Compositions including endophytes for improving nutrition, growth and performance of plants and methods of using same

Abstract

Endophyte inoculum compositions, methods of making such compositions, methods of using such compositions, and physiologically altered plant treatments with such compositions are disclosed. The endophyte inoculum compositions may include one or more of endophyte strains WW5, WW6, WW7, and PTD1 that, when applied to non-natural host plants, promote rapid acquisition and uptake of plant mineral nutrients, vigor, health, growth, and yield.

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 318,549, filed on March 10, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to manufactures, compositions and methods for improving plant performance. The novel compositions include non-natural endophytes applied to plants, which incorporation of the endophytes results in measurable plant benefits, such as uptake of nitrogen and other macro- and micronutrients, nutrient use efficiency, photosynthesis, growth, yield, carbon sequestration, tolerance to biotic and abiotic stresses, disease resistance (excluding biological control mechanisms), and general plant health. The invention generally has a wide range of applications for plants, including applications in agricultural crop management, reducing fertilizer and pesticide use, reducing crop carbon footprint, improving crop mineral nutrition status, thereby reducing plant pathogen burden and consequently reducing pesticide use, improving food quality and safety, increasing plant health and biomass growth rate, for landscaping and ornamental plants, and forestry. Background Art

[0003] Endophytes are microorganisms (e.g., fungi and bacteria) that can form symbiotic relationships with trees and plants, through which they can improve plant growth, fruit and seed production, overall health, and other traits. See, e.g., AghaiMatthew M., Khan Zareen, Joseph Matthew R., Stoda Aubrey M., Sher Andrew W., EttlGregory 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.10doi10.3389 / fmicb.2019.01353; RhoHyungmin, Van Epps Victor, Wegley Nicholas, Doty Sharon L., Kim Soo-Hyung, 2018. Salicaceae Endophytes Modulate Stomatal Behavior and Increase Water UseEfficiency in Rice.Frontiers in Plant Science, 9 doi10.3389 / fpls.2018.00188. Endophytes are integrated into plant tissues and can become a heritable part of the plant. Endophytes can insert themselves into plant tissues by inserting between and inside plant cells. Once integrated into the plant and associated with the plant root surface, endophytes can improve plant nutrition by providing and increasing 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.

[0004] Despite the benefits that endophytes provide, their practical application in agriculture is very limited. Consumers and agricultural operators are becoming increasingly aware of the environmental damage caused by fertilizers and pesticides, and are now using more natural alternatives to improve the long-term sustainability of agricultural, livestock and forestry production. Summary of the invention

[0005] The present invention provides novel and inventive compositions comprising bacterial endophytes for non-native plant species, methods of making and using the same, and novel plants produced therefrom. Specifically, one or more of the following endophyte strains may be included in an inoculant composition and applied to a non-native host plant:

[0006]

[0007]

[0008] The species identified in the above table were submitted to the ARS Culture Collection (NRRL) at 1815 N. University Street, Peoria, IL 61604 on November 9, 2021. The 16S rDNA sequence of each of the above endophyte strains is Figure 1-4 Listed in. These strains were found in the branches of willows and poplars, which are not nodulating plants. Therefore, it was unexpected that potential nitrogen-fixing endophytes were present in all branches of these plant species. Through the process of screening a large number of endophytic tree bacterial strain libraries, the endophytic bacterial strains identified above were selected after the following discovery that they were able to grow on nitrogen-free and nitrogen-limited media and enhance the root-to-shoot uptake of N and P. It was further found that these strains provide additional mineral nutrient uptake capacity from roots to shoots when inoculated, and these strains are present in a large number of crop plants that have been specifically tested and screened. Subsequently, these unique strains were identified and isolated for this purpose. In addition, by optimizing the most effective nitrogen fixation, ammonium excretion strains, and specific strains that mobilize insoluble forms of macronutrients (e.g., phosphorus), it was found that the resulting new inoculant formulations increased the plant's acquisition of macronutrient ions and micronutrient ions required by the plant. In addition, the new microbial inoculants disclosed in the present invention were applied to non-native plants, resulting in increased crop growth, biomass and yield. The new inoculant formulations also similarly improved crop yield and quality under scarce, abundant and highly optimized agronomic conditions. These endophyte strains and combinations thereof are not normally present in crop plant varieties and soils used in agricultural production.

[0009] Two of the above endophyte strains have been shown to be newly discovered endophyte species: Curtobacterium salicaceae (WW7) and Rhizobium populi (PTD1). Examples 1B-1C present experimental results demonstrating that the WW7 and PTD1 endophyte strains are new species.

[0010] Whole genome sequence and safety analysis

[0011] All four genomes of the four endophyte strains were fully sequenced and analyzed for genes encoding proteins and / or phenotypes known to be harmful to plants or animals. The whole genome sequences of the endophyte strains were analyzed using the method of Varghese et al. See https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4538840 / . This method reliably classifies strains as safe or potential pathogens by comparing the genome sequence with the genome similarities of known harmful and pathogenic microorganisms. If the organism belongs to a cluster represented by human or plant pathogens, the presence of virulence factor antibiotic resistance genes was evaluated. This pathogen species association was not found in any of the strains. To further confirm that these strains are safe for agricultural food crops, the genomes of the endophyte strains were analyzed for genes known to be associated with plant and human pathogens. No genes associated with pathogens were found in any of the four selected endophyte strains.

[0012] Biochemical analysis of safety

[0013] In addition, endophyte strains are analyzed to determine if they show any biochemical similarities to known pathological biochemistry in mammalian bacterial pathogens. The MacConkey agar test is often used to test whether a bacterial species is Gram-negative. Bacteria that grow well on the agar for more than 24 hours are likely to be Gram-negative. MacConkey agar also uses a neutral pH indicator to test the ability of bacteria to ferment lactose. Fermentation of lactose by bacteria lowers the pH, causing the colonies to turn pink and causing the surrounding medium to become hazy. Bacteria that cannot utilize lactose use peptone and form ammonia, which raises the pH and causes the colonies to turn white or colorless. Some organisms (e.g., Klebsiella and Enterobacter) produce slime-like colonies that appear very wet and slimy. This occurs because the organism is producing a capsule, which is formed primarily from the lactose in the agar.

[0014] Endophyte strains were also subjected to a 24-hour hemolysis test (blood agar lysis) to test the ability of pathogenic bacteria to produce toxins that lyse erythrocytes. Possible results of this test can be no growth, gamma hemolysis (growth without lysis), alpha hemolysis (partial lysis and dark green coloration) or beta hemolysis (clear yellow areas of complete lysis). Mannitol salt agar was used to confirm the presence of Staphylococci, as it is unfavorable to most bacteria except Staphylococci.

[0015] The endophyte strain produced tan colonies when grown on MacConkey agar that were not moist or slimy in appearance. Therefore, the endophyte strain was negative for lactose fermentation, lactone fermentation, and mucus capsule production. The results of the MacConkey agar test indicated that the endophyte strain did not have (1) lactose fermentation as pathogenic species such as Escherichia coli, Enterobacter, or Klebsiella; (2) peptone utilization as pathogenic species such as Salmonella, Proteus, Yersinia, Pseudomonas aeruginosa, or Shigella; or mucus capsule production as pathogenic species such as Klebsiella or Enterobacter. The mannitol salt agar results showed that the growth of the endophyte strain was inhibited, indicating that the endophyte cannot ferment mannitol and therefore does not share this phenotype with any Staphylococcus species. Testing using a hemolysis assay showed that all four strains were gamma-hemolytic (grow but do not lyse any blood cells). The above results cumulatively confirm that no similarities to mammalian bacterial pathogens were found in the endophytic strains and that these four endophytic bacterial strains are safe for agricultural food crop applications and other applications. Figure 5 The results of the above bioassays are provided in.

[0016] The screened, identified endophyte strains are developed into stocks through novel fermentation growth methods for novel inoculant formulations. These formulations are used to treat and improve non-natural host plant species (species where endophytes do not naturally occur). The inoculant compositions of the present invention include additional ingredients that promote long-term stability (long shelf life), delivery, colonization in the host plant, and efficacy. The inoculant compositions of the present invention may include liquid seed treatments, seed coatings, freeze-dried rehydratable seed treatments, encapsulated dry beads, foliar sprays, in-groove liquid products, and other formulations.

[0017] Compositions including non-natural endophytes can 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 can be treated heterologously with the endophyte strains of the present invention. A basic principle of traditional as well as genetically modified plant breeding is to start with the production of "clean" germplasm. This generally means free of microorganisms. Therefore, most propagation material for annual crops is microbially vacuumed. For perennial plants, modern propagation methods use various cleaning procedures (STG), which also result in germplasm free of microorganisms. In contrast to traditional practices, the invention disclosed herein provides bacterial endophytes to heterologous monocots and dicots, resulting in plants that perform better than plants that do not contain endophytes.

[0018] In some embodiments, the host plant treated with the endophyte inoculants disclosed herein can be a plant cultivated by humans for food, feed, fiber, fuel and / or industrial purposes, and can include, but is not limited to, 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, Gossypium truncatum, Gossypium truncatum, Gossypium truncatum, Gossypium truncatum, Gossypium truncatum, Gossypium truncatum, Gossypium truncatum, Gossypium truncatum, Gossypium truncatum, Gossypium truncatum, Gossypium truncatum, barbadense), broccoli (e.g., Brassica oleracea italica), kale (e.g., Brassica oleracea acephala), tomato (e.g., Solanum lycopersicum), rice (e.g., Oryza sativa), barley (e.g., Hordeum vulgare), sugar beets (e.g., Beta vulgaris), peas (e.g., Pisum sativum), potatoes (e.g., Solanum tuberosum), sugar cane (e.g., Saccharum officinarum), bananas (e.g., Musa acuminata and Musa balbisiana), spinach (e.g., Spinacia oleracea), lettuce (e.g., Lactuca sativa), zucchini (e.g., Cucurbita pekinensis), pepo), peppers (e.g., Capsicum annuum), rapeseed (e.g., Brassicanapus), alfalfa (e.g., Medicago sativa), conifers (e.g.,Douglas fir (Pseudotsugamenziesii), loblolly pine (Pinus taeda), and red alder (Alnus rubra), Salicaceae (e.g., Salixsitchensis, Salix nigra), poplars (e.g., Populus trichocarpa, Populus nigra, Populus deltoides, and all hybrid poplar hybrids DxT TxN DxNxT DxN, etc.), eucalyptus (e.g., Eucalyptus rostrata, Eucalyptusstereticornas, Eucalyptus cladocalyx, and Eucalyptus globulus), Rosaceae (e.g., Apple (Malus domestica), Pear (Pyrus communis), European sweet cherry (Prunus avium), Almond (Prunus dulcis), Peach (Prunus persica), Apricot (Prunus armeniaca), and American plum (Prunusamericana). Endophyte strains can be applied to a variety of environments, including host plants grown under greenhouse or field conditions and in a variety of culture methods. Endophyte strains can be applied mechanically, manually, by irrigation, by artificial inoculation, and generally by placement on or in a plant, plant element, plant tissue, seed, seedling, or on or in a plant growth medium so that the treatment is present on or in the plant, plant element, plant tissue, seed, seedling, or plant growth medium in a manner not found in nature.

[0019] In various embodiments, heterologous application can be application to a non-natural host plant variety, application to a plant at a stage of plant development where the endophyte strain does not occur naturally, or in a growth environment where the same endophyte strain does not occur naturally. For example, an endophyte strain naturally found in willow stem tissue is considered heterologous to any tissue of corn, spring wheat, cotton, soybean plants that naturally lack such endophyte strain. In some embodiments, non-naturally occurring application can be the presence of the non-natural endophyte in a host plant tissue or tissue of a different plant element, tissue, cell type, or other physical location in or on a plant that is different from that found in nature. For example, if the endophyte species or strains are heterologously arranged, where the endophyte is normally found in the root tissue of a plant element, but not in the leaf tissue, it is applied to the leaves.

[0020] "Host plant" includes any plant, particularly agronomically important plants, to which non-natural endophytes can be heterologously applied. Endophyte strains detectable in host plants can improve the growth characteristics, stress resistance and / or other characteristics of the host plant. Endophyte strains also improve agronomic traits of crop varieties, such as yield and nutritional content of harvested crop parts, compared to untreated plants without the non-natural endophyte strain. When a non-natural endophyte can be stably detected in a plant or plant element for a period of time, such as days, weeks, months or years, it can colonize the host plant or its element.

[0021] Specific formulations of heterologous endophyte strains provide unique inoculant benefits to plant hosts, enhancing overall plant growth, health, yield and quality, reducing plant resistance to biotic and abiotic stresses, and preventing infection by inducing plant resistance to plant / seed diseases and pests. The ability of endophyte strains to colonize non-native plant hosts has been experimentally demonstrated by various methods, including polymerase chain reaction (PCR) analysis of specific genetic markers on host plant tissues and 16s sequencing of each endophyte strain, detection of specifically selected nitrogen-fixing colony forming units (CFU) isolated from apparently sterile host plant tissues, genetic RFP or GFP fluorescent marker tagging for localization by laser fluorescence confocal microscopy, and other appropriate methods. The presence of endophyte strain genetic material in host plant tissues, CFU in host plant tissues, and other physiological metrics such as increased chlorophyll leaf content, enhanced root branching and growth, increased aboveground biomass, and enhanced leaf mineral nutrient ion content all demonstrate successful colonization of the host plant by the endophyte strain. In addition, biochemical and molecular analyses have characterized the mode of action of endophyte strains to improve performance, yield, mineral nutrition and health of host plants. These analyses include, but are not limited to, acetylene reduction assays, isotope dilution assays, growth on nitrogen-free media, measurement of exogenous ammonium production using quantitative chemical test kits, ICPMS ion concentration profiles of leaf tissue, quantification of exogenous insoluble phosphorus mobilization in liquid cultures using fluorescent dyes, measurement of Fe-siderophore production on CAS plates, bioinformatics and genomics of the genetic pathways responsible for these biochemical traits. Endophyte colonization in non-natural heterologous host plants results in measurable improvements in nitrogen and other macro- and micronutrient uptake, nutrient use efficiency, photosynthesis, growth, yield, carbon capture, tolerance to biotic and abiotic stresses, and general plant health.

[0022] Host plants containing one or more endophyte strains in their tissues exhibit a detectable change in the content of at least one nutritional trait, and this improvement can be transmitted by asexual reproduction (e.g., stem, root or leaf cuttings, layering, splitting, separation, grafting, budding and micropropagation) or seed. Progeny produced by endophyte-associated host plants or tissues thereof, which may have one or more endophyte strains present in their tissues, have an increase in at least one nutritional quality trait compared to untreated plants of the same species. Progeny of cultivars produced from rootstocks, cuttings or tissue cultures may exhibit such phenotypic traits and enhanced performance due to the presence of heterologous endophyte strains in their tissues. The level of the nutritional trait can be measured in asexually propagated progeny, seeds, or progeny grown from seeds of the host plant and compared to the level of the nutritional quality trait in comparable tissues of a reference agricultural plant that does not contain the heterologous endophyte strains. The presence or improvement of a phenotypic trait in asexually propagated or germinated progeny of a host plant as compared to untreated control plants of the same species grown under similar conditions can be measured by various methods including, but not limited to, increased height, total biomass, root mass, aboveground biomass, seed germination, seedling survival, photosynthetic efficiency, seed / fruit number or mass, fruit yield, leaf chlorophyll content, photosynthetic rate, root length, abiotic stress resistance, biotic stress resistance, disease resistance, recovery from blight, turgor pressure, or any combination thereof.

[0023] Selected endophyte strains WW5, WW6, WW7 and PTD1 for treating host plants can be developed as strains through a microbial fermentation process. Microbial strain maintenance and fermentation methods can be used to increase the expression of the nitrogenase gene and maintain the active form of the plasmid. Endophyte strains can be grown on bacterial growth media with nitrogen limitation and other special characteristics (e.g., chelated iron and / or magnesium) to enhance atmospheric nitrogen fixation, macro- and micronutrient solubilization and acquisition, and other beneficial properties of the endophyte strains. Improved nitrogen fixation, P solubilization and acquisition, and micronutrient solubilization and acquisition can provide higher levels of such nutrients to the treated host plants. Nitrogenase upregulation can be induced by growing and fermenting endophyte strains in nitrogen-limited or nitrogen-free growth media, which prompts the endophyte strains to increase absorption and assimilation of atmospheric N2. See, for example, the following reference for a nitrogen-free 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. This upregulation results in increased levels of the active subunit Nif H of the nitrogenase gene, which can be measured by RT-PCR analysis. This analysis was performed on WW5, WW6, WW7 and PTD1 endophyte strains that were subjected to a selective fermentation process and confirmed increased expression of the Nif H subunit.

[0024] Considering the growth and health of the endophyte strain during the fermentation process, the fermentation broth used to ferment the endophyte strain can include various components. The nitrogen-limited medium used in the fermentation process can include 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 does not include nitrates, ammonium salts and other nitrogen sources. The fermentation solution can also include other suitable ingredients, such as yeast extract, agar and other suitable ingredients. The resulting composition can be used as a liquid composition for treating host plants. See, for example, the following references on examples of nitrogen-limited medium: RJ Rennie, Asingle medium for the isolation of acetylene reducing (dinitrogen-fixing) bacteria from soils, Canadian Journal of Microbiology, vol. 27, no. 1, pp. 8–14, 1981. In order to drive the upregulation of microbial nitrogenase genes in endophyte strains, the nitrogen-limited medium can be almost free of nitrogen. However, after fermentation in NLM fermentation broth, the resulting novel composition includes one or more nitrogen components in limited amounts, such as 30-100 mg NH4 in the physiological range. + / L, common amino acids such as glutamic acid, glutamine, histidine, nitrate, nitrite, amino acid and other nutrients. Figure 6 Specific examples of other ingredients that may be present in the fermentation composition are provided.

[0025] At least one endophyte strain (WW7) produces a range of organic acids or other insoluble phosphorus (P) mobilizing compounds (malic acid and citric acid). The endophytes live extracellularly in the apoplast, between plant cells in the vasculature of roots, culms, stems, and branches. Endophytes in culms and stems that exude these compounds also help prevent P from binding to other metals. This strain and another also help solubilize potassium (K) and convert it to soluble forms, similar to how P is mobilized and acquired. Inoculated bacterial endophytes affect P and K mobility through acidification, chelation, and ion exchange reactions.

[0026] Some endophyte strains (WW7, WW5, WW6) also produce exogenous extracellular siderophore compounds, which the plant then further exudes from its roots for mobilizing insoluble micronutrient mineral ions or metals such as iron, magnesium, zinc, copper, nickel, manganese, and other divalent macronutrient cations such as calcium Ca 2+ .

[0027] Combinations of endophyte strains, including co-fermented combinations of two or more endophyte strains disclosed herein, can be applied to a host plant to provide an increased benefit or additional benefit to the host plant compared to the benefit provided by the application of a single endophyte. For example, one endophyte strain that induces a benefit in a host plant may induce that benefit equally well in a plant that is also colonized by a different endophyte strain, which also induces the same benefit or additional benefit in the host plant. In some cases, when two or more endophyte strains are applied heterologously to the same host plant, the host plant may experience greater improvements in specific nutritional traits, growth traits, stress tolerance, and overall health, exceeding the expected improvement in the trait, indicating a synergistic effect of applying multiple endophyte strains to the host plant. Examples 41-51 provide data demonstrating the synergistic effect of multiple endophyte strains in heterologous application. The presently disclosed endophyte strain combinations do not exhibit incompatibility in host plants, which may occur with endophyte strains other than the endophyte strains disclosed herein.

[0028] One or more additional ingredients may be included in the inoculum composition to improve the performance of the heterologous endophyte strain and enhance effective application and colonization of a variety of host plants. The endophyte strains of the present invention may be used to heterologously colonize non-natural host plants. Molecular and microbiological analyses of treated host plant tissues have demonstrated that the endophyte strains heterologously applied to non-natural host plants via the inoculum composition of the present invention have colonized the host plant and established in the tissues of the host plant.

[0029] Composition

[0030] The inoculant compositions of the present invention are provided as liquid suspensions, seed treatments and coatings, foliar sprays, freeze-dried reconstitutable formulations, and solid forms (e.g., in-groove, granular spray-dried / air-dried beads). The inoculant compositions of the present invention may include heterologous endophyte strains WW5, WW6, WW7, PTD1, and combinations thereof. More specifically, the inoculant compositions may include an effective amount of one or more of the WW5, WW6, WW7, and PTD1 strains and one or more additional ingredients to stabilize and enhance the uptake and viability of the endophyte strains, enabling practical use and application of the endophyte strains to seeds, roots, stems, leaves, flowers, bulbs, and other structures of non-natural host plants. In some embodiments, the compositions of the present invention may include additional endophytes or microbial species, such as additional Rhizobium strains, Mycorrhizae species, Bacillus species, Azotobacter species, Azospirillum species, Sphingobium species, Herbiconjux species, biological control 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 are used to promote plant vigor, health, growth, yield, and abiotic and biotic stress resistance.

[0031] The composition may comprise one or more ingredients that promote the delivery, shelf life and / or efficacy of the administered endophyte strain and may include surfactants, buffers, carriers, viscosity enhancers, microbial stabilizers, mineral or clay particles, nutrients, excipients, wetting agents and / or salts. Additional ingredients may exclude compounds including amines, amides and other nitrogen groups to maintain a low nitrogen or substantially nitrogen-free environment for the endophyte strain.

[0032] The present composition can be formulated to be shelf-stable, including liquid, suspension and solid preparations. Shelf-stable preparations can include suspension preparations, dry preparations, powder preparations and preparations containing dry endophyte strains. Under predetermined conditions, the composition can be stably stored for at least 3 weeks or longer. For example, the composition can be stable for 10 weeks or longer at various temperatures, including low temperatures (freezing temperature or near freezing temperature), continuous high temperatures, or at room temperature under standard temperature and pressure (STP) conditions.

