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

Abstract

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

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 318,549, filed March 10, 2022, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to compositions, compositions and methods for improving plant performance. The novel compositions include non-native endophytes that are applied to plants and that, when incorporated into the plant, result in measurable plant benefits such as nitrogen and other macro- and micronutrient uptake, nutrient use efficiency, photosynthesis, growth, yield, carbon sequestration, resistance to biotic and biotic stress factors, disease resistance (excluding biological control mechanisms) and general plant health. The present invention has a wide range of applications to plants in general, including crop management applications, reduced fertilizer and pesticide use, reduced carbon footprint of crops, improved mineral nutritional status of crops to reduce plant pathogen load and associated pesticide use, improved food quality and safety, improved plant health and biomass growth rates for landscaping and ornamental plants and forestry use. [Background technology]

[0003] 2. Background of the Invention Endophytes are microorganisms (e.g. fungi and bacteria) that can form a symbiotic relationship with trees and plants, which can improve plant growth, fruit and seed yield, general health, and other characteristics. For example, Aghai Matthew M., Khan Zareen, Joseph Matthew R., Stoda Aubrey M., Sher Andrew W., Ettl Gregory J., Doty Sharon L. (2019) The Effect of Microbial Endophyte Consortia on Pseudotsuga menziesii and Thuja plicata Survival, Growth, and Physiology Across Edaphic Gradients.Frontiers in Microbiology.10 doi10.3389 / fmicb.2019.01353;Rho Hyungmin, Van Epps Victor, Wegley Nicholas, Doty Sharon L., Kim Soo-Hyung, 2018. Salicaceae Endophytes Modulate Stomatal Behavior and Increase Water Use Efficiency in Rice. Frontiers in Plant Science, 9 See doi10.3389 / fpls.2018.00188. Endophytes can be incorporated into plant tissues and become a genetic part of the plant. Endophytes can penetrate between and inside plant cells and become incorporated into plant tissues. Once incorporated into plants and living on the surface of plant roots, endophytes can improve the nutritional status of plants by providing and enhancing the supply of macronutrients such as nitrogen, potassium, phosphorus, calcium, and sulfur, and micronutrients such as iron, zinc, and magnesium, increasing photosynthesis, improving water use efficiency, and enhancing resistance to biotic and abiotic stresses.

[0004] Despite the benefits endophytes bring, their practical use in agriculture is very limited: consumers and farmers are becoming more aware of the damage that chemical fertilizers and pesticides cause to the environment and are turning to alternatives that are more natural and enhance the long-term sustainability of agricultural, livestock and forestry production. Summary of the Invention [Means for solving the problem]

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

[0006] [Table 1]

[0007] The species identified in the above table were submitted to the ARS Culture Collection (NRRL, 1815 N. University Street, Peoria, IL 61604) on November 9, 2021. The 16S rDNA sequences of each of the above endophytic strains are listed in Figures 1-4. These strains were found in the branches of willow and poplar, plants that do not form root nodules. Thus, the presence of potential diazotrophic endophytes living inside all the branches of these plant species was unexpected. In the process of screening a large library of endophytic tree bacteria, the set of endophytic bacterial strains identified above was selected after they were found to be capable of growing in nitrogen-free and nitrogen-limited media and to enhance root-to-shoot uptake of N and P. These strains were further found to provide additional root-to-shoot uptake capacity of mineral nutrients when inoculated, and these strains are present in a wide variety of crop plants that were specifically tested and screened. These unique strains were then identified and isolated specifically for this purpose. Furthermore, by optimizing with the most efficient nitrogen fixing, ammonium excreting strains, and specific strains that mobilize insoluble forms of macronutrients (e.g., phosphorus), the resulting novel inoculant formulations have been found to increase plant acquisition of macronutrient ions and micronutrient ions required by the plant. Furthermore, application of the novel microbial inoculants disclosed herein to non-native plants results in increased growth, biomass and yield in crop plants. The novel inoculant formulations improve crop plant yield and quality under both poor, well-rounded and highly optimized agronomic conditions. These endophytic strains and combinations thereof are not typically present in crop plant varieties and soils used for agricultural production.

[0008] Of the above endophytic strains, two strains have been demonstrated to be newly discovered endophytic species: Curtobacterium salicis (WW7) and Rhizobium populi (PTD1). Examples 1B-1C show experimental results indicating that the WW7 and PTD1 endophytic strains are novel species.

[0009] Whole genome sequence and safety analysis The genomes of all four of these endophytic strains were fully sequenced and analyzed for genes encoding proteins and / or phenotypes known to be harmful to plants or animals. To analyze the entire genome sequences of the endophytic strains, the method of Varghese et al. was used. See https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4538840 / . This method reliably classifies strains as safe or potential pathogens by comparing the genome sequence with known harmful and pathogenic microorganisms for similarity in the genome. If the organism falls into a cluster representative of human or plant pathogens, the presence of virulence factors and antibiotic resistance genes is evaluated. No close affiliation to such pathogen species was found for any of these strains. To further confirm that these strains are safe for agricultural crops, the genomes of the endophytic strains were analyzed and investigated for genes known to be associated with plant and human pathogens. No pathogen-associated genes were found in any of the four selected endophytic strains.

[0010] Biochemical analysis for safety Additionally, endophytic strains were analyzed to determine whether they exhibited biochemical similarities to known pathological biochemistry in mammalian bacterial pathogens. The MacConkey agar test is often used to determine whether a bacterial species is gram negative. Bacteria that grow well on this agar medium for 24 hours are likely to be gram negative. MacConkey agar also uses a neutral pH indicator to test for the ability of bacteria to ferment lactose sugar. Fermentation of lactose sugar by bacteria lowers the pH, causing the colony to turn pink and the surrounding medium to become cloudy. Bacteria that cannot utilize lactose use peptone to produce ammonia, which raises the pH and causes the colony to turn white or colorless. Some microorganisms (e.g., Klebsiella and Enterobacter) form mucoid colonies that appear very moist and sticky. This occurs because the bacteria produce a capsule made primarily from the lactose in the agar medium.

[0011] A hemolysis assay (blood agar lysis) was also performed on endophytic strains for 24 hours to test the pathogenic bacteria's ability to produce toxins that lyse red blood cells. The outcome of this test can be no growth, gamma hemolysis (growth without hemolysis), alpha hemolysis (partial hemolysis and dark green coloration), or beta hemolysis (yellow clear zones of complete hemolysis). Mannitol salt agar is used to confirm the presence of Staphylococci, as it is harmful to most bacteria other than Staphylococci.

[0012] The endophytic strains produced yellow-brown colonies that were neither wet nor sticky when grown on MacConkey agar. Thus, the endophytic strains were negative for lactose fermentation, lactone fermentation, and mucoid capsule production. The results of the MacConkey agar test indicate that the endophytic strains do not share the following characteristics with pathogenic species: (1) they do not share lactose fermentation with pathogenic species such as Escherichia coli, Enterobacter, or Klebsiella, (2) they do not share peptone utilization with pathogenic species such as Salmonella, Proteus species, Yersinia, Pseudomonas aeruginosa, or Shigella, and they do not share mucoid capsule production with pathogenic species such as Klebsiella or Enterobacter. Mannitol salt agar results demonstrated growth inhibition in the endophytic strains, suggesting that the endophytes are unable to ferment mannitol and therefore do not share this phenotype with any Staphylococcus species. Testing using a hemolysis assay showed that all four strains were gamma-hemolytic (grow but did not lyse hemocytes). Taken together, these results confirm that the endophytic strains share no similarities with mammalian bacterial pathogens and that the four endophytic bacterial strains are safe for crop and other applications. The results of the above biochemical assays are shown below in Figure 5.

