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

Abstract

Disclosed are endophyte inoculant compositions, methods of making such compositions, methods of 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] 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 that, when incorporated into the plant, result in measurable plant benefits such as nitrogen and other macro- and micronutrient uptake, nutrient use efficiency, photosynthesis, growth, yield, carbon sequestration, resistance to biotic and biotic stress factors, disease resistance (excluding biological control mechanisms) and general plant health. The technology has a wide range of applications to plants in general, including crop management applications, reduced fertilizer and pesticide use, reduced carbon footprint of crops, improved mineral nutritional status of crops to reduce plant pathogen load and associated pesticide use, improved food quality and safety, improved plant health and biomass growth rates for use in landscaping and ornamentals, and 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. Endophytes can be incorporated into plant tissues and become a genetic part of the plant. Endophytes can penetrate between and inside plant cells and become incorporated into plant tissues. Once incorporated into plants and living on the surface of plant roots, endophytes can improve the nutritional status of plants 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 resistance 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 enhance 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 of 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 a non-native host plant:

[0005] [Table 1]

[0006] The species identified in the above table 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 above endophytic strains are listed in Figures 1-4. These strains were found in the branches of willow and poplar, plants that do not form root nodules. Thus, the presence of potential diazotrophic endophytes living inside all the branches 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 root-to-shoot uptake of N and P. These strains were further found to provide additional root-to-shoot uptake capacity of mineral nutrients when inoculated, and these strains are present in a wide variety of crop plants that were specifically tested and screened. These unique strains were then identified and isolated specifically for this purpose. Furthermore, by optimizing with the most efficient nitrogen fixing, ammonium excreting strains, and specific strains that mobilize insoluble forms of macronutrients (e.g., phosphorus), the resulting novel inoculant formulation was found to increase the plant's acquisition of macronutrient ions and micronutrient ions required by the plant. Furthermore, application of the novel microbial inoculants disclosed herein to non-native plants results in increased growth, biomass and yield in crop plants. The novel inoculant formulations improve crop plant yield and quality under both poor, well-rounded and highly optimized agronomic conditions. These endophytic strains and combinations thereof are not typically present in crop plant varieties and soils used for agricultural production.

[0007] Of the above endophytic strains, two strains have been demonstrated to be newly discovered endophytic species: Curtobacterium salicaceae (WW7) and Rhizobium populi (PTD1). Examples 1B-1C show experimental results indicating that the WW7 and PTD1 endophytic strains are novel species.

[0008] Whole genome sequence and safety analysis The genomes of all four of these 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, the method of Varghese et al. was used. See https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4538840 / . This method reliably classifies strains as safe or potential pathogens by comparing the genome sequence with known harmful and pathogenic microorganisms for similarity in the genome. If the organism falls into a cluster representative of human or plant pathogens, the presence of virulence factors and antibiotic resistance genes is evaluated. No close affiliation to such pathogen species was found for any of these strains. To further confirm that these strains are safe for agricultural crops, the genomes of the endophytic strains were analyzed and investigated for genes known to be associated with plant and human pathogens. No pathogen-associated 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 medium for 24 hours are likely to be gram negative. MacConkey agar also uses a neutral pH indicator to test for the ability of bacteria to ferment lactose sugar. Fermentation of lactose sugar by bacteria lowers the pH, causing the colony to turn pink and the surrounding medium to become cloudy. Bacteria that cannot utilize lactose use peptone to produce ammonia, which raises the pH and causes the colony to turn white or colorless. Some microorganisms (e.g., Klebsiella and Enterobacter) form mucoid colonies that appear very moist and sticky. This phenomenon occurs because the bacteria produce a capsule made primarily from the lactose in the agar medium.

[0010] A hemolysis assay (blood agar lysis) was also performed on endophytic strains for 24 hours to test the pathogenic bacteria's ability to produce toxins that lyse red blood cells. The outcome 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] The endophytic strains produced yellow-brown colonies that did not have a wet or sticky appearance when grown on MacConkey agar. Thus, the endophytic strains were negative for lactose fermentation, lactone fermentation, and mucoid capsule production. The results of the MacConkey agar test indicate that the endophytic strains do not share the following characteristics with pathogenic species: (1) they do not share lactose fermentation with pathogenic species such as Escherichia coli, Enterobacter, or Klebsiella, and (2) they do not share peptone utilization with pathogenic species such as Salmonella, Proteus species, Yersinia, Pseudomonas aeruginosa, or Shigella, and they do not 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 (grow but did not lyse hemocytes). Taken together, these results confirm that the endophytic strains share no similarities to 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 inoculant formulation. The 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 powdered 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 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 may be treated heterologously with the endophyte strains of the present technology. The basic principle of plant breeding, including transgenic plant breeding, begins with the creation of "clean" germplasm, which typically means that there are no microorganisms present. For this reason, most propagation material of annual crops is microbiologically empty. For perennial plants, modern propagation techniques 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 endophyte-free plants.

[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, 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), sugar cane (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). The 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 such 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 development 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, a non-naturally occurring application may be the presence of a non-native endophyte in a tissue of a host plant, or in a tissue of a different plant element, tissue, cell type, or physical location of the plant other than the physical location where it naturally occurs. 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 located, is 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. The detectable inclusion of an endophyte strain in a host plant can result in improved growth characteristics, stress resistance 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 crop plants, 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 combinations of heterologous endophytic strains provide unique inoculant benefits to plant hosts that improve plant growth, health, yield and quality, reduce plant resistance to biotic and abiotic stresses, and prevent infection through plant induced resistance to plant / seed diseases and pests. The ability of endophytic strains to colonize non-native plant hosts has been experimentally demonstrated by various 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 suitable methods. The presence of genetic material of the endophytic strain in the host plant, increased CFUs and chlorophyll leaf content in host plant tissues, increased chlorophyll content in roots, and increased chlorophyll content in roots. Other physiological measurements such as enhanced branching and growth, increased shoot biomass, and increased mineral nutrient ion content in leaves all demonstrate successful colonization of host plants by endophytic strains. Furthermore, biochemical and molecular analyses have revealed the mode of action by which endophytic strains improve host plant performance, yield, mineral nutrition, and health. 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 a spectrophotometric plate reader, 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 uptake of nitrogen and other macro- and micronutrients, nutrient use efficiency, photosynthesis, growth, yield, carbon sequestration, tolerance to biotic and abiotic stressors, and general health of the plants.

[0018] Host plants containing one or more endophyte strains in their tissues show detectable changes in the content of at least one nutritional trait, and this improvement may be passed on via asexual reproduction (e.g., stem, root or leaf cuttings, layering, division, segregation, grafting, budding, micropropagation) or seeds. Progeny resulting from an endophyte-associated host plant or tissues thereof may have one or more endophyte strains in their tissues and may have increased at least one nutritional quality trait compared to untreated plants of the same species. Progeny of cultivars produced from rootstocks (root stocks), cuttings or tissue culture may show such phenotypic traits and improved performance due to the presence of a heterologous endophyte strain in their tissues. The level of the nutritional trait may 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 tissues 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 an untreated control plant of the same species grown under similar conditions.

[0019] The endophytic strains WW5, WW6, WW7 and PTD1 selected for use in treating host plants were developed as stocks through a microbial fermentation process. The microbial stock maintenance and fermentation methods may be used to increase the expression of the nitrogenase gene and maintain the plasmid in an active form. The endophytic strains are nitrogen-limited and have other special properties (e.g., chelated iron and / or magnesium) to enhance atmospheric nitrogen fixation, macro- and micronutrient solubilization and acquisition, and other beneficial characteristics of the endophytic strains. The endophytic strains may be grown in bacterial growth media with a nitrogen-restricted or nitrogen-free growth medium, which induces the endophytic strains to absorb and assimilate atmospheric N2. For an example of nitrogen-free, 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.

[0020] The fermentation broth utilized to ferment the endophytic strain may contain various components that allow for the growth and health of the endophytic strain during the fermentation process. The nitrogen-limited medium utilized 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., MgSO4), chloride salts (e.g., CaCl2), and other suitable salts, but excluding 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 utilized as a liquid composition for treating the host plant. For examples of nitrogen-limited media, 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. The nitrogen-limited medium may be substantially free of nitrogen to promote the upregulation of microbial nitrogenase genes in the endophytic strain. However, after fermentation in NLM broth, the resulting novel composition contained 30-100 mg NH4 + / The fermentation composition may include one or more of the following nutrients: nitrogen components in limited amounts, such as within the physiological range of L, common amino acids, such as glutamic acid, glutamine, histidine, nitrates, nitrites, carbamates, and other nutrients. Figure 6 shows examples of other components that may be included in the fermentation composition.

[0021] 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 in the apoplast between plant cells of vascular bundles in roots, stems, stalks and branches. Endophytes in stems and trunks that exude these compounds also help prevent phosphorus from binding with other metals. This and other strains aid in the solubilization and conversion of potassium (K) to soluble forms, as well as the solubilization and acquisition of P. Mobility of P and K is influenced by the inoculated bacterial endophyte through acidification, chelation and ion exchange reactions.

[0022] Some endophytic strains (WW7, WW5, WW6) also produced exogenous extracellular iron siderophore compounds, which plants further excreted through the roots to release insoluble micronutrient mineral ions or metals (e.g., iron, magnesium, zinc, copper, nickel, manganese), calcium Ca 2 + to mobilize other divalent macronutrient cations.

[0023] Combinations of endophyte strains, including co-fermenting combinations of two or more endophyte strains disclosed herein, may be applied to a host plant 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 that is also colonized by 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 in certain nutritional traits, growth traits, stress resistance, and overall health of the host plant beyond the expected trait improvement, indicating a synergistic effect of application of multiple endophyte strains to a host plant. Examples 41-51 provide data showing synergistic effects in heterologous application of multiple endophyte strains. The combinations of endophyte strains disclosed herein do not exhibit incompatibility in host plants that may occur with endophyte strains other than those disclosed herein.

[0024] The inoculant composition may include one or more additional components 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 biological 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 became established within the host plant tissues.

[0025] 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 / air-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 allow practical use and application to the 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 endophytes or microbial species, such as additional Rhizobium strains, Mycorrhizae species, Bacillus species, Azotobacter species, Azospirillum species, Sphingobium species, Herbiconjux species, biocontrol bacterial species (e.g., Erwinia, Rhanella, Paraburkholderia, Curtobacteria, etc.), 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 for the promotion of plant vigor, health, growth, yield, and abiotic and biotic stress tolerance.

[0026] The composition may include one or more components that facilitate the delivery, preservation and / or efficacy of the applied endophytic strain, and may include surfactants, buffers, carriers, adhesives, microbial stabilizers, mineral or clay granules, nutrients, excipients, wetting agents 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 endophytic strain.

[0027] The composition may be formulated as storage stable, including liquid, suspension and solid formulations. Storage stable formulations include suspension, dry, powder and formulations containing dried endophyte strains. The composition may be storage stable for at least 3 weeks or more under predetermined conditions. For example, the composition may be stable for 10 weeks or more at various temperatures, including low temperatures (near freezing), sustained high temperatures, or room temperature under standard temperature and pressure (STP) conditions.

[0028] The formulation may comprise one or more dried endophyte strains, in which the moisture content of the endophyte strains is 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, the formulation comprises a dried endophyte strain and 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. 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 a hard, approximately dehydrated round or oval bead. The beads range in size from about 400 nm to about 5 mm in average diameter and may additionally or alternatively include thickeners, starches, carbohydrates, or mineral thickeners, stabilizers and / or carriers.

[0029] In some embodiments, the inoculant composition may contain stabilizers that are compatible with the endophyte strain and promote the viability of the strain as well as the application to and colonization of the heterologous endophyte strain in the host plant. 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. HPO 4), hydroxypropyl guar (HP-Guar), xantham gum, polyvinylpyrrolidone, polyvinylpyrrolidone / vinyl acetate (PVP-VA), non-reducing sugars or sugar alcohols such as mannitol or sorbitol, or other suitable materials. The amount of stabilizer in the composition can be in the range of about 5 wt% to about 50 wt% (e.g., between about 10 wt% to about 40 wt%, between about 15 wt% to about 35 wt%, between about 20 wt% to about 30 wt%, or any value or range of values ​​therein).

[0030] In some embodiments, the composition may include a carrier, such as an agriculturally acceptable carrier, which may be any material that can be added to plant components without causing or having an adverse effect 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 materials. The carrier may be any one or more of several carriers that impart various properties, such as improved stability, wettability, flowability, and / or dispersibility.

[0031] In some embodiments, the agricultural carrier is a solid, such as diatomaceous earth, loam, silica, magnesium silicate, alginates (e.g., sodium, calcium or magnesium salts of alginic acid), 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 (major 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, rice bran, etc. may also be used as solid carriers. Mixtures of any of the above components are also contemplated as carriers, such as, but not limited to, pasta (wheat flour and kaolin clay) or flour-based pellets in loam, sand or clay. 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 in 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, in-furrow applications.

[0032] 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 compositions such as vegetable oils, such as soybean oil, neem oil, cottonseed oil, and glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, and 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, a water-in-oil emulsion 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, oils derived from plants, humectants, or combinations thereof. The composition may 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.

[0033] In some embodiments, the surfactant that can be included in the composition can include nonionic surfactant and / or anionic surfactant.Examples of nonionic surfactant include alkylphenol alkoxylate, alcohol alkoxylate, polyoxyethylene glycerol fatty acid ester, castor oil alkoxylate, fatty acid alkoxylate, fatty amide alkoxylate, fatty polydiethanolamide, lanolin ethoxylate, fatty acid polyglycol ester, isotridecyl alcohol, fatty amide, methylcellulose / hemicellulose, fatty acid ester, alkyl polyglycoside, glycerol fatty acid ester, polyethylene glycol, polypropylene glycol, polyethylene glycol / polypropylene glycol block copolymer, polyethylene glycol alkyl ether, polypropylene glycol alkyl ether, polyethylene glycol / polypropylene glycol ether block copolymer, polyethylene oxide / polypropylene oxide block copolymer and mixtures thereof. Examples of anionic surfactants include alkylarylsulfonates, phenylsulfonates, alkyl sulfates, arylalkylsulfonates, alkylether sulfates, alkylarylether sulfates, alkyl-polyglycoletherphosphates, polyarylphenyletherphosphates, alkylsulfosuccinates, 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 with urea, and also with phenolsulfonic acid, with formaldehyde and urea, lignosulfite waste liquors and lignosulfonates, alkyl phosphates, alkylaryl phosphates (e.g. tristyryl phosphates), and also polycarboxylates (e.g. polyacrylic acid, maleic anhydride / olefin copolymers) (including alkali metals, alkaline earth metals and mixtures thereof).

[0034] 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., those used for seed coating) and help maintain the heterologous endophyte and other materials in contact with the plant or plant element. In some embodiments, the adhesive agent may include one or more of alginates, gums, starches, maltodextrins, lecithin, formononetin, polyvinyl alcohol, alkaline formononetinate, hesperetin, polyvinyl acetate, cephalin, gum arabic, xanthan gum, carrageenan, PGA, other biopolymers, mineral oil, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), arabino-galactan, methylcellulose, PEG400, chitosan, polyacrylamide, polyacrylates, polyacrylonitrile, glycerol, triethylene glycol, vinyl acetate, gellan gum, polystyrene, polyvinyl, carboxymethylcellulose, hemicellulose, gum ghatti, polyoxyethylene-polyoxybutylene block copolymers, and other suitable agents.