[0033] The formulation may include one or more dried endophyte strains, the water content of the endophyte strains being reduced to 30% or less compared to the undried endophyte strains. In some embodiments, the one or more endophyte strains included in the inoculum composition may be freeze-dried. In other embodiments, the endophyte strains may be dried using other methods, such as air drying, drying and / or spray drying. The dried endophyte strains in the inoculum composition may enhance the stability of the endophyte strains therein. In some embodiments, the formulation may contain dried endophyte strains that are substantially stable at temperatures between about -20°C and about 50°C for at least about 4 weeks, and up to one year or more. In some embodiments, the formulation contains a partially hydrated shell that encloses the endophyte in a carbohydrate carrier, such as sodium alginate, calcium alginate or magnesium alginate or other suitable carbohydrate carriers (e.g., Scogin LDH), providing hard, mostly dehydrated round or oval beads. The beads may have an average diameter in the range of about 400 nm to about 5 mm, and may additionally or alternatively contain thickeners, starch, carbohydrate or mineral thickeners, stabilizers and / or carriers.

[0034] In some embodiments, the inoculum composition can include a stabilizer compatible with the endophyte strain, which promotes the viability of the strain, application to the host plant and the colonization of the heterologous endophyte strain in the host plant. Examples of suitable stabilizers include guar gum, xanthan gum, agarose, sucrose, glucose, polysucrose, phytogel, sodium alginate, calcium alginate, magnesium alginate, glycine betaine, methylcellulose, maltodextrin, molasses and mixtures thereof. Other available stabilizers include trehalose, sucrose, glycerol and methylene glycol, glucose, sucrose mineral oil, soy lecithin, peptone, monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), hydroxypropyl-guar gum (HP-Guar), xanthan gum polyvinyl pyrrolidone, polyvinyl pyrrolidone / vinyl acetate (PVP-VA), non-reducing sugars and sugar alcohols such as one or more of mannitol or sorbitol, and 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 therein).

[0035] In some embodiments, the composition can include a carrier such as an agriculturally acceptable carrier, which can be any material that can be added to a plant element without causing or adversely affecting the host plant or its element. The carrier can be a solid carrier or a liquid carrier, and can be in any form, including microspheres, powders, emulsions, various polymers, dry powdered fertilizers such as potash, potassium phosphate, potassium nitrate or other suitable materials. The carrier can be any one or more of several carriers that impart various properties, such as increased stability, wettability, fluidity and / or dispersibility.

[0036] In some embodiments, the agricultural carrier can be a solid, such as diatomaceous earth, loam, silica, magnesium silicate, alginates (e.g., sodium alginate, calcium or magnesium), glycine betaine (natural or synthetic), clay, bentonite, biochar, vermiculite, seed boxes, peat, wheat, bran, talc, lime, starch, cellulose (methylcellulose, hemicellulose), Fuller's earth, pasteurized soil, fertilizer powder or fertilizer salts (macro and micronutrients), other plants, animals or non-biological products, or combinations thereof, including particles, pellets or suspensions. In some embodiments, the solid carrier of the treatment formulation includes, for example, a mineral carrier, such as dolomite, kaolin, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acid clay, vermiculite and perlite, and an inorganic salt, such as calcium carbonate. In addition, organic fine powders such as wheat flour, wheat bran and rice bran can also be used as solid carriers. Mixtures of any of the ingredients are also contemplated as carriers, such as, but not limited to, pasta (flour and kaolin) or flour-based pellets in loam, sand, or clay. In some embodiments, the agricultural carrier may be soil or a plant growth medium, and / or a food source for the cultured organism. In a particular embodiment, the endophyte strain may be encapsulated in calcium alginate, magnesium alginate, agarose, or other suitable material, with or without one or more carbohydrate stabilizers, such as sucrose, glucose, or other suitable sugars. The encapsulated endophyte strain may be included in a suspension or solid formulation, and may be used for seed treatment and coating, foliar application, in-furrow application, as a secreted fertilizer coating for all fertilizers, macronutrients, and micronutrients, including, but not limited to, granular urea, ammonium nitrate, potassium nitrate, potassium phosphate, calcium phosphate, and other embodiments.

[0037] In some embodiments, agricultural carriers can be liquid carriers that impart various characteristics, such as increased stability, wettability, fluidity and / or dispersibility.Liquid carriers can include vegetable oils, such as soybean oil, neem oil, cottonseed oil, and other compositions, such as glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol and other suitable liquids.In some embodiments, carriers can be combinations of liquid ingredients, such as water-in-oil emulsions, or other suitable preparations.For example, water-in-oil emulsions can be prepared to include wettable powders, granules, gels, agar, thickeners, biopolymers, microcapsule particles, etc.Other agricultural carriers that can be used include water, vegetable oils, humectants or combinations thereof.Described compositions can include wetting agents, such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or combinations thereof can be included in compositions of the present invention.When such preparations are used as wettable powders, biocompatible dispersants can be used, such as nonionic, anionic, amphoteric or cationic dispersants and emulsifiers.

[0038] In some embodiments of the present invention, the surfactant that can be included in the composition can include nonionic and / or anionic surfactants. Examples of nonionic surfactants include alkylphenol alkoxylates, alcohol alkoxylates, polyoxyethylene glycerol fatty acid esters, castor oil alkoxylates, fatty acid alkoxylates, fatty amide alkoxylates, fatty polydiethanolamides, lanolin ethoxylates, fatty acid polyethylene glycol esters, isotridecanol, fatty amides, methylcellulose / hemicellulose, fatty acid esters, alkyl polyglycosides, glycerol fatty acid esters, polyethylene glycol, polypropylene glycol, polyethylene glycol / polypropylene glycol block copolymers, polyethylene glycol alkyl ethers, polypropylene glycol alkyl ethers, polyethylene glycol / polypropylene glycol ether block copolymers, polyethylene oxide / polypropylene oxide block copolymers, and mixtures thereof. Examples of anionic surfactants include alkylarylsulfonates, phenylsulfonates, alkyl sulfates, alkylsulfonates, arylalkylsulfonates, alkyl ether sulfates, alkylaryl ether sulfates, alkyl-polyethylene glycol ether phosphates, polyarylphenyl ether phosphates, alkyl-sulfosuccinates, olefin sulfonates, paraffin sulfonates, petroleum sulfonates, taurides, sarcosides, fatty acid salts, alkyl-naphthalenesulfonic acids, naphthalenesulfonic acid and ligninsulfonic acids, condensates of sulfonated naphthalene with formaldehyde or with formaldehyde and phenol, where appropriate with urea, and condensates of phenolsulfonic acid, formaldehyde and urea, ligninsulfite waste liquors and ligninsulfonates, alkyl phosphates, alkylaryl phosphates, for example triphenylethylene phosphates, and polycarboxylates, for example, polyacrylates, maleimide hydride / olefin copolymers, including alkali metals, alkaline earth metals and mixtures thereof.

[0039] In some embodiments, the inoculum composition may include a viscosity enhancer or adhesive to assist in binding the endophyte strain to a carrier that may contain other non-biological compounds. Such compositions may be useful in forming a coating around a plant or plant element (e.g., for seed coating) to maintain contact between the heterologous endophyte and other materials and the plant or plant element. In some embodiments, the adhesive may include one or more alginates, gums, starches, maltodextrins, lecithin, formononetin, polyvinyl alcohol, alkali formononetinate, hesperidin, polyvinyl acetate, cephalin, gum arabic, xanthan gum, carrageenan, PGA, other biopolymers, mineral oil, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), arabinogalactan, methylcellulose, PEG 400, chitosan, polyacrylamide, polyacrylate, polyacrylonitrile, glycerol, triethylene glycol, vinyl acetate, gellan gum, polystyrene, polyethylene, carboxymethylcellulose, hemicellulose, gum ghatti, polyoxyethylene-polyoxybutylene block copolymers, and other suitable agents.

[0040] In some embodiments, one or more adjuvants can be used in the inoculum composition to help improve the delivery and performance of the endophyte strain. The composition of the present invention can be combined with adjuvants to produce a specific product form or mixture, such as a liquid mixture for foliar application. The composition of the present invention may further include other agronomically suitable excipients such as solvents, pH adjusters, viscosity modifiers (rheology modifiers), crystallization inhibitors, defoamers, dispersants, wetting agents, humectants, anti-caking agents, suspending agents, spray droplet regulators, pigments, antioxidants, UV protectants, compatibilizers, chelating agents, neutralizers, corrosion inhibitors, dyes, odorants, spreading agents, penetration aids, lubricants, adhesives, thickeners, freezing point depressants, antimicrobial agents, etc. The composition content of these auxiliary excipients is not particularly limited and can be determined by those skilled in the art according to conventional protocols.

[0041] In some embodiments of the invention, suitable pH adjusters that may be included in the inoculum composition include buffers such as alkali metal salts of weak inorganic or organic acids, for example, 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.

[0042] The inoculant composition may further include a food source for the cultured organism, such as barley, rice, wheat, or other biological material, such as seeds, plant parts, bagasse, husks or straw from grain processing, ground plant material or wood from construction site waste, sawdust or small fibers from paper, fabric or wood recycling.

[0043] The inoculant composition may further include additional components for enhancing plant health and microbial survival, such as biostimulants, prebiotics, amino acids, fatty acids, plant proteins, fungicides, insecticides, nematicides, plant microbial enhancers (prebiotics), plant hormones and inducers, mineral macronutrients and micronutrients (liquid and dry), seed treatment polymers, common dyes, carbohydrates and gels (alginate, mucilage, agarose, guar gum, xanthan gum, etc.) powdered carriers (soy protein, talc, lime, starch biochar, cellulose / hemicellulose, silica, clay, nanotechnology structures including mineral nutrients, such as carbon dot buckyballs, carbon cages or other forms of nanotechnology, etc.). Exemplary biostimulants may include amino acid combinations (e.g., one or more of L-glutamine, L-lysine, L-methionine, L-arginine and L-threonine), fatty acids, vitamins, plant proteins, phosphorus sources, betaine, plant growth factors and other suitable ingredients. Formulations of the endophyte strains discussed herein were combined with commercial biocides and nutraceuticals to test the viability of the endophytes under such conditions. The endophytes were found to be compatible with several of these commercial products in both laboratory and field settings. Based on the compatibility testing results, several new formulations were developed.

[0044] The endophyte strains described herein can be combined with one or more of the above agents to produce a composition suitable for application to a plant or its tissue, seedling, seed or other plant element. Endophyte populations can be obtained from selection and growth in culture as described herein and added to the composition. Endophytes in different growth stages can be used. For example, endophytes in delayed phase, early log phase, mid-log phase, late log phase or stationary phase can be used.

[0045] The above ingredients may be combined in a liquid composition that may include an effective amount of one or more of the endophyte strains WW5, WW6, WW7 and PTD1. For example, the inoculant composition of the invention may include the endophyte strain in an amount of about 0.1 to 90% by weight of the wet weight of the composition, for example, between about 1% and 80%, between about 5% and 70%, between about 10% and 60%, between about 15% and 50%. The inoculant composition may include at least about 10 3 CFU / mL, for example, 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 of values ​​therein.

[0046] An exemplary liquid formulation according to the present invention may include two or more dried or undried endophyte strains (e.g., WW6 and WW7 prepared by co-fermentation), each endophyte strain having a concentration of about 10 8 CFU / mL to about 10 9 CFU / mL; one or more monosaccharides or disaccharides (e.g., 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, % to about 0.05 wt %; CaCl2 in an amount of about 0.005 wt % to about 0.05 wt %; Na2FeEDTA 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. Another exemplary liquid formulation may include one or more dried or undried endophyte strains, each strain having a concentration ranging from about 10 4 -10 10 CFU / mL, alone or in combination with one or more of the following: low viscosity alginates (e.g., sodium alginate, magnesium alginate, calcium alginate, LDH (Dupont or other high purity alginate) in an amount of about 0.1% v / v to about 5% wt%; glycerol in an amount of about 0.1% v / v to about 5% v / v; and monosaccharides and disaccharides (e.g., glucose and lactose) in an amount of about 1 wt% to about 10 wt%. Another exemplary liquid formulation may include one or more dried or undried endophyte strains, each strain in a concentration range of about 10 4 -10 10 CFU / mL, alone or in combination with one or more of the following: low viscosity alginates (e.g., sodium alginate, magnesium alginate, calcium alginate, LDH (Dupont or other low viscosity high purity alginate) 0.5-50% w / v; gelatin in an amount of about 1% v / v to about 5% wt%, PEG in an amount of about 1% v / v to about 10% v / v; and monosaccharides and disaccharides (e.g., glucose and lactose) in an amount of about 1 wt% to about 10 wt%.

[0047] In some embodiments, the composition may be a suspension formulation comprising the aforementioned ingredients in the aforementioned proportions. In some embodiments, the composition may further comprise one or more solid carriers, thickeners or fillers. Such ingredients may include inorganic mineral soils 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 hydrophilic colloids, polymers, cellulose, methylcellulose and / or hemicellulose powders and combinations thereof. The suspension composition may further comprise a wetting agent, an emulsifier, an anti-caking agent, a suspending agent, a freezing point depressant, and the like. In some embodiments, the suspension formulation may include one or more endophyte strains WW5, WW6, WW7 and PTD1, the concentrations of which are disclosed in the preceding paragraph. An exemplary suspension formulation according to the present invention may include one or more dried endophyte strains, each endophyte strain having a concentration of about 10 8 CFU / mL to about 10 9 CFU / mL, microencapsulated in sodium alginate, calcium or magnesium (e.g., by a spray drying process). The formulation may further include one or more monosaccharides or disaccharides (e.g., sucrose) in an amount of about 1 wt% to about 10 wt%; and glycerol in an amount of about 1 wt% to about 20 wt%.

[0048] In some embodiments, the composition can be a solid composition. The solid composition can be a dry, granular or flowable composition for dispersion or suspension in an aqueous solution prior to delivery to the plant. The dry fertilizer composition can form a fully dispersed suspension. In other cases, the dry fertilizer composition can provide a slow release (such as by low water solubility or by encapsulation, such as sodium alginate), for example when a stable or controlled delivery of nutrients over time is required. The solid composition can include an amount of endophyte strains WW5, WW6, WW7 and PTD1, which ranges from about 10 3 CFU / mL to at least about 10 10 CFU / mL, or any value or range of values ​​therein. For example, the solid composition may include one or more endophyte strains WW5, WW6, WW7, and PTD1, each endophyte strain having a concentration of about 10 8 CFU / mL to about 10 9CFU / mL. A solid formulation according to the present invention may include 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 50 wt%, or any value or range of values ​​therein). An exemplary solid formulation according to the present invention may include one or more dried endophyte strains, each endophyte strain having a concentration of about 10 4 CFU / mL to about 10 10 CFU / mL, microencapsulated in alginate beads (sodium, calcium or magnesium 0.1-10% w / v) and solids (starch 0.1-10% w / v), and dripped by proprietary slurry preparation, batch dripping, ion exchange and fluidized bed drying process. The formulation may further include one or more monosaccharides or disaccharides (e.g., sucrose) in an amount of about 1wt% to about 10wt%; and clay (e.g., zeolite, bentonite and / or other clay materials) in an amount of about 30wt% to about 50wt%.

[0049] Application

[0050] The compositions described herein comprising one or more endophyte strains can be applied to plants to increase the growth characteristics, health, stress resistance and improve other characteristics of plants. The compositions of the present invention can be mechanically or manually or artificially inoculated into plants or their elements by any of many methods, such as but not limited to seed treatment, root washing, seedling soaking, soil inoculant, in-ditch application, leaf spray, leaf coating, side application, wound inoculation, irrigation, fertilization, soaking, injection, osmotic initiation, hydroponics, aeroponics, aeroponics or any combination thereof. In some embodiments, the compositions of the present invention can also be directly applied to plants or parts of plants, such as leaves, roots, foliars, tillers, flowers, plant cells, plant tissues or combinations thereof. The compositions of the present invention can be applied to seeds (e.g., as coatings or by spraying or soaking to treat seeds, etc.), and / or before emergence (before the seedlings emerge or appear on the ground). The compositions of the present invention can also be applied to other propagation materials of plants, such as grains, some fruits, tubers, spores, cuttings, grafts, meristems, plant cells, nuts or embryos. In some examples, the composition can be applied as part of a dip to the host plant roots and / or other tissues, as a seed coating, as a coating applied to the host plant leaves and / or other elements, as a powder applied to the surface of the host plant leaves and / or other elements, as a spray applied to the host plant leaves and / or other elements, as part of a dropper applied to the host plant soil and / or roots, or other appropriate methods. The composition of the present invention can also be applied to the growth medium (e.g., by applying to the soil surrounding the plant).

[0051] Compared to untreated control plants of the same species grown under similar conditions, the inoculum compositions of the present invention can improve phenotypic traits measured by various methods, including but not limited to increased height, total biomass, total carbon, root mass, aboveground biomass, seed germination rate, seedling survival rate, photosynthetic efficiency, seed / fruit quantity or quality, fruit yield, chlorophyll content of leaves, photosynthetic rate, root length, abiotic stress resistance, biotic stress resistance, disease resistance, wilt recovery, turgor pressure or any combination thereof. Rootstocks, cuttings or tissue cultures of host plants can be used to produce cultivars that exhibit such phenotypic traits and enhanced performance. In addition, the application of inoculum compositions can increase carbon fixation of treated host plants. This is an economically attractive benefit because it results in the removal of carbon dioxide from the atmosphere, as well as increased biomass. Indicators of plant acquisition of more carbon include greater CO2 fixation activity, greater dry weight to fresh weight ratio, and total biomass.

[0052] Treatment with the composition of the present invention can result in increased uptake of macro- and micronutrients from the soil and atmosphere. Treatment with the composition of the present invention can result in increased nitrogen uptake rates. Faster nitrogen uptake rates also contribute to significant improvements in utilization efficiency. Host plants treated heterologously with the composition of the present invention show greater total nitrogen uptake, with high levels of nitrogen utilization efficiency, resulting in more protein production, and promoting increased biomass yield. Application of the composition of the present invention also results in greater nutrient uptake and utilization of other macro- and micronutrients. Experimental results show that in host plants treated with the inoculum composition of the present invention, macronutrient potassium, phosphorus, calcium and magnesium increased, as well as micronutrient boron, copper, iron, manganese, molybdenum, nickel, sulfur and zinc uptake increased - see, for example, Examples 5-7 below. Heterologous endophytes can take fixed or stubborn forms of certain nutrients (including phosphorus) in the soil and convert them into soluble forms that can be more efficiently utilized by host plants. Endophyte strains are also able to produce siderophores that are beneficial to host plants, which chelate Fe in plant tissues to assist in Fe uptake by host plants.

[0053] Increases in macro- and micronutrient uptake are accompanied by increases in catabolism, carbon uptake, and carbon capture. Host plants treated with the compositions of the present invention show greater total carbon uptake and associated increases in RuBisCo carboxylation activity, carbon quality, and biomass. Host plants may also exhibit increased aromatic amino acid production via the shikimate pathway. These aromatic amino acids serve as precursors for a wide range of secondary metabolites that are important for plant resistance to biotic and abiotic stresses (e.g., oxidative, drought, and / or salt stress).

[0054] Therefore, the application of the composition can lead to an increase in the adaptive tolerance of the host plant to abiotic and biotic stresses such as disease, cold, salinity, etc. In an exemplary embodiment, the innate and adaptive tolerance to stress is significantly improved. Abiotic stress factors include extreme temperatures, high salinity, drought and other causes. Abiotic stress factors can significantly reduce yield and biomass, and often kill plants. Lower growth temperatures are often encountered in cereal agricultural production, especially in the early stages of the growing season, and can be used to stress plants in several ways, first by poor germination, then by stunted seedling growth, yellowing of leaves, reduced leaf expansion, wilting and tissue death. Cold stress severely inhibits the development of plant reproductive parts. Crop yields are reduced due to cold stress, which is proportional to the degree of damage to the plant. As salt accumulates in irrigated soil, high salt concentrations in soil or water are an increasingly serious problem, and irrigation water with higher salt concentrations must be used. The impact of high salt concentrations can be referred to as osmotic stress, because high salt concentrations in soil and water interfere with the transport of ions and water in plants. Symptoms of high salt stress include growth inhibition, wilting, chlorosis, leaf drop, senescence and death. The increased nutrient uptake and utilization efficiency resulting from the presence of the endophyte strains in the host plant protected the host plant in a stress-inducing environment, and the host plants exhibited greater growth and biomass even under abiotic stress conditions.

[0055] Application of the composition to the host plant also improves adaptive tolerance through advanced mineral nutrition, thereby generating biotic stress tolerance that inhibits viruses, bacteria and fungi. The endophyte strains of the present invention provide tolerance in the host plant, which may be a result of activation of induced systemic resistance (ISR) and / or other metabolic mechanisms in the plant. Thus, the endophyte strains may be applied to the host plant or its seeds as a nutritional treatment to help defend against pathogenic fungi, viruses and bacteria.

[0056] Other aspects, objects and advantages of the invention will be apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 SEQ ID NO. 1 is shown.

[0058] Figure 2 SEQ ID NO. 2 is shown.

[0059] Figure 3 SEQ ID NO. 3 is shown.

[0060] Figure 4 SEQ ID NO. 4 is shown.

[0061] Figure 5 It is a table that provides microbiological measurement data.

[0062] Figure 6 is a table providing exemplary nutritional ingredients for fermented compositions.

[0063] Figure 7 A provides images related to the experiments of Example 1A.

[0064] Figure 7 B is a table providing data related to the experiments of Example 1A.

[0065] Figure 7 C is a table providing data related to the experiments of Example 1A.

[0066] Fig. 8A This is a table related to the experiment of Example 1B.

[0067] Figure 8B This is a table related to the experiment of Example 1B.

[0068] Figure 8C This is a table related to the experiment of Example 1B.

[0069] Fig. 9 is a table related to the experiment of Example 1C.

[0070] Fig. 10A This is a table related to the experiment of Example 2B.

[0071] Fig. 10B This is a table related to the experiment of Example 2B.

[0072] Fig.11 is a table related to the experiment of Example 2C.

[0073] Fig.12 This is a table related to the experiment of Example 2D.

[0074] Fig.13A Enzyme pathways relevant to the experiments in Example 3 are provided.

[0075] Fig. 13B This is a table related to the experiment of Example 3.

[0076] Fig. 13C This is a diagram related to the experiment of Example 3.

[0077] Fig.14A Enzyme pathways relevant to the experiments in Example 4 are provided.

[0078] Fig. 14B Images related to the experiments of Example 4 are provided.

[0079] Fig. 14C This is a table related to the experiment of Example 4.

[0080] Fig.14D This is a diagram related to the experiment of Example 4.

[0081] FIG. 15 is a diagram related to the experiment of Example 5.

[0082] Fig.16 Images related to the experiments of Example 6 are provided.

[0083] Fig.17A Images related to the experiments of Example 7 are provided.