[0013] The screened and identified endophyte strains were developed as stocks by a novel fermentation growth method for use in a novel inoculant formulation. The formulation is used to treat and improve non-native host plant species in which the endophyte does not naturally occur. The inoculant compositions of the present invention contain additional ingredients that enhance long-term stability (long shelf life), delivery, colonization and efficacy in host plants. The inoculant compositions of the present invention may include liquid seed treatments, seed coatings, freeze-dried reconstitutable seed treatments, encapsulated dry beads, foliar sprays, in-furrow liquid products and other formulations.

[0014] Compositions containing non-native endophytes may be applied "heterologously" to various plant species and crops, meaning that the applied endophyte strain is not naturally present in the treated host plant. Important agricultural, ornamental and other host plant varieties and species can be heterologously treated with the endophyte strains of the present invention. A fundamental principle of traditional and transgenic plant breeding begins with the creation of "clean" germplasm, which typically means that there are no microorganisms present. For this reason, most propagation material of annual crops is microbiologically empty. For perennial plants, modern propagation techniques employ various purification procedures (STG), which also result in microorganism-free germplasm. Contrary to conventional practice, the invention disclosed herein provides bacterial endophytes for heterologous monocotyledonous and dicotyledonous plants, resulting in plants that are superior to endophyte-free plants.

[0015] In some embodiments, the host plant treated with the endophyte inoculant disclosed herein can be a plant cultivated by humans for food, feed, fiber, fuel, and / or industrial purposes, such as wheat (e.g., Triticum aestivum, Triticum spelta, Triticum monococcum, Triticum dicoccum, Triticum durum, Triticum turgidum, and Triticum rigidum), corn (e.g., Zea mays, including subspecies such as Zea mays indenata, Zea mays indurata, Zea mays amylacea, Zea mays saccharata, and Zea mays everta), soybean (e.g., Glycine max), cotton (e.g., Gossypium arboretum, Gossypium herbaceum, Gossypium hirsutum, Gossypium barbadense), broccoli (e.g., Brassica oleracea italica), kale (e.g., Brassica oleracea acephala), tomato (e.g. Solanum lycopicum), rice (e.g. Oryza sativa), barley (e.g. Hordeum vulgare), beet (e.g. Beta vulgaris), pea (e.g. Pisum sativum), potato (e.g. Solanum tuberosum), sugarcane (e.g. Saccharum officinarum), banana (e.g. Musa acuminata and Musa balbisiana), spinach (e.g. Spinacia oleracea), lettuce (e.g. Lactuca sativa), zucchini (e.g. Cucurbita pepo), pepper (e.g. Capsicum annuum), rapeseed (e.g. Brassica napus), alfalfa (e.g. Medicago sativa), conifers (e.g. Pseudotsuga menziesii, Pinus taeda and Alnus rubra), Salicaceae (e.g., SalixThe endophytic strains may include, but are not limited to, host plants (e.g., Populus trichocarpa, Populus nigra, Populus deltoides and all hybrid poplar hybrids DxT, TxN, DxNxT, DxN, etc.), Eucalyptus (e.g., Eucalyptus rostrata, Eucalyptus tereticornas, Eucalyptus cladocalyx and Eucalyptus globulus), Rosaceae (e.g., Malus domestica, Pyrus communis, Prunus avium, Prunus dulcis, Prunus persica, Prunus armeniaca and Prunus americana). The endophytic strains may be applied in a variety of settings, including host plants grown in greenhouse or field conditions and various cultivation methods. The endophytic strains may be applied mechanically, manually, through irrigation, by artificial inoculation, and generally by placement on the plant, plant element, plant tissue, seed, seedling or plant growth medium such that the treatment is present on the plant, plant element, plant tissue, seed, seedling or plant growth medium in a manner not found in nature.

[0016] In various embodiments, this heterologous application may be to a non-native host plant variety or plant development stage where the endophyte strain does not naturally occur, or in a growing environment where the same endophyte strain does not naturally occur. For example, an endophyte strain naturally occurring in the stem tissue of a willow would be considered heterologous to any tissue of a corn, spring wheat, cotton, or soybean plant that naturally lacks the endophyte strain. In some embodiments, a non-naturally occurring application may be the presence of a non-native endophyte in a tissue of a host plant, or in a tissue of a different plant element, tissue, cell type, or physical location of the plant other than the physical location where it naturally occurs. For example, if an endophyte is normally found in the root tissue of a plant element but not in the leaf tissue, the endophyte species or strain, when heterologously located, is applied to the leaves.

[0017] "Host plant" includes any plant to which a non-native endophyte can be heterologously applied, particularly an agriculturally important plant. The detectable inclusion of an endophyte strain in a host plant can result in improved growth characteristics, stress resistance and / or other properties of the host plant. The endophyte strain also improves agronomic traits in crop plant varieties, such as yield and nutritional composition of harvested parts of crop plants, compared to untreated plants that do not have the non-native endophyte strain. A non-native endophyte can colonize a host plant or an element thereof if the endophyte is stably detectable within the host plant or an element thereof over a period of time, such as days, weeks, months or years.

[0018] Specific formulations of heterologous endophytic strains provide unique inoculant benefits to plant hosts that improve plant growth, health, yield and quality, reduce plant resistance to biotic and abiotic stresses, and prevent infection through plant induced resistance to plant / seed diseases and pests. The ability of endophytic strains to colonize non-native plant hosts has been experimentally demonstrated by various methods including polymerase chain reaction (PCR) analysis for specific genetic markers in host plant tissues and 16s sequencing of each endophytic strain, detection of specifically selected diazotrophic colony forming units (CFUs) isolated from surface-sterile host plant tissues, gene RFP or GFP fluorescent marker tagging for localization by laser fluorescence confocal microscopy, and other suitable methods. The presence of genetic material of endophytic strains in host plants, increased CFUs and chlorophyll leaf content in host plant tissues, enhanced root branching and growth, increased shoot biomass, and increased mineral nutrient ion content in leaves all demonstrate successful colonization of host plants by endophytic strains. Furthermore, biochemical and molecular analyses have revealed the mode of action by which endophytic strains improve the performance, yield, mineral nutrition and health of host plants. These analyses include acetylene reduction assays, 15These include, but are not limited to, N isotope dilution assays, growth in nitrogen-free media, measurement of exogenous ammonium production using quantitative chemical test kits, ICPMS ion concentration profiling of leaf tissue, quantification of exogenous insoluble phosphorus mobilization in liquid cultures using fluorescent dyes, measurement of Fe-siderophore production on CAS plates, and bioinformatics and genomics of the genetic pathways responsible for these biochemical traits.Endophyte colonization of non-native heterologous host plants results in measurable improvements in uptake of nitrogen and other macro- and micronutrients, nutrient use efficiency, photosynthesis, growth, yield, carbon sequestration, tolerance to biotic and abiotic stressors, and general plant health.

