Compositions containing endophytes for improving plant nutrition, growth and performance and methods of using the compositions

Abstract

Disclosed are endophyte inoculant compositions, methods for making such compositions, methods for using such compositions, and physiologically altered plants treated with such compositions. The endophyte inoculant compositions may include one or more of the endophytic species WW5, WW6, WW7, and PTD1, and when applied to a non-native host plant, promote plant mineral nutrient acquisition and uptake, vigor, health, growth, and yield.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 390,387, filed July 19, 2022, which is incorporated herein by reference in its entirety. Technical Field The technology relates to preparations, compositions, and methods for improving plant performance. The novel compositions include non-native endophytes that are applied to plants and, when incorporated into plants, result in measurable plant benefits, such as increased nitrogen and other macro- and micronutrient uptake, nutrient use efficiency, photosynthesis, growth, yield, carbon sequestration, resistance to biotic and biotic stressors, disease resistance (excluding biological control mechanisms), and overall plant health. The technology has broad applications to plants generally, including crop management applications, reduced fertilizer and pesticide use, reduced carbon footprint of crops, improved mineral nutritional status of crops to reduce plant pathogen burden and therefore pesticide use, improved food quality and safety, and improved plant health and biomass growth rates for use in landscaping and ornamentals, as well as forestry. [Background technology]

[0002] background Endophytes are microorganisms (e.g., fungi and bacteria) that can form a symbiotic relationship with trees and plants, which can improve plant growth, fruit and seed yield, general health, and other characteristics. For example, Aghai Matthew M., Khan Zareen, Joseph Matthew R., Stoda Aubrey M., Sher Andrew W., Ettl Gregory J., Doty Sharon L. (2019) The Effect of Microbial Endophyte Consortia on Pseudotsuga menziesii and Thuja plicata Survival, Growth, and Physiology Across Edaphic Gradients.Frontiers in Microbiology.10 doi10.3389 / fmicb.2019.01353;Rho Hyungmin, Van Epps Victor, Wegley Nicholas, Doty Sharon L., Kim Soo-Hyung, 2018. Salicaceae Endophytes Modulate Stomatal Behavior and Increase Water Use Efficiency in Rice. Frontiers in Plant Science, 9 See doi10.3389 / fpls.2018.00188. After inoculation, endophytes can be incorporated into plant tissues and become a genetic part of the plant. Endophytes can penetrate between and within plant cells and become incorporated into plant tissues. Once incorporated into plants and even living on the surface of plant roots, endophytes can improve the plant's nutritional status by providing and enhancing the supply of macronutrients such as nitrogen, potassium, phosphorus, calcium, and sulfur, and micronutrients such as iron, zinc, and magnesium, increasing photosynthesis, improving water-use efficiency, and enhancing tolerance to biotic and abiotic stresses.

[0003] Despite the benefits endophytes bring, their practical use in agriculture is very limited. Consumers and farmers are becoming more aware of the damage that chemical fertilizers and pesticides cause to the environment and are turning to alternatives that are more natural and increase the long-term sustainability of agricultural, livestock and forestry production. Summary of the Invention [Means for solving the problem]

[0004] Summary of the Invention The present disclosure provides novel and inventive compositions comprising bacterial endophytes for application to non-native plant species, methods for making and using the compositions, and the resulting novel plants. Specifically, one or more of the following endophyte strains can be included in an inoculant composition for application to non-native host plants:

[0005] [Table 1]

[0006] The species identified in the table above were submitted to the ARS Culture Collection (NRRL, 1815 N. University Street, Peoria, IL 61604) on November 9, 2021. The 16S rDNA sequences of each of the endophytic strains are listed in Figures 1-4 and can be found in the Sequence Listing XML file submitted with this application. These strains were discovered in the branches of willow and poplar, plants that do not form root nodules. Thus, the presence of a potential diazotrophic endophyte living within the branches of all of these plant species was unexpected. In the process of screening a large library of endophytic tree bacteria, the set of endophytic bacterial strains identified above was selected after being found to be able to grow in nitrogen-free and nitrogen-limited media and to enhance nitrogen (N) and phosphorus (P) uptake from roots to shoots. These strains, when inoculated, were further found to provide additional root-to-shoot uptake of mineral nutrients, and these strains are present in a wide variety of crop plants that have been specifically tested and screened. These unique strains were then identified and isolated specifically for this purpose. Furthermore, by optimizing the most efficient nitrogen-fixing, ammonium-excreting strains, along with specific strains that mobilize insoluble forms of macronutrients (e.g., phosphorus), the resulting novel inoculant formulation was found to synergistically increase plant acquisition of macronutrient ions and plant-required micronutrient ions. Furthermore, application of the novel microbial inoculants disclosed herein to non-native plants resulted in increased growth, biomass, and yield in crop plants. This novel inoculant formulation improved crop plant yield and quality under both deficient, competent, and highly optimized agricultural conditions. These endophytic strains and combinations thereof are not typically present in crop plant varieties and soils used in agricultural production.

[0007] Of the above endophytic strains, three strains have been proven to be newly discovered endophytic species: Sphingobium sp. (WW5), Curtobacterium salicaceae (WW7), and Rhizobium populi (PTD1). Examples 1B-1C and 74 present experimental results demonstrating that the WW5, WW7, and PTD1 endophytic strains are novel species.

[0008] Whole genome sequencing and safety analysis The genomes of all four endophytic strains were fully sequenced and analyzed for genes encoding proteins and / or phenotypes known to be harmful to plants or animals. To analyze the entire genome sequences of the endophytic strains, we used the method described by Varghese et al. (See https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4538840 / ). This method reliably classifies strains as safe or potential pathogens by comparing genome sequences with those of known harmful and pathogenic microorganisms. If the organism falls into a cluster representative of human or plant pathogens, the presence of virulence factors and antibiotic resistance genes is assessed. No close relationship to such pathogenic species was found for any of these strains. To further confirm that these strains are safe for crops, the genomes of the endophytic strains were analyzed for genes known to be associated with plant and human pathogens. No pathogenesis-related genes were found in any of the four selected endophytic strains.

[0009] Biochemical analysis for safety Additionally, endophytic strains were analyzed to determine whether they exhibited biochemical similarities to known pathological biochemistry in mammalian bacterial pathogens. The MacConkey agar test is often used to determine whether a bacterial species is Gram-negative. Bacteria that grow well on this agar for 24 hours are likely Gram-negative. MacConkey agar also uses a neutral pH indicator to test for the ability of bacteria to ferment lactose sugar. Bacterial fermentation of lactose sugar lowers the pH, turning colonies pink and the surrounding medium cloudy. Bacteria that cannot utilize lactose use peptone to produce ammonia, which raises the pH and turns colonies white or colorless. Some microorganisms (e.g., Klebsiella and Enterobacter) form mucoid colonies that appear very moist and sticky. These microorganisms produce a mucoid capsule from the metabolism of lactose sugar in the agar medium.

[0010] A hemolysis assay (blood agar lysis) was also performed on endophytic strains over a 24-hour period to test the pathogenic bacteria's ability to produce toxins that lyse red blood cells. The results of this test can be no growth, gamma hemolysis (growth without hemolysis), alpha hemolysis (partial hemolysis and dark green coloration), or beta hemolysis (yellow clear zones of complete hemolysis). Mannitol salt agar is used to confirm the presence of Staphylococci, as it is harmful to most bacteria other than Staphylococci.

[0011] When grown on MacConkey agar, the endophytic strains produced yellow-brown colonies that were neither wet nor sticky. Thus, the endophytic strains were negative for lactose fermentation, lactone fermentation, and mucoid capsule production. The results of the MacConkey agar test indicated that the endophytic strains did not share the following characteristics with pathogenic species: (1) they did not share lactose fermentation with pathogenic species such as Escherichia coli, Enterobacter, or Klebsiella; and (2) they did not share peptone utilization with pathogenic species such as Salmonella, Proteus species, Yersinia, Pseudomonas aeruginosa, or Shigella, nor did they share mucoid capsule production with pathogenic species such as Klebsiella or Enterobacter. Mannitol salt agar results demonstrated growth inhibition in the endophytic strains, suggesting that the endophytes are unable to ferment mannitol and therefore do not share this phenotype with any Staphylococcus species. Testing using a hemolysis assay showed that all four strains were gamma-hemolytic (they grew but did not lyse blood cells). Taken together, these results confirm that the endophytic strains share no similarities with mammalian bacterial pathogens and that the four endophytic bacterial strains are safe for crop and other applications. The results of the above biochemical assays are shown below in Figure 5.

[0012] The screened and identified endophyte strains were developed as stocks by a novel fermentation growth method for use in a novel inoculum formulation. This formulation is used to treat and improve non-native host plant species (in which the endophyte does not naturally occur). The inoculant compositions of the present technology contain additional ingredients that promote long-term stability (long shelf life), delivery, colonization and efficacy in host plants. The inoculant compositions of the present technology may include liquid seed treatments, seed coatings, lyophilized powder reconstitutable seed treatments, encapsulated dry beads, foliar sprays, in-furrow liquid products and other formulations.

[0013] Compositions containing non-native endophytes may be applied "heterologously" to various plant species and agricultural crops, meaning that the applied endophyte strain is not naturally present in the treated host plant. Important agricultural, ornamental, and other host plant varieties and species can be heterologously treated with the endophyte strains of the present technology. A fundamental principle of plant breeding, including transgenic plant breeding, begins with the creation of "clean" germplasm, which typically means the absence of microorganisms. For this reason, most propagation material of annual crops is microbiologically free. For perennial plants, modern breeding methods employ various purification procedures, which also result in microorganism-free germplasm. Contrary to conventional practice, the technology disclosed herein provides bacterial endophytes to heterologous monocotyledonous and dicotyledonous plants, resulting in plants that are superior to plants that do not contain these beneficial endophyte strains.

[0014] In some embodiments, the host plant treated with the endophyte inoculant disclosed herein can be a plant cultivated by humans for food, feed, fiber, fuel, and / or industrial purposes, such as wheat (e.g., Triticum aestivum, Triticum spelta, Triticum monococcum, Triticum dicoccum, Triticum durum, Triticum turgidum, and Triticum rigidum), corn (e.g., Zea mays, including subspecies such as Zea mays indenata, Zea mays indurata, Zea mays amylacea, Zea mays saccharata, and Zea mays everta), soybean (e.g., Glycine max), cotton (e.g., Gossypium arboretum, Gossypium herbaceum, Gossypium hirsutum, and Gossypium barbadense), broccoli (e.g., Brassica oleracea italica), kale (e.g., Brassica oleracea acephala), tomato (e.g., Solanum lycopicum), rice (e.g., Oryza sativa), barley (e.g., Hordeum vulgare), beet (e.g., Beta vulgaris), pea (e.g., Pisum sativum), potato (e.g., Solanum tuberosum), sugarcane (e.g., Saccharum officinarum), banana (e.g., Musa acuminata and Musa balbisiana), spinach (e.g., Spinacia oleracea), lettuce (e.g., Lactuca sativa), zucchini (e.g., Cucurbita pepo), pepper (e.g., Capsicum annuum), rapeseed (e.g., Brassica napus), alfalfa (e.g., Medicago sativa), conifers (e.g., Pseudotsuga menziesii, Pinus taeda, and Alnus rubra), Salicaceae (e.g., Salix sitchensis, Salixnigra), Populus (e.g., Populus trichocarpa, Populus nigra, Populus deltoides, and all hybrid poplar hybrids DxT, TxN, DxNxT, DxN, etc.), Eucalyptus (e.g., Eucalyptus rostrata, Eucalyptus tereticornas, Eucalyptus cladocalyx, and Eucalyptus globulus), Rosaceae (e.g., Malus domestica, Pyrus communis, Prunus avium, Prunus dulcis, Prunus persica, Prunus armeniaca, and Prunus americana). Endophytic strains can be applied in a variety of settings, including host plants grown in greenhouse or field conditions and various cultivation methods. The endophytic strains may be applied mechanically, manually, through irrigation, by artificial inoculation, and generally by placement on the plant, plant element, plant tissue, seed, seedling, or plant growth medium so that the treatment is present on the plant, plant element, plant tissue, seed, seedling, or plant growth medium in a manner not found in nature.

[0015] In various embodiments, this heterologous application may be to a non-native host plant variety or plant developmental stage where the endophyte strain does not naturally occur, or in a growing environment where the same endophyte strain does not naturally occur. For example, an endophyte strain naturally occurring in the stem tissue of a willow would be considered heterologous to any tissue of a corn, spring wheat, cotton, or soybean plant that naturally lacks the endophyte strain. In some embodiments, non-naturally occurring application can mean that the non-native endophyte is present in the tissue of the host plant, or in the tissue of a different plant element, tissue, or cell type, or in a physical location of the plant other than its naturally occurring physical location. For example, if an endophyte is normally found in the root tissue of a plant element but not in the leaf tissue, the endophyte species or strain, when heterologously disposed, would be applied to the leaves.

[0016] "Host plant" includes any plant to which a non-native endophyte can be heterologously applied, particularly an agriculturally important plant. Detectably containing an endophyte strain in a host plant can result in improved growth characteristics, stress tolerance, and / or other properties of the host plant. The endophyte strain also improves agronomic traits in crop plant varieties, such as yield and nutritional composition of harvested parts of the crop plant, compared to untreated plants that do not have the non-native endophyte strain. A non-native endophyte can colonize a host plant or an element thereof if the endophyte is stably detectable within the host plant or an element thereof over a period of time, such as days, weeks, months, or years.

[0017] Specific formulations of heterologous endophytic strains provide unique inoculant benefits to plant hosts, improving plant growth, health, yield, and quality; reducing plant resistance to biotic and abiotic stresses; and preventing infection through induced plant resistance to plant / seed diseases and pests. The ability of endophytic strains to colonize non-native plant hosts has been experimentally demonstrated by a variety of methods, including polymerase chain reaction (PCR) analysis for specific genetic markers in host plant tissues and 16S sequencing of each endophytic strain; detection of specifically selected diazotrophic colony-forming units (CFUs) isolated from surface-sterile host plant tissues; gene RFP or GFP fluorescent marker tagging for localization by laser fluorescence confocal microscopy; and other appropriate methods. The presence of endophytic strain genetic material in host plants, as well as other physiological measurements such as increased CFU and chlorophyll content in host plant tissues, enhanced root branching and growth, increased shoot biomass, and increased mineral nutrient ion content in leaves, all demonstrate successful host plant colonization by endophytic strains.

[0018] Furthermore, biochemical and molecular analyses have revealed the mode of action by which endophytic strains improve the performance, yield, mineral nutrition, and health of host plants. These analyses include acetylene reduction assays, 15These include, but are not limited to, N isotope dilution assays, growth in nitrogen-free media, measurement of exogenous ammonia and ammonium production using quantitative probe plus meters and chemical test kits, ICPMS ion concentration profiling of leaf tissue, quantification of exogenous insoluble phosphorus mobilization in liquid cultures using fluorescent dyes in spectrophotometric plate readers, measurement of Fe-siderophore production on CAS plates, and bioinformatics and genomics of the genetic pathways responsible for these biochemical traits.Endophyte colonization of non-native heterologous host plants results in measurable improvements in nitrogen and other macro- and micronutrient uptake, nutrient use efficiency, photosynthesis, growth, yield, carbon sequestration, tolerance to biotic and abiotic stressors, and general plant health.

[0019] Host plants containing one or more endophyte strains in their tissues exhibit a detectable change in the content of at least one nutritional trait, and this improvement can be passed on through asexual propagation (e.g., stem, root, or leaf cuttings, layering, division, separation, grafting, budding, micropropagation) or seeds. Progeny resulting from an endophyte-associated host plant or its tissues have one or more endophyte strains in their tissues and can exhibit increased at least one nutritional quality trait compared to untreated plants of the same species. Progeny of cultivars produced by rootstock (rootstock), cuttings, or tissue culture can exhibit such phenotypic traits and improved performance due to the presence of heterologous endophyte strains in their tissues. The level of a nutritional trait can be measured in asexually propagated progeny, seeds, or progeny grown from seeds of the host plant and compared to the level of the nutritional quality trait in equivalent tissue of a reference agricultural plant that does not contain the heterologous endophyte strain. The presence or improvement of a phenotypic trait in the asexually propagated or germinated progeny of a host plant may be measured by a variety of methods, including, but not limited to, increased height, overall biomass, root mass, shoot biomass, seed germination rate, seedling survival rate, photosynthetic efficiency, seed / fruit number or mass, fruit yield, leaf chlorophyll content, photosynthetic rate, root length, abiotic stress tolerance, biotic stress tolerance, disease resistance, wilt recovery, turgor pressure, or any combination thereof, compared to untreated control plants of the same species grown under similar conditions.

[0020] The endophytic strains WW5, WW6, WW7, and PTD1 selected for use in host plant treatment were developed as stocks through a microbial fermentation process. Microbial stock maintenance and fermentation methods may be used to increase nitrogenase gene expression and maintain the plasmid in an active form. The endophytic strains may be grown in nitrogen-limited bacterial growth media with other special properties (e.g., chelated iron and / or magnesium) to enhance atmospheric nitrogen fixation and confer other beneficial characteristics of the endophytic strains. Growing and fermenting the endophytic strains in nitrogen-limited medium (NLM: a microbiological medium containing multiple carbon sources, buffer salts, essential metals, acidification inhibitors, and a limited nitrogen source from amino acids) or nitrogen-free growth medium (NFM: a microbiological medium containing multiple carbon sources, buffer salts, essential metals, and acidification inhibitors, but no nitrogen source) may induce upregulation of nitrogenase, thereby enhancing the endophytic strain's absorption and assimilation of atmospheric N2. For an example of nitrogen-free medium, see, for example, RJ Rennie, "A single medium for the isolation of acetylene-reducing (dinitrogen-fixing) bacteria from soils," Canadian Journal of Microbiology, vol. 27, no. 1, pp. 8-14, 1981. Such upregulation results in higher levels of nitrogenase genes (e.g., Nif H, D, K, E, N, B), which can be measured by PCR analysis. In another embodiment, a rich medium, which is a microbiological medium containing higher concentrations of at least one carbon source, buffer salts, higher concentrations of amino acids, and nitrogen than NFM or NLM medium, may be used for fermenting endophytic strains.

[0021] The fermentation broth used to ferment the endophytic strain may contain various components that enable the growth and health of the endophytic strain during the fermentation process. The nitrogen-free or nitrogen-limited medium used in the fermentation process may contain one or more salts, such as sodium chloride, phosphates (e.g., monopotassium phosphate, dipotassium phosphate, and other phosphates), sulfates (e.g., MgSO), chloride salts (e.g., CaCl), and other suitable salts, but excludes nitrates, ammonium salts, and other nitrogen sources. The fermentation broth may further contain other suitable components, such as yeast extract, agar, and other suitable ingredients. The resulting composition may be used as a liquid composition for treating host plants. For examples of nitrogen-limited media, see, for example, R. J. Rennie, "A single medium for the isolation of acetylene-reducing (dinitrogen-fixing) bacteria from soils," Canadian Journal of Microbiology, vol. 27, no. 1, pp. 8-14, 1981. To promote the upregulation of microbial nitrogenase genes in endophytic strains, the nitrogen-free medium or nitrogen-limited medium may be substantially free of nitrogen. However, after fermentation in NLM broth, the resulting novel composition contains 30-100 mg NH3 or NH4. + / The fermentation composition may contain one or more of the following: nitrogen components in limited amounts, such as within the physiological range of L; amino acids, such as glutamic acid, glutamine, and histidine; other nitrogen-containing compounds, such as nitrates, nitrites, and carbamates; and other nutrients. Figure 6 shows specific examples of other components that may be included in the fermentation composition.

[0022] At least one endophytic strain (WW7) produces a series of organic acids or other compounds (malate and citrate) that mobilize insoluble phosphorus (P). The endophyte lives extracellularly within the apoplast between plant cells in the vascular bundles in roots, stems, stalks, and branches. The endophyte in stems and trunks exuding these compounds also helps prevent P from binding with other metals. This and other strains assist in the solubilization and conversion of potassium (K) and other macronutrient cations to soluble forms, as well as P solubilization and acquisition by the endophyte and roots. The mobility of P and K relative to the roots is affected by the inoculated bacterial endophyte through acidification, chelation, and ion exchange reactions.

[0023] Some endophytic strains (WW7, WW5, WW6) also produce exogenous extracellular iron siderophore compounds, which plants further excrete through their roots to convert insoluble micronutrient mineral ions or metals (e.g., iron, magnesium, zinc, copper, nickel, manganese), calcium, etc., from the soil. 2 + and helps to keep them mobile within the root.

[0024] Combinations of endophyte strains, including co-fermenting combinations of two or more endophyte strains disclosed herein, can be applied to host plants to provide increased or additional benefits compared to those provided by the application of a single endophyte. For example, one endophyte strain that induces a benefit in a host plant may also induce that benefit in a plant colonized with another endophyte strain that induces the same or additional benefit in the host plant. When two or more endophyte strains are heterologously applied to the same host plant, the host plant may experience significant improvements beyond the expected trait improvement in certain nutritional traits, growth traits, stress tolerance, and overall health of the host plant, demonstrating synergistic effects of application of multiple endophyte strains to a host plant, including synergistic increases in increased nitrogen fixation in combination with endophyte strains that increase nitrogen assimilation in the host plant. Examples 41-51 provide data demonstrating synergistic effects in heterologous application of multiple endophyte strains. The combinations of endophyte strains disclosed herein do not exhibit the incompatibilities in host plants that may occur with endophyte strains other than those disclosed herein.

[0025] The inoculant composition may contain one or more additional ingredients to improve the performance of the heterologous endophyte strain and promote effective application and colonization of various host plants. The endophyte strains of the present technology are capable of heterologously colonizing non-native host plants. Molecular and microbiological analyses performed on treated host plant tissues demonstrate that the endophyte strains heterologously applied to non-native host plants via the inoculant composition of the present technology colonized the host plants and established themselves within the host plant tissues.

[0026] composition The inoculant compositions of the present technology are provided in liquid suspensions, seed treatments and coatings, foliar sprays, reconstitutable freeze-dried formulations, and solid forms (e.g., in-furrow, granular spray-dried / fluid-dried beads). The inoculant compositions of the present invention may comprise heterologous endophyte strains WW5, WW6, WW7, PTD1, and combinations thereof. More specifically, the inoculant compositions may comprise an effective amount of one or more of the WW5, WW6, WW7, and PTD1 strains, and one or more additional components that stabilize and improve the uptake and survival of the endophyte strains and enable practical use and application to seeds, roots, stems, leaves, flowers, bulbs, and other structures of non-native host plants. In some embodiments, the compositions of the present technology may include additional endophyte or microbial species, such as additional beneficial Rhizobium strains, Curtobacterium species, Mycorrhizae species, Bacillus species, Priestia species, Azotobacter species, Azospirillum species, Sphingobium species, Herbiconjux species, Rhanella species, Pseudomonas species, or biocontrol bacterial species (e.g., Erwinia, Rhanella, Pseudomonas, Bacillus, Paraburkholderia) endophytic yeast strains and other beneficial microbial strains. In some embodiments, the inoculant composition may include the endophyte Rhodotorula graminis yeast strain WP1. The inoculant compositions of the present invention provide enhanced plant vigor, health, growth, yield and abiotic and biotic stress tolerance.

[0027] The composition may include one or more components that facilitate the delivery, preservation, and / or efficacy of the applied endophyte strain, and may include surfactants, buffers, carriers, adhesives, microbial stabilizers, mineral or clay granules, nutrients, excipients, humectants, and / or salts. Additional components may exclude amines, amides, and other nitrogen-containing compounds to maintain a low-nitrogen or substantially nitrogen-free environment for the endophyte strain.

[0028] The compositions may be formulated to be shelf-stable, including liquid, suspension, and solid formulations. Shelf-stable formulations include suspension, dry, and powder formulations, as well as formulations containing dried endophyte strains. The compositions may be shelf-stable for at least three weeks or longer under predetermined conditions. For example, the compositions may be stable for ten weeks or longer at various temperatures, including low temperatures (near freezing), sustained high temperatures, or room temperature under standard temperature and pressure (STP) conditions. In some instances, liquid concentrate formulations of endophyte fermentate or inoculant may be prepared using a microbial filter, light heating to evaporate water from the liquid, or bulk centrifugation to create a semi-solid bacterial paste.

[0029] The formulation may include one or more dried endophyte strains, in which the moisture content of the endophyte strains has been reduced to 30% or less compared to the undried endophyte strains. In some embodiments, one or more endophyte strains included in the inoculant composition may be freeze-dried. In other embodiments, the endophyte strains may be dried using other methods, such as air-drying, (complete) drying, and / or spray drying. The dried endophyte strains in the inoculant composition may enhance the stability of the endophyte strains therein. In some embodiments, a formulation comprising a dried endophyte strain may be substantially stable at temperatures between about -20°C and about 50°C for at least about four weeks and up to one year or more. The dried endophyte may be used in a variety of formulations that can be administered to a host plant. In some embodiments, the dried endophyte may be used in encapsulated granules. The dried endophyte formulation may be mixed with a dry granular solid or seed, and then the dry formulation may be encapsulated using a binder such as hydroxypropyl cellulose to bind the endophyte to the dry granular solid (e.g., phosphate fertilizer, biochar, calcined clay, or seeds). In other embodiments, the dried endophyte may be mixed with a dry carrier (e.g., insoluble inert ingredients such as dolomite, kaolin clay, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acid clay, vermiculite, and perlite, and inorganic salts such as calcium carbonate) to form a suspended granule. In other embodiments, the dried endophyte may be mixed with a dry carrier, a finely divided solid carrier such as silicates or aluminosilicates, including single- or double-lattice clays, and other suitable carriers to form water-dispersible granules. In other embodiments, dried endophytes may be pulverized to a particle size of about 0.200 μm to about 500 μm and then mixed with dry powders (e.g., graphite, talc, soy protein powder) to form a dustable powder operable to lubricate and reduce static electricity and friction on seeds or other substrates.In another embodiment, the dried endophyte may be pulverized to a particle size of about 0.200 μm to about 500 μm and passed through an ion-rich region with an applied voltage (0-20 kV) to charge the endophyte formulation, causing individual particles of the formulation to repel each other and forming an electrostatic powder operable to enhance coating uniformity on dry granular solids. In another embodiment, the dried endophyte may be pulverized to a particle size of about 0.200 μm to about 500 μm and mixed with a wetting carrier, surfactant, wetting agent, dispersant, and / or adjuvant described herein to form a wettable powder formulation.

[0030] In some embodiments, the formulation comprises a partially hydrated shell surrounding the endophyte within a carbohydrate carrier such as sodium alginate, calcium alginate, magnesium alginate, or other suitable carbohydrate carrier (e.g., Scogin LDH), providing hard, approximately dehydrated, round or oval beads. The beads range in size from about 400 nm to about 5 mm in average diameter, including nanobeads, microbeads, and capsule beads. The beads may additionally or alternatively comprise thickeners, starches, carbohydrates, or mineral thickeners, stabilizers, and / or carriers.

[0031] In some embodiments, the inoculant composition may contain stabilizers that are compatible with the endophyte strain and that promote the viability of the strain as well as its application to and colonization of the host plant by the heterologous endophyte strain. Examples of suitable stabilizers include guar gum, xantham gum, agarose, sucrose, glucose, ficoll, phytogel, sodium alginate, calcium alginate, magnesium alginate, glycine betaine, methylcellulose, maltodextrin, molasses, and mixtures thereof. Additional stabilizers that may be used include trehalose, sucrose, glycerol, and methylene glycol, glucose, sucrose mineral oil, soy lecithin, peptone, monopotassium phosphate (KH2PO4), dipotassium phosphate (K2PO4), and mixtures thereof. HPO4), hydroxypropyl guar (HP-Guar), xanthan gum, polyvinylpyrrolidone, polyvinylpyrrolidone / vinyl acetate (PVP-VA), a non-reducing sugar or sugar alcohol such as mannitol or sorbitol, or other suitable material. The amount of stabilizer in the composition can range from about 5 wt% to about 50 wt% (e.g., from about 10 wt% to about 40 wt%, from about 15 wt% to about 35 wt%, from about 20 wt% to about 30 wt%, or any value or range therein).

[0032] In some embodiments, the composition may include a carrier, such as an agriculturally acceptable carrier, which may be any substance that can be added to plant components without causing or having adverse effects on the host plant or its components. The carrier may be a solid or liquid carrier and may be in a variety of forms, including microspheres, powders, emulsions, various polymers, dry powder fertilizers such as potash, potassium phosphate, potassium nitrate, or other suitable substances. The carrier may be any one or more of several carriers that impart various properties, such as improved stability, wettability, flowability, and / or dispersibility.

[0033] In some embodiments, the agricultural carrier is a solid, such as diatomaceous earth, loam, silica, magnesium silicate, alginates (e.g., sodium, calcium, or magnesium alginate), glycine betaine (natural or synthetic), clay, bentonite, biochar, vermiculite, seed husks, peat, wheat, bran, talc, lime, starch, cellulose (methylcellulose, hemicellulose), Fuller's earth, heat-sterilized soil, fertilizer powder or fertilizer salts (macro- and micronutrients), other plant, animal, or non-biological products, or combinations thereof, including granules, pellets, or suspensions. In some embodiments, the solid carrier of the treatment formulation includes, for example, mineral carriers such as dolomite, kaolin clay, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acid clay, vermiculite, and perlite, and inorganic salts such as calcium carbonate. Organic fine powders such as wheat flour, wheat bran, and rice bran may also be used as solid carriers. Mixtures of any of the above ingredients, such as, but not limited to, pasta (wheat flour and kaolin clay) or flour-based pellets in loam, sand, or clay, are also contemplated as carriers. In some embodiments, the agricultural carrier may be soil or plant growth medium and / or a food source for cultured organisms. In certain embodiments, the endophyte strains may be encapsulated in calcium alginate, magnesium alginate, agarose, or sucrose, glucose, or other suitable materials, with or without one or more carbohydrate stabilizers, such as other suitable sugars. The encapsulated endophyte strains may be included in suspensions or solid formulations for use as powder fertilizer coatings (including, but not limited to, granular urea, ammonium nitrate, potassium nitrate, potassium phosphate, calcium phosphate, etc.) for all fertilizers, macronutrients, and micronutrients in seed treatments and coatings, foliar sprays, and in-furrow applications.

[0034] In some embodiments, the agricultural carrier may be a liquid carrier that imparts various properties, such as improved stability, wettability, flowability, and / or dispersibility. Liquid carriers may include vegetable oils, such as soybean oil, neem oil, cottonseed oil, and compositions containing glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, or other suitable liquids. In some embodiments, the carrier may be a combination of liquid components, such as a water-in-oil emulsion or other suitable formulation. For example, water-in-oil emulsions may be prepared to include wettable powders, granules, gels, agar, thickeners, biopolymers, microencapsulated particles, and the like. Other agricultural carriers that may be used may include water, vegetable oils, humectants, or combinations thereof. The composition may also include a wetting agent, such as a natural or synthetic surfactant, which may be a nonionic or ionic surfactant, or a combination thereof. When such formulations are used as wettable powders, biologically compatible dispersants, such as nonionic, anionic, amphoteric, or cationic dispersants and emulsifiers, may be used.

[0035] In some embodiments, the surfactant that can be included in the composition can include nonionic surfactants and / or anionic surfactants.Examples of nonionic surfactants include alkylphenol alkoxylates, alcohol alkoxylates, polyoxyethylene glycerol fatty acid esters, castor oil alkoxylates, fatty acid alkoxylates, fatty amide alkoxylates, fatty polydiethanolamides, lanolin ethoxylates, fatty acid polyglycol esters, isotridecyl alcohol, fatty amides, methylcellulose / hemicellulose, fatty acid esters, alkyl polyglycosides, glycerol fatty acid esters, polyethylene glycol, polypropylene glycol, polyethylene glycol / polypropylene glycol block copolymers, polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, polyethylene glycol / polypropylene glycol ether block copolymers, polyethylene oxide / polypropylene oxide block copolymers, and mixtures thereof. Examples of anionic surfactants include alkylarylsulfonates, phenylsulfonates, alkyl sulfates, arylalkylsulfonates, alkyl ether sulfates, alkylarylether sulfates, alkyl-polyglycol ether phosphates, polyarylphenyl ether phosphates, alkyl sulfosuccinates, olefinsulfonates, paraffin sulfonates, petroleum sulfonates, taurides, sarcosides, fatty acid salts, alkylnaphthalenesulfonates, naphthalenesulfonates and lignosulfonates, condensates of sulfonated naphthalene with formaldehyde or with formaldehyde and phenol and optionally urea, further condensates of phenolsulfonic acid, formaldehyde and urea, lignosulfite waste liquor and lignosulfonates, alkyl phosphates, alkylaryl phosphates (e.g. tristyryl phosphates), and polycarboxylates (e.g. polyacrylic acid, maleic anhydride / olefin copolymers) containing alkali metals, alkaline earth metals and mixtures thereof.

[0036] In some embodiments, the inoculant composition may include a sticking or adhesive agent to aid in combining the endophyte strain with a carrier, which may include other non-biological compounds. Such compositions form a coating around the plant or plant element (e.g., one used in seed coating) and help maintain contact between the heterologous endophyte and other materials and the plant or plant element. In some embodiments, the adhesive agent may include one or more of alginate, gum, starch, maltodextrin, lecithin, formononetin, polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinyl acetate, cephalin, gum arabic, xanthan gum, carrageenan, PGA, other biopolymers, mineral oil, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), arabino-galactan, methylcellulose, PEG 400, chitosan, polyacrylamide, polyacrylate, polyacrylonitrile, glycerol, triethylene glycol, vinyl acetate, gellan gum, polystyrene, polyvinyl, carboxymethylcellulose, hemicellulose, gum ghatti, polyoxyethylene-polyoxybutylene block copolymers, and other suitable agents.