[0035] In some embodiments, one or more adjuvants may be used in the inoculant composition to help improve the delivery and performance of the endophyte strain. The composition may be combined with an adjuvant to create a specific product form or mixture, such as a liquid mixture for foliar application. The composition may further include other agriculturally suitable excipients, such as solvents, pH adjusters, viscosity 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, antimicrobial agents, etc. The compositional content of these auxiliary excipients is not particularly limited and may be determined by one skilled in the art according to conventional protocols.

[0036] In some embodiments, suitable pH adjusting agents that may 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).

[0037] 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.

[0038] 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, xantham gum, etc.), powder carriers (soy protein, talc, lime, starch biochar, cellulose / hemicellulose, silica, clay, nanotechnology structures containing mineral nutrients (e.g., carbon dots, buckyboil, etc.), and the like. Exemplary biostimulants may include combinations of amino acids (e.g., one or more of L-glutamine, L-lysine, L-methionine, L-arginine, 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 nutrient products to test the viability of the endophyte under such 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.

[0039] The endophyte strains described herein can be combined with one or more of the agents described above to obtain compositions suitable for application to plants or their tissues, seedlings, seeds or other plant elements. Endophyte populations can be obtained from selection and propagation in culture as described herein and added to the composition. Endophytes in different growth stages can be used. For example, lag phase, early log phase, mid log phase, late log phase or stationary phase endophytes can be used.

[0040] The aforementioned components may be combined in a liquid composition comprising an effective amount of one or more of endophyte strains WW5, WW6, WW7, and PTD1. For example, the inoculant compositions described herein may comprise an 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, between about 15% and 50% by weight, based on the wet weight of the composition. The inoculant composition may comprise 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 about 10 5 CFU / mL, at least about 10 6 CFU / mL, at least about 10 7 CFU / mL, at least about 10 8 CFU / mL, at least about 10 9 CFU / mL, at least about 10 10 CFU / mL or any value or range within said range.

[0041] An exemplary liquid formulation according to the present invention comprises two or more dried or wet 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%, CaCl2 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 concentrations 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 wet endophyte strains in an amount of about 10 wt% to about 100 wt%. 4 ~10 10 The compositions may contain a range of concentrations of each strain in CFU / mL, either alone or in combination with the following: 0.5-50 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%.

[0042] In some embodiments, the composition may be a suspension formulation comprising the above components in the above proportions. In such embodiments, the composition may further comprise one or more solid carriers, thickeners or extenders. Such components 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 and 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 comprise humectants, emulsifiers, anti-caking agents, suspending agents, freezing point depressants, etc. In some embodiments, the suspension formulation may comprise one or more of the endophyte strains WW5, WW6, WW7 and PTD1 at the concentrations disclosed in the previous paragraph. An exemplary suspension formulation according to the present invention comprises approximately 10% glycerol 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 include one or more dried endophyte strains at a concentration of each endophyte strain in CFU / mL. The formulation may further include 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.

[0043] In some embodiments, the composition may be a solid composition. The solid composition may be a dry composition, a granular composition, or a flowable composition intended to be dispersed or suspended in an aqueous solution before being fed to the plant. The dry fertilizer composition may form a fully dispersed suspension. In other contexts, the dry fertilizer composition may provide sustained release (e.g., by low water solubility or encapsulation, e.g., by sodium alginate), for example, when a constant or controlled delivery of nutrients over time is desired. The solid composition may be a fertilizer composition having a concentration of about 103 CFU / mL ~ at least about 10 10 The solid composition may contain endophyte strains WW5, WW6, WW7, and PTD1 in an amount in the range of CFU / mL or any value or range of values ​​therein. For example, the solid composition may contain one or more of endophyte strains WW5, WW6, WW7, and PTD1 in an amount in the range of about 10 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., an amount ranging from about 40 wt% to about 55 wt%, an amount ranging from 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 wt% to about 60 wt% (e.g., an amount ranging from about 40 wt% to about 55 wt%, an amount ranging from about 45 wt% to about 99.9 wt%, or any value or range of values ​​therein). 4 CFU / mL ~ approx. 10 10 The formulations may contain a concentration of each endophyte strain in 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 fluid 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%.

[0044] Applicable The compositions described herein, including one or more endophytic strains, may be applied to plants to enhance growth characteristics, health, stress resistance, and improve other characteristics of the plants. The compositions described herein may advantageously be mechanically or manually applied to the plant or its elements, or artificially inoculated, by any one of a number of means, including, but not limited to, seed treatment, root washing, seedling dipping, soil inoculation, furrow application, foliar spray, foliar coating, side-row application, wound inoculation, irrigation, fertilizer irrigation, soaking, injection, osmotic priming, hydroponics, aquaponics, aeroponics, or any combination thereof. In some embodiments, the compositions may also be applied directly to the plant 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 the seeds (e.g., as a coating or by treating the seeds by spraying or soaking) and / or pre-emergence (before the seedlings emerge or appear above ground). 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 on the soil and / or roots of the host plant, or in any other suitable manner. The composition may also be applied to the growing medium (e.g., by applying to the soil surrounding the plant).

[0045] 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 resistance, biotic stress resistance, disease resistance, wilt recovery, turgor pressure, or any combination thereof, compared to untreated control plants of the same species grown under similar conditions. Rootstocks, root stocks, cuttings, or tissue cultures of the host plants may be used to produce cultivars that exhibit such phenotypic traits and enhanced performance. Application of the inoculant composition may also increase carbon fixation in the treated host plants. This is an economically attractive benefit, as it results in the removal of carbon dioxide from the atmosphere and an increase in biomass. Indicators of increased carbon acquisition by plants include increased CO2 fixation activity, increased dry weight to fresh weight ratio, and overall biomass.

[0046] 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 rates of nitrogen uptake. Increased rates of nitrogen uptake 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 more protein production and promoting increased biomass production. Application of the compositions described herein also increases uptake and utilization of other macro- and micronutrients. Experimental results demonstrate increased uptake of the macronutrients potassium, phosphorus, calcium, and magnesium, as well as increased uptake of 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 take up 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.

[0047] Increased macronutrient and micronutrient uptake is accompanied by increased catabolism, carbon uptake and carbon sequestration. Host plants treated with the compositions of the present invention show greater total carbon uptake, with associated increases in RuBisCo carboxylation activity, carbon content and biomass. Host plants can also show increased production of aromatic amino acids via the shikimate pathway. These aromatic amino acids function as precursors for a wide range of secondary metabolites that are important for plant resistance to biotic and abiotic stresses (e.g., oxidative stress, drought stress and / or salt stress).

[0048] Thus, application of the composition may result in enhanced adaptive resistance of the host plant to abiotic and biotic stresses such as disease, cold, salinity, etc. 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 kill plants. In agricultural production of crops, low growth temperatures are often encountered, especially during the early growing season, which can stress plants in a variety of ways, starting with poor germination, followed by stunted seedling growth, yellowing of leaves, reduced leaf expansion, wilting, and tissue death. Cold stress severely inhibits the development of reproductive parts of plants. Crop yields are reduced in response to cold stress in proportion to the extent of damage to the plant. High salinity in soil or water is becoming an increasing problem, as salts accumulate in irrigated soils and irrigation water with high salinity must be used. The effects of high salinity in soil and water are called osmotic stress because high salinity impedes the transport of ions and water within the plant. 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 the host plant protects the host plant under stress-induced environments, and the host plant exhibits greater growth and biomass even under abiotic stress conditions.

[0049] Application of the composition to a host plant enhances adaptive resistance through advanced mineral nutrition and provides biological stress resistance that inhibits viruses, bacteria, and fungi. The endophytic strains described herein provide the host plant 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 may be applied to the host plant or its seeds as a nutritional treatment to help protect against pathogenic fungi, viruses, and bacteria.

[0050] Other aspects, objects and advantages of the presently described technology will become apparent from the following detailed description. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 shows SEQ ID NO:1. [Diagram 2] FIG. 2 shows SEQ ID NO:2. [Diagram 3] FIG. 3 shows SEQ ID NO:3. [Figure 4] FIG. 4 shows sequence number 4. [Diagram 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 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 relating to the experiments of Example 1B. [Figure 8B] FIG. 8B is a table relating to the experiments of Example 1B. [Figure 8C] FIG. 8C is a table relating to the experiments of Example 1B. [Figure 9] FIG. 9 is a table relating to the experiments of Example 1C. [Figure 10A]FIG. 10A is a table relating to the experiments of Example 2B. [Figure 10B] FIG. 10B is a table relating to the experiments of Example 2B. [Figure 11] FIG. 11 is a table relating to the experiments of Example 2C. [Figure 12] FIG. 12 is a table relating to the experiments of Example 2D. [Figure 13A] FIG. 13A provides the enzymatic pathways relevant to the experiments in Example 3. [Figure 13B] FIG. 13B is a table relating 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 relevant to 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 of 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 experiments in Example 6. [Figure 17A] FIG. 17A provides images related to the experiment of Example 7. [Figure 17B] FIG. 17B provides images related to the experiment of 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 of Example 9. [Figure 20] FIG. 20 is a table relating to the experiments of Example 10. [Figure 21] FIG. 21 is a table relating to the experiments of Example 11. [Figure 22] FIG. 22 is a table relating to the experiments of 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 of 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 of 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 of 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 of Example 20. [Figure 30B] FIG. 30B is a table relating to the experiments of 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. [Diagram 32] FIG. 32 is a table relating to the experiments of 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 of Example 24. [Diagram 35] FIG. 35 provides images related to the experiment of 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 of Example 26. [Figure 36D] FIG. 36D is a graph related to the experiment of Example 26. [Figure 36E] FIG. 36E is a graph related to the experiment of 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 of Example 30. [Figure 40B] FIG. 40B provides images related to the experiment of Example 30. [Diagram 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 of Example 33. [Figure 43B] FIG. 43B is a graph related to the experiment of Example 33. [Diagram 44] FIG. 44 is a graph related to the experiment of Example 34. [Figure 45A] FIG. 45A is a graph related to the experiment of Example 35. [Figure 45B] FIG. 45B provides images related to the experiment of Example 35. [Figure 46] FIG. 46 is a table relating to the experiments of 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 of Example 43. [Figure 53B] FIG. 53B is a graph related to the experiment of 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.

[0052] 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. Although the present invention will be described with reference to these embodiments, it will be understood that they are not intended to limit the present invention. On the contrary, the present invention is intended to cover alternatives, modifications, and equivalents that are included within the spirit and scope of the present invention as defined by the appended claims. In the following disclosure, specific details are given to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without these specific details. EXAMPLES

[0053] method Selection and propagation methods and compositions The present invention includes methods for the propagation and selection of nitrogen-fixing endophyte strains. These methods include inoculating specialized nitrogen-limited and nitrogen-free 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.

[0054] Example 1A Selection and propagation of endophytes Each endophyte strain, WW5, WW6, WW7 and PTD1, was tested and found to be positive for the ability to grow on nitrogen-limited medium (NLM), with each strain growing to various extents on nitrogen-limited medium, as shown in the images below: Each endophyte strain was tested and found to be positive for the ability to grow on plant tissue culture grade agarose plates with nitrogen-limited medium (NLM, pH 7.6), with each strain growing to various extents on plant tissue culture grade agarose plates with nitrogen-free medium (NFCCM, pH 7.6), as shown in the images below:

[0055] Fermentation mixtures containing 10 mL broth cultures were prepared in 50 ml conical tubes. Each culture was inoculated with 100 μL of standardized QC broth culture of the endophyte strain. The conical tubes were placed on a shaker at a 45° angle and incubated for 72 hours at room temperature with shaking at 200 rpm. 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 inoculated onto NLM agar plates. The plates were then incubated at 25° C. for 72 hours. The CFUs formed on the plates were observed and recorded. Figure 7A shows visual evidence of colony growth on the NLM plates.

[0056] 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 showed that each endophytic strain was able to produce ammonium under such conditions. The assays demonstrated the ability of the endophytic strains to participate in the N2 fixation mechanism in the plant and correlated with the growth effect observed after inoculation by treatment with the endophytic fermentate.

[0057] Each strain was tested for exogenous ammonium (NH4) 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), as shown in Figure 7B. + All sterile media tested were negative for ammonium concentrations below 0 mg / L.

[0058] Ammonium NH4 + In addition to their ability to produce ammonia, 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 for 72 hours at room temperature with shaking at 125 rpm.

[0059] 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 endophyte strains were able to produce ammonia under these conditions. Ammonia production data for each strain is shown in Figure 7C.

[0060] The ability of endophytic strains to fix nitrogen in tree or plant tissues is made possible 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.

[0061] The ability of the endophyte strains to fix nitrogen was further measured by acetylene reduction assay, which 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 ability, as the functional presence of nitrogenase enzyme correlates with ethylene production. WW6, WW7 and PTD1 endophyte strains exhibited acetylene reduction activity, as shown in Figure 7D.

[0062] The selected WW5, WW6, WW7 and PTD1 cells were grown in nitrogen-limited medium for 1–3 days to grow endophyte strains at approximately 10 7 CFU / mL to about 10 10 They were grown individually until they were present in the medium at concentrations ranging from 0.1 to 1.5 CFU / mL. In some runs, two or more endophyte strains were combined and co-fermented to produce approximately 10 3 CFU / mL to about 10 9 CFU / mL range, e.g., at least about 10 4 CFU / mL, at least about 10 5 CFU / mL, at least about 10 6 CFU / mL, at least about 10 7 CFU / mL, at least about 10 8 CFU / mL, at least about 10 9 The fermentation process conditions can include a predetermined incubation temperature in the range of 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 of the fermenter at a speed in the range of 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).

[0063] To drive upregulation of microbial nitrogenase genes in endophytic strains, the nitrogen-limited medium can be substantially free of nitrogen, but can contain one or more sugars, such as mannitol, mannose, sucrose, glucose, fructose, lactose, and other suitable sugars. The nitrogen-limited medium can also contain one or more salts, such as sodium chloride, phosphates (e.g., monopotassium phosphate, dipotassium phosphate, and other phosphates), sulfates (e.g., MgSO4), chlorides (e.g., CaCl2), and other suitable salts, but excluding 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 utilized to treat host plants as a liquid composition. For examples of nitrogen-limited media, see, for example, 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.

[0064] 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 and citric acids that can solubilize insoluble forms of phosphate, as well as Fe-siderophores that solubilize insoluble forms of iron. Curtobacterium salicaceae was isolated from the vasculature of the trunks of willow (Sitka sitchenses) trees.

[0065] WW7 was sequenced by the US Department of Energy (DOE) Joint Genome Institute (JGI) using an 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 one flow cell. The library was barcoded, mixed with 11 samples, and sequenced using a 2 × 250-bp format. MiSeq was performed using MiSeq Reagent Kit v3 (600 cycles) chemistry. Shotgun sequencing yielded 1,530,321 reads. After trimming, quality filtering, and removal of potential contamination using the BBMap package, 1,436,665 read pairs were used as input to the SPAdes v3.13.0 genome assembler. The final assembly was generated using a multi-k-mer approach (k = 77, 95, and 127).

[0066] The genome of Curtobacterium salicaceae strain (WW7) consists of 18 scaffolds (N 50 The genome is represented by a sequence division multiplex (Seq. 1.0, n = 329,216 bp), has a length of 3,489,963 bp, and a G+C content of 71.35%, which corresponds to a coverage of approximately 84×. Completion 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, 3286 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 (COG), 1,082 were annotated with enzyme identifiers (EC), and 1,722 were assigned to KEGG Orthology (KO).