[0084] Fig. 17B Images related to the experiments of Example 7 are provided.

[0085] Fig.18A This is a table related to the experiment of Example 8.

[0086] Fig.18B This is a table related to the experiment of Example 8.

[0087] Fig.19 This is a table related to the experiment of Example 9.

[0088] Fig. 20 This is a table related to the experiment of Example 10.

[0089] Fig.21 This is a table related to the experiment of Example 11.

[0090] Fig. 22 This is a table related to the experiment of Example 12.

[0091] Fig.23 This is a table related to the experiment of Example 13.

[0092] Fig.24 This is a table related to the experiment of Example 14.

[0093] Fig.25A This is a table related to the experiment of Example 15. Fig.25B This is a diagram related to the experiment of Example 15.

[0094] Fig.25C This is a diagram related to the experiment of Example 15.

[0095] Fig.26A This is a table related to the experiment of Example 16.

[0096] Fig.26B This is a diagram related to the experiment of Example 16.

[0097] Fig. 27 This is a diagram related to the experiment of Example 17.

[0098] Fig.28A This is a diagram related to the experiment of Example 18.

[0099] Fig.28B This is a diagram related to the experiment of Example 18.

[0100] Fig.29 This is a diagram related to the experiment of Example 19.

[0101] Fig. 30A This is a table related to the experiment of Example 20.

[0102] Fig. 30B This is a table related to the experiment of Example 20.

[0103] Fig.31A This is a table related to the experiment of Example 21. Fig.31B This is a diagram related to the experiment of Example 21.

[0104] Fig.31C This is a diagram related to the experiment of Example 21.

[0105] Fig.32 This is a table related to the experiment of Example 22.

[0106] Fig.33A This is a table related to the experiment of Example 23.

[0107] Fig.33B This is a diagram related to the experiment of Example 23.

[0108] Fig.34A This is a table related to the experiment of Example 24.

[0109] Fig.34B Images related to the experiments of Example 24 are provided.

[0110] Fig.35 Images related to the experiments of Example 25 are provided.

[0111] Fig.36A This is a table related to the experiment of Example 26. Fig.36B This is a diagram related to the experiment of Example 26. Fig.36C Images related to the experiments of Example 26 are provided.

[0112] Fig.36D This is a diagram related to the experiment of Example 26.

[0113] Fig.36E This is a diagram related to the experiment of Example 26.

[0114] Fig.37A This is a table related to the experiment of Example 27.

[0115] Fig.37B This is a diagram related to the experiment of Example 27.

[0116] Fig.38 This is a diagram related to the experiment of Example 28.

[0117] Fig.39 This is a diagram related to the experiment of Example 29.

[0118] Fig.40A Images related to the experiments of Example 30 are provided.

[0119] Fig.40B Images related to the experiments of Example 30 are provided.

[0120] Fig.41 This is a diagram related to the experiment of Example 31.

[0121] Fig.42A This is a diagram related to the experiment of Example 32.

[0122] Fig.42B This is a diagram related to the experiment of Example 32.

[0123] Fig.43A This is a diagram related to the experiment of Example 33.

[0124] Fig.43B This is a diagram related to the experiment of Example 33.

[0125] Fig.44 This is a diagram related to the experiment of Example 34.

[0126] Fig.45A This is a diagram related to the experiment of Example 35.

[0127] Fig.45B Images related to the experiments of Example 35 are provided.

[0128] Fig.46 This is a table related to the experiment of Example 36.

[0129] Fig.47 This is a table related to the experiment of Example 37.

[0130] Fig.48 This is a table related to the experiment of Example 38.

[0131] Fig.49 This is a diagram related to the experiment of Example 39.

[0132] Fig.50 This is a diagram related to the experiment of Example 40.

[0133] Fig.51 This is a diagram related to the experiment of Example 41.

[0134] Fig.52 This is a diagram related to the experiment of Example 42.

[0135] Fig.53A This is a diagram related to the experiment of Example 43.

[0136] Fig.53B This is a diagram related to the experiment of Example 43.

[0137] Fig.54 This is a diagram related to the experiment of Example 44.

[0138] Fig.55 This is a diagram related to the experiment of Example 45.

[0139] Fig.56 This is a diagram related to the experiment of Example 46.

[0140] Fig.57 This is a diagram related to the experiment of Example 47.

[0141] Fig.58 This is a diagram related to the experiment of Example 48.

[0142] Fig.59 This is a diagram related to the experiment of Example 49.

[0143] Fig.60A This is a diagram related to the experiment of Example 50.

[0144] Fig.60B This is a diagram related to the experiment of Example 50.

[0145] Fig.60C This is a diagram related to the experiment of Example 50.

[0146] Fig.61 This is a diagram related to the experiment of Example 51.

[0147] Fig.62 This is a diagram related to the experiment of Example 52.

[0148] Fig.63 This is a diagram related to the experiment of Example 53.

[0149] Fig.64 This is a diagram related to the experiment of Example 54. DETAILED DESCRIPTION OF THE INVENTION

[0151] Reference will now be made in detail to certain embodiments of the present invention, as well as to the exemplary compositions and applications of these embodiments. Although the present invention will be described with reference to these embodiments, it should 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 included within the spirit and scope of the present invention as defined by the claims. In the following disclosure, specific details are given to provide a comprehensive understanding of the present invention. However, it will be appreciated by those skilled in the art that the present invention can be implemented without these specific details.

[0152] method:

[0153] Selection and growth methods and compositions

[0154] The invention includes a method for growing and selecting nitrogen-fixing endophyte strains. The method includes inoculating a special nitrogen-limited or nitrogen-free growth medium and selecting colonies that can reproduce on the special growth medium. The ability of the endophyte strains to grow on nitrogen-free and nitrogen-limited media is evaluated.

[0155] Example 1A. Endophyte Selection and Growth

[0156] Each of the WW5, WW6, WW7 and PTD1 endophyte strains was tested and found to be positive for the ability to grow on nitrogen limited medium (NLM), and each strain grew to varying degrees on nitrogen limited medium, as shown in the figure below. Each endophyte strain was tested and found to be positive for the ability to grow on plant tissue culture grade agarose plates containing nitrogen limited medium (NLM pH 7.6), and each strain grew to varying degrees on plant tissue culture grade agarose plates containing nitrogen free medium (NFCCM pH 7.6), as shown in the figure below.

[0157] A fermentation mixture containing 10 mL of broth culture was prepared in a 50 ml conical tube. Each culture was inoculated with 100 μL of a standardized QC broth culture of an endophyte strain. The conical tube was placed on a shaker at a 45° angle and incubated at room temperature, shaking at 200 rpm for 72 hours and measuring the optical density (OD) of each culture at 600 nm. 100 μL of 10-5 and 10-6 dilutions of each endophyte strain were inoculated on NLM agar plates. The plates were then incubated at 25°C for 72 hours. The CFU formed on the plates was observed and recorded. Figure 7 A shows visual evidence of colony growth on NLM plates.

[0158] It was also analyzed whether the endophyte strains could produce ammonium in liquid fermentation under aerobic conditions. Individual assays for each of the WW5, WW6, WW7, and PTD1 endophyte strains showed that each was able to produce ammonium under such conditions. These assays demonstrated the ability of the endophyte strains to participate in the N2 fixation mechanism in plants and correlated with the growth effects observed after inoculation with endophyte fermentation broth treatments.

[0159] Each strain was tested and found to produce ammonium (NH 4+ ) were positive; MGL (mannitol-glutamate / Luria-Bertani), NLM (nitrogen-limited medium), MCDY (M series yeast medium nitrogen base with amino acid supplement) and CS+KNO3 (corn syrup + KNO3), such as Figure 7 As shown in B. The ammonium concentration of all sterile culture media is negative, less than or equal to 0 mg / L.

[0160] The ability of endophyte strains to fix nitrogen within tree or plant tissues is achieved by the microbial nitrogenase gene within the endophytic bacteria. The presence of the nitrogenase gene in strains WW5, WW6, WW7 and PTD1 was detected by PCR using primers specific for Nif H. Each endophyte strain was found to include a copy of an active Nif H gene subunit.

[0161] The ability of the endophyte strains to fix nitrogen was further measured by an acetylene reduction assay. The acetylene reduction assay measures the ability of nitrogenase to reduce acetylene gas to ethylene using gas chromatography to quantify the amount of ethylene produced. This is an indirect measure of N2 fixation capacity, measuring the functional presence of nitrogenase through its associated production. Endophyte strains WW6, WW7, and PTD1 exhibited acetylene reduction activity, as Figure 7 As shown in C.

[0162] For administration purposes, the selected WW5, WW6, WW7 and PTD1 cells were grown in nitrogen-limited medium for 1-3 days until the endophyte strains were approximately 10 7 CFU / mL to about 10 10 CFU / mL concentration in the culture medium. In some embodiments, two or more endophyte strains can be combined and co-fermented to produce a combined concentration of about 10 3 CFU / mL to about 10 9 CFU / mL of fermentation broth, for example, 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 108 CFU / mL, at least about 10 9 CFU / mL or any value or value range therein. Fermentation process conditions can include a predetermined incubation temperature within 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 value range therein), a fermentation vessel is shaken at a speed within the range of about 25rpm to about 300rpm (e.g., about 75rpm to about 250rpm, about 125rpm to about 225rpm, about 200rpm or any value or value range therein), and a fermentation volume of about 1L to about 10L (e.g., about 2L to about 8L, about 4L to about 6L, about 4L, about 2L or any value or value range therein).

[0163] To drive upregulation of microbial nitrogenase genes in endophytic strains, the nitrogen-limited medium may contain almost no nitrogen, but may include one or more sugars, such as mannitol, mannose, sucrose, glucose, fructose, lactose, and other suitable sugars. The nitrogen-limited medium may also include 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 not nitrates, ammonium salts, and other nitrogen sources. The fermentation solution may also include other suitable ingredients, such as yeast extract, agar, and other suitable ingredients. The resulting composition may be used as a liquid composition for treating host plants. See, for example, the following references to examples of nitrogen-limited medium: RJ Rennie, A single medium for the isolation of acetylenereducing (dinitrogen-fixing) bacteria from soils, Canadian Journal of Microbiology, vol. 27, no. 1, pp. 8–14, 1981.

[0164] Example 1B, Genome Analysis of Curtobacterium salicaceae (WW7)

[0165] Curtobacterium salicaceae (WW7) is a novel species of nitrogen-fixing bacteria that occurs naturally in willow, grass phyllosphere (leaves), leaf litter / soil, and corn roots. WW7 also produces the organic acids malic and citric acid, which solubilize insoluble forms of phosphate, and Fe siderophores, which solubilize insoluble forms of iron. Curtobacterium salicaceae was isolated from the trunk vascular system of willow (Sitka sitchenses).

[0166] WW7 was sequenced by the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) using the Illumina MiSeq platform. Paired-end libraries were constructed from 376 ng of gDNA using the Nextera DNA Flex Library Preparation Kit and loaded into a flow cell. The libraries were barcoded for mixing with 11 samples and sequenced using a 2×250-bp format. MiSeq runs were performed using the MiSeq Reagent Kit v3 (600 cycles) chemistry. Shotgun sequencing yielded 1,530,321 reads. After trimming, quality filtering, and removal of possible 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 the multi-k-mer method (k=77, 95, and 127).

[0167] The genome of Curtobacterium salicaceae (WW7) strain consists of 18 scaffolds (N 50 =329,216 bp), with a length of 3,489,963 bp and a G+C content of 71.35%, corresponding to ~84× coverage. The WW7 genome completeness was calculated using CheckM v1.0.8 based on the presence of Actinomycetales lineage-specific single-copy marker genes. In this regard, 99% completeness was achieved. Gene prediction was performed on the assembly draft using Prokka v1.11 (7). Of the 3,363 predicted genes, 3286 were protein-coding genes, 53 were tRNAs, 12 miscRNAs, 1 tmRNA and 11 rRNAs. A total of 1,114 genes were assigned to clusters of orthologous groups (COGs), 1,082 were annotated with enzyme commission (EC) numbers, and 1,722 were assigned to KEGG orthologs (KOs).

[0168] To classify WW7 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). Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. Doi:10.1093 / bioinformatics / btu153), and (ii) calculation of within-species probabilities using the genome-wide average nucleotide identity (gANI) strategy described by Varghese et al. (see Richter M and Rossello-Mora R. (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA. 106:19126-19131). In this regard, the WW7 genome was compared with all publicly available Curtobacterium genome assemblies in the NCBI GenBank database: there are a total of 107 Curtobacterium genomes. By using the ANIm algorithm, all genomes were aligned to each other and the ANI values ​​were used to construct an adjacency matrix. This matrix was converted into a similarity matrix ( Fig. 8A), and a cutoff of 0.9 (corresponding to 90% ANI) was used to extract closely related genome clusters. WW7 is closely related to Curtobacterium herbarum DSM 14013 (ASM1690733v1) and seven new Brevibacterium strains isolated from leaf litter in Southern California (NCBI BioProject accession number: PRJNA391502): Brevibacterium MCPF17_052 (NCBI Assembly ID: ASM323408v1), Brevibacterium MCPF17_047 (NCBI Assembly ID: ASM323404v1), Brevibacterium MCPF17_031 (NCBI Assembly ID: ASM323403v1), Brevibacterium MCPF17_011 (NCBI Assembly ID: ASM323414v1), Brevibacterium MCPF17_001 (NCBI Assembly ID: ASM323461v1), Brevibacterium MCLR17_032 (NCBI Assembly ID: ASM323462v1), and Brevibacterium MCLR17_033 (NCBI Assembly ID: ASM323403v1). Assembly ID: ASM323479v1), Brevibacterium MCBD17_030 (NCBI Assembly ID: ASM322425v1) - see Fig. 8A In addition, WW7 is distantly related (84%) to two strains isolated from leaf litter in Massachusetts (MCBA15_007 (ASM186490v1), MCBA15_005 (ASM186485v1)) and Curtobacterium pusillum isolated from corn roots (NCBI Assembly ID: ASM202564v1). <ANI)。

[0169] To further evaluate the relationship between WW7 strain and Brevibacterium type strains, the paired digital DNA-DNA hybridization value (dDDH) of WW7 strain was calculated to determine its interspecies relatedness with Brevibacterium representatives (type strains). The paired dDDH value between WW7 and Brevibacterium type strains was less than 70%, indicating that WW7 strain is a representative of a new species of Brevibacterium, such as Figure 8BSee Kim MK, Kim YJ, Kim HB, Kim SY, Yi TH, Yang DC 2008. Curtobacterium ginseng isoli sp. nov., isolated from soil of a ginseng field. Int J Syst Evol Microbiol. 58(10): 2393–7. Similarly, the Genomic Taxonomy Database (GTDB) identified WW7 as the only member of the new species cluster, Curtobacterium flaccumfacies (https: / / gtdb.ecogenomic.org / species?id=Curtobacterium%20flaccumfaciens), further supporting that the strain has no relationship with any known species of Curtobacterium.

[0170] Phylogenetic distances were also calculated according to the Anvi'o pan-genome pipeline using 605 concatenated single-copy protein-coding genes assigned to clusters conserved among all GTDB Pumilus representatives. The resulting phylogenetic tree showed a clear separation of strain WW7 from other Pumilus species, identifying C. herbarum as the closest type strain. Figure 8C . The Anvi'o pan-genome analysis pipeline was used to identify 605 single-copy core genes and generate partition files. Partition analysis was then performed with 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 Brevibacterium strains according to the List of Permanent Prokaryotic Names (LPSN) database. Clavibacter michiganensis was used as an outgroup.

[0171] Example 1C. Genome Analysis of Rhizobium populi (PTD1)

[0172] To evaluate the relationship between strain PTD1 and Rhizobium-type strains, the pairwise digital DNA-DNA hybridization values ​​(dDDH) of strain PTD1 were calculated to determine its interspecific relatedness with representatives of the genus Rhizobium (type strains). The pairwise dDDH values ​​between PTD1 and Rhizobium-type strains were less than 70%, indicating that strain PTD1 was a representative of a new species of the genus Rhizobium, such as Fig. 9 shown.

[0173] Example 2A: Fermentation preparation of dual-strain product under nitrogen-limited medium conditions.

[0174] Cultivation of two endophyte strains WW6 and WW7: Each strain is first plated on a nitrogen limited medium (NLM) semi-solid medium. Three to four colonies can be selected and used to inoculate a 2-liter seed culture of fresh sterile NLM medium. The 2-liter flask can be grown for 72 hours at about 25°C to about 30°C with constant agitation at about 200 rpm and about 500 rpm. Upon completion, the two strains can be pooled into a large bottle and stored at 4°C before inoculating 4500 liters of NLM medium (pH 7.6). Commercial scale fermentation medium can be sterilized in a 30,000 liter commercial steam jacketed fermentor. Co-fermentation can be carried out for 3 days at an aeration rate of 20 PSI. When plated on tryptic soy broth agar (TSBA), the colony forming units obtained for the two strains were 2.2 x 10 8 CFU / mL to 1.23 x 10 9 CFU / mL WW6 and 1.03x10 8 CFU / mL to 1.4 x 10 9 CFU / mL WW7. This procedure is applicable to other combinations of endophyte strains, such as any combination of WW5, WW6, WW7, and PTD1.

[0175] Example 2B: An endophyte composition prepared by mixing two commonly used dry fertilizers and dry powder is used in combination in agriculture.

[0176] Two new compositions were prepared containing WW6 and WW7 endophyte strains, sodium alginate (DuPont Nutrition USA, IN) and dry fertilizer; triple superphosphate 0-28-0 (OCP group), and single superphosphate plus dolomitic lime (Down To Earth, Inc). 3 mL of the mixture containing WW6+WW7+0.5% alginate was added to 5 grams of dry fertilizer respectively. The composition was dried at room temperature for 24 hours and stored. The bacterial counts of colony forming units (CFU / gram) of the two strains were then determined by plating on NLM semi-solid medium.

[0177] Fig. 10A The results presented in showed that the viability of both endophyte strains was slightly reduced when mixed with different fertilizers in the novel composition. These results suggest that the liquid endophyte composition can be used in novel compositions to be put into commercial agricultural practices as NUE endophyte-enhanced agricultural fertilizers.

[0178] In addition, a liquid fermentation composition containing WW6 and WW7 endophyte strains plus sodium alginate (DuPont Nutrition USA, In) was used to produce a new composition that, when combined with whey protein (Chemital técnicas alimentarias), sodium bentonite (Specialty Minerals, Inc) and coconut bran (W. Atlee Burpee & Co), contained three different powdered dry carriers. The compositions were prepared by adding 3 ml of a liquid composition containing a mixture of WW6+WW7+0.5% alginate to 5 grams of different dry powder carriers. The powdered carrier endophyte compositions were dried separately at room temperature for 24 hours and then stored, and then counted for colony forming units (CFU / gram) by plating on NLM semi-solid medium.

[0179] The results are Fig. 10B The viability of both endophyte strains was slightly reduced when mixed with different dry powder carriers. This suggests that endophyte compositions can be prepared and used for mixing, coating and delivery to commercial agricultural practices when used with different dry carriers.

[0180] Example 2C: The survival rate of endophyte strains (WW6 and WW7) was determined after freeze drying and combination with different powdered carriers and mycorrhizal powders.

[0181] The compatibility of various powdered carrier mixtures (maltodextrin, sucrose, glucose, whey) with the FD powder mixture of WW6+WW7 was determined. The mixture ratios tested were as follows: powdered carrier 2.09 g (~95 wt%), freeze-dried, powdered WW6+WW7 0.11 g (~5 wt%), mycorrhiza 0.0022 g (~0.1 wt%). The results of the compatibility test showed that Fig.11 The following results are shown in .

[0182] Compatibility testing of freeze-dried WW6+WW7 mixed with four possible bulking agents (maltodextrin, sucrose, glucose, and whey) and mycorrhizae gave positive results. The one powdered carrier that reduced CFU the most was a whey product, which only slightly inhibited WW6. Maltodextrin and glucose slightly reduced the CFU of the WW6 strain. The other powdered products did not reduce the CFU much at all. Sucrose did not reduce the CFU of either strain in the assay and was the best powdered carrier.

[0183] The example results demonstrate the ability to mix freeze-dried endophyte powders into various carriers that can dilute the freeze-dried inoculum to lower levels to enable use in fertilizer coatings or as a reconstituted powder for subsequent resuspension and various aqueous foliar applications.

[0184] Example 2D. Short-term and long-term survival of endophyte strains (WW5, WW6, WW7, PTD1 and WP1) after 1-2 days and two weeks when combined with powdered carrier biochar alone and with a biochar plus carbohydrate molasses composition.

[0185] Biochar was treated with co-fermented inoculum compositions of WW5, WW6, WW7, PTD1, and WP1, alone and in combination with 1 / 10X molasses solution. Powdered biochar material was dried in open bags at room temperature and stored at 25°C. After two weeks, dried biochar (0.1 g) was resuspended in 1 mL potassium phosphate buffer and the survival of endophytes was determined by strain, with results reported as CFU / ml. Data are available at Fig.12 Available in.

[0186] The results showed that using 50 μL of the co-fermented endophyte mixture and 950 μL of 1 / 10x molasses per 1 g of biochar was an effective ratio for the viability and stability of four of the five tested endophyte strains on the biochar powder carrier for at least 2 weeks.

[0187] Example 3: Endophytic bacterial strain WW7 can solubilize insoluble phosphate.

[0188] The ability of endophyte strains to enhance endophyte metabolism allows them to utilize insoluble forms of phosphorus (P) in soil or soil solution, translocate P in the plant, and enhance P uptake relative to other soil particles or internal metal ions. Heterologous application of the endophyte strain WW7 has been shown to have an enhancing effect on P uptake in host plants. Heterologous WW7 was apparently able to solubilize different insoluble P forms that were insoluble in the mixture of culture medium solutions. Genomic analysis of WW7 indicated that the underlying genetic mechanisms for the biosynthesis of Krebs cycle intermediates such as organic oxoacids malate and citrate may be responsible for solubilizing insoluble phosphate forms in the soil, allowing for better uptake by the plant. In addition, the endophyte may have exudates that, once inside the plant, help keep phosphate ligands free by competing with other metals that may tightly bind phosphate, rendering it again insoluble and unassimilable. WW7 genomic data were used to identify protein-coding genes involved in the reactions of interest, and the predicted proteome of WW7 was functionally annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using the KofamKOALA genomejp tool (https: / / www.genome.jp / tools / kofamkoala / ). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used as a functional database to query the WW7 proteome for enzymatic reactions and pathways that catalyze malate and citrate, which are secreted by roots and may solubilize insoluble P. A total of 1602 proteins in WW7 were mapped according to the KEGG database. The enzymes involved in the synthesis of malate and citrate are citrate synthase (gene id: 2821609409) and fumarate hydratase (gene id: 2821609475), respectively. Malate can also be synthesized by the assimilation and conversion of aspartate and glutamate. 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, subsequently releasing a fumarate molecule for use in the citric acid cycle pathway (see Fig.13A ). Glutamate is the first amino acid product in the GS-GOGAT pathway, which produces 1 mol of glutamate for every 1 mol of NH3 in the GOGAT pathway responsible for atmospheric nitrogen fixation in bacteria. Fig.13A The WW7 enzymatic pathway involved in the synthesis of fumarate from glutamate and aspartate is shown, as well as the enzymatic reactions catalyzed by the WW7 enzyme (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).