[0037] In some embodiments, one or more adjuvants may be used in the inoculant composition to help improve delivery and performance of the endophyte strain. The composition can be combined with an adjuvant to create a specific product form or mixture, such as a liquid mixture for foliar application. The composition can further include other agriculturally suitable excipients, such as solvents, pH adjusters, rheology modifiers, crystallization inhibitors, antifoaming agents, dispersants, wetting agents, humectants, anti-caking agents, suspending agents, spray droplet conditioners, pigments, antioxidants, UV protection agents, compatibilizers, chelating agents, neutralizing agents, corrosion inhibitors, dyes, fragrances, spreading agents, penetration aids, lubricants, adhesives, thickeners, freezing point depressants, and antimicrobial agents. The compositional content of these adjuvants is not particularly limited and can be determined by one of ordinary skill in the art according to conventional protocols.

[0038] In some embodiments, suitable pH adjusting agents that can be included in the inoculant composition include buffers, such as alkali metal salts of weak inorganic or organic acids (e.g., phosphoric acid, phosphorous acid, boric acid, acetic acid, propionic acid, citric acid, fumaric acid, tartaric acid, oxalic acid, malic acid, oxaloacetic acid, and succinic acid).

[0039] The inoculant composition may further comprise a food source for the cultured organisms such as barley, rice, wheat, or other biological material such as seeds, plant parts, sugarcane bagasse, husks or stalks from grain processing, crushed plant material from construction sites, or sawdust or small fibers from recycled wood, paper, cloth, or wood.

[0040] The inoculant composition may further comprise additional ingredients used to enhance plant health and microbial survival, such as biostimulants, prebiotics, amino acids, fatty acids, plant proteins, fungicides, insecticides, nematicides, plant microbial boosters (prebiotics), plant hormones and elicitors, mineral macro- and micronutrients (liquid and dry), seed treatment polymers, commonly used dyes, carbohydrates and gels (alginate, mucilage, agarose, guar, xanthan gum, etc.), powder carriers (soy protein, talc, lime, starch biochar, cellulose / hemicellulose, silica, clay, nanotechnology structures (e.g., carbon dots, buckybodies) containing mineral nutrients, etc. Biostimulants may include biostimulants such as steroids, steroid hormones, carbon cages, or other forms of nanotechnology. Exemplary biostimulants may include combinations of amino acids (e.g., one or more of L-glutamine, L-lysine, L-methionine, L-arginine, and L-threonine), fatty acids, vitamins, plant proteins, phosphorus sources, betaine, plant growth factors, and other suitable ingredients. Formulations of the endophyte strains described herein were combined with commercially available biocides and nutritional products to test the viability of the endophyte under these conditions. The endophyte was found to be compatible with several commercial products in the laboratory and in the field. Based on the results of the compatibility tests, several new formulations were developed.

[0041] The endophyte strains described herein can be combined with one or more of the above-mentioned agents to obtain compositions suitable for application to plants or their tissues, seedlings, seeds, or other plant components. Endophyte populations can be obtained from selection and propagation in culture as described herein and added to the compositions. Endophytes in different growth stages can be used. For example, lag-phase, early-logarithmic, mid-logarithmic, late-logarithmic, or stationary phase endophytes can be used.

[0042] The foregoing components may be combined in a liquid composition comprising an effective amount of one or more of the endophyte strains WW5, WW6, WW7, and PTD1. For example, the inoculant compositions described herein may comprise the endophyte strain in an amount of between about 0.1 and 90% by weight, e.g., between about 1% and 80% by weight, between about 5% and 70% by weight, between about 10% and 60% by weight, or between about 15% and 50% by weight, based on the wet weight of the composition. The inoculant composition may contain at least about 10% by weight of the endophyte strain per mL. 3 CFU, e.g., at least about 10 4 CFU / mL, at least approximately 10 5 CFU / mL, at least approximately 10 6 CFU / mL, at least approximately 10 7 CFU / mL, at least approximately 10 8 CFU / mL, at least approximately 10 9 CFU / mL, at least approximately 10 10 CFU / mL or any value or range within said range.

[0043] An exemplary liquid formulation according to the present invention comprises two or more dried or undried endophyte strains (e.g., WW6 and WW7 prepared by co-fermentation) in a concentration of about 10 8 CFU / mL ~ approx. 10 9At a concentration of each endophyte strain in CFU / mL, one or more mono- or disaccharides (sucrose) in an amount of about 1 wt% to about 10 wt%, mannitol in an amount of about 1 wt% to about 10 wt%, sodium lactate in an amount of about 0.01% v / v to about 0.1% v / v, potassium phosphate salts (e.g., K2HPO4 and KH2PO4) in an amount of about 0.05 wt% to about 0.5 wt%, sodium molybdate in an amount of about 0.001 wt% to about 0.01 wt%, NaCl in an amount of about 0.005 wt% to about 0.05 wt%, and CaCl2 in an amount of about 0.005 wt% to about 0.05 wt%. % to about 0.05 wt%, NaFeEDTA in an amount of about 0.001 wt% to about 0.01 wt%, magnesium sulfate in an amount of about 0.01 wt% to about 0.1 wt%, yeast extract in an amount of about 0.005 wt% to about 0.05 wt%, and agar in an amount of about 1 wt% to about 10 wt%, all in distilled water. 4 ~10 10 A range of CFU / mL of each strain may be included, alone or in combination with the following: low viscosity alginate (e.g., sodium alginate, magnesium alginate, calcium alginate, Scogin® LDH (Dupont) or other high purity alginate) in an amount of about 0.1% v / v to about 5% v / v, glycerol in an amount of about 0.1% v / v to about 5% v / v, and one or more mono- and disaccharides (e.g., glucose and lactose) in an amount of about 1 wt% to about 10 wt%. Yet another exemplary liquid formulation comprises one or more dried or undried endophyte strains in an amount of about 10 wt% to about 10% v / v. 4 ~10 10 The formulation may contain a concentration range of CFU / mL for each strain, either alone or in combination with the following: 0.5 to 50 w / v low-viscosity alginate (e.g., sodium alginate, magnesium alginate, calcium alginate, Scogin® LDH (Dupont) or other low-viscosity, high-purity alginate), gelatin in an amount of about 1% v / v to about 5% v / v, PEG in an amount of about 1% v / v to about 10% v / v, and one or more mono- and disaccharides (e.g., glucose and lactose) in an amount of about 1 wt% to about 10 wt%.

[0044] In some embodiments, the composition may be a suspension formulation containing the above-mentioned components in the above-mentioned proportions. In such embodiments, the composition may further contain one or more solid carriers, thickeners, or extenders. Such ingredients may include inorganic mineral earths such as silica gel, silicates, talc, kaolin, Atta clay, limestone, lime, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate and magnesium sulfate, magnesium oxide, attapulgite, montmorillonite, mica, vermiculite, synthetic silicic acid, amorphous silicic acid, synthetic calcium silicate, or mixtures thereof; and / or organic carriers such as hydrocolloids, polymers, cellulose, methylcellulose, and / or hemicellulose powders, and combinations thereof. The suspension composition may further include a humectant, an emulsifier, an anti-caking agent, a suspending agent, a freezing point depressant, etc. In some embodiments, the suspension formulation may contain one or more of the endophyte strains WW5, WW6, WW7, and PTD1 at the concentrations disclosed in the preceding paragraph. An exemplary suspension formulation according to the present invention comprises approximately 10 microcrystalline cellulose microcapsules microencapsulated in sodium alginate, calcium alginate, or magnesium alginate (e.g., by a spray drying process). 8 CFU / mL ~ approx. 10 9 The formulation may contain one or more dried endophyte strains at a concentration of each endophyte strain in CFU / mL. The formulation may further contain one or more mono- or disaccharides (e.g., sucrose) in an amount of about 0.1 wt% to about 10 wt%, and glycerol in an amount of about 0.1 wt% to about 20 wt%, all in distilled water.

[0045] In some embodiments, the composition may be a solid composition. The solid composition may be a dry, granular, or flowable composition intended to be dispersed or suspended in an aqueous solution before application to plants. The dry fertilizer composition may form a fully dispersed suspension. In other contexts, the dry fertilizer composition may provide sustained release (e.g., through low water solubility or encapsulation, e.g., with sodium alginate), for example, when a constant or controlled delivery of nutrients over time is desired. The solid composition may be about 103 CFU / mL ~ at least about 10 10 The solid composition may contain one or more of the endophyte strains WW5, WW6, WW7, and PTD1 in an amount of about 10 CFU / mL or any value or range of values ​​therein. 8 CFU / mL ~ approx. 10 9 The solid formulation may contain one or more solid carriers in an amount ranging from about 30 wt% to about 60 wt% (e.g., about 40 wt% to about 55 wt%, about 45 wt% to about 99.9 wt%, or any value or range of values ​​therein). Exemplary solid formulations according to the present invention contain one or more dried endophyte strains in an amount ranging from about 10 4 CFU / mL ~ approx. 10 10 The formulations contain a concentration of each endophyte strain at CFU / mL, which may be microencapsulated in alginate beads (sodium, calcium, or magnesium alginate 0.1-10% w / v) and solids (starch 0.1-10% w / v) and dripped through unique slurry formulation, batch drip, ion exchange, and fluidized bed drying processes. The formulations may further include one or more mono- or disaccharides (e.g., sucrose) in an amount of about 1 wt% to about 10 wt% and clay (e.g., zeolite, bentonite, and / or other clay materials) in an amount of about 30 wt% to about 50 wt%.

[0046] manufacturing The endophytic strains of the presently disclosed methods and formulations (WW5, WW6, WW7, and PTD1) may be prepared for inclusion in various formulations, including liquid and dry formulations, which can be applied to foliar tissues, stems, seeds, roots, and / or soil adjacent to seeds or plants. Specifically, the inoculant compositions of the present technology may include liquid seed treatments, seed coatings, freeze-dried wettable powders reconstituted for seed treatment or other uses, spray-dried wettable powders reconstituted for seed treatment or other uses, encapsulated dry beads, foliar sprays, in-furrow liquid products, and other formulations. The endophytic strains may be fermented individually or in various combinations. The endophytic strains were developed as stocks using a novel fermentation growth method for use in the inoculant compositions. Sufficient mineral nutrients, vitamins, carbon source, aeration, temperature, and duration may be utilized to stabilize the endophytic strains during fermentation. Additionally, components that promote long-term stability (long shelf life), delivery, colonization and efficacy in host plants may be included in the inoculant compositions of the present technology.

[0047] In some embodiments, the selected WW5, WW6, WW7, and PTD1 cells are cultured at a target concentration of about 10 7 CFU / mL ~ approx. 10 10 For application purposes, they may be grown individually in nitrogen-free or nitrogen-limited media for 1-3 days until present in the range of CFU / mL. In some implementations, two or more endophyte strains are combined and co-fermented to produce approximately 10 3 CFU / mL to approximately 10 9 CFU / mL range, e.g., at least about 10 4 CFU / mL, at least approximately 10 5 CFU / mL, at least approximately 10 6 CFU / mL, at least approximately 10 7 CFU / mL, at least approximately 10 8 CFU / mL, at least approximately 10 9The fermentation process conditions can include a predetermined incubation temperature ranging from about 20° C. to about 30° C. (e.g., about 23° C. to about 26° C., about 25° C., or any value or range of values ​​therein), shaking the fermenter at a speed ranging from about 25 rpm to about 300 rpm (e.g., about 75 rpm to about 250 rpm, about 125 rpm to about 225 rpm, about 200 rpm, or any value or range of values ​​therein), and a fermentation volume of about 1 L to about 10 L (e.g., about 2 L to about 8 L, about 4 L to about 6 L, about 4 L, about 2 L, or any value or range of values ​​therein).

[0048] To drive upregulation of microbial nitrogenase genes in endophytic strains, the fermentation medium can be substantially free of nitrogen but may be a nitrogen-free (NF) or nitrogen-limited medium (NLM) that can contain one or more sugars, such as mannitol, mannose, sucrose, glucose, fructose, lactose, and any other sugars. NF / NLM can also contain one or more salts, such as sodium chloride, phosphates (e.g., monopotassium phosphate, dipotassium phosphate, and other phosphates), sulfates (e.g., MgSO), chlorides (e.g., CaCl), and other suitable salts, but excludes nitrates, ammonium salts, and other nitrogen sources. The fermentation broth can further contain other suitable components, such as yeast extract, agar, and other suitable ingredients. The resulting composition can be used as a liquid composition to treat host plants. For example, for examples of nitrogen-limited media, see the following reference: RJ Rennie, A single medium for the isolation of acetylene-reducing (dinitrogen-fixing) bacteria from soils, Canadian Journal of Microbiology, Vol. 27, No. 1, pp. 8-14, 1981.

[0049] The fermentation process can be terminated when the endophytic strain reaches a stationary phase where microbial growth slows and the microorganisms become semi-dormant. To enhance the preservation of the microorganisms, sterile water may be added to the fermentate to dilute the remaining sugars and nutrients, thereby placing the microorganisms in a dormant stationary phase and reducing CO2 production.

[0050] Methods for producing inoculants of the present technology, including those for foliar and stem treatment, seed treatment, root treatment, and soil and in-furrow treatment, are described in detail below in the Examples, particularly Examples 2A-2D and 65-75.

[0051] Applicable The compositions described herein, including one or more endophytic strains, may be applied to plants to enhance plant growth characteristics, health, stress resistance, and improve other plant traits. The compositions described herein may advantageously be mechanically or manually applied to plants or their components, or artificially inoculated, by any one of a number of means, including, but not limited to, seed treatment, root wash, seedling dipping, soil inoculation, furrow application, foliar spray, foliar coating, side-row application, wound inoculation, drench, fertilizer irrigation, soaking, injection, osmotic priming, hydroponics, aquaponics, aeroponics, or any combination thereof. In some embodiments, the compositions may also be applied directly to plants or plant parts, such as leaves, roots, foliage, foliage, tufts, tillers, flowers, plant cells, plant tissues, or combinations thereof. The compositions may be applied to seeds (e.g., as a coating or by treating the seeds by spraying or dipping) and / or pre-emergence (before the seedling emerges or appears). The composition may also be applied to other propagation materials of the plant, such as grains, fruits, tubers, spores, cuttings, slips, meristems, plant cells, nuts, or embryos. In some instances, the composition may be applied as part of a soak of the roots and / or other tissues of the host plant, as a seed coating, as a coating applied to the leaves and / or other elements of the host plant, as a powder on the surface of the leaves and / or other elements of the host plant, as a spray on the leaves and / or other elements of the host plant, as part of a drip onto the soil and / or roots of the host plant, or any other suitable method. The composition may also be applied to the growing medium (e.g., by applying to the soil surrounding the plant).

[0052] The application methods developed were specific for different uses depending on the particular inoculant formulation, successful synergistic combination of crop plants, and specific application rates and methods for each crop type; these methods used are summarized and provided in the table shown in Figure 6A.

[0053] The presently described inoculant compositions can improve phenotypic traits measured by various methods, including, but not limited to, increased height, total biomass, total carbon, root mass, shoot biomass, seed germination rate, seedling survival rate, photosynthetic efficiency, seed / fruit number or mass, fruit yield, leaf chlorophyll content, photosynthetic rate, root length, abiotic stress tolerance, biotic stress tolerance, disease resistance, wilt recovery, turgor pressure, or any combination thereof, compared to untreated control plants of the same species grown under similar conditions. Host plant rootstocks, rootstocks, cuttings, or tissue cultures may be used to produce cultivars exhibiting such phenotypic traits and enhanced performance. Application of the inoculant composition can also increase carbon fixation in treated host plants, an economically attractive benefit because it results in carbon dioxide removal from the atmosphere and increased biomass. Indicators of increased carbon acquisition by plants include increased CO2 fixation activity, increased dry weight to fresh weight ratio, and overall biomass.

[0054] Treatment with the compositions described herein can result in increased uptake of macro- and micronutrients from the soil and atmosphere. Treatment with the compositions can result in increased nitrogen uptake rates. Increased nitrogen uptake rates promote significant improvements in utilization efficiency. Host plants heterologously treated with the compositions incorporate more total nitrogen and assimilate it at higher levels of nitrogen utilization efficiency, resulting in greater protein production and promoting increased biomass production. Application of the compositions described herein also increases the uptake and utilization of other macro- and micronutrients. Experimental results demonstrate increased uptake of the macronutrients potassium, phosphorus, calcium, and magnesium, as well as the micronutrients boron, copper, iron, manganese, molybdenum, nickel, sulfur, and zinc in host plants treated with the inoculant compositions—see, e.g., Examples 5-7 below. Heterologous endophytes can uptake fixed or poorly soluble forms of certain nutrients, including phosphorus, in soil and convert them to soluble forms that can be more efficiently utilized by the host plant. Endophytic strains can also produce iron siderophores that are beneficial to the host plant, chelating iron in plant tissues and aiding in iron uptake into the host plant.

[0055] Increased macronutrient and micronutrient uptake is accompanied by increased catabolism, carbon uptake, and carbon sequestration. Host plants heterologously treated with the compositions of the present invention exhibit greater total carbon uptake, with associated increases in RuBisCo carboxylation activity, carbon content, and biomass. Host plants may also exhibit increased production of aromatic amino acids via the shikimate pathway. These aromatic amino acids serve as precursors for a wide range of secondary metabolites important for plant resistance to biotic and abiotic stresses (e.g., oxidative stress, drought stress, and / or salt stress).

[0056] Thus, application of the present compositions can result in enhanced adaptive resistance of host plants to abiotic and biotic stresses, such as disease, cold, and salinity. In exemplary embodiments, the level of innate and adaptive resistance to stress is significantly enhanced. Abiotic stressors include extreme temperatures, high salinity, drought, and other causes. Abiotic stressors dramatically reduce yield and biomass and often result in plant death. In agricultural production of crops, low growth temperatures are often encountered, especially during the early growing season, and can stress plants in various ways, beginning with poor germination and followed by stunted seedling growth, leaf yellowing, reduced leaf expansion, wilting, and tissue death. Cold stress significantly inhibits the development of reproductive parts of plants. Crop yields decrease in response to cold stress in proportion to the extent of damage to the plant. High salt concentrations in soil or water are becoming increasingly problematic due to salt accumulation in irrigated soils and the need for irrigation water with high salinity. High salt concentrations in soil and water hinder the transport of ions and water within plants, and the effects of high salt concentrations are called osmotic stress. Symptoms of high salt stress include stunted growth, wilting, yellowing, leaf drop, senescence, and death. The increased efficiency of nutrient uptake and utilization brought about by the presence of endophytic strains in host plants protects the host plants under stress-inducing environments, and the host plants exhibit greater growth and biomass even under abiotic stress conditions.

[0057] Application of the present compositions to host plants enhances adaptive resistance through advanced mineral nutrition, resulting in biological stress tolerance that suppresses viruses, bacteria, and fungi. The endophytic strains described herein provide host plants with resistance that may be the result of activation of induced systemic resistance (ISR) and / or other metabolic mechanisms in the plant. Thus, the endophytic strains can be applied to host plants or their seeds as nutritional treatments to help protect against pathogenic fungi, viruses, and bacteria.

[0058] Other aspects, objects and advantages of the presently described technology will be apparent from the following detailed description. [Brief explanation of the drawings]

[0059] [Figure 1] FIG. 1 shows SEQ ID NO:1. [Figure 2] FIG. 2 shows SEQ ID NO:2. [Figure 3] FIG. 3 shows SEQ ID NO:3. [Figure 4] FIG. 4 shows SEQ ID NO:4. [Figure 5] FIG. 5 is a table providing data from the microbial assays. [Figure 6] FIG. 6 is a table providing exemplary nutritional components of the fermentation composition. [Figure 6A] FIG. 6A is a table providing exemplary application methods and rates. [Figure 7A] FIG. 7A provides images related to the experiment of Example 1A. [Figure 7B] FIG. 7B is a table providing data related to the experiments of Example 1A. [Figure 7C] FIG. 7C is a table providing data related to the experiments of Example 1A. [Figure 8A] FIG. 8A is a table related to the experiments in Example 1B. [Figure 8B] FIG. 8B is a table related to the experiments in Example 1B. [Figure 8C] FIG. 8C is a table related to the experiments in Example 1B. [Figure 9] FIG. 9 is a table related to the experiments in Example 1C. [Figure 10A] FIG. 10A provides gel data related to the experiment of Example 2B. [Figure 10B] FIG. 10B is a table related to the experiments in Example 2B. [Figure 10C] FIG. 10C is a table related to the experiments in Example 2B. [Figure 11] FIG. 11 is a table related to the experiments in Example 2C. [Figure 12] FIG. 12 is a table related to the experiments of Example 2D. [Figure 13A] FIG. 13A provides the enzymatic pathways associated with the experiments in Example 3. [Figure 13B] FIG. 13B is a table related to the experiments in Example 3. [Figure 13C] FIG. 13C is a graph related to the experiment of Example 3. [Figure 14A] FIG. 14A provides the enzymatic pathways associated with the experiments in Example 4. [Figure 14B] FIG. 14B provides images related to the experiment in Example 4. [Figure 14C] FIG. 14C is a table relating to the experiments in Example 4. [Figure 14D] FIG. 14D is a graph related to the experiment in Example 4. [Figure 15] FIG. 15 is a graph related to the experiment of Example 5. [Figure 16] FIG. 16 provides images related to the experiment in Example 6. [Figure 17A] FIG. 17A provides images related to the experiment in Example 7. [Figure 17B] FIG. 17B provides images related to the experiment in Example 7. [Figure 18A] FIG. 18A is a table relating to the experiments in Example 8. [Figure 18B] FIG. 18B is a table relating to the experiments in Example 8. [Figure 19] FIG. 19 is a table relating to the experiments in Example 9. [Figure 20] FIG. 20 is a table relating to the experiments in Example 10. [Figure 21] FIG. 21 is a table relating to the experiments in Example 11. [Figure 22] FIG. 22 is a table relating to the experiments in Example 12. [Figure 23] FIG. 23 is a table relating to the experiments in Example 13. [Figure 24] FIG. 24 is a table relating to the experiments in Example 14. [Figure 25A] FIG. 25A is a table relating to the experiments in Example 15. [Figure 25B] FIG. 25B is a graph related to the experiment of Example 15. [Figure 25C] FIG. 25C is a graph related to the experiment in Example 15. [Figure 26A] FIG. 26A is a table relating to the experiments in Example 16. [Figure 26B] FIG. 26B is a graph related to the experiment in Example 16. [Figure 27] FIG. 27 is a graph related to the experiment of Example 17. [Figure 28A] FIG. 28A is a graph related to the experiment of Example 18. [Figure 28B] FIG. 28B is a graph related to the experiment of Example 18. [Figure 29] FIG. 29 is a graph related to the experiment of Example 19. [Figure 30A] FIG. 30A is a table relating to the experiments in Example 20. [Figure 30B] FIG. 30B is a table relating to the experiments in Example 20. [Figure 31A] FIG. 31A is a table relating to the experiments in Example 21. [Figure 31B] FIG. 31B is a graph related to the experiment of Example 21. [Figure 31C] FIG. 31C is a graph related to the experiment of Example 21. [Figure 32]FIG. 32 is a table relating to the experiments in Example 22. [Figure 33A] FIG. 33A is a table relating to the experiments in Example 23. [Figure 33B] FIG. 33B is a graph related to the experiment of Example 23. [Figure 34A] FIG. 34A is a table relating to the experiments in Example 24. [Figure 34B] FIG. 34B provides images related to the experiment in Example 24. [Figure 35] FIG. 35 provides images related to the experiment in Example 25. [Figure 36A] FIG. 36A is a table relating to the experiments in Example 26. [Figure 36B] FIG. 36B is a graph related to the experiment of Example 26. [Figure 36C] FIG. 36C provides images related to the experiment in Example 26. [Figure 36D] FIG. 36D is a graph related to the experiment in Example 26. [Figure 36E] FIG. 36E is a graph related to the experiment in Example 26. [Figure 37A] FIG. 37A is a table relating to the experiments in Example 27. [Figure 37B] FIG. 37B is a graph related to the experiment of Example 27. [Figure 38] FIG. 38 is a graph related to the experiment of Example 28. [Figure 39] FIG. 39 is a graph related to the experiment of Example 29. [Figure 40A] FIG. 40A provides images related to the experiment in Example 30. [Figure 40B] FIG. 40B provides images related to the experiment in Example 30. [Figure 41] FIG. 41 is a graph related to the experiment of Example 31. [Figure 42A] FIG. 42A is a graph related to the experiment of Example 32. [Figure 42B]FIG. 42B is a graph related to the experiment of Example 32. [Figure 43A] FIG. 43A is a graph related to the experiment in Example 33. [Figure 43B] FIG. 43B is a graph related to the experiment in Example 33. [Figure 44] FIG. 44 is a graph related to the experiment of Example 34. [Figure 45A] FIG. 45A is a graph related to the experiment in Example 35. [Figure 45B] FIG. 45B provides images related to the experiment in Example 35. [Figure 46] FIG. 46 is a table relating to the experiments in Example 36. [Figure 47] FIG. 47 is a table relating to the experiments in Example 37. [Figure 48] FIG. 48 is a table relating to the experiments in Example 38. [Figure 49] FIG. 49 is a graph related to the experiment of Example 39. [Figure 50] FIG. 50 is a graph related to the experiment of Example 40. [Figure 51] FIG. 51 is a graph related to the experiment of Example 41. [Figure 52] FIG. 52 is a graph related to the experiment of Example 42. [Figure 53A] FIG. 53A is a graph related to the experiment in Example 43. [Figure 53B] FIG. 53B is a graph related to the experiment in Example 43. [Figure 54] FIG. 54 is a graph related to the experiment of Example 44. [Figure 55] FIG. 55 is a graph related to the experiment of Example 45. [Figure 56] FIG. 56 is a graph related to the experiment of Example 46. [Figure 57] FIG. 57 is a graph related to the experiment of Example 47. [Figure 58] FIG. 58 is a graph related to the experiment of Example 48. [Figure 59] FIG. 59 is a graph related to the experiment of Example 49. [Figure 60A] FIG. 60A is a graph related to the experiment of Example 50. [Figure 60B] FIG. 60B is a graph related to the experiment of Example 50. [Figure 60C] FIG. 60C is a graph related to the experiment of Example 50. [Figure 61] FIG. 61 is a graph related to the experiment of Example 51. [Figure 62] FIG. 62 is a graph related to the experiment of Example 52. [Figure 63] FIG. 63 is a graph related to the experiment of Example 53. [Figure 64] FIG. 64 is a graph related to the experiment of Example 54. [Figure 65] FIG. 65 provides graphs related to the experiments in Example 55. [Figure 66] FIG. 66 provides graphs related to the experiments in Example 56. [Figure 67A] FIG. 67A provides a table related to the experiments in Example 57. [Figure 67B] FIG. 67B provides a table related to the experiments in Example 57. [Figure 67C] FIG. 67C provides a table related to the experiments in Example 57. [Figure 67D] FIG. 67D provides graphs related to the experiments in Example 57. [Figure 68A] FIG. 68A provides a table related to the experiments in Example 58. [Figure 68B] FIG. 68B provides a table related to the experiments in Example 58. [Figure 69A] FIG. 69A provides graphs related to the experiments of Example 59. [Figure 69B] FIG. 69B provides graphs related to the experiments in Example 59. [Figure 70] FIG. 70 provides graphs related to the experiments in Example 60. [Figure 71A] FIG. 71A provides graphs related to the experiments of Example 61. [Figure 71B] FIG. 71B provides graphs related to the experiments in Example 61. [Figure 72] FIG. 72 provides graphs related to the experiments in Example 62. [Figure 73A] FIG. 73A provides images related to the experiment in Example 63. [Figure 73B] FIG. 73B provides images related to the experiment in Example 63. [Figure 73C] FIG. 73C provides graphs related to the experiments in Example 63. [Figure 73D] FIG. 73D provides graphs related to the experiments in Example 63. [Figure 73E] FIG. 73E provides graphs related to the experiments in Example 63. [Figure 73F] FIG. 73F provides graphs related to the experiments in Example 63. [Figure 73G] FIG. 73G provides graphs related to the experiments in Example 63. [Figure 74] FIG. 74 provides graphs related to the experiments in Example 64. [Figure 75A] FIG. 75A is a table providing vacuum application for preparing inoculum. [Figure 75B] FIG. 75B is a table providing vacuum application for preparing inoculum. [Figure 76] FIG. 76 is a table relating to the experiments in Example 71. [Figure 77] FIG. 77 is a table related to the experiments in Example 72. [Figure 78] FIG. 78 provides graphs related to the experiments in Example 75. [Figure 79] FIG. 79 provides gel data related to the experiment in Example 76. [Figure 80] FIG. 80 is a table relating to the experiments in Example 77. [Figure 81] FIG. 81 provides gel data related to the experiment in Example 78.

[0060] Detailed Description of the Invention Reference will now be made in detail to certain embodiments of the present invention and exemplary compositions and applications of such embodiments. While the present invention will be described with reference to these embodiments, it will be understood that they are not intended to limit the invention. On the contrary, the present invention is intended to cover alternatives, modifications, and equivalents included within the spirit and scope of the present invention as defined by the appended claims. The following disclosure sets forth specific details to provide a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced without these specific details. [Example]

[0061] method Selection and propagation methods and compositions The present invention includes methods for the propagation and selection of nitrogen-fixing endophyte strains. These methods involve inoculating specialized nitrogen-limited, nitrogen-free, or optionally rich medium (RM) growth media and selecting colonies that can grow in the specialized growth media. The ability of endophyte strains to grow in nitrogen-free and nitrogen-limited media was evaluated.

[0062] Example 1A Endophyte selection and testing Each of the endophyte strains WW5, WW6, WW7, and PTD1 were tested and found to be positive for the ability to grow on nitrogen-limited medium (NLM), with each strain growing to varying degrees on nitrogen-limited medium, as shown in the images below: Each of the endophyte strains were tested and found to be positive for the ability to grow on plant tissue culture grade agarose plates containing nitrogen-limited medium (NLM, pH 7.6), with each strain growing to varying degrees on plant tissue culture grade agarose plates containing nitrogen-free medium (NFCCM, pH 7.6), as shown in the images below:

[0063] Fermentation mixtures containing 10 mL of broth culture were prepared in 50 ml conical tubes. Each culture was inoculated with 100 μL of a standardized QC broth culture of the endophyte strain. The conical tubes were placed on a shaker at a 45° angle and incubated at room temperature with shaking at 200 rpm for 72 hours. The optical density (OD) of each culture was measured at 600 nm. 100 μL of the 10-5 and 10-6 dilutions of each endophyte strain were plated onto NLM agar plates. The plates were then incubated at 25°C for 72 hours. The plates were incubated for 1 h. The CFU formed on the plates were observed and recorded. Figure 7A shows visual evidence of colony growth on NLM plates.

[0064] The endophytic strains were also analyzed for their ability to produce ammonium in liquid fermentation under aerobic conditions. Separate assays of each of the WW5, WW6, WW7, and PTD1 endophytic strains demonstrated that each strain was capable of producing ammonium under such conditions. The assays demonstrated the ability of the endophytic strains to participate in the N fixation mechanism within the plant and correlated with the growth effects observed after inoculation with the endophytic fermentation product.

[0065] Each strain was tested in nitrogen-limited media: MGL (mannitol-glutamate / Luria-Bertani), NLM (nitrogen-limited medium), MCDY (M series Yeast Media nitrogen base with amino acid supplements), and CS+KNO3 (corn syrup+KNO3) with exogenous ammonium (NH4 + All sterile media tested were negative for ammonium concentrations below 0 mg / L.

[0066] Ammonium NH4 +In addition to their ability to produce NH3, endophyte strains WW5, WW6, WW7, and PTD1 were evaluated to determine whether they could also produce ammonia (NH3) in liquid fermentation under aerobic conditions. Fermentation mixtures containing 1 liter of nitrogen-limited medium (NLM) broth culture were prepared in 2-liter flasks. Each culture was inoculated with 1–3 colonies from an NLM agar plate containing a single endophyte strain. The flasks were placed on a shaker and incubated at room temperature with shaking at 125 rpm for 72 hours.

[0067] The endophyte strains were then analyzed for their ability to produce ammonia in liquid fermentation under aerobic conditions. Separate assays of each of the WW5, WW6, WW7, and PTD1 endophyte strains showed that all strains were able to produce ammonia under these conditions. Ammonia production data for each strain are shown in Figure 7C.

[0068] The ability of endophytic strains to fix nitrogen within tree or plant tissues is enabled by the microbial nitrogenase gene within the endophytic bacteria. Each of the WW5, WW6, WW7, and PTD1 strains was assayed for the presence of the nitrogenase gene by PCR using specific primers for the Nif gene. Each endophytic strain was found to contain at least one copy of the Nif gene subunit.

[0069] The ability of the endophyte strains to fix nitrogen was further measured by an acetylene reduction assay. The acetylene reduction assay measures the ability of nitrogenase enzyme to reduce acetylene gas to ethylene using gas chromatography to quantify the amount of ethylene produced. This is an indirect method of measuring N2 fixation capacity, and the functional presence of nitrogenase enzyme is measured by its correlating ethylene production. The WW6, WW7, and PTD1 endophyte strains exhibited acetylene reduction activity, as shown in Figure 7D.