[0067] To classify WW7 phylogenetically down to the species level, two different strategies were used: (i) average nucleotide identity (ANI) analysis using the ANIm algorithm (see 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 intra-species probabilities (Printra-species) using the genome-wide average nucleotide identity (gANI) strategy described in (see NCBI GenBank 2010, 19126-19131). In this regard, the WW7 genome was compared to all published 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 the 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, which corresponds to 90% ANI.WW7 was identified as a novel Curtobacterium species, including Curtobacterium herbarum DSM 14013 (ASM1690733v1), as well as 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. WW7 was closely related (ANI > 90%) to MCPF17_001 (NCBI assembly ID: ASM323461v1), Curtobacterium sp. MCLR17_032 (NCBI assembly ID: ASM323479v1), and Curtobacterium sp. MCBD17_030 (NCBI assembly ID: ASM322425v1). See Figure 8A. Additionally, WW7 was 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。

[0068] To further evaluate the affiliation of WW7 to the Curtobacterium type strains, pairwise digital DNA-DNA hybridization values ​​(dDDH) were calculated for WW7 to determine intraspecific relatedness with representative strains (type strains) of the genus Curtobacterium. The pairwise dDDH values ​​between WW7 and the Curtobacterium type strains were lower than 70%, as shown in FIG. 8B, indicating that 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.

[0069] Phylogenetic distances were also calculated using 605 linked single-copy protein-coding genes assigned to clusters conserved in 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 type 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 type strains according to the List of Prokaryotic names with Standing in Nomenclature (LPSN) database. Clavibacter michiganensis was used as an outgroup.

[0070] Example 1C Genome analysis of Rhizobium populi (PTD1) To assess the affiliation of strain PTD1 to the Rhizobium type strains, pairwise digital DNA-DNA hybridization values ​​(dDDH) were calculated for strain PTD1 to determine its intraspecific relatedness to the closest representatives of the Rhizobium genus (type strains). The pairwise dDDH values ​​between strain PTD1 and the Rhizobium type strains were lower than 70%, as shown in Figure 9, indicating that strain PTD1 is a representative of a novel Rhizobium species.

[0071] Example 2A Two-strain product fermentation mix under nitrogen-limited medium conditions Cultivation of the two endophyte strains WW6 and WW7: Each strain was first inoculated into Nitrogen Limited Medium (NLM) semi-solid medium. Three to four colonies can be selected and used to inoculate a 2-liter seed train culture containing fresh sterile NLM medium. The 2-liter flasks can be grown for 72 hours at about 25°C to about 30°C under constant agitation at about 200 rpm and about 500 rpm. Upon completion, the two strains can be pooled in a single carboy and stored at 4°C before inoculating 4500 liters of NLM medium (pH 7.6). The industrial scale fermentation medium can be sterilized in a 30,000 liter industrial steam jacketed fermenter. The co-fermentation can be carried out at an aeration rate of 20 PSI for three days. The resulting colony forming units of the two strains can be 2.2 x 10 when inoculated onto Tryptic Soy Broth Agar (TSBA). 8 CFU / mL to 1.23 × 10 9 CFU / mL WW6, and 1.03 x 10 8 CFU / mL to 1.4 × 10 9 CFU / mL WW7. This procedure can be used for other combinations of endophyte strains, for example, any combination of WW5, WW6, WW7 and PTD1.

[0072] Example 2B Endophyte compositions made by mixing with two commonly used dry fertilizers and dry powders for combined use in agriculture Two novel compositions were made, containing WW6 and WW7 endophyte strains, sodium alginate (DuPont Nutrition USA, In), and dry fertilizers triple superphosphate 0-28-0 (OCP group) and another one dolomitic lime (Down To Earth, Inc). 3mL of the mixture containing WW6+WW7+0.5% alginate was added separately to 5 grams of dry fertilizer. The compositions were dried 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.

[0073] The results shown in Figure 10A indicated that the viability of both endophyte strains was slightly reduced when mixed with different fertilizers in the new composition. These results demonstrate that the liquid endophyte composition can be used to create a new composition for use in commercial agricultural practice as a NUE endophyte-enhanced agricultural fertilizer.

[0074] Additionally, a liquid fermentation composition containing 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+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.

[0075] The results are shown in Figure 10B 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.

[0076] Example 2C The survival of endophytic strains (WW6 and WW7) was assayed together with Mycorrhizae powder after freeze-drying and 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 mixtures tested were as follows: powder carrier 2.09g (approximately 95% by weight), lyophilized powder WW6+WW7 0.11g (approximately 5% by weight), Mycorrhizae 0.0022g (approximately 0.1% by weight). The results of the compatibility test demonstrated the following results, which are shown in Figure 11.

[0077] Compatibility testing of freeze-dried WW6+WW7 mixed with Mycorrhizae with four potential bulking agents (maltodextrin, sucrose, dextrose, and whey) 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 reduce CFU very much. Sucrose was the best powder carrier, not reducing CFU of either strain in the assay.

[0078] The results of this example demonstrate that the freeze-dried endophyte powder can be mixed with a variety of carriers that allow the freeze-dried inoculum 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.

[0079] 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 1 / 10X molasses solution. Powdered biochar material was dried in open baggies at room temperature and stored at 25°C. After 2 weeks, dried biochar (0.1 g) was resuspended in 1 mL potassium phosphate buffer and assayed for endophyte survival by strains with results expressed as CFU / ml. Data is shown in Figure 12.

[0080] The results demonstrated that 50 μL of co-fermented endophyte mix with 950 μL of 1 / 10x molasses applied per gram of biochar was an effective ratio for the viability and stability of four out of five tested endophyte strains on the biochar powder carrier for at least two weeks.

[0081] 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 planta, and enhance P uptake for other soil particles or internal metal ions. Heterologous use of 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 points to possible genetic mechanisms for the biosynthesis of Krebs cycle intermediates, such as the organic oxyacids malate and citrate, that may be responsible for solubilizing insoluble forms of phosphate from soil allowing better plant uptake. Furthermore, endophytes may have exudates that keep phosphate ligands free once inside the plant by competing with other metals that tightly bind phosphate and make it insoluble again and unavailable for assimilation. WW7 genome data was used for identification of 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 with KofamKOALA genome jp tools (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 that catalyze the synthesis of malate and citrate, which can be exuded from roots and solubilize insoluble P. A total of 1602 proteins in WW7 were mapped against 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 has 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 FIG. 13A). Glutamate is the first amino acid product of the GS-GOGAT pathway, which produces one mole of glutamate from one mole of NH3, respectively. The GOGAT pathway is responsible for atmospheric nitrogen fixation in bacteria. FIG. 13A shows the WW7 enzyme 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.

[0082] Phosphate solubilization genes According to the KEGG annotation, genes involved in the solubilization of inorganic and organic phosphates in other species were also detected in WW7. See FIG. 13B. The results show the presence of acid phosphatase (AcPase) genes, as well as genes involved in the synthesis of acetate and glucuronic acid. AcPase has been shown in other species to be involved in the solubilization of phosphate from phosphomonoesters, and acetate and glucuronic acid have been shown in other species to be involved in the solubilization of phosphate from inorganic forms.

[0083] The ability of endophytic strains to solubilize insoluble phosphorus (P) from soil or soil solution through exogenous production of various mobilizing compounds is important for helping plants acquire needed P, other nutrients such as potassium K, and trace nutrient ions more efficiently 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 aid in uptake and translocation from the plant roots to the shoot.

[0084] To prove the ability of WW7 to solubilize insoluble forms of phosphate by secretion of exogenous compounds, an insoluble phosphate solubilization assay was performed in liquid medium using phosphate-sensitive dyes and a microplate reader method 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 modified National Botanical Research Institute (NBRIP) broth without phosphate to deplete residual internal phosphate. Five milliliters of 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). Supernatants from each sample were added at different dilutions to a 96-well plate. Additionally, phosphate standards provided in 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.

[0085] 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. The WW7 strain 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.

[0086] 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 the treated host plant, which could lead to enhanced biomass, stress tolerance and other favorable characteristics.

[0087] 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 by the production of various solubilizing compounds and / or heme-associated binding factors. Endophytic strains can also enhance the host plant to acquire the required Fe and improve its ability to acquire other metal and micronutrient ions, especially positively charged divalent cations, from the soil. These Fe-siderophore chelating compounds produced by some bacterial and yeast endophytes can help the plant to better compete with the high cation exchange capacity of soil clay particles and aid in the uptake and translocation of metal ions from the roots to the shoots of the plant. To confirm the capabilities of the WW7 strain, a genome-wide analysis of the WW7 genes was performed. WW7 was shown to have the genetic equipment required to make Fe-siderophores that can transport or scavenge Fe ions. InterProscan results show the presence of a gene cluster efeUOB involved in the salvage 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, an enzymatic reaction that liberates the bound ferrous iron.

[0088] 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 ferric siderophores.

[0089] Assay for Fe-siderophore production in all four endophyte strains Endophytic strains WW5, WW6, WW7 and PTD1 for Fe-siderophore production ability. Fe-siderophore production was demonstrated in three of the endophytic bacterial strains tested. The four strains were assayed using microbial growth solid agar plate-based Fe-siderophore CAS medium. Agar plates were prepared using Chromeazurol as a color indicator and a CAS agar plate preparation containing FeCl3. Endophytic strains were applied to individual plates.

[0090] Fe solubilization was evidenced by the presence of decolorized areas developed on the CAS test plates that appeared as a clear white color around the streaks of bacterial growth on the plate. 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 strains to solubilize Fe was photographed, measured, and compared. See Figure 14B.

[0091] The in vitro results in Figures 14B-14D demonstrate that WW7 produces extracellular compounds that scavenge insoluble Fe. WW7 showed the greatest Fe solubilization ability of all strains. Both WW5 and WW6 also showed significant Fe solubilization. However, the PTD1 Rhizobium populi strain showed little or no Fe solubilization 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 the production of Fe siderophores is greatest in WW7, followed by WW5 and WW6. Endophyte strains producing these compounds can assist the host plant in solubilizing and mobilizing Fe. The Fe solubilization activity of each strain is quantified in Figure 14C and shown graphically in Figure 14D.

[0092] 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 highly specific and unique FAME ID chemical identification chromatograms for each endophyte strain. This allows these microbial isolates to be individually tracked and each one unambiguously identified based on their unique fatty acid methyl ester signature. The unique chromatograms for each strain are shown in Figure 15.

[0093] How to use The formulations disclosed herein can be advantageously applied to plants by several means, including, without limitation, spraying, irrigating, covering, dipping, injecting, in-furrow, or any combination thereof. The compositions according to the invention can be used on leaves, roots, foliars, foliage, tillers, flowers, plant cells, plant tissues, seeds (e.g., as a cover or by seed treatment such as by spraying or dipping), pre-emergence (before the seedling emerges or emerges above ground), grains, fruits, 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 surfaces 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 into 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.

[0094] The compositions according to the present invention are effective for improving the metabolism of the host plant (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 lead to increased yields of harvestable crops and more robust plants. Exemplary methods are described below.

[0095] Example 6 PCR analysis of endophyte colonization of host plants following root dip inoculation The ability of the endophyte strains to heterologously colonize host crop plants was tested using PCR techniques. Results conclusively demonstrated that the endophyte was present within surface-sterilized plant tissues. In planta PCR clearly demonstrated successful colonization in agriculturally important wheat, rice and barley species inoculated with the WW6 (Pseudomonas siliginis) endophyte strain by seed treatment.

[0096] 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, 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 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 in trays as needed with a 25 ppm concentration of nitrogen reduced Hoagland's solution 2-3 times a week to maintain moist-slightly dry soil. 14 days after transplanting, plants were individually harvested. Plants were then removed from the soil and processed. DNA was isolated from treated samples using the PureLink™ Microbiome DNA Purification Kit DNA Isolation Kit (Thermo Fisher Scientific).

[0097] 20 microliter PCR reactions were set up using 2X HotStart PCR Master Mix (MCLAB), 1 uL of template DNA and 1 uM of PCR primers specific for the WW6 gene. PCR cycle 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, and finally at 72°C for 5 minutes. 10 μL of each PCR reaction was loaded onto a 1.2% agarose gel and subjected to DNA QS710 electrophoresis (IBISCI) at 120V. Figure 16 shows that WW6 is present in plants of all three test host plants (wheat, rice and barley) inoculated pre-emergence and is absent in the controls of 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 evident that the WW6 endophyte strain is able to effectively colonize several host plants.

[0098] Example 7 PCR analysis of endophyte colonization of host plants after seed coating The ability of the endophyte strains to heterologously colonize host crop plants when used with seed coatings was tested using PCR methods. Shoots and roots treated with WW6 (Pseudomonas siliginis) of winter wheat (Triticum aestivum) and broccoli (Brassica oleracea) plants were evaluated for colonization and uptake of the WW6 endophyte strain into plant tissues. Seeds were treated with WW6 as follows before growing plants: 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.

[0099] 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 in trays as needed with a 25 ppm concentration of Nitrogen Reduced Hoagland's Solution 2-3 times a week to maintain moist-slightly dry soil. 14 days after transplanting, plants were then removed from the soil and processed. Root and shoot tissues were collected from the plants. DNA was isolated from the treated samples using the PureLink™ Microbiome DNA Purification Kit DNA Isolation Kit (Thermo Fisher Scientific).

[0100] PCR was performed as described in Example 5 above. Figures 17A and 17B show electrophoretic gel data of 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 the control. Figure 17A shows electrophoretic gel data demonstrating that WW6 is present in the shoot and root tissues of winter wheat host plants grown from treated seeds by the presence of genome-specific PCR primers designed for the protein that yield DNA bands that are specifically present in the WW6 strain and not present in the shoot and root tissues of the control. Thus, it is clear that the WW6 endophyte strain can effectively colonize the root and shoot tissues of wheat host plants after seed inoculation.

[0101] Figure 17B shows electrophoretic PCR gel data demonstrating that WW6 is present in the root tissue of seedlings inoculated with WW6 seed treatment after surface sterilization of the root tissue. Several treatment groups were prepared using the WW6 endophyte strain: a first treatment of WW6 liquid fermentation, a second treatment in which broccoli seeds were treated with 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 WW6 liquid fermentation mixed with 1% sodium alginate and seed coating material. Different groups of broccoli seeds were coated with the three treatments. Plant growth, DNA isolation and PCR were performed as described in Example 5 above.

[0102] Figure 17B shows electrophoretic gel data demonstrating that WW6 is present in the shoot and root tissues of winter wheat, rice, soybean, broccoli and corn host plants grown from treated seeds by the presence of genome-specific PCR primers designed for proteins that yield unique DNA bands present in the WW6 strain and absent in the control shoot and root tissues, thus demonstrating that the WW6 endophyte strain can effectively colonize the root and shoot tissues of several host plants following seed inoculation.

[0103] 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) method. Shoots and roots of barley (Hordeum vulgare) plants were evaluated for colonization of WW6 and WW7 after the seeds were coated with the seed treatment composition. WW6 and WW7 fermentates were blended with 0.5% by weight sodium alginate (Scogin™ LDH) and the composition was used to coat untreated barley seeds, which were air-dried at room temperature and stored for one month. The same seed coating composition without the endophyte strains was used for the control seeds.

[0104] An appropriate number of seeds were placed in an 11 cm x 11 cm seed germination box with sterile filter paper. Seeds were germinated for 5 days in deionized water 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 2-3 times a week in trays as needed to maintain moist soil. 21 days after transplanting, plants were then removed from the soil and roots and shoots were treated by surface sterilization with 2% bleach followed by rinsing with sterile water. Shoots and roots were separated and then flash frozen in liquid nitrogen and then stored at -20°C. Plant material was then ground to a fine powder in a mortar and pestle using liquid nitrogen. 100 mg of each tissue sample was used to isolate DNA using the DNeasy Plant Pro DNA isolation kit (QIAGEN, Inc).

