag contains a unit weight or count of the plant elements comprising the endophytic population as described herein, and further comprises a label. In an embodiment, the bag or container contains at least 100 plant elements, between 100 and 1,000 plant elements, 1,000 plant elements, between 1,000 and 5,000 plant elements, for example, at least 5,000 plant elements, between 5,000 and 10,000 plant elements, at least 10,000 plant elements, between 10,000 and 20,000 plant elements, at least 20,000 plant elements, between 20,000 and 30,000 plant elements, at least 30,000 plant elements, between 30,000 and 50,000 plant elements, at least 50,000 plant elements, between 50,000 and 70,000 plant elements, at least 70,000 plant elements, between 70,000 and 80,000 plant elements, at least 80,000 plant elements, between 80,000 and 90,000, at least 90,000 plant elements or more. In another embodiment, the bag or container can comprise a discrete weight of plant elements, for example, at least 1 lb, between 1 and 2 lbs, at least 2 lbs, between 2 and 5 lbs, at least 5 lbs, between 5 and 10 lbs, at least 10 lbs, between 10 and 30 lbs, at least 30 lbs, between 30 and 50 lbs, at least 50 lbs, between 50 and 70 lmbs, at least 70 lbs or more. The bag or container comprises a label describing the plant elements and/or said endophytic population. The label can contain additional information, for example, the information selected from the group consisting of: net weight, lot number, geographic origin of the plant elements, test date, germination rate, inert matter content, and the amount of noxious weeds, if any. Suitable containers or packages include those traditionally used in plant seed commercialization. The invention also contemplates other containers with more sophisticated storage capabilities (e.g., with microbiologically tight wrappings or with gas- or water-proof containments).

In some cases, a sub-population of seeds comprising the endophytic population is further selected on the basis of increased uniformity, for example, on the basis of uniformity of microbial population. For example, individual plant elements of pools collected from individual cobs, individual plants, individual plots (representing plants inoculated on the same day) or individual fields can be tested for uniformity of microbial density, and only those pools meeting specifications (e.g., at least 80% of tested plant elements have minimum density, as determined by quantitative methods described elsewhere) are combined to provide the agricultural seed sub-population.

The methods described herein can also comprise a validating step. The validating step can entail, for example, growing some plant elements collected from the inoculated plants into mature agricultural plants, and testing those individual plants for uniformity. Such validating step can be performed on individual seeds collected from cobs, individual plants, individual plots (representing plants inoculated on the same day) or individual fields, and tested as described above to identify pools meeting the required specifications.

In some embodiments, methods described herein include planting a synthetic composition described herein. Suitable planters include an air seeder and/or fertilizer apparatus used in agricultural operations to apply particulate materials including one or more of the following, seed, fertilizer and/or inoculants, into soil during the planting operation. Seeder/fertilizer devices can include a tool bar having ground-engaging openers thereon, behind which is towed a wheeled cart that includes one or more containment tanks or bins and associated metering means to respectively contain and meter therefrom particulate materials.

In certain embodiments, a composition described herein may be in the form of a liquid, a slurry, a solid, or a powder (wettable powder or dry powder). In another embodiment, a composition may be in the form of a seed coating. Compositions in liquid, slurry, or powder (e.g., wettable powder) form may be suitable for coating plant elements. When used to coat plant elements, the composition may be applied to the plant elements and allowed to dry. In embodiments wherein the composition is a powder (e.g., a wettable powder), a liquid, such as water, may need to be added to the powder before application to a seed.

In still another embodiment, the methods can include introducing into the soil an inoculum of one or more of the endophyte populations described herein. Such methods can include introducing into the soil one or more of the compositions described herein. The inoculum(s) or compositions may be introduced into the soil according to methods known to those skilled in the art. Non-limiting examples include in-furrow introduction, spraying, coating seeds, foliar introduction, etc. In a particular embodiment, the introducing step comprises in-furrow introduction of the inoculum or compositions described herein.

In an embodiment, plant elements may be treated with composition(s) described herein in several ways but preferably via spraying or dripping. Spray and drip treatment may be conducted by formulating compositions described herein and spraying or dripping the composition(s) onto a seed(s) via a continuous treating system (which is calibrated to apply treatment at a predefined rate in proportion to the continuous flow of seed), such as a drum-type of treater. Batch systems, in which a predetermined batch size of seed and composition(s) as described herein are delivered into a mixer, may also be employed.

In another embodiment, the treatment entails coating plant elements. One such process involves coating the inside wall of a round container with the composition(s) described herein, adding plant elements, then rotating the container to cause the plant elements to contact the wall and the composition(s), a process known in the art as “container coating.” Plant elements can be coated by combinations of coating methods. Soaking typically entails using liquid forms of the compositions described. For example, plant elements can be soaked for about 1 minute to about 24 hours (e.g., for at least 1 min, between 1 and 5 min, 5 min, between 5 and 10 min, 10 min, between 10 and 20 min, 20 min, between 20 and 40 min, 40 min, between 40 and 80 min, 80 min, between 80 min and 3 hrs, 3 hrs, between 3 hrs and 6 hrs, 6 hr, between 6 hrs and 12 hrs, 12 hr, between 12 hrs and 24 hrs, 24 hrs).

Population of Plants and Agricultural Fields

A major focus of crop improvement efforts has been to select varieties with traits that give, in addition to the highest return, the greatest homogeneity and uniformity. While inbreeding can yield plants with substantial genetic identity, heterogeneity with respect to plant height, flowering time, and time to seed, remain impediments to obtaining a homogeneous field of plants. The inevitable plant-to-plant variability is caused by a multitude of factors, including uneven environmental conditions and management practices. Another possible source of variability can, in some cases, be due to the heterogeneity of the endophyte population inhabiting the plants. By providing endophyte populations onto plant reproductive elements, the resulting plants generated by germinating the plant reproductive elements have a more consistent endophyte composition, and thus are expected to yield a more uniform population of plants.