[0189] Phosphate solubilization gene

[0190] According to the KEGG annotation, genes involved in inorganic and organic phosphate solubilization in other species were also detected in WW7. Fig. 13BThe results indicated the presence of an acid phosphatase (AcPase) gene as well as genes involved in the synthesis of acetic acid and gluconic acid. AcPases have been shown in other species to be involved in the solubilization of phosphate from phosphomonoesters, and acetic acid and gluconic acid have been shown in other species to be involved in the solubilization of phosphate from inorganic forms.

[0191] The ability of endophyte strains to solubilize insoluble phosphorus (P) from soil or soil solution by exogenously producing various mobilizing compounds is important in helping plants more efficiently acquire the required P, other nutrients such as potassium K, and micronutrient ions from the soil. These chelated or pH-lowering acidic compounds produced by endophytes can apparently help plant roots better acquire these minerals from the soil and aid in plant uptake and translocation from roots to aerial parts.

[0192] To demonstrate the ability of WW7 to solubilize insoluble forms of phosphate by secreting exogenous compounds, an insoluble phosphate solubilization assay was performed in liquid culture using a phosphate-sensitive dye and microplate reader method to further confirm the biochemical ability of WW7 to solubilize different phosphate species. A physiological assay was also developed and modified from the previous method of Varga et al., Endophyte-Promoted Phosphorus Solubilization in Populus, Frontiers in Plant Science, 11; 2020; 1585 ("Varga"). WW7 cells were grown in modified National Institute of Plant Research (NBRIP) broth without phosphate to consume residual phosphate in vivo. As previously described by Varga, 5 ml of modified NBRIP broth culture with or without phosphate was added to 10 mL of WW7 cell culture. These 10 mL tubes were then incubated at 25 ° C for 3 days and shaken at 220 rpm. The 10 mL tubes were then removed from the shaker and allowed to settle for 90 min. 1 mL of the culture was rotated at 5,000 rpm for 5 min at 25 ° C, and the supernatant was used to measure dissolved phosphate. The absorbance of dissolved phosphate was measured at 650 nm (A650) using a phosphate colorimetric assay kit (Sigma-Aldrich, MAK030). The supernatant of each sample was added to a 96-well plate at different dilution rates. In addition, the phosphate standard provided in the kit was prepared to calculate a linear equation for determining the dissolved phosphate in each sample. Four to five technical replicates were used to determine the statistical differences between samples.

[0193] Fig. 13CThe results presented in show that the growth and molecular activity of the endophytic strain WW7 resulted in a statistically significant increase (p<0.01) in the phosphate solubility of insoluble suspended aluminum phosphate and tricalcium phosphate, but not in the solubility of iron phosphate. Strain WW7 increased the phosphate solubility of insoluble aluminum phosphate by an average of 29% and of insoluble tricalcium phosphate by an average of 100%. These results demonstrate that WW7 mobilizes a large amount of insoluble phosphate.

[0194] WW7 can be mixed with other nitrogen-fixing microorganisms to mobilize P, so that the conversion of insoluble phosphate by WW7 and the potential uptake of soluble P by the host plant from soil or rock can be combined with enhanced nitrogen acquisition (e.g., from the atmosphere). Together, these mechanisms can greatly enhance the metabolic performance of the treated host plants, which can translate into enhanced biomass, stress tolerance, and other favorable traits.

[0195] Example 4. Genomic and biochemical assays for siderophore production

[0196] Endophyte strains may be able to solubilize iron (Fe) from soil or soil solution. This may be achieved by the production of various solubilizing compounds and / or heme-associated binding factors. Endophyte strains may also enhance the host plant to have a better ability to acquire the required Fe from the soil, as well as to acquire other metal and micronutrient ions, especially positively charged divalent cations. These Fe-siderophore chelating compounds produced by some bacterial and yeast endophytes may help plants better compete with the high cation exchange capacity of soil clay particles and may aid in the uptake of metal cations in the plant and their translocation from roots to aerial parts. To determine the capabilities of strain WW7, a whole genome analysis of the WW7 genes was performed. The results showed that WW7 possesses the genetic machinery required to make Fe siderophores that can transport or scavenge Fe ions. InterProscan results indicated the presence of the gene cluster efeUOB, which is involved in the recovery of ferrous and ferric iron from exogenous heme, and the presence of three genes encoding NADPH-dependent siderophore reductases. See Fig.14A The latter catalyzes the reduction of ferric iron complexed to various iron carriers such as ferric triscatecholate and ferric dicitrate. This enzymatic reaction releases the bound ferrous iron.

[0197] Furthermore, according to anti-smash analysis, the WW7 NADPH-dependent siderophore reductase (Ga0372474_197) was in a cluster with the gene encoding the enzyme nonribosomal peptide synthetase-like protein (Ga0372474_207), which is usually found in biosynthetic gene clusters (BGCs) involved in siderophore synthesis.

[0198] Determination of Fe siderophore production in all four endophyte strains

[0199] Fe-siderophore production capacity of endophyte strains WW5, WW6, WW7 and PTD1. Three of the endophyte bacterial strains tested demonstrated Fe-siderophore production. The four strains were assayed using microbial growth solid agar plates based on Fe-siderophore CAS medium. Agar plates were prepared using CAS agar plate preparation, including chrome azurol and FeCl3 as color indicators. Endophyte strains were plated on individual plates.

[0200] Fe solubilization is evidenced by the appearance of discolored areas in the CAS test plates that appear as clear white around streaks of bacteria growing on the plates. The areas are 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 is photographed, measured, and compared. See Fig. 14B .

[0201] Figures 14B-14D The in vitro results in , demonstrated that WW7 produces an extracellular compound that scavenge insoluble Fe. WW7 showed the greatest Fe solubilizing activity among all strains. WW5 and WW6 also showed significant Fe solubilizing effects. However, the PTD1Rhizobium populi strain had almost no Fe solubilizing activity, and insoluble Fe was observed immediately near the colony streaks. The test results demonstrated that WW5, WW6, and WW7 produce Fe siderophores. The test results demonstrated that WW5, WW6, and WW7 can all solubilize Fe, and showed that the production of Fe siderophores was the greatest in WW7, followed by WW5 and WW6. Endophytic strains that produce these compounds can assist the host plant in the solubilization and mobilization of Fe. The Fe solubilizing activity of each strain was measured in Fig. 14C quantified in Fig.14D Represented graphically.

[0202] Example 5, gas chromatography analysis and identification

[0203] Bacterial identification was performed by gas chromatography analysis of fatty acid methyl esters (GC-FAME). GC-FAME analysis provides a unique FAME ID chemical identification chromatogram that is highly specific for each endophyte strain. This allows us to track these microbial isolates individually and confirm them one by one based on their unique fatty acid methyl ester signature. The unique chromatogram for each strain is shown in Figure 15.

[0204] How to use

[0205] The preparation of the present invention can be advantageously applied to plants in a variety of ways, including but not limited to spraying, irrigation, coating, soaking, injection, furrow application or any combination thereof. The composition of the present invention can be applied to leaves, roots, leaves, branches, tillers, flowers, plant cells, plant tissues, seeds (e.g., as coatings or by spraying or soaking to treat seeds, etc.), pre-emergence (before the seedlings emerge or appear on the ground), cereals, fruits, tubers, spores, cuttings, grafts, meristems, plant cells, nuts or embryos. In certain embodiments, the composition can be applied as part of an impregnation agent for host plant roots and / or other tissues, as a seed coating, as a coating applied to host plant leaves and / or other elements, as a powder applied to the surface of host plant leaves and / or other elements, as a spray applied to host plant leaves and / or other elements, as a part of drops applied to host plant soil and / or roots, as a dry alginate bead that encapsulates endophytes and delivers them to roots, or other appropriate methods or inoculations.

[0206] The compositions of the present invention are effective in improving the metabolism (e.g., nutrient uptake, carbon uptake, growth, etc.) of the host plant. Therefore, the compositions and methods of the present invention have significant economic advantages because the increase in growth characteristics can lead to an increase in harvestable crop yields and more robust plants. Exemplary methods are discussed below.

[0207] Example 6: PCR analysis of endophyte colonization in host plants after root immersion inoculation

[0208] The ability of the endophyte strains to heterologously colonize host crop plants was tested using PCR technology. The results clearly showed that the endophytes were present inside 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.

[0209] Four groups of plants were planted after the seeds were treated as follows: an appropriate number of seeds were placed in a seed germination box with a bottom of 11 cm by 11 cm, and 20 mL of inoculation solution (co-fermented endophytes WW6+WW7 in NLM medium, with concentrations of ~10 7 CFU / ml). Seeds were germinated for 5 days and transplanted into 3.5" pots containing a potting mix of washed play sand, vermiculite and perlite. Plants were grown in a growth chamber at 25°C under sodium halide lamps with a photoperiod of 14hr light / 10hr dark. In addition, plants were watered and fertilized in trays with 25ppm reduced nitrogen Hoagland solution 2-3 times per week as needed to keep the soil moist to slightly dry. Plants were harvested individually 14 days after transplanting. Plants were then removed from the soil and processed. Use PureLinkTM Microbiome DNA Purification Kit DNA Isolation Kit (Thermo Fisher Scientific) was used to isolate DNA from the processed samples.

[0210] A 20 microliter PCR reaction was set up using 2X HotStart PCR Master Mix (MCLAB), 1uL template DNA and 1uM primers specific to the hypothetical protein gene in WW6. The PCR cycling conditions were as follows: 1 cycle at 95°C for 10 minutes; 25 PCR cycles at 95°C for 30 seconds, 65°C for 30 seconds and 72°C for 45 seconds; and finally 72°C for 5 minutes. 10uL of each PCR reaction was loaded on a 1.2% agarose gel and run at 120V by DNA QS710 electrophoresis (IBISCI). Fig.16 WW6 was shown to be present in plants of all three host plants tested (wheat, rice and barley) inoculated before germination, while it was absent in the controls of the three tested plants. The bands in lanes 2 (wheat treated with WW6), 4 (rice treated with WW6) and 6 (barley treated with WW6) indicate the presence of the WW6-specific gene. Thus, the WW6 endophyte strain is able to effectively colonize several host plants.

[0211] Example 7: PCR analysis of endophyte colonization in host plants after seed coating

[0212] The ability of endophyte strains to heterologously colonize host crop plants when applied as seed coatings was tested using PCR technology. The aerial parts and roots of winter wheat (Triticum aestivum) and broccoli (Brassica oleracea) plants treated with WW6 (Pseudomonas siliginis) were evaluated for colonization and incorporation of WW6 endophyte strains into plant tissues. Plants were grown after seeds were treated with WW6 as follows: WW6 was fermented according to the methods disclosed herein, mixed with 0.5% sodium alginate (Scogin TM LDH), and then used to coat raw 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 1 month after the coating treatment.

[0213] Place an appropriate number of seeds in a seed germination box with an 11 cm by 11 cm bottom. Germinate the seeds for 5 days before transplanting them into 3.5 inch pots containing a potting mix of washed play sand, vermiculite and perlite. Plants are grown in a growth room at 25°C under sodium halide lamps with a photoperiod of 14 hr light / 10 hr dark. In addition, water and fertilize the plants in trays with a 25 ppm reduced nitrogen Hoagland solution 2-3 times a week as needed to keep the soil moist to slightly dry. 14 days after transplanting, remove the plants from the soil and process. Harvest the roots and aerial tissues of the plants. Use PureLink TM Microbiome DNA Purification Kit DNA Isolation Kit (Thermo Fisher Scientific) was used to isolate DNA from the processed samples.

[0214] PCR was performed as described in Example 5 above. Fig.17A and 17B Electrophoresis gel data are provided for DNA encoding an unknown hypothetical protein present only in WW6. The gel demonstrates that WW6-specific DNA is present in aerial and root tissues of winter wheat and broccoli host plants grown from treated seeds, but not in aerial and root tissues of controls. Fig.17A Electrophoresis gel data are provided to demonstrate the presence of WW6 in the aerial and root tissues of winter wheat host plants grown from treated seeds, due to the presence of genome-specific PCR primers designed for an unknown hypothetical protein, which produced DNA bands indicating the specific presence of the WW6 strain, but not in the aerial and root tissues of the control. Thus, the WW6 endophyte strain is able to effectively colonize the root and aerial tissues of the wheat host plant after seed inoculation.

[0215] Fig. 17B Electrophoretic PCR gel data are provided demonstrating the presence of WW6 in the root tissue of seedlings inoculated with WW6 seed treatments after surface disinfection of the root tissue. Several treatments using the WW6 endophyte strain were prepared: a first treatment with WW6 liquid fermentation broth, a second treatment in which broccoli seeds were treated with liquid fermentation broth 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 broth mixed with 1% sodium alginate and seed coating material. These three treatments were coated on different groups of broccoli seeds. Plant growth, DNA isolation, and PCR were performed as described in Example 5 above.

[0216] Fig. 17BElectrophoresis gel data are presented demonstrating the presence of WW6 in aerial and root tissues of winter wheat, rice, soybean, broccoli, and maize host plants grown from treated seeds due to the presence of genome-specific PCR primers designed for an unknown hypothetical protein, which produced unique DNA bands indicating the presence of the WW6 strain, but not in aerial and root tissues of controls. Thus, the WW6 endophyte strain is able to effectively colonize the root and aerial tissues of several host plants following seed inoculation.

[0217] Example 8: Analysis of endophyte colonization of host plants after foliar application

[0218] Spinach plants (Spinacia oleracea) treated with WW6 and WW7 endophytes in foliar sprays were tested for their performance in the field and the ability of the endophyte strains to heterologously colonize the host plants was also measured.

[0219] A foliar inoculum composition comprising co-fermented WW6 and WW7 freeze-dried powders resuspended in an aqueous composition was applied to spinach plants at commercial rates along with a 10-5-3CaO liquid fertilizer formulation, Greenstim TM (concentrated glycine betaine extracted from beetroot, 12% total nitrogen, from Massó, SAAgro Department). Spinach was cultivated using a full conventional nutritional regimen with 550 L of 10-5-3 3% CaO fertilizer applied daily to each plot via fertigation (715 kg fertilizer, 18 kg / day). The foliar composition was applied at the four true leaf stage at a leaf rate of 1 L per hectare with a WW6+WW7 endophyte concentration of 20 g / L. The experimental plots were divided into three blocks, each with six beds. Sampling was carried out in the central portion of the four central beds in each block. The canopy cover was measured using a software tool (Canopeo TM ) Canopy cover was assessed 35 days after application. At the same time, leaves were harvested for nutrient analysis and surface disinfection, followed by in-plant endophyte quantification. Results for plant cover on plot bed data showed that the endophyte leaf treatment increased spinach leaf canopy cover by 30.92% statistically significantly (p<0.05) compared to commercial fertilizer alone and by 58% statistically significantly (p<0.05) compared to the control treatment. Fig.18A .

[0220] To determine and quantify the presence of plant endophytes in spinach tissues, leaves and roots were harvested 35 days after leaf inoculation, and cleaned and surface sterilized to detect strains WW6 and WW7 in plant tissues. All samples were weighed and photographed to calculate CFU associated with sample weight and leaf or root area. The ends of leaves and roots were sealed in plastic bags before surface sterilization in a laminar flow hood. Surface cleaning was performed with distilled water to remove dust, soil and other contaminants. Subsequently, the samples were placed in sterile flasks, 70% ETOH alcohol was added, and the flasks were shaken for 2 minutes (150rpm). After shaking, the alcohol solution was removed and 1% sodium hypochlorite bleach solution was added. The mixture was shaken for another 2 minutes. Subsequently, the sodium hypochlorite solution was also removed and the samples were cleaned 3 times by manually shaking in sterile water for 1 minute.

[0221] Leaves and roots were ground in 50 ml of 0.9% saline solution. The extract was placed in a sterile tube and allowed to stand for 1 hour to allow the release of endophytes from the ground tissue. Several serial dilutions of the extract were prepared using sterile water. 100 μl of each dilution was spread on potato dextrose agar (PDA) plates and actinomycete isolation agar containing glycerol.

[0222] The agar plates were incubated until bacterial growth was visible to the naked eye. The bacterial concentration was measured and the CFU / g and CFU / cm 2 The results of the analysis material are shown.

[0223] Fig.18B The data demonstrate that endophytes (WW6 and WW7) were present in surface sterilized plant leaves and roots of spinach host plants inoculated with the foliar inoculum composition. The endophyte strains were absent from spinach plants treated with Greenstim liquid fertilizer alone (see detailed discussion above).

[0224] After 35 days of treatment with the foliar inoculum composition containing WW6 and WW7 endophytes, WW6 and WW7 endophytes were detected in both leaves and roots of the host plants. No endophytes were present in the standard treated control plants and no endophytes were detected. When the endophyte concentrations were expressed as CFU / g, the concentrations in leaves were higher than in roots, but the difference was so small that the concentrations in leaves and roots can be considered to be the same in both parts. The CFU / cm 2 The surface area was analyzed visually and calculated from the measurements.

[0225] The above data indicate that 35 days after foliar application, the endophytes had successfully colonized and improved leaf growth and biomass of the treated host spinach plants. The results demonstrate the efficacy of the composition and foliar application method, which increased yield by improving establishment, growth and soil coverage of the spinach host plants.

[0226] Example 9: Effect of single endophyte strain seed treatment on total nutrient accumulation

[0227] Corn seeds (Zea mays) were treated with a seed inoculum composition containing a heterologous endophyte strain selected from WW5, WW6 and WW7 and compared to control corn seeds treated with an inoculum composition without endophytes. Four groups of corn plants were planted as follows: an appropriate amount of corn seeds were placed in the bottom of a large gallon-sized Ziplock bag and sealed. Three groups of corn seeds were treated with a specific endophyte culture (WW5, WW6 or WW7). WW5, WW6 and WW7 cultures were prepared in NLM medium plus sodium alginate (0.5% w / v) (containing ~10 7 CFU / ml of endophyte) and then refrigerated. The culture was removed from refrigeration, mixed, and 1 mL drops were carefully placed onto the seeds in a sterile laminar flow hood at a rate of 3.4 mL / lb of seed. After each 1 mL addition, the bag and seeds were manually inverted and carefully massaged. After the entire 3.4 mL / lb application was added, the seeds were massaged, shaken, and inverted for 2-3 minutes until all corn seeds in the bag were visibly wet. The bag was then opened and air-dried in a sterile laminar flow hood – air flowed into the hood during the air-drying process. After drying, the seeds were stored at room temperature for 3 weeks and then grown in 1-gallon felt Smart Pots in a growth chamber at 25°C under halogen lamps (photon flux of 710 mol m -2 s -1 ), planted and germinated in cleaned game sand, vermiculite and perlite potting mix under a photoperiod of 14hr light / 10hr dark. After germination, water and fertilize the plants 2-3 times a week in a tray with 50ppm Hoagland solution modified for nitrogen reduction to keep the soil moist and slightly dry. Cultivate the control under the same conditions without pretreatment before planting. Harvest plants separately at 24 days, dry and weigh. The tissue is sent for inductively coupled plasma mass spectrometry (ICP-MS) analysis to determine the ion content of the tissue. The nutrient accumulation of the aboveground biomass is calculated by multiplying the total aboveground dry weight by the aboveground concentration of each sample.

[0228] like Fig.19 As shown, when all plants were grown in Hoagland de-N nutrient solution supplemented with 50 ppm bioavailable nitrogen, corn plants inoculated with endophyte strains WW5, WW6 or WW7 accumulated significantly higher levels of macro- and trace mineral nutrients in the important mineral ion profile, as measured by the % change in total nutrient content of aboveground biomass relative to untreated control plants.

[0229] Fig.19The data in the study showed that heterologous endophytes successfully increased macro- and micronutrient uptake and incorporation in host maize plants grown from treated seeds under reduced nitrogen conditions. The results demonstrate the efficacy of heterologous endophytes in improving physiological performance and nitrogen acquisition from the air in non-native host plants. The endophyte seed treatment compositions increased nitrogen in aerial parts of maize as follows: WW5 47%, WW6 45%, and WW7 29%.

[0230] Example 10. Endophyte screening assay for crop yield when grown under limited bioavailable forms of nitrogen and phosphorus.

[0231] The WW5, WW6, WW7 and PTD1 endophyte strains were further screened in a greenhouse pot study, where plants were inoculated with endophyte strains encapsulated in alginate beads either alone or as a mixture of all four strains. Alginate beads encapsulated with endophytes in calcium alginate were placed next to the seeds, using 1 bead per seed, and the pots were evenly watered using controlled drip irrigation to allow the seeds to germinate. Plants were specifically grown under limited nutrient concentrations in the potting medium, purposefully reducing bioavailable soluble nitrogen and phosphorus forms, where the potting medium included nitrate <13ppm, ammoniacal N <6ppm and phosphate <11ppm.

[0232] Fig. 20 The results in the present study showed that when commercially available, agronomically relevant, commonly used seeds were used, the four selected endophytes had positive responses in a variety of crops, increasing yield under limited bioavailable nitrogen and phosphorus.