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

[0020] The endophytic strains WW5, WW6, WW7 and PTD1 selected for use in treating host plants were developed as stocks through a microbial fermentation process. The microbial stock maintenance and fermentation methods may be used to increase the expression of the nitrogenase gene and maintain the plasmid in an active form. The endophytic strains may be grown in a bacterial growth medium that is nitrogen-limited and has other special properties (e.g., chelated iron and / or magnesium) to enhance atmospheric nitrogen fixation, macro- and micronutrient solubilization and acquisition, and other beneficial characteristics of the endophytic strains. Improved nitrogen fixation, P solubilization and acquisition, and micronutrient solubilization and acquisition may result in enhanced levels of these nutrients in the treated host plants. Nitrogenase upregulation may be induced by growing and fermenting the endophytic strains in a nitrogen-limited or nitrogen-free growth medium, which induces the endophytic strains to absorb and assimilate atmospheric N2. For an example of nitrogen-free, see for example RJ Rennie, A single medium for the isolation of acetylene reducing (dinitrogen-fixing) bacteria from soils, Canadian Journal of Microbiology, vol.27, no.1, pp.8-14, 1981. Such upregulation results in higher levels of the active subunit Nif H of the nitrogenase gene, which can be measured by RT-PCR analysis. Such an analysis was performed on WW5, WW6, WW7 and PTD1 endophytic strains subjected to a selective fermentation process, confirming increased expression of the Nif H subunit.

[0021] The fermentation broth utilized to ferment the endophytic strain may contain various components that allow for the growth and health of the endophytic strain during the fermentation process. The nitrogen-limited medium utilized in the fermentation process may contain one or more salts such as sodium chloride, phosphates (e.g., monopotassium phosphate, dipotassium phosphate, and other phosphates), sulfates (e.g., MgSO4), chloride salts (e.g., CaCl2), and other suitable salts, but excluding nitrates, ammonium salts, and other nitrogen sources. The fermentation broth may further contain other suitable components such as yeast extract, agar, and other suitable ingredients. The resulting composition may be utilized as a liquid composition for treating the host plant. For examples of nitrogen-limited media, see, for example, RJ Rennie, A single medium for the isolation of acetylene reducing (dinitrogen-fixing) bacteria from soils, Canadian Journal of Microbiology, vol.27, no.1, pp.8-14, 1981. The nitrogen-limited medium may be substantially free of nitrogen to promote the upregulation of microbial nitrogenase genes in the endophytic strain. However, after fermentation in NLM broth, the resulting novel composition contained 30-100 mg NH4 + / The fermentation composition may include one or more of the following nutrients: nitrogen components in limited amounts, such as within the physiological range of L, common amino acids, such as glutamic acid, glutamine, histidine, nitrates, nitrites, carbamates, and other nutrients. Figure 6 shows examples of other components that may be included in the fermentation composition.

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

[0023] Some endophytic strains (WW7, WW5, WW6) also produced exogenous extracellular iron siderophore compounds, which plants further excreted through the roots to release insoluble micronutrient mineral ions or metals (e.g., iron, magnesium, zinc, copper, nickel, manganese), calcium Ca 2 + to mobilize other divalent macronutrient cations.

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

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

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

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

[0028] The composition may be formulated as storage stable, including liquid, suspension and solid formulations. Storage stable formulations include suspension, dry, powder and formulations containing dried endophyte strains. The composition may be storage stable for at least 3 weeks or more under predetermined conditions. For example, the composition may be stable for 10 weeks or more at various temperatures, including low temperatures (near freezing), sustained high temperatures, or room temperature under standard temperature and pressure (STP) conditions.

[0029] The formulation may comprise one or more dried endophyte strains, in which the moisture content of the endophyte strains is reduced to 30% or less compared to the undried endophyte strains. In some embodiments, one or more endophyte strains included in the inoculant composition may be freeze-dried. In other embodiments, the endophyte strains may be dried using other methods, such as air drying, (complete) drying and / or spray drying. The dried endophyte strains in the inoculant composition may enhance the stability of the endophyte strains therein. In some embodiments, the formulation comprises a dried endophyte strain and may be substantially stable at temperatures between about -20°C and about 50°C for at least about four weeks and up to one year or more. In some embodiments, the formulation comprises a partially hydrated shell surrounding the endophyte within a carbohydrate carrier, such as sodium alginate, calcium alginate, magnesium alginate, or other suitable carbohydrate carrier (e.g., Scogin LDH), providing a hard, approximately dehydrated round or oval bead. The beads range in size from about 400 nm to about 5 mm in average diameter and may additionally or alternatively include thickeners, starches, carbohydrates, or mineral thickeners, stabilizers and / or carriers.

[0030] In some embodiments, the inoculant composition may contain stabilizers that are compatible with the endophyte strain and promote the viability of the strain as well as the application to and colonization of the heterologous endophyte strain in the host plant. Examples of suitable stabilizers include guar gum, xantham gum, agarose, sucrose, glucose, ficoll, phytogel, sodium alginate, calcium alginate, magnesium alginate, glycine betaine, methylcellulose, maltodextrin, molasses, and mixtures thereof. Additional stabilizers that may be used include trehalose, sucrose, glycerol, and methylene glycol, glucose, sucrose mineral oil, soy lecithin, peptone, monopotassium phosphate (KH2PO4), dipotassium phosphate (K2PO4), and mixtures thereof. HPO 4), hydroxypropyl guar (HP-Guar), xantham gum, polyvinylpyrrolidone, polyvinylpyrrolidone / vinyl acetate (PVP-VA), non-reducing sugars or sugar alcohols such as mannitol or sorbitol, or other suitable materials. The amount of stabilizer in the composition can be in the range of about 5 wt% to about 50 wt% (e.g., between about 10 wt% to about 40 wt%, between about 15 wt% to about 35 wt%, between about 20 wt% to about 30 wt%, or any value or range of values ​​therein).

[0031] In some embodiments, the composition may include a carrier, such as an agriculturally acceptable carrier, which may be any material that can be added to plant components without causing or having an adverse effect on the host plant or its components. The carrier may be a solid or liquid carrier and may be in a variety of forms, including microspheres, powders, emulsions, various polymers, dry powder fertilizers such as potash, potassium phosphate, potassium nitrate, or other suitable materials. The carrier may be any one or more of several carriers that impart various properties, such as improved stability, wettability, flowability, and / or dispersibility.