[0105] A novel primer set was designed and multiplexed with our gene-specific PCR primers with the goal of allowing for simultaneous analysis and differential quantification of both strains. Strain-specific primers were created with fluorophores (FAM and Hex). 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 Bio-Rad Laboratories, Inc.

[0106] Results shown in Figure 17C show dot plot graphs of quantification of WW6 strain PCR target using FAM and Hex fluorescent signals for the WW6 strain assay. Figure 17D shows dot plot graphs of quantification of WW7 strain PCR target using FAM and Hex fluorescent signals. Experimental results were quantified using QuantaSoft software (Bio-Rad Laboratories, Inc.).

[0107] 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 FIG. 17E. The results demonstrated quantitative in planta detection of both strains, WW6 in roots and shoots, and WW7 in roots and shoots. The highest detection levels were quantified for WW6 in barley shoots relative to control uninoculated plants.

[0108] 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 demonstrated by the presence of genome-specific primers designed to hybridize to unique DNA bands present in the WW6 strain and absent from the control shoot and root tissues, thus demonstrating that the WW6 and WW7 endophyte strains can effectively colonize the root and shoot tissues of several crop plants following seed inoculation.

[0109] Example 8 Analysis of endophyte colonization of host plants following 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.

[0110] A foliar inoculant composition containing co-fermented WW6 and WW7 freeze-dried powders resuspended in water was applied to spinach plants together with 10-5-3 CaO liquid fertilizer formulation Greenstim™ (concentrated glycine betaine extracted from beet roots with 12% total nitrogen, from Masso, SA Agro Department) according to industrial rates. Spinach was grown hydroponically using a complete nutrition regimen with daily application of 550 L of 10-5-3 3% CaO fertilizer (715 kg of fertilizer, 18 kg / day) to each plot. The foliar composition was applied at the 4-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 containing six beds each. Sampling was performed in the center of the four middle beds of each block. Percentage canopy coverage was assessed 35 days after application using a software tool for analyzing and measuring canopy coverage in photographs (Canopeo™). At the same time, leaves were collected for nutrient analysis and for surface sterilization followed by in planta endophyte quantification. Percentage plant coverage results for plot bed data showed that endophyte foliar treatments resulted in a statistically significant (p<0.05) increase of 30.92% in spinach leaf canopy coverage compared to the industrial fertilizer alone and a statistically significant (p<0.05) increase of 58% over the control treatment. See Figure 18A.

[0111] To assay and quantify the presence of endophytes in planta in spinach tissues, leaves and roots were collected 35 days after foliar inoculation treatment, washed, and surface sterilized for detection of WW6 and WW7 strains inside plant tissue. All samples were weighed and photographed to perform CFU calculations for sample weight and leaf surface or root area. Leaf and root ends were sealed separately in plastic bags before surface sterilization in a laminar flow fume hood. Surfaces were washed with distilled water to remove dust, soil, and other contaminants. Samples were then placed in sterile flasks, 70% ETOH alcohol was added, and the flasks were shaken (150 rpm) for 2 min. After shaking, the alcohol solution was removed and 1% sodium hypochlorite bleach solution was added. The mixture was shaken for another 2 min. The sodium hypochlorite solution was then also removed, and the samples were cleaned three times by shaking manually in sterile water for 1 min.

[0112] Leaves and roots were ground in 50 ml of 0.9% saline. Extracts were placed in sterile tubes and left for 1 h to release endophytes from the ground tissue. Several serial dilutions of the extracts were made in sterile water. 100 μl of each dilution was plated on potato dextrose agar (PDA) plates with glycerol and on actinomycete isolation agar.

[0113] The agar plates were incubated until bacterial growth was visually confirmed. Bacterial concentrations were performed to determine CFU / g and CFU / cm of the assay material. 2 The result expressed as follows was obtained.

[0114] The data in Figure 18B demonstrated 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 not present in spinach plants that received the Greenstim liquid fertilizer treatment alone (see detailed discussion above).

[0115] WW6 and WW7 endophytes were detected in both leaves and roots of the host plants 35 days after treatment with the foliar inoculum composition containing WW6 and WW7 endophytes. In the standard treated control plants, endophytes were absent and no endophytes were detected. When expressing endophyte concentrations in CFU / g, higher endophyte concentrations were found in leaves than in roots, but the differences were small and the concentrations in leaves and roots can be considered to be the same in both sites. CFU / cm 2 Surface areas were visually analyzed and calculated from measurements using Adobe Photoshop software.

[0116] The above data demonstrate that the endophyte was 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 improved establishment, growth and soil coverage of the spinach host plants, resulting in increased production.

[0117] Example 9 Effect of single endophyte strain seed treatments on total nutrient accumulation Corn seeds (Zea mays) were treated with a seed inoculum composition containing one heterologous endophyte strain selected from WW5, WW6 and WW7 and compared to a control corn seed treated with an inoculum composition without 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 ziplock 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 7Cultures were prepared at 100°C (containing endophytes in CFU / ml) and then refrigerated. 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 seed dispersed into 1 mL droplets. After each 1 mL addition, the bag and seeds were manually inverted and carefully massaged. Once all 3.4 mL / lb had been added, the seeds were massaged, shaken, and inverted for 2-3 minutes to ensure all corn seeds were visibly wet in 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 mixture of washed play sand, vermiculite, and perlite potting mix in 1 gallon felt Smart Pots 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-h light / 10-h dark cycle under a photon flux rate of 100 Hz. Once germinated, plants were watered and fertilized in trays with Hoagland's solution amended with 50 ppm nitrogen reduction 2-3 times a week to maintain moist-slightly dry soil as needed. 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 for each sample.

[0118] As shown in FIG. 19, corn plants inoculated with endophyte strains WW5, WW6, or WW7 accumulated significantly higher levels of major and trace mineral nutrients across key mineral ion profiles, as measured by percent change in total nutrient content of shoot biomass relative to untreated control plants, all grown in Hoagland's Dropout N nutrient solution supplemented with 50 ppm bioavailable nitrogen.

[0119] The data in Figure 19 demonstrate that the heterologous endophyte was successful in improving 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 to enhance 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%.

[0120] 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 either individually or as a mix of all four strains. Alginate beads encapsulating the endophytes inside calcium alginate were placed next to the seeds, one bead per seed, and the pots were watered equally 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 nitrate <13 ppm, ammoniacal N <6 ppm and phosphate <11 ppm.

[0121] The results in Figure 20 demonstrate that the four selected endophytes responded positively to increased yields of a wide variety of crop plants under limited nitrogen and phosphorus bioavailability when using commercially available, agriculturally relevant and commonly used seeds.

[0122] Example 11 Effect 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 co-fermented WW6 and WW7 heterologous endophyte strains were grown and compared to control canola plant seeds treated with an inoculant composition without endophytes. Treated seeds were grown as follows: an appropriate amount of canola seeds was industrially treated with Integral pro (BASF) and prebiotic UBS 016 (Unium Bioscience Ltd.) (both according to the manufacturer's instructions) at a rate of 500 mL of 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 establishment of the host plant. The prebiotic includes a microbial nutrient package, plant biostimulants, osmoprotectants, buffers and seed lubricants. Endophyte survival was confirmed on the seeds 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 canola seeds received the same crop protection package. Seeds were then stored for one month under normal industrial seed storage conditions (4°C-15°C) and planted industrially in autumn in Cuxwold, Lincolnshire, United Kingdom using a seed drill in a large-scale replicated CRO field experiment.

[0123] Shoots were collected in mid-spring during the early growth period and sent for agronomic mineral nutrient analysis. Shoot biomass nutrient accumulation was calculated by multiplying total shoot weight by shoot ion concentration. As shown in Figure 21, canola seeds inoculated with 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 % change in total nutrient content of shoot biomass from the control.

[0124] 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 the co-fermenting heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0125] 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 plant seeds treated with an inoculant composition without endophytes. The inoculant composition was combined with a prebiotic composition UBS 016 from Unium Bioscience Ltd. to support endophyte survival, colony growth and establishment of the host plant. The 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 sedaxane, which protects against seed-borne diseases, was also added. Vibrance Duo® from Syngenta AG was used as the crop protection package, which contains 25 g / l sedaxane and 25 g / l fludioxonil. The Vibrance Duo® product was used at 2 L per metric ton.

[0126] Control seeds of the same cultivars received the same crop 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-15°C) and planted industrially in autumn at conventional rates using a seed drill in a large-scale replicated CRO field experiment at Cuxwold, Lincolnshire, United Kingdom.

[0127] Shoots were collected in late spring during the growing season, 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 macro- 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.

[0128] The above data demonstrate that the co-fermenting heterologous endophytes WW6 and WW7 successfully improved the uptake and incorporation of macro- and micronutrients in winter wheat plants grown from inoculated seeds. The results demonstrate the efficacy of the co-fermenting heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0129] Example 13 Effect of combined WW6 and WW7 strains on total nutrient accumulating shoot biomass Co-fermentation WW6 and WW7 fermentates were mixed for 10 min. 7 Spring oat seeds (Avena sativa var Elyann and SO1) treated with seed inoculant compositions engineered with CFU / ml heterologous endophyte strains were grown and compared to control seeds treated with inoculant compositions without endophytes. Treated seeds were grown as follows: an appropriate amount of oat seeds was industrially treated with the following ratio per metric ton of seeds: 500 mL mixed co-ferment in water, 500 mL 1% alginate and 1000 ml 10% prebiotic composition UBS 016 (from Unium Bioscience Ltd.) and seed disease protectant Redigo (Bayer) according to the manufacturer's instructions. Survival of the endophyte 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 variety received the same prebiotic treatment but without endophytes. The oat seeds were then stored for one month under normal industrial seed storage conditions and planted industrially in Suffolk, UK in May in a large-scale replicated CRO field trial with conventional fertiliser rates.

[0130] Shoots were collected in the early growth phase, in mid-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 23, spring oat plants inoculated with co-fermented WW6+WW7 accumulated significantly higher levels of macro- 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.

[0131] 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 the co-fermenting heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0132] Example 14 Effect of combined WW5+WW6+WW7+PTD1 strains 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 the seeds after the seeds had been coated with two fungicide / insecticide products, GA3 (gibberellic acid), a dye, and a pre-treatment with a flowable zinc micronutrient coating. 500 mL of the endophyte fermentate and 1% alginate were added to 2,205 pounds of rice seeds. Examination of the seed coating quality of 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 2.0×10 6 ;WW6 8.0×10 5 ;WW7 3.6×10 6 ;PTD1 1.2×10 6The control seeds were treated with the pretreatment but without the seed inoculant composition. The seeds were then stored for 2 months under normal industrial seed storage conditions and planted industrially in spring 2020 by a large-scale grower in Clay County Arkansas, USA. The paddy fields were fertilized with 400 lbs of urea per acre, which is equivalent to 184 lbs of N per acre. The shoots with 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 macro- and micronutrients, and these differences are expressed as % change in leaf nutrient concentration from the control.

[0133] Example 15 Effect of (WW5, WW6, WW7 and PTD1) used as seed treatments on yield under reduced and normal rates of nitrogen fertiliser in the field. Broccoli seeds were first industrially treated with a mixture of endophyte ferment and 1% alginate, applied at different rates using two different crop protection packages and industry representative methods. The seed rates of endophyte used were 10 mL, 50 mL and 100 mL of ferment mixed with industrial slurry per kg of broccoli seeds. Control seeds of the same variety received the same crop protection package without endophyte. Endophyte survival was then assayed for the presence of each microorganism. The microorganism mix demonstrated the survival of strains (WW5, WW6, WW7, 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 plating 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.

[0134] Broccoli seeds were then stored under normal industrial conditions (<25°C in dark packages) for 2 months and planted industrially in the fall using industrial methods in a production field fertilized at normal and 25% reduced nitrogen fertilizer rates compared to 4 industrial nitrogen applications by trickle in Salinas, CA in a large-scale replicated CRO field experiment. 12 gal / acre of calcium ammonium nitrate (17-0-0) was applied twice and 5 gal / acre of (17-0-0) was applied two more times for the total amount of fertilizer over the season. In the 25% reduced nitrogen fertilizer rate treatment, the amount of nitrogen was reduced by 25% per fertilizer application. In addition, all treatments were applied with 0-0-6-3% Ca fertilizer for a total of 8 times over the season. Irrigation was controlled at the discretion of the farm manager according to industrial farm management standards. After one month of growth, there was a clear difference in plant size between the treatments that received 100% nitrogen rate and those that received less than 25% nitrogen. None of the treatment groups had 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 with 100% nitrogen and those with less than 25% nitrogen. The differences in uniformity and industrial quality were then measured at harvest for all treatments. In selecting industrial broccoli tips, growers took into account different criteria, such as tip size (diameter in inches), tip smoothness, dark green color, and firmness. The maximum mean industrial head diameter at collection was observed for the two highest endophyte treatment rates, 100 mL (used for the I4WP-20F and I4WP-20D groups) and 50 mL (used for the I4WP-10F and I4WP-10D groups), under both 25% less N fertilizer and regular 100% fertilizer, as seen in Figure 25B for 75% N and 100% N rates, respectively.

[0135] 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.

[0136] 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 on shoot growth of WW6 and WW7 endophytes applied individually to Brassica species under limited nitrogen in a controlled environment were tested. Endophyte strains WW6 and WW7 were applied individually inside 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 grown in an indoor growth room under greenhouse light at 710 μmol / m 2 s 1 The experiment was carried out at an ambient temperature of 25° C. and 50% humidity, with exposure to 14 hours per day of UV light. 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.

[0137] At harvest (27 days old), total plant seedling fresh weights were obtained for all treatments and the results are reported below. The WW7 treatment (RD12378) showed a significant increase of 30% in seedling weight over the control. The WW6 treatment (RD12381) showed a highly significant increase of 47% over the control. See Figure 26B.

[0138] Example 17 Effects of WW5, WW6, WW7 and co-fermented mixtures used as seed treatments under reduced nitrogen fertilizer in a controlled environment growth chamber WW5, WW6 and WW7 endophytes applied individually to corn seeds under limited nitrogen in a controlled environment were tested for their effect on shoot growth. Endophyte strain formulations were applied to corn seeds in combination with 1% w / v sodium alginate (Scogin LDH) and 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 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 during the experimental period with Hoagland's N-Dropout solution modified to contain nitrogen at 50 ppm total N. Two control groups were included, each treated with modified Hoagland's solution but not with endophytes. The first control group was treated with Hoagland's solution modified to contain 75 ppm total N, and the second control and endophyte treatment groups were treated with Hoagland's solution modified to contain 50 ppm total N. The study was conducted in an indoor growth chamber with greenhouse light at 710 μmol / m 2 s 1The experiment was 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 after emergence, the plants were harvested, washed and dried in individual paper bags at 45°C for one month. The dry weight of the shoots was then measured and recorded. The results are shown in Figure 27. The endophyte seed coat formulations increased the dry weight biomass of corn shoots as follows; WW6 +102% p>0.05, WW5 84% p>0.05 and WW7 49% p>0.01.