[0070] Example 1B Genome analysis of Curtobacterium salicaceae (WW7) Curtobacterium salicaceae (WW7) is a new nitrogen-fixing bacterial species found naturally in willow trees, grass phyllosphere (leaves), leaf litter / soil, and maize roots. WW7 also produces the organic acids malic acid and citric acid, which can solubilize insoluble phosphate, as well as Fe-siderophores, which solubilize insoluble iron. Curtobacterium salicaceae was isolated from the vasculature of willow (Sitka sitchenses) tree trunks.

[0071] WW7 was sequenced by the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) using the Illumina MiSeq platform. A paired-end library was constructed from 376 ng of gDNA using the Nextera DNA Flex Library Preparation Kit and loaded onto a single flow cell. The library was barcoded, mixed with 11 samples, and sequenced using a 2 × 250-bp format. MiSeq was performed using the MiSeq Reagent Kit v3 (600 cycle) chemistry. Shotgun sequencing yielded 1,530,321 reads. After trimming, quality filtering, and removal of potential contaminants using the BBMap package, 1,436,665 read pairs were used as input for the SPAdes v3.13.0 genome assembler. The final assembly was generated using a multi-k-mer approach (k = 77, 95, and 127).

[0072] The genome of Curtobacterium salicaceae strain (WW7) consists of 18 scaffolds (N 50The genome is 3,489,963 bp in length and has a G+C content of 71.35%, corresponding to approximately 84× coverage. The completeness of the WW7 genome was calculated based on the presence of Actinomycetales lineage-specific single-copy marker genes using CheckM v1.0.8. At this point, a completion rate of 99% was achieved. Gene prediction for the draft assembly was performed using Prokka v1.11 (7). Of the 3,363 predicted genes, 3,286 were protein-coding genes, 53 were tRNAs, 12 were miscRNAs, 1 was tmRNA, and 11 were rRNAs. A total of 1,114 genes were assigned to Clusters of Orthologous Groups (COGs), 1,082 were annotated with enzyme codes (ECs), and 1,722 were assigned to KEGG Orthology (KO).

[0073] To phylogenetically classify WW7 to the species level, two different strategies were used: (i) average nucleotide identity (ANI) analysis using the ANIm algorithm (Seemann T. (2014). Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068-2069. doi:10.1093 / bioinformatics / btu153). Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068-2069. doi:10.1093 / bioinformatics / btu153), and (ii) Varghese et al. (Richter M and Rossello-Mora R. (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA. 106: Calculation of intraspecific probabilities (Printra-species) using the genome-wide average nucleotide identity (gANI) strategy described in (see refs. 19126-19131). In this regard, the WW7 genome was compared to all publicly available Curtobacterium genome assemblies in the NCBI GenBank database: a total of 107 Curtobacterium genomes. All genomes were aligned to each other using the ANIm algorithm, and ANI values ​​were used to construct an adjacency matrix. This matrix was converted to a similarity matrix (Figure 8A), and clusters of closely related genomes were extracted using a cutoff of 0.9 (corresponding to a 90% ANI).WW7 includes Curtobacterium herbarum DSM 14013 (ASM1690733v1) and seven new Curtobacterium strains isolated from leaf litter in Southern California (NCBI BioProject accession number: PRJNA391502): Curtobacterium sp. MCPF17_052 (NCBI assembly ID: ASM323408v1), Curtobacterium sp. MCPF17_047 (NCBI assembly ID: ASM323404v1), Curtobacterium sp. MCPF17_031 (NCBI assembly ID: ASM323403v1), Curtobacterium sp. MCPF17_011 (NCBI assembly ID: ASM323414v1), and Curtobacterium sp. MCPF17_001 (NCBI assembly ID: ASM323461v1), Curtobacterium sp. MCLR17_032 (NCBI assembly ID: ASM323479v1), and Curtobacterium sp. MCBD17_030 (NCBI assembly ID: ASM322425v1) were closely related (ANI > 90%). See Figure 8A. WW7 was also distantly related (84%) to two strains isolated from leaf litter in Massachusetts (MCBA15_007 (ASM186490v1), MCBA15_005 (ASM186485v1)) and to Curtobacterium pusillum (NCBI assembly ID: ASM202564v1) isolated from maize roots. <ANI)であった。

[0074] To further evaluate the affiliation of strain WW7 to the Curtobacterium type strain, pairwise digital DNA-DNA hybridization (dDDH) values ​​were calculated for strain WW7 to determine its intraspecific relationship with representative strains (type strains) of the genus Curtobacterium. As shown in Figure 8B, the pairwise dDDH values ​​between strain WW7 and the Curtobacterium type strain were lower than 70%, indicating that strain WW7 is a representative strain of a novel Curtobacterium species. See Kim MK, Kim YJ, Kim HB, Kim SY, Yi TH, Yang DC 2008. Curtobacterium ginsengisoli sp. nov., isolated from soil of a ginseng field. Int J Syst Evol Microbiol. 58(10):2393-7. Similarly, the Genome Taxonomy database (GTDB) identified WW7 as the only member of a novel species cluster, namely, Curtobacterium flaccumfacies (https: / / gtdb.ecogenomic.org / species?id=Curtobacterium%20flaccumfaciens), which supports the lack of affiliation of this strain to any known Curtobacterium species.

[0075] Phylogenetic distances were also calculated using 605 linked single-copy protein-coding genes assigned to clusters conserved among all GTDB Curtobacterium representatives according to the Anvi'o pangenomic pipeline. The resulting phylogenetic tree showed that strain WW7 was clearly separated from other Curtobacterium species, with C. herbarum identified as the closest reference strain (see Figure 8C). Using the Anvi'o pangenomic analysis pipeline, 605 single-copy core genes were identified and partition files were generated. Partitioning analysis was then performed using IQ-TREE to calculate the best substitution model for each single-copy core gene. Bootstrap values ​​were calculated based on 1000 replicates, and only nodes with bootstrap values ​​>80% are shown. Asterisks indicate Curtobacterium reference strains according to the List of Prokaryotic Names with Standing in Nomenclature (LPSN) database. Clavibacter michiganensis was used as the outgroup.

[0076] Example 1C Genome analysis of Rhizobium populi (PTD1) To assess the affiliation of strain PTD1 to the Rhizobium type strain, pairwise digital DNA-DNA hybridization values ​​(dDDH) were calculated for strain PTD1 to determine its intraspecific relatedness to the closest representative strain (type strain) of the genus Rhizobium. The pairwise dDDH values ​​between strain PTD1 and the Rhizobium type strain were lower than 70%, as shown in Figure 9, indicating that strain PTD1 is a representative strain of a novel Rhizobium species.

[0077] Example 2A Two-strain product co-fermentation and blending Co-fermentation and co-cultivation of the two endophyte strains WW6 and WW7 was achieved using nitrogen-limited medium under tightly controlled fermentation conditions. Each strain was first inoculated into nitrogen-limited semi-solid medium (NLM). Three to four colonies were selected and used to inoculate a 2-liter seed starter culture containing fresh, sterile NLM medium. The 2-liter flasks were grown for 72 hours at approximately 25°C to approximately 30°C under constant agitation ranging from 200 rpm to approximately 500 rpm. Upon completion, the two strains were pooled into a single carboy and stored at 4°C before being used to inoculate 4,500 liters of NLM medium (pH 7.6). The fermentation medium was sterilized in a steam-jacketed fermentor ranging in size from approximately 1,000 to approximately 30,000 liters. Co-fermentation / co-cultivation was carried out for 3 to 4 days with an aeration rate of approximately 10 to approximately 30 PSI. The range of colony forming units obtained for the two strains was 2.2 × 10 when plated on Tryptic Soy Broth Agar (TSBA). 8 CFU / mL to 1.23 × 10 9 CFU / mL WW6, and 1.03 × 10 8 CFU / mL to 1.4 × 10 9 The CFU / mL was WW7.

[0078] To demonstrate the efficacy of the co-fermentation method, we applied it to strains WW6 and WW7. Fermentations containing both strains WW6 and WW7 were prepared using the large-scale co-fermentation method described above. Samples taken from the fermentation tank were tested in triplicate sterile dilutions using a neutral pH-7.0 buffer solution. Serial dilutions from 10-4 to 10-9 were plated using standard microbiology spread plate techniques. Media spread plates used included nitrogen-free medium (NFM), nitrogen-limited medium (NLM), tryptic soy agar (TSA), potato dextrose agar (PDA), and / or other media. Colonies were incubated at approximately 75°F to approximately 86°F for 48 to 72 hours. Colonies on the plates were then counted based on their morphology, and CFU / ml was calculated.

[0079] Furthermore, two colonies of each morphology were examined by PCR using primers specific to each strain. Figure 10A shows a PCR gel demonstrating the presence of two endophyte strains, WW6 and WW7.

[0080] The co-fermentation procedure described above can be used for other combinations of endophyte strains, for example, any combination of WW5, WW6, WW7 and PTD1.

[0081] Example 2B Endophyte compositions prepared by mixing with dry fertilizers and dry powders for combined use in agriculture Two novel compositions were created containing WW6 and WW7 endophyte strains, sodium alginate (DuPont Nutrition USA, Inc.), and dry fertilizers, one containing triple superphosphate 0-28-0 (OCP group) and the other containing dolomite lime (Down To Earth, Inc.). A 3 mL mixture containing WW6, WW7, and 0.5% alginate was added to 5 grams of dry fertilizer. The compositions were allowed to dry and stored at room temperature for 24 hours. The bacterial counts of colony-forming units (CFU / gram) of the two strains were then determined by plating on NLM semi-solid medium.

[0082] The results shown in Figure 10B indicated that the viability of both endophyte strains was slightly reduced when mixed with different fertilizers in the new compositions. These results demonstrate that the liquid endophyte composition can be used to create new compositions for use in commercial agricultural practices as NUE endophyte-enhanced agricultural fertilizers.

[0083] Additionally, a liquid fermentation composition containing the WW6 and WW7 endophyte strains and sodium alginate (DuPont Nutrition USA, Inc.) was used to create new compositions containing three different powdered dry carriers when combined with whey protein (Chemital tecnicas alimentarias), sodium bentonite (Specialty Minerals, Inc.), and coconut coir (W. Atlee Burpee & Co.). The compositions were created by adding 3 ml of a liquid composition containing a mixture of WW6, WW7, and 0.5% alginate to 5 grams of the different dry powdered carriers. The powdered carrier endophyte compositions were dried separately at room temperature for 24 hours and then stored before counting colony-forming units (CFU / gram) by plating on NLM semi-solid medium.

[0084] The results are shown in Figure 10C and indicate that the viability of both endophyte strains was slightly reduced when mixed with different dry powder carriers, demonstrating that endophyte compositions can be made and used for formulation, coating, and delivery in commercial agricultural practices when used with different dry carriers.

[0085] Example 2C The survival of endophyte strains (WW6 and WW7) was assayed along with lyophilized Mycorrhizae powder after combining with different powder carriers. Various powder carrier mixtures (maltodextrin, sucrose, dextrose, whey) were assayed for compatibility with the WW6+WW7 FD powder mixture. The proportions of the mixture tested were as follows: powder carrier 2.09 g (approximately 95% by weight), lyophilized powder WW6+WW7 0.11 g (approximately 5% by weight), Mycorrhizae 0.0022 g (approximately 0.1% by weight). The results of the compatibility test demonstrated the following results, shown in Figure 11.

[0086] Compatibility testing of freeze-dried WW6+WW7 mixtures of four potential bulking agents (maltodextrin, sucrose, dextrose, and whey) with Mycorrhizae yielded positive results. The one powder carrier that reduced CFU the most was a whey product, which only slightly inhibited WW6. Maltodextrin and dextrose slightly reduced CFU of the WW6 strain. The other powder products did not significantly reduce CFU. Sucrose was the best powder carrier, reducing CFU of either strain in the assay.

[0087] The results of this example demonstrate that freeze-dried endophyte powder can be mixed with a variety of carriers that allow the freeze-dried inoculant to be diluted to lower levels and used as a fertilizer coating or as a reconstituted powder for subsequent resuspension and use as a variety of aqueous foliar treatments.

[0088] Example 2D Short-term and long-term survival of endophyte strains (WW5, WW6, WW7, PTD1, and WP1) over 1–2 days and after 2 weeks on powdered carrier biochar alone and in combination with biochar and hydrocarbon molasses. Biochar was treated with inoculum compositions of co-fermented WW5, WW6, WW7, PTD1, and WP1 alone and in combination with a 1 / 10X molasses solution. The powdered biochar material was dried in open baggies at room temperature and stored at 25°C. After two weeks, dried biochar (0.1 g) was resuspended in 1 mL of potassium phosphate buffer and assayed for endophyte survival by strain, resulting in CFU / ml. The data are shown in Figure 12.

[0089] Results demonstrated that 50 μL of co-fermented endophyte mix with 950 μL of 1 / 10x molasses applied per 1 g of biochar was an effective rate for the viability and stability of four of the five tested endophyte strains on the biochar powder carrier for at least two weeks.

[0090] Example 3 Endophytic strain WW7 solubilizes insoluble phosphate The ability of endophytic strains to enhance endophyte metabolism and utilize insoluble forms of phosphorus (P) from soil or soil solution, mobilize P in the plant, and enhance P uptake for other soil particles or internal metal ions. Heterologous application of the endophytic strain WW7 has been shown to be effective in enhancing P uptake in host plants. Heterologous WW7 can apparently solubilize different insoluble forms of P that are insoluble in the medium solution mixture. Genomic analysis of WW7 indicates possible genetic mechanisms for the biosynthesis of Krebs cycle intermediates, such as the organic oxyacids malate and citrate, which may be responsible for solubilizing insoluble forms of phosphate from soil, allowing for better plant uptake. Furthermore, endophytes may have exudates that leave phosphate ligands free once inside the plant by tightly binding phosphate and competing with other metals, which would reinsolve it and make it unavailable for assimilation. The WW7 genome data was used to identify protein-coding genes involved in the reactions of interest, and the predicted proteome of WW7 was functionally annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the KofamKOALA genome jp tool (https: / / www.genome.jp / tools / kofamkoala / ). Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database as a functional database, the WW7 proteome was searched for enzymatic reactions and pathways catalyzing the synthesis of malate and citrate, which can be exuded from roots and solubilize insoluble P. A total of 1,602 proteins in WW7 were mapped to the KEGG database. The enzymes involved in the synthesis of malate and citrate were citrate synthase (gene ID: 2821609409) and fumarate hydratase (gene ID: 2821609475), respectively. Malic acid can also be synthesized by the assimilation and conversion of aspartic acid and glutamic acid.In this regard, WW7 possesses a complete set of enzymes that catalyze the conversion of glutamate and aspartate to L-argininosuccinate, which is further converted to arginine, resulting in the release of a molecule of fumarate for use in the citric acid cycle pathway (see Figure 13A). Glutamate is the first amino acid product of the GS-GOGAT pathway, which produces one mole of glutamate from each mole of NH3. The GOGAT pathway is responsible for atmospheric nitrogen fixation in bacteria. Figure 13A shows the WW7 enzymatic pathway involved in the synthesis of fumarate from glutamate and aspartate (reaction numbers 2.3.1.1, 2.7.2.8, 1.21.38, 2.6.1.11, 2.3.1.35, 2.1.3.3, 6.3.4.5, and 4.3.2.1), along with the enzymatic reactions catalyzed by the WW7 enzymes.

[0091] phosphate solubilization genes According to KEGG annotation, genes involved in the solubilization of inorganic and organic phosphates in other species were also detected in WW7 (see Figure 13B). The results indicate the presence of acid phosphatase (AcPase) genes and genes involved in the synthesis of acetate and glucuronic acid. AcPase is shown in other species to be involved in the solubilization of phosphate from phosphomonoesters, and acetate and glucuronic acid are shown in other species to be involved in the solubilization of phosphate from inorganic forms.

[0092] The ability of endophytic strains to solubilize insoluble phosphorus (P) from soil or soil solution through the exogenous production of various mobilizing compounds is important for helping plants more efficiently acquire necessary P, other nutrients such as potassium K, and trace nutrient ions from the soil. These chelating, or pH-reducing, acidic compounds produced by endophytes can apparently help plant roots better access these minerals from the soil and can aid in uptake and translocation from the plant roots to the shoot.

[0093] To demonstrate the ability of WW7 to solubilize insoluble phosphate through the secretion of exogenous compounds, an insoluble phosphate solubilization assay was performed in liquid medium using a phosphate-sensitive dye and a microplate reader to further confirm the biochemical ability of WW7 to solubilize different species of phosphate. The physiological assay method was developed and modified from a previous method performed by Varga et al., Endophyte-Promoted Phosphorus Solubilization in Populus, Frontiers in Plant Science, 11; 2020; 1585 ("Varga"). WW7 cells were grown in phosphate-free modified National Botanical Research Institute (NBRIP) broth to deplete residual internal phosphate. Five milliliters of the modified NBRIP broth culture, with or without phosphate, as previously described by Varga, was added to 10 mL of WW7 cell culture. These 10 mL tubes were then incubated for 3 days at 25°C with shaking at 220 rpm. The 10 mL tubes were then removed from the shaker and allowed to settle for 90 minutes. One mL of culture was spun down at 5,000 rpm for 5 minutes at 25°C, and the supernatant was used to measure solubilized phosphate. The absorbance of solubilized phosphate was measured at 650 nm (A650) using a phosphate colorimetric assay kit (Sigma-Aldrich, MAK030). Supernatant from each sample was added at different dilutions to a 96-well plate. Additionally, phosphate standards provided with the kit were prepared to calculate the linear equation used to determine solubilized phosphate in each sample. Four to five technical replicates were used to determine statistical differences between samples.

[0094] The results shown in Figure 13C demonstrate that the growth and molecular activity of endophyte strain WW7 resulted in a statistically significant (p<0.01) increase in phosphate solubilization of both insoluble suspended aluminum phosphate and tricalcium phosphate, but not iron phosphate. Strain WW7 increased phosphate solubilization from insoluble aluminum phosphate by an average of 29% and from insoluble tricalcium phosphate by an average of 100%. These results demonstrate substantial mobilization of insoluble phosphate by WW7.

[0095] WW7 can be mixed with other nitrogen-fixing microorganisms to mobilize P such that the transformation of insoluble phosphate by WW7 and the potential uptake of solubilized P by the host plant from soil or rock can be combined with enhanced nitrogen acquisition (e.g., from the atmosphere). Taken together, these mechanisms can greatly enhance the metabolic performance of treated host plants, which could lead to increased biomass, stress tolerance, and other favorable traits.

[0096] Example 4 Genomic and biochemical iron siderophore production assays Endophytic strains may be able to solubilize iron (Fe) from soil or soil solution. This can be achieved through the production of various solubilizing compounds and / or heme-associated binding factors. Endophytic strains can also enhance host plants to acquire necessary Fe and improve their ability to acquire other metal and micronutrient ions, especially positively charged divalent cations, from soil. These Fe-siderophore chelating compounds produced by some bacterial and yeast endophytes can help plants better compete with the high cation exchange capacity of soil clay particles and aid in the uptake and translocation of metal ions from plant roots to shoots. To confirm the capabilities of strain WW7, we performed a genome-wide analysis of the WW7 gene. WW7 was shown to possess the genetic machinery necessary to produce Fe-siderophores that can transport or scavenge Fe ions. InterProscan results indicate the presence of a gene cluster, efeUOB, involved in the recovery of ferrous and ferric iron from exogenous heme, and three genes encoding NADPH-dependent ferric siderophore reductases. See Figure 14A. The latter catalyzes the reduction of ferric iron complexed with different siderophores, such as ferric tricatechol and ferric dicitrate. This enzymatic reaction liberates the bound ferrous iron.

[0097] Furthermore, anti-smash analysis revealed that the WW7 NADPH-dependent ferric siderophore reductase (Ga0372474_197) was found in a cluster with a gene encoding a nonribosomal peptide synthetase-like protein (Ga0372474_207), an enzyme typically found in biosynthetic gene clusters (BGCs) involved in the synthesis of iron siderophores.

[0098] Assay for Fe-siderophore production in all four endophyte strains Endophytic strains WW5, WW6, WW7, and PTD1 for Fe-siderophore production. Fe-siderophore production was demonstrated in three of the endophytic bacterial strains tested. Four strains were assayed using a microbial growth solid agar plate-based Fe-siderophore CAS medium. Agar plates were prepared using a CAS agar plate preparation containing Chromeazurol as a color indicator and FeCl3. Endophytic strains were applied to individual plates.

[0099] Fe solubilization was evidenced by the presence of decolorized areas on the CAS test plates, which appeared as a clear white color around the streaks of bacterial growth on the plate. The areas were measured and quantified using image analysis software (e.g., ImageJ, a publicly available image analysis program provided by the National Institutes of Health, available at http: / / rsb.info.nih.gov / ij / ). The ability of the strains to solubilize Fe was photographed, measured, and compared. See Figure 14B.

[0100] The in vitro results in Figures 14B–14D demonstrate that WW7 produces extracellular compounds that scavenge insoluble Fe. WW7 exhibited the greatest Fe-solubilizing activity of all strains. WW5 and WW6 also exhibited significant Fe solubilization. However, the PTD1 Rhizobium populi strain exhibited little or no Fe-solubilizing activity, and insoluble Fe was observed immediately adjacent to the colony streaks. The results demonstrate the production of Fe-siderophores by WW5, WW6, and WW7. The results demonstrate Fe solubilization by each of the WW5, WW6, and WW7 strains, suggesting that Fe-siderophore production was greatest in WW7, followed by WW5 and WW6. Endophytic strains producing these compounds can assist host plants in solubilizing and mobilizing Fe. The Fe-solubilizing activity of each strain is quantified in Figure 14C and graphically depicted in Figure 14D.

[0101] Example 5 Gas chromatographic analysis and identification Bacterial identification was performed by gas chromatography analysis of fatty acid methyl esters (GC-FAME). GC-FAME analysis provides a unique FAME ID chemical identification chromatogram that is highly specific for each endophyte strain. This allows these microbial isolates to be individually tracked and unambiguously identified based on their unique fatty acid methyl ester signatures. The unique chromatograms for each strain are shown in Figure 15.

[0102] How to use The formulations disclosed herein can be advantageously applied to plants by several means, including, but not limited to, spraying, irrigating, coating, dipping, injecting, in-furrow, or any combination thereof. The compositions according to the invention can be used on leaves, roots, foliar, foliage, tillers, flowers, plant cells, plant tissues, seeds (e.g., as a coating or by seed treatment such as by spraying or dipping), pre-emergence (before the seedling emerges or emerges above ground), grain, fruit, tubers, spores, cuttings, cuttings, meristems, plant cells, nuts, or embryos. In some instances, the composition can be used as part of a dip for the roots and / or other tissues of the host plant, as a seed coating, as a coating applied to the leaves and / or other elements of the host plant, as a powder on the surface of the leaves and / or other elements of the host plant, as a spray on the leaves and / or other elements of the host plant, as part of a trickle to the soil and / or roots of the host plant, as dry alginate beads that encapsulate the endophytes and deliver them to the roots, or any other suitable method or inoculation.

[0103] The compositions of the present invention are effective for improving the metabolism of host plants (e.g., nutrient uptake, carbon uptake, growth, etc.). Thus, the compositions and methods of the present invention can be significantly economically advantageous, as increased growth characteristics can result in increased yields of harvestable crops and more robust plants. Exemplary methods are described below.

[0104] Example 6 PCR analysis of endophyte colonization of host plants after root dip inoculation The ability of the endophyte strains to heterologously colonize host crop plants was tested using PCR. Results conclusively demonstrated that the endophyte was present within surface-sterilized plant tissue. In planta PCR clearly demonstrated successful colonization in agriculturally important wheat, rice, and barley species inoculated with the WW6 (Pseudomonas siliginis) endophyte strain via seed treatment.

[0105] Four sets of plants were grown after treating the seeds as follows: an appropriate number of seeds were placed in the bottom of an 11 cm x 11 cm seed germination box and 20 mL of inoculation solution was added (both in NLM medium for approximately 10 7 CFU / ml of co-fermenting endophytes (WW6+WW7). Seeds were allowed to germinate for 5 days before being transplanted into 3.5-inch pots containing a washed play sand, vermiculite, and perlite potting mix. Plants were grown in a 25°C growth room under a 14-hour light / 10-hour dark cycle of sodium halide lights. Additionally, plants were watered and fertilized with a 25 ppm nitrogen-reduced Hoagland's solution in trays as needed two to three times a week to maintain moist to slightly dry soil. 14 days after transplanting, plants were individually harvested. Plants were then removed from the soil and processed. DNA was isolated from the treated samples using the PureLink™ Microbiome DNA Purification Kit DNA Isolation Kit (Thermo Fisher Scientific).

[0106] Twenty microliter PCR reactions were set up using 2X HotStart PCR Master Mix (MCLAB), 1 μL of template DNA, and 1 μM PCR primers specific to the WW6 gene. PCR cycling conditions were as follows: 1 cycle at 95°C for 10 minutes; 25 PCR cycles at 95°C for 30 seconds, 65°C for 30 seconds, and 72°C for 45 seconds, followed by a final cycle at 72°C for 5 minutes. Ten μL of each PCR reaction was loaded onto a 1.2% agarose gel and subjected to DNA QS710 electrophoresis (IBISCI) at 120 V. Figure 16 shows that WW6 was present in pre-emergently inoculated plants from all three test host plants (wheat, rice, and barley) but absent from the control plants for each of the three test plants. Bands in lanes 2 (WW6-treated wheat), 4 (WW6-treated rice), and 6 (WW6-treated barley) indicate the presence of the WW6-specific gene. Thus, it is clear that the WW6 endophyte strain can effectively colonize several host plants.

[0107] Example 7 PCR analysis of endophyte colonization of host plants after seed coating The ability of the endophyte strain to heterologously colonize host crop plants when used in conjunction with seed coatings was tested using PCR. Shoots and roots of winter wheat (Triticum aestivum) and broccoli (Brassica oleracea) plants treated with WW6 (Pseudomonas siliginis) were evaluated for colonization and uptake of the WW6 endophyte strain into plant tissues. Seeds were treated with WW6 before growing the plants as follows: WW6 was fermented according to the method disclosed herein, then blended with 0.5% sodium alginate (Scogin™ LDH), and then used to coat untreated wheat seeds. The same seed coating material without the endophyte strain was applied to control plants. The coated seeds were air-dried at room temperature and stored for one month after coating treatment.

[0108] An appropriate number of seeds were placed in the bottom of an 11 cm x 11 cm seed germination box. Seeds were allowed to germinate for 5 days before being transplanted into 3.5-inch pots containing a mixture of washed play sand, vermiculite, and perlite potting mix. Plants were grown in a growth room at 25°C under a 14-hour light / 10-hour dark cycle of sodium halide lights. Additionally, plants were watered and fertilized with a 25 ppm concentration of reduced nitrogen Hoagland's solution two to three times a week in trays as needed to maintain moist to slightly dry soil. 14 days after transplanting, plants were then removed from the soil and fertilized. The plants were then removed from the soil and processed. Root and shoot tissues were harvested from the plants. DNA was isolated from the processed samples using the PureLink™ Microbiome DNA Purification Kit DNA Isolation Kit (Thermo Fisher Scientific).

[0109] PCR was performed as described in Example 5 above. Figures 17A and 17B show electrophoresis gel data for DNA encoding a protein present only in WW6. The gels demonstrate that WW6-specific DNA is present in the shoot and root tissues of winter wheat and broccoli plants grown from treated seeds, but not in the shoot and root tissues of control plants. Figure 17A shows electrophoresis gel data demonstrating the presence of WW6 in the shoot and root tissues of winter wheat host plants grown from treated seeds, based on the presence of genome-specific PCR primers designed for the protein that yield DNA bands specific to the WW6 strain and absent from the shoot and root tissues of control plants. Thus, it is clear that the WW6 endophyte strain can effectively colonize the root and shoot tissues of wheat host plants after seed inoculation.

[0110] Figure 17B shows electrophoresis PCR gel data demonstrating that WW6 is present in the root tissue of seedlings inoculated with the WW6 seed treatment after surface sterilization of the root tissue. Several treatment groups using the WW6 endophyte strain were prepared: a first treatment of WW6 liquid fermentation, a second treatment in which broccoli seeds were treated with the liquid fermentation mixed with 0.5% sodium alginate (Scogin™ LDH from DuPont) and seed coating material, and a third treatment in which broccoli seeds were treated with the WW6 liquid fermentation mixed with 1% sodium alginate and seed coating material. Different groups of broccoli seeds were coated in the three treatments. Plant growth, DNA isolation, and PCR were performed as described in Example 5 above.

[0111] Figure 17B shows electrophoresis gel data demonstrating the presence of WW6 in the shoot and root tissues of winter wheat, rice, soybean, broccoli, and corn host plants grown from treated seeds, as evidenced by the presence of genome-specific PCR primers designed for proteins that yield unique DNA bands present in the WW6 strain but absent in the control shoot and root tissues. Thus, it is clear that the WW6 endophyte strain can effectively colonize the root and shoot tissues of several host plants after seed inoculation.

[0112] Example 7A PCR analysis of endophyte colonization of host plants after seed coating The ability of heterologous endophyte strains to colonize host crop plants when used with seed coatings was tested using quantitative ddPCR (digital droplet PCR). Shoots and roots of barley (Hordeum vulgare) plants were evaluated for colonization by WW6 and WW7 after the seeds were coated with the seed treatment composition. WW6 and WW7 fermentates were blended with 0.5% by weight of sodium alginate (Scogin™ LDH), and this composition was used to coat untreated barley seeds. The seeds were air-dried at room temperature and stored for one month. The same seed coating composition without the endophyte strains was used for control seeds.

[0113] An appropriate number of seeds were placed in an 11 cm x 11 cm seed germination box with sterile filter paper. Seeds were germinated in deionized water for 5 days before being transplanted into 3.5-inch pots containing a mixture of washed play sand, vermiculite, and perlite potting mix. Plants were grown in a growth room at 25°C under a 14-hour light / 10-hour dark cycle of sodium halide lights. Additionally, plants were watered and fertilized with reduced-nitrogen (25 ppm N) Hoagland's solution two to three times a week in trays as needed to maintain moist soil. 21 days after transplanting, the plants were then removed from the soil, and the roots and shoots were treated by surface sterilization with 2% bleach, followed by rinsing with sterile water. Shoots and roots were separated, flash-frozen in liquid nitrogen, and then stored at -20°C. Plant material was then ground to a fine powder using a mortar and pestle with liquid nitrogen. 100 mg of each tissue sample was used to isolate DNA using the DNeasy Plant Pro DNA isolation kit (QIAGEN, Inc).

[0114] We designed a novel primer set and multiplexed it with our gene-specific PCR primers, aiming to enable simultaneous analysis and differential quantification of both strains. Strain-specific primers were created using fluorophores (FAM and Hex). The strain-specific primers were validated for strains WW6 and WW7 using gBlock, a double-stranded synthetic sequence of a specific fragment from each strain. Bacterial genomic DNA was used as a positive control. The validated strain-specific primers were used for PCR analysis of samples from root and shoot tissues of treated barley seeds for the presence of strains WW6 and WW7 using a droplet digital PCR (ddPCR) instrument from Bio-Rad Laboratories, Inc.

[0115] The results shown in Figure 17C show a dot plot graph of the quantification of the WW6 strain PCR target using FAM and Hex fluorescent signals for the WW6 strain assay. Figure 17D shows a dot plot graph of the quantification of the WW7 strain PCR target using FAM and Hex fluorescent signals. Experimental results were quantified using QuantaSoft software (Bio-Rad Laboratories, Inc.).

[0116] The total copies of hybridizing DNA isolated per mg of plant tissue, calculated from copy number concentrations provided by QuantaSoft software (Bio-Rad Laboratories, Inc.), are summarized in the table shown in Figure 17E. Results demonstrated quantitative in planta detection of both strains, WW6 in roots and shoots and WW7 in roots and shoots. The highest detection level was quantified for WW6 in barley shoots relative to control uninoculated plants.

[0117] The data demonstrated that the WW6 and WW7 strains were present in the shoot and root tissues of barley host plants grown from treated seeds, as evidenced by the presence of genome-specific primers designed to hybridize to a unique DNA band present in the WW6 strain but absent from the control shoot and root tissues. Thus, it is clear that the WW6 and WW7 endophyte strains can effectively colonize the root and shoot tissues of several crop plants after seed inoculation.

[0118] Example 8 Analysis of endophyte colonization of host plants after foliar treatment The performance of spinach plants (Spinacia oleracea) in the field treated with WW6 and WW7 endophytes in a foliar spray was tested, and the ability of the endophyte strains to heterologously colonize the host plant was also determined.

[0119] A foliar inoculant composition containing co-fermented WW6 and WW7 freeze-dried powders resuspended in water was applied to spinach plants according to industrial rates along with a 10-5-3 CaO liquid fertilizer formulation, Greenstim™ (concentrated glycine betaine extracted from beet roots with 12% total nitrogen, from the Masso, SA Agro Department). Spinach was grown hydroponically using a complete nutrient regimen, with 550 L of 10-5-3 3% CaO fertilizer (715 kg of fertilizer, 18 kg / day) applied daily to each plot. The foliar composition was applied at the four-leaf stage at a foliar rate of 1 L per hectare, with a WW6+WW7 endophyte concentration of 20 g / L. The experimental plot was divided into three blocks, each containing six beds. Sampling was performed in the center of the four central beds in each block. Percent canopy cover was assessed 35 days after application using a software tool (Canopeo™) for analyzing and measuring canopy cover in photographs. At the same time, leaves were collected for nutrient analysis and for surface sterilization followed by in-plant endophyte quantification. Percent plant cover versus plot bed data showed that endophyte foliar treatment resulted in a statistically significant 30.92% (p<0.05) increase in spinach leaf canopy cover compared to industrial fertilizer alone, and a statistically significant 58% (p<0.05) increase over the control treatment. See Figure 18A.