[0032] In some embodiments, the agricultural carrier is a solid, such as diatomaceous earth, loam, silica, magnesium silicate, alginates (e.g., sodium, calcium or magnesium salts of alginic acid), glycine betaine (natural or synthetic), clay, bentonite, biochar, vermiculite, seed husks, peat, wheat, bran, talc, lime, starch, cellulose (methylcellulose, hemicellulose), fuller's earth, heat-sterilized soil, fertilizer powder or fertilizer salts (major and micronutrients), other plant, animal or non-biological products, or combinations thereof, including granules, pellets or suspensions. In some embodiments, the solid carrier of the treatment formulation includes, for example, mineral carriers such as dolomite, kaolin clay, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acid clay, vermiculite and perlite, and inorganic salts such as calcium carbonate. Organic fine powders such as wheat flour, wheat bran, rice bran, etc. may also be used as solid carriers. Mixtures of any of the above components are also contemplated as carriers, such as, but not limited to, pasta (wheat flour and kaolin clay) or flour-based pellets in loam, sand or clay. In some embodiments, the agricultural carrier may be soil or plant growth medium, and / or a food source for cultured organisms. In certain embodiments, the endophyte strains may be encapsulated in calcium alginate, magnesium alginate, agarose, or in sucrose, glucose, or other suitable materials, with or without one or more carbohydrate stabilizers, such as other suitable sugars. The encapsulated endophyte strains may be included in suspensions or solid formulations operable for use as powder fertilizer coatings (including, but not limited to, granular urea, ammonium nitrate, potassium nitrate, potassium phosphate, calcium phosphate, etc.) for all fertilizers, macronutrients and micronutrients, in seed treatments and coatings, foliar sprays, in-furrow applications.

[0033] In some embodiments, the agricultural carrier may be a liquid carrier that imparts various properties such as improved stability, wettability, flowability and / or dispersibility. Liquid carriers may include compositions such as vegetable oils, such as soybean oil, neem oil, cottonseed oil, and glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, and other suitable liquids. In some embodiments, the carrier may be a combination of liquid components, such as a water-in-oil emulsion or other suitable formulation. For example, a water-in-oil emulsion may be prepared to include wettable powders, granules, gels, agar, thickeners, biopolymers, microencapsulated particles, and the like. Other agricultural carriers that may be used may include water, oils derived from plants, humectants, or combinations thereof. The composition may include a wetting agent, such as a natural or synthetic surfactant, which may be a nonionic or ionic surfactant, or a combination thereof. When such formulations are used as wettable powders, biologically compatible dispersants, such as nonionic, anionic, amphoteric, or cationic dispersants and emulsifiers, may be used.

[0034] In some embodiments of the present invention, the surfactant that can be included in the composition can include nonionic surfactant and / or anionic surfactant.Examples of nonionic surfactant include alkylphenol alkoxylate, alcohol alkoxylate, polyoxyethylene glycerol fatty acid ester, castor oil alkoxylate, fatty acid alkoxylate, fatty amide alkoxylate, fatty polydiethanolamide, lanolin ethoxylate, fatty acid polyglycol ester, isotridecyl alcohol, fatty amide, methylcellulose / hemicellulose, fatty acid ester, alkyl polyglycoside, glycerol fatty acid ester, polyethylene glycol, polypropylene glycol, polyethylene glycol / polypropylene glycol block copolymer, polyethylene glycol alkyl ether, polypropylene glycol alkyl ether, polyethylene glycol / polypropylene glycol ether block copolymer, polyethylene oxide / polypropylene oxide block copolymer and mixtures thereof. Examples of anionic surfactants include alkylarylsulfonates, phenylsulfonates, alkyl sulfates, arylalkylsulfonates, alkylether sulfates, alkylarylether sulfates, alkyl-polyglycoletherphosphates, polyarylphenyletherphosphates, alkylsulfosuccinates, olefinsulfonates, paraffin sulfonates, petroleum sulfonates, taurides, sarcosides, fatty acid salts, alkylnaphthalenesulfonates, naphthalenesulfonates and lignosulfonates, condensates of sulfonated naphthalene with formaldehyde or with formaldehyde and phenol and optionally with urea, and also with phenolsulfonic acid, with formaldehyde and urea, lignosulfite waste liquors and lignosulfonates, alkyl phosphates, alkylaryl phosphates (e.g. tristyryl phosphates), and also polycarboxylates (e.g. polyacrylic acid, maleic anhydride / olefin copolymers) (including alkali metals, alkaline earth metals and mixtures thereof).

[0035] In some embodiments, the inoculant composition may include a sticking or adhesive agent to aid in combining the endophyte strain with a carrier, which may include other non-biological compounds. Such compositions form a coating around the plant or plant element (e.g., those used for seed coating) and help maintain the heterologous endophyte and other materials in contact with the plant or plant element. In some embodiments, the adhesive agent may include one or more of alginates, gums, starches, maltodextrins, lecithin, formononetin, polyvinyl alcohol, alkaline formononetinate, hesperetin, polyvinyl acetate, cephalin, gum arabic, xanthan gum, carrageenan, PGA, other biopolymers, mineral oil, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), arabino-galactan, methylcellulose, PEG400, chitosan, polyacrylamide, polyacrylates, polyacrylonitrile, glycerol, triethylene glycol, vinyl acetate, gellan gum, polystyrene, polyvinyl, carboxymethylcellulose, hemicellulose, gum ghatti, polyoxyethylene-polyoxybutylene block copolymers, and other suitable agents.

[0036] In some embodiments, one or more adjuvants may be used in the inoculant composition to help improve the delivery and performance of the endophyte strain. The compositions of the present invention may be combined with adjuvants to create specific product forms or mixtures, such as liquid mixtures for foliar application. The compositions according to the present invention may further include other agriculturally suitable excipients, such as solvents, pH adjusters, viscosity adjusters (rheology modifiers), crystallization inhibitors, antifoaming agents, dispersants, wetting agents, humectants, anti-caking agents, suspending agents, spray droplet conditioners, pigments, antioxidants, UV protection agents, compatibilizers, chelating agents, neutralizing agents, corrosion inhibitors, dyes, fragrances, spreading agents, penetration aids, lubricants, adhesives, thickeners, freezing point depressants, antimicrobial agents, etc. The compositional content of these auxiliary excipients is not particularly limited and may be determined by one skilled in the art according to conventional protocols.

[0037] In some embodiments of the invention, suitable pH adjusting agents that may be included in the inoculant composition include buffers, such as alkali metal salts of weak inorganic or organic acids (e.g., phosphoric acid, phosphorous acid, boric acid, acetic acid, propionic acid, citric acid, fumaric acid, tartaric acid, oxalic acid, malic acid, oxaloacetic acid, and succinic acid).

[0038] The inoculant composition may further comprise a food source for the cultured organisms such as barley, rice, wheat or other biological material such as seeds, plant parts, sugarcane bagasse, husks or stalks from grain processing, crushed plant material from construction sites or sawdust or small fibers from recycled wood, paper, cloth or wood.