Therefore, in another embodiment, the invention provides a substantially uniform population of plants. The population can include at least 10 plants, between 10 and 100 plants, for example, at least 100 plants, between 100 and 300 plants, at least 300 plants, between 300 and 1,000 plants, at least 1,000 plants, between 1,000 and 3,000 plants, at least 3,000 plants, between 3,000 and 10,000 plants, at least 10,000 plants, between 10,000 and 30,000 plants, at least 30,000 plants, between 30,000 and 100,000 plants, at least 100,000 plants or more. The plants are derived from plant reproductive elements comprising endophyte populations as described herein. The plants are cultivated in substantially uniform groups, for example in rows, groves, blocks, circles, or other planting layout.

The uniformity of the plants can be measured in a number of different ways. In one embodiment, there is an increased uniformity with respect to endophytes within the plant population. For example, in one embodiment, a substantial portion of the population of plants, for example at least 10%, between 10% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 90%, at least 90%, between 90% and 95%, at least 95% or more of the plant elements or plants in a population, contains a threshold number of an endophyte population. The threshold number can be at least 10 CFU, between 10 and 100 CFU, at least 100 CFU, between 100 and 300 CFU, for example at least 300 CFU, between 300 and 1,000 CFU, at least 1,000 CFU, between 1,000 and 3,000 CFU, at least 3,000 CFU, between 3,000 and 10,000 CFU, at least 10,000 CFU, between 10,000 and 30,000 CFU, at least 30,000 CFU, between 30,000 and 100,000 CFU, at least 100,000 CFU or more, in the plant or a part of the plant. Alternatively, in a substantial portion of the population of plants, for example, in at least 1%, between 1% and 10%, at least 10%, between 10% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 90%, at least 90%, between 90% and 95%, at least 95% or more of the plants in the population, the endophyte population that is provided to the seed or seedling represents at least 0.1%, between 0.1% and 1% at least 1%, between 1% and 5%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 70%, at least 70%, between 70% and 80%, at least 80%, between 80% and 90%, at least 90%, between 90% and 95%, at least 95%, between 95% and 99%, at least 99%, between 99% and 100%, or 100% of the total endophyte population in the plant/seed.

In an embodiment, there is increased genetic uniformity of a substantial proportion or all detectable endophytes within the taxa, genus, or species of a component relative to an uninoculated control. This increased uniformity can be a result of the endophyte being of monoclonal origin or otherwise deriving from a population comprising a more uniform genome sequence and plasmid repertoire than would be present in the endophyte population a plant that derives its endophyte community largely via assimilation of diverse soil symbionts.

In another embodiment, there is an increased uniformity with respect to a physiological parameter of the plants within the population. In some cases, there can be an increased uniformity in the height of the plants when compared with a population of reference agricultural plants grown under the same conditions. For example, there can be a reduction in the standard deviation in the height of the plants in the population of at least 5%, between 5% and 10%, for example, at least 10%, between 10% and 15%, at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60% or more, when compared with a population of reference agricultural plants grown under the same conditions. In other cases, there can be a reduction in the standard deviation in the flowering time of the plants in the population of at least 5%, between 5% and 10%, for example, at least 10%, between 10% and 15%, at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60% or more, when compared with a population of reference agricultural plants grown under the same conditions.

Commodity Plant Products

The present invention provides a commodity plant product, as well as methods for producing a commodity plant product, that is derived from a plant of the present invention. As used herein, a “commodity plant product” refers to any composition or product that is comprised of material derived from a plant, seed, plant cell, or plant element of the present invention. Commodity plant products may be sold to consumers and can be viable or nonviable. Nonviable commodity products include but are not limited to nonviable plant elements and grains; processed seeds, seed parts, and plant elements; dehydrated plant tissue, frozen plant tissue, and processed plant tissue; seeds and plant elements processed for animal feed for terrestrial and/or aquatic animal consumption, oil, meal, flour, flakes, bran, fiber, paper, tea, coffee, silage, crushed of whole grain, and any other food for human or animal consumption such as the fruit or other edible portion of the plant; and biomasses and fuel products; and raw material in industry.

Industrial uses of oils derived from the agricultural plants described herein include ingredients for paints, plastics, fibers, detergents, cosmetics, lubricants, and biodiesel fuel. Plant oils may be split, inter-esterified, sulfurized, epoxidized, polymerized, ethoxylated, or cleaved. Designing and producing plant oil derivatives with improved functionality and improved oliochemistry is a rapidly growing field. For example, a mixture of triglycerides is usually split and separated into pure fatty acids, which are then combined with petroleum-derived alcohols or acids, nitrogen, sulfonates, chlorine, or with fatty alcohols derived from fats and oils to produce the desired type of oil or fat. Commodity plant products also include industrial compounds, such as a wide variety of resins used in the formulation of adhesives, films, plastics, paints, coatings and foams.

Although the present invention has been described in detail with reference to examples below, it is understood that various modifications can be made without departing from the spirit of the invention. For instance, while the particular examples below may illustrate the methods and embodiments described herein using a specific plant, the principles in these examples may be applied to any agricultural crop. Therefore, it will be appreciated that the scope of this invention is encompassed by the embodiments of the inventions recited herein and the specification rather than the specific examples that are exemplified below. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety.

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