[0120] To assay and quantify the presence of intraplantar endophytes in spinach tissues, leaves and roots were collected 35 days after foliar inoculation, washed, and surface-sterilized for detection of WW6 and WW7 strains within the plant tissue. All samples were weighed and photographed to calculate CFU for sample weight and leaf or root area. Leaf and root ends were individually sealed in plastic bags and then surface-sterilized in a laminar flow fume hood. The surfaces were washed with distilled water to remove dust, soil, and other contaminants. The samples were then placed in sterile flasks, and 70% ETOH alcohol was added. The flasks were shaken (150 rpm) for 2 minutes. After shaking, the alcohol solution was removed, and a 1% sodium hypochlorite bleach solution was added. The mixture was shaken for an additional 2 minutes. The sodium hypochlorite solution was then removed, and the samples were cleaned three times by manually shaking in sterile water for 1 minute.

[0121] Leaves and roots were ground in 50 ml of 0.9% saline. The extract was placed in a sterile tube and left for 1 hour to release endophytes from the ground tissue. Several serial dilutions of the extract were made in sterile water. 100 μl of each dilution was plated on potato dextrose agar (PDA) plates containing glycerol and on actinomycete isolation agar.

[0122] The agar plates were incubated until bacterial growth was visually confirmed. Bacterial concentrations were performed to determine CFU / g and CFU / cm of the analyte. 2 The result expressed as follows was obtained.

[0123] The data in Figure 18B demonstrate that endophytes (WW6 and WW7) were present in the leaves and roots of surface-sterilized spinach host plants inoculated with the foliar inoculation composition. Endophyte strains were absent from spinach plants that received the Greenstim liquid fertilizer treatment alone (see detailed discussion above).

[0124] WW6 and WW7 endophytes were detected in both the leaves and roots of the host plants 35 days after treatment with the foliar inoculation composition containing WW6 and WW7 endophytes. In the standard-treated control plants, endophytes were absent and no endophytes were detected. When endophyte concentrations were expressed in CFU / g, higher endophyte concentrations were found in the leaves than in the roots, but the difference was small and the concentrations in the leaves and roots can be considered to be the same in both locations. CFU / cm 2 Surface areas were visually analyzed and calculated from measurements using Adobe Photoshop software.

[0125] The above data demonstrate that the endophyte successfully established and improved leaf growth and biomass of treated host spinach plants 35 days after foliar treatment. The results demonstrate the efficacy of the composition and foliar treatment method, which resulted in enhanced production due to better establishment, growth, and soil coverage of the spinach host plants.

[0126] Example 9 Effect of single endophyte strain seed treatment on total nutrient accumulation Corn seeds (Zea mays) were treated with a seed inoculant containing one heterologous endophyte strain selected from WW5, WW6, and WW7, and compared with control corn seeds treated with an inoculant containing no endophyte. Four sets of corn seeds were grown as follows: an appropriate amount of corn seeds was placed in the bottom of a large gallon-sized Ziploc bag and sealed. Three groups of corn seeds were each treated with a specific endophyte culture (WW5, WW6, or WW7). The WW5, WW6, and WW7 cultures were grown under conditions (approximately 10 7The cultures were prepared in a sterile laminar flow fume hood (containing endophytes at CFU / ml) and then refrigerated. The cultures were removed from refrigeration, mixed well, and carefully pipetted onto the seeds in a sterile laminar flow fume hood at a rate of 3.4 mL / lb of seed dispersed in 1 mL droplets. After each 1 mL addition, the bag and seeds were manually inverted and carefully kneaded. Once all 3.4 mL / lb had been added, the seeds were kneaded, shaken, and inverted for 2-3 minutes until all corn seeds were visibly wet within the bag. The bag was then opened for air drying in a sterile laminar flow fume hood, with air flowing through the fume hood to air-dry the corn seeds. After drying, the seeds were stored at room temperature for 3 weeks and then grown in a 1-gallon felt Smart Pot at 25°C in a growth room under sodium halide light (710 μmol m -2 s -1 Seeds were sown and germinated under a 14-hour light / 10-hour dark cycle under a photon flux rate of 1000 kJ / s. Once germinated, plants were watered and fertilized in trays with a 50 ppm nitrogen-reduced Hoagland's solution two to three times a week, as needed, to maintain moist to slightly dry soil. Controls were grown under the same conditions without pretreatment before planting. Plants were individually harvested on day 24, dried, and weighed. Tissues were sent for inductively coupled plasma mass spectrometry (ICP-MS) to determine tissue iron content. Nutrient accumulation in shoot biomass was calculated by multiplying the total shoot dry weight by the shoot concentration in each sample.

[0127] As shown in Figure 19, corn plants inoculated with endophyte strains WW5, WW6, or WW7 accumulated significantly higher levels of macro- and trace mineral nutrients across key mineral ion profiles, as measured by the percent change in total nutrient content of shoot biomass relative to untreated control plants, when all were grown in Hoagland's Dropout N nutrient solution supplemented with 50 ppm bioavailable nitrogen.

[0128] The data in Figure 19 demonstrate that the heterologous endophyte successfully improved both macro- and micronutrient uptake and incorporation in host corn plants grown under reduced nitrogen from treated seeds. The results demonstrate the efficacy of the heterologous endophyte in enhancing the physiological performance of non-native host plants and air-sourced nitrogen. The endophyte seed treatment composition increased nitrogen in corn shoots as follows: WW5 47%, WW6 45%, and WW7 29%.

[0129] Example 10 Endophyte screen assay of crop plant yield when grown with limited bioavailable forms of both nitrogen and phosphorus The WW5, WW6, WW7, and PTD1 endophyte strains were further screened in greenhouse pot studies in which plants were inoculated with endophyte strains encapsulated in alginate beads individually or as a mix of all four strains. Alginate beads encapsulating the endophyte within calcium alginate were placed next to the seeds, one bead per seed, and the pots were watered evenly using controlled drip irrigation to allow the seeds to germinate. Plants were grown specifically under limiting nutrient concentrations in potting medium that intentionally reduced the bioavailability of soluble forms of nitrogen and phosphorus, where the potting medium contained <13 ppm nitrate, <6 ppm ammoniacal N, and <11 ppm phosphate.

[0130] The results in Figure 20 demonstrate that the four selected endophytes responded positively to increased yields of a wide variety of crop plants in the presence of limited nitrogen and phosphorus bioavailability when using commercially available, agriculturally relevant, and commonly used seeds.

[0131] Example 11 Effects of combined endophyte strain (WW6+WW7) seed treatments on total nutrient accumulation and shoot biomass Canola seeds (Brassica napus) treated with a seed inoculant composition containing the co-fermented WW6 and WW7 heterologous endophyte strains were grown and compared with control canola seeds treated with an endophyte-free inoculant composition. Treated seeds were grown as follows: an appropriate amount of canola seeds was industrially treated with Integral pro (BASF) and the prebiotic UBS 016 (Unium Bioscience Ltd.) (both according to the manufacturer's instructions) at a rate of 500 mL of the mixed co-fermented WW6 and WW7 strains and 500 mL of 1% alginate per metric ton of seeds to support endophyte survival, colony growth, and host plant establishment. The prebiotic contains a microbial nutrient package, plant biostimulants, osmoprotectants, buffers, and seed lubricants. Endophyte survival was confirmed on the seeds by adding them to a 0.2 M phosphate resuspension solution and then planting them in NLM semi-solid medium at appropriate dilutions. Control canola seeds received the same crop protection package. Seeds were then stored for one month under normal industrial seed storage conditions (4°C to 15°C) and planted industrially in autumn using a seed drill in a large-scale replicated CRO field experiment in Cuxwold, Lincolnshire, United Kingdom.

[0132] Shoots were collected in mid-spring during the early growth season and sent for agronomic mineral nutrient analysis. Shoot biomass nutrient accumulation was calculated by multiplying the total shoot weight by the shoot ion concentration. As shown in Figure 21, canola seeds inoculated with the co-fermented WW6 and WW7 strains accumulated significantly higher levels of macro- and micronutrients, and the increase in nutrient accumulation relative to the control is expressed as the percent change in total nutrient content of the shoot biomass from the control.

[0133] The above data demonstrate that the co-fermenting heterologous endophytes WW6 and WW7 successfully improved the uptake and incorporation of macro- and micronutrients in canola plants grown from treated seeds. The results demonstrate the efficacy of co-fermenting heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0134] Example 12 Effect of combined WW6+WW7 strains on total nutrient accumulation and biomass Winter wheat seeds (Triticum aestivum) treated with a seed inoculant composition containing the co-fermented WW6 and WW7 heterologous endophyte strains were grown and compared to control winter wheat seeds treated with an endophyte-free inoculant composition. The inoculant composition was combined with the prebiotic composition UBS 016 from Unium Bioscience Ltd. to support endophyte survival, colony growth, and host plant establishment. Treated seeds were grown as follows: an appropriate amount of wheat seeds was industrially treated with 500 mL of the mixed co-ferment, 500 mL of 1% alginate, and 1000 mL of 10% UBS 016 in water per metric ton of seeds. A crop protection package containing fludioxonil and sedaxan, which protects against seed-borne diseases, was also added. Vibrance Duo® from Syngenta AG was used as the crop protection package, containing 25 g / L sedaxan and 25 g / L fludioxonil. Vibrance Duo® product was used at 2 L per metric ton.

[0135] Control seeds of the same cultivars received the same plant protection package, and endophyte survival was confirmed on the seeds by adding them to a 0.2 M phosphate resuspension solution and then planting them at appropriate dilutions on NLM semi-solid medium. The seeds were then stored for one month under normal industrial conditions (4°C to 15°C) and planted industrially in autumn at conventional rates using a seed drill in a large-scale replicated CRO field experiment in Cuxwold, Lincolnshire, United Kingdom.

[0136] Shoots were collected during the growing season in late spring, 5 months after planting, and sent for agronomic mineral nutrient analysis. Shoot biomass nutrient accumulation was calculated by multiplying the total shoot weight by the shoot ion concentration. As shown in Figure 22, winter wheat plants inoculated with co-fermented WW6+WW7 accumulated significantly higher levels of macronutrients and micronutrients, and the increase in nutrient accumulation relative to the control is expressed as the percent change in total nutrient content of the shoot biomass from the control.

[0137] The above data demonstrate that the co-fermenting heterologous endophytes WW6 and WW7 successfully improved macro- and micronutrient uptake and incorporation in winter wheat plants grown from inoculated seeds. The results demonstrate the efficacy of co-fermenting heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0138] Example 13 Effect of combined WW6 and WW7 strains on total nutrient-accumulating shoot biomass Co-fermented WW6 and WW7 fermentates for 10 7 Spring oat seeds (Avena sativa var. Elyann and SO1) treated with seed inoculant compositions engineered with heterologous endophyte strains were grown and compared with control seeds treated with endophyte-free inoculant compositions. Treated seeds were grown as follows: appropriate amounts of oat seeds were industrially treated with 500 mL of mixed co-ferment in water, 500 mL of 1% alginate, and 1000 mL of 10% prebiotic composition UBS 016 (from Unium Bioscience Ltd.) in water per metric ton of seeds, along with the seed disease protectant Redigo (Bayer) according to the manufacturer's instructions. Endophyte survival on the seeds was confirmed by adding the seeds to a 0.2 M phosphate resuspension solution and then planting them in NLM semi-solid medium at appropriate dilutions. Control seeds of the same varieties received the same prebiotic treatment but without the endophyte. The oat seeds were then stored under normal industrial seed storage conditions for one month and planted industrially in May in Suffolk, UK, in a large-scale replicated CRO field experiment using conventional fertiliser rates.

[0139] Shoots were collected during the early growth season, in mid-spring, five months after planting, and sent for agronomic mineral nutrient analysis. Shoot biomass nutrient accumulation was calculated by multiplying the total shoot weight by the shoot ion concentration. As shown in Figure 23, spring oat plants inoculated with co-fermented WW6+WW7 accumulated significantly higher levels of macronutrients and micronutrients, and the increase in nutrient accumulation relative to the control is expressed as the % change in total nutrient content of shoot biomass from the control.

[0140] The above data demonstrate that the co-fermenting heterologous endophytes WW6 and WW7 successfully improved the uptake and incorporation of macro- and micronutrients in spring oat plants grown from inoculated seeds. The results demonstrate the efficacy of co-fermenting heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0141] Example 14 Effect of strain combination WW5+WW6+WW7+PTD1 on nutrient concentrations Asian rice (Oryza sativa) hybrid XP753 seeds were treated with a seed inoculant composition containing co-fermented WW5+WW6+WW7+PTD1 heterologous endophyte strains top-coated on seeds after seed coating with two fungicide / insecticide products, GA3 (gibberellic acid), dye, and a pre-treatment with a flowable zinc micronutrient coating. 500 mL of endophyte fermentate and 1% alginate were added to 2,205 pounds of rice seeds. Examination of the quality of the seed coating on the industrially treated seeds showed survival of the WW5+WW6+WW7+PTD1 endophyte strains, expressed in colony-forming units (CFU) per seed, as follows: WW5 2.0 x 10 6 ;WW6 8.0×10 5 ;WW7 3.6×10 6 ;PTD1 1.2×10 6Control seeds were treated with the pretreatment but without the seed inoculant composition. The seeds were then stored under normal industrial seed storage conditions for two months and planted industrially in the spring of 2020 by a large-scale grower in Clay County, Arkansas, USA. Paddy fields were fertilized with 400 lbs of urea per acre, equivalent to 184 lbs of N per acre. Shoots containing leaves were pooled at mid-growing stage (early booting) and separated again at late-growing stage (booting), air-dried, and sent for agronomic mineral nutrient content analysis. As shown in Figure 24, plants inoculated with (WW5 + WW6 + WW7 + PTD1) accumulated higher levels of plant-related macronutrients and micronutrients; these differences are expressed as the percent change in leaf nutrient concentration from the control.

[0142] Example 15 Effect of seed treatments (WW5, WW6, WW7 and PTD1) on yield under reduced and normal nitrogen fertilizer rates in the field Broccoli seeds were first industrially treated with a mixture of endophyte ferment and 1% alginate at different rates using two different crop protection packages and industry-representative methods. The endophyte seed rates used were 10 mL, 50 mL, and 100 mL of fermentation mixed with the industrial slurry per kg of broccoli seeds. Control seeds of the same cultivar received the same crop protection package without the endophyte. Endophyte survival was then assayed for the presence of each microorganism. The microorganism mixes demonstrated viability of strains (WW5, WW6, WW7, and PTD1) on the seeds after drying; the four-strain mix is ​​shown as "I4WP" in Figure 25A. Seed coat counts were performed using 10 seeds that were washed with 10 mL of water to remove the seed coat and then assayed. The results of the dilution plantings showed clear survival with bacterial titers expressed as colony forming units / seed (CFU / seed) of each strain still viable on the seeds in Figure 25, successfully dehydrated, and then becoming dormant.

[0143] Broccoli seeds were then stored under normal industrial conditions (<25°C in dark packages) for two months and then planted industrially in the fall in a large-scale replicated CRO field experiment in Salinas, CA, using industrial methods on a production field fertilized with normal and 25% reduced nitrogen fertilizer rates compared to four industrial applications of nitrogen via trickle. Two applications of 12 gallons / acre of calcium ammonium nitrate (17-0-0) were applied, followed by two additional applications of 5 gallons / acre of ammonium nitrate (17-0-0) for the full fertilizer application rate over the season. In the 25% reduced nitrogen fertilizer rate treatment, the amount of nitrogen was reduced by 25% with each application. Additionally, all treatments received 0-0-6-3% Ca fertilizer for a total of eight applications over the season. Irrigation was controlled at the discretion of the farm manager in accordance with industrial farm management standards. After one month of growth, there was a clear difference in plant size between treatments receiving 100% nitrogen and those receiving less than 25% nitrogen. None of the treatment groups showed any signs of disease or damage from pests during the trial. At the time of harvest, there was still a clear difference in the leaves between the treatments that received 100% nitrogen and those that received less than 25% nitrogen. Differences in uniformity and industrial quality were then measured for all treatments at the time of harvest. In selecting industrial broccoli tips, growers take into account different criteria, such as tip size (diameter in inches), tip smoothness, dark green color, and firmness. The largest mean industrial ear diameters at collection were observed for both seed crop protection packages under both 25% less N fertilizer and conventional 100% fertilizer at the two highest endophyte treatment rates, 100 mL per kg (used for the I4WP-20F and I4WP-20D groups) and 50 mL per kg (used for the I4WP-10F and I4WP-10D groups), as seen in Figure 25B for the 75% N and 100% N rates, respectively.

[0144] The highest mean industrial head weights at collection were observed for the two highest endophyte treatment rates, 100 mL and 50 mL per kg of seed, under both 25% less N fertilizer and regular 100% fertilizer, for both seed crop protection packages, as seen in Figure 25C for 75% N and 100% N rates, respectively.

[0145] Example 16 Effects of WW6 and WW7 Used Individually as Seed Treatments Under Reduced Nitrogen Fertilizer (32 ppm N) in a Hoagland Dropout Indoor Growing Chamber Pot Study The early effects of WW6 and WW7 endophytes applied individually to Brassica species under limited nitrogen conditions in a controlled environment were tested for their effects on shoot growth. Endophyte strains WW6 and WW7 were applied individually within an industrial seed coating process using both clay and dip coatings on broccoli seeds in fermentation, and the seeds were germinated and grown in flats using an artificial soilless medium. Controls were applied with only the industrial coating without the endophyte fermentation mixture. Eight seeds per treatment were planted ½ inch deep in 2" mineral planting medium (all washed with DI water; ⅓ play sand, ⅓ perlite, ⅓ vermiculite) in 10" x 20" plastic growing trays. Trays were watered on Mondays, Wednesdays, and Fridays for the duration of the experiment with Hoagland's N Dropout solution at pH 7, modified to contain 32 ppm total N, a 70% reduction from the optimum nitrogen. The study lasted 27 days and was maintained in an indoor growth room under greenhouse light at 710 μmol / m 2 s 1 The experiments were conducted at an ambient temperature of 25°C and 50% humidity, with 14 hours of exposure per day. Seed coating viability and dilution sowing were assayed to determine the survival of the endophyte composition used in conjunction with the crop protection product after dehydration of the industrially coated seeds. See Figure 26A.

[0146] At the time of harvest (27 days old), whole plant seedling fresh weights were obtained for all treatments and the results are reported below. The WW7 treatment (RD12378) showed a significant 30% increase in seedling weight over the control. The WW6 treatment (RD12381) showed a highly significant 47% increase over the control. See Figure 26B.

[0147] Example 17 Effects of WW5, WW6, WW7 and co-fermented mixtures used as seed treatments under reduced nitrogen fertilization in a controlled environment growth chamber The effects of WW5, WW6, and WW7 endophytes applied individually to corn seeds under limited nitrogen in a controlled environment were tested for their effects on shoot growth. The endophyte strain formulations were applied to corn seeds in combination with 1% w / v sodium alginate (Scogin LDH), and the seeds were germinated and grown in 1-gallon felt smart pots using an artificial soilless medium. Eight seeds per treatment group were planted 1 / 2 inch deep in a mineral planting medium (washed with DI water; 1 / 3 play sand, 1 / 3 perlite, 1 / 3 vermiculite). Pots were watered on Mondays, Wednesdays, and Fridays throughout the experimental period using Hoagland's N-dropout solution modified to contain 50 ppm total N. Two control groups were included, each treated with the modified Hoagland's solution but not the endophyte. The first control group was treated with Hoagland's solution modified to contain 75 ppm total N, and the second control group and the endophyte treatment group were treated with Hoagland's solution modified to contain 50 ppm total N. The study was conducted in an indoor growth chamber under greenhouse light conditions of 710 μmol / m 2 s 1The experiments were carried out at an ambient temperature of 25°C and 50% humidity, with 14 hours of exposure per day to UV light. At 24 days post-emergence, plants were harvested, washed, and dried in individual paper bags at 45°C for one month. Shoot dry weights were then measured and recorded. The results are shown in Figure 27. The endophyte seed coat formulations increased corn shoot dry weight biomass as follows: WW6 +102% p>0.05, WW5 84% p>0.05, and WW7 49% p>0.01.

[0148] Example 18 Effects of WW6 and WW7 as seed treatments on winter wheat in the field. Winter wheat seeds were treated with a co-fermented WW6 + WW7 endophyte inoculum. The co-fermented WW6 + WW7 endophyte mixture was fermented in a low-nitrogen medium and then freeze-dried to a powder. Five grams of the freeze-dried fermentate was mixed with 5 grams of dry sodium alginate in 1 liter of water. The mixture was then combined with commercially available prebiotic composition UBS 016 (from Unium Bioscience Ltd.) in a ratio of about 3:1 to about 5:1 mixture to prebiotic composition. This combination resulted in a final seed slurry, which was used at a rate of about 4 L to about 6 L per metric ton of winter wheat seeds. A control group was treated with the prebiotic without the ferment. Seeds were planted industrially in the field in early November, and the trial continued until the following mid-summer regular harvest, at which time the wheat kernels were weighed and the final yield results recorded. The treatment group showed a substantial 10% increase in crop yield as shown in Figure 28A.

[0149] Additionally, nitrogen accumulation was measured in the shoots of winter wheat plants from February through June. Plants were collected per square meter at each time point, dried, and sent for total nitrogen measurement by Kjeldahl. A steady increase in total nitrogen per hectare of wheat shoots was measured, as shown in Figure 28B, which demonstrates a +30% increase in total nitrogen accumulation in wheat shoots in kg / ha when measured at the final sampling in June. This result clearly demonstrates the impact of the nitrogen-fixing, N2-fixing endophyte strain on this wheat species in the field. Endophyte treatments increased total plant shoot nitrogen per hectare throughout the vegetative season, demonstrating a +100% increase in nitrogen shoot accumulation in early May and a +30% increase in nitrogen shoot accumulation per hectare in early June.

[0150] Example 19 Carbon accumulation in plants treated with endophytic strains The ability of the WW5, WW6, WW7, and PTD1 endophytic bacterial strains to increase whole-plant carbon accumulation as a mixed association when grown under field conditions was assayed using fast-growing poplar trees planted in soil in a Lower Mississippi Alluvial Valley field. Trees were inoculated with approximately 20 endophyte-encapsulating calcium alginate beads, which were applied to the base of the Populus cuttings at the time of planting. Field replicate blocks were planted at field locations (3 trees × 5 trees) containing five replicate blocks. Dormant, unrooted, 22.86 cm long hybrid poplar cuttings were obtained from Greenwood Resources (Portland, Oregon, USA) and treated with Admire® Pro (Bayer Corp., Whippany, NJ, USA).

[0151] For carbon sampling, leaf samples were brought back to the laboratory, dried in an oven at 60°C, ground to a fine powder, and then placed in tin capsules. Samples were analyzed with an ECS 4010 CHNS-O analyzer (Costech Analytical Technologies Inc. Valencia, CA, USA) to estimate total C and N concentrations.

[0152] Statistically significant results demonstrated that endophyte inoculation increased total plant carbon content by 71.01% with a p-value of 0.063, as shown in Figure 29 below. Total carbon was calculated by multiplying the percent carbon by dry weight to total biomass dry weight in treated (n=12 trees) and treated (n=12 trees). The results, shown in Figure 29 below, demonstrate a significant increase in carbon accumulation in the treatment group relative to the control.

[0153] Example 20 Compatibility with commonly used crop protection chemistries for seed treatment Endophyte strains WW5, WW6, WW7, and PTD1 were tested for their ability to survive when combined with various commonly used industrial seed crop protection chemicals. The viability of the endophyte strains was evaluated when added to five different seed crop protection chemical solutions: Beret Gold® (Syngenta), Raxil star® (Bayer CropScience), Redigro Pro® (Bayer CropScience), Vibrance Duo® (Syngenta), and Latitude® (Bayer CropScience). Solution mixes were prepared according to the manufacturer's specifications. Five to six minutes after creating the mixture, the colony-forming units (CFU / ml) of the strains were determined by complete dilution and plating onto NLM semi-solid medium. The results, shown in Figure 30A, demonstrate that all strains were able to survive in the five different solution mixes.

[0154] The viability of WW5, WW6, WW7, and PTD1 was evaluated on seeds of different crops when used with different seed crop protection chemical active ingredients: mefonoxam, fludioxonil, azoxystrobin, sedaxan, thiabendazole-thiram, metalaxyl, hymexazole, penthiopyrad, ponchobeta, and thiamethoxam. Solution mixes were prepared according to the manufacturer's specifications, and then the different strains were added to the solution before application to the seeds using an industrial seed treatment device. The colony-forming units (CFU / ml) of the strains that survived on the seeds were assessed by adding the seeds to a 0.2 M phosphate resuspension solution and then planting them in NLM semi-solid medium at appropriate dilutions. The results, shown in Figure 30B, demonstrated that the endophyte strain compositions were tolerant and viable when mixed with various industrial products and survived the temperature and dryness conditions encountered in industrial seed treatment processes.

[0155] Example 21 Compatibility with commonly used fertilizers, micronutrients and herbicides during in-furrow and foliar applications The feasibility of adding endophyte strains to various commonly used tank-mix solutions for in-furrow and foliar treatments was evaluated based on the viability of the strains for different periods ranging from 3 hours to 1 month. The viability of WW5 and WP1 strains was evaluated in a liquid starter fertilizer, ammonium polyphosphate (10-34-0) and / or a micronutrient product (4% ammoniacal nitrogen, 3% water-soluble nitrogen, 9.0% chelated zinc), used for in-furrow fertilizer applications. For experimental purposes, the volume was scaled down from 5 gallons / acre to 50 ml. The mixture composition included: 32 fl oz / acre of micronutrients, 5 gallons / acre of 10-34-0 fertilizer, 16 fl oz / acre of WW5 strain inoculum, and water. Three hours after the mixture was created, the colony-forming units (CFU / ml) of the two strains were determined by planting them on NLM semi-solid medium. The results shown in Figure 31A demonstrate that both WW5 and WP1 survived when 10-34-0 was present in the aqueous furrow plus endophyte tank mix.

[0156] Following these results, a tank mix of 3 gallons / acre of 10-34-0 fertilizer and 16 fl oz / acre of WW5+WP1 strains was evaluated in the field in a replicated block trial using the tank mix as an in-furrow tank mix for growing corn. The tank mix was dripped over the seed, and in-furrow controls received only the fertilizer tank mix at the same rate but without the endophyte. The fields received either no N or a full rate of N at 180 lb N per acre.

[0157] The results shown in Figure 31B show a +12% increase in crop grain yield at industrial harvest when the WW5+WP1 strain was combined with 10-34-0 fertilizer and corn was grown at conventional Midwest WI USA rates under full NPK fertilizer.

[0158] Additionally, mineral nutrient content was measured in leaves of V9 corn plants. Figure 31C shows consistent increases in total nitrogen, potassium, and phosphorus (NPK) when WW5 and WP1 lines were applied in-furrow in the fertilizer tank mix at planting. Importantly, potassium (K) showed a significant increase of +9.5% in leaves from blocks that did not receive nitrogen, demonstrating increased uptake and assimilation of K into the shoot in addition to N and P.

[0159] Example 22 Compatibility with commonly used fertilizers, micronutrients and herbicides during in-furrow and foliar applications The effects of endophyte fertilizer and micronutrient compatibility were tested using strains WW6 and WW7, which were evaluated for long-term viability when mixed with 6-22-6-4, a common liquid starter fertilizer frequently used for in-furrow fertilizer applications. The test solution mix was scaled down from 5 gallons / acre to 50 ml for experimental container size purposes. The composition included: 5 gallons / acre of 6-22-6-4 fertilizer and 40 fl oz / acre of the WW6 + WW7 microbial composition. The colony-forming units (CFU / ml) of the two strains were determined by inoculation into NLM semi-solid medium over periods ranging from 1 week to 5.5 months. The results, shown in Figure 32, indicate that the viability of both strains was slightly reduced when 6-22-6-4 was present in the solution mix.

[0160] Example 23 Endophyte compositions tested for foliar herbicide compatibility The viability of WW6 and WW7 strains was evaluated in a composition containing the broad-spectrum systemic herbicide glyphosate used for foliar treatment and / or adjuvants (modified vegetable oil, polyoxyethylene sorbitan fatty acid ester, vegetable oil, and ethoxylated soybean oil) and / or a micronutrient composition (3.6% sulfur, 0.1% boron, 3.0% manganese, and 4.0% zinc). For experimental purposes, the solution mix was scaled down from 10 gallons per acre to 50 ml. The mix contained a unique combination of the following products: 32 fl oz / acre of micronutrients, 16 fl oz / acre of adjuvant, 24 fl oz / acre of glyphosate, and 16 fl oz / acre of a WW6 + WW7 microbial fermentation composition prepared according to the method described herein and having a pH of 5.3. Twenty-four hours after the mixture was created, the colony-forming units (CFU / ml) of the two strains, WW6 + WW7, were determined by inoculating them onto NLM semi-solid medium. The results shown in Figure 33A show that the viability of both strains was not reduced in the different compositions.

[0161] A field study was conducted to demonstrate the effects of the WW6 and WW7 compositions when applied as a foliar spray to corn with a glyphosate herbicide and adjuvant. A tank mix was prepared by adding 32 oz / acre of the WW6 and WW7 inoculant compositions, 32 oz / acre of Cornerstone 5 Plus (a glyphosate herbicide from WinField® United), 32 oz / acre of MasterLock (an adjuvant from WinField® United), and 10 gallons / acre of water. The field study included 27.5 ft. x 5 ft. plots of high- and low-yielding corn varieties. Three plots were treated with the tank mix described above, and three plots were treated with a tank mix of the same materials without the endophyte strain. The results shown in Figure 33B show an increase in crop yield of 10.6 bu / acre for the low-yielding corn variety, while the high-yielding variety increased by 11.3 bu / acre with the WW6+WW7 endophyte strains compared to the control that received no endophyte treatment.

[0162] Example 24 Effects of WW5, WW6, WW7, and PTD1 on enhancing plant tolerance to waterlogged and saturated soils Waterlogging of agricultural soils is a major problem in food production systems in the United States and worldwide, frequently resulting in large-scale plant mortality and crop loss. To test the ability of endophytes to confer waterlogging tolerance to crops, sugar beets (Beta vulgaris) were industrially treated with and without heterologous endophyte applications (WW5, WW6, WW7, and PTD1) using industrial seed treatment methods. Seed coatings were used to control the plants, and seed coatings combined with the co-fermenting endophyte strains WW5, WW6, WW7, and PTD1 were used on experimental seeds. The coated seeds were then washed, and experimental groups were tested for endophyte strain survival thereon. Bacterial counts were assayed by dilution plating 10 seeds onto NLM agar medium after vortexing and washing in 10 mL of potassium phosphate (KP) buffer to remove and dissolve the coating. The assay showed clear endophyte survival, with titers shown as CFU / seed in Figure 34A.

[0163] Seeds treated with endophytes for 24 hours were planted in separate cells, and 24-hour control seeds were planted in separate cells. Seeds were constantly overwatered in fully saturated soil and germinated in standard industrial transplant potting medium. The medium was overwatered daily until saturated and grown at 25°C under natural circadian light conditions. Once germinated, the plants were continuously overwatered and subjected to flooding-like conditions. After this exposure to flooding, the plants were evaluated for symptoms of flooding stress, germination, and overall growth effects. Plants inoculated with the endophyte strains exhibited faster germination and establishment, less waterlogging damage, and better growth than controls under continuously flooded, saturated soil conditions, as shown in Figure 34B.

[0164] The above data demonstrate that the co-fermenting heterologous endophyte WW5+WW6+WW7+PTD1 successfully improved beet plant response to abiotic stress (flooding) induced by overwatering saturated medium, increasing early germination rate and improving biomass growth compared to the control. The visual results clearly demonstrate the efficacy of the co-fermenting heterologous endophyte and its ability to enhance the physiological performance of non-native host crop plants.

[0165] Example 25 Effect of combined endophytic strain (WW6+WW7) seed treatment on plant cold tolerance Two sets of broad beans (Vicia faba) were prepared: a control group treated with an industrial seed treatment, and an experimental group treated with the industrial seed treatment, co-fermented heterologous endophyte strains WW6 and WW7, and the prebiotic UBS 016 (from Unium Bioscience Ltd.). The co-fermented WW6 + WW7 endophyte mixture was fermented in low-nitrogen medium and then freeze-dried to a powder. Five grams of the freeze-dried fermentate was mixed with 5 grams of dried sodium alginate in 500 ml of water. The mixture was then combined with 500 ml of prebiotic UBS 016 in a 1:1 ratio of endophyte sodium alginate mixture to prebiotic composition. This combination was then incorporated into the industrial seed treatment slurry, which was used at a rate of approximately 4 L to approximately 6 L per metric ton of broad bean seeds. The control group of seeds was treated with the prebiotic alone. Seeds were germinated in standard potting medium and grown in 6-inch pots in a greenhouse under natural circadian light conditions with daytime minimum temperatures ranging from 45°F to 55°F and maximum temperatures ranging from 65°F to 70°F. Once germinated, plants were fertilized with a 90-day delayed-release complete fertilizer (Osmocote 15-9-12 coated granular fertilizer). Six weeks after emergence, both sets of plants were exposed to a 6-hour cold shock treatment at 34°F. After this exposure, plants were photographed and evaluated for cold stress symptoms and damage. As shown in Figure 35, the experimental plants inoculated with WW6+WW7 exhibited less wilting and cold damage than the control plants, which were significantly wilted. Furthermore, the endophyte-treated plants fully recovered from the cold stress, whereas the non-endophyte-treated plants did not fully recover and showed symptoms of chlorosis and necrosis.