[0233] Example 11 Effects of seed treatment with combined endophyte strains (WW6+WW7) on total nutrient accumulation and aboveground biomass

[0234] Rapeseed (Brassica napus) treated with a seed inoculum composition containing co-fermented WW6 and WW7 heterologous endophyte strains was grown and compared to control rapeseed plant seeds treated with an inoculum composition without endophytes. The treated seeds were planted as follows: The appropriate amount of rapeseed seeds were commercially treated with 500 mL of a mixture of co-fermented WW6 and WW7 strains, 500 mL of 1% alginate, and Integral pro (BASF) and prebiotics UBS 016 (Unium Bioscience Ltd.) per metric ton of seeds to aid endophyte survival, colony growth, and host plant colonization according to the manufacturer's instructions. Prebiotics included microbial nutrient packages, plant biostimulants, osmoprotectants, buffers, and seed lubricants. Endophyte survival on seeds was confirmed by adding seeds to a 0.2 M phosphate resuspension solution and then plating on NLM semi-solid medium at appropriate dilutions. Control rapeseed seeds received the same crop protection package. The seeds were then stored for 1 month under normal industrial seed storage conditions (4°C to 15°C) and planted commercially in a large-scale replicated CRO field trial using a seed drill in Cuxwold, Lincolnshire, UK, in the autumn.

[0235] The aboveground biomass was harvested during the early spring vegetative period and sent for agronomic mineral nutrient analysis. Nutrient accumulation of aboveground biomass was calculated by multiplying the total aboveground weight by the aboveground ion concentration. Fig.21 As shown, rapeseed plants inoculated with co-fermented WW6 and WW7 strains accumulated significantly higher levels of macro- and micronutrients, with increased nutrient accumulation compared to the control expressed as % change in total nutrient content of aboveground biomass compared to the control.

[0236] The above data indicate that co-fermented heterologous endophytes WW6 and WW7 successfully increased macro- and micronutrient uptake and incorporation in rapeseed plants grown from treated seeds. The results demonstrate the efficacy of co-fermented heterologous endophytes in improving the physiological performance of non-natural host plants.

[0237] Example 12: Effect of combined strain WW6+WW7 on total nutrient accumulation and biomass

[0238] Winter wheat seeds (Triticum aestivum) treated with a seed inoculum composition comprising co-fermented WW6 and WW7 heterologous endophyte strains were grown and compared to control winter wheat plant seeds treated with an inoculum composition that did not contain any endophytes. The inoculum composition was combined with a prebiotic composition UBS016 from Unium Bioscience Ltd. to aid endophyte survival, colony growth and host plant colonization. The treated seeds were planted as follows: The appropriate amount of wheat seeds were commercially treated at a ratio of 500 mL of mixed co-fermentation broth, 500 mL of 1% alginate and 1000 ml of 10% UBS 016 aqueous solution per 1 metric ton of seeds. A crop protection package containing fludioxonil and fluazifop was also added to prevent seed-borne diseases. Vibrance from Syngenta AG For use as a crop protection bag containing 25 g / l cyfluthrin and 25 g / l fludioxonil. The application rate of the product is 2L per metric ton.

[0239] Control seeds of the same variety received the same crop protection package and endophyte survival on seeds was confirmed by adding seeds to a 0.2M phosphate resuspension solution and then plating on NLM semi-solid medium at appropriate dilutions. The seeds were then stored for 1 month under normal industrial conditions (4°C to 15°C) and commercially planted in a large-scale replicated CRO field trial at conventional rates using a seed drill in Cuxwold, Lincolnshire, UK in the autumn.

[0240] In late spring, five months after planting, the aerial parts were harvested during the vegetative period and sent for agronomic mineral nutrient analysis. Nutrient accumulation of aerial biomass was calculated by multiplying the total aerial weight by the aerial ion concentration. Fig. 22 As shown, winter wheat plants inoculated with co-fermented WW6+WW7 accumulated significantly higher levels of macro- and micronutrients, with increased nutrient accumulation compared to the control expressed as % change in total nutrient content of aboveground biomass compared to the control.

[0241] The above data indicate that co-fermented heterologous endophytes WW6 and WW7 successfully increased macro- and micronutrient uptake and incorporation in winter wheat plants grown from inoculated seeds. The results demonstrate the efficacy of co-fermented heterologous endophytes in improving the physiological performance of non-natural host plants.

[0242] Example 13: Effect of combined strain WW6+WW7 on total nutrient accumulation aboveground biomass

[0243] Spring oat seeds (Avena sativa vars. Elyann and S01) were grown using a seed inoculant composition treated with 10 7 CFU / ml of heterologous endophyte strains were incorporated into co-fermented WW6 and WW7 broths and compared to control seeds treated with an inoculum composition that did not contain endophytes. Treated seeds were planted as follows: appropriate amounts of oat seeds were commercially treated with 500 mL of mixed co-fermentation broth, 500 mL of 1% alginate and 1000 ml of a 10% prebiotic composition UBS 016 (from Unium Bioscience Ltd.) in water per tonne of seed according to the manufacturer's instructions, and seed disease protectant Redigo (Bayer) was used. Endophyte survival on seeds was confirmed by adding seeds to a 0.2 M phosphate resuspension solution and then plating on NLM semi-solid medium at appropriate dilutions. Control seeds of the same variety received the same prebiotic treatment but did not receive endophyte application. Oat seeds were then stored under normal industrial seed storage conditions for 1 month and commercially planted in Suffolk, UK in May for a large-scale replicated CRO field trial with conventional fertilizer rates.

[0244] In mid-spring, five months after planting, the aerial parts were harvested at an early vegetative stage and sent for agronomic mineral nutrient analysis. Nutrient accumulation of aerial biomass was calculated by multiplying the total aerial weight by the aerial ion concentration. Fig.23 As shown, spring oat plants inoculated with co-fermented WW6+WW7 accumulated significantly higher levels of macro- and micronutrients, with increased nutrient accumulation compared to the control expressed as % change in total nutrient content of aboveground biomass compared to the control.

[0245] The above data indicate that co-fermented heterologous endophytes WW6 and WW7 successfully increased macro- and micronutrient uptake and incorporation in spring oat plants grown from inoculated seeds. The results demonstrate the efficacy of co-fermented heterologous endophytes in improving the physiological performance of non-natural host plants.

[0246] Example 14: Effect of the combined strain WW5+WW6+WW7+PTD1 on nutrient concentration

[0247] After pre-treating the seeds with two fungicide / insecticide products, GA3 (gibberellic acid), a dye, and a flowable zinc micronutrient coating, seeds of Asian rice (Oryza sativa) hybrid XP753 were treated with a seed inoculum composition comprising co-fermented WW5+WW6+WW7+PTD1 heterologous endophyte strains coated on the seeds. 500 mL of endophyte fermentation broth and 1% alginate were added to 2,205 pounds of rice seeds. Examination of the seed coat quality of the commercially treated seeds indicated survival of the WW5+WW6+WW7+PTD1 endophyte strains, expressed as colony forming units (CFU) per seed, as follows: WW5 2.0 x 10 6 ;WW68.0x 10 5 ;WW7 3.6x 10 6 ;PTD1 1.2x 10 6 . Control seeds were treated with a pretreatment but not with the seed inoculant composition. The seeds were then stored under normal industrial seed storage conditions for 2 months and planted commercially in the spring of 2020 by a large grower in Clay County, Arkansas, USA. The rice fields were fertilized with 400 pounds of urea per acre, equivalent to 184 pounds of N per acre. The aboveground parts, including leaves, were collected at mid-vegetative stage (pre-booting) and late-vegetative stage (booting), air-dried and sent for agronomic mineral nutrient content analysis. Fig.24 As shown, 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 concentrations compared to the control.

[0248] Example 15. Effects of (WW5, WW6, WW7 and PTD1) used as seed treatments at reduced nitrogen fertilizer and normal nitrogen fertilizer ratios on field harvest yield.

[0249] Broccoli seeds were first commercially treated with a mixture of endophyte fermentation broth plus 1% alginate and applied at different rates using two different crop protection packages and industry-leading conventional methods. Seed application rates of endophytes were 10mL, 50mL and 100mL of fermentation broth per 1kg of broccoli seeds mixed into the commercial slurry and applied. Control seeds of the same variety received the same crop protection package minus the endophytes. Endophyte survival assays were then performed for the presence of each microorganism. The microbial mixtures demonstrated survival of the strains (WW5, WW6, WW7, PTD1) on the seeds after drying – the four strain mixtures were Fig.25AThe seed coat counts were performed using seeds washed in 10 mL of water to remove the seed coat prior to assay. The dilution plate results showed clear survival, and the bacterial titers were provided as colony forming units / seed (CFU / seed) for each strain that remained viable and successfully dehydrated and then dormant on the seed ( Fig.25A ).

[0250] The broccoli seeds were then stored for 2 months under normal industrial conditions (<25°C in light-proof packaging) and commercially planted in the fall in Salinas, CA using commercial methods. A large-scale replicated CRO field trial was conducted on a production farm with fertilization at normal and 25% reduced nitrogen fertilizer rates compared to commercial, and four nitrogen fertilizers were applied through drip irrigation. Two 12 gal / acre calcium ammonium nitrate (17-0-0) applications were applied with full fertilizer throughout the season, and two 5 gal / acre applications of (17-0-0). In the 25% reduced nitrogen fertilizer treatment, the amount of nitrogen was reduced by 25% at each fertilizer application. In addition, all treatments received a total of 8 applications of 0-0-6-3% Ca fertilizer throughout the season. Irrigation was at the discretion of the farm manager according to commercial farming standards. After one month of growth, there was a significant difference in plant size between the treatment that received the 100% nitrogen rate and the treatment that received 25% less nitrogen. No symptoms of any disease or insect infestation occurred in any treatment group during the trial. At harvest, there were still significant differences in the leaves between the treatments that received 100% nitrogen and the treatments that received 25% less nitrogen. All treatments were then measured for uniformity and differences in commercial quality at harvest. When selecting commercial broccoli heads, growers consider different criteria such as head size (diameter in inches), head smoothness, dark green color, and firmness. Fig.25B As shown, the largest average commercial head diameters at harvest were observed for the highest two endophyte treatment rates of 100 mL and 50 mL per 1 kg for both seed crop protection packages under 25% less N fertilizer and normal 100% fertilizer (expressed as 75% N and 100% N rates, respectively).

[0251] like Fig.25C As shown, the maximum average commercial head weights at harvest were observed for the highest two endophyte treatment rates of 100 mL and 50 mL per 1 kg for both crop protection packages under 25% less N fertilizer and normal 100% fertilizer (expressed as 75% N and 100% N rates respectively).

[0252] Example 16 Effect of WW6 and WW7 applied alone as seed treatments under reduced nitrogen fertilizer (32 ppm N) in the Hoagland breakaway indoor planting pot study

[0253] The early effects of WW6 and WW7 endophytes applied alone to Brassica species on aerial growth were tested in a controlled environment under nitrogen-limited conditions. Endophyte strains WW6 and WW7 were applied alone to broccoli seeds as a fermentation broth in a commercial seed coating process using clay and dip coatings, and broccoli seeds were germinated and grown on plates using artificial soilless substrates. The control was the commercial coating applied alone without the endophyte fermentation broth mixture. Eight seeds per treatment were planted at a depth of 1 / 2 inch in 2” inorganic planting media (all DI washed; 1 / 3 play sand, 1 / 3 perlite, 1 / 3 vermiculite) in 10” x 20” plastic planting trays. During the experimental period, the trays were watered on Monday, Wednesday, and Friday with a modified Hoagland de-N solution that has 70% less optimal nitrogen, 32 ppm total N, and a pH of 7. The study lasted 27 days and was conducted in an indoor growth chamber with greenhouse lights on for 14 hours per day at 710 μmol / m 2 , ambient temperature was 25°C and humidity was 50%. Seed coat viability and dilution plates were measured to determine the survival of endophyte combinations and crop protection products after dehydration on commercially coated seeds. Fig.26A .

[0254] At harvest (27 days old), total plant seedling fresh weights were obtained for all treatments and the results are reported below. The seedling weights of the WW7 treatment (RD12378) were significantly increased by 30% over the control. The seedling weights of the WW6 treatment (RD12381) were significantly increased by 47% over the control. Fig.26B .

[0255] Example 17 Effect of WW5, WW6, WW7 and co-fermented mixtures applied as seed treatments under reduced nitrogen fertilizer in a controlled environment growth chamber

[0256] Endophytes WW5, WW6 and WW7 applied alone to corn seeds were tested for their effects on aerial growth under nitrogen-limited conditions in a controlled environment. Endophyte strain preparations were applied to corn seeds in combination with 1% w / v sodium alginate (Scogin LDH) and the corn seeds were germinated and grown in 1-gallon felt smart pots with artificial soilless substrate. Eight seeds of each treatment were planted at a depth of 1 / 2 inch in an inorganic planting substrate (DI washed; 1 / 3 play sand, 1 / 3 perlite, 1 / 3 vermiculite). During the experiment, the pots were watered on Mondays, Wednesdays and Fridays using a modified Hoagland de-N solution that included 50 ppm of nitrogen with total N. Two control groups were included, each treated with a modified Hoagland solution without endophytes. The first control group was treated with a modified Hoagland that included 75 ppm total N, and the second control group and endophyte treatment group were treated with a modified Hoagland that included 50 ppm total N. The study was conducted in an indoor growth chamber with greenhouse lighting for 14 hours per day at a concentration of 710 μmol / m 2 s 1 , the ambient temperature was 25°C and the humidity was 50%. On the 24th day after emergence, the plants were harvested, washed, and dried in individual paper bags at 45°C. The dry weight of the aerial parts was then measured and recorded. Fig. 27 Endophyte seed coat preparations resulted in increases in aerial dry weight biomass of maize as follows: WW6+ 102% p>0.05, WW5 84% p>0.05, and WW7 49% p>0.01.

[0257] Example 18 Effect of WW6 and WW7 applied as seed treatment to winter wheat in the field

[0258] Winter wheat was treated with a co-fermented WW6+WW7 endophyte inoculum. The co-fermented WW6+WW7 endophyte mixture was fermented in a low nitrogen medium and then freeze-dried into a powder. 5 grams of freeze-dried fermentate was mixed with 5 grams of dry sodium alginate in 1 liter of water. The mixture was then combined with a commercially available prebiotic composition UBS 016 (from Unium Bioscience Ltd.) in a ratio of about 3:1 to about 5:1 of the mixture to the prebiotic composition. The combination produced a final seed slurry that was applied at a rate of about 4L to about 6L per metric ton of winter wheat seeds. A control group was treated with prebiotics without fermentate. The seeds were commercially planted in the field in early November and the trial continued until the normal harvest in mid-summer of the following year, when the wheat kernels were weighed and the final yield results at harvest were recorded. Fig.28A As shown, the crop yield of the treatment group increased significantly by 10%.

[0259] In addition, nitrogen accumulation in the aerial parts of winter wheat plants was measured from February to June. At each time point, 1 square meter of plants was harvested, dried and sent for total nitrogen measurement using the Kjeldahl method. The continuous increase in total nitrogen per hectare of aerial parts of wheat was measured, as Fig.28B The results presented show that the total nitrogen accumulation in Kg / Ha of the wheat aerial parts increased by +30% at the last sampling measurement in June. The results clearly demonstrate the impact of nitrogen-fixing endophytes on this wheat variety in the field. The endophyte treatment increased the total nitrogen content of the aerial parts of the plants per hectare throughout the vegetative growing season and showed an increase of +100% in the aerial accumulation of nitrogen in early May and +30% in the aerial accumulation of nitrogen per hectare in early June.

[0260] Example 19. Carbon accumulation in plants treated with endophyte strains.

[0261] The ability of WW5, WW6, WW7, and PTD1 endophyte bacterial strains to increase total plant carbon accumulation when grown under field conditions was determined using fast-growing poplar (populus) trees planted in field soils of the Lower Mississippi Alluvial Valley. Trees were inoculated with approximately 20 calcium alginate beads containing encapsulated endophytes, applied to the base of poplar cuttings at planting. Replicate blocks were planted in the field (3 trees × 5 trees), with five replicate blocks in the field. Dormant, rootless poplar cuttings 22.86 cm long were obtained from (Portland, Oregon, USA) and inoculated with ( Pro, Bayer Corp., Whippany, NJ, USA).

[0262] For carbon sampling, leaf samples were returned to the laboratory, dried in a 60°C oven and ground into a fine powder, then placed in tin capsules. Samples were analyzed in an ECS 4010CHNS-O analyzer (Costech Analytical Technologies Inc. Valencia, CA, USA) to estimate C and N concentrations.

[0263] Statistically significant results showed that endophyte inoculation increased total plant carbon content by 71.01%, p value = 0.063, as follows Fig.29 Total carbon was calculated by multiplying the percentage of carbon in dry weight by the total biomass dry weight in n = 12 treated trees and n = 12 control trees. Fig.29 The results shown indicate that carbon accumulation in the treatment groups was significantly increased compared to the control.

[0264] Example 20: Compatibility with Crop Protection Products Commonly Used for Seed Treatment

[0265] Endophyte strains WW5, WW6, WW7 and PTD1 were tested for their ability to survive in combination with various commonly used commercial seed crop protection chemicals. Endophyte strains were evaluated for their ability to survive when added to 5 different seed crop protection chemical solutions: Beret (Syngenta), Raxil (Bayer CropScience), Redigro (Bayer CropScience), Vibrance (Syngenta) and (Bayer CropScience). The solution mixtures were prepared according to the manufacturer's specifications. The colony forming units (CFU / ml) of the strains were determined by complete dilution and plating on NLM semi-solid medium 5 to 6 minutes after the mixtures were generated. Fig. 30A The results presented in show that all strains could survive in the five different solution mixtures.

[0266] The viability of WW5, WW6, WW7 and PTD1 was evaluated when applied to seeds of different crops and using different seed crop protection chemical active ingredients: Metalaxyl-M, Fludioxonil, Azoxystrobin, Cyclofoamid, Thiabendazole Thiram, Metalaxyl, Hydromycin, Penthiopyrad, Poncho Beta and Thiamethoxam. The solution mixture was prepared according to the manufacturing specifications and then the different strains were added to the solution and then applied to the seeds through a commercial seed treatment machine. The colony forming units (CFU / ml) of the surviving strains on the seeds were evaluated by adding the seeds to a 0.2 M phosphate resuspension solution and then plating on NLM semi-solid medium at appropriate dilutions. Fig. 30B The results presented in show that the combination of endophyte strains is resistant and survives when mixed with various commercial products and can survive the temperature and dryness conditions found in commercial seed treatments.

[0267] Example 21. Compatibility of commonly used fertilizers, micronutrients and herbicides in in-furrow and foliar applications.

[0268] Endophyte strains were evaluated for their ability to be added to various commonly used tank mix solutions for in-furrow and foliar applications based on their viability over different periods of time ranging from 3 hours to 1 month. WW5 and WP1 viability were evaluated in ammonium polyphosphate (10-34-0), a liquid starter fertilizer for in-furrow nutrient applications, and / or a micronutrient product (4% ammoniacal nitrogen, 3% water-soluble nitrogen, 9.0% chelated zinc). For experimental purposes, the volume was scaled down from 5 gallons / acre to 50 ml. The mix composition contained: 32 fl oz / acre of micronutrients, 5 gallons / acre of 10-34-0 fertilizer, 16 fl oz / acre of strain WW5 inoculum, and water. Colony forming units (CFU / ml) of both strains were determined by plating on NLM semi-solid medium 3 hours after the mixture was generated. Fig.31A The results presented in show that both WW5 and WP1 survived when 10-34-0 was present in the aqueous in-ditch fertilizer + endophyte tank mix.

[0269] Based on the above results, 3 gal / acre of 10-34-0 fertilizer and 16 fl oz / acre of strain WW5+WP1 were evaluated in a replicated plot trial applied as an in-furrow tank mix to corn. The tank mix was dripped onto the seed in an in-furrow control that received only the fertilizer tank mix at the same rate and no endophytes. The field was either fertilized with no N or with a full N rate of 180 lb. N per acre.

[0270] Fig.31B Results presented in , show that when strain WW5+WP1 was combined with 10-34-0 fertilizer and corn was grown under full NPK fertilizer at conventional Midwestern WI USA rates, crop grain yield was increased by +12% at commercial harvest.

[0271] In addition, mineral nutrient content in leaves of maize plants was measured at the V9 growth stage. Fig.31C It was shown that total nitrogen, potassium and phosphorus (NPK) continued to increase when WW5 and WP1 strains were applied in-furrow in a fertilizer tank mix at planting. Importantly, potassium (K) in leaves collected from the block without nitrogen increased significantly by +9.5%, demonstrating increased K uptake and assimilation into the aerial parts in addition to N and P.

[0272] Example 22. Compatibility of commonly used fertilizers, micronutrients and herbicides in in-furrow and foliar applications.

[0273] The WW6 and WW7 strains were used to test the effectiveness and micronutrient compatibility of endophyte fertilizers and their long-term viability when mixed with 6-22-6-4, a common liquid starter fertilizer commonly used for in-furrow nutrient applications, was evaluated. For experimental container size purposes, the test solution mixture was scaled down from 5 gallons / acre to 50 ml. The composition contained: 5 gallons / acre of 6-22-6-4 fertilizer, 40 fl oz / acre of WW6+WW7 microbial composition. The colony forming units (CFU / ml) of both strains were determined by placing on NLM semi-solid medium for periods ranging from 1 week to 5.5 months. Fig.32 The results presented in show that the viability of both strains was slightly reduced when 6-22-6-4 was present in the solution mixture.

[0274] Example 23: Testing of endophyte compositions for compatibility with foliar herbicides

[0275] The viability of WW6 and WW7 was evaluated in a composition including glyphosate (a broad-spectrum systemic herbicide for foliar application) and / or adjuvants (modified vegetable oils, polyoxyethylene sorbitan fatty acid esters, vegetable oils, and ethoxylated soybean oils) and / or a micronutrient composition (sulfur 3.6%, boron 0.1%, manganese 3.0%, and zinc 4.0%). For experimental purposes, the solution mixture was scaled down from 10 gallons / acre to 50 ml. The mixture 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 a WW6+WW7 microbial fermentation composition prepared according to the methods described herein, at a pH of 5.3. 24 hours after the mixture was generated, the colony forming units (CFU / ml) of the two strains (WW6+WW7) were determined by plating on NLM semi-solid medium. Fig.33A The results presented in show that the viability of both strains was not reduced in the different compositions.