[0039] The inoculant composition may further comprise additional ingredients used to enhance plant health and microbial survival, such as biostimulants, prebiotics, amino acids, fatty acids, plant proteins, fungicides, insecticides, nematicides, plant microbial boosters (prebiotics), plant hormones and elicitors, mineral macro- and micronutrients (liquid and dry), seed treatment polymers, commonly used dyes, carbohydrates and gels (such as alginate, mucilage, agarose, guar, xantham gum, etc.), powder carriers (soy protein, talc, lime, starch biochar, cellulose / hemicellulose, silica, clay, nanotechnology structures containing mineral nutrients (such as carbon dots, buckyballs, carbon cages or other forms of nanotechnology, etc.). Exemplary biostimulants may include combinations of amino acids (e.g., one or more of L-glutamine, L-lysine, L-methionine, L-arginine, L-threonine), fatty acids, vitamins, plant proteins, phosphorus sources, betaine, plant growth factors, and other suitable ingredients. Formulations of the endophyte strains described herein were combined with commercially available biocide and nutrient products to test the viability of the endophyte under such conditions. The endophyte was found to be compatible with several commercial products in the laboratory and in the field. Based on the results of the compatibility tests, several new formulations were developed.

[0040] The endophyte strains described herein can be combined with one or more of the agents described above to obtain compositions suitable for application to plants or their tissues, seedlings, seeds or other plant elements. Endophyte populations can be obtained from selection and propagation in culture as described herein and added to the composition. Endophytes in different growth stages can be used. For example, lag phase, early log phase, mid log phase, late log phase or stationary phase endophytes can be used.

[0041] The aforementioned components may be combined in a liquid composition comprising an effective amount of one or more of the endophyte strains WW5, WW6, WW7, and PTD1. For example, an inoculant composition of the present invention may comprise an endophyte strain in an amount of between about 0.1-90% by weight, e.g., between about 1%-80% by weight, between about 5%-70% by weight, between about 10%-60% by weight, between about 15%-50% by weight, based on the wet weight of the composition. An inoculant composition may comprise at least about 10% by weight of the endophyte strain per mL. 3 CFU, e.g., at least about 10 4 CFU / mL, at least about 10 5 CFU / mL, at least about 10 6 CFU / mL, at least about 10 7 CFU / mL, at least about 10 8 CFU / mL, at least about 10 9 CFU / mL, at least about 10 10 CFU / mL or any value or range within said range.

[0042] An exemplary liquid formulation according to the present invention comprises two or more dried or wet endophyte strains (e.g., WW6 and WW7 prepared by co-fermentation) in a concentration of about 10 8 CFU / mL ~ approx. 10 9At a concentration of each endophyte strain in CFU / mL, one or more mono- or disaccharides (sucrose) in an amount of about 1 wt% to about 10 wt%, mannitol in an amount of about 1 wt% to about 10 wt%, sodium lactate in an amount of about 0.01% v / v to about 0.1% v / v, potassium phosphate salts (e.g., K2HPO4 and KH2PO4) in an amount of about 0.05 wt% to about 0.5 wt%, sodium molybdate in an amount of about 0.001 wt% to about 0.01 wt%, NaCl in an amount of about 0.005 wt% to about 0.05 wt%, CaCl2 in an amount of about 0.005 wt% to about 0.05 wt%, and CaCl2 in an amount of about 0.005 wt% to about 0.05 wt%. % to about 0.05 wt%, NaFeEDTA in an amount of about 0.001 wt% to about 0.01 wt%, magnesium sulfate in an amount of about 0.01 wt% to about 0.1 wt%, yeast extract in an amount of about 0.005 wt% to about 0.05 wt%, and agar in an amount of about 1 wt% to about 10 wt%, all in distilled water. 4 ~10 10 A range of CFU / mL concentrations of each strain may be included, alone or in combination with the following: low viscosity alginate (e.g., sodium alginate, magnesium alginate, calcium alginate, Scogin® LDH (Dupont) or other high purity alginate) in an amount of about 0.1% v / v to about 5% v / v, glycerol in an amount of about 0.1% v / v to about 5% v / v, and one or more mono- and disaccharides (e.g., glucose and lactose) in an amount of about 1 wt% to about 10 wt%. Yet another exemplary liquid formulation comprises one or more dried or wet endophyte strains in an amount of about 10 wt% to about 100 wt%. 4 ~10 10 The compositions may contain a range of concentrations of each strain in CFU / mL, either alone or in combination with one or more of the following: low viscosity alginate (e.g., sodium alginate, magnesium alginate, calcium alginate, Scogin® LDH (Dupont) or other low viscosity high purity alginate) at 0.5-50 w / v, gelatin in an amount of about 1% v / v to about 5% v / v, PEG in an amount of about 1% v / v to about 10% v / v, and mono- and disaccharides (e.g., glucose and lactose) in an amount of about 1 wt% to about 10 wt%.

[0043] In some embodiments, the composition may be a suspension formulation comprising the above components in the above proportions. In such embodiments, the composition may further comprise one or more solid carriers, thickeners or extenders. Such components may include inorganic mineral earths such as silica gel, silicates, talc, kaolin, Atta clay, limestone, lime, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate and magnesium sulfate, magnesium oxide, attapulgite, montmorillonite, mica, vermiculite, synthetic silicic acid, amorphous silicic acid and synthetic calcium silicate or mixtures thereof; and / or organic carriers such as hydrocolloids, polymers, cellulose, methylcellulose and / or hemicellulose powders and combinations thereof. The suspension composition may further comprise humectants, emulsifiers, anti-caking agents, suspending agents, freezing point depressants, etc. In some embodiments, the suspension formulation may comprise one or more of the endophyte strains WW5, WW6, WW7 and PTD1 at the concentrations disclosed in the previous paragraph. An exemplary suspension formulation according to the present invention comprises approximately 10% glycerol microencapsulated in sodium alginate, calcium alginate, or magnesium alginate (e.g., by a spray drying process). 8 CFU / mL ~ approx. 10 9 The formulation may include one or more dried endophyte strains at a concentration of each endophyte strain in CFU / mL. The formulation may further include one or more mono- or disaccharides (e.g., sucrose) in an amount of about 1 wt% to about 10 wt%, and glycerol in an amount of about 1 wt% to about 20 wt%, all in distilled water.

[0044] In some embodiments, the composition may be a solid composition. The solid composition may be a dry composition, a granular composition, or a flowable composition intended to be dispersed or suspended in an aqueous solution before being fed to the plant. The dry fertilizer composition may form a fully dispersed suspension. In other contexts, the dry fertilizer composition may provide sustained release (e.g., by low water solubility or encapsulation, e.g., by sodium alginate), for example, when a constant or controlled delivery of nutrients over time is desired. The solid composition may be a fertilizer composition having a concentration of about 103 CFU / mL ~ at least about 10 10 The solid composition may contain endophyte strains WW5, WW6, WW7, and PTD1 in an amount in the range of CFU / mL or any value or range of values ​​therein. For example, the solid composition may contain one or more of endophyte strains WW5, WW6, WW7, and PTD1 in an amount in the range of about 10 8 CFU / mL ~ approx. 10 9 A solid formulation according to the present invention may contain one or more solid carriers in an amount ranging from about 30 wt% to about 60 wt% (e.g., an amount ranging from about 40 wt% to about 55 wt%, an amount ranging from about 45 wt% to about 50 wt%, or any value or range of values ​​within said ranges). An exemplary solid formulation according to the present invention may contain one or more dried endophyte strains in an amount ranging from about 10 wt% to about 60 wt% (e.g., an amount ranging from about 40 wt% to about 55 wt%, an amount ranging from about 45 wt% to about 50 wt%, or any value or range of values ​​within said ranges). 4 CFU / mL ~ approx. 10 10 The formulations may contain a concentration of each endophyte strain in CFU / mL, which may be microencapsulated in alginate beads (sodium, calcium or magnesium alginate 0.1-10% w / v) and solids (starch 0.1-10% w / v) and dripped through unique slurry formulation, batch drip, ion exchange and fluid bed drying processes. The formulations may further include one or more mono- or disaccharides (e.g., sucrose) in an amount of about 1 wt% to about 10 wt% and clay (e.g., zeolite, bentonite and / or other clay materials) in an amount of about 30 wt% to about 50 wt%.