[0139] Example 18 Effects of WW6 and WW7 as seed treatments on winter wheat in the field. Winter wheat seeds were treated with co-fermented WW6+WW7 endophyte inoculum. The co-fermented WW6+WW7 endophyte mixture was fermented in low nitrogen medium and then freeze-dried to powder. Five grams of freeze-dried fermentate were mixed with 5 grams of dried sodium alginate in 1 liter of water. The mixture was then combined with an industrially available prebiotic composition UBS 016 (from Unium Bioscience Ltd.) in a ratio of about 3:1 to about 5:1 of mixture to prebiotic composition. This combination resulted in a final seed slurry, which was used in a ratio of about 4L to about 6L per metric ton of winter wheat seeds. The control group was treated with prebiotic without fermentate. Seeds were industrially planted 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 were recorded. The treatment group showed a substantial 10% increase in crop yield as shown in Figure 28A.

[0140] Furthermore, nitrogen accumulation was measured in the shoots of winter wheat plants from February to June. Plants were collected per square meter at each time point, dried, and sent for total nitrogen measurement by Kjeldahl method. A steady increase in total nitrogen per hectare of wheat shoots was measured, as shown in the results shown in Figure 28B, which demonstrated a +30% increase in Kg / Ha of total nitrogen accumulation in wheat shoots when measured at the final sampling in June. This result clearly demonstrated the impact of the nitrogen fixing N2 fixing endophyte strain on this wheat species in the field. The endophyte treatment 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.

[0141] Example 19 Carbon accumulation in plants treated with endophytic strains The ability of WW5, WW6, WW7 and PTD1 endophytic bacterial strains to increase total plant carbon accumulation as a mixed association when grown under field conditions was assayed using fast-growing poplar trees planted in soil from the Lower Mississippi Alluvial Valle field. Trees were inoculated with approximately 20 calcium alginate beads encapsulating the endophyte and applied to the base of the Populus cuttings at the time of planting. Replicate blocks in the field were planted (3 trees x 5 trees) at field locations containing 5 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).

[0142] 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.

[0143] 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 n=12 trees. The results shown in Figure 29 below demonstrate a significant increase in carbon accumulation in the treatment group versus the control.

[0144] Example 20 Compatibility with commonly used crop protection chemistries for seed treatment Endophytic strains WW5, WW6, WW7 and PTD1 were tested for their ability to survive when combined with various commonly used industrial seed crop protection chemistries. The survival of the endophytic 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). The 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 on NLM semi-solid medium. The results shown in FIG. 30A show that all strains can survive in the five different solution mixes.

[0145] 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, sedaxane, thiabendazole thiram, metalaxyl, hymexazole, penthiopyrad, ponchobeta and thiamethoxam. The solution mix was prepared according to the manufacturer's specifications and then applied to the seeds by an industrial seed treatment machine after adding the different strains to the solution. The colony forming units (CFU / ml) of the strains that survived on the seeds were evaluated by adding the seeds to a 0.2M phosphate resuspension solution and then planting them in NLM semi-solid medium at appropriate dilutions. The results shown in Figure 30B showed that the composition of endophyte strains was tolerant and viable when mixed with various industrial products and survived the temperature and dryness conditions found in the industrial seed treatment process.

[0146] Example 21 Compatibility with commonly used fertilizers, micronutrients and herbicides during in-furrow and foliar applications The possibility of adding endophytic strains to tank mix solutions for various commonly used in-furrow and foliar treatments was evaluated based on the survival of the strains for different periods of time ranging from 3 hours to 1 month. The survival of WW5 and WP1 was evaluated in ammonium polyphosphate (10-34-0) and / or micronutrient products (4% ammoniacal nitrogen, 3% water-soluble nitrogen, 9.0% chelated zinc), a liquid starter fertilizer used for in-furrow nutrient application. For the purpose of the experiment, the volume was scaled down from 5 gal / acre to 50 ml. The mixture composition included: 32 fl oz / acre of micronutrients, 5 gal / acre of 10-34-0 fertilizer, 16 fl oz / acre of WW5 strain inoculum, and water. Three hours after creating the mixture, the colony forming units (CFU / ml) of the two strains were determined by inoculating NLM semi-solid medium. The results shown in FIG. 31A demonstrate that both WW5 and WP1 survived when 10-34-0 was present in the aqueous furrow plus endophyte tank mix.

[0147] Following these results, a tank mix of 10-34-0 fertilizer at 3 gal / acre and WW5+WP1 at 16 fl oz / acre was evaluated in a replicated block study used as an in-furrow tank mix for growing corn in the field. 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 field received either no N or full rate N at 180 lb N per acre.

[0148] The results shown in FIG. 31B show a +12% increase in crop grain yield when WW5+WP1 strains were combined with 10-34-0 fertilizer and corn was grown at conventional Midwest WI USA rates under full NPK fertilizer at industrial harvest.

[0149] Additionally, mineral nutrient content was measured in the leaves of V9 vegetative corn plants. Figure 31C shows consistent increases in total nitrogen, potassium, and phosphorus (NPK) when WW5 and WP1 strains were used in furrow in fertilizer tank mix at planting. Importantly, potassium (K) showed a significant increase of +9.5% in leaves from blocks not receiving nitrogen, demonstrating increased uptake and assimilation of K in the shoots in addition to N and P.

[0150] Example 22 Compatibility with commonly used fertilizers, micronutrients and herbicides during in-furrow and foliar applications The impact of endophyte fertilizer and micronutrient compatibility was tested with WW6 and WW7 strains, which were evaluated for long-term viability when mixed with 6-22-6-4, a common liquid starter fertilizer frequently used for in-furrow nutrient applications. The test solution mix was scaled down from 5 gal / acre to 50 ml for purposes of the size of the experimental container. The composition included: 5 gal / acre of 6-22-6-4 fertilizer, 40 fl oz / acre of WW6+WW7 microbial composition. The colony forming units (CFU / ml) of the two strains were determined by inoculating NLM semi-solid medium over a period between 1 week and 5.5 months. The results shown in FIG. 32 indicate that the viability of both strains is slightly reduced when 6-22-6-4 is present in the solution mix.

[0151] Example 23 Endophyte compositions tested for foliar herbicide compatibility The viability of WW6 and WW7 was evaluated in a composition containing glyphosate, a broad-spectrum systemic herbicide 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 (sulfur 3.6%, boron 0.1%, manganese 3.0% and zinc 4.0%). For the purpose of the experiment, the solution mix was scaled down from 10 gallons / 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 adjuvants, 24 fl oz / acre of glyphosate and 16 fl oz / acre of WW6+WW7 microbial fermentation composition prepared according to the method described herein and having a pH of 5.3. 24 hours after the mixture was created, the colony forming units (CFU / ml) of the two strains WW6+WW7 were determined by inoculating NLM semi-solid medium. The results, shown in FIG. 33A, show that the viability of both strains was not reduced in the different compositions.

[0152] A field study was conducted to demonstrate the impact of the WW6 and WW7 compositions when applied as a foliar spray on corn with 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 (glyphosate herbicide from WinField® United), 32 oz / acre of MasterLock (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 material without the endophyte strain. The results shown in FIG. 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 using the WW6+WW7 endophyte strains compared to the control that received no endophyte treatment.

[0153] Example 24 Effect 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, beets (Beta vulgaris) were industrially treated with and without application of heterologous endophytes (WW5, WW6, WW7, PTD1) using industrial seed treatment methods. Seed coatings were used to control plants, and seed coatings combined with co-fermenting endophyte strains WW5, WW6, WW7, and PTD1 were used for experimental seeds. The coated seeds were then washed, and the experimental groups were tested for survival of the endophyte strains thereon. Bacterial counts were assayed by dilution plating on NLM agar medium after vortexing and washing 10 seeds in 10 mL of potassium phosphate (KP) buffer to remove and dissolve the coating. The assay showed clear survival of the endophyte, with titers shown as CFU / seed in Figure 34A.

[0154] Seeds treated with endophytes for 24 hours were planted in separate cells, and control seeds for 24 hours were planted in separate cells. Seeds were constantly overwatered and germinated in standard industrial transplant potting medium in fully saturated soil. The medium was overwatered daily to saturation 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 endophyte strains showed faster germination and establishment, less waterlogging damage, and better growth compared to the control under continuously flooded saturated soil conditions, as shown in Figure 34B.

[0155] The above data demonstrate that the co-fermenting heterologous endophyte WW5+WW6+WW7+PTD1 successfully improved beet plants' response to abiotic stress (flooding) induced by over-watering 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.

[0156] Example 25 Effects of combined endophytic strain (WW6+WW7) seed treatments on plant cold tolerance Two sets of broad beans (Vicia faba) were prepared, a control group was treated with an industrial seed treatment and an experimental group was 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 into a powder. Five grams of freeze-dried fermentate were 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 about 4 L to about 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 daily minimum temperatures ranging from 45°F to 55°F and maximum temperatures ranging from 65°F to 70°F. Once germinated, the 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 cold shock treatment at 34°F for 6 hours. After this exposure, the plants were photographed and evaluated for cold stress symptoms and damage. As shown in FIG. 35, the experimental plants inoculated with WW6+WW7 had less wilting and cold damage than the control plants which were severely wilted. Furthermore, the endophyte-treated plants fully recovered from the cold stress, while the non-endophyte-treated plants did not fully recover and showed symptoms of chlorosis and necrosis.

[0157] The above data demonstrate that the co-fermenting heterologous endophytes WW6+WW7 successfully improved broad bean plants in response to abiotic stress (cold shock) compared to the control. The results demonstrate the efficacy of the co-fermenting heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0158] Example 26 Effect 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 that was once suitable for cultivation is now unsuitable for agriculture due to saline 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 that included 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 according to FIG. 37A.

[0159] Seeds of 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 high saline / saline soils and high boron and chlorine levels was selected for the field trial. The site was Five Points, California. An exchangeable sodium percentage (ESP) of greater than 6% is considered a saline soil, 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, but levels of about 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.

[0160] Using a rototiller, three identical 72-inch beds were created in a homogenous high salinity 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 beds with 6 ft width and two rows (2 ft from the edge of each bed and 2 ft between rows). 290 plants were planted from each of the two endophyte treatment groups and the untreated control seed. At the time of collection, a photograph of the beds was taken 91 days after transplanting and is shown in Figure 36C. The salt tolerance of the endophyte-enhanced plants (designated WW7 and Phase A in the figure) can be easily seen in Group 2 (Phase A).

[0161] 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 FIG. 36D shows the 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 FIG. 36E.

[0162] The data demonstrate that (1) the co-fermented heterologous endophyte WW5+WW6+WW7+PTD1+WP1 successfully improved broccoli fresh and dry weights compared to the control, and (2) the endophyte strains improved broccoli dry weights compared to the control under abiotic stress (saline / saline soil conditions). The results demonstrate the efficacy of heterologous endophytes in enhancing the physiological performance of non-native host plants.

[0163] Example 27 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. The occurrence of drought is increasing and drought induces many physiological and molecular biochemical changes in plants. Internal processes that help plants to tolerate drought stress include scavenging of reactive oxygen species (ROS), osmoregulation (OA), stomatal closure and synthesis of defense molecules including inducible dehydrins. Plant recovery after drought stress involves a series of steps occurring over time that may be aided or facilitated by internal beneficial endophytes. To prove the effect of endophytes on increasing tolerance to drought, a fermentation mixture was prepared containing endophyte strains WW5+WW6+WW7+PTD1 together with the fungal yeast endophyte WP1, referred to as "Phase A mix". The Phase A mix fermentation, along with an industrial seed treatment incorporating polymer and talc powder as carriers, was applied to tall fescue seeds (Festuca aramdinacea, a forage grass used in livestock animal production) and allowed to dry to a husk-like natural hard covering. 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 tonne of seeds;

[0164] Control groups received an industrial seed treatment that did not contain an endophyte strain. These treatments resulted in endophyte survival on the seeds assayed and shown in Figure 37A.

[0165] 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. The seeds were watered three times a week for four weeks with a modified low nitrogen Hoagland nutrient solution containing 65 ppm nitrogen. The herbs were grown at 30°C and then subjected to a 14 day waterless drought stress period which allowed the growth medium to completely dry out. After the drought stress treatment was stopped, watering of the flats was resumed to give the herbs a chance to recover. Each group was then harvested and weighed. Groups 1 and 2 showed a statistically significant increase (p<0.05) in dry weight of 42% and 67%, respectively. The total weight results are shown in the graph in Figure 37B below.

[0166] The above data demonstrate that the co-fermenting heterologous endophytes 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.

[0167] Example 28 The endophyte increases seedling germination, seedling emergence and seedling biomass weight. A series of tests were conducted in which endophyte strains were applied to seeds to determine the effect of application on increased seedling germination, increased seedling emergence from seed coating and soil, and increased seedling biomass weight. The first experiment involved treating romaine lettuce seeds with 10 treatment groups and a control group, as identified in FIG. 38. For each treatment, romaine lettuce seeds were applied with a clay seed coating and a nitrogen-limited endophyte ferment mixture in 1% w / v alginate solution. The control did not contain endophyte ferment. Seeds, including the control seeds, were coated using an industrial method that included a clay coating and a 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. Seeds were then germinated and seedlings were weighed after growing in DI water under light and fluorescent light without nutrients for 14 days. Results showed that the inoculated seedlings were larger and able to grow better under nitrogen-limited conditions. This result strongly suggests that the inoculated plants were better able to fix atmospheric nitrogen and utilize nutrients from the seedling germination paper than the controls. With regard to 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.

[0168] 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 (WW7 strain only and a mix of WW5, WW6, WW7 and PTD1) and a control group, as shown in Figure 39. In each treatment, broccoli seeds were applied with a clay seed coating and a mixture of nitrogen-limited endophyte fermentate in 1% w / v alginate solution. The control did not contain endophyte fermentate. Seeds, including the control seeds, were coated using an industrial method that included a clay coating and a 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.

[0169] Seeds were germinated in square petri dishes containing seed germination paper moistened with 14 mL of deionized sterile water per sterile seed germination container 4×5”. Seedlings were then grown under fluorescent light with no nutrients in light for 14 days before being weighed. Results showed that the inoculated seedlings were larger and better able to grow under limiting conditions. These inoculated plants were able to fix atmospheric nitrogen and utilize nutrients from the seedling germination paper better than the control, which is likely why they produced the greatest seedling weights as shown in FIG. 39.

[0170] 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 and controls treated with co-fermentation WW6+WW7. For each treatment, barley seeds were applied with a clay seed coating and a mixture of nitrogen-limited endophyte fermentate in 1% w / v alginate solution. The control did not receive endophyte fermentate. Seeds, including the control seeds, were coated with an industrial seed treatment containing polymers and crop protection agents, including 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.

[0171] 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 noticeably larger, as shown in Figure 40A, and continued to grow larger after 4 days, as shown in Figure 40B.

[0172] 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, treated with WW7 fermentation and a four-strain mix (I4WP), as well as a control group. For each treatment, broccoli seeds were applied with a clay seed coating and a nitrogen-limited endophyte fermentation mixture in 1% w / v alginic acid solution. The control did not contain endophyte fermentation. Seeds, including the control seeds, were coated with an industrial seed treatment that included a clay coating and a polymer and crop protection agent, including a seed coating polymer.

[0173] 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 treatment group. Plants inoculated with the endophyte strain showed faster germination (see Figure 41) and establishment as well as better growth.

[0174] Example 32 An experiment was carried out in which endophytic strains were applied to sugar beet varieties and seeds (C578 and M5) were treated with the seed inoculant composition. The experiment included two treatment groups, one treated with WW7 fermentate and one treated with a four-strain mix (I4WP), as well as a control group. Seeds in the treatment groups were industrially treated at the rate of 50 mL of fermentate applied per kg of seeds together with the crop protection products Thiram, Metalaxyl, Hymexazol, Penthiopyrad and Poncho Beta (Clothianidin). Control seeds of the same varieties were prepared in the same way but without the fermentate. Seeds were then stored for one month under normal industrial conditions and planted industrially.