[0276] A field study was conducted to demonstrate the efficacy of the WW6 and WW7 compositions when applied to corn with glyphosate herbicides and adjuvants. United's glyphosate herbicide), 32 oz / acre MasterLock (from United's adjuvant) and 10 gal / acre of water to prepare the tank mix. The field study included 27.5 ft. x 5 ft. plots of high and low yielding corn varieties. Three plots were treated with the above tank mix and three plots were treated with a tank mix with the same ingredients except for the endophyte strain. Fig.33B Results presented in showed that crop yield increased by 10.6 bu / acre in low-yielding corn varieties treated with WW6+WW7 endophyte strains, while high-yielding varieties increased by 11.3 bu / acre, compared to controls that did not receive endophyte treatment.

[0277] Example 24 Effects of WW5, WW6, WW7, and PTD1 on improving plant tolerance to flooding and saturated soil

[0278] Flooding of agricultural soils is a major problem in the U.S. and global food production systems, often resulting in large-scale plant mortality and crop losses. To test the ability of endophytes to provide flooding tolerance to crops, sugar beets (Beta vulgaris) were commercially treated with or without heterologous endophytes (WW5, WW6, WW7, PTD1) using a commercial seed treatment method. Seed coatings were applied to control plants, and seed coatings were applied to experimental seeds in combination with co-fermented endophyte strains WW5, WW6, WW7, and PTD1. The coated seeds were then washed and the survival of the endophyte strains on the seeds of the experimental groups was tested. Bacterial counts were determined using 10 seeds, vortexed and washed in 10 mL potassium phosphate (KP) buffer to remove and dissolve the coating, and then dilutions were plated on NLM agar medium. This assay showed significant survival of the endophytes, with titers as high as 10%. Fig.34A The CFU / seed is shown in Figure 2.

[0279] Twenty-four endophyte-treated seeds were planted in separate units and twenty-four control seeds were planted in separate units. Seeds were continuously overwatered in fully saturated soil and germinated in standard commercial transplant potting media. The media was overwatered daily until saturated and grown in natural daylight conditions at 25°C. Once germinated, plants were continuously overwatered and subjected to flood-like conditions. Following this flood exposure, plants were evaluated for flood stress symptoms, germination, and overall growth effects. Fig.34B As shown, plants inoculated with endophyte strains exhibited faster germination and establishment, less flood damage, and better growth under continuously flooded saturated soil conditions compared to controls.

[0280] The above data show that the co-fermented heterologous endophytes WW5+WW6+WW7+PTD1 successfully improved the response of sugar beet plants to abiotic stress (flooding) caused by overwatering of saturated substrates, increased early germination rate, and improved biomass growth compared to the control. The visual results clearly demonstrate the efficacy of the co-fermented heterologous endophytes and their ability to improve the physiological performance of non-natural host crop plants.

[0281] Example 25 Effect of seed treatment with combined endophyte strains (WW6+WW7) on plant cold tolerance

[0282] Two groups of Vicia faba were prepared, a control group treated with a commercial seed treatment and an experimental group treated with a commercial seed treatment and 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 a low nitrogen medium and then freeze-dried into a powder. Five grams of the freeze-dried fermentate was mixed with 5 grams of dried sodium alginate in 500 ml of water. The mixture was then combined with 500 ml of the prebiotic UBS 016, with a ratio of 1:1 between the endophyte sodium alginate mixture and the prebiotic composition. The combination was then incorporated into a commercial seed treatment slurry and applied at a rate of about 4L to about 6L per metric ton of faba bean seeds. The control group seeds were treated with prebiotics alone. Seeds were germinated in standard potting media and grown in 6-inch pots in a greenhouse with daily minimum temperatures ranging from 45°F to 55°F and maximum temperatures ranging from 65°F to 70°F under natural daylight conditions. After germination, the plants were fertilized with a 90-day slow-release complete fertilizer (Osmocote 15-9-12 Coated Granule Fertilizer). Six weeks after germination, both groups 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 symptoms of cold stress and damage. Fig.35 As shown, experimental plants inoculated with WW6+WW7 showed less wilt and chilling injury than severely wilted control plants. Furthermore, endophyte-treated plants fully recovered from chilling stress, whereas non-endophyte-treated plants never fully recovered and showed signs of chlorosis and necrosis.

[0283] The above data indicate that co-fermented heterologous endophytes WW6+WW7 successfully improved the response of broad bean plants to abiotic stress (cold shock) compared to the control. The results demonstrate the efficacy of co-fermented heterologous endophytes in improving the physiological performance of non-natural host plants.

[0284] Example 26. Effects of endophyte strains alone and in combination on plant tolerance to sodium / saline soils high in boron and chloride.

[0285] Soils characterized by sodium and / or salinity restrict plant growth and often result in plant mortality, crop losses, and reduced yields. The USDA estimates that 40% of the world's once arable land is now unusable for agriculture due to high soil salinity. The endophyte strains of the present invention were tested for their ability to promote crop plant growth in sodium and saline soils after seed treatment. Control and experimental groups of conventional broccoli (Brassica oleracea) seeds were prepared as follows: two experimental groups were coated using a commercial seed treatment method, including a polymer dip coating in combination with WW7 (Group 1) or endophyte strains WW5+WW6+WW7+PTD1+WP1, and the control group was treated with a polymer dip coating and a broccoli protector bag. The seeds were then tested for total endophyte survival in the commercial seed coat, and the total counts were as follows. Fig.37A shown.

[0286] Seeds of both endophyte treatments (Group 1 and Group 2) and control seeds were planted in a commercial transplanting greenhouse in Santa Monica, California prior to planting into the field trial. A site identified by the USDA as having high sodium / salty soils and high boron and chloride levels was selected for the field trial. The site was in Five Points, California. An exchangeable sodium percentage (ESP) of more than 6% is considered a sodium soil and an ESP of 15% is considered a high sodium soil. The ESP value represents the percentage of sodium that is present in the soil cation exchange capacity (CEC). Poor quality soils have the following chemical profile. As Fig.36B As shown, the soil used in the test had an ESP of 12.9. The soil also included 25 ppm of boron, where levels around 3-5 ppm are harmful to plants. The soil also had a very high chloride level of 68 ppm. These characteristics indicate that the soil used in the test was very poor.

[0287] Using a rotary tiller, three identical 72 inch beds were created in a homogenous high-salinity field called (RRR West) and two drip tape irrigation lines were carefully installed under each bed and tested. The bed dimensions were 100 ft long bed, 6 ft wide 2 rows (2 ft at the edge of each row of beds and 2 ft between each row). 290 plants were planted from each of the 2 endophyte treatments and an untreated control seed. At harvest, photos of the beds were taken 91 days after transplanting, which are provided as Fig.36C The salt tolerance of the endophyte-enhanced plants (denoted as WW7 and stage A in the figure) can be easily seen in group 2 (stage A).

[0288] Seven-week-old broccoli plants were harvested and weighed 91 days after transplantation. Compared with the control group, the fresh weight of the plants in group 2 increased statistically significantly by 13.24% (p<0.05). Fig.36DThe figures provided in provide data from a broccoli field trial. Broccoli florets were dried in a drying oven after harvest. The total dry weight of plants in group 2 (stage A) was statistically significantly increased by 47.06% (p<0.05) and that of plants in group 1 (WW7) was statistically significantly increased by 16.81% (p<0.05) compared to the control group. See Fig.36E .

[0289] The data showed that the co-fermented heterologous endophytes WW5+WW6+WW7+PTD1+WP1 successfully increased the fresh and dry weights of broccoli compared to the control, and (2) under abiotic stress (sodium / saline soil conditions), the endophyte strains increased the dry weight of broccoli compared to the control. The results demonstrate the efficacy of heterologous endophytes in improving the physiological performance of non-natural host plants.

[0290] Example 27 Effect of combined heterologous endophyte strains on improving plant tolerance and recovery under drought conditions

[0291] Drought stress has negative effects on crops, plants, grasses and trees, often leading to plant mortality and crop losses. Droughts are becoming more frequent and induce many physiological and molecular biochemical changes in plants. Internal processes that help plants tolerate drought stress include scavenging of reactive oxygen species (ROS), osmoregulation (OA), stomatal closure and the synthesis of protective molecules including inducible dehydrins. Plant recovery after drought stress involves a series of steps that occur over time and may be aided or promoted by internal beneficial endophytes.

[0292] To demonstrate the role of endophytes in improving drought tolerance, a fermentation mixture was prepared, including endophyte strains WW5+WW6+WW7+PTD1 and the fungal yeast endophyte WP1, named "Phase A Mix". The Phase A mixed ferment was applied together with a commercial seed treatment that incorporated a polymer with talc as a carrier into tall fescue seeds (Festuca arundinacea - a forage grass used in livestock production) and dried into a shell that resembled a natural hard membrane. Two treatment groups were used:

[0293] Group 1, treated with 0.5 L of fermentation liquid per ton of seeds, and

[0294] Group 2, each ton of seeds was treated with 1.0 L (0.5 L fermentation broth + 0.5 L 2% alginate).

[0295] The control group received a commercial seed treatment that did not include any endophyte strains. These treatments were assayed and resulted in Fig.37A Endophyte survival on seeds is shown.

[0296] The control and experimental groups 1 and 2 were planted at equal density on 3 flats containing a low carbon growth substrate consisting of washed play sand, perlite and vermiculite. The seeds were watered 3 times per week for 4 weeks with a modified low nitrogen Hoagland nutrient solution containing 65 ppm nitrogen. The grass was grown at 30°C and then subjected to a drought stress period of 14 days without watering, causing the growth substrate to completely dry out. After the drought stress treatment was stopped, watering of the flats was resumed to give the grass a chance to recover. Each group was then harvested and weighed. There was a statistically significant (p<0.05) increase in dry weight for Groups 1 and 2: 42% and 67%, respectively. Total weight results are given below. Fig.37B is provided in the figure.

[0297] The above data showed that under abiotic stress conditions (drought conditions), the co-fermented heterologous endophytes WW5+WW6+WW7+PTD1+WP1 successfully increased the fresh weight of fescue compared to the control. The results demonstrate the effectiveness of heterologous endophytes in improving the physiological performance of non-natural host plants.

[0298] Example 28: Endophytes increase seedling germination rate, emergence rate and seedling biomass weight

[0299] A series of experiments were conducted in which the endophyte strains were applied to seeds to determine the effect of application on increasing seedling germination rate, increasing seedling emergence from the seed coat and soil, and increasing seedling biomass weight. The first experiment involved treating lettuce seeds with 10 treatments and one control group, e.g. Fig.38 As shown. Each treatment was applied to lettuce seeds with a clay seed coat and a fermentation mixture of nitrogen-limited endophytes in a 1 w / v% alginate solution. The control did not include the endophyte ferment. Seeds were coated using commercial methods, including clay coatings and seed coat polymers, including control seeds. Seeds were germinated in square petri dishes, and each 4in. x 5in. sterile seed germination container was wetted with 14mL of deionized sterile water on the seed germination paper. The seeds were then germinated and the seedlings were grown under light in DI water without any nutrients under fluorescent light for 14 and then weighed. The results showed that the inoculated seedlings were larger and able to grow better under nitrogen-limited conditions. The results strongly suggest that the inoculated plants are able to fix atmospheric nitrogen and utilize nutrients on the seedling germination paper better than the control. In terms of nitrogen fixation, it was observed that those strains that fixed the most nitrogen and grew best under nitrogen-free bacterial medium conditions also resulted in the largest lettuce seedling weights in the order WW6>WW5>WW6 / WW7>endophyte mixture (WW5, WW6, WW7, PTD1+WP1)>PTD1>WW7.

[0300] Embodiment 29

[0301] An experiment was conducted to apply the endophyte strains to broccoli seeds to determine the effect of this application on the biomass weight of the seedlings. The experiment included 4 treatment groups (WW7 strain only and a mixture of WW5, WW6, WW7 & PTD1) and a control group, such as Fig.39 Each treatment was applied to broccoli seeds with a clay seed coat and a fermentation mixture of nitrogen-limiting endophytes in a 1% w / v alginate solution. The control did not include the endophyte fermentation. Seeds were coated using commercial methods, including clay coating and seed coat polymer, including control seeds. Seeds were germinated in square petri dishes, with 14 mL of deionized sterile water moistened seed germination paper per 4 in. x 5 in. sterile seed germination container.

[0302] Seeds were germinated in square petri dishes with 14 ml of sterile deionized water per 4x5" sterile seed germination container to wet the seed germination paper. The seedlings were then grown under light without any nutrients for 14 and then weighed. The results showed that the inoculated seedlings were larger and able to grow better under limited conditions. This inoculated plant may have fixed atmospheric nitrogen better than the control and utilized nutrients from the seedling germination paper, resulting in the largest seedling weights, as Fig.39 shown.

[0303] Embodiment 30

[0304] An experiment was conducted to apply endophyte strains to barley seeds to determine the effect of such application on emergence. The experiment included treatment groups treated with co-fermented WW6+WW7 and a control group. Each treatment was applied to barley seeds with a clay seed coat and a fermentation mixture of nitrogen-limited endophytes in a 1 w / v% alginate solution. The control did not include the endophyte fermentate. Seeds were coated with a commercial seed treatment comprising a polymer and a crop protection agent, including a clay coating and a seed coat polymer, including control seeds. Seeds were germinated in square petri dishes, and each 4in. x 5in. sterile seed germination container was moistened with 14mL of deionized sterile water.

[0305] The seedlings were then allowed to germinate for four days under white light without any nutrients before the first photograph was taken. The results showed that the inoculated seedlings were significantly larger, e.g. Fig.40A As shown, after four days, it continued to grow over time, as shown in Fig.40B shown.

[0306] Embodiment 31

[0307] Experiments were conducted in which endophyte strains were applied to broccoli seeds to determine the effect of this application on seedling emergence. The experiment included two treatments, one with WW7 fermentation broth and the second with a four-strain mixture (I4WP), and a control group. Each treatment was applied to broccoli seeds with a clay seed coat and a fermentation mixture of nitrogen-limited endophytes in a 1 w / v% alginate solution. The control did not include the endophyte ferment. Seeds were coated using a commercial seed treatment comprising a polymer and a crop protection agent, including a clay coating and a seed coat polymer, including control seeds.

[0308] Treated and control broccoli seedlings were planted in separate units. Seeds were germinated in standard commercial transplant potting medium. The medium was watered daily and grown under natural daylight conditions at 25°C for 15 days. Plants were then evaluated for germination. An increase in the percentage of germination at 15 days was observed in the endophyte treated group. Plants inoculated with the endophyte strains showed faster germination (see Fig.41 ) and build and better growth.

[0309] Embodiment 32

[0310] An experiment was conducted in which endophyte strains were applied to sugar beet variety seeds (C578 and M5) treated with a seed inoculant composition. The experiment included two treatment groups, one treated with WW7 fermentation broth, the second treated with a four-strain mixture (I4WP), and a control group. The treatment group seeds were commercially treated at a ratio of 50 mL of fermentation broth, applied with the crop protection products Thiram, Metalaxyl, Hydrazol, Penthiopyrad and Poncho Beta (thiamethoxam) per 1 kg of seeds. Control seeds of the same variety received the same preparation without fermentation broth. The seeds were then stored under normal industrial conditions for 1 month and planted commercially.

[0311] Long-term measurements of sugar beet emergence after sewing. The endophyte seed treatment composition allowed survival of all four strains WW5, WW6, WW7, PTD1 (I4WP), increasing the emergence time of sugar beet varieties by an average of 29 days compared to the control. In this trial, where 200 plants were considered equivalent to a 75% plant stand in sugar beets, the endophytes increased the average emergence rate at 13, 16 and 29 days after planting. Fig.42A and 42B As shown, the C578 variety control had 187 seedlings after 29 days, while the four strains (I4WP) had 200 seedlings (13 more), and the M5 variety control had 161 seedlings after 29 days, while the treatment group treated with the I4WP mixture had 177 beet plants (16 more).

[0312] Embodiment 33

[0313] An experiment was conducted to apply endophyte strains to seeds of wheat varieties, treated with a seed inoculant composition. The experiment included a treatment group of seeds treated with co-fermented WW6 / WW7 broth and a control group. The treated seeds were commercially treated at a rate of 500 mL of broth, applying 500 mL of 1% alginate and prebiotic UBS 016 per 1 metric ton of seeds according to the manufacturer's instructions. Control seeds of the same variety received the same preparation without broth. The seeds were then stored under normal industrial conditions for 1 month and commercially planted.

[0314] In the CRO field trial, wheat emergence was measured over time. Treatments showed improved growth of post-emergence wheat seedlings over untreated controls, which were randomly harvested over time from appropriate field plots. After tracking weights for 15 days, final seedling fresh weights for the WW6 / WW7 combination were 1.054 g for leaves and 0.781 g for roots, compared to 0.95 g for leaves and 0.44 g for roots for the controls. Fig.43A and 43B The fresh weight of new plants pulled out each day is shown in .

[0315] Example 34. Effect of WW6 or WW7 applied as a seed treatment on cereal yield in the field

[0316] Endophytes were applied as seed treatments to improve field yields under highly optimized nutritional conditions. Spring wheat seeds were treated with WW6 or WW7 endophyte NLM ferments. The ferments were freeze dried to powder prior to seed treatment. Seed treatment slurry was prepared by adding 5 grams of freeze dried ferment and 5 grams of dry sodium alginate per liter of water. A 6 L volume of seed slurry was used to treat 1 metric ton of spring wheat seeds, variety Tybalt. Fig.44 The results presented in showed that spring wheat seeds treated with WW6 had a 12% increase in crop yield and seeds treated with WW7 had a statistically significant 23% increase in crop yield compared to controls that did not receive endophyte treatment.

[0317] Example 35. Effect of WW6 or WW7 applied as a seed treatment on improving the tolerance of the host plant treated with the inoculant composition to plant diseases.

[0318] Field trials were set up in Lincolnshire, UK. Wheat seeds (variety Skyfall) were treated with the following: Vibrance (Syngenta, Basel, Switzerland), plus WW6+WW7 endophyte LNM ferment freeze-dried powder (5 grams applied in combination with 5 grams of dry sodium alginate suspended in 1L H2O), and combined with the commercially available prebiotic UBS 016 at a ratio of 100 ml diluted to 1 liter (Unium Biosciences, Brind, Howden, UK). The final seed treatment slurry volume was 4 L and applied to 1 ton of wheat seeds. In the fall of 2020, the seeds were sown at a rate of 200 Kg / ha. Untreated blocks received only commercial crop protection agent seed treatments.

[0319] This experiment covered two nitrogen treatments. A commercial rate of 220Kg nitrogen and a lower rate of 180Kg / ha (supplied as ammonium nitrate). In both cases 50 units were provided at planting and the remainder the following spring. Each treatment had four replicates, comprising 10 x 25m blocks. An industry standard plant protection foliar fungicide was applied following the UK protocol recommended by Farmers Weekly Journal (Guide to tackling Septoria disease in wheat; https: / / www.fwi.co.uk / arable / crop-management / disease-management / a-guide-to-tackling-sept oria-in-wheat).

[0320] Wheat Septoria is the most destructive wheat disease in Northern Europe. In these trials, Septoria assessments were made on flag and second leaves over the course of the season. Fig.45A It showed that the infection rate of wheat plants treated with endophyte seeds was significantly lower than that of the conventionally treated controls. Fig.45B Visual, qualitative differences in the health of leaf 2 specimens from endophyte-treated plants compared to leaf 2 specimens from control plants are shown.

[0321] Example 36. Compatibility of endophyte herbicides commonly used in agricultural foliar applications to control weeds in various monocot and dicot crops.

[0322] Three liquid foliar herbicide chemistries; Enlist One (Corteva Agriscience, LLC), Impact (AMVAC Chemical Corporation), and Callisto (Syngenta Crop Protection, LLC) were evaluated for compatibility as tank mixes with WW6 and WW7 strains. For experimental purposes, the solution mix was scaled down from 20 gallons / acre to 10 ml. The mixture contained 32 fl oz / acre of WW6+WW7 endophyte fermentation composition in combination 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 (mefenacet 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. Colony forming units (CFU / ml) of both strains were determined by plating on NLM semi-solid medium 4 hours after the mixture was generated. Fig.46 .

[0323] Fig.46 The results presented in show that the viability of both endophyte strains was not significantly reduced over a long period of time in different solution mixtures, so these formulations that improve nutrient use efficiency, biomass and stress tolerance of endophytes could be combined with commercial agricultural herbicide foliar sprays.

[0324] Example 37. Examples of commonly used agricultural foliar-applied fertilizer-biostimulant compatibility in various monocot and dicot crops.

[0325] Two foliar fertilizers or biostimulants Isabion (Syngenta Agro AG) and Megafol (Syngenta Crop Protection AG) were tested for their compatibility as foliar tank mixtures with a combination of WW6 and WW7 strains. For experimental purposes, the tank mixture was scaled down from 400 l / ha to 10 ml. The mixture contained 2.4 l / ha of the WW6+WW7 microbial combination in combination with the following products: 6 l / ha of Isabion, 3 l / ha of Megafol or water. The colony forming units (CFU / ml) of both strains were assessed by plating on NLM semi-solid medium 4 and 24 h after the mixture was generated. See Fig.47 .

[0326] Fig.47The results presented in the present study showed that the viability of both strains was not significantly reduced over a long period of time in different solution mixtures, thus making it possible to use this endophyte composition for commercial agricultural fertilizer and biostimulant foliar spray applications.

[0327] Example 38. Examples of compatibility of stable freeze-dried powder endophytes with foliar herbicide applications commonly used in various agricultural and monocot cereal crops.

[0328] The compatibility of strains WW6 and WW7 was tested and evaluated by reconstituting them as freeze-dried compositions in water into aqueous solutions and mixing them with different standard herbicide tank mixes commonly used in cereal crops. Compatibility was tested with the products Azimut (Comercial Química Masso, SA), Guadana (Comercial Química Masso, SA) and Tower (Comercial Química Masso, SA). For experimental purposes, the solution mixture was scaled down from 400 liters / ha to 10 ml. The mixture contained 0.25% of WW6+WW7 microbial product (10 grams of freeze-dried product mixed with 1 liter), combined with the following products: 0.13% of Azimut (bifenazolidinone 5g / L (0,5% w / v) + chloramphenicol (potassium salt) 10g / L (1% w / v) + 2,4-D (ester-2-etilhexil) 180g / L (18% w / v)), 0.15% of Guadana (flufenacil 40% w / v (400g / l) (32.4% w / w) + diflufenacil 20% w / v (200g / l) (16.2% w / w)), 0.50% of Tower (difenacil 4% + chlorotoluron 25% + pendimethalin 30% (SC)) or water as a control. After the mixture was generated and stored for 4 hours, the colony forming units (CFU / ml) of the two strains were determined by plating on NLM semi-solid medium. Fig.48 .