[0045] Applicable The compositions described herein, including one or more endophytic strains, may be applied to plants to enhance plant growth characteristics, health, stress resistance, and improve other characteristics. The compositions of the present invention may advantageously be mechanically or manually applied to the plant or its elements, or artificially inoculated, by any one of a number of means, including, but not limited to, seed treatment, root washing, seedling dipping, soil inoculation, furrow application, foliar spray, foliar coating, side-row application, wound inoculation, irrigation, fertilizer irrigation, soaking, injection, osmotic priming, hydroponics, aquaponics, aeroponics, or any combination thereof. In some embodiments, the compositions of the present invention may also be applied directly to the plant or plant parts, such as leaves, roots, foliage, foliage, tufts, tillers, flowers, plant cells, plant tissues, or combinations thereof. The compositions of the present invention may be applied to the seed (e.g., as a coating or by treating the seed by spraying or soaking) and / or pre-emergence (before the seedling emerges or appears above ground). The compositions of the present invention may also be applied to other propagation materials of plants, such as grains, fruits, tubers, spores, cuttings, slips, meristems, plant cells, nuts or embryos. In some instances, the compositions may be applied as part of a soak of the roots and / or other tissues of the host plant, as a seed coating, as a coating applied to the leaves and / or other elements of the host plant, as a powder on the surface of the leaves and / or other elements of the host plant, as a spray on the leaves and / or other elements of the host plant, as part of a drip on the soil and / or roots of the host plant, or in other suitable manner. The compositions of the present invention may also be applied to the growing medium (e.g., by applying to the soil surrounding the plant).

[0046] The inoculant composition 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, shoot biomass, seed germination rate, seedling survival rate, photosynthetic efficiency, seed / fruit number or mass, fruit yield, leaf chlorophyll content, photosynthetic rate, root length, abiotic stress resistance, biotic stress resistance, disease resistance, wilt recovery, turgor pressure, or any combination thereof, compared to untreated control plants of the same species grown under similar conditions. Rootstocks, root stocks, cuttings, or tissue cultures of the host plant may be used to produce cultivars that exhibit such phenotypic traits and enhanced performance. Application of the inoculant composition may also increase carbon fixation of the treated host plant. This is an economically attractive benefit, as it results in the removal of carbon dioxide from the atmosphere and an increase in biomass. Indicators of increased carbon acquisition by the plant include increased CO2 fixation activity, increased dry weight to fresh weight ratio, and overall biomass.

[0047] Treatment with the compositions of the present invention may result in increased uptake of macro- and micronutrients from soil and air. Treatment with the compositions of the present invention may result in increased rates of nitrogen uptake. Increased rates of nitrogen uptake promote significant improvements in utilization efficiency. Host plants heterologously treated with the compositions of the present invention have more total nitrogen uptake and assimilated at higher levels of nitrogen utilization efficiency, resulting in more protein production and promoting increased biomass production. Application of the compositions of the present invention also increases uptake and utilization of other macro- and micronutrients. Experimental results demonstrate increased uptake of the macronutrients potassium, phosphorus, calcium, and magnesium, as well as increased uptake of the micronutrients boron, copper, iron, manganese, molybdenum, nickel, sulfur, and zinc in host plants treated with the inoculant compositions of the present invention - see, for example, Examples 5-7 below. Heterologous endophytes can take up fixed or poorly soluble forms of certain nutrients, including phosphorus, in soil and convert them to soluble forms that can be more efficiently utilized by the host plant. Endophytic strains can also produce iron siderophores that are beneficial to the host plant, chelating iron in plant tissues and aiding in iron uptake into the host plant.

[0048] Increased macronutrient and micronutrient uptake is accompanied by increased catabolism, carbon uptake and carbon sequestration. Host plants treated with the compositions of the present invention show greater total carbon uptake, with associated increases in RuBisCo carboxylation activity, carbon content and biomass. Host plants can also show increased production of aromatic amino acids via the shikimate pathway. These aromatic amino acids function as precursors for a wide range of secondary metabolites that are important for plant resistance to biotic and abiotic stresses (e.g., oxidative stress, drought stress and / or salt stress).

[0049] Thus, application of the composition may result in enhanced adaptive resistance of the host plant to abiotic and biotic stresses such as disease, cold, salinity, etc. In exemplary embodiments, the level of innate and adaptive resistance to stress is significantly enhanced. Abiotic stressors include extreme temperatures, high salinity, drought, and other causes. Abiotic stressors dramatically reduce yield and biomass, and often kill plants. In agricultural production of crops, low growth temperatures are often encountered, especially during the early growing season, which can stress plants in a variety of ways, starting with poor germination, followed by stunted seedling growth, yellowing of leaves, reduced leaf expansion, wilting, and tissue death. Cold stress severely inhibits the development of reproductive parts of plants. Crop yields are reduced in response to cold stress in proportion to the extent of damage to the plant. High salinity in soil or water is becoming an increasing problem, as salts accumulate in irrigated soils and irrigation water with high salinity must be used. The effects of high salinity in soil and water are called osmotic stress because high salinity impedes the transport of ions and water within the plant. Symptoms of high salt stress include stunted growth, wilting, yellowing, leaf drop, senescence and death. The increased efficiency of nutrient uptake and utilization brought about by the presence of endophytic strains in the host plant protects the host plant under stress-induced environments, and the host plant exhibits greater growth and biomass even under abiotic stress conditions.

[0050] Application of the composition to a host plant enhances adaptive resistance through advanced mineral nutrition and provides biological stress resistance that inhibits viruses, bacteria, and fungi. The endophytic strains of the present invention provide host plants with resistance that may be the result of activation of induced systemic resistance (ISR) and / or other metabolic mechanisms in the plant. Thus, the endophytic strains may be applied to host plants or their seeds as a nutritional treatment to help protect against pathogenic fungi, viruses, and bacteria.