[0166] The above data demonstrate that the co-fermenting heterologous endophyte WW6+WW7 successfully improved broad bean plants' response to abiotic stress (cold shock) compared to the control. The results demonstrate the efficacy of co-fermenting heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0167] Example 26 Effects of individual and combined endophyte strains on plant tolerance to saline / saline soils rich in boron and chlorine Soils with saline and / or saline characteristics restrict plant growth, frequently resulting in plant death, crop loss, and reduced yields. Globally, the USDA estimates that 40% of agricultural land formerly suitable for cultivation is now unsuitable for agriculture due to excessive salt content in soils. The novel endophyte strains disclosed herein were tested for their ability to increase crop plant growth after seed treatment in saline and saline soils. Control and experimental groups of heritage broccoli (Brassica oleracea) seeds were prepared as follows: two experimental groups were coated using an industrial seed treatment method involving a polymer dip coating combined with WW7 (Group 1) or endophyte strains WW5+WW6+WW7+PTD1+WP1 (referred to as "Phase A"), and a control group was treated with a polymer dip coating and a broccoli disease prevention package. Seeds were then assayed for total endophyte survival in the industrial seed coating, and total counts are shown in Figure 37A.

[0168] Seeds from the two endophyte treatments (Groups 1 and 2) and control seeds were then grown in an industrial transplant greenhouse in Santa Monica, California, before being planted in a field trial. A site characterized by the USDA as having highly saline / saline soil and high boron and chlorine levels was selected for the field trial. The site was Five Points, California. An exchangeable sodium percentage (ESP) greater than 6% is considered saline, and an ESP of 15% is considered highly saline. The ESP value indicates the percentage of the soil's cation exchange capacity (CEC) that is accounted for by sodium. The poor-quality soil had the following chemical profile: As shown in Figure 36B, the soil used in the test had an ESP of 12.9. This soil also contained 25 ppm boron, although levels of approximately 3-5 ppm are harmful to plants. This soil also had very high chlorine levels of 68 ppm. These characteristics indicated that the quality of the soil used in the test was very poor.

[0169] Using a rototiller, three identical 72-inch beds were created in a homogeneously saline field site designated (RRR West), and two drip tape irrigation lines were carefully lowered into each bed for testing. Bed size was 100 ft long, with two 6 ft wide rows (2 ft from the edge of each bed and 2 ft between rows). 290 plants were planted from each of the two endophyte-treated groups and the untreated control seed. At harvest, photographs of the beds were taken 91 days after transplanting and are shown in Figure 36C. The salt tolerance of the endophyte-enhanced plants (designated WW7 and Phase A in the figure) is readily visible in Group 2 (Phase A).

[0170] Seven-week-old broccoli plants were harvested and weighed 91 days after transplanting. Group 2 plants showed a statistically significant increase of 13.24% (p<0.05) in fresh weight compared to the control group. The graph shown in Figure 36D shows data for the broccoli field trial. Broccoli florets were then dried in a drying oven after harvesting. The total dry weight of Group 2 plants (Phase A) showed a statistically significant increase of 47.06% (p<0.05) and Group 1 plants (WW7) showed a statistically significant increase of 16.81% (p<0.05) compared to the control group. See Figure 36E.

[0171] The data demonstrate that (1) the co-fermented heterologous endophyte WW5+WW6+WW7+PTD1+WP1 successfully improved broccoli fresh and dry weight compared to the control, and (2) the endophyte strain improved broccoli dry weight compared to the control under conditions of abiotic stress (saline / saline soil conditions). The results demonstrate the efficacy of heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0172] Example 27 The effect of combining heterologous endophyte strains on increasing plant tolerance and recovery under drought conditions Drought stress negatively affects crops, plants, herbs, and trees, frequently resulting in plant death and crop loss. Drought occurrence is increasing, and drought induces numerous physiological and molecular biochemical changes in plants. Internal processes that help plants tolerate drought stress include reactive oxygen species (ROS) scavenging, osmoregulation (OA), stomatal closure, and the synthesis of defense molecules, including inducible dehydrins. Plant recovery after drought stress involves a series of steps occurring sequentially that can be aided or facilitated by internal beneficial endophytes. To demonstrate the influence of endophytes on increasing drought tolerance, a fermentation mixture called the "Phase A mix" was prepared containing endophyte strains WW5+WW6+WW7+PTD1 together with the fungal yeast endophyte WP1. The Phase A mix fermentation, along with an industrial seed treatment incorporating polymer and talc powder as a carrier, was applied to tall fescue seeds (Festuca aramdinacea, a forage grass used in livestock production) and allowed to dry to a husk-like natural hard coating. Two treatment groups were used: Group 1, treated with 0.5 L of fermentation per ton of seeds; Group 2, treated with 1.0 L (0.5 L fermentate + 0.5 L 2% alginate) per ton of seeds;

[0173] Control groups received an industrial seed treatment that did not contain the endophyte strain. These treatments resulted in endophyte survival on the seeds assayed, as shown in Figure 37A.

[0174] The control group and experimental groups 1 and 2 were planted at the same density in three flats containing a low-carbon growth medium consisting of washed play sand, perlite, and vermiculite. Seeds were watered three times a week for four weeks with a modified low-nitrogen Hoagland nutrient solution containing 65 ppm nitrogen. The plants were grown at 30°C and then subjected to a 14-day waterless drought stress period, which allowed the growth medium to dry out completely. After the drought stress treatment was stopped, watering of the flats was resumed to give the plants an opportunity to recover. Each group was then harvested and weighed. Groups 1 and 2 showed a statistically significant increase in dry weight of 42% and 67%, respectively (p<0.05). Total weight results are shown in the graph below in Figure 37B.

[0175] The above data demonstrate that the co-fermenting heterologous endophyte WW5+WW6+WW7+PTD1+WP1 successfully improved fescue fresh weight compared to the control under abiotic stress (drought) conditions. The results demonstrate the efficacy of heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0176] Example 28 The endophyte increases seedling germination, seedling emergence and seedling biomass weight. Endophyte strains were applied to seeds in a series of experiments to determine the effects of application on increased seedling germination, seed coating and seedling emergence from the soil, and seedling biomass weight. The first experiment involved treating romaine lettuce seeds with 10 treatment groups and a control group, as identified in Figure 38. For each treatment, romaine lettuce seeds were applied with a clay seed coating and a nitrogen-limited endophyte fermentation mixture in a 1% w / v alginate solution. The control seeds did not contain endophyte fermentation. Seeds, including control seeds, were coated using an industrial method involving clay coating and seed coating polymer. Seeds were germinated in square Petri dishes containing seed germination paper moistened with 14 mL of deionized sterile water per 4 in x 5 in sterile seed germination container. The seeds were then germinated, and seedlings were grown in DI water under light and fluorescent light without nutrients for 14 days before being weighed. Results indicated that inoculated seedlings were larger and able to grow better under nitrogen-limited conditions. This result strongly suggests that the inoculated plants were able to fix atmospheric nitrogen and utilize nutrients from the seedling germination paper better than the control. Regarding nitrogen fixation, it was also observed that the strains that fixed the most nitrogen and appeared to grow best under nitrogen-free bacterial medium conditions resulted in the highest lettuce seedling weights, in the following order: WW6 > WW5 > WW6 / WW7 > endophyte mix (WW5, WW6, WW7, PTD1 + WP1) > PTD1 > WW7.

[0177] Example 29 An experiment was conducted in which endophyte strains were applied to broccoli seeds to determine the effect of application on seedling biomass weight. The experiment included four treatment groups (strain WW7 alone and a mix of WW5, WW6, WW7, and PTD1) and a control group, as shown in Figure 39. For each treatment, broccoli seeds were applied with a clay seed coating and a nitrogen-limited endophyte fermentation mixture in a 1% w / v alginate solution. The control seeds did not contain endophyte fermentation. Seeds, including control seeds, were coated using an industrial method involving clay coating and seed coating polymer. Seeds were germinated in square Petri dishes containing seed germination paper moistened with 14 mL of deionized sterile water per 4 in x 5 in sterile seed germination container.

[0178] Seeds were germinated in square Petri dishes containing seed germination paper moistened with 14 mL of deionized sterile water per 4 x 5" sterile seed germination container. The seedlings were then grown under fluorescent light without nutrients in light for 14 days before being weighed. The results showed that the inoculated seedlings were larger and able to grow better under limiting conditions. These inoculated plants were likely better at fixing atmospheric nitrogen and utilizing nutrients from the seedling germination paper than the controls, resulting in the greatest seedling weights, as shown in Figure 39.

[0179] Example 30 An experiment was conducted in which endophyte strains were applied to barley seeds to determine the effect of application on seedling emergence. The experiment included treatments with the co-fermentation WW6 + WW7 and a control group. For each treatment, barley seeds were treated with a clay seed coating and a mixture of nitrogen-limited endophyte fermentate in a 1% w / v alginate solution. The control seeds did not receive endophyte fermentate. Seeds, including the control seeds, were coated with an industrial seed treatment containing a clay coating, a polymer containing a seed coating polymer, and a crop protection agent. Seeds were germinated in square Petri dishes containing seed germination paper moistened with 14 mL of deionized sterile water per 4 in x 5 in sterile seed germination container.

[0180] The seedlings were then germinated without nutrients under white light for 4 days before being photographed first. The results showed that the inoculated seedlings were significantly larger, as shown in Figure 40A, and continued to grow larger after 4 days, as shown in Figure 40B.

[0181] Example 31 An experiment was conducted in which endophyte strains were applied to broccoli seeds to determine the effect of application on seedling emergence. The experiment included two treatment groups: one treated with WW7 fermentation and one treated with a four-strain mix (I4WP), as well as a control group. For each treatment, broccoli seeds were treated with a clay seed coating and a nitrogen-limited endophyte fermentation mixture in a 1% w / v alginate solution. The control did not receive the endophyte fermentation. Seeds, including the control seeds, were coated with an industrial seed treatment containing a clay coating, a polymer containing a seed coating polymer, and a crop protection agent.

[0182] Treatment and control broccoli seedlings were planted in individual cells. Seeds were germinated in standard commercial transplant potting medium. The medium was watered daily and grown at 25°C under natural circadian light conditions for 15 days. Plants were then evaluated for germination. An increased germination rate at 15 days was observed for the endophyte-treated group. Plants inoculated with the endophyte strain showed faster germination (see Figure 41) and establishment, as well as better growth.

[0183] Example 32 An experiment was conducted in which endophytic strains were applied to sugar beet cultivars, and seeds (C578 and M5) were treated with the seed inoculant composition. The experiment included two treatment groups: one treated with WW7 fermentation and one treated with a four-strain mix (I4WP), as well as a control group. Seeds in the treatment groups were industrially treated at a rate of 50 mL of fermentation, along with the crop protection products Thiram, Metalaxyl, Hymexazol, Penthiopyrad, and Poncho Beta (Clothianidin) per kg of seed. Control seeds of the same cultivars were prepared in the same way, but without the fermentation. The seeds were then stored under normal industrial conditions for one month and then planted industrially.

[0184] The emergence of sugar beet seedlings was measured over time after sowing. The endophyte seed treatment composition enabled the survival of all four strains (I4WP)—WW5, WW6, WW7, and PTD1—and, on average, improved plant emergence of the sugar beet varieties at 29 days compared to the control group. In this study, 200 seedlings were considered equivalent to 75% establishment of the sugar beet plants. The endophyte treatment improved average emergence at 13, 16, and 29 days after planting. The C578 control strain had 187 emergent plants at 29 days, compared to 200 (an increase of 13 plants) for the four strains (I4WP). The M5 control strain had 161 emergent plants at 29 days, compared to 177 emergent plants (an increase of 16 plants) for the I4WP mixture treatment group, as shown in Figures 42A and 42B.

[0185] Example 33 An experiment was conducted in which endophyte strains were applied to wheat seeds as a seed inoculant composition. The experiment included a seed treatment group treated with the co-fermented WW6 / WW7 fermentate and a control group. Seeds in the treatment group were industrially treated with 50 mL of fermentate and 500 mL of 1% alginic acid, and applied per ton of seed along with the prebiotic UBS 016 according to the manufacturer's instructions. Control seeds of the same variety were prepared in the same way but without the fermentate. The seeds were then stored under normal industrial conditions for one month and then planted industrially.

[0186] Wheat seedling emergence was measured over time in a CRO field trial. Treatment groups improved wheat seedling growth after emergence compared to untreated controls, randomly harvested from appropriate field plots over time. Final seedling fresh weights after 15 days of follow-up were 1.054 g leaf and 0.781 g root for the WW6 / WW7 combo, compared to 0.95 g leaf and 0.44 g root for the control. Fresh weights of newly pulled seedlings on each day are shown below in Figures 43A and 43B.

[0187] Example 34 Effects of WW6 or WW7 applied as a seed treatment on grain yield in the field. To improve field yield under a highly optimized nutrient system, endophytes were applied as seed treatments. Spring wheat seeds were treated with WW6 or WW7 endophyte NLM ferment. Prior to seed treatment, the fermentation was freeze-dried to a powder. A seed treatment slurry was prepared by adding 5 grams of freeze-dried fermentation with 5 grams of dry sodium alginate per liter of water. Six liters of seed slurry were used to treat one metric ton of spring wheat cultivar Tybalt seeds. The results in Figure 44 show a 12% increase in crop yield for spring wheat seeds treated with WW6 and a statistically significant 23% increase in crop yield for seeds treated with WW7 compared to the untreated control.

[0188] Example 35 Reducing nitrogen fertilizer requirements in wheat crops while maintaining yield A field trial was designed to demonstrate the ability of an endophyte seed treatment composition to provide substantial crop yield under reduced nitrogen fertilization conditions. Bluerock romaine lettuce (Lactuca sativa, Vilmorin-Mikado USA) and corn (Zea mays) seeds were treated with the WW6+WW7 fermentation product and mixed with 0.5% sodium alginate (Scogin™ LDH) as a seed coating inoculant.

[0189] Lettuce seeds were planted in the fall in Fresno, California, in fields fertilized with conventional and 33% reduced nitrogen fertilizer rates, as is typical for CRO field trials. Field plots were divided into groups and fertilized via drip irrigation at different rates: (1) 25 lb / acre calcium ammonium nitrate (17-0-0); (2) 50 lb / acre 0-0-30 and 50 lb / acre 0-46-0. The conventionally fertilized control plots also received 50 lb / acre of UN-32 nitrogen fertilizer. Irrigation was adjusted at the farm manager's discretion, following general agricultural practices. At harvest, 10 heads of lettuce were collected from each plot, with six plots per treatment. Mean head weights were calculated for each plot, and an overall average was calculated. Figure 45A shows that the WW6+WW7 treatment under 33% reduced nitrogen resulted in a 3.3% increase in head weight compared to full-nitrogen control plants. Additionally, plant tissue samples were collected from four of the six harvest plots for nitrogen tissue concentration assays. Figure 45B presents data showing that leaf nitrogen concentrations of WW6+WW7 treatment plants grown in reduced nitrogen were statistically significantly increased compared to control plants under the same reduced nitrogen fertilizer regime.

[0190] The WW5+WW6+WW7 fermentation product was mixed with 0.5% sodium alginate (Scogin™ LDH), and this composition was used to over-treat corn (Channel 216-36 STX RIB) seed that had been pre-treated with prothioconazole, metalaxyl, fluoxastrobin, clothianidin, LCO SP104, and Bacillus firmus 1-1582. The seed was planted in Clay, Nebraska, on fields fertilized at conventional and 25% reduced nitrogen fertilizer rates, as is typical for university field trials. The trial was conducted in a split-block and split-plot design, with each treatment containing six plots fertilized at different rates: (1) 165 lbs / acre of anhydrous ammonia nitrogen at the 25% reduced rate prior to planting; and (2) 220 lbs / acre of anhydrous ammonia nitrogen at the conventional rate. Grain yields were collected from the two center planted rows of each plot when grain moisture reached approximately 15.5%. Average grain yields were calculated for the six plots per treatment. Figure 45C shows that the WW5+WW6+WW7 treatment under 25% reduced nitrogen had a 0.2% decrease in average grain yield compared to full-nitrogen control plants, while the reduced-nitrogen control plants had a 2.7% decrease in average grain yield.

[0191] Additionally, the WW5+WW6+WW7 fermentation product was mixed with 0.5% sodium alginate (Scogin™ LDH), and this composition was used to treat corn (Channel 213-19 VT2P RIB) seed that had been pre-treated with prothioconazole, metalaxyl, fluoxastrobin, clothianidin, and LCO SP104. The seed was planted in Saunders, Nevada, using standard and 25% reduced nitrogen fertilizer rates, using typical university field trials. The trial was conducted in a split-block and split-plot design, with six plots fertilized at different rates: (1) 25% reduced rate, with liquid UAN 32-0-0 at 112.5 lbs / acre of nitrogen prior to planting; and (2) standard rate, with 150 lbs / acre of nitrogen. Grain yields were collected from the center two planted rows of each plot when the grain moisture reached approximately 15.5%. Average grain yield was calculated for six plots per treatment. Figure 45D shows that the WW5+WW6+WW7 treatment under 25% reduced nitrogen increased average grain yield by 7.67% compared to the full nitrogen control plants, while the control plants under reduced nitrogen decreased average grain yield by 8.4%.

[0192] Example 36 Suitability of commonly used endophytic herbicides for agricultural foliar application to control weeds in a wide range of monocotyledonous and dicotyledonous crops Three liquid foliar herbicides were evaluated for compatibility as tank mixes with strains WW6 and WW7: Enlist One (Corteva Agriscience, LLC), Impact (AMVAC Chemical Corporation), and Callisto (Syngenta Crop Protection, LLC). Solution mixtures were scaled down from 20 gallons / acre to 10 ml for experimental purposes. The mixture contained 32 fl oz / acre of the WW6+WW7 endophyte fermentation composition and was combined with 16 fl oz / acre of Enlist One (2,4-D choline salt 55.7% w / w, glycerol ≥ 3% ≤ - 10% w / w, dipropylene glycol monoethyl ether >= 3% - < 10% w / w, residuals > 20% w / w), 1 fl oz / acre of Impact (Topramezone 29.7% w / v, inactive ingredients 70.3% w / v), 3 fl oz / acre of Callisto (ethylene glycol < 15% w / v, other ingredients > 45% w / v, mesotrione 40% w / v), or water. Four hours after preparation, the colony-forming units (CFU / ml) of the two strains were determined by plating on NLM semi-solid medium (Figure 46).

[0193] The results in Figure 46 show that there was no significant decrease in viability of both endophyte strains in the different solution mixtures over an extended period of time, indicating that these endophyte nutrient utilization efficiency, biomass, and stress tolerance enhancing formulations can be used in combination with commercial agricultural foliar herbicide applications.

[0194] Example 37 Examples of fertilizer-biostimulant suitability for commonly used agricultural foliar applications in a wide range of monocotyledonous and dicotyledonous crops Two foliar fertilizers or biostimulants, Isabion (Syngenta Agro AG) and Megafol (Syngenta Crop Protection Ag), were tested for compatibility with the WW6 and WW7 strain compositions as foliar tank mixes. The tank mixes were scaled down from 400 liters / ha to 10 ml for experimental purposes. The mixes contained 2.4 liters / ha of the WW6 + WW7 endophyte microbial composition and were combined with the following products: 6 liters / ha of Isabion, 3 liters / ha of Megafol, or water. The colony-forming units (CFU / ml) of the two strains were assessed 4 and 24 hours after the mixes were made by plating on NLM semi-solid medium. See Figure 47.

[0195] The results in Figure 47 show that there was no significant decrease in the viability of both strains in the different solution mixtures over an extended period of time, making this endophyte composition suitable for commercial agricultural fertilizer and foliar application of biostimulants.

[0196] Example 38 Examples of compatibility of stable freeze-dried powder endophytes with commonly used foliar herbicide applications across a wide range of field crops and monocotyledonous cereals. Compatibility testing and evaluation was performed on strains WW6 and WW7, which were reconstituted as lyophilized compositions in water to form aqueous solutions and mixed separately with different standard herbicide tank mixes commonly used in cereal crops. Compatibility with Azimut (Comercial Quimica Masso, SA), Guadana (Comercial Quimica Masso, SA), and Tower (Comercial Quimica Masso, SA) products was all tested separately. The solution mixtures were scaled down from 400 liters / ha to 10 ml / ha for experimental purposes. The mixture contained 0.25% of WW6 + WW7 microbial products (10 grams of lyophilized product mixed per liter) and was formulated separately with the following products: Azimut (Florasulam 5g / L (0.5% w / v) + Aminopyralid (potassium salt) 10g / L (1% w / v) + 2,4-D (ester-2-etilhexil) 180g / L (18% w / v)) at 0.13%, Guadana (Flufenacet 40% w / v (400g / L) (32.4% w / w) + Diflufenican 20% w / v (200g / L) (16.2% w / w)) at 0.15%, Tower (Diflufenican 4% + Chlortoluron 25% + Pendimethalin 30% (SC)) at 0.50% or water as a control. After 4 hours of making and storing the mixtures, the colony forming units (CFU / ml) of the two strains were determined by plating on NLM semi-solid medium. See Figure 48.

[0197] The results in Figure 48 show that there was no significant decrease in viability of both strains in the different product mixtures over time, indicating that this endophyte composition can be used in combination with commercial agricultural foliar herbicide applications to formulate products that enhance nutrient utilization efficiency, biomass, and stress tolerance.

[0198] Example 39 Ability to enhance atmospheric nitrogen fixation after treatment with endophyte inoculant seed coat suspensions A seed treatment suspension mixed with a 0.5% w / v nutrient additive sodium alginate carbohydrate suspension was used to demonstrate the ability of different endophytes to fix atmospheric nitrogen in crop plants germinated from inoculant-coated hybrid maize seeds. Individual endophyte inoculants, WW5, WW6, and PTD1, were cultured using the adjusted fermented inoculum until the culture reached a titer of at least 1.0E8 cells / mL, and then combined with the carbohydrate nutrient additive. Seed treatment inoculants were QC checked for viability, and colony morphology of the appropriate species and strain was genetically confirmed using specific primers for colony PCR. This endophyte solution seed treatment composition was mixed with widely used chemical seed treatments, including the fungicides Fludioxonil and Mefenoxam, and the neonicotinoid insecticide Thiamethoxam. The endophyte inoculant seed treatment solution was applied to corn seeds at a rate of 2.4 mL / 1800 kernels and added to the chemical seed treatment according to the manufacturer's recommended rate and corn seed application instructions. The treated seeds were then dried and assayed for viable microbial colony counts by plating seed coat wash and KP buffer count dilutions onto nitrogen-limited medium NLM plus agar. The counts revealed microbial viability on the seeds one month after application of the mixed slurry seed coat: approximately 400 CFU / seed for WW6, approximately 40 CFU / seed for WW5, and approximately 40 CFU / seed for PTD1.

[0199] Seeds were then planted in 2-gallon pots containing 2.5 kg of field soil amended with perlite and allowed to germinate and grow for 4-5 weeks in soil containing 1,200 ppm total N and 10 ppm soluble N (NO3 + NH3) without additional fertilizer until they reached the V6 stage. Plants were grown in a greenhouse and analyzed for biological nitrogen fixation (BNF) using a 15N isotope dilution assay, specifically measuring the proportion of nitrogen derived from air. The proportional dependence of inoculated corn plants on atmospheric and soil nitrogen was estimated by comparing the natural 15N content of the inoculated plant biomass with that of adjacent, uninoculated control plants living on soil nitrogen alone. Concentrations of total N and 15N isotopes in maize shoot tissues were measured at the V6 growth stage, 4–5 weeks after planting; total N was measured using an Elementar EA Vario Pyrocube, followed by 15N using an Elementar IRMS GeoVisION isotope ratio mass spectrometer (IRMS). To quantify the percentage of air-derived nitrogen (NDFA%) in plant shoots, the 15N isotope nitrogen amount measured by the IRMS was subtracted from the total nitrogen concentration, and the difference was reported as NDFA, a percentage of the total N pool.

[0200] The validation results, shown in Figure 49, indicated that seed treatment with the endophyte seed treatment inoculant resulted in the majority of total nitrogen in corn shoots coming from the air: PTD1 42% NDFA, WW5 36% NDFA, and WW6 69% NDFA. This result clearly demonstrates that the corn endophyte seed treatment can be stabilized after drying and used for seed inoculation, and that subsequent crop plants germinated from the seeds enhance biological nitrogen fixation at the V6 plant growth stage of corn plants.

[0201] Example 40 Ability of formulated liquid endophyte inoculum treatments to enhance atmospheric nitrogen fixation after direct application to the roots of young wheat plants. To demonstrate the ability of various endophytes to fix atmospheric nitrogen in crop plants after soil or furrow rhizosphere inoculation, endophyte liquid inoculum cultures were prepared for strains WW5, WW6, PTD1, and a synergistic mixture of a co-culture of strains WW6 + WW7. Culture suspensions were simultaneously prepared using nitrogen-limited NLM medium supplemented with nutrient additives until the cultures reached a titer of at least 1.0E8 cells / mL. The nutrient-supplemented inoculum was then analyzed for viable cells and colony morphology. The presence of the WW5, WW6, PTD1, and WW6 + WW7 co-culture mixture in each inoculum was confirmed using specific primers for colony PCR. The endophyte suspension inoculum was then inoculated into the roots of 2-week-old wheat plants. One mL of the inoculum suspension was applied per plant from the potting soil to the roots of the transplanted plant. Transplants were planted in 1-gallon greenhouse pots filled with 1.5 kg of unfertilized perlite-amended field soil supplemented with 1,200 ppm total N and 10 ppm soluble N (NO3 + Nh3). Plants were grown in the greenhouse until the zygote growth stage, approximately 4-5 weeks after transplanting. Tissues were then harvested and analyzed for biological nitrogen fixation (BNF), measured as airborne nitrogen, using an Elementar EA Vario Pyrocube for total N and an Elementar IRMS GeoVisION isotope ratio mass spectrometer (IRMS).

[0202] The results are shown in Figure 50 and demonstrate that seed treatment with the endophyte seed treatment inoculant resulted in significant amounts of air-derived nitrogen in wheat shoots: 43% NDFA for PTD1, 51% NDFA for WW5, 38% NDFA for WW6, and 47% NDFA for the synergistic combination of WW6 + WW7. The results clearly demonstrate that endophyte inoculation of wheat roots, both in single strains and in co-fermented nutrient additive solutions, enhances the biological nitrogen fixation capacity, which carries over to the main growth stages of wheat plants.

[0203] Example 41 Efficacy of the endophyte synergistic combination WW6+WW7 compared to single strains when applied as a freeze-dried reconstituted seed treatment formulation to barley The objective of this study was to determine whether a synergistic endophyte mixed consortium could increase total plant biomass (shoots + roots) in barley more than a single strain used alone. Dry weight biomass was measured after 26 days of growth under reduced nitrogen in a controlled environment. A formulated seed treatment slurry of bacterial inoculants, first freeze-dried, stored, and then resuspended at the moisture content of the original growth solution, was used as the endophyte seed inoculum (500 mL resuspended endophyte freeze-dried culture at 1.1% w / v + sterile sodium alginate solution at 2% w / v). A sterile control nitrogen-limited medium (NLM) with a final alginate concentration of 2% w / v was added alone without the endophyte. All treatments were applied at a rate of 1 L per metric ton of seed. Spring barley was treated with three seed formulations using endophyte strains WW6, WW7, and the co-fermented WW6+WW7 strains and allowed to dry overnight in a laminar flow cabinet. Four pots were prepared with one plant per pot, and n = 4 plants were grown per treatment group. Control plants received Hoagland's nitrogen dropout solution formulated at 65 ppm N (100%) and reduced at 32 ppm N (50%). Experimental treatments inoculated with endophytes received reduced at 32 ppm N (50%). Hoagland's nutrients were applied to trays on Mondays, Wednesdays, and Fridays, with the trays drained 1 / 2 hour later. Plants were grown in a plant growth incubator at 25°C under artificial lighting with a 12-hour light / 12-hour dark cycle for 26 days after germination, then harvested and dried in paper bags at 45°C for 48 days. The results shown in Figure 51 demonstrate the synergistic effect of the combined treatment WW6+WW7, which produced the greatest increase in total dry weight biomass of barley under 50% reduced nitrogen, with a significant increase in total biomass weight of 75%, while WW6 alone produced a non-significant 17% increase in total biomass and WW7 alone produced a non-significant 17% increase in total dry weight biomass over the control when grown at the same reduced nitrogen rate.

[0204] Example 42 Stacking endophyte strains for synergistic application to increase grain yield biomass by inoculant seed treatment of industrial field-grown spring wheat. To test the ability of specific endophyte combinations to confer synergistic benefits to cereal crops, spring wheat varieties (Sy Ingmar) were industrially treated with seed treatments containing WW6 + WW7 co-fermentation and WW5 + WW6 + WW7 co-fermentation. No other seed treatments were used. Seeds were coated by mixing a seed treatment slurry containing a prepared solution (>1.0 E6 CFU / mL NLM fermentation + 1-0.5% w / v carbohydrate solution) and applying the slurry at a rate of 0.23 mL per lb of wheat seeds using a seed treatment device. Controls were not inoculated with the endophyte fermentation. Wheat was planted in late May in Berthold, North Dakota, USA, and harvested in September after 117 days of field growth. Plots measured 5 ft x 30 ft and contained Williams Silt Loam soil. Four rows of wheat were planted per plot, with 10 seeds evenly spaced per row. A rate of 1.5 million seeds per acre was planted with four replicates per treatment in a completely randomized block design. Grain yield data was adjusted for 14% moisture. Fertilizer was applied at planting and consisted of a blend of 10-34-0 at 15 gal / ac and 28-0-0 at 44.5 gal / ac. Soil tests were conducted on soils 0-24" deep and showed the following properties: pH: 6.2, N: 16 lb / ac, P: 9 ppm, K: 345 ppm, OM 3.7%, CEC=20.83, Ca: 2427 ppm, Mg: 547 ppm, S: 404 lb / ac, and Zn: 1.11 ppm.

[0205] The results of this study are shown in Figure 52. The two-strain treatment (WW6 + WW7) increased the average yield by 0.7 bu / ac, while the three-strain stack treatment produced a synergistic effect, increasing the average yield by an additional 3.33 bu / ac over the control, which was statistically significant at p < 0.1.

[0206] Example 43 Stacking endophyte strains for synergistic application to increase biomass yield by inoculant seed treatment of industrial field-grown romaine lettuce. To test the ability of specific endophyte combinations applied to lettuce crops to benefit yield, romaine seeds (River Road CVS) were industrially treated with single bacterial endophyte strains and with two-, three-, and four-strain combinations in which strains WW5, WW6, WW7, and PTD1 were co-fermented with the endophytic yeast WP1. All fermentations were mixed into a treatment slurry with the following formulation: CFU / mL >1.0E6 cells in NLM fermentation + 1% w / v carbohydrate solution. The treatment slurry was also mixed into clay seed coatings using a seed treatment device and then applied to seeds using standard industrial techniques. The endophyte solution was applied at the following rate: a final volume of 10 mL of endophyte inoculant per 1 / 3 lb of romaine seeds. For co-fermentations containing two endophyte strains, 5 mL of each strain was applied. For co-fermentations containing five endophyte strains, 2 mL of each strain was co-applied. Industry standard field planting parameters included 80-inch beds planted with 142,000 seeds on approximately one acre near Spreckels, California. As shown in Figures 53A and 53B, single-strain treatments improved yield. The combined treatment of strains WW5, WW6, WW7, and PTD1 with yeast strain WP1 significantly increased shoot biomass weight by 43% after industrial field growth.

[0207] Example 44 Efficacy of the endophytic synergistic combination WW6+WW7 compared to the single strains when mixed with a prebiotic carrier composition in the form of a compatible biostimulant and then applied as a seed treatment to canola.

[0208] A study was conducted to determine whether a mixed synergistic consortium of endophytes combined with a prebiotic plant microbe booster could increase canola plant total biomass (shoots + roots) more than a single strain used alone with a prebiotic plant microbe booster. Fresh weight biomass was measured after 21 days of growth under reduced nitrogen in a controlled environment. The bacterial inoculant was freeze-dried, stored, and then resuspended at the moisture content of the original growth solution and incorporated into a formulated seed treatment slurry. This slurry was used to prepare an endophyte seed inoculum with the following formulation: 500 mL of 1.1% w / v resuspended endophyte freeze-dried culture, 500 mL of prebiotic and microbial biostimulants, and a 4% w / v sterile sodium alginate solution made in HO. A sterile control nitrogen-limited medium (NLM) containing prebiotic and microbial biostimulants and 4% w / v sterile sodium alginate was used. Treatments were applied at a rate of 1 L per metric ton of seed. Seeds (untreated spring canola 'Atomic TT') were treated with three seed formulations containing endophyte strains WW6, WW7, and WW6+WW7. After application, the seeds were allowed to dry overnight in a laminar flow cabinet. Three pots were prepared, each with one plant, and n = 3 plants were grown in each of the four treatment groups. Control plants received Hoagland's nitrogen dropout solution formulated at 65 ppm N (100%) and 32 ppm N (50%). Experimental plants received 32 ppm N (50%). Nutrients were applied to the trays on Mondays, Wednesdays, and Fridays, after which the tray water was discarded. Plants were grown for 21 days after germination in a plant growth incubator at 25°C under artificial lighting with a 12-hour light / 12-hour dark cycle, and then harvested.