[0329] Fig.48 The results presented in show that the viability of both strains was not significantly reduced over a long period of time in different product mixtures, so this endophyte combination can be used in product formulations that enhance nutrient use efficiency, biomass and stress tolerance in combination with commercial agricultural herbicide foliar spray applications.

[0330] Example 39. Enhanced atmospheric nitrogen fixation capacity after treatment with an endophyte inoculum seed bark suspension.

[0331] Tests were conducted to demonstrate the ability of different endophytes to fix atmospheric nitrogen in crop plants germinated from inoculum-coated hybrid maize seeds using a seed treatment suspension mixed with a 0.5% w / v nutrient supplement sodium alginate carbohydrate suspension. Endophyte inoculum individual strains WW5, WW6 and PTD1 were cultured using a custom fermentation inoculum until the cultures reached at least 1.0 E 8 The seed treatment inoculum was titrated to 100 cells / mL and then combined with a nutrient additive carbohydrate. The seed treatment inoculum was QC checked for viable cells and the appropriate species and strain colony morphology was genetically determined using specific primers for colony PCR. This endophyte solution seed treatment composition was then mixed with a widely used chemical seed treatment containing the fungicides fludioxonil, metalaxyl-M and the neonicotinoid insecticide thiamethoxam. The endophyte inoculum seed treatment solution was applied at a rate of 2.4 mL / 1800 corn seeds and added to the chemical seed treatment according to the manufacturer's recommended corn seed ratio and application instructions. The treated seeds were then dried and the colony counts of viable microorganisms were determined on nitrogen-limited medium NLM plus agar using seed coat wash KP buffer count dilution plating. The count results showed that the microbial survival rates on the seeds one month after the application of the mixed slurry seed coat were as follows: WW6~400 CFU / seed, WW5~40 CFU / seed, and PTD1~40 CFU / seed.

[0332] The seeds were then potted in 2-gallon pots containing 2.5 kg of field soil amended with perlite, germinated and grown for 4-5 weeks until the V6 stage with no fertilizer added to the soil, which contained 1,200 ppm total N plus 10 ppm soluble N (NO3+NH3). Plants were grown in a greenhouse and treated with 15 Biological nitrogen fixation (BNF) was analyzed using an N isotope dilution assay, which specifically measures the percentage of nitrogen that comes from the air. 15 N content compared with the natural nitrogen content of adjacent reference non-inoculated plants that depended solely on soil nitrogen for survival 15 The total N and nitrogen content in the aerial tissues of maize were measured at the V6 growth stage 4-5 weeks after planting. 15 N isotopes, total N measured using the Elementar EAVario Pyrocube, followed by N using the Elementar IRMS GeoVisION isotope ratio mass spectrometer (IRMS) 15 To quantify the percentage of nitrogen in the aerial parts of plants that came from the air (NDFA%), the IRMS-measured nitrogen concentration was subtracted from the total nitrogen concentration. 15 The difference between the N isotope nitrogen content and the NDFA content is the percentage of NDFA in the total N pool.

[0333] Test results such as Fig.49 As shown, it was shown that seed treatment with endophyte seed treatment inoculum resulted in the major percentage of total nitrogen in the aerial parts of corn coming from the air as follows; PTD1 42% NDFA, WW5 36% NDFA and WW6 69% NDFA. The results clearly showed that endophyte seed treatment of corn can be stabilized after drying and used to inoculate seeds, and the crop plants subsequently germinated from the seeds have enhanced biological nitrogen fixation at the vegetative growth stage V6 of the corn plant.

[0334] Example 40: Treatment with formulated liquid endophyte inoculum directly applied to the roots of wheat seedlings enhances the ability to fix atmospheric nitrogen.

[0335] To demonstrate the ability of different endophytes to fix atmospheric nitrogen in crop plants after inoculation in soil or in-furrow rhizosphere, endophyte liquid inocula of individual strains of WW5, WW6, PTD1 and a co-cultured synergistic mixture of WW6+WW7 strains were cultured. Culture suspensions were prepared simultaneously using a customized nitrogen-limited NLM medium with nutrient supplements until the cultures reached at least 1.0 E 8 The titer of cells / mL was obtained. The nutrient additive inoculum was then analyzed for viable cells and colony morphology. The presence of co-cultures of WW5, WW6, PTD1 and WW6+WW7 strains in the respective inocula was confirmed using specific primers for colony PCR. The roots of 2-week-old wheat plants were then inoculated with the endophyte suspension inoculum. After transplanting from the potting soil flat, 1 mL of the inoculum suspension was applied to the roots at the base of the plant per plant. The transplants were planted in 1-gallon greenhouse pots containing 1.5 kg of field soil amended with perlite, without any fertilizer added to the field soil, with a total N of 1,200 ppm, plus 10 ppm soluble N (NO3+NH3). The plants were grown in the greenhouse until the jointing growth stage, approximately 4-5 weeks after transplanting. The tissues were then harvested and analyzed for biological nitrogen fixation (BNF), measured as nitrogen from air, total N using an Elementar EA Vario Pyrocube, and nitrogen from nitrogen using an Elementar IRMS GeoVisION isotope ratio mass spectrometer (IRMS). 15 N.

[0336] The results are as follows Fig.50As shown, it was shown that seed treatment with endophyte seed treatment inoculum resulted in a significant percentage of nitrogen from air in the aerial parts of wheat as follows; 43% NDFA for PTD1, 51% NDFA for WW5, 38% NDFA for WW6, and 47% NDFA for the synergistic combination of WW6+WW7. The results clearly indicate that endophyte inoculation of wheat roots in both single strain and co-fermented nutrient additive solutions can enhance biological nitrogen fixation and maintain this capacity throughout the main vegetative growth stages of the wheat plant.

[0337] Example 41: The synergistic combination of endophytes WW6+WW7 is superior to the effect of a single strain when applied to barley as a freeze-dried recombinant seed treatment formulation.

[0338] The purpose of this trial was to determine whether a mixed synergistic community of endophytes could increase total barley plant biomass (aerial parts + roots) better than using a single strain alone. Dry weight biomass was measured after 26 days of growth under reduced nitrogen in a controlled environment. The bacterial inoculum, which was first freeze-dried, stored and then resuspended at its original growth solution water content, was prepared as a formulated seed treatment slurry and used as an endophyte seed inoculum (500 mL resuspended endophyte freeze-dried culture 1.1% w / v plus the addition of sterile sodium alginate solution 2% w / v), and a sterile control nitrogen-limited medium (NLM) with a final alginate 2% w / v alone, without endophytes. Both treatments were applied at a rate of 1 L per 1 metric ton of seed. Spring barley was treated with 3 different seed preparation solutions using endophyte strains WW6, WW7 and co-fermented WW6+WW7 and dried overnight in a laminar flow cabinet. Four pots were planted in each of the four treatment groups, with one plant per pot and n=4 plants per treatment. Control plants were given Hoagland denitrification solution at 65 ppm N (100%) and reduced 32 ppm N (50%). Endophyte-inoculated experimental treatments were also given reduced 32 ppm N (50%). Hoagland nutrients were given at M, W, and F by tray watering, and the trays were drained after 1 / 2 hr. After germination in a plant growth incubator at 25°C, plants were grown for 26 days with artificial lighting set at 12h light / 12h dark, then harvested and dried in paper bags at 45°C for 48 days.

[0339] Fig.51 The results shown demonstrate the synergistic effect of the combined treatment WW6+WW7, which increased barley dry weight biomass under 50% nitrogen reduction, resulting in a significant increase in total biomass weight by up to 75%, while WW6 alone increased total biomass by a non-significant 17% and WW7 alone increased total dry weight biomass by a non-significant 17% when grown under the same nitrogen reduction conditions compared to the control.

[0340] Example 42. Synergistic application of stacked endophyte strains to increase grain yield biomass via inoculum seed treatment of commercial field-grown spring wheat.

[0341] To test the ability of specific endophyte combinations to provide synergistic benefits to cereal crops, a variety of spring wheat (Sy Ingmar) was commercially treated with co-ferment of WW6+WW7 and co-ferment of WW5+WW6+WW7 using a seed treatment approach. No other seed treatments were applied. Seed treatment slurries (>1.0 E 6 CFU / mL NLM fermentation broth plus carbohydrate solution 1-0.5% w / v), and the slurry was applied at a rate of 0.23mL of bacteria per 1lb of wheat seeds using seed treatment equipment to coat the seeds. The control was not inoculated with endophyte fermentation broth. Wheat was planted in Berthold, ND USA in late May and harvested in September after 117 days of field growth. The plot size was: 5ft x 30ft, containing Williams Silt Loam soil. Each plot was planted with 4 rows of wheat, 10 seeds per row, evenly spaced. In a randomized complete block design, each treatment was replicated 4 times and 1,500,000 seeds were planted per acre. Grain yield data was adjusted to 14% moisture. Fertilizer was applied at planting, consisting of a mixture of 15gal / ac of 10-34-0 and 44.5gal / ac of 28-0-0. Soil testing was conducted on soil from 0 to 24" depth and showed the following characteristics: 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.

[0342] The research results are as follows Fig.52 The two-strain treatment (WW6+WW7) increased the average yield by 0.7 bu / ac, while the stacking of three strains in the strain treatment had a synergistic effect, further increasing the average yield by 3.33 bu / ac compared with the control, and was statistically significant, p<0.1.

[0343] Example 43. Synergistic application of stacked endophyte strains to enhance biomass yield via inoculum seed treatment of commercial field grown romaine lettuce.

[0344] To test the ability of the applied characteristic endophyte combinations to provide yield benefits to lettuce crops, a commercial treatment of lettuce (River Road CVS) was performed using single bacterial endophyte strains as well as co-fermentation strain combinations (2, 3, and 4 strains) of strains WW5, WW6, WW7, and PTD1 with the endophytic yeast WP1. All fermentations were mixed into treatment slurries of the following formulations: CFU / mL>1.0E in NLM fermentations 6 cells plus a carbohydrate solution 1% w / v. The treatment slurry was also mixed into a clay seed coat using a seed treatment device and then applied to the seeds using standard commercial techniques. The endophyte solution was applied at the following rate: 10 mL of final volume of endophyte inoculum per 1 / 3 lb of lettuce seeds. In cases where the co-fermenter included two endophyte strains, 5 mL of each strain was applied. In cases where the co-fermenter included five endophyte strains, a total of 2 mL of each strain was applied. Commercial standard field planting parameters included an 80" seedbed planted with 142,000 seeds on approximately one acre of land near Spreckles, CA.

[0345] like Fig.53A and 53B As shown, single strain treatments increased yield. The combined mixed treatment of strains WW5, WW6, WW7 and PTD1 with yeast strain WP1 showed a significant increase of 113% in aboveground biomass weight after growth in commercial fields.

[0346] Example 44: The synergistic combination of endophytes WW6+WW7 was superior to the single strain when mixed with a prebiotic carrier composition in the form of a compatible biostimulant and applied to rapeseed as a seed treatment.

[0347] Tests were conducted to determine whether a mixed synergistic community of endophytes could better increase total rapeseed plant biomass (aerial + root) when mixed with a prebiotic plant microbial enhancer than using a single strain and prebiotic plant microbial enhancer alone. Fresh weight biomass was measured after 21 days of growth under reduced nitrogen in a controlled environment. The bacterial inoculum was freeze-dried, stored, resuspended at its original growth solution water content, and then incorporated into the formulated seed treatment slurry. The slurry was used to prepare an endophyte seed inoculum of the following formulation: 500 mL of a 1.1% w / v resuspended endophyte freeze-dried culture, 500 mL of a prebiotic program and microbial biostimulant, and 4% w / v sterile sodium alginate in water. The sterile control nitrogen-limited medium (NLM) included the prebiotic program and microbial biostimulant, and 4% w / v sterile sodium alginate. Treatments were applied at a rate of 1 L per 1 metric ton of seed. Seeds (untreated spring rapeseed "Atomic TT") were treated with 3 different seed preparation solutions using endophyte strains WW6, WW7 and WW6+WW7. The seeds were dried overnight in a laminar flow cabinet after application. Three pots with 1 plant were planted in each of the four treatment groups, and n=3 plants per treatment. Control plants were given Hoagland denitrification solution at concentrations of 65ppm N (100%) and 32ppm N (50%). The experimental group was given 32ppm N (50%). Nutrients were given by watering trays at M, W, and F, after which the trays were drained. After germination in a plant growth incubator at 25°C, the plants were grown for 21 days with artificial lighting set to 12h light / 12h dark, and then harvested.

[0348] like Fig.54 As shown, the combined synergistic mixed treatment WW6+WW7+prebiotics increased total biomass the most, significantly increasing the total biomass weight by 82%, while WW6+prebiotics increased total biomass by 20% and WW7 increased total biomass by 55%.

[0349] Example 45. Use of three synergistic endophyte strains for foliar application on aerial parts of corn to reduce fertilizer requirements and increase harvest grain yield.

[0350] Tests were conducted to determine the efficacy of a novel endophyte combination (WW5+WW6+WW7) applied as a foliar spray to enhance nitrogen fixation in a hybrid corn variety (Channel 113day213-19VT2PRIB). The corn variety was first treated commercially with the seed chemical Acceleron according to the manufacturer's protocol. Corn was planted in early May in Mead, Nebraska, USA. The soil was Tomek Silt Loam with pre-planting nutrient levels including 11.1 ppm P, 344 ppm K, and 7.4 ppm S, pH 5.8, OM 4.1%, and CEC 17.6. Corn plants were treated at V6 using a pressurized sprayer to apply a foliar spray, spraying either the control or the experimental treatment including the endophyte strains WW5, WW6, and WW7. Plot size was 10ft x 40ft with 4 rows of corn planted per plot. A block design was used with 6 replications per treatment. Fertilizers were also applied and included a soil nitrate pre-fertilizer at 17 lb / acre, a liquid formulation of UAN 32-0-0 applied at 75 lb / acre prior to planting; and an additional 37.5 lb / acre of nitrogen for a total of 130 lb N / ac. The nitrogen application rate for the treatment group was 75% of the standard nitrogen application rate for the planting area. A secondary control treatment was also included in the study and received 170 lb of nitrogen (100% of the standard nitrogen application rate). Herbicides were also applied. Pre-Acuron+Roundup was applied to the plants on May 13, shortly after planting. The middle two rows were harvested in late October after 163 days of growth in the field, and the grain was weighed and statistically analyzed. The results are summarized in Fig.55 middle.

[0351] Results at 75% N fertilizer rate showed that the synergistic three strain endophyte foliar application applied at V6 significantly increased average corn grain yield by +44.5 bu / ac (p=0.03). Endophyte foliar results at V6 increased average yield compared to the full 100% N control treatment of 35 bu / ac.

[0352] Example 46. Stacking of two endophyte strains for synergistic in-furrow application to increase harvested grain yield at 75% nitrogen reduction.

[0353] Tests were conducted to determine the efficacy of a specific endophyte combination WW5+WP1 applied as an in-furrow liquid composition to improve nitrogen fixing inoculum and yield in a hybrid corn variety (Channel 113day213-19VT2PRIB). The corn variety was first commercially 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-planting nutrient levels including 11.1 ppm P, 344 ppm K and 7.4 ppm S, pH 5.8, OM of 4.1%, and CEC of 17.6. Corn plants were treated with the WW5+WP1 endophyte solution at planting, which was applied over the seeds in the furrow using a dropper. Plot size was 10ft x 40ft with 4 rows of corn planted per plot. A split design was used with 6 replications per treatment. Fertilizers were also applied and included a soil nitrate pre-fertilizer at 17 lb / acre, a liquid formulation of UAN 32-0-0 applied at 75 lb / acre prior to planting; and an additional 37.5 lb / acre of nitrogen for a total of 130 lb N / ac. The nitrogen application rate for the treatment group was 75% of the standard nitrogen application rate for the planting area. A secondary control treatment was also included in the study and received 170 lb of nitrogen (100% of the standard nitrogen application rate). Herbicides were also applied. Pre-Acuron+Roundup was applied to the plants on May 13, shortly after planting. The middle two rows were harvested in late October after 163 days of growth in the field, and the grain was weighed and statistically analyzed. The results are summarized in Fig.56 middle.

[0354] Results at 75% N fertilizer rate showed that in-furrow application of the synergistic two strain endophyte applied at planting increased average corn grain yield by +30.2 bu / ac (p=0.07). The in-furrow endophyte results at planting increased average yield compared to the full 100% N control treatment of 20 bu / ac.

[0355] Example 47. Stacking endophyte strains for synergistic application to enhance biomass yield of strawberry plants using liquid root sprays of transplants prior to field planting.

[0356] Testing was conducted at an organic strawberry farm in Salinas CA to determine the efficacy of a specific endophyte combination WW5+WP1 applied as a liquid root spray to increase biomass yield in Albion strawberry plants. Transplant roots were sprayed until covered with a mist of the different endophyte inocula. Spray treatments included WW5 alone, WW6 alone, WW7 alone, PTD1 alone, and a mixture of WW5, WW6, WW7, and PTD1. The control group did not include the endophyte strains. Treatments were applied using standard methods and plants were planted in early November 2016. Strawberries were planted in beds containing two rows of strawberry plants spaced 25-30 cm apart (200-240 plants / row) with 3 beds per treatment spaced 120 cm apart. Strawberries were fertilized using standard methods under the guidance of a registered CCA and harvested on May 20, 2017 after 28 weeks of growth.

[0357] like Fig.57 As shown, the treatment group including a mixture of all 4 strains WW5, WW6, WW7 and PTD1 performed best, increasing fruit yield measured in fresh weight by 25% compared to the control. Single strain inoculation treatments resulted in smaller increases over the control.

[0358] Example 48. Combination of endophyte strains in partially hydrated hard beads as dry granular carrier for synergistic application to increase tomato transplant biomass.

[0359] Tests were conducted to determine the efficacy of individual WW5, WW6 and WW7 combinations and the combination of the four endophyte strains WW5, WW6, WW7 and PTD1 in increasing tomato biomass yield. The ferment suspension was mixed into a sodium alginate slurry and dropped into a calcium chloride 100 mM water bath where a cation exchange reaction occurred to produce fully hydrated but hard calcium alginate beads, which were then dried to a final moisture content of about 4% to about 6% and a final bead size of 2 mm. Quality 47 (Q47) hybrid tomato seeds were then placed on or near individual beads containing endophyte treatments. Controls were treated with beads without endophyte strains. Plants were then germinated and grown for 3 weeks in a commercial transplant potting mix containing high organic matter consisting primarily of peat and perlite in a 125-grid transplant pot tray under normal commercial greenhouse fertilization rates and normal light at 25°C. After 21 days, eight replicates in each treatment were weighed and the total plant biomass dry weight was collected. The survival rate of Q47 tomato plants inoculated with the mixture was 100% compared to the uninoculated control, which had a suboptimal germination rate of 88%. Fig.58 shown.

[0360] The results clearly showed that there was a synergistic effect on the growth of tomato plants inoculated with alginate beads containing a combination of four endophyte strains WW5, WW6, WW7 and PTD1 compared to the control. The mixture of endophytes resulted in a statistically significant increase of +35% in total plant weight (aerial and root). Single endophyte treatments WW5 and the combination of WW6 and WW7 provided smaller increases.

[0361] Example 49. Combination of endophyte strains in partially hydrated hard beads as a dry granular carrier for synergistic inoculant application to leaf lettuce grown in the absence of bioavailable N and P.

[0362] Tests were conducted to determine the efficacy of endophyte strains in dry calcium alginate beads to reduce nitrate and phosphate application in loose leaf lettuce (Lactuca sativa, variety Refugio) while increasing edible harvest yield. In the experiment, we reduced the bioavailable forms of N and P to deficiency and conducted the experiment in a greenhouse using a specific soilless substrate consisting of Terragreen, a baked calcined clay gravel mixture consisting of 9 kg Terragreen, 0.66 kg peat moss, 40 g of 8-3-5 organic fertilizer, producing nutrient concentrations, and a well-drained organic-rich soil profile, including 12 ppm nitrate, 5 ppm ammonia, 11 ppm phosphate, 328 ppm potassium, 690 ppm sulfate, 2.71 SAR, pH 7.28, 2.89 dS / m EC, 18.76 meq / 100 g TEC and 1831 ppm total nitrogen, mainly composed of amino acids. The greenhouse trial was harvested 106 days after planting. Each seed was planted in a pint-sized starter pot with a bead on each seed, both placed 1 / 2 inch in the growth medium and watered. Alginate beads were applied adjacent to or near the germinating seeds in the soil. The plants were then carefully transplanted with a hand shovel at 3 weeks to remove all beaded roots and keep the surrounding soil intact. Roots, beads and soil were placed in holes of the same size and placed in 2-gallon felt smart pots. All plants were automatically watered with the same amount of water every 12 hours through a controlled drip irrigation system. From 4:15pm to 7:15pm, greenhouse lights (high-pressure sodium halide lamps) were used to supplement the lack of evening sunlight to ensure a complete 12-hour growth cycle. Lettuce plants were inoculated with beads containing a single WW7 or PTD1, or a mixture of all four bacterial endophytes (WW5+WW6+WW7+PTD1). The control received alginate beads without endophytes. All treatments were repeated 6 times, with n=6 pots for each treatment.

[0363] Fig.59Results of the fresh weight aboveground biomass analysis are shown. The treatment that included a mixture of all four endophytes performed best, with an average aboveground weight of 4.23 g per plant, a 191% increase in aboveground weight compared to the uninoculated control plants, a statistically significant result at p < 0.1. WW7 beads had an average yield of 3.96 g per plant and a 173% increase in average aboveground weight. PTD1 bead inoculation increased aboveground biomass by an average of 2.74 g per plant, resulting in an 89% increase in average yield.

[0364] Example 50: Combining endophyte strains in a lyophilized powder for reconstitution and application in the field as a foliar spray on Mexican peppers

[0365] To determine the efficacy of endophyte inoculants from a freeze-dried endophyte mixture on the growth and biomass of Mexican pepper plants (variety RPP7042). In late spring, approximately 420 Mexican pepper plants were inoculated by foliar spray at the beginning of flowering. Five individual endophyte strains (WW5, WW6, WW7, PTD1 and WP1) resuspended from freeze-dried powders were used. A mixture of the five resuspended endophyte strains was also prepared. 1 gram of freeze-dried endophytes was added per liter of DI water to rehydrate the freeze-dried endophytes and then placed in a foliar sprayer. Mexican pepper seeds were planted in May to early June. The harvest date was mid-September to early October. The reconstituted mixture was applied to the leaves along each 50-foot treatment block. A 50-foot control treatment separated each treatment, with three treatments per row. 50 feet is approximately equivalent to 75 plants. A series of growth and biomass analyses were performed at harvest 2 months later.