[0051] Other aspects, objects and advantages of the present invention will become apparent from the following detailed description. [Brief description of the drawings]

[0052] [Figure 1] FIG. 1 shows SEQ ID NO:1. [Diagram 2] FIG. 2 shows SEQ ID NO:2. [Diagram 3] FIG. 3 shows SEQ ID NO:3. [Figure 4] FIG. 4 shows sequence number 4. [Diagram 5] FIG. 5 is a table providing data from the microbial assays. [Figure 6] FIG. 6 is a table providing exemplary nutritional components of the fermentation composition. [Figure 7A] FIG. 7A provides images related to the experiment of Example 1A. [Figure 7B] FIG. 7B is a table providing data related to the experiments of Example 1A. [Figure 7C] FIG. 7C is a table providing data related to the experiments of Example 1A. [Figure 8A] FIG. 8A is a table relating to the experiments of Example 1B. [Figure 8B] FIG. 8B is a table relating to the experiments of Example 1B. [Figure 8C] FIG. 8C is a table relating to the experiments of Example 1B. [Figure 9] FIG. 9 is a table relating to the experiments of Example 1C. [Figure 10A] FIG. 10A is a table relating to the experiments of Example 2B. [Figure 10B] FIG. 10B is a table relating to the experiments of Example 2B. [Figure 11] FIG. 11 is a table relating to the experiments of Example 2C. [Figure 12] FIG. 12 is a table relating to the experiments of Example 2D. [Figure 13A] FIG. 13A provides the enzymatic pathways relevant to the experiments in Example 3. [Figure 13B] FIG. 13B is a table relating to the experiments in Example 3. [Figure 13C]FIG. 13C is a graph related to the experiment of Example 3. [Figure 14A] FIG. 14A provides the enzymatic pathways relevant to the experiments in Example 4. [Figure 14B] FIG. 14B provides images related to the experiment in Example 4. [Figure 14C] FIG. 14C is a table relating to the experiments in Example 4. [Figure 14D] FIG. 14D is a graph related to the experiment of Example 4. [Figure 15] FIG. 15 is a graph related to the experiment of Example 5. [Figure 16] FIG. 16 provides images related to the experiments in Example 6. [Figure 17A] FIG. 17A provides images related to the experiment of Example 7. [Figure 17B] FIG. 17B provides images related to the experiment of Example 7. [Figure 18A] FIG. 18A is a table relating to the experiments in Example 8. [Figure 18B] FIG. 18B is a table relating to the experiments in Example 8. [Figure 19] FIG. 19 is a table relating to the experiments of Example 9. [Figure 20] FIG. 20 is a table relating to the experiments of Example 10. [Figure 21] FIG. 21 is a table relating to the experiments of Example 11. [Figure 22] FIG. 22 is a table relating to the experiments of Example 12. [Diagram 23] FIG. 23 is a table relating to the experiments in Example 13. [Figure 24] FIG. 24 is a table relating to the experiments of Example 14. [Figure 25A] FIG. 25A is a table relating to the experiments in Example 15. [Figure 25B] FIG. 25B is a graph related to the experiment of Example 15. [Figure 25C] FIG. 25C is a graph related to the experiment of Example 15. [Figure 26A] FIG. 26A is a table relating to the experiments in Example 16. [Figure 26B] FIG. 26B is a graph related to the experiment of Example 16. [Figure 27] FIG. 27 is a graph related to the experiment of Example 17. [Figure 28A] FIG. 28A is a graph related to the experiment of Example 18. [Figure 28B] FIG. 28B is a graph related to the experiment of Example 18. [Figure 29] FIG. 29 is a graph related to the experiment of Example 19. [Figure 30A] FIG. 30A is a table relating to the experiments of Example 20. [Figure 30B] FIG. 30B is a table relating to the experiments of Example 20. [Figure 31A] FIG. 31A is a table relating to the experiments in Example 21. [Figure 31B] FIG. 31B is a graph related to the experiment of Example 21. [Figure 31C] FIG. 31C is a graph related to the experiment of Example 21. [Diagram 32] FIG. 32 is a table relating to the experiments of Example 22. [Figure 33A] FIG. 33A is a table relating to the experiments in Example 23. [Figure 33B] FIG. 33B is a graph related to the experiment of Example 23. [Figure 34A] FIG. 34A is a table relating to the experiments in Example 24. [Figure 34B] FIG. 34B provides images related to the experiment of Example 24. [Diagram 35] FIG. 35 provides images related to the experiment of Example 25. [Figure 36A] FIG. 36A is a table relating to the experiments in Example 26. [Figure 36B] FIG. 36B is a graph related to the experiment of Example 26. [Figure 36C]FIG. 36C provides images related to the experiment of Example 26. [Figure 36D] FIG. 36D is a graph related to the experiment of Example 26. [Figure 36E] FIG. 36E is a graph related to the experiment of Example 26. [Figure 37A] FIG. 37A is a table relating to the experiments in Example 27. [Figure 37B] FIG. 37B is a graph related to the experiment of Example 27. [Figure 38] FIG. 38 is a graph related to the experiment of Example 28. [Figure 39] FIG. 39 is a graph related to the experiment of Example 29. [Figure 40A] FIG. 40A provides images related to the experiment of Example 30. [Figure 40B] FIG. 40B provides images related to the experiment of Example 30. [Diagram 41] FIG. 41 is a graph related to the experiment of Example 31. [Figure 42A] FIG. 42A is a graph related to the experiment of Example 32. [Figure 42B] FIG. 42B is a graph related to the experiment of Example 32. [Figure 43A] FIG. 43A is a graph related to the experiment of Example 33. [Figure 43B] FIG. 43B is a graph related to the experiment of Example 33. [Diagram 44] FIG. 44 is a graph related to the experiment of Example 34. [Figure 45A] FIG. 45A is a graph related to the experiment of Example 35. [Figure 45B] FIG. 45B provides images related to the experiment of Example 35. [Diagram 46] FIG. 46 is a table relating to the experiments of Example 36. [Figure 47] FIG. 47 is a table relating to the experiments in Example 37. [Figure 48]FIG. 48 is a table relating to the experiments in Example 38. [Figure 49] FIG. 49 is a graph related to the experiment of Example 39. [Figure 50] FIG. 50 is a graph related to the experiment of Example 40. [Figure 51] FIG. 51 is a graph related to the experiment of Example 41. [Figure 52] FIG. 52 is a graph related to the experiment of Example 42. [Figure 53A] FIG. 53A is a graph related to the experiment of Example 43. [Figure 53B] FIG. 53B is a graph related to the experiment of Example 43. [Figure 54] FIG. 54 is a graph related to the experiment of Example 44. [Figure 55] FIG. 55 is a graph related to the experiment of Example 45. [Figure 56] FIG. 56 is a graph related to the experiment of Example 46. [Figure 57] FIG. 57 is a graph related to the experiment of Example 47. [Figure 58] FIG. 58 is a graph related to the experiment of Example 48. [Figure 59] FIG. 59 is a graph related to the experiment of Example 49. [Figure 60A] FIG. 60A is a graph related to the experiment of Example 50. [Figure 60B] FIG. 60B is a graph related to the experiment of Example 50. [Figure 60C] FIG. 60C is a graph related to the experiment of Example 50. [Figure 61] FIG. 61 is a graph related to the experiment of Example 51. [Figure 62] FIG. 62 is a graph related to the experiment of Example 52. [Figure 63] FIG. 63 is a graph related to the experiment of Example 53. [Figure 64] FIG. 64 is a graph related to the experiment of Example 54.