[0209] As shown in Figure 54, the combined synergistic mixture treatment of WW6 + WW7 + prebiotic significantly increased the total biomass weight by 82%, resulting in the greatest increase in total biomass, while WW6 + prebiotic increased the total biomass by 20% and WW7 increased the total biomass by 55%.

[0210] Example 45 Use of three synergistic endophyte strains for foliar treatment of maize shoots to reduce fertilizer requirements and increase harvested grain yield.

[0211] A study was conducted to determine the efficacy of a novel endophyte combination (WW5+WW6+WW7) applied as a foliar spray to improve nitrogen fixation in a hybrid corn variety (Channel 113 day213-19VT2PRIB). The corn variety was first industrially treated with the seed chemical Acceleron according to the manufacturer's method. Corn was planted in early May in Mead, Nebraska, USA. The soil was Tomek Silt Loam, and preplant nutrient levels were 11.1 ppm P, 344 ppm K, 7.4 ppm S, pH 5.8, 4.1% OM, and CEC 17.6. Corn plants were treated with either a control or experimental treatment containing endophyte strains WW5, WW6, and WW7 as a foliar spray applied at V6 using a pressurized mist sprayer. Plot size was 10 ft × 40 ft, and four rows of corn were planted per plot. Six replicates were run per treatment in a split-block design. Fertilizer was applied as a pre-application of soil nitrate at 17 lbs per acre, followed by 75 lbs per acre of nitrogen in the liquid formulation UAN32-0-0 before planting, for a total of 130 lb N / acre. N application to treatments was 75% of the standard nitrogen application rate for the acreage. A secondary control checkpoint treatment was included in the trial, receiving 170 lbs of nitrogen (100% of the standard nitrogen rate). Herbicide was also applied. Pre-Acuron + Roundup was applied on May 13, immediately after planting. The center two rows were harvested in late October after 163 days of field growth, and grain was weighed for statistical analysis. Results are summarized in Figure 55. At 75% N fertilization, the synergistic effect of foliar treatments of the three endophyte strains at V6 significantly increased average corn grain yield by +44.5 bu / ac (p=0.03). Foliar treatments of endophyte at V6 resulted in an increase in average yield compared to the full 100% N control treatment of 35 bu / ac.

[0212] Example 46 Stacking of two endophytic strains in synergistic in-furrow applications used to increase harvested grain yield with reduced nitrogen at 75%.

[0213] A study was conducted to investigate the efficacy of a specific endophyte combination, WW5+WP1, applied as an in-furrow liquid composition to improve nitrogen fixation and yield in a hybrid corn cultivar (Channel 113 day213-19VT2PRIB). This corn cultivar was first commercially treated with the seed chemical Acceleron according to the manufacturer's instructions. Corn was planted in early May in Mead, Nebraska, USA. The soil was Tomek silt loam, and preplant nutrient levels were 11.1 ppm P, 344 ppm K, 7.4 ppm S, pH 5.8, 4.1% OM, and CEC 17.6. At planting, corn plants were treated with the WW5+WP1 endophyte solution applied as an in-furrow overlay over the seeds using a dribble tube. Plot dimensions were 10 ft x 40 ft, with four rows of corn planted per plot. A split-block design was used with six replicates per treatment. Fertilizer was applied as a pre-application of soil nitrate at 17 lbs / acre, followed by 75 lbs / acre of liquid nitrogen formulation UAN 32-0-0 before planting, for a total of 37.5 lbs / acre of nitrogen, for a total of 130 lb N / ac. N application to treatments was 75% of the standard nitrogen application rate for the acreage. A secondary control checkpoint treatment was included in the trial, receiving 170 lbs of nitrogen (100% of the standard nitrogen rate). Herbicide was also applied. Pre-Acuron + Roundup was applied to plants on May 13, immediately after planting. The center two rows were harvested in late October after 163 days of field growth, and grain weights were measured for statistical analysis. Results are summarized in Figure 56.

[0214] Results at the 75% N fertilizer rate demonstrated that synergistic two-plant endophyte in-furrow applications applied at planting increased average corn grain yield by +30.2 bu / ac (p=0.07). Endophyte in-furrow applications at planting resulted in an increase in average yield compared to the full 100% N control treatment of 20 bu / ac.

[0215] Example 47 Stacking of endophyte strains for synergistic application to increase biomass yield in strawberry plants using a liquid root spray on transplanted seedlings before planting in the field.

[0216] A study was conducted at an organic strawberry farm in Salinas, California, to examine the efficacy of endophyte inoculants applied as a liquid root spray to improve biomass yield in Albion strawberry plants. The roots of transplanted seedlings were sprayed until covered with a thin mist of different endophyte inoculants. Spray treatments included WW5 alone, WW6 alone, WW7 alone, PTD1 alone, and a mixture of WW5, WW6, WW7, and PTD1. The control group contained no endophyte strains. Treatments were planted using standard methods in early November 2016. Strawberries were planted in beds containing 200–240 strawberry plants spaced 25–30 cm apart in two rows, with three beds per treatment spaced 120 cm apart. Strawberries were fertilized using standard methods under the guidance of a registered CCA and harvested on May 20, 2017, after 28 weeks of growth.

[0217] The treatment containing a mixture of all four strains, WW5, WW6, WW7, and PTD1, performed best, with a 25% increase in fruit yield measured by fresh weight compared to the control, as shown in Figure 57. Single-strain inoculation treatments produced smaller increases than the control.

[0218] Example 48 Combining endophyte strains within hard partially hydrated beads as dry granular carriers for synergistic application to increase transplant biomass in tomato.

[0219] A study was conducted to examine the efficacy of WW5 alone, the combination of WW6 and WW7, and the combination of four endophyte strains (WW5, WW6, WW7, and PTD1) in increasing biomass yield in tomato. The fermentation suspension was blended with sodium alginate slurry and added dropwise to a 100 mM calcium chloride water bath for a cation exchange reaction to produce fully hydrated but rigid calcium alginate beads. These beads were dried to a final moisture content of approximately 4% to 6% with a final bead size of 2 mm. Quality 47 (Q47) hybrid tomato seeds were then placed on or adjacent to a single bead containing the endophyte treatment. Controls were treated with beads containing no endophyte strains. Plants were then germinated in commercially available transplanting medium (rich in organic matter, primarily peat and perlite) in 125-cell transplanting planter trays and grown for 3 weeks at 25°C under normal lighting and a standard industrial greenhouse fertilization rate. After 21 days, eight replicate plants per treatment were weighed and the total dry weight of plant biomass was collected. The survival rate of Q47 tomato plants inoculated with the mixed fungi was 100%, while the germination rate of uninoculated controls was suboptimal at less than 88%. Figure 58 shows the results of endophyte-enhanced biomass (shoot + root) growth in transplanted plants.

[0220] The results clearly demonstrated a synergistic effect on growth in tomato plants inoculated with alginate beads containing a combination of four endophyte strains, WW5, WW6, WW7, and PTD1, compared to the control. The endophyte mixture resulted in a statistically significant increase of +35% in total plant weight (shoots and roots). The single endophyte treatments WW5 and the combination of WW6 and WW7 resulted in smaller increases.

[0221] Example 49 Combining endophyte strains in hard, partially hydrated beads as dry granular carriers for synergistic inoculant application to loose-leaf lettuce grown under conditions deficient in bioavailable N and P.

[0222] A study was conducted to investigate the efficacy of an endophyte strain in dry calcium alginate beads to reduce nitrate and phosphate application rates and increase edible yield in loose-leaf lettuce (Lactuca sativa, cv. Refugio). Experiments were conducted in greenhouses using a specific soilless medium with the Terragreen formulation: a baked clay-gravel mixture containing 9 kg of Terragreen, 0.66 kg of peat moss, 40 g of 8-3-5 organic fertilizer to reduce bioavailable forms of N and P to a nutrient concentration of 12 ppm nitrate, 5 ppm ammonia, 11 ppm phosphate, 328 ppm potassium, 690 ppm sulfate, SAR of 2.71, pH of 7.28, EC of 2.89 dS / m, TEC of 18.76 meq / 100 g, and 1831 ppm total nitrogen (mostly composed of amino acids). Greenhouse experiments were harvested 106 days after planting. Each seed was planted in a pint-sized starter pot, with one bead per seed placed 1 / 2 inch into the growing medium, and watered. Alginate beads were applied adjacent to or close to germinating seeds in soil. At three weeks, plants were then carefully transplanted, with all roots removed using a hand trowel and the surrounding soil intact. The roots, beads, and soil were placed in holes of equal size and placed in 2-gallon felt Smart Pots. All plants were automatically watered every 12 hours with the same volume of water using a controlled drip system. Low evening light was supplemented with greenhouse lighting (high-pressure sodium halide lamps) starting at 4:15 PM and ending at 7:15 PM, allowing for a full 12-hour growth cycle. Lettuce plants were inoculated with beads containing WW7 or PTD1 individually or a mixture of all four bacterial endophytes (WW5 + WW6 + WW7 + PTD1). Controls were inoculated with alginate beads containing no endophytes. Each treatment was replicated six times, with n = 6 pots per treatment.

[0223] Figure 59 shows the results of shoot biomass analysis of fresh weight. The treatment containing a mixture of all four endophytes performed best, averaging 4.23 g shoot weight per plant, a 191% increase in shoot weight compared to uninoculated control plants, a statistically significant result at p < 0.1. WW7 beads produced an average yield of 3.96 g per plant, increasing average shoot weight by 173%. Inoculation with PTD1 beads increased shoot biomass by an average of 2.74 g per plant, increasing average yield by 89%.

[0224] Example 50 Combining endophyte strains in a freeze-dried powder for reconstitution and foliar spray on jalapeños in the field.

[0225] To examine the efficacy of endophyte inoculation from a freeze-dried endophyte mixture on the growth and biomass of jalapeño plants (cv. RPP7042). Approximately 420 jalapeño plants were inoculated in late spring by foliar spray application at flowering. Five individual endophyte strains (WW5, WW6, WW7, PTD1, and WP1) suspended from freeze-dried powder were used. A resuspended mixture of the five endophyte strains was also prepared. The freeze-dried endophyte was rehydrated by adding 1 gram of freeze-dried endophyte to 1 liter of purified water and then placed in a foliar sprayer. Jalapeño seeds were planted in May or early June. Harvest occurred mid-September to early October. The reconstituted mixture was applied to the foliage along each 50-foot treatment block. Each treatment was separated by a 50-foot control treatment, with three treatments per row. Each 50-foot block represented approximately 75 plants. At harvest two months later, a series of growth and biomass analyses were performed.

[0226] As shown in Figure 60A, all inoculation treatments using lyophilized powder reconstituted at first flowering, except for the WP1 treatment, increased the average unripe pepper yield per plant compared to the control. A mixture of all five strains (Phase A) was found to be the best inoculant and to have a synergistic effect. Phase A increased the total unripe pepper yield by +44%, favoring RPP7042 jalapeño plants.

[0227] Additionally, each treatment was assayed for its ability to increase the total number of peppers, and the results are shown in Figure 60B. Phase A inoculations also performed best, with the exception of WP1, which initially increased the average number of peppers per plant by 66% over the control. Phase A mixtures, PTD1, and WW7 were all statistically significant, indicating the early positive impact of the freeze-dried endophyte on the jalapeño plants.

[0228] Total yield of ripe peppers was also assayed 3 months after initial inoculation and 4 months after planting and is reported in Figure 60C. Results indicated that endophyte inoculation using reconstituted freeze-dried powder applied as a foliar spray at first flowering can be used to increase average yield per jalapeño plant compared to the control. Phase A inoculant increased pepper biomass yield more than other treatments. While all bacterial endophyte strains tested increased yield relative to the control, Phase A showed increased synergy. These results suggest that reconstituting freeze-dried endophyte into solution and spraying it on flowers and leaves can have an early positive impact on total yield of jalapeño plants.

[0229] Example 51 Effect of different endophyte seed treatment compositions using WW5, WW6, WW7, PTD1, and WP1 on leaf chlorophyll.

[0230] The endophyte synergistic mixture was freeze-dried, resuspended in water, and then used to treat canola seeds. After 36 days of growth under reduced nitrogen, we investigated whether the resuspended endophyte fermentation product could increase canola leaf chlorophyll after 36 days of growth. The endophyte seed inoculant contained endophyte freeze-dried powder resuspended in 500 mL of water at a concentration of 1.1% w / v, mixed with 500 mL of 4% sterile aqueous sodium alginate solution, and applied at a rate of 1 L per metric ton of seed. Endophyte inoculant compositions were prepared for the following strains and strain combinations: WP1, GWW6 + WW7, and WW5 + WW6 + WW7 + WP1 + PTD1. The seed variety used in the study was Spring Canola "Atomic TT." Seeds were treated with the prepared endophyte treatment compositions: WP1, WW6 + WW7, WW5 + WW6 + WW7 + WP1 + PTD1. The control group was treated with NLM alone without the endophyte. After seed treatment, all groups were dried overnight at room temperature in a laminar flow cabinet. Each of the five treatment groups had four pots with two plants per pot (n = 8 plants per treatment). Control plants were treated with Hoagland's nitrogen dropout solution formulated at 65 ppm N (100%) and 32 ppm N (50%). The experimental groups were treated with 32 ppm N (50% N). Nutrients were added to the trays three days a week (Monday, Wednesday, and Friday), and the water was removed 30 minutes later. Plants were grown in a plant growth incubator at 25°C under artificial lighting with a 12-hour light / 12-hour dark cycle for 36 days after germination before harvesting.

[0231] The results showed that the endophyte compositions used in canola seed treatments had a synergistic effect on chlorophyll, with the two strains WW6 + WW7 increasing leaf chlorophyll by 14.3% (p < 0.1), while the synergistic five-strain endophyte consortium composition increased leaf chlorophyll by 21.0% (p < 0.05) (see Figure 61).

[0232] Example 52 Stacking endophyte strains in alginate beads for synergistic application to increase leaf chlorophyll in strawberry plants.

[0233] A study was conducted to examine the efficacy of an endophyte inoculant applied as a composition to increase leaf chlorophyll in Albion strawberry plants. The study was conducted at an organic strawberry farm in Salinas, California. Strawberry plants were transplanted onto five 2 mm calcium alginate beads placed in holes before planting. The calcium alginate beads were prepared in one of the following treatment groups: endophyte inoculant containing WW5 alone, endophyte inoculant containing WW6 alone, endophyte inoculant containing WW7 alone, endophyte inoculant containing PTD1 alone, endophyte inoculant containing WW5, WW6, WW7, and PTD1, or a control without endophyte strains. Plants were planted in early November 2016 using standard application methods. Strawberries were planted in beds containing two rows of strawberry plants (200–240 plants / row) spaced 25–30 cm apart, with three beds per treatment spaced 120 cm apart. Strawberries were fertilized using standard methods under the guidance of a registered CCA and harvested on May 20, 2017, after 28 weeks of growth.

[0234] As shown in Figure 62, the treatment containing a mixture of all four strains, WW5, WW6, WW7 and PTD1, produced the greatest leaf chlorophyll, significantly increasing leaf chlorophyll by 5.5% p < 0.05.

[0235] Example 53 Effect of different endophyte seed treatment compositions with WW6+WW7 versus market leading biologicals and biostimulants on total chlorophyll in winter wheat leaves.

[0236] An experiment was conducted to determine whether application of a freeze-dried and resuspended endophyte composition to seeds could increase leaf chlorophyll in treated host plants. The endophyte was applied to seeds of the winter wheat cultivar AWC13. A freeze-dried endophyte seed inoculation composition was prepared by suspending freeze-dried WW6+WW7 endophyte strains in 500 mL of water at a concentration of 1.1% w / v and mixing this suspension with 500 mL of 4% sterile sodium alginate solution. A control was prepared without the endophyte, consisting of sodium alginate solution and 500 mL of water. The treatment composition was applied at a rate of 1 L per metric ton of seed. Winter wheat plants were grown under optimal nitrogen conditions in a field experiment and harvested at the vegetative stage. Chlorophyll was extracted from 1 cm2 leaf sections using acetone. The results in Figure 63 showed an average increase of 6% compared to the untreated control.

[0237] Example 54 Enhancement of glutamic acid / glutamine Glx in maize leaves after treatment with endophyte inoculant seed coat compositions.

[0238] A study was conducted to determine whether seed coatings containing heterologous endophyte compositions could fix atmospheric nitrogen gas (N2), generate ammonium, and convert it into glutamate and glutamine, the primary amino acid end products, via the GOGAT GS and GDH ammonium assimilation pathways in host plants. A seed treatment containing WW6 fermentation product and 0.5% w / v sodium alginate was prepared. Corn seeds were treated with the seed treatment, then dried and stored for one month. Treated seeds and untreated control seeds were then planted in one-gallon pots containing 1.5 kg of sand, vermiculite, and perlite and allowed to germinate and grow for three weeks. During the growth period, the seeds and resulting plants were watered with Hoagland's nitrogen dropout nutrient solution supplemented with 50 ppm N. Plants were grown in a greenhouse, harvested, and freeze-dried. Common amino acids were analyzed at AAA Labs Inc. USA using a Shimadzu HPLC with post-column ninhydrin derivatization. The results, shown in Figure 64, indicate that WW6-inoculated maize seeds developed plants with significantly (p=0.6) +46% higher glutamate and glutamine amino acid content in leaves compared to controls. Furthermore, analysis of the WW6 genome for the presence of genes encoding glutamine synthetase (GS) revealed a total of six distinct copies of the GS enzyme gene, located adjacent to other genes related to nitrogen assimilation, quorum sensing, and motility. These results further support other findings related to atmospheric N fixation and enhanced N concentrations in maize shoots treated with WW6. These results also provide a mechanistic basis for the efficacy of WW6 as a biological inoculant to increase nitrogen assimilation of the amino acids glutamate and glutamine directly from the atmosphere in crop plants.

[0239] Example 55 Detection and quantification of glutamine synthetase (GS) activity in plant tissues Glutamine synthetase (GS) is a key enzyme in bacterial atmospheric nitrogen assimilation via the GOGAT pathway, which converts N2 into ammonia / ammonium and then synthesizes the amino acid glutamine. The in planta effects of endophytic strains WW6 and WW7 on this process were evaluated in wheat (Triticum aestivum) shoots. Zenda wheat seeds were first treated with (1) Cruiser Maxx Vibrance Cereals (Syngenta Crop Protection, LLC) at 5 fl oz per 100 lbs of seed and (2) Cruiser 5FS at 0.75 fl oz per 100 lbs of seed. WW6 and WW7 fermented inoculants blended with 0.5% sodium alginate by weight were applied to the wheat seeds at a rate of 500 ml per 2000 lbs of seed. Treated seeds were air-dried at room temperature and stored for one month. Controls were prepared with a seed coating solution containing only alginate and growth medium without the endophytic fungus.

[0240] Five seeds per treatment group were planted in five different 3.5-inch pots. The pots contained a mixture of washed play sand, vermiculite, and perlite potting mix. The pots were maintained at 4°C for 24 hours to induce vernalization, after which they were transferred to a growth chamber maintained at 25°C under LED lighting with a 14-hour light / 10-hour dark growth cycle. After 7 days, the number of seedlings per pot was thinned to three. Additionally, the plants were watered and fertilized with Hoagland's Dropout Hydroponic Solution with reduced nitrogen (25 ppm N), applied as needed to the trays two to three times a week, to keep the soil moist. Thirty-one days after transplanting, the plants were removed from the soil. Shoots and roots were separated and flash-frozen in liquid nitrogen, and the tissues were stored in a -80°C freezer. Plant material from each pot was individually ground to a fine powder using a mortar and pestle with liquid nitrogen. Approximately 100 mg samples of ground tissue from each treatment were then transferred to five separate 1.5 ml tubes, frozen in liquid nitrogen, and stored in a −80°C freezer for later use.

[0241] The glutamine synthetase (GS) enzyme activity in the prepared samples was detected and quantified using a Glutamine Synthetase Microplate Assay Kit (MyBioSource, Inc.). Shoot tissue samples from three potted control plants and three experimental plants (treated with WW6 and WW7) were used for the GS enzyme assay. Three technical replicates were prepared for each sample, and the mean values ​​of all replicates were then used to calculate GS synthetase activity (U / g).

[0242] Figure 65 shows the quantification of GS (U / gU is the amount of enzyme that catalyzes the reaction of 1 μmol of substrate in 1 min), adjusted for the amount of tissue examined. Data were analyzed using ANOVA analysis with post-hoc Tukey's test. Results showed that the endophyte inoculant used as a seed treatment had an average of 55% higher glutamine synthetase GS enzyme activity than uninoculated control plants, and the increase in activity was statistically significant at p<0.05.

[0243] In summary, WW6 and WW7 treated seeds significantly increased glutamine synthetase activity in wheat shoots. This result related to the GS enzyme GOGAT correlates with data from the field study in Example 62 herein showing nitrogen accumulation in endophyte-inoculated wheat shoots and the greenhouse study in Example 57 herein showing increased assimilation of airborne nitrogen in inoculated wheat.

[0244] Example 56 Reducing nitrogen fertilizer requirements in corn crops while maintaining yield A field study was designed to demonstrate the ability of an endophyte seed treatment composition to produce substantial crop yields under reduced nitrogen fertilization conditions. Corn seeds were treated with WW6 endophyte inoculant and 0.5% w / v sodium alginate and then coated. Treated seeds were planted in urea-fertilized field soil in Kansas, USA. Field plots were divided into groups and fertilized at different rates via spray application: (1) a normal rate of 201.75 kg / ha urea (100% rate), (2) a reduced rate of 141.22 kg / ha urea (70% rate), (3) a second reduced rate of 100.875 kg / ha urea (50% rate), and (4) a no-urea group (0% rate).

[0245] Grain was harvested from each plot when the grain moisture reached approximately 15.5%, with four plots per treatment. The average grain yield was calculated for all plots representing each treatment. Figure 66 shows the average shoot nitrogen uptake of corn plants inoculated with the endophyte compared to the control. This graph demonstrates the reduction in corn fertilizer requirements due to the endophyte, i.e., how much less fertilizer was required by the endophyte to maintain the same grain yield compared to uninoculated control corn plants. As a result, inoculation with either the WW6 or WW5 endophyte reduced the nitrogen fertilizer requirement per hectare by 87 kg.

[0246] Example 57 Enhancement of nitrogen uptake by single- and double-seed treatments of wheat seeds. To demonstrate the ability of endophyte seed treatment compositions to increase nitrogen uptake in crop plants, winter wheat seeds (Everest) were treated with endophyte inoculants and then grown in pots under three different conditions: (1) field soil (Kansas, USA) amended with perlite alone; (2) nitrogen-sufficient field soil amended with perlite; and (3) field soil fertilized with urea. The field soil chemical composition profile is shown in Figure 67A.

[0247] As shown in Figure 67B, seeds were treated with an endophyte seed treatment inoculant composition consisting of a bacterial ferment combined with a 0.5% w / v sodium alginate solution applied at a rate of 1 ml / kg of seeds and a standard seed treatment (Cruiser Maxx Vibrance™ seed treatment) applied at a rate of 5.7 fl oz / 100 lbs.

[0248] Endophyte viability was counted on the treated seeds after dehydration and one month of storage, and the results, shown in Figure 67C, indicated that bacterial endophytes survived on the seeds for each strain. Seeds were washed and sampled for seed wash counts. No microorganisms with morphology similar to the different endophyte strains were found in the washed samples of winter wheat control seeds. The correct strains were clearly present in all treated seeds, indicating survival and compatibility after Cruiser Maxx Vibrance treatment and storage. The WW6 treatment alone yielded the highest CFU viability per wheat seed. The CFU levels detected on the seeds may not represent the actual microbial load due to limited detection using this seed coat washing and count plating method.

[0249] A potted wheat plant growth trial was conducted in a controlled greenhouse maintained at a temperature range of 60°F to 75°F. Five winter wheat seedlings were potted and later thinned to three. One milliliter of WW5, WW6, PTD1, and WW6+WW5 solutions was applied to the seedlings, which were harvested at flowering three months later (Feekes 10.5). The results, shown in Figure 67D, indicate that the endophyte-inoculated seed treatment composition significantly increased shoot N uptake in both unfertilized, zero-nitrogen potted plants and 188 mg N (urea) potted plants.

[0250] Under fertilization conditions of 188 mg N urea / potted plant, the double strain WW6+WW7 and single strain WW6 increased the nitrogen content. Under urea treatment and non-fertilization conditions, single strains WW6 and PTD1 were most effective in increasing the nitrogen content of shoots. In addition, inoculation with a combination of two strains WW6+WW7 significantly increased the nitrogen content in the shoots of treated plants under urea treatment and non-fertilization conditions.

[0251] Example 58 Enhancement of nitrogen uptake by single- and double-seed seed treatments in maize. To demonstrate the ability of endophyte seed treatment compositions to increase nitrogen uptake in crop plants, a greenhouse study was designed in which corn seeds were first treated and then wheat plants were grown in potted field soil from Kansas, USA, amended with perlite only (see Tables XYZ below) and in nitrogen-sufficient field soil amended with perlite and urea in fertilized field soil.

[0252] Corn seeds were treated with each of the individual endophyte (WW5, WW6, PTD1) seed treatment inoculants mixed with 0.5% w / v aqueous sodium alginate and applied at a rate of 2.4 ml / 1800 kernels, and then treated with a standard seed treatment (Cruiser Maxx™ seed treatment - Syngenta) applied at a rate of 5.7 fl oz / 100 lbs. Seeds were air-dried at room temperature and stored for one month before planting.

[0253] The treated seeds were dehydrated and stored for one month before being counted for endophyte viability. Results, as shown in Figure 68A, indicated that bacterial endophytes survived on the seeds for each strain. The seeds were washed and sampled for seed wash counts. No microorganisms with morphology similar to the different endophyte strains were found in the washed samples of corn control seeds. All treated seeds clearly contained the correct strains, demonstrating survival and fitness after Cruiser Maxx treatment and storage. The WW6 treatment alone yielded the highest CFU viability per corn seed. Both WW5 and PTD1 seeds showed perfectly consistent colony morphology at 40 CFU / seed, with the WW6 treatment yielding the highest CFU viability at 400 WW6 CFU / seed. The CFU levels detected on the seeds may not represent the actual microbial load due to limited detection using this seed coat washing and count plating method.

[0254] A potted corn plant growth trial was conducted in a controlled greenhouse maintained at a temperature range of 60°F to 75°F. Three corn seeds per pot were planted and later thinned to one seedling. Corn seeds were treated with individual endophyte (WW5, WW6, PTD1) seed treatment inoculants mixed with 0.5% w / v aqueous sodium alginate and applied at a rate of 2.4 ml / 1800 seeds, followed by a standard seed treatment (Cruiser Maxx™ seed treatment - Syngenta) applied at a rate of 5.7 fl oz / 100 lbs. Plants were harvested approximately one and a half months later, during the eighth growing season. The results, shown in Figure 68B, indicate that the endophyte inoculant seed treatment composition significantly increased shoot N uptake in both unfertilized, zero-nitrogen potted plants and 318 mg N (urea) potted plants. The WW6 treatment increased total shoot nitrogen uptake by ∼12 mg per plant shoot compared to the control under unfertilized field soil conditions. PTD1 and WW5 did not increase shoot nitrogen in the greenhouse without added fertilizer.

[0255] Under fertilized conditions with 318 mg N urea / pot, treatments with individual strains PTD1, WW5, and WW6 resulted in increased nitrogen content. Under unfertilized conditions, treatment with the single strain WW6 increased shoot nitrogen content. Under fertilized conditions, inoculation of seeds with the endophyte via seed coating resulted in an increase of ~100 mg of nitrogen per shoot in all three treatments, with WW6 being the most effective, followed by PTD1, and these increases were statistically significant at the p<0.05 level. WW5 seed treatment also increased nitrogen content in corn shoots by ~85 mg compared to the control.

[0256] Example 59 Use of two and three synergistic endophyte strain compositions in seed coating applications on corn shoots to increase harvested grain yield.

[0257] This study examined whether specific endophyte combinations (WW6 + WW7) and (WW5 + WW6 + WW7) applied as seed coats to hybrid corn varieties treated with endophyte strain combinations resulted in synergistic effects on nitrogen fixation. Corn was planted in various states in the Midwestern United States by various CRO field testing organizations from early May 2020 through 2022. Plots measured 10 ft x 40 ft, with four rows of corn plants planted, evenly spaced with 30 plants per row. The study included six block replicates per treatment. Standard fertilizer applications were used. Residual soil nitrate before fertilization was 17 lbs / acre, and 170 lbs of supplemental nitrogen was applied. The herbicide Roundup was applied to plots at growth stages 3–6, depending on environmental conditions. Soil type varied by site, with organic matter content ranging from 2.1 to 4.1%. The two center rows were harvested in late October, and the grain was weighed. Yield results for the WW6+WW7 treatment, summarized as average bushels / acre compared to the uninoculated control, are shown in Figure 69A. Yield results for the WW5+WW6+WW7 treatment, summarized as average bushels / acre compared to the uninoculated control, are shown in Figure 69B. Each numbered bar in Figures 69A and 69B represents an individual field trial and indicates the difference in bushels per acre from the untreated control for that trial, with the average across all trials shown in the far right bar. Results confirmed an average yield increase of 6.2 bushels / acre for the WW6+WW7 treated seed and 11.9 bushels / acre for the WW5+WW6+WW7 treated seed. These results demonstrate the synergistic, beneficial effects of stacking multiple strain compositions together for use as seed treatments.

[0258] Example 60 Effect of endophyte seed treatment compositions using WW6+WWW7 on total leaf chlorophyll across various trials testing different cultivation programs for UK winter wheat.

[0259] A freeze-dried endophyte resuspended in water was used to treat seeds of winter wheat cultivar GS59 to determine whether the endophyte strain could increase leaf chlorophyll. WW6+WW7: A freeze-dried endophyte seed inoculation composition consisting of 1.1% w / v freeze-dried endophyte powder resuspended in 500 ml of water was added to 500 mL of a 4% sterile sodium alginate solution made in H2O and applied at a rate of 1 L per metric ton of seed. Field trials were conducted in which wheat plants were grown under optimal nitrogen conditions and harvested during the growing season. Chlorophyll was extracted with acetone from 1 cm2 leaf sections. Differences in leaf chlorophyll content (mg / cm2) in winter wheat GS59 under different programs were observed. As shown in Figure 70, in 21 of 26 field trials conducted using different industry-standard grower programs, endophyte application increased leaf chlorophyll in the experimental groups compared to the untreated control.

[0260] Example 61 Nitrogen-limited biomass enhancement in maize and teosinte inbred genetic lines. Effects of WW5 endophyte application as a coating on Zeasyn maize seeds on shoot growth. The inbred seeds of the Zeasyn population used in this study were from a synthetic population containing both maize and Teosinte alleles. The Zeasyn population was created through several generations of random crossing between nested association mapping (NAM) founders and 11 geographically distinct teosinte individuals. The final genome composition was approximately 38% B73 (maize parent breeding line), approximately 2% NAM parent line + Mo17 (maize parent breeding line), and approximately 1% teosinte. Seeds were treated with a WW5 solution containing 1.0E8 CFU WW5 per mL and 1% w / w sodium alginate. The seed treatment process was as follows: The cooled WW5 single-strain culture was thoroughly mixed and carefully pipetted onto seeds placed in Ziploc® bags at a rate of 3.4 mL per pound of seed. The seeds were dispersed dropwise in 1 mL increments in a sterile laminar flow fume hood. After each 1 mL addition, the seeds were carefully loosened by manually rolling them in the bag. Once all 3.4 mL / lb had been added, the seeds were loosened, shaken, and rolled for 2–3 minutes until all corn seeds were visibly wet inside the bag. The bag was then opened, turned upside down, and air-dried overnight at room temperature in a sterile laminar flow fume hood. The dried seeds were stored for one month before being washed and bacterial viability was counted by plating. Plating assays showed variation in endophyte coverage (WW5 CFU / seed) among the treated seeds. This variation in endophyte concentration resulted in a wide range of CFU per seed, ranging from 0 to 1,000 WW5 CFU / seed.

[0261] Seeds were tested in two groups: low-nitrogen soil (50 ppm N) and nitrogen-sufficient soil (100 ppm N) in a greenhouse experiment maintained at temperatures ranging from 70°F to 80°F and a 12- to 14-hour photoperiod. Seeds were grown for three weeks prior to leaf biomass analysis. A physiological camera was used to photograph the shoot area of ​​treated plants as an indicator of biomass. Images were taken using a phenotypic screening instrument after the treated plants had grown for three weeks. The results of the biomass phenotypic screen, shown in Figures 71A-71B, demonstrate a clear correlation between WW5 CFU / seed concentration and corn biomass shoot production, regardless of inbred line genotype. The collected data revealed a correlation between biomass growth and endophyte concentration. The optimal CFU / seed was approximately 400 WW5 CFU / seed when plants were grown in nitrogen-limited soil and approximately 300 WW5 CFU / seed when plants were grown in nitrogen-sufficient soil. Seed treatment with WW5 increased shoot biomass by approximately 2.3 times in nitrogen-deficient soil, and by approximately 1.7 times in nitrogen-sufficient soil.