[0175] The emergence of sugar beet seedlings after sowing was measured over time. The endophyte seed treatment composition allowed survival of all four strains (I4WP), WW5, WW6, WW7 and PTD1, and on average improved the 29-day plant emergence of the sugar beet varieties compared to the control group. In this test, 200 seedlings were treated as equivalent to 75% of the sugar beet plants, and the endophyte improved the average emergence at 13, 16 and 29 days after planting. The C578 variety control had 187 seedlings emergent after 29 days, compared to 200 seedlings (13 more), and the M5 variety control had 161 seedlings emergent after 29 days, compared to 177 seedlings emergent in the treatment group treated with the I4WP mixture (16 more), as shown in Figures 42A and 42B.

[0176] Example 33 An experiment was carried out in which endophyte strains were applied as a seed inoculant composition to wheat seeds. 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 at a rate of 50 mL of fermentate and 500 mL of 1% alginic acid, applied per ton of seeds together 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. Seeds were then stored for one month under normal industrial conditions and planted industrially.

[0177] Wheat seedling emergence was measured over time in a CRO field trial. Treatments improved wheat seedling growth after emergence compared to untreated controls, which were randomly harvested over time from appropriate field plots. Final seedling fresh weights after 15 days of follow-up were 1.054g leaf and 0.781g root for the WW6 / WW7 combo, compared to 0.95g leaf and 0.44g root for the control. Fresh weights of freshly pulled seedlings on each day are shown below in Figures 43A and 43B.

[0178] Example 34 Effect of WW6 or WW7 applied as a seed treatment on field grain yields. To improve field yield under a highly optimized nutrient system, endophytes were applied as seed treatments and spring wheat seeds were treated with WW6 or WW7 endophyte NLM ferment. Prior to seed treatment, the ferment was freeze-dried to powder. Five grams of freeze-dried ferment along with 5 grams of dried sodium alginate were added per liter of water to prepare a seed treatment slurry. Six liters of seed slurry were used to treat one metric ton of spring wheat cultivar Tybalt seeds. The results in FIG. 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 control not treated with endophytes.

[0179] Example 35 Reducing nitrogen fertilizer requirements in wheat crops while maintaining yield A field trial was designed to demonstrate the ability of the 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 WW6+WW7 fermentation and mixed with 0.5% sodium alginate (Scogin™ LDH) as a seed coat inoculant.

[0180] Lettuce seeds were planted in the fall in Fresno, California, on fields fertilized with normal and 33% reduced nitrogen fertilizer rates, as is typical for CRO field trials. Field plots were divided into groups and fertilized by 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 normal fertilized control plots also received 50 lb / acre of UN-32 nitrogen fertilizer. Irrigation was adjusted at the discretion of the farm manager, following general agricultural practices. At harvest, 10 heads of lettuce were collected from each plot, six plots per treatment. The average head weight was calculated for each plot, and an overall average was calculated. Figure 45A shows that the WW6+WW7 treatment under 33% reduced nitrogen increased head weight by 3.3% compared to the full nitrogen control plants. Additionally, plant tissue samples were collected from 4 of the 6 harvest plots for nitrogen tissue concentration assays. Figure 45B shows 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.

[0181] The WW5+WW6+WW7 fermentation was mixed with 0.5% sodium alginate (Scogin™ LDH) and the composition was used to over-treat corn (Channel 216-36 STX RIB) seed that had been previously treated with Prothioconazole, Metalaxyl, Fluoxastrobin, Clothianidin, LCO SP104, and Bacillus firmus 1-1582. The seed was planted in Clay, Nebraska, on fields fertilized with normal 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 including six plots fertilized at different rates: (1) 165 lbs / acre anhydrous ammonia nitrogen prior to planting at the 25% reduced rate, and (2) 220 lbs / acre anhydrous ammonia nitrogen prior to planting at the normal rate. Grain yields were collected from the center two 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 treatments under 25% reduced nitrogen had a 0.2% reduction in average grain yield compared to the full nitrogen control plants, while the reduced nitrogen control plants had a 2.7% reduction in average grain yield.

[0182] Additionally, the WW5+WW6+WW7 fermentation was mixed with 0.5% sodium alginate (Scogin™ LDH) and the composition was used to treat corn (Channel 213-19 VT2P RIB) seed that had been previously treated with Prothioconazole, Metalaxyl, Fluoxastrobin, Clothianidin, and LCO SP104. The seed was planted in Saunders, Nevada, on fields fertilized with normal and 25% reduced nitrogen fertilizer rates using typical practices in university field trials. The trial was conducted in a split block and split plot design, with each treatment including six plots fertilized at different rates: (1) 112.5 lbs / acre of liquid UAN 32-0-0 nitrogen prior to planting at the 25% reduced rate, and (2) 150 lbs / acre of liquid UAN 32-0-0 nitrogen prior to planting at the normal rate. Grain yields were collected from the center two planted rows of each plot when grain moisture reached approximately 15.5%. Average grain yield was calculated for the six plots for each treatment. Figure 45D shows that the WW5+WW6+WW7 treatment under 25% reduced nitrogen had a 7.67% increase in average grain yield compared to the full nitrogen control plants, while the control plants under reduced nitrogen had an 8.4% decrease in average grain yield.

[0183] 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 the WW6 and WW7 strains: Enlist One (Corteva Agriscience, LLC), Impact (AMVAC Chemical Corporation), and Callisto (Syngenta Crop Protection, LLC). The solution mixtures were scaled down from 20 gallon / acre to 10 ml for experimental purposes. The mixture contained 32 fl oz / acre of WW6+WW7 endophyte fermentation composition combined with the following products: 16 fl oz / acre of Enlist One (2,4-D choline salt 55.7% w / w, Glycerol >= 3 -< 10% w / w, Dipropylene glycol monomethyl ether >= 3 -< 10% w / w, Balance > 20% w / w), 1 fl oz / acre of Impact (Topramezone 29.7% w / v, Inert 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 the mixture was prepared, it was plated on NLM semi-solid medium and the colony forming units (CFU / ml) of the two strains were confirmed (FIG. 46).

[0184] 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 herbicide foliar applications.

[0185] Example 37 Examples of the suitability of fertilizer-biostimulants 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 mixtures. The tank mixtures were scaled down from 400 liters / ha to 10 ml for experimental purposes. The mixtures 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 mixtures were made by plating on NLM semi-solid medium. See Figure 47.

[0186] The results in Figure 47 show that there was no significant decrease in the viability of both strains in the different solution mixtures over a long period of time, indicating that this endophyte composition can be used in commercial agricultural fertilizers and foliar applications of biostimulants.

[0187] 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 verification and evaluation was performed for WW6 and WW7 strains, which were reconstituted from the 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 verified separately. The solution mixtures were scaled down from 400 liters / ha to 10 ml 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) 10 g / L (1 % w / v) + 2,4-D (ester-2-ethylhexyl) 180 g / L (18% w / v)) at 0.13%, Guadana (Flufenacet 40% w / v (400 g / l) (32.4% w / w) + Diflufenican 20% w / v (200 g / 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 making and storing the mixtures for 4 hours, the colony forming units (CFU / ml) of the two strains were confirmed by plating on NLM semi-solid medium. See Figure 48.

[0188] 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.

[0189] Example 39 Ability to enhance atmospheric nitrogen fixation following treatment with endophyte inoculant seed coat suspensions A test was conducted to demonstrate the ability of different endophytes to fix atmospheric nitrogen in crop plants germinated from inoculant-coated hybrid maize seeds using seed treatment suspensions mixed with 0.5% w / v nutritional additive sodium alginate carbohydrate suspension. Cultivation of endophyte inoculants as individual strains WW5, WW6 and PTD1 was performed with adjusted fermented inoculants until the cultures reached a titer of at least 1.0E8cell / mL and then combined with the nutritional additive carbohydrate. Seed treatment inoculants were QC checked for viable cells and genetically confirmed for colony morphology of the appropriate species and strains using specific primers for colony PCR. This endophyte solution seed treatment composition was mixed with widely used chemical seed treatments including fungicides Fludioxonil, Mefenoxam and 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 application instructions for corn seeds. The treated seeds were then dried and assayed for viable microbial colony counts by plating seed coat wash and KP buffer count dilutions on nitrogen-limited medium NLM plus agar. Counts showed that microbial viability on the seeds one month after the mixed slurry seed coat was applied was as follows: WW6 - 400 CFU / seed, WW5 - 40 CFU / seed, and PTD1 - 40 CFU / seed.

[0190] 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 with 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 air and soil nitrogen was estimated by comparing the native 15N content of the inoculated plant biomass with the native 15N content of adjacent reference non-inoculated plants living on soil nitrogen only. Concentrations of total N and 15N isotopes in corn shoot tissues were measured at the V6 growth stage, 4–5 weeks after planting, where total N was measured using an Elementar EA Vario Pyrocube and 15N was measured using an Elementar IRMS GeoVisION, an isotope ratio mass spectrometer (IRMS). To quantify the percentage of nitrogen derived from air (NDFA%) in plant shoots, the amount of 15N isotope nitrogen measured by the IRMS was subtracted from the total nitrogen concentration, and the difference was the NDFA reported as a percentage of the total N pool.

[0191] The validation results are shown in Figure 49, which indicate that the major proportion of total nitrogen in maize shoots was derived from air by seed treatment with endophyte seed treatment inoculant; PTD1 42% NDFA, WW5 36% NDFA, and WW6 69% NDFA. This result clearly indicates that the maize endophyte seed treatment can be stabilized after drying and used for inoculation into seeds, and the subsequent crop plants sprouted from the seeds enhance biological nitrogen fixation at the V6 plant growth stage of maize plants.

[0192] Example 40 Ability of formulated liquid endophyte inoculum treatments to enhance atmospheric nitrogen fixation following direct application to the roots of young wheat plants To demonstrate the ability of different endophytes to fix atmospheric nitrogen in crop plants after soil or furrow rhizosphere inoculation, endophyte liquid inoculum cultures were prepared for each strain WW5, WW6, PTD1 and a synergistic mixture of co-cultures of WW6+WW7. Culture suspensions were prepared simultaneously using nitrogen-limited NLM medium prepared with nutrient additives until the cultures reached a titer of at least 1.0E8 cells / mL. Nutrient-added inoculum was then analyzed for viable cells and colony morphology. The presence of the co-culture mixtures of WW5, WW6, PTD1 and WW6+WW7 strains in each inoculum was confirmed using specific primers for colony PCR. The endophyte suspension inoculum was inoculated into the roots of 2-week-old wheat plants. One mL of the inoculum suspension was used for each plant and applied to the roots of the plant substrate after transplantation from the potted soil. Transplants were planted in 1-gallon greenhouse pots filled with 1.5 kg of field soil amended with 1,200 ppm total N and 10 ppm soluble N (NO3 + Nh3) without fertilizer and amended with perlite. 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 air-derived nitrogen, using an Elementar EA Vario Pyrocube for total N analysis and an Elementar IRMS GeoVisION, isotope ratio mass spectrometer (IRMS) for 15N.

[0193] The results are shown in Figure 50 and demonstrate that seed treatment with endophyte seed treatment inoculants results in significant amounts of air-derived nitrogen in wheat shoots; 43% NDFA in PTD1, 51% NDFA in WW5, 38% NDFA in WW6, and 47% NDFA in the synergistic combination of WW6+WW7. The results clearly indicate that endophyte inoculation of wheat roots in both single strains and co-fermented nutrient additive solutions enhances the biological nitrogen fixation capacity, which is carried over to the main growth stages of wheat plants.

[0194] 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 mixed consortium of endophytes could increase the total plant biomass (shoot + root) of 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 inoculant (500 mL resuspended endophyte freeze-dried culture 1.1% w / v + sterile sodium alginate solution 2% w / v added) and a sterile control nitrogen-limited medium (NLM) with final alginate 2% w / v added alone without endophyte. All treatments were applied at a rate of 1 L per metric ton of seed. Spring barley was treated with three seed formulation solutions using endophyte strains WW6, WW7 and co-fermented WW6+WW7 and dried overnight in a laminar flow cabinet. Four pots were grown with one plant per pot, with n=4 plants per treatment group. Control plants received Hoagland's nitrogen dropout solution made with 65 ppm N (100%) and reduced 32 ppm N (50%), and the experimental treatments inoculated with endophytes received reduced 32 ppm N (50%). Hoagland's nutrients were added to the trays on Mondays, Wednesdays, and Fridays, and the tray water was discarded after ½ hour. Plants were grown for 26 days after germination in a plant growth incubator at 25°C with artificial lighting set at 12 hours light / 12 hours dark, then harvested and dried in paper bags at 45°C for 48 days. The results shown in Figure 51 indicate a synergistic effect of the combined treatment WW6+WW7 which increased barley total dry weight biomass the most under 50% reduced nitrogen, significantly increasing total biomass weight by 75%, while WW6 alone showed a non-significant 17% increase in total biomass and WW7 alone showed a non-significant 17% increase in total dry weight biomass over the control when grown at the same reduced nitrogen rate.

[0195] 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 certain endophyte combinations to confer synergistic benefits to cereal crops, spring wheat varieties (Sy Ingmar) were industrially treated with seed treatments of WW6+WW7 co-ferment and WW5+WW6+WW7 co-ferment. No other seed treatments were used. Seeds were coated by mixing seed treatment slurries of the prepared solutions (>1.0 E6 CFU / mL NLM ferment + 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 endophyte ferment. Wheat was planted in late May in Berthold, North Dakota, USA, and harvested in September after 117 days of field growth. Plot size was 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. 1.5 million seeds were planted per acre 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 performed on soil from 0-24" depth 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.

[0196] 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.

[0197] 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 mono-strain combinations (2-strain, 3-strain, and 4-strain) 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 a clay seed coating using a seed treatment machine and then applied to the seeds using standard industrial techniques. The endophyte solutions were applied at the following rates: 10 mL final volume of endophyte inoculant per 1 / 3 lb of romaine seed. For co-fermentations with two endophyte strains, 5 mL of each strain was applied. For co-fermentations with 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. WW5, WW6, WW7 and PTD1 strains in combination with yeast strain WP1 significantly increased shoot biomass weight by 43% after industrial field growth.

[0198] Example 44 Efficacy of the endophyte 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.

[0199] A study was conducted to determine whether a mixed synergistic consortium of endophytes could increase the total biomass (shoots + roots) of canola plants when mixed with a prebiotic plant microbial booster compared to a single strain used alone with a prebiotic plant microbial 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 inoculant with the following formulation: 500 mL of endophyte freeze-dried culture resuspended at 1.1% w / v, 500 mL of prebiotic and microbial biostimulants, 4% w / v sterile sodium alginate solution made in HO. Sterile control nitrogen-limited medium (NLM) with prebiotic and microbial biostimulants and 4% w / v sterile sodium alginate. 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 formulation solutions using endophyte strains WW6, WW7 and WW6+WW7. Seeds were dried overnight in a laminar flow cabinet after application. Three pots were grown with one plant per pot, with n=3 plants per each of the four treatment groups. Control plants were fed Hoagland's Nitrogen Dropout solution made at 65 ppm N (100%) and 32 ppm N (50%). Experimental groups were fed 32 ppm N (50%). Nutrients were added 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 with artificial lighting set at 12 hours light / 12 hours dark and then harvested.