[0366] like Fig.60A As shown, all inoculation treatments using the recombinant freeze-dried powder at the time of first flowering increased the average immature pepper yield per plant compared to the control, except for the treatment with the WP1 strain. The mixture of all five strains (Phase A) was found to be the best performing inoculum and produced a synergistic effect. Phase A increased the total immature pepper yield by +44% and had a positive effect on the RPP7042 Mexican pepper plants.

[0367] In addition, the ability of each treatment to increase the total number of peppers was determined, and the results were as follows Fig.60B The A phase inoculation also demonstrated the best results, with the exception of WP1, where the average number of peppers per plant increased by 66% over the initial control. The mixture of A phase, PTD1, and WW7 all showed statistical significance, indicating that the freeze-dried endophytes had an early positive impact on the Mexican pepper plants.

[0368] The total yield of mature peppers was also measured three months after the initial inoculation and four months after planting and Fig.60CThe results showed that endophyte inoculation using reconstituted freeze-dried powder applied as a foliar spray during the early flowering stage could be used to increase the average yield of jalapeno pepper per plant compared to the control. The A-stage inoculum increased pepper biomass yield more than the other treatments. All bacterial endophyte strains tested increased yield compared to the control, while the A-stage showed an increased synergistic effect. The results support that freeze-dried endophytes, once reconstituted into a solution and sprayed on flowers and leaves, have an early positive impact on total jalapeno pepper yield.

[0369] Example 51. Effects of different endophyte seed treatment compositions using WW5, WW6, WW7, PTD1 and WP1 on leaf chlorophyll.

[0370] The mixed synergistic compositions of endophytes were freeze dried and resuspended in water and used to treat rapeseed seeds to determine whether the resuspended endophyte fermentation could increase chlorophyll in rapeseed leaves after 36 days of growth under reduced nitrogen conditions. The endophyte seed inoculum consisted of endophyte freeze dried powder resuspended in 500 mL of water at a concentration of 1.1% w / v, mixed with 500 ml of a 4% sterile sodium alginate aqueous solution and applied at a rate of 1 L per metric ton of seeds. Endophyte inoculum 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 rapeseed "Atomic TT". Seeds were treated with the prepared endophyte treatment compositions: WP1, WW6+WW7, WW5+WW6+WW7+WP1+PTD1. The control group was treated with NLM alone without endophytes. After seed treatment, all groups were dried overnight at room temperature in a laminar flow cabinet. Four pots were planted in each of the five treatments, with two plants per pot (n = 8 plants / treatment). Control plants were given Hoagland denitrification solution at concentrations of 65 ppm N (100% N) and 32 ppm N (50% N). The experimental group was given 32 ppm N (50% N). Nutrients were given three days a week (M, W, F) by watering the trays, and the trays were drained after 30 minutes. After germination in a plant growth incubator at 25°C, the plants were grown for 36 days with artificial lighting set to 12 h light / 12 h dark before harvesting.

[0371] The results showed that the endophyte composition used as rapeseed seed treatment had a synergistic effect on leaf chlorophyll, with the two strains WW6+WW7 increasing leaf chlorophyll by 14.3% p<0.1, while the synergistic 5-strain endophyte community composition increased leaf chlorophyll by 21.0% p<0.05. Fig.61 .

[0372] Example 52. Stacking of endophyte strains in alginate beads for synergistic application to increase chlorophyll in leaves of strawberry plants.

[0373] Tests were conducted to determine the efficacy of endophyte inoculants applied as a composition to increase leaf chlorophyll in Albion strawberry plants. Tests were conducted at an organic strawberry farm in Salinas CA. Prior to planting, strawberry plants were transplanted onto five 2 mm calcium alginate beads placed in a hole. The calcium alginate beads were prepared with one of the following treatment groups: an endophyte inoculant including WW5 alone, an endophyte inoculant including WW6 alone, an endophyte inoculant including WW7 alone, an endophyte inoculant including PTD1 alone, an endophyte inoculant including WW5, WW6, WW7, and PTD1, or a control without an endophyte strain. Plants were planted in early November 2016 using standard methods. Strawberries were planted in beds containing two rows of strawberry plants spaced 25-30 cm apart (200-240 plants / row), with three beds per treatment spaced 120 cm apart. The strawberries were fertilized using standard methods under the guidance of a registered CCA and harvested on May 20, 2017, after 28 weeks of growth.

[0374] like Fig.62 As shown, the treatment group including a mixture of all four strains WW5, WW6, WW7 and PTD1 performed best, providing more leaf chlorophyll and significantly increased leaf chlorophyll by 5.5% p<0.05.

[0375] Example 53. Effect of different endophyte seed treatment compositions of WW6+WW7 with market leading bioagents and biostimulants on total chlorophyll in winter wheat leaves.

[0376] Experiments were conducted to determine if a freeze-dried and resuspended endophyte composition could increase leaf chlorophyll in treated host plants when applied to seeds. Endophytes were applied to seeds of winter wheat variety AWC13. A freeze-dried endophyte seed inoculum composition was prepared by resuspending freeze-dried WW6+WW7 endophyte strains in 500 mL of water at a concentration of 1.1% w / v and mixing the resuspension with 500 mL of a 4% sterile sodium alginate aqueous solution. A control was prepared with the sodium alginate solution and 500 mL of water without endophytes. The treatment composition was applied at a rate of 1 L per 1 metric ton of seed. In the field trial, winter wheat plants were grown under optimal nitrogen conditions and then harvested during the vegetative period. From 1 cm 2 Chlorophyll was extracted from leaf slices of different sizes and extracted with acetone. Fig.63 The results in showed an average increase of 6% compared to the untreated controls.

[0377] Example 54. Increased glutamate / glutamate Glx in maize leaves after treatment with an endophyte inoculum seed coat composition.

[0378] Tests were conducted to determine if seed coats containing heterologous endophyte compositions could fix N2 atmospheric nitrogen, produce ammonium, which was then converted to the first amino acid end products glutamate and glutamine by the GOGAT GS and GDH ammonium assimilation pathways in the host plant. A seed treatment comprising WW6 fermentate and sodium alginate 0.5% w / v was prepared. The seed treatment was applied to corn seeds, which were then dried and stored for 1 month. The treated seeds and control untreated seeds were then planted in 1 gallon pots containing 1.5 kg sand, vermiculite and perlite, germinated and grown for 3 weeks. During the growth period, the seeds and resulting plants were watered with Hoagland denitrified nutrient solution supplemented with 50 ppm N. Plants were grown in a greenhouse, harvested, freeze-dried, and analyzed for common amino acids by AAAlabs Inc USA using a Shimadzu HPLC with post-column ninhydrin derivatization. Fig.64 The results shown in the 2014 study showed that the glutamate and glutamine amino acid content in the leaves of plants grown from maize seeds inoculated with WW6 was significantly (p=0.6) increased by +46% compared to the control. In addition, when the genome of WW6 was analyzed for the presence of genes encoding the glutamine synthetase GS enzyme, a total of 6 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, as well as the increase in N concentrations in the aboveground parts of WW6-treated maize. These results also demonstrate the mechanistic basis for the efficacy of WW6 as a biological inoculant in crop plants to increase the assimilation of nitrogen directly from the atmosphere into the amino acids glutamate and glutamine.

[0379] The above descriptions of specific embodiments of the present invention have been given for the purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in view of the above teachings. The embodiments are selected and described to best explain the principles of the invention and its practical application, thereby enabling those skilled in the art to best utilize the invention and the various embodiments and to make various modifications as may be required for the particular use contemplated.

Claims

1. A plant inoculant composition for treating a host plant, comprising at least one heterologous endophyte bacterial strain selected for its ability to fix atmospheric nitrogen, increase nitrogen uptake by the plant, and a culture medium composition containing at least one nutrient additive operable to enhance the survival and colonization of the at least one heterologous endophyte bacterial strain in the host plant.

2. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain is selected for its ability to fix atmospheric nitrogen and to grow on nitrogen-limited and nitrogen-free growth media.

3. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain is selected to have the ability to produce exogenous ammonium (NH4 + ) ability.

4. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain increases solubilization of various forms of insoluble phosphorus in a liquid bacterial growth culture.

5. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain has a genetic mechanism to produce an Fe siderophore.

6. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain comprises a plurality of endophyte bacterial strains selected to fix atmospheric nitrogen.

7. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain increases total plant carbon in a plant in which it colonizes.

8. The composition of claim 1, wherein said at least one heterologous endophyte bacterial strain increases the acquisition of essential macronutrients and micronutrients from soil and air without substantially disrupting the plant nutrient ion stoichiometry of said host plant.

9. The composition of claim 1, wherein at least one of the heterologous endophyte bacterial strains is resistant to a biocide.

10. The composition of claim 1, wherein the biocide comprises a fungicide, an insecticide, a nematicide, and a herbicide.

11. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain is tolerant to plant hormones, plant elicitors, mineral micronutrients, and mineral macronutrients.

12. The composition of claim 1, wherein the composition is mixed with one or more organic or synthetic biostimulants, fatty acids, carbohydrates, amino acids, mineral nutrients or chemical prebiotics to produce a combined seed treatment, a combined foliar application or a combined in-furrow application.

13. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain comprises at least one of Pseudomonassiliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

14. The composition of claim 13, wherein the at least one heterologous endophyte bacterial strain is a plurality of heterologous endophyte bacterial strains comprising at least two of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

15. The composition of claim 13, wherein the plurality of heterologous endophyte bacterial strains comprises at least three of Pseudomonassiliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

16. The composition of claim 13, wherein the plurality of heterologous endophyte bacterial strains comprises Pseudomonassiliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

17. The composition of claim 1, wherein the endophytic bacterial strains Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae can all be traced and positively identified using gene specific PCR primers.

18. The composition of claim 1, wherein the endophyte bacterial strains Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae can all be traced and positively identified in planta using gene specific PCR primers.

19. The composition of claim 1, wherein the endophyte bacterial strains Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi and Sphingobium yanoikuyae can all be traced and positively identified by using FAME fatty acid methyl ester GC-FAME ID.

20. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain reduces acetylene.

21. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain has an active nitrogenase gene subunit Nif H.

22. The composition of claim 1, wherein said at least one heterologous endophyte bacterial strain is operable to increase biomass in said host plant treated with said composition.

23. The composition of claim 1, wherein said at least one heterologous endophyte bacterial strain is operable to increase amino acid production in said host plant treated with said composition.

24. The composition of claim 1, wherein said at least one heterologous endophyte bacterial strain exhibits fixation of atmospheric nitrogen in said host plant treated with said composition when said host plant is planted in nitrogen-deficient soil or when said host plant is planted in nitrogen-enriched soil.

25. The composition of claim 1, wherein said inoculation with at least one heterologous endophyte bacterial strain increases total nitrogen accumulation in aerial parts of the plant.

26. The composition of claim 1, wherein said inoculant composition increases chlorophyll in leaves of said host plant treated with said inoculant composition.

27. The composition of claim 1, wherein said inoculant composition increases tolerance to abiotic stress of said host plant treated with said inoculant composition.

28. The composition of claim 1, wherein said inoculant composition increases the tolerance of said host plant treated with said inoculant composition to a plant disease.

29. The composition of claim 1, wherein the inoculant composition increases root biomass, root branching, root hairs, or any root ultrastructural changes.

30. The composition of claim 1, wherein said inoculant composition increases seedling germination, seedling emergence, and seedling biomass weight of said host plant treated with said inoculant composition.

31. The composition of claim 1 for use in short-term or long-term inoculation of dormant seed embryos, inoculated with one or more species of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi and Sphingobiumyanoikuyae.

32. The composition of claim 1, further comprising one or more Rhizobium species.

33. The composition of claim 1, further comprising one or more mycorrhizal species.

34. The composition of claim 1, further comprising an endophytic yeast strain, including but not limited to WP1.

35. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain is selected for use in combination with a plant-microbe enhancer or prebiotic comprising one or more of an osmoprotectant, an amino acid, an antioxidant, a mineral nutrient, a plant hormone or plant growth inducer, a carbohydrate, and a mucilage gel, applied to the host plant as a separate application or as a mixture.

36. The composition of claim 1, further comprising one or more fungicides, insecticides, herbicides, biostimulants, plant growth regulators, adjuvants and fertilizers, applied to the host plant as separate applications or as a mixture.

37. The composition of claim 1, wherein said composition comprises a carrier composition that enables said plant inoculant composition to be applied to seeds.

38. The composition of claim 1, wherein said composition comprises a carrier composition enabling said plant inoculant composition to be applied to foliar portions of said host plant.

39. The composition of claim 1, wherein said composition comprises a carrier composition that enables said plant inoculant composition to be applied to soil adjacent to said host plant.

40. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain is freeze-dried into a flowable powder prior to incorporation into the inoculum composition.

41. The composition of claim 1, wherein the at least one heterologous endophyte bacterial strain is encapsulated in micro- or macro-beads incorporated into the inoculum composition.

42. The composition of claim 38, wherein the inoculum composition is a suspension composition.

43. The composition of claim 39 or 40, wherein the inoculum composition is a dry composition.

44. The composition of claim 39 or 40, wherein the inoculum composition comprises a dry particulate composition.

45. The composition of claim 1, wherein the inoculant composition comprises a dry particulate composition.

46. ​​The composition of claim 1, wherein the inoculant composition comprises a liquid composition operable to be applied as a foliar spray.

47. The composition of claim 1, wherein the inoculant composition comprises a liquid composition operable for in-furrow application.

48. The composition of claim 1, wherein the inoculant composition comprises a liquid composition operable for coating a fertilizer.

49. The composition of claim 1, wherein the inoculant composition comprises a liquid composition operable to be applied to a dry powder carrier for coating a fertilizer.

50. The composition of claim 1, wherein the inoculant composition comprises a beaded composition operable for coating a fertilizer.

51. A plant inoculant composition comprising at least one heterologous endophyte bacterial strain selected from the group consisting of Pseudomonassiliginis, Curtobacterium salicaceae, Rhizobium populi and Sphingobium yanoikuyae for heterologous application to a host plant.

52. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain is selected for its ability to fix atmospheric nitrogen and to grow on nitrogen-limited and nitrogen-free growth media.

53. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain is selected to have the ability to produce exogenous ammonium (NH4 + ) ability.

54. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain increases solubilization of multiple forms of insoluble phosphorus in a liquid bacterial growth culture.

55. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain has a genetic mechanism to produce an Fe siderophore.

56. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain comprises a plurality of endophyte bacterial strains selected to fix atmospheric nitrogen.

57. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain increases total plant carbon in a plant in which it colonizes.

58. The composition of claim 51, wherein the inoculant composition comprises a prebiotic composition operable to enhance colonization of a host plant by the at least one heterologous endophyte bacterial strain.

59. The composition of claim 51, wherein the prebiotic composition comprises at least one of a microbial nutrient package, a plant biostimulant, an osmoprotectant, a buffer, and a seed lubricant.

60. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain has the nitrogenase gene subunit Nif H.

61. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain comprises a plurality of heterologous endophyte bacterial strains including at least two of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

62. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain comprises at least three of Pseudomonassiliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

63. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain comprises Pseudomonassiliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

64. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain reduces acetylene.

65. The composition of claim 51, wherein said at least one heterologous endophyte bacterial strain is operable to increase biomass in said host plant treated with said composition.

66. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain is operable to increase amino acid production in the host plant.

67. The composition of claim 51, wherein said at least one heterologous endophyte bacterial strain exhibits fixation of atmospheric nitrogen in said host plant treated with said composition when said host plant is planted in nitrogen-deficient soil or when said host plant is planted in nitrogen-enriched soil.

68. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain produces an Fe siderophore.

69. The composition of claim 51, wherein said inoculant composition increases the acquisition of essential macronutrients and micronutrients from soil and air without substantially disrupting the plant nutrient ion stoichiometry of said host plant.

70. The composition of claim 51, wherein said inoculant composition increases chlorophyll in leaves of said host plant treated with said inoculant composition.

71. The composition of claim 51, wherein said inoculant composition increases tolerance to abiotic stress of said host plant treated with said inoculant composition.

72. The composition of claim 51, wherein said inoculant composition increases the tolerance of said host plant treated with said inoculant composition to a plant disease.

73. The composition of claim 51, wherein said inoculant composition increases seedling germination, seedling emergence, and seedling biomass weight of said host plant treated with said inoculant composition.

74. The composition of claim 51, wherein the inoculant composition further comprises one or more Rhizobium species.

75. The composition of claim 51, wherein the inoculant composition further comprises one or more mycorrhizal species.

76. The composition of claim 51, wherein the inoculum composition further comprises endophytic yeast strain WP1.

77. The composition of claim 51, wherein the inoculant composition further comprises one or more of a fungicide, an insecticide, an herbicide, a biostimulant, a plant growth regulator, a prebiotic, an adjuvant, and a fertilizer, applied to the host plant as a single application or as a mixture.

78. The composition of claim 51, wherein the inoculant composition further comprises a carrier composition for enhancing application of the plant inoculant composition to seeds.

79. The composition of claim 51, wherein said inoculant composition further comprises a carrier composition enabling said plant inoculant composition to be applied to foliar portions of said host plant.

80. The composition of claim 51, wherein said inoculant composition further comprises a carrier composition that enables said plant inoculant composition to be applied to soil adjacent to said host plant.

81. The composition of claim 51, wherein the at least one heterologous endophyte bacterial strain is encapsulated in micro- or macro-beads incorporated into the inoculum composition.

82. The composition of claim 81, wherein the inoculum composition is a suspension composition.

83. The composition of claim 81, wherein the inoculum composition is a dry composition.

84. The composition of claim 81, wherein the inoculum composition comprises a dry particle composition.

85. The composition of claim 51, wherein the inoculant composition comprises a dry particulate composition.

86. The composition of claim 51, wherein the inoculant composition comprises a liquid composition operable to be applied as a foliar spray.

87. A plant inoculant composition comprising at least two co-fermented heterologous endophyte bacterial strains selected from the group consisting of Pseudomonas siliginis, Curtobacterium salicaceae, Rhizobium populi and Sphingobiumyanoikuyae for heterologous application to a host plant.

88. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains are selected for their ability to fix atmospheric nitrogen.

89. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains are selected to have the ability to produce exogenous ammonium (NH4 + ) ability.

90. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains increase solubilization of multiple forms of insoluble phosphorus in a liquid bacterial growth culture.

91. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains have a genetic mechanism to produce Fe siderophores.

92. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains comprise a plurality of endophyte bacterial strains selected to fix atmospheric nitrogen.

93. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains increase total plant carbon in the plant they colonize.

94. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains are resistant to a biocide.

95. The composition of claim 93, wherein the biocide comprises a fungicide, an insecticide, a nematicide, and a herbicide.

96. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains are tolerant to plant hormones, plant elicitors, mineral micronutrients, and mineral macronutrients.

97. The composition of claim 87, wherein the at least one heterologous endophyte bacterial strain has the nitrogenase gene subunit Nif H.

98. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains comprise at least three of Pseudomonassiliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

99. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains comprise Pseudomonassiliginis, Curtobacterium salicaceae, Rhizobium populi, and Sphingobium yanoikuyae.

100. The composition of claim 87, wherein each of the at least two heterologous endophyte bacterial strains is capable of reducing acetylene.

101. The composition of claim 87, wherein said at least two heterologous endophyte bacterial strains are operable to increase biomass in said host plant treated with said composition.

102. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains are operable to increase amino acid production in the host plant.

103. The composition of claim 87, wherein said at least two heterologous endophyte bacterial strains exhibit fixation of atmospheric nitrogen in said host plant treated with said composition when said host plant is planted in nitrogen-deficient soil or when said host plant is planted in nitrogen-enriched soil.

104. The composition of claim 87, wherein the at least two heterologous endophyte bacterial strains produce Fe siderophores.

105. The composition of claim 87, wherein said inoculant composition increases the acquisition of essential macronutrients and micronutrients from soil and air without substantially disrupting the plant nutrient ion stoichiometry of said host plant.

106. The composition of claim 87, wherein said inoculant composition increases chlorophyll in leaves of said host plant treated with said inoculant composition.

107. The composition of claim 87, wherein said inoculant composition increases tolerance to abiotic stress of said host plant treated with said inoculant composition.

108. The composition of claim 87, wherein said inoculant composition increases the tolerance of said host plant treated with said inoculant composition to a plant disease.

109. The composition of claim 87, wherein said inoculant composition increases seedling germination, seedling emergence, and seedling biomass weight of said host plant treated with said inoculant composition.

110. The composition of claim 87, wherein the inoculant composition further comprises one or more Rhizobium species.

111. The composition of claim 87, wherein the inoculant composition further comprises one or more mycorrhizal species.

112. The composition of claim 87, wherein the inoculum composition further comprises endophytic yeast strain WP1.

113. The composition of claim 87, wherein the inoculant composition further comprises one or more fungicides, insecticides, herbicides, biostimulants, plant growth regulators, prebiotics, adjuvants and fertilizers, applied to the host plant as a single application or as a mixture.

114. The composition of claim 87, wherein the inoculant composition further comprises a carrier composition for enhancing application of the plant inoculant composition to seeds.

115. The composition of claim 87, wherein said inoculant composition further comprises a carrier composition enabling said plant inoculant composition to be applied to foliar portions of said host plant.

116. The composition of claim 87, wherein said inoculant composition further comprises a carrier composition that enables said plant inoculant composition to be applied to soil adjacent to said host plant.

117. The composition of claim 87, wherein the at least one heterologous endophyte bacterial strain is encapsulated in micro- or macro-beads incorporated into the inoculum composition.

118. The composition of claim 87, wherein the inoculum composition is a suspension composition.

119. The composition of claim 87, wherein the inoculum composition is a dry composition.

120. The composition of claim 87, wherein the inoculum composition comprises a dry particle composition.

121. The composition of claim 87, wherein the inoculum composition comprises a dry particle composition.

122. The composition of claim 87, wherein the inoculant composition comprises a liquid composition operable to be applied as a foliar spray.

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