[0262] Example 62 Reducing nitrogen fertilizer requirements in wheat crops while maintaining yield A field study was designed to demonstrate the ability of an endophyte seed treatment composition to produce substantial crop yields under reduced nitrogen fertilization conditions. Wheat seeds were treated with WW6 endophyte fermentation in combination with 0.5% w / v sodium alginate as a seed coat. Treated seeds were planted in field soil in Kansas, USA, fertilized with urea. Field plots were divided into groups (four groups per treatment) and fertilized at different rates via spray application: (1) 112 kg / ha urea (100% rate), (2) 78.4 kg / ha reduced urea (70% rate), (3) 56 kg / ha second reduced urea (50% rate), and (4) no urea (0% rate).

[0263] Wheat grains from each plot were harvested when fully ripe. Average grain yield was calculated for all plots representing each treatment. Figure 72 shows the average shoot nitrogen uptake of wheat plants inoculated with the endophyte compared to the control. This graph illustrates the reduction in fertilizer requirements of wheat due to the endophyte, i.e., how much less fertilizer was required with the endophyte compared to uninoculated control wheat. As a result, inoculation with WW6 endophyte reduced the nitrogen requirement of wheat fertilizer by 23 kg per hectare.

[0264] Example 63 Enhanced root and shoot growth in grapevine cutting production and long-term presence of endophyte-derived traits after inoculation with endophyte beads.

[0265] In a new grapevine production scenario, the persistence of endophyte-derived traits long after inoculation is beneficial for establishing nurseries for clonal propagation. We planned a long-term study to track the physiological status of cuttings inoculated with endophyte beads in the field. Approximately 20 endophyte beads containing calcium alginate beads were placed adjacent to commercial Cabernet wine grape cuttings pressed 5–7 cm deep into potting soil (Sunshine Mix #4). The endophyte beads contained an endophyte consortium inoculant containing strains WW5, WW6, WW7, and PTD1 (the mixture is referred to as "Phase B"). After rooting for two weeks in pots in the greenhouse, the cuttings inoculated with endophyte beads exhibited more developed root systems and larger shoots than the control, as shown in Figure 73A. Plant growth continued for an additional two weeks in a greenhouse maintained at temperatures ranging from 70° to 80° F. and a 12- to 14-hour photoperiod. The plants exhibited a persistence of the enhanced shoot and root growth phenotype during the additional growth period, as shown in Figure 73B.

[0266] An uninoculated control group of Cabernet wine grapes is shown on the right side of Figure 73B, and inoculated cuttings are shown on the left. Biomass and cutting length were measured at the stage shown in Figure 73B, and the plants were then transplanted into field plots. Collected biomass data showed that the total biomass of the inoculated cuttings grew significantly 40% greater than the control group, as shown in Figure 73C. Additionally, the endophyte-inoculated Cabernet cuttings grew 31% higher in height than the uninoculated control, as shown in Figure 73D.

[0267] Two years after planting the endophyte-inoculated cuttings in the field, the cuttings matured into grapevines and an increased chlorophyll phenotype was also observed. Figure 73E shows data showing that the inoculated cuttings had a higher mean leaf chlorophyll.

[0268] Additionally, the average bunch weight of 2-year-old wine grapes increased by an average of 7% on inoculated vines relative to controls. Figure 73F shows data demonstrating increased grape bunch weight on inoculated cuttings.

[0269] In a separate field trial conducted at the same site, approximately 20 beads of endophyte strains WW5, WW6, WW7, and PTD1 (Phase B) in calcium alginate were pressed into potting soil (Sunshine Mix #4) at a depth of 5–7 cm adjacent to Grignolino wine grape cuttings. Field results from a year and three months after planting showed a similar effect, with endophyte-inoculated (Phase B) cuttings experiencing an average 9% increase in stem base diameter after two years compared to uninoculated controls. Results are shown in Figure 73G.

[0270] Example 64 Endophyte rooting effects in greenhouse cutting clone production of Cabernet wine grapes

[0271] Root production from cuttings is an important factor for clonal propagation in greenhouse settings. We examined the effect of treating cuttings with an endophyte strain on root production. Commercially available Cabernet wine grape cuttings were inoculated with a slurry dip made from freeze-dried WW6+WW7 powder, added with sodium alginate, and reconstituted with well water. To promote root growth, the cuttings were dipped in the endophyte slurry immediately before being placed on the mister bench. The root growth results, shown in Figure 74, demonstrate a statistically significant 41% increase in root growth in cuttings treated with WW6+WW7 endophyte inoculation, demonstrating a significant increase or improvement in clonal propagation of cuttings from production.

[0272] The combined results of the grapevine cutting propagation experiment and field transplanting in Example 65 demonstrated improved clonal propagation of cuttings and continued phenotypic improvement after transplanting, supporting the ability to propagate new varietal characteristics within plants for production purposes, thereby creating new varieties, through endophyte inoculation, clonal, or vegetative propagation.

[0273] Example 65 Methods for preparing inoculant formulations. Starter cultures for the fermentation of the endophyte strains of the present technology are prepared by aseptically pipetting 1–2 ml of a stock sample of the endophyte strain in glycerol into a flask or bioreactor (e.g., 500 ml–10 liter volume) containing nitrogen-limited medium (NLM), nitrogen-free medium (NFM), or rich liquid medium, to a final concentration of approximately 1 × 10 6 CFU ~ approx. 1 x 10 10 The starter culture may be grown aerobic at 23-30°C for 2-4 days to reach CFU. If necessary, the starter culture may be scaled up to intermediate volumes of 50-200 liters for each endophyte strain. The starter culture volume for each strain should be 0.01-1% of the total volume of the subsequent fermentation process.

[0274] Large-scale fermentation may begin with starter preparation by adding a starter culture of an endophyte strain (e.g., WW5, WW6, WW7, or PTD1) to a tank containing 50 to 10,000 liters of pasteurized NLM, NFM, or rich liquid medium and incubating aerobically at 23°C to 30°C for 6 to 24 hours with agitation using sparged airflow at a rate of 0.1 to 0.5 volumes of air per volume of growth medium per minute (VVM).

[0275] The second strain for the co-fermentation process may be added to the first fermentation 10-24 hours after the first endophyte strain growth. A starter culture of the second endophyte strain (e.g., prepared as described above in this example) is added to the fermentation medium, and the growth of the co-fermented two-strain batch is continued for an additional 24-72 hours or until the total CFU reaches 1 x 10 7 ~1×10 12 It can be fermented until soft.

[0276] Example 66 Method for preparing co-fermentation inoculant formulation. Separate starter cultures for each of the endophyte strains of the present technology (e.g., WW5, WW6, WW7, or PTD1) are prepared by aseptically pipetting 1–2 ml of a stock sample of the endophyte strain in glycerol into separate flasks or bioreactors (e.g., 500 ml–10 liter volume) containing nitrogen-limited medium (NLM), nitrogen-free medium (NFM), or rich liquid medium, to a final concentration of approximately 1 × 10 6 CFU ~ approx. 1 x 10 10 The starter culture may be grown aerobic at 23-30°C for 2-4 days to reach CFU. If necessary, the starter culture may be scaled up to intermediate volumes of 50-200 liters for each endophyte strain. The starter culture volume for each strain should be 0.01-1% of the total volume of the subsequent fermentation process.

[0277] Large-scale fermentation may begin with a starter preparation by adding one of the endophyte strains (e.g., WW5, WW6, WW7, or PTD1) to a tank containing 50-10,000 liters of pasteurized NLM, NFM, or rich liquid medium and incubating aerobically at 23°C to 30°C for 6-24 hours with agitation using sparged airflow at a rate of 0.1 VVM to 0.5 VVM.

[0278] After 10-24 hours of fermentation of the first endophyte strain, starter cultures of one or more additional endophyte strains (e.g., WW5 and / or WW6) may be added to the initial fermentation to perform the co-fermentation process. Starter cultures of the additional endophyte strains (e.g., prepared as described above in this example) are added to the fermentation medium, and growth of the co-fermented multi-strain batch is continued for an additional 24-72 hours or until a total CFU of 1 x 10 is reached. 7 ~1×10 12 It can be fermented until soft.

[0279] Example 67 Method for preparing co-fermentation inoculant formulation. Separate starter cultures for each of the endophyte strains of the present technology (e.g., WW5, WW6, WW7, etc.) are prepared by aseptically pipetting 1–2 ml of a stock sample of the endophyte strain in glycerol into separate flasks or bioreactors (e.g., 500 ml–10 liter volume) containing nitrogen-limited medium (NLM), nitrogen-free medium (NFM), or rich liquid medium, to a final concentration of approximately 1 × 10 6 CFU ~ approx. 1 x 10 10 The starter culture may be grown aerobic at 23-30°C for 2-4 days to reach CFU. If necessary, the starter culture may be scaled up to intermediate volumes of 50-200 liters for each endophyte strain. The starter culture volume for each strain should be 0.01-1% of the total volume of the subsequent fermentation process.

[0280] Large-scale fermentation involves adding starter preparations of multiple endophyte strains (e.g., WW5, WW6, WW7, and / or PTD1) to tanks containing 50–10,000 liters of pasteurized NLM, NFM, or rich liquid medium at 23–30°C for 6–24 hours or until a total CFU of 1×10 7 ~1×10 12 The culture may be started by culturing aerobically with agitation using sparged airflow at a rate of 0.1 VVM to 0.5 VVM until the

[0281] Example 68 Liquid microbial stabilization in packaging immediately after fermentation. After fermenting the inoculum according to the methods disclosed herein, the microbial population of the inoculum may stabilize, with a minimum of about 1 x 10 CFU of each inoculated strain. 7 ~Approx. 1×10 9 Once this is achieved, sterile purified water is added in a ratio of 1:1, 1:2, 1:3, 2:1, or 3:1. For example, 380 liters of purified water may be added to 380 liters of fermentation broth to produce a total volume of 760 liters (~200 gallons) of final shelf-stable inoculum mix.

[0282] As an alternative or additional stabilization step, the fermentate may be combined with an additional liquid stabilizing osmotic protectant or carbohydrate solution (e.g., a sterile alginate solution having an alginate concentration of about 0.05% to about 1.5% by weight). For example, the alginate solution may be made by mixing alginate salt with water, sterilizing the mixture, and cooling it to 25°C. The fermentate may then be mixed with the alginate solution in various ratios: 1:1, 1:2, 1:3, 2:1, or 3:1.

[0283] The alginate solution may then be transferred into bladder bags (e.g., up to 275 gallon bags) and the endophyte inoculum may be prepared for storage stability. Bladder bags may be partially filled to leave 15-25% available headspace for expansion.

[0284] Example 69 Monitoring and quantification of liquid formulations by count plating of PCR confirmed growth. Microplating - Liquid inoculum formulations may be tested for the presence of active endophytes after fermentation, storage, or other circumstances. The inoculum may be sampled aseptically and tested in triplicate sterile dilution series using dilutions in a neutral pH buffer of approximately 7.0. -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 and / or 10 -9 Serial dilutions of the 1000-kJ / ml microbial strain were plated using standard microbiology spread plate techniques. Media spread plates used include NFM (nitrogen-free medium), NLM (nitrogen-limited medium), TSA (tryptic soy agar), PDA (potato dextrose agar), and / or other media. Colonies were incubated at approximately 75°F to approximately 86°F for 48–72 hours, depending on the plate type and strain. Colonies on the plates were then counted based on their morphology, and a CFU / mL reading was calculated and converted to CFU / g.

[0285] Colony PCR and 16S-colonies may be further validated using two molecular confirmation steps. First, 2-5 colonies of each morphology are used for colony PCR using primers specific to each strain. Next, 1-3 colonies from each morphology are selected for 16S PCR. The selected samples are sent to a DNA sequencing company for sequencing. This sequence may be compared to the known 16S sequences of each endophyte strain expected to be included in the inoculant composition.

[0286] Digital CFU-DNA may be isolated from the liquid formulation or a dilution of the formulation. The DNA is then used for quantitative PCR, such as qPCR or DDPCR. Digital PCR allows for direct calculation of the CFU of an endophyte strain. Quantitative digital PCR data is used in combination with a formula for the surviving colonies predicted by microbiological plating on culture media to provide a digital CFU value.

[0287] Propidium Monoazide (PMA) Dye - PMA dye is a photoreactive dye that binds to exposed DNA. PMA may be added to a sample of the inoculum composition or a dilution thereof, which can then be exposed to light. DNA not currently present within cells is bound by PMA and becomes unavailable for PCR, so DNA from dead bacteria does not contaminate PCR results. DNA is isolated from this sample and then used in quantitative PCR, such as qPCR or DDPCR. This PCR value can be considered as digital CFU.

[0288] Example 70 Preparation of freeze-dried powder inoculant formulation. To allow for the creation of stable freeze-dried (FD) microbial preparations, the fermentation may be subjected to an initial cold shock of a liquid inoculum at 44°F in a refrigerator or refrigerator for at least 24 hours. To preserve the endophytic fungi in ice prior to freeze-drying, the cooled fermentation may be flash-frozen as a solid block in an evaporator tray at -40°F. Alternatively, the cooled fermentation preparation may be flash-frozen in liquid nitrogen and frozen to a 0.13-in. 2 Alternatively, the endophyte fermentation may be first concentrated using a microbial filter or bulk centrifuge to produce a semi-solid bacterial paste, which is flash frozen and then freeze-dried.

[0289] The frozen fermentate may then be freeze-dried using a variety of equipment and techniques. For example, the frozen fermentate preparation may be dried in a vacuum chamber using the following 30 hour drying cycle, as shown in Figure 75A.

[0290] As a further example, frozen ferment may be dried in a vacuum chamber using the following 30 hour drying cycle, as shown in Figure 75B: At the end of the cycle, the pressure is slowly released over an hour or more to prevent disturbance of the blend.

[0291] Example 71 Spray drying of powder inoculants. Endophyte liquid fermentations as described herein (e.g., those described in Examples 65-67) may be used to produce spray-dried powder inoculant formulations. The liquid fermentation may be mixed in a low- to high-shear mixer with approximately equal amounts of additional carbohydrate, sugar, and / or protein solids, at about 0.1% to about 10% w / v (e.g., about 0.5% to about 6%, and other values ​​therebetween), to produce a slurry. For example, sodium alginate, maltodextrin, whey protein, mannitol, starch, sucrose, PEG, talc, or other compatible solids may be added as components at 0.1% to 10% w / v (0.5% to 6%). The slurry may be spray-dried at a target outlet temperature of about 50°C to about 120°C, with a final target moisture content of 4% to 10%, depending on the strain used. The spray-dried composition provides a dry, stable endophyte microbial inoculant composition that can be easily reconstituted.

[0292] As an example, a liquid fermentation inoculant composition containing the WW7 endophyte strain was used to generate a new composition comprising a spray-dried powder combined with sodium alginate (DuPont Nutrition USA, Inc.). 9.46 liters of a liquid composition containing WW7 and 1.1-2.2% w / v sodium alginate were mixed using a rotor-stator mixer under low-energy (2700 rpm) or high-energy (7000 rpm) mixing to produce the spray-dried composition. The mixture was then spray-dried at a target outlet temperature of approximately 80°C. The colony-forming units (CFU / gram) of the spray-dried product were quantified by plating the product on NLM semi-solid medium as shown in the table in Figure 76. The results shown in the table demonstrate the viability of the WW7 endophyte strain when mixed with different percentages of alginate and mixing energy. The data demonstrate that an endophyte composition can be produced and used for formulation, coating, and delivery in commercial agricultural practices.

[0293] Example 72 Method for producing encapsulated bead formulations The following process may be used to produce partially hydrated encapsulated calcium alginate beads of various sizes, ranging from about 200 μm to about 5 mm in diameter. The endophytes thus encapsulated are semi-dormant and have a final bacterial titer of about 10. 4 CFU / gram to approximately 10 10 CFU / gram of beads. The endophyte strains of the present technology may be incorporated into the beads as a single strain or in multiple strains (e.g., two, three, or four strains of WW5, WW6, WW7, and PTD1), and may further include the yeast strain WP1.

[0294] Alginate beads containing endophyte strains may be produced as follows: Deionized water, cornstarch, and high-purity, low-viscosity sodium alginate extracted from seaweed may be mechanically blended. A liquid endophyte strain culture (e.g., containing one or more of WW5, WW6, WW7, and PTD1, with WP1 added as needed) is mechanically mixed into a final slurry in the following weight proportions: 70-90% water, 1-8% w / v starch, 2-10% v / v bacterial fermentation mixture, and 1-4% w / v alginic acid. The slurry is then constantly stirred and dripped from a height of 6 inches into a 1.1% w / v CaCl2 cationic salt bath (MgCl2 or other suitable salts may also be used) using a peristaltic pump and a large bank of 6-gauge stainless steel dispensing needles to form individual beads. Depending on the rate of calcium depletion, the bath may be circulated and the salt replenished. The volume of the slurry may vary from 300 gallons to 5,000 gallons. After the beads have finished dripping and cation exchange cross-linking has occurred, the beads are mechanically lifted from the bath using a collection tote and drained thoroughly. The beads may then be weighed into 100 lb aliquots and dried at 50°C in a tray or fluidized bed. The beads are dried to a moisture content of <10% and measured with a Karl Fischer titrator for accurate moisture determination.

[0295] Using the method described above, alginate beads containing fermentation products containing a combination of WW5, WW6, WW7, and PTD1 endophyte strains were produced. This method yielded approximately 138,800 beads / kg. The average size and weight of beads produced with the large needle gauge set was as follows: the 5-gauge needle produced 7.7 mg of beads with a diameter of 2.6 mm, the 6-gauge needle produced 7.7 mg of beads with a diameter of 2.1 mm, and the 9-gauge needle produced 7.2 mg of beads with a diameter of 1.8 mm. The CFU / g titers of 6-gauge, 2 mm beads for the four-strain mixture of WW5, WW6, WW7, and PTD1 are shown in the table in Figure 77.

[0296] After production, the beads were stored at temperatures below 110° F, with the recommended storage temperature being 40° F to 80° F. Under these conditions, the endophyte strains remained viable within the partially hydrated shells of the encapsulated alginate beads for up to three years.

[0297] Example 73 Dry carrier powder inoculant mixture and fertilizer coating method. Dry powder formulations on various carriers may be produced by spraying a liquid endophyte formulation or by mixing a liquid endophyte formulation with a dry powder formulation. The carrier may be an inert powder carrier or a mineral fertilizer. Carriers may include sodium alginate, powdered or crude biochar, dolomitic lime, triple superphosphate, maltodextrin, whey protein, cellulose / hemicellulose, tapioca flour, sucrose, or other dry carriers. In other embodiments, the endophyte formulation or a dry powder formulation containing the endophyte formulation may be used to coat other fertilizers, such as triple superphosphate (dibasic calcium phosphate + monobasic calcium phosphate), tricalcium phosphate, urea, DAP, KNO, NH-NO, and / or other suitable fertilizer compositions. Dry powder carrier mixture formulations may be produced by spraying a liquid endophyte formulation onto powder moving on a conveyor belt or by blending in a mechanical mixer. In another embodiment, powdered freeze-dried or spray-dried endophyte inoculant (eg, as described in Examples 68 and 69) is mixed with a carrier and / or fertilizer.

[0298] Example 74 Methods and protocols for seed treatment with endophyte inoculants. The seed coating and nutrient formulation may include one or more endophyte strain inoculants (e.g., WW5, WW6, WW7, and / or PTD1), about 0.01% to about 2% w / v of a carbohydrate such as sodium alginate, a nitrogen-limited medium, amino acids, mineral nutrients, sucrose, and mannitol. The seed coating and nutrient formulation may be applied to the seeds as a first step. Thereafter, one or more of the following specific seed chemicals may be applied: mefenoxam, fludioxonil, azoxystrobin, sedaxan, thiabendazole thiram, metalaxyl, hymexazole, penthiopyrad, clothianidin, beta-cyfluthrin, thiamethoxam, tebuconazole, prothioconazole, imidacloprid, clothianidin, acibenzolar-S-methyl, difenoconazole, cyantraniliprole, spinosad, oxathiapiprolin, pydiflumetofen, cyromazine, picarbutrazox, mandipropamid, mancozeb, thiodicarb, flupyradifurone, penflufen, prothioconazole, trifloxystrobin, beta-cyfluthrin, triadimenol, pyraclostrobin, boscalid, abamectin, captan and fluopyram. The seed coating polymer may be applied along with the seed chemical until the seeds are sufficiently coated with both formulations and then dried under the lowest possible forced air temperature. In other embodiments, the endophyte strain inoculant can be applied after coating the seeds with the seed chemical and / or seed polymer.

[0299] In some embodiments, the seed chemical and seed coating polymer may be simultaneously applied to the seeds along with the endophyte strain inoculant in a seed treater using separate blend streams. The blend streams may be thoroughly mixed in a seed treatment bowl or container until the seeds are well coated with both blends, and then dried under the lowest possible forced air temperature.

[0300] In a further embodiment, the seed chemical, seed coating polymer, and endophyte strain inoculant may be premixed until the mixture is homogeneous before application to the seeds as a slurry. The mixture may be applied to the seeds within 24 hours in a seed treater. The seeds are thoroughly mixed in a bowl or container until well coated with both formulations and allowed to dry under the lowest possible forced air temperature.

[0301] Example 75 Use of powdered endophyte formulations mixed directly into powdered cellulose hemicellulose seed coatings and liquid endophyte formulations applied to dry carrier seed coatings.

[0302] Endophyte-encapsulated beads were produced using a liquid endophyte inoculant nutrient formulation containing Sphingomonas sp. (WW5) and mixed with sodium alginate to produce calcium alginate beads with a 2 mm diameter and 10% moisture content using methods described herein (e.g., Example 70). 200 g of the beads were finely ground and mixed with a cellulose and hemicellulose carrier mixture for application as a natural seed coat, followed by application of a white seed polymer and used to coat 250,000 tomato seeds (Syngenta Qual-47). The powdered carrier and inoculant mixture was dusted onto the seeds in a mixing bowl, resulting in WW5 present on the treated seeds at 3.68E+07 CFU / seed.

[0303] In a separate treatment group, 490 mL of the WW5 liquid seed treatment vegetative inoculant formulation was mixed with the same hemicellulose powdered carrier and used to treat 250,000 tomato seeds (Syngenta Qual-47) with a white seed coating polymer. The liquid seed treatment vegetative inoculant + powdered carrier formulation produced a similar seed CFU of 8.33E+07 CFU / seed.

[0304] Because the CFU / seed were similar, the two groups of tomato seeds treated with WW5 endophyte were pooled together and designated (inoculated). A control group of seeds treated with hemicellulose plus polymer without the endophyte was also prepared. The seed groups were grown in a California tomato transplant house for 40 days before commercial growers planted the seed groups in a field trial near Huron, California. Data at harvest demonstrated that WW5 produced significantly more plant biomass than the uninoculated control in the large-scale field trial, as shown in Figure 78.

[0305] Example 76 Methods for using endophyte inoculants to create new stable endophyte-inoculated crop varieties.

[0306] Peach shoot cuttings were prepared and cultured overnight with an endophyte inoculum containing strains WW5 and WW6. The shoot cuttings were then grown and maintained for production purposes. Two weeks after inoculation, root callus formed, and new leaves emerged from the peach shoot cuttings. The tissue was carefully harvested, surface sterilized with 2% bleach, and then rinsed with sterile DI water. PCR studies of undifferentiated root callus and leaf tissue were performed. Results indicated the presence of the endophyte in the root callus and leaf tissue.

[0307] Figure 79 shows PCR gel data showing that endophyte strains WW5 and WW6 were present in both tissues and were more prevalent within the undifferentiated root callus.

[0308] Example 77 WW5 is a novel species based on dDDH analysis. To assess the affiliation of strain WW5 to the Sphingobium type strain, pairwise digital DNA-DNA hybridization values ​​(dDDH) were calculated for strain WW5 to determine its intraspecific relatedness to representative strains (type strains) of the genus Sphingobium. The pairwise dDDH percentage values ​​between strain WW5 and the Sphingobium type strain were lower than 70%, as shown in Figure 80, indicating that strain WW5 is a representative strain of a novel Sphingobium species.

[0309] Example 78 PCR analysis demonstrating the presence of the nif D gene in WW5. The nif D gene encodes the α subunit of nitrogenase, which contains the catalytic site for reducing nitrogen gas to ammonia. It binds to the FeMo-cofactor required for nitrogen fixation and is involved in electron transfer from ferredoxin to nitrogenase, reducing N2 to NH3. Colony PCR and DNA sequence analysis of the WW5 endophytic strain were performed to determine the presence of nif D in the genome. PCR demonstrated that nif D is also present in WW5. Further sequence analysis of WW5 amplicons using primers constructed from degenerate sequences demonstrated that the WW5 nif D sequence is 91% identical to the nif D gene of Pseudacidovorax intermedius (NCBI Sequence ID: KM103913). PCR analysis was performed according to the method described by Darcy L. McRose, Xinning Zhang, Anne ML Kraepiel, and Francois MM Morel, Diversity and Activity of Alternative Nitrogenases in Sequenced Genomes and Coastal Environments, Frontiers in Microbiology, 8, 2017.

[0310] Figure 81 shows gel data for the presence of nif D in strain WW5. According to the method of Darcy et al., the presence of 300 bp and 500 bp bands indicates the presence of the nif D gene.

[0311] Example 79 Additional nitrogen assimilation pathway genes in WW6. It has been experimentally demonstrated that the WW6 strain has the following genes in its genome involved in the following nitrogen metabolism and assimilation processes: As listed in the table in Figure 82, the WW6 genome contains genes involved in the assimilatory and dissimilatory nitrate reduction pathways, including NasA (assimilatory nitrate reductase catalytic subunit), which catalyzes the reduction of nitrate to nitrite: Nitrite + acceptor + H2O <=> Nitrate + reduced acceptor

[0312] The WW6 genome also contains Nir (nitrite reductase), which is involved in the reduction of nitrite to ammonium using the following reaction: Nitrite + 3 NADH + 5 H + → Ammonia + 3 NAD + +2 H2O

[0313] The WW6 genome also contains nitric oxide dioxygenase, which means that WW6 also has the ability to couple the detoxification of NO by Nas-Nir enzymes to the production of ammonia. 2NO+2O2+NAD(P)H→2nitrate+NAD(P) + +H +

[0314] Finally, the WW6 nitrilase protein can catalyze the hydrolysis of CN organic compounds in the environment, converting them to the corresponding carboxylic acids and ammonia, and mobilizing the ammonia for nitrogen uptake by the host plant. The table shown in Figure 82 shows the KEGG database identification of genes present in WW6 involved in the nitrogen assimilation pathway.

[0315] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and many modifications and variations are possible in light of the above teachings. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, so as to enable others skilled in the art to best utilize the invention and its various embodiments, with various modifications suited to the particular uses contemplated.

Claims

1. 1. A method for enhancing nitrogen fixation in a host plant, comprising applying an inoculant composition to tissues or seeds of the host plant, the inoculant composition comprising at least one heterologous endophytic bacterial strain selected for its ability to fix atmospheric nitrogen, and at least one nutrient additive operable to enhance survival and colonization of the at least one heterologous endophytic bacterial strain in the host plant.

2. The at least one heterologous endophytic strain is grown in a nitrogen-limited growth medium supplemented with exogenous ammonium (NH 4 + 10. The method of claim 1, further comprising selecting for the ability to produce

3. 10. The method of claim 1, further comprising selecting the at least one heterologous endophyte strain for its ability to increase the solubilization of multiple forms of insoluble phosphorus in a liquid bacterial growth culture.

4. The method of claim 1 , wherein the inoculant composition increases nitrogen uptake in the host plant.

5. The method of claim 1 , wherein the at least one heterologous endophytic bacterial strain comprises a plurality of endophytic bacterial strains selected to fix atmospheric nitrogen.

6. 10. The method of claim 1, wherein the inoculant composition increases total plant carbon in the host plant colonized thereby.

7. The method of claim 1 , wherein the at least one heterologous endophyte strain has the nitrogenase gene subunit Nif H.

8. 10. The method of claim 1, wherein the inoculant composition comprises a prebiotic composition operable to enhance colonization of the host plant by the at least one heterologous endophytic bacterial strain.

9. 10. The method of claim 9, wherein the prebiotic composition comprises at least one of a microbial nutrient package, a plant biostimulant, an osmotic protectant, a buffering agent, and a seed lubricant.

10. 2. The method of claim 1, wherein the at least one heterologous endophyte strain comprises at least one of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

11. The method of claim 1, wherein the at least one heterologous endophyte strain is a plurality of heterologous endophyte strains including at least two of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

12. The method of claim 11, wherein the plurality of heterologous endophyte strains comprises at least three of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

13. The method of claim 11 , wherein the plurality of heterologous endophyte strains comprises Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

14. The method of claim 1 , wherein the at least one heterologous endophyte strain reduces acetylene.

15. 10. The method of claim 1, wherein the inoculant composition increases biomass in the host plant treated with the composition.

16. 10. The method of claim 1, wherein the inoculant composition increases amino acid production in the host plant.

17. 2. The method of claim 1, wherein the at least one heterologous endophytic strain exhibits fixation of atmospheric nitrogen in the host plant treated with the composition when the host plant is planted in nitrogen-depleted soil or when the host plant is planted in nitrogen-rich soil.

18. The method of claim 1 , wherein the at least one heterologous endophyte strain produces an Fe siderophore.

19. 10. The method of claim 1, wherein the inoculant composition increases the acquisition of essential macro- and micronutrients from the soil and atmosphere without substantially disrupting the plant nutrient ion stoichiometry of the host plant.

20. 10. The method of claim 1, wherein the inoculant composition increases leaf chlorophyll in the host plant treated with the inoculant composition.

21. 10. The method of claim 1, wherein the inoculant composition increases tolerance to abiotic stress in the host plant treated with the inoculant composition.

22. 10. The method of claim 1, wherein the inoculant composition increases the resistance of the host plant to a plant disease.

23. 10. The method of claim 1, wherein the inoculant composition increases germination, emergence, and seedling biomass weight in the host plant treated with the inoculant composition.

24. 10. The method of claim 1, wherein the inoculant composition increases total nitrogen accumulation in plant shoots of the host plant.

25. 10. The method of claim 1, wherein the inoculant composition increases germination, emergence, and seedling biomass weight in the host plant treated with the inoculant composition.

26. The inoculant composition may be applied to the root biomass, root branches, root hairs and / or root nuclei of the host plant.

2. The method of claim 1, wherein the method increases any desired ultrastructural changes.

27. The method of claim 1 , wherein the at least one heterologous endophyte strain is resistant to a biocide.

28. 28. The method of claim 27, wherein the biocides include fungicides, insecticides, nematicides and herbicides.

29. The method of claim 1 , wherein the at least one heterologous endophytic strain is resistant to plant hormones, plant elicitors, mineral micronutrients and mineral macronutrients.

30. 10. The method of claim 1, further comprising mixing the composition with an organic or synthetic nutrient or chemical prebiotic for the purpose of a combined seed treatment, combined foliar application, or combined in-furrow application.

31. 10. The method of claim 1, wherein the inoculant composition further comprises one or more Rhizobium species.

32. 10. The method of claim 1, wherein the inoculant composition further comprises one or more Mycorrhizae species.

33. 10. The method of claim 1, wherein the inoculant composition further comprises endophytic yeast strain WP1.

34. 10. The method of claim 1, wherein the inoculant composition further comprises one or more of a fungicide, an insecticide, a herbicide, a biostimulant, a plant growth regulator, a prebiotic, an adjuvant, and a fertilizer applied to the host plant as separate applications or as a mixture.

35. 10. The method of claim 1, wherein the inoculant composition further comprises a carrier composition that enables the plant inoculant composition to be applied to a seed.

36. 10. The method of claim 1, wherein the inoculant composition further comprises a carrier composition that enables application of the plant inoculant composition to leaf portions of the host plant.

37. 10. The method of claim 1, wherein the inoculant composition further comprises a carrier composition that enables the plant inoculant composition to be applied to soil adjacent to the host plant.

38. The method of claim 1 , wherein the at least one heterologous endophyte strain is encapsulated in microbeads that are incorporated into the inoculant composition.

39. 36. The method of claim 35, wherein the inoculant composition is a suspension composition.

40. 36. The method of claim 35, wherein the inoculant composition is a dry composition.

41. 36. The method of claim 35, wherein the inoculant composition comprises a dry granular composition.

42. 10. The method of claim 1, wherein the inoculant composition comprises a dry granular composition.

43. 10. The method of claim 1, wherein the inoculant composition comprises a liquid carrier and is applicable as a foliar spray.

44. 10. The method of claim 1, wherein applying the inoculant composition to the tissue or seed of the host plant comprises short-term or long-term inoculation of a dormant seed embryo.

45. 2. The method of claim 1, wherein the at least one heterologous endophytic bacterial strain is selected for a plant-microbe booster or prebiotic and is used in combination with a plant-microbe booster or prebiotic comprising one or more of an osmoprotectant, an amino acid, an antioxidant, a mineral nutrient, a plant hormone or plant growth elicitor, a carbohydrate, and a mucilaginous gel applied to the host plant as separate applications or as a mixture.

46. The method of claim 1 , wherein the at least one heterologous endophyte strain has the genetic machinery to produce Fe siderophores.