[0200] As shown in Figure 54, the combined synergistic mixed treatment of WW6+WW7+prebiotic significantly increased the total biomass weight by 82%, which increased the total biomass the most, while WW6+prebiotic increased the total biomass by 20% and WW7 increased the total biomass by 55%.

[0201] Example 45 Use of three synergistic endophytic strains for foliar treatment of maize shoots to reduce fertilizer requirements and increase harvested grain yield.

[0202] A trial 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 Mead, Nebraska, USA in early May. The soil was Tomek Silt Loam with pre-plant nutrient levels of 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 a foliar spray applied at V6 using a pressurized sprayer misting either the control or experimental treatments containing endophyte strains WW5, WW6 and WW7. Plot size was 10 ft x 40 ft with four rows of corn planted per plot. Six replicates were run for each treatment in a split block design. Fertilizer was applied as a pre-applied soil nitrate at 17 lbs per acre, 75 lbs of nitrogen per acre as a liquid formulation UAN 32-0-0 prior to planting, and an additional 37.5 lbs of nitrogen per acre for a total of 130 lb N / ac. N application to treatments was 75% of the standard nitrogen application method for the acreage. A secondary control check treatment was included in the trial with 170 lbs of nitrogen (100% of the standard nitrogen application rate). Herbicide was also applied. Pre-Acuron + Roundup was applied on May 13th, immediately after planting. The center two rows were harvested in late October after 163 days in the field, and grain was weighed and statistically analyzed. Results are summarized in Figure 55. At 75% N fertilization, the synergistic effect of foliar treatments with the three endophyte strains at V6 significantly increased corn mean grain yield by +44.5 bu / ac (p=0.03). Foliar treatments with endophyte at V6 increased mean yield compared to the full 100% N control treatment of 35 bu / ac.

[0203] Example 46 Stacking of two endophytic strains in synergistic in-furrow applications used to increase harvested grain yield with reduced nitrogen at 75%.

[0204] A trial was conducted to determine the efficacy of a specific endophyte combination, WW5+WP1, applied as an in-furrow liquid composition to improve nitrogen fixing inoculation and yield in a corn hybrid variety (Channel 113 day213-19VT2PRIB). The corn variety was first commercially 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 with pre-plant nutrient levels of 11.1 ppm P, 344 ppm K, 7.4 ppm S, pH 5.8, 4.1% OM, and 17.6 CEC. Corn plants were treated at planting with the WW5+WP1 endophyte solution applied as an overlay over the seeds in the furrow using a dribble tube. Plot size was 10 ft × 40 ft, with 4 rows of corn planted per plot. A split block design was used with six replicates per treatment. Fertilizer was applied as a pre-applied soil nitrate application at 17 lbs / acre, a liquid nitrogen formulation UAN 32-0-0 at 75 lbs / acre prior to planting, and an additional 37.5 lbs / acre of N for a total of 130 lb N / ac. N application to treatments was 75% of the standard N application method for the acreage. A secondary control check treatment was included in the trial with 170 lbs of N (100% of the standard N application rate). Herbicide was also applied. Pre-Acuron + Roundup was applied to plants on May 13th immediately after planting. The center two rows were harvested in late October after 163 days in the field and grains were weighed for statistical analysis. Results are summarized in Figure 56.

[0205] Results at a 75% N fertilizer rate demonstrated that a synergistic two-plant endophyte in-furrow application applied at planting increased average corn grain yield by +30.2 bu / ac (p=0.07). Endophyte in-furrow applications at planting resulted in increased average yield compared to the full 100% N control treatment of 20 bu / ac.

[0206] 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.

[0207] A trial was conducted at an organic strawberry farm in Salinas, California, to determine the efficacy of endophyte inoculants applied as liquid root sprays to improve biomass yield in Albion strawberry plants. The roots of transplanted seedlings were sprayed until covered with a light mist of the different endophyte inoculants. Spray treatments included WW5 alone, WW6 alone, WW7 alone, PTD1 alone, and a mix of WW5, WW6, WW7, and PTD1. The control group contained no endophyte strains. Treatments were applied and plants were planted using standard methods in early November 2016. Strawberries were planted in beds containing strawberry plants (200-240 plants / row) spaced 25-30 cm apart in two rows, with three beds for each 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.

[0208] The treatment containing a mixture of all four lines, 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 line inoculation treatments showed smaller increases than the control.

[0209] Example 48 Combining endophyte strains within hard partially hydrated beads as dry granular carriers for synergistic application to increase transplant biomass of tomato.

[0210] A study was conducted to determine the efficacy of WW5 alone, the combination of WW6 and WW7, and the combination of four endophyte strains, WW5, WW6, WW7, and PTD1, to increase biomass yield in tomato. The fermentation suspension was blended into a sodium alginate slurry and dripped into a 100 mM calcium chloride water bath to allow a cation exchange reaction to occur, producing fully hydrated but rigid calcium alginate beads that were dried to a final moisture content of about 4% to about 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 commercial transplanting soil (high in organic matter, consisting mainly of peat and perlite) in 125-cell transplant planter trays and grown for 3 weeks at 25°C under normal industrial greenhouse fertilization rates and normal lighting. 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%, whereas the germination rate of the non-inoculated controls was suboptimal at less than 88%. Figure 58 shows the results of endophyte-enhanced biomass (shoot + root) in growing transplants.

[0211] The results clearly demonstrated a synergistic effect of 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.

[0212] Example 49 Combining endophyte strains in rigid partially hydrated beads as dry granular carriers for synergistic inoculant application to loose-leaf lettuce grown under conditions deficient in bioavailable N and P.

[0213] A study was conducted to determine 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). The experiments were conducted in a greenhouse using a specific soilless medium with the composition of Terragreen, a baked clay gravel mixture that reduces the biologically available forms of N and P to a deficient state, and results in nutrient concentrations of 40g of 8-3-5 organic fertilizer and a well-drained, organic matter-rich soil profile with 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 / 100g, and 1831 ppm total nitrogen (mostly composed as amino acids). The greenhouse trials 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 or close to the germinating seeds in soil. Plants were then carefully transplanted at 3 weeks by removing all roots with a hand trowel, leaving the surrounding soil intact. Roots, beads, and soil were placed in equal sized holes and placed in 2 gallon felt Smart Pots. All plants were automatically watered with the same amount of water every 12 hours with 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 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 without endophytes. All treatments were replicated 6 times, with n=6 pots each.

[0214] 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 the average shoot weight by 173%. Inoculation with PTD1 beads increased shoot biomass by an average of 2.74 g per plant, increasing the average yield by 89%.

[0215] Example 50 Combining the endophyte strains in a lyophilized powder for reconstitution and foliar spray on jalapenos in the field.

[0216] To determine the efficacy of endophyte inoculation from a freeze-dried endophyte mixture on the growth and biomass of jalapeno plants (cv. RPP7042). Approximately 420 jalapeno plants were inoculated in late spring with a foliar spray application at flowering. Five individual endophyte strains (WW5, WW6, WW7, PTD1, and WP1) suspended from freeze-dried powder were used. A mixture of the five resuspended endophyte strains was also prepared. One gram of freeze-dried endophyte was added per liter of pure water, and the freeze-dried endophyte was rehydrated and then placed in a foliar sprayer. Jalapeno seeds were planted in May to early June. Harvest time was 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. The 50 feet represented approximately 75 plants. At harvest two months later, a series of growth and biomass analyses were performed.

[0217] As shown in Figure 60A, all inoculation treatments using freeze-dried powder reconstituted at first flowering, except for the treatment with strain WP1, increased the average non-ripe pepper yield per plant compared to the control. The 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 non-ripe pepper yield by +44%, positively impacting RPP7042 jalapeno plants.

[0218] Additionally, the ability to increase the total number of peppers per treatment was assayed and the results are shown in Figure 60B. Phase A inoculations also performed best, with an initial increase of 66% in the average number of peppers per plant over the control, except for WP1. Phase A mixtures, PTD1 and WW7 were all statistically significant, indicating that the freeze-dried endophyte has an early positive impact on the jalapeno plants.

[0219] Total yield of ripe peppers was also assayed 3 months after initial inoculation and 4 months after planting and reported in Figure 60C. Results showed that endophyte inoculation with reconstituted freeze-dried powder applied at first flowering as a foliar spray can be used to increase the average yield per jalapeno 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. This result suggests that freeze-dried endophyte reconstituted in solution and sprayed on flowers and leaves can have an early positive impact on total yield of jalapeno plants.

[0220] Example 51 Effect of different endophyte seed treatment compositions using WW5, WW6, WW7, PTD1 and WP1 on leaf chlorophyll.

[0221] The endophyte synergistic mixed composition was freeze-dried and resuspended in water and then used to treat canola seeds to determine whether the resuspended endophyte ferment could increase canola leaf chlorophyll after 36 days when grown under reduced nitrogen. The endophyte seed inoculant included 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 composition: WP1, WW6+WW7, WW5+WW6+WW7+WP1+PTD1. The control group was treated with NLM alone without endophytes. After seed treatment, all groups were dried overnight at room temperature in a laminar flow cabinet. Four pots were prepared with two plants per pot for each of the five treatment groups (n=8 plants per treatment). The control plants were fed with Hoagland's nitrogen dropout solution made with 65 ppm N (100%) and 32 ppm N (50%). The experimental groups were fed with 32 ppm N (50% N). Nutrients were added to the trays three days a week (Monday, Wednesday, Friday) and the water was discarded after 30 minutes. The plants were grown for 36 days after germination in a plant growth incubator at 25°C with artificial lighting set to 12 hours light / 12 hours dark, and then harvested.

[0222] The results showed that the endophyte compositions used in canola seed treatments showed synergistic effects 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.

[0223] Example 52 Stacking endophyte strains in alginate beads for synergistic application to increase leaf chlorophyll in strawberry plants.

[0224] A study was conducted to determine the efficacy of endophyte inoculants 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 prior to 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 for each 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.

[0225] 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.

[0226] Example 53 Effect of different endophyte seed treatment compositions using WW6+WW7 versus market leading biologicals and biostimulants on total chlorophyll in winter wheat leaves.

[0227] An experiment was conducted to determine whether a freeze-dried and resuspended endophyte composition could increase leaf chlorophyll in treated host plants when applied to seeds. The endophyte was applied to seeds of winter wheat cultivar AWC13. The 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 endophyte, with 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 trial and harvested at the vegetative stage. Chlorophyll was extracted from 1 cm2 sized leaf sections using acetone. The results in Figure 63 showed an average increase of 6% compared to the untreated control.

[0228] Example 54 Enhancement of glutamic acid / glutamine Glx in maize leaves following treatment with endophyte inoculant seed coat composition.

[0229] A study was conducted to determine whether seed coatings containing heterologous endophyte compositions could fix N2 atmospheric nitrogen gas, generate ammonium, and convert it to the primary amino acid end products glutamic acid and glutamine in the host plant via the GOGAT GS and GDH ammonium assimilation pathways. A seed treatment containing WW6 fermentate and sodium alginate 0.5% w / v was prepared. Corn seeds were seed treated, then the seeds were dried and stored for one month. Treated seeds and control untreated seeds were then planted in one gallon pots containing 1.5 kg 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. The plants were grown in a greenhouse, harvested, freeze-dried, and then analyzed for common amino acids using a Shimadzu HPLC with post-column ninhydrin derivatization at AAA labs Inc USA. The results shown in Figure 64 indicate that WW6-inoculated maize seeds grew into plants with significantly (p=0.6) +46% higher glutamic acid and glutamine amino acid content in leaves when compared to the control. Furthermore, the genome of WW6 was analyzed for the presence of a gene encoding glutamine synthetase GS, and a total of six different copies of the GS enzyme gene were found adjacent to other genes related to nitrogen assimilation, quorum sensing, and motility. These results further support other findings related to atmospheric N2 fixation and enhanced N concentration 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 into the amino acids glutamic acid and glutamine directly from the atmosphere in crop plants.

[0230] 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 leads from N2 to ammonia / ammonium and then to the synthesis of the amino acid glutamine. The in planta effects of endophytic strains WW6 and WW7 on this process were evaluated in shoots of wheat plants (Triticum aestivum). Wheat seeds of the Zenda variety 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. Wheat seeds were applied with WW6 and WW7 fermented inoculants blended with 0.5% sodium alginate by weight at a rate of 500 ml per 2000 lbs. of seed, and the treated seeds were air-dried at room temperature and stored for one month. Controls were prepared with seed coating solution containing only alginate and growth medium without endophytic fungi.

[0231] Five seeds were planted in five different 3.5 inch pots for each treatment group. The pots were filled with a mixture of washed play sand, vermiculite, and perlite potting mix. The pots were kept at 4°C for 24 hours to induce vernalization and then transferred to a growth room 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 at reduced nitrogen concentration (25 ppm N) applied as needed in trays 2-3 times per week to keep the soil moist. Thirty-one days after transplanting, the plants were removed from the soil. Shoots and roots were separated and then flash frozen in liquid nitrogen, and the tissues were stored in a -80°C freezer. The plant material from each pot was individually ground to a fine powder in a mortar and pestle with liquid nitrogen. Approximately 100 mg samples of ground tissue from each treatment were then transferred into five separate 1.5 ml tubes, frozen in liquid nitrogen, and then stored in a −80° C. freezer for later use.

[0232] The enzyme activity of glutamine synthetase (GS) in the prepared samples was detected and quantified using a Glutamine Synthetase Microplate Assay Kit (MyBioSource, Inc.). Shoot tissue samples of three potted plants for control plants and three experimental plants (treated with WW6+WW7) were used for GS enzyme assay. Three technical replicates were prepared for each sample, and then the average value of all replicates was used to calculate the GS synthetase activity U / g.

[0233] Figure 65 shows the GS quantification (U / gU units are the amount of enzyme that catalyzes the reaction of 1 μmol of substrate in 1 min) adjusted according to the amount of tissue examined. Data were analyzed using ANOVA analysis with post-hoc Tukey test. The results showed that the endophyte inoculant composition used as seed treatment had an average of 55% higher glutamine synthetase GS enzyme activity than the uninoculated control plants, and the increase in activity was statistically significant at p<0.05.

[0234] In summary, WW6 and WW7 treated seeds had a significant effect on increasing glutamine synthetase activity in wheat shoots. This result related to the GS enzyme GOGAT correlates with the 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 that air-derived nitrogen assimilation was increased in inoculated wheat.

[0235] 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 provide substantial crop yields under reduced nitrogen fertilization conditions. Corn seeds were treated with WW6 endophyte inoculant and 0.5% w / v sodium alginate and seed coated. Treated seeds were planted and grown in field soil in Kansas, USA, fertilized with urea. Field plots were divided into groups and fertilized at different rates by spraying: (1) normal rate of 201.75 kg / ha urea (100% rate), (2) 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) no urea group (0% rate).

[0236] Grain was harvested from each plot when the grain moisture reached approximately 15.5%, 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 endophytes compared to the control. This graph shows the reduction in fertilizer requirements of corn plants with endophytes, i.e., how much less fertilizer is required by endophytes to maintain the same grain yield compared to uninoculated control corn plants. The result was that inoculation with either WW6 or WW5 endophytes reduced the nitrogen fertilizer requirement per hectare by 87 Kg.

[0237] Example 57 Enhancement of nitrogen uptake by single- and double-stock seed treatments in wheat. 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.

[0238] As shown in FIG. 67B, seeds were treated with an endophyte seed treatment inoculant composition consisting of a bacterial ferment combined with 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.