47. A method for enhancing nitrogen fixation in a host plant, comprising applying an inoculant composition comprising at least one heterologous endophytic bacterial strain to tissues or seeds of the plant, wherein the at least one heterologous endophytic bacterial strain comprises at least one of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

48. 48. The method of claim 47, further comprising selecting the at least one heterologous endophytic bacterial strain for the ability to fix atmospheric nitrogen.

49. 48. The method of claim 47, wherein the inoculant composition comprises at least one nutritional additive operable to enhance survival and colonization of the at least one heterologous endophytic bacterial strain in the host plant.

50. The at least one heterologous endophytic strain is grown in a nitrogen-limited growth medium supplemented with exogenous ammonium (NH 4 + 10. The method of claim 1, further comprising selecting for the ability to produce

51. 48. The method of claim 47, further comprising selecting the at least one heterologous endophyte strain for its ability to increase the solubilization of multiple forms of insoluble phosphorus in a liquid bacterial growth culture.

52. 48. The method of claim 47, wherein said inoculant composition increases nitrogen uptake in said host plant.

53. 48. The method of claim 47, wherein the at least one heterologous endophytic bacterial strain comprises a plurality of endophytic bacterial strains selected to fix atmospheric nitrogen.

54. 48. The method of claim 47, wherein the inoculant composition increases total plant carbon in the host plant colonized thereby.

55. 48. The method of claim 47, wherein the at least one heterologous endophyte strain has the nitrogenase gene subunit Nif H.

56. 48. The method of claim 47, wherein the inoculant composition comprises a prebiotic composition operable to enhance colonization of the host plant by the at least one heterologous endophytic bacterial strain.

57. 48. The method of claim 47, wherein the prebiotic composition comprises at least one of a microbial nutrient package, a plant biostimulant, an osmoprotectant, a buffering agent, and a seed lubricant.

58. 48. The method of claim 47, wherein the at least one heterologous endophyte strain comprises at least one of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

59. 48. The method of claim 47, wherein the at least one heterologous endophyte strain comprises a plurality of heterologous endophyte strains including at least two of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

60. 60. The method of claim 59, wherein the plurality of heterologous endophyte strains comprises at least three of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

61. 60. The method of claim 59, wherein the plurality of heterologous endophyte strains comprises Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

62. 48. The method of claim 47, wherein the at least one heterologous endophyte strain reduces acetylene.

63. 48. The method of claim 47, wherein the inoculant composition increases biomass in the host plant treated with the composition.

64. 48. The method of claim 47, wherein the inoculant composition increases amino acid production in the host plant.

65. 48. The method of claim 47, wherein the at least one heterologous endophytic strain exhibits fixation of atmospheric nitrogen in the host plant treated with the composition when the host plant is planted in nitrogen-depleted soil or when the host plant is planted in nitrogen-rich soil.

66. 48. The method of claim 47, wherein the at least one heterologous endophyte strain produces an Fe siderophore.

67. 48. The method of claim 47, wherein said inoculant composition increases the acquisition of essential macro- and micronutrients from the soil and atmosphere without substantially disrupting the plant nutrient ion stoichiometry of said host plant.

68. 48. The method of claim 47, wherein the inoculant composition increases leaf chlorophyll in the host plant treated with the inoculant composition.

69. 48. The method of claim 47, wherein the inoculant composition increases tolerance to abiotic stress in the host plant treated with the inoculant composition.

70. 48. The method of claim 47, wherein the inoculant composition increases the resistance of the host plant to a plant disease.

71. 48. The method of claim 47, wherein the inoculant composition increases germination, emergence and seedling biomass weight in the host plant treated with the inoculant composition.

72. 48. The method of claim 47, wherein the inoculant composition increases total nitrogen accumulation in plant shoots of the host plant.

73. 48. The method of claim 47, wherein the inoculant composition increases germination, emergence and seedling biomass weight in the host plant treated with the inoculant composition.

74. The inoculant composition may be applied to the root biomass, root branches, root hairs and / or root nuclei of the host plant.

48. The method of claim 47, wherein the method increases any desired ultrastructural changes.

75. 48. The method of claim 47, wherein the at least one heterologous endophyte strain is resistant to a biocide.

76. 76. The method of claim 75, wherein the biocides include fungicides, insecticides, nematicides and herbicides.

77. 48. The method of claim 47, wherein the at least one heterologous endophytic strain is resistant to plant hormones, plant elicitors, mineral micronutrients and mineral macronutrients.

78. 48. The method of claim 47, further comprising mixing the composition with an organic or synthetic nutrient or chemical prebiotic for the purpose of a combined seed treatment, combined foliar application, or combined in-furrow application.

79. 48. The method of claim 47, wherein the inoculant composition further comprises one or more Rhizobium species.

80. 48. The method of claim 47, wherein the inoculant composition further comprises one or more Mycorrhizae species.

81. 48. The method of claim 47, wherein the inoculant composition further comprises endophytic yeast strain WP1.

82. 48. The method of claim 47, wherein the inoculant composition further comprises one or more of a fungicide, an insecticide, a herbicide, a biostimulant, a plant growth regulator, a prebiotic, an adjuvant, and a fertilizer applied to the host plant as separate applications or as a mixture.

83. 48. The method of claim 47, wherein the inoculant composition further comprises a carrier composition that enables the plant inoculant composition to be applied to a seed.

84. 48. The method of claim 47, wherein the inoculant composition further comprises a carrier composition that enables application of the plant inoculant composition to leaf portions of the host plant.

85. 48. The method of claim 47, wherein the inoculant composition further comprises a carrier composition that allows the plant inoculant composition to be applied to soil adjacent to the host plant.

86. 48. The method of claim 47, wherein the at least one heterologous endophyte strain is encapsulated in microbeads that are incorporated into the inoculant composition.

87. 85. The method of claim 84, wherein the inoculant composition is a suspension composition.

88. 85. The method of claim 84, wherein the inoculant composition is a dry composition.

89. 85. The method of claim 84, wherein the inoculant composition comprises a dry granular composition.

90. 48. The method of claim 47, wherein the inoculant composition comprises a dry granular composition.

91. 48. The method of claim 47, wherein the inoculant composition comprises a liquid carrier and is applicable as a foliar spray.

92. 48. The method of claim 47, wherein applying the inoculant composition to the tissue or seed of the host plant comprises short-term or long-term inoculation of a dormant seed embryo.

93. 48. The method of claim 47, wherein the at least one heterologous endophytic bacterial strain is selected for a plant-microbe booster or prebiotic and is used in combination with a plant-microbe booster or prebiotic comprising one or more of an osmoprotectant, an amino acid, an antioxidant, a mineral nutrient, a plant hormone or plant growth elicitor, a carbohydrate, and a mucilaginous gel applied to the host plant as separate applications or as a mixture.

94. 48. The method of claim 47, wherein the at least one heterologous endophyte strain has the genetic machinery to produce Fe siderophores.

95. 1. A method for enhancing nitrogen fixation in a host plant, comprising applying a seed inoculant treatment composition to seeds of the host plant, the inoculant composition comprising at least one heterologous endophytic bacterial strain, at least one nutrient additive operable to enhance survival and colonization of the at least one heterologous endophytic bacterial strain in the host plant, and a carrier composition that enables the plant inoculant composition to be applied to seeds.

96. 96. The method of claim 95, wherein the inoculant composition comprises a seed adhesive comprising one or more of alginate, gum, starch, maltodextrin, lecithin, formononetin, polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinyl acetate, cephalin, gum arabic, xanthan gum, carrageenan, PGA, other biopolymers, mineral oil, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), arabinogalactan, methylcellulose, PEG 400, chitosan, polyacrylamide, polyacrylate, polyacrylonitrile, glycerol, triethylene glycol, vinyl acetate, gellan gum, polystyrene, polyvinyl, carboxymethylcellulose, hemicellulose, gum ghatti, polyoxyethylene-polyoxybutylene block copolymer, and other suitable agents.

97. 96. The method of claim 95, wherein the inoculant composition comprises sodium alginate, calcium alginate and / or magnesium alginate.

98. 96. The method of claim 95, wherein the inoculant composition comprises one or more of a plant biostimulant, a systemic protectant, a buffering agent, and a seed lubricant.

99. 96. The method of claim 95, wherein the inoculant composition comprises one or more of mefenoxam, fludioxonil, azoxystrobin, sedaxan, thiabendazole thiram, metalaxyl, hymexazole, penthiopyrad, clothianidin, beta-cyfluthrin, thiamethoxam, tebuconazole, prothioconazole, imidacloprid, clothianidin, acibenzolar-S-methyl, difenoconazole, cyantraniliprole, spinosad, oxathiapiprolin, pydiflumetofen, cyromazine, picarbutrazox, mandipropamid, mancozeb, thiodicarb, flupyradifurone, penflufen, prothioconazole, trifloxystrobin, beta-cyfluthrin, triadimenol, pyraclostrobin, boscalid, abamectin, captan, and fluopyram.

100. 96. The method of claim 95, wherein applying the inoculant composition comprises applying by coating, spraying, and / or dipping.

101. 96. The method of claim 95, wherein the seeds are surface sterilized before applying the inoculant composition to the seeds.

102. 96. The method of claim 95, wherein at least 500,000 CFU, at least 1,000,000 CFU, at least 2,000,000 CFU, at least 3,000,000 CFU, at least 4,000,000 CFU, at least 5,000,000 CFU, at least 6,000,000 CFU, at least 7,000,000 CFU, at least 8,000,000 CFU or at least 9,000,000 CFU of the at least one heterologous endophyte is applied to the surface of the seed.

103. 96. The method of claim 95, further comprising drying the seeds treated with the inoculant composition before sowing and germinating.

104. 96. The method of claim 95, further comprising storing the seeds treated with the inoculant composition for a predetermined period of time before sowing and allowing them to germinate.

105. 96. The method of claim 95, wherein the at least one heterologous endophyte strain survives on the surface of the seed.

106. 96. The method of claim 95, further comprising confirming survival of the at least one heterologous endophyte strain on the surface of the seeds, comprising placing the seeds treated with the inoculant composition in a resuspension solution and plating them on a nutrient medium.

107. 106. The method of claim 105, wherein at least 1,000 CFU, at least 2,000 CFU, at least 3,000 CFU, at least 4,000 CFU, at least 5,000 CFU, at least 6,000 CFU, at least 7,000 CFU, at least 8,000 CFU, at least 9,000 CFU or at least 10,000 CFU of viable cells of the at least one heterologous endophyte are present on the surface of seeds treated with the inoculant composition.

108. 106. The method of claim 105, wherein the nutrient medium is a nitrogen-limited medium.

109. The at least one heterologous endophytic strain is grown in a nitrogen-limited growth medium supplemented with exogenous ammonium (NH 4 + 96. The method of claim 95, further comprising selecting for the ability to produce

110. 96. The method of claim 95, further comprising selecting the at least one heterologous endophyte strain for its ability to increase the solubilization of multiple forms of insoluble phosphorus in a liquid bacterial growth culture.

111. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase nitrogen uptake in the host plant.

112. 96. The method of claim 95, wherein the at least one heterologous endophyte bacterial strain comprises a plurality of endophytic bacterial strains selected to fix atmospheric nitrogen.

113. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase total plant carbon in the host plant colonized by the strain.

114. 96. The method of claim 95, wherein the at least one heterologous endophyte strain has the nitrogenase gene subunit Nif H.

115. 96. The method of claim 95, wherein the inoculant composition comprises a prebiotic composition operable to enhance colonization of the host plant by the at least one heterologous endophytic bacterial strain.

116. 96. The method of claim 95, wherein the prebiotic composition comprises at least one of a microbial nutrient package, a plant biostimulant, an osmotic protectant, a buffering agent, and a seed lubricant.

117. 96. The method of claim 95, wherein the at least one heterologous endophyte strain comprises at least one of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

118. 96. The method of claim 95, wherein the at least one heterologous endophyte strain comprises a plurality of heterologous endophyte strains including at least two of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

119. 119. The method of claim 118, wherein the plurality of heterologous endophyte strains comprises at least three of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

120. 119. The method of claim 118, wherein the plurality of heterologous endophyte strains comprises Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

121. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculum composition in an amount effective to increase biomass in the host plant treated with the composition.

122. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase amino acid production in the host plant.

123. 96. The method of claim 95, wherein the at least one heterologous endophyte strain exhibits fixation of atmospheric nitrogen in the host plant treated with the composition when the host plant is planted in nitrogen-depleted soil or when the host plant is planted in nitrogen-rich soil.

124. 96. The method of claim 95, wherein the at least one heterologous endophyte strain produces an Fe siderophore.

125. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase the acquisition of essential macro- and micronutrients from soil and atmosphere without substantially disrupting the plant nutrient ion stoichiometry of the host plant.

126. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase leaf chlorophyll in the host plant treated with the inoculant composition.

127. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase tolerance to abiotic stress in the host plant treated with the inoculant composition.

128. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase the resistance of the host plant to a plant disease.

129. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase germination, emergence, and seedling biomass weight in the host plant treated with the inoculant composition.

130. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase total nitrogen accumulation in plant shoots of the host plant.

131. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase germination, emergence, and seedling biomass weight in the host plant treated with the inoculant composition.

132. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase root biomass, root branching, root hairs and / or any root ultrastructural changes in the host plant.

133. 96. The method of claim 95, wherein the at least one heterologous endophyte strain is resistant to a biocide.

134. 134. The method of claim 133, wherein the biocides include fungicides, insecticides, nematicides and herbicides.

135. 96. The method of claim 95, wherein the at least one heterologous endophytic strain is resistant to plant hormones, plant elicitors, mineral micronutrients and mineral macronutrients.

136. 96. The method of claim 95, further comprising mixing the composition with an organic or synthetic nutrient or chemical prebiotic for the purpose of a combined seed treatment, combined foliar application, or combined in-furrow application.

137. 96. The method of claim 95, wherein the inoculant composition further comprises one or more Rhizobium species.

138. 96. The method of claim 95, wherein the inoculant composition further comprises one or more Mycorrhizae species.

139. 96. The method of claim 95, wherein the inoculant composition further comprises endophytic yeast strain WP1.

140. 96. The method of claim 95, wherein the inoculant composition further comprises one or more of a fungicide, an insecticide, a herbicide, a biostimulant, a plant growth regulator, a prebiotic, an adjuvant, and a fertilizer applied to the host plant as separate applications or as a mixture.

141. 96. The method of claim 95, wherein the inoculant composition further comprises a carrier composition that enables the plant inoculant composition to be applied to a seed.

142. 96. The method of claim 95, wherein the inoculant composition further comprises a carrier composition that allows the plant inoculant composition to be applied to a leaf portion of the host plant.

143. 96. The method of claim 95, wherein the at least one heterologous endophytic bacterial strain is selected for a plant-microbe booster or prebiotic and is used in combination with a plant-microbe booster or prebiotic comprising one or more of an osmoprotectant, an amino acid, an antioxidant, a mineral nutrient, a plant hormone or plant growth elicitor, a carbohydrate, and a mucilaginous gel applied to the host plant as separate applications or as a mixture.

144. 1. A method for enhancing nitrogen fixation in a host plant, comprising applying a foliar inoculant treatment composition to one or more leaf parts of the host plant, wherein the inoculant composition comprises at least one heterologous endophytic bacterial strain, at least one nutrient additive operable to enhance survival and colonization of the at least one heterologous endophytic bacterial strain in the host plant, and a carrier composition that enables the plant inoculant composition to be applied to the leaf parts.

145. 145. The method of claim 144, wherein the inoculant composition comprises one or more vegetable oils, such as soybean oil, neem oil, cottonseed oil, and other components, such as glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, and / or other suitable liquids.

146. 145. The method of claim 144, wherein the inoculant composition comprises a non-ionic or ionic surfactant or a combination thereof.

147. 145. The method of claim 144, wherein the inoculant composition comprises a nonionic, anionic, amphoteric or cationic dispersant and emulsifier.

148. 145. The method of claim 144, wherein the inoculant composition comprises one or more of a plant biostimulant, an osmoprotectant, and a buffering agent.

149. 145. The method of claim 144, wherein the inoculant composition comprises one or more of mefenoxam, fludioxonil, azoxystrobin, sedaxan, thiabendazole thiram, metalaxyl, hymexazole, penthiopyrad, clothianidin, beta-cyfluthrin, thiamethoxam, tebuconazole, prothioconazole, imidacloprid, clothianidin, acibenzolar-S-methyl, difenoconazole, cyantraniliprole, spinosad, oxathiapiprolin, pydiflumetofen, cyromazine, picarbutrazox, mandipropamid, mancozeb, thiodicarb, flupyradifurone, penflufen, prothioconazole, trifloxystrobin, beta-cyfluthrin, triadimenol, pyraclostrobin, boscalid, abamectin, captan, and fluopyram.

150. 145. The method of claim 144, wherein applying the inoculant composition comprises applying the inoculant composition to the portion of the host plant by spraying.

151. 146. The method of claim 145, wherein at least 100 CFU, at least 250 CFU, at least 400 CFU, at least 500 CFU, at least 750 CFU, at least 1,000 CFU, at least 1,100 CFU, at least 1,200 CFU, at least 1,500 CFU or at least 2,000 CFU of the at least one heterologous endophyte strain are present in the treated leaf portion of the host plant.

152. 145. The method of claim 144, wherein the at least one heterologous endophytic bacterial strain survives on the surface of the leaf portion of the host plant.

153. 145. The method of claim 144, further comprising confirming survival of the at least one heterologous endophyte strain on the surface of the seeds, comprising placing the seeds treated with the inoculant composition in a resuspension solution and plating them on a nutrient medium.

154. 154. The method of claim 153, wherein the nutrient medium is a nitrogen-limited medium.

155. The at least one heterologous endophytic strain is grown in a nitrogen-limited growth medium supplemented with exogenous ammonium (NH 4 + 145. The method of claim 144, further comprising selecting for the ability to produce

156. 145. The method of claim 144, further comprising selecting the at least one heterologous endophyte strain for its ability to increase the solubilization of multiple forms of insoluble phosphorus in a liquid bacterial growth culture.

157. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase nitrogen uptake in the host plant.

158. 146. The method of claim 145, wherein said at least one heterologous endophyte strain comprises a plurality of endophyte bacterial strains selected to fix atmospheric nitrogen.

159. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase total plant carbon in the host plant colonized by the strain.

160. 145. The method of claim 144, wherein said at least one heterologous endophyte strain has the nitrogenase gene subunit Nif H.

161. 145. The method of claim 144, wherein the inoculant composition comprises a prebiotic composition operable to enhance colonization of the host plant by the at least one heterologous endophytic bacterial strain.

162. 145. The method of claim 144, wherein the at least one heterologous endophyte strain comprises at least one of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

163. The method of claim 144, wherein the at least one heterologous endophyte strain comprises a plurality of heterologous endophyte strains including at least two of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

164. 164. The method of claim 163, wherein the plurality of heterologous endophyte strains comprises at least three of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

165. 164. The method of claim 163, wherein the plurality of heterologous endophyte strains comprises Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

166. 166. The method of any one of claims 163 to 165, wherein at least two of the plurality of heterologous endophytic bacterial strains are co-fermented prior to application to the leaf parts of the host plant.

167. 145. The method of claim 144, wherein the at least one heterologous endophyte strain comprises a plurality of co-fermented heterologous endophyte strains.

168. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculum composition in an amount effective to increase biomass in the host plant treated with the composition.

169. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase amino acid production in the host plant.

170. 145. The method of claim 144, wherein the at least one heterologous endophyte strain exhibits fixation of atmospheric nitrogen in the host plant treated with the composition when the host plant is planted in nitrogen-depleted soil or when the host plant is planted in nitrogen-rich soil.

171. 145. The method of claim 144, wherein the at least one heterologous endophyte strain produces an Fe siderophore.

172. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase the acquisition of essential macro- and micronutrients from soil and atmosphere without substantially disrupting the plant nutrient ion stoichiometry of the host plant.

173. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase leaf chlorophyll in the host plant treated with the inoculant composition.

174. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase tolerance to abiotic stress in the host plant treated with the inoculant composition.

175. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase the resistance of the host plant to a plant disease.

176. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase germination, emergence, and seedling biomass weight in the host plant treated with the inoculant composition.

177. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase total nitrogen accumulation in plant shoots of the host plant.

178. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase root biomass, root branching, root hairs and / or any ultrastructural changes in roots in the host plant.

179. 145. The method of claim 144, wherein the at least one heterologous endophyte strain is resistant to a biocide.

180. 180. The method of claim 179, wherein the biocides include fungicides, insecticides, nematicides and herbicides.

181. 145. The method of claim 144, wherein the at least one heterologous endophytic strain is resistant to plant hormones, plant elicitors, mineral micronutrients and mineral macronutrients.

182. 145. The method of claim 144, further comprising mixing the composition with an organic or synthetic nutrient or chemical prebiotic for the purpose of a combined seed treatment, combined foliar application, or combined in-furrow application.

183. 145. The method of claim 144, wherein the inoculant composition further comprises one or more Rhizobium species.

184. 145. The method of claim 144, wherein the inoculant composition further comprises one or more Mycorrhizae species.

185. 145. The method of claim 144, wherein the inoculant composition further comprises endophytic yeast strain WP1.

186. 145. The method of claim 144, wherein said inoculant composition further comprises one or more of a fungicide, an insecticide, a herbicide, a biostimulant, a plant growth regulator, a prebiotic, an adjuvant, and a fertilizer applied to said host plant as separate applications or as a mixture.

187. 145. The method of claim 144, wherein the inoculant composition further comprises a carrier composition that allows the plant inoculant composition to be applied to leaf tissue of the host plant.

188. 145. The method of claim 144, wherein the inoculant composition further comprises a carrier composition that enables application of the plant inoculant composition to leaf portions of the host plant.

189. 145. The method of claim 144, wherein the at least one heterologous endophytic bacterial strain is selected for a plant-microbe booster or prebiotic and is used in combination with a plant-microbe booster or prebiotic comprising one or more of an osmoprotectant, an amino acid, an antioxidant, a mineral nutrient, a plant hormone or plant growth elicitor, a carbohydrate, and a mucilaginous gel applied to the host plant as separate applications or as a mixture.

190. 1. A method for enhancing nitrogen fixation in a host plant, comprising applying an inoculant treatment composition to soil adjacent to the host plant, wherein the inoculant composition comprises at least one heterologous endophytic bacterial strain, at least one nutrient additive operable to enhance survival and colonization of the at least one heterologous endophytic bacterial strain in the host plant, and a carrier composition that enables the plant inoculant composition to be applied to the soil.

191. 191. The method of claim 190, wherein the inoculant composition comprises a seed adhesive comprising one or more of alginate, gum, starch, maltodextrin, lecithin, formononetin, polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinyl acetate, cephalin, gum arabic, xanthan gum, carrageenan, PGA, other biopolymers, mineral oil, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), arabinogalactan, methylcellulose, PEG 400, chitosan, polyacrylamide, polyacrylate, polyacrylonitrile, glycerol, triethylene glycol, vinyl acetate, gellan gum, polystyrene, polyvinyl, carboxymethylcellulose, hemicellulose, gum ghatti, polyoxyethylene-polyoxybutylene block copolymers, and other suitable agents.

192. 191. The method of claim 190, wherein the inoculant composition comprises sodium alginate, calcium alginate, and / or magnesium alginate.

193. 191. The method of claim 190, wherein the inoculant composition comprises one or more of a plant biostimulant, an osmoprotectant, and a buffering agent.

194. 191. The method of claim 190, wherein the inoculant composition comprises one or more of mefenoxam, fludioxonil, azoxystrobin, sedaxan, thiabendazole thiram, metalaxyl, hymexazole, penthiopyrad, clothianidin, beta-cyfluthrin, thiamethoxam, tebuconazole, prothioconazole, imidacloprid, clothianidin, acibenzolar-S-methyl, difenoconazole, cyantraniliprole, spinosad, oxathiapiprolin, pydiflumetofen, cyromazine, picarbutrazox, mandipropamid, mancozeb, thiodicarb, flupyradifurone, penflufen, prothioconazole, trifloxystrobin, beta-cyfluthrin, triadimenol, pyraclostrobin, boscalid, abamectin, captan, and fluopyram.

195. The at least one heterologous endophytic strain is grown in a nitrogen-limited growth medium supplemented with exogenous ammonium (NH 4 + 191. The method of claim 190, further comprising selecting for the ability to produce

196. 191. The method of claim 190, further comprising selecting the at least one heterologous endophyte strain for its ability to increase the solubilization of multiple forms of insoluble phosphorus in a liquid bacterial growth culture.

197. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase nitrogen uptake in the host plant.

198. 191. The method of claim 190, wherein said at least one heterologous endophyte strain comprises a plurality of endophyte bacterial strains selected to fix atmospheric nitrogen.

199. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase total plant carbon in the host plant colonized by the strain.

200. 191. The method of claim 190, wherein the at least one heterologous endophyte strain has the nitrogenase gene subunit Nif H.

201. 191. The method of claim 190, wherein the inoculant composition comprises a prebiotic composition operable to enhance colonization of the host plant by the at least one heterologous endophytic bacterial strain.

202. 191. The method of claim 190, wherein the prebiotic composition comprises at least one of a microbial nutrient package, a plant biostimulant, an osmoprotectant, and a buffering agent.

203. 191. The method of claim 190, wherein the at least one heterologous endophyte strain comprises at least one of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

204. 191. The method of claim 190, wherein the at least one heterologous endophyte strain comprises a plurality of heterologous endophyte strains including at least two of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

205. 205. The method of claim 204, wherein the plurality of heterologous endophyte strains comprises at least three of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

206. 205. The method of claim 204, wherein the plurality of heterologous endophyte strains comprises Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

207. 207. The method of any one of claims 204-206, wherein at least two of the plurality of heterologous endophytic bacterial strains are co-fermented prior to application to the soil.

208. 191. The method of claim 190, wherein the at least one heterologous endophyte strain comprises a plurality of co-fermented heterologous endophyte strains.

209. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculum composition in an amount effective to increase biomass in the host plant treated with the composition.

210. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase amino acid production in the host plant.

211. 191. The method of claim 190, wherein the at least one heterologous endophyte strain exhibits fixation of atmospheric nitrogen in the host plant treated with the composition when the host plant is planted in nitrogen-depleted soil or when the host plant is planted in nitrogen-rich soil.

212. 191. The method of claim 190, wherein the at least one heterologous endophyte strain produces Fe siderophore.

213. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase the acquisition of essential macro- and micronutrients from soil and atmosphere without substantially disrupting the plant nutrient ion stoichiometry of the host plant.

214. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase leaf chlorophyll in the host plant treated with the inoculant composition.

215. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase tolerance to abiotic stress in the host plant treated with the inoculant composition.

216. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase the resistance of the host plant to a plant disease.

217. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase germination, emergence, and seedling biomass weight in the host plant treated with the inoculant composition.

218. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase total nitrogen accumulation in plant shoots of the host plant.

219. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is present in the inoculant composition in an amount effective to increase root biomass, root branching, root hairs and / or any root ultrastructural changes in the host plant.

220. 191. The method of claim 190, wherein the at least one heterologous endophyte strain is resistant to a biocide.

221. 221. The method of claim 220, wherein the biocides include fungicides, insecticides, nematicides and herbicides.

222. 191. The method of claim 190, wherein the at least one heterologous endophytic strain is resistant to plant hormones, plant elicitors, mineral micronutrients and mineral macronutrients.

223. 191. The method of claim 190, further comprising mixing the composition with an organic or synthetic nutrient or chemical prebiotic.

224. 191. The method of claim 190, wherein the inoculant composition further comprises one or more Rhizobium species.

225. 191. The method of claim 190, wherein the inoculant composition further comprises one or more Mycorrhizae species.

226. 191. The method of claim 190, wherein the inoculant composition further comprises endophytic yeast strain WP1.

227. 191. The method of claim 190, wherein the inoculant composition further comprises one or more of a fungicide, an insecticide, a herbicide, a biostimulant, a plant growth regulator, a prebiotic, an adjuvant, and a fertilizer applied to the soil as separate applications or as a mixture.

228. 191. The method of claim 190, wherein the inoculant composition further comprises a carrier composition that enables the plant inoculant composition to be applied to the soil.

229. 191. The method of claim 190, wherein the inoculant composition is applied to the soil furrow adjacent to the plant.

230. 191. The method of claim 190, wherein the inoculant composition is applied to the soil adjacent to the plants prior to emergence.

231. The at least one heterologous endophytic bacterial strain is a plant-microbe booster or pre- 191. The method of claim 190, wherein the biotic is selected for and used in combination with a plant-microbe booster or prebiotic comprising one or more of an osmoprotectant, an amino acid, an antioxidant, a mineral nutrient, a plant hormone or plant growth elicitor, a carbohydrate, and a mucilaginous gel applied to the soil as a separate application or as a mixture.

232. A plant treated with an inoculant composition comprising at least one heterologous endophytic bacterial strain selected for its ability to fix atmospheric nitrogen, wherein said at least one heterologous endophytic bacterial strain is incorporated into the tissue of the plant.

233. 233. The plant of claim 232, wherein said at least one heterologous endophytic bacterial strain comprises a plurality of endophytic bacterial strains selected for the ability to fix atmospheric nitrogen.

234. 233. The plant of claim 232, wherein said at least one heterologous endophytic strain has increased expression of the nitrogenase gene subunit Nif H.

235. 233. The plant of claim 232, wherein the at least one heterologous endophyte strain comprises at least one of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

236. 233. The plant of claim 232, wherein the at least one heterologous endophyte strain is a plurality of heterologous endophyte strains including at least two of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

237. 237. The plant of claim 236, wherein the plurality of heterologous endophytic strains includes at least three of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

238. 237. The plant of claim 236, wherein the plurality of heterologous endophytic strains include Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

239. 233. The plant of claim 232, wherein the at least one heterologous endophyte strain reduces acetylene.

240. 233. The plant of claim 232, wherein colonization of the plant with the at least one heterologous endophytic bacterial strain results in an increase in biomass in the plant.

241. 233. The plant of claim 232, wherein colonization of the plant with the at least one heterologous endophytic bacterial strain results in increased amino acid production in the plant.

242. 233. The plant of claim 232, wherein the at least one heterologous endophytic strain exhibits atmospheric nitrogen fixation in the host plant when the host plant is planted in nitrogen-depleted soil or when the host plant is planted in nitrogen-rich soil.

243. The method of claim 1 , wherein the at least one heterologous endophytic strain increases the enzymatic activity of glutamine synthetase (GS) in the plant.

244. 48. The method of claim 47, wherein the at least one heterologous endophytic strain increases the enzymatic activity of glutamine synthetase (GS) in the plant.

245. 96. The method of claim 95, wherein the at least one heterologous endophytic strain increases the enzyme activity of glutamine synthetase (GS) in the plant.

246. 145. The method of claim 144, wherein the at least one heterologous endophytic strain increases the enzyme activity of glutamine synthetase (GS) in the plant.

247. 2. The method of claim 1, wherein at least two of the plurality of heterologous endophytic bacterial strains are co-fermented prior to application to the foliage of the host plant, and wherein the application of the at least two co-fermented heterologous endophytic bacterial strains increases one of the following characteristics compared to treatment with only one of the heterologous endophytic bacterial strains: nitrogen uptake, total leaf chlorophyll, glutamine synthetase (GS) enzyme activity in plants, total biomass, total plant carbon, amino acid production, resistance to abiotic stress of the host plant treated with the inoculant composition, resistance to plant diseases, total nitrogen accumulation in plant shoots, increased root biomass, root branching, root hairs, germination, emergence, and seedling biomass weight.

248. 48. The method of claim 47, wherein at least two of the plurality of heterologous endophytic bacterial strains are co-fermented prior to application to the foliage of the host plant, and wherein the application of the at least two co-fermented heterologous endophytic bacterial strains increases one of the following characteristics compared to treatment with only one of the heterologous endophytic bacterial strains: nitrogen uptake, total leaf chlorophyll, glutamine synthetase (GS) enzyme activity in plants, total biomass, total plant carbon, amino acid production, resistance to abiotic stress of the host plant treated with the inoculant composition, resistance to plant diseases, total nitrogen accumulation in plant shoots, increased root biomass, root branching, root hairs, germination, emergence, and seedling biomass weight.

249. 96. The method of claim 95, wherein at least two of the plurality of heterologous endophytic bacterial strains are co-fermented prior to application to the foliage of the host plant, and wherein the application of the at least two co-fermented heterologous endophytic bacterial strains increases one of the following characteristics compared to treatment with only one of the heterologous endophytic bacterial strains: nitrogen uptake, total leaf chlorophyll, glutamine synthetase (GS) enzyme activity in plants, total biomass, total plant carbon, amino acid production, resistance to abiotic stress of the host plant treated with the inoculant composition, resistance to plant diseases, total nitrogen accumulation in plant shoots, increased root biomass, root branching, root hairs, germination, emergence, and seedling biomass weight.

250. 145. The method of claim 144, wherein at least two of the plurality of heterologous endophytic bacterial strains are co-fermented prior to application to the foliage of the host plant, and wherein the application of the at least two co-fermented heterologous endophytic bacterial strains increases one of the following characteristics compared to treatment with only one of the heterologous endophytic bacterial strains: nitrogen uptake, total leaf chlorophyll, glutamine synthetase (GS) enzyme activity in plants, total biomass, total plant carbon, amino acid production, resistance to abiotic stress of the host plant treated with the inoculant composition, resistance to plant diseases, total nitrogen accumulation in plant shoots, increased root biomass, root branching, root hairs, germination, emergence, and seedling biomass weight.

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