[0239] Endophyte viability was counted on treated seeds after dehydration and storage for one month, and the results showed that bacterial endophytes were viable on the seeds for each strain, as shown in Figure 67C. 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. All treated seeds clearly had the correct strains present, indicating survival and compatibility after treatment and storage with Cruiser Maxx Vibrance. WW6 treatment alone had the highest CFU viability per wheat seed. CFU levels detected on seeds may not represent the actual microbial load due to limited detection using this seed coat washing and count plating method.

[0240] A pot growth trial of wheat plants was conducted in a controlled greenhouse maintained at a temperature range of 60° F. to 75° F. where five winter wheat seedlings were potted and later thinned to three. 1 ml of WW5, WW6, PTD1 and WW6+WW5 solutions were applied to the seedlings and harvested at anthesis 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.

[0241] Under fertilized conditions of 188 mg N urea / pot, double strains WW6+WW7 and single strain WW6 increased the nitrogen content. Under urea treatment and non-fertilized conditions, single strains WW6 and PTD1 were the most effective in increasing the nitrogen content in shoots. Also, inoculation with a combination of two strains WW6+WW7 significantly increased the nitrogen content in shoots of treated plants under urea treatment and non-fertilized conditions.

[0242] Example 58 Enhancement of nitrogen uptake by single- and double-stalk 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) as well as in nitrogen-sufficient field soil amended with perlite and urea in fertilized field soil.

[0243] Corn seeds were treated with each of the individual endophyte (WW5, WW6, PTD1) seed treatment inoculant compositions mixed with 0.5% w / v aqueous sodium alginate and applied at a rate of 2.4 ml / 1800 kernels, and 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.

[0244] Endophyte viability was counted on treated seeds after dehydration and storage for one month, and the results showed that bacterial endophytes were viable on the seeds for each strain, as shown in Figure 68A. Seeds were washed and sampled for seed wash counts. No microorganisms with similar morphology were found in the washed samples of corn control seeds. All treated seeds clearly had the correct strains demonstrating survival and fitness after treatment and storage with Cruiser Maxx. WW6 treatment alone had the highest CFU viability per corn seed. Both WW5 and PTD1 seeds had perfect match in colony morphology with 40 CFU / seed, and WW6 treated seeds had the highest with 400 WW6 CFU / seed. CFU levels detected on seeds may not represent the actual microbial load due to limited detection using this seed coat washing and count plating method.

[0245] A pot growth trial of corn plants was conducted in a controlled greenhouse maintained at a temperature range of 60°F to 75°F, with 3 corn seeds per pot planted and later thinned to one seedling. Corn seeds were treated with each of the individual endophyte (WW5, WW6, PTD1) seed treatment inoculant compositions mixed with 0.5% w / v aqueous sodium alginate solution and applied at a rate of 2.4 ml / 1800 seeds, and treated with 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 in the eighth growing season. The results, as shown in the results in Figure 68B, indicate that the endophyte inoculant seed treatment composition caused a significant increase in shoot N uptake in both unfertilized zero nitrogen pots and 318 mg N (urea) pots. WW6 treatment increased total shoot N uptake by ~12 mg per plant shoot compared to the control under unfertilized field soil conditions. PTD1 and WW5 did not increase shoot N in the absence of added fertilizer in the greenhouse.

[0246] Under fertilized conditions with 318 mg N urea / pot, treatments with individual strains PTD1, WW5, and WW6 increased nitrogen content. Under unfertilized conditions, single strain WW6 treatment increased shoot nitrogen content. Under fertilized conditions, inoculation of seeds with endophytes via seed coating increased nitrogen content per shoot by ~100 mg for all three treatments, with WW6 being the best, followed by PTD1, and these increases were statistically significant at the p<0.05 level. WW5 seed treatment also increased nitrogen content in maize shoots by ~85 mg compared to the control.

[0247] Example 59 Use of two and three synergistic endophyte strain compositions in seed coat applications on corn shoots to increase harvested grain yield.

[0248] This study examined whether combinations of endophyte strains could have synergistic effects on nitrogen fixation in hybrid corn varieties treated with specific endophyte combinations (WW6+WW7) and (WW5+WW6+WW7) applied as seed coats. Corn was planted in various states in the Midwestern United States by various CRO field testing agencies from early May 2020 to 2022. Plot size was 10 ft x 40 ft with four rows of corn planted in each plot, with 30 plants evenly spaced per row. The study included six block replicates per treatment. Standard fertilizer applications were utilized. 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 location, with organic matter content ranging from 2.1-4.1%. The two middle rows were harvested in late October and the grain was weighed. Yield results for the WW6+WW7 treatments were summarized as average bushels / acre compared to the uninoculated control and are shown in Figure 69A. Yield results for the WW5+WW6+WW7 treatments were summarized as average bushels / acre compared to the uninoculated control and are shown in Figure 69B. Each numbered bar in Figures 69A and 69B is an individual field trial and represents 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. The results confirmed an average yield increase of 6.2 bushels / acre for the WW6+WW7 treated seeds and 11.9 bushels / acre for the WW5+WW6+WW7 treated seeds. These results demonstrate the synergistic beneficial effects of stacking multiple strain compositions together for use as seed treatments.

[0249] Example 60 Effect of endophyte seed treatment compositions using WW6+WWW7 on total leaf chlorophyll across a range of trials testing different cultivation programmes for UK winter wheat.

[0250] A freeze-dried endophyte resuspended in water composition was used to treat seeds of winter wheat cultivar GS59 to test whether the endophyte strains could increase leaf chlorophyll. WW6+WW7 A freeze-dried endophyte seed inoculation composition consisting of 1.1% w / v endophyte freeze-dried powder resuspended in 500 ml water was supplemented with 500 mL of 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 from 1 cm2 sized leaf sections using acetone. Differences in leaf chlorophyll content mg / cm2 of winter wheat GS59 under different programs. 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.

[0251] Example 61 Nitrogen-limited biomass enhancement in maize and teosinte inbred genetic lines. Effects of application of WW5 endophyte 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 mating with nested association mapping (NAM) founders and 11 geographically distinct teosinte individuals, resulting in a final genome composition of approximately 38% B73 (maize parent breeding line), approximately 2% NAM parent + Mo17 (maize parent breeding line), and approximately 1% teosinte. Seeds were treated with 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 mixed well and carefully pipetted into the seeds in a Ziploc® bag at a rate of 3.4 mL per pound of seed and dispersed dropwise in a sterile laminar flow fume hood in 1 mL increments. The seeds were carefully loosened by rolling them manually in the bag after each 1 mL addition. Once all 3.4 mL / lb had been added, the seeds were loosened, shaken, and rolled for 2-3 minutes to ensure that all corn seeds were visibly wet in the bag. The bag was then opened, turned upside down, and blown dry 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 counted by plating. In plating assays, treated seeds showed variation in endophyte coverage (WW5 CFU / seed). This variation in endophyte concentration resulted in a wide range of CFU per seed, from 0 to 1000 WW5 CFU / seed.

[0252] Seeds were tested in two groups, low nitrogen soil (50 ppm N) and nitrogen sufficient soil (100 ppm N), in a greenhouse trial with temperatures ranging from 70°F to 80°F and a photoperiod of 12 to 14 hours. 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 been grown for three weeks. The results of the biomass phenotypic screen, as shown in Figures 71A-71B, indicate a clear correlation between WW5 CFU / seed concentration and corn biomass shoot production, regardless of inbred genotype. The data collected revealed a correlation between biomass increase 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-fold in nitrogen-deficient soil, and by approximately 1.7-fold in nitrogen-sufficient soil.

[0253] 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 provide substantial crop yield under reduced nitrogen fertilization conditions. Wheat seeds were treated with WW6 endophyte ferment in combination with 0.5% w / v sodium alginate as a seed coat. Treated seeds were planted and grown in field soil in Kansas, USA, fertilized with urea. Field plots were divided into groups (4 groups per treatment) and fertilized at different rates by spraying: (1) 112 kg / ha urea rate (100% rate), (2) reduced rate of 78.4 kg / ha urea (70% rate), (3) a second reduced rate of 56 kg / ha urea (50% rate), and (4) no urea group (0% rate).

[0254] Wheat grains from each plot were harvested when fully ripe. The average grain yield was calculated for all plots representing each treatment. Figure 72 shows the average shoot nitrogen uptake of wheat plants inoculated with endophytes compared to the control. This graph shows the reduction in fertilizer requirements of wheat due to the endophyte, i.e., how much less fertilizer is required with the endophyte compared to the uninoculated control wheat. As a result, inoculation with WW6 endophyte reduced the nitrogen requirement of wheat fertilizer by 23 kg per hectare.

[0255] Example 63 Enhanced root and shoot growth in grapevine cutting production and long-term presence of endophyte-derived traits after inoculation with endophyte beads.

[0256] In a new grapevine production scenario, the presence of endophyte-derived traits for a long period after inoculation is beneficial for the creation of nurseries for clonal propagation. A long-term validation study was planned to plant cuttings inoculated with endophyte beads in the field and track their physiological status. Approximately 20 endophyte beads containing calcium alginate beads were placed adjacent to commercial Cabernet wine grape cuttings pressed into potting soil (Sunshine Mix #4) at a depth of 5–7 cm. The endophyte beads contained an endophyte consortium inoculum containing strains WW5, WW6, WW7, and PTD1 in calcium alginate beads (the mixture is referred to as “Phase B”). After rooting in pots in the greenhouse for 2 weeks, the cuttings inoculated with endophyte beads had more developed root systems and larger shoots compared to the control, as shown in Figure 73A. Plant growth was continued for an additional two weeks in a greenhouse maintained at temperatures ranging from 70° F. to 80° F. and a 12- to 14-hour photoperiod, and the plants showed persistence of the enhanced shoot and root growth phenotype during the additional growth period, as shown in Figure 73B.

[0257] An uninoculated control of Cabernet wine grapes is shown on the right side of Figure 73B, and the inoculated cuttings are shown on the left. Biomass and length of the cuttings were measured at the stage shown in Figure 73B, and the plants were then transplanted into field plots. Biomass data collected showed that the total biomass of the inoculated cuttings grew significantly 40% greater than the control, as shown in Figure 73C. Additionally, the endophyte-inoculated Cabernet cuttings increased in height by 31% compared to the uninoculated control, as shown in Figure 73D.

[0258] Two years after planting the endophyte-inoculated cuttings in the field, the cuttings matured into vines and an increased chlorophyll phenotype was also observed. Figure 73E shows data showing that the inoculated cuttings had a higher mean leaf chlorophyll.

[0259] Additionally, the average bunch weight of 2-year-old wine grapes increased by an average of 7% on inoculated vines versus controls. Figure 73F shows data demonstrating increased grape bunch weight on inoculated cuttings.

[0260] In another field trial conducted at the same location, approximately 20 beads of endophyte strains WW5, WW6, WW7, and PTD1 (Phase B) in calcium alginate were pressed into potting soil (Sunshine Mix #4) 5-7 cm deep adjacent to Grignolino wine grape cuttings. Field results 1 year and 3 months after planting showed a similar effect, with endophyte-inoculated (Phase B) cuttings showing an average 9% increase in stem base diameter compared to non-inoculated controls after 2 years. Results are shown in Figure 73G.

[0261] Example 64 Effect of endophyte rooting on greenhouse clonal production of Cabernet wine grape cuttings

[0262] Root production of cuttings is an important factor for clonal propagation in a greenhouse setting. The effect of treating cuttings with an endophyte strain on root production was examined. Commercially available Cabernet wine grape cuttings were inoculated with a slurry dip of freeze-dried WW6+WW7 powder with sodium alginate and reconstituted with well water. The cuttings were dipped in the endophyte slurry immediately before being placed on the mister bench to promote root growth. The root growth results are shown in Figure 74 and demonstrate that cuttings treated with WW6+WW7 endophyte inoculation had a statistically significant 41% increase in root growth, demonstrating a significant increase or improvement in clonal propagation of cuttings from production.

[0263] The combined results of the grapevine cutting propagation experiment and field transplants in Example 65 demonstrated improved clonal propagation of cuttings and continued phenotypic improvement after transplantation, supporting the ability to propagate new varietal traits within plants for production purposes, thereby creating new varieties, through endophyte inoculation and clonal or vegetative propagation.

[0264] 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 have been 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. A method for enhancing nitrogen fixation in a host plant, comprising applying an inoculant composition to the tissue or seeds of the host plant, wherein the inoculant composition comprises at least one heterologous endophyte bacterial strain selected from strains deposited in the ARS Culture Collection under accession numbers NRRL B-68081, NRRL B-68078, NRRL B-68080, and NRRL B-68079.

2. The method according to claim 1, wherein the inoculant composition further comprises at least one nutrient additive that is activating to enhance the survival and colonization of the at least one heterologous endophyte bacterial strain in the host plant.

3. The aforementioned at least one heterologous endophyte strain was grown in a nitrogen-restricted growth medium with exogenous ammonium (NH₃). 4 + The method according to claim 1 or 2, further comprising selecting the ability to produce ).

4. The method according to claim 1 or 2, 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.

5. The method according to claim 1 or 2, wherein the inoculant composition increases nitrogen uptake in the host plant.

6. The method according to claim 1 or 2, wherein the at least one heterologous endophyte strain fixes atmospheric nitrogen.

7. The method according to claim 1 or 2, wherein the at least one heterologous endophyte strain comprises a plurality of heterologous endophyte strains selected from the group consisting of strains deposited with the ARS Culture Collection under accession numbers NRRL B-68081, NRRL B-68078, NRRL B-68080 and NRRL B-68079.

8. The method according to claim 7, wherein the plurality of heterologous endophyte strains include at least two, at least three, or at least four strains independently selected from the group of strains defined in claim 7.

9. The method according to claim 1 or 2, wherein the inoculant composition further comprises at least one additional microbial component selected from Rhizobium species, Mycorrhizae species, and endogenous yeast strains.

10. The method according to claim 1 or 2, wherein the inoculant composition further comprises one or more fungicides, insecticides, herbicides, biostimulants, plant growth regulators, prebiotics, adjuvants, or fertilizers, which are applied to the host plant as separate applications or as a mixture.

11. The method according to claim 1 or 2, wherein the inoculant composition further comprises a carrier composition that enables the application of the plant inoculant composition to seeds.

12. The method according to claim 1 or 2, further comprising encapsulating the at least one heterologous endophyte strain in microbeads and incorporating the microbeads into the inoculant composition.

13. The method according to claim 1 or 2, wherein the inoculant composition comprises a liquid carrier and is applied as a foliar spray.

14. The method according to claim 1 or 2, further comprising applying the inoculant composition in combination with one or more of the following to the host plant, used as separate applications or as a mixture: a penetrating protective agent, an amino acid, an antioxidant, a mineral nutrient, a plant hormone or plant growth elicitor, a carbohydrate, and a mucilaginous gel.

15. The method according to claim 1 or 2, wherein the at least one heterologous endophyte strain increases the enzymatic activity of glutamine synthetase (GS) in the plant body.

16. The method according to claim 1 or 2, wherein at least two of the plurality of heterologous endophyte bacterial strains are co-fermented before application to the leaves of the host plant, and the application of the at least two co-fermented heterologous endophyte strains results in an increase in one of the following characteristics compared to treatment with only one of the heterologous endophyte strains: nitrogen uptake, total chlorophyll in the leaves, glutamine synthetase (GS) enzyme activity in the plant body, total biomass, total plant carbon, amino acid production, resistance of the host plant treated with the inoculant composition to abiotic stress, resistance to plant diseases, total nitrogen accumulation in plant shoots, increased root biomass, increased root branching, root hairs, germination, sprouting, and seedling biomass weight.

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