[0053] Detailed Description of the Invention Reference will now be made in detail to certain embodiments of the present invention and exemplary compositions and applications of such embodiments. Although the present invention will be described with reference to these embodiments, it will be understood that they are not intended to limit the present invention. On the contrary, the present invention is intended to cover alternatives, modifications, and equivalents that are included within the spirit and scope of the present invention as defined by the appended claims. In the following disclosure, specific details are given to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without these specific details. EXAMPLES

[0054] method: Selection and growth methods and compositions The present invention includes a method for the propagation and selection of diazotrophic endophyte strains. The method involves inoculating specialized nitrogen-limited and nitrogen-free growth media and selecting colonies capable of growing on the specialized growth media. The growth ability of the endophyte strains on nitrogen-free and nitrogen-limited media was evaluated.

[0055] Example 1A Selection and propagation of endophytes Each of the endophytic strains WW5, WW6, WW7 and PTD1 were tested and found to be positive for the ability to grow on nitrogen-limited medium (NLM), each of which grew to different extents in nitrogen-limited medium as shown in the image below. Each of the endophytic strains were tested and found to be positive for the ability to grow on plant tissue culture grade agarose plates with nitrogen-limited medium (NLM pH 7.6), each of which grew to different extents in plant tissue culture grade agarose plates with nitrogen-free medium (NFCCM pH 7.6), as shown in the image below.

[0056] Fermentation mixtures containing 10 mL of broth culture were prepared in 50 mL conical tubes. Each culture was inoculated with 100 μL of standardized QC broth culture of an endophytic strain. The conical tubes were placed on a shaker at a 45° angle and incubated at room temperature with shaking at 200 rpm for 72 h. The optical density (OD) of each culture was measured at 600 nm. 10% of each endophytic strain was inoculated with 100 μL of standardized QC broth culture of the endophytic strain. -5 and 10 -6 100 μL of the dilutions were plated on NLM agar plates. The plates were then incubated at 25° C. for 72 hours. The CFU formed on the plates were observed and recorded. Figure 7A shows visual evidence of colony growth on the NLM plates.

[0057] The endophytic strains were also analyzed for their ability to produce ammonium in liquid fermentation under aerobic conditions. Individual assays of endophytic strains WW5, WW6, WW7 and PTD1 showed that each was capable of producing ammonium under these conditions. This assay indicates the ability of the endophytic strains to participate in N2 fixation mechanisms within the plant and relates to the growth effects observed after inoculation with treatments of endophytic fermentates.

[0058] Each strain was tested and found to be positive for the ability to exogenously produce ammonium (NH4+) in nitrogen-limited media; MGL (Mannitol-Glutamate / Luria-Bertani), NLM (Nitrogen-Limited Media), MCDY (M Series Yeast Medium Nitrogen Base, Supplemented with Amino Acids) and CS+KNO3 (Corn Syrup + KNO3), as shown in Figure 7B. All axenic media tested negative at ammonium concentrations below 0 mg / L.

[0059] The ability of endophytic strains to fix nitrogen in tree or plant tissues is made possible by the microbial nitrogenase gene within the endophytic fungus. Strains WW5, WW6, WW7, and PTD1 were assayed for the presence of the nitrogenase gene by PCR using specific primers for Nif H. Each endophytic strain was found to contain a copy of an active Nif H gene subunit.

[0060] Furthermore, the nitrogen fixation ability of the endophytic strains was measured by acetylene reduction assay. Acetylene reduction assay measures the ability of nitrogenase enzyme to reduce acetylene gas to ethylene and quantifies the amount of ethylene produced using gas chromatography. This is an indirect method to measure nitrogen fixation ability and measures the functional presence of nitrogenase enzyme through correlating ethylene production. WW6, WW7 and PTD1 endophytic strains exhibited acetylene reduction activity as shown in Figure 7C.

[0061] The selected WW5, WW6, WW7 and PTD1 cells were cultured with endophytic strains ca. 7 CFU / mL to about 10 10 For application purposes, the strains were individually cultured in nitrogen-limited medium for 1-3 days until they were present in the medium at a concentration range of CFU / mL. In some embodiments, two or more endophytic strains were combined and co-fermented to produce a total of about 10 3 CFU / mL ~ approx. 10 9 CFU / mL range, e.g., at least about 10 4 CFU / mL, at least about 10 5 CFU / mL, at least about 10 6 CFU / mL, at least about 10 7 CFU / mL, at least about 10 8 CFU / mL, at least about 10 9The fermentation process conditions may include a predetermined incubation temperature in the range of about 20° C. to about 30° C. (e.g., about 23° C. to about 26° C., about 25° C., or any value or range within said range), shaking of the fermentation vessel at a speed in the range of about 25 rpm to about 300 rpm (e.g., about 75 rpm to about 250 rpm, about 125 rpm to about 225 rpm, about 200 rpm, or any value or range within said range), and fermentation in a volume of about 1 L to about 10 L (e.g., about 2 L to about 8 L, about 4 L to about 6 L, about 4 L, about 2 L, or any value or range within said range).

[0062] To promote upregulation of microbial nitrogenase genes in endophytic strains, the nitrogen-limited medium is substantially free of nitrogen and may contain one or more sugars, such as mannitol, mannose, sucrose, glucose, fructose, lactose, and other suitable sugars. The nitrogen-limited medium may also contain one or more salts, such as sodium chloride, phosphates (e.g., monopotassium phosphate, dipotassium phosphate, and other phosphates), sulfates (e.g., MgSO4), chlorides (e.g., CaCl2), and other suitable salts, excluding nitrates, ammonium salts, and other nitrogen sources. The fermentation broth may further contain other suitable components, such as yeast extract, agar, and other suitable ingredients. The resulting composition may be utilized as a liquid composition for treating host plants. For examples of nitrogen-limited media, see, for example, RJ Rennie, A single medium for the isolation of acetylene reducing (dinitrogen-fixing) bacteria from soils, Canadian Journal of Microbiology, vol.27, no.1, pp.8-14, 1981.

[0063] Example 1B Genome analysis of Curtobacterium salicis (WW7) Curtobacterium salicis (WW7) is a new nitrogen-fixing diazotrophic bacterial species found naturally in willow trees, grass foliage (leaves), mulch / soil, and corn roots. WW7 also produces the organic acids malate and citrate, which solubilize insoluble forms of phosphate, and iron siderophore, which solubilizes insoluble forms of iron. Curtobacterium salicis was isolated from the trunk vasculature of willow (Sitka sitchenses).

[0064] WW7 was sequenced by the US Department of Energy (DOE) Integrated Genomics Institute (JGI) using the Illumina MiSeq platform. A paired-end library was constructed from 376 ng of gDNA using the Nextera DNA Flex library preparation kit and loaded onto one flow cell. Libraries were barcoded to mix with 11 samples and sequenced in 2 × 250 bp format. The MiSeq run was performed using MiSeq Reagent Kit v3 (600 cycles) chemistry. Shotgun sequencing yielded 1,530,321 reads. After trimming, quality filters and removal of potential contaminants using the BBMap package, 1,436,665 read pairs were used as input for the SPAdes v3.13.0 genome assembler. The final assembly was created using a multi-k-mer approach (k=77, 95 and 127).

[0065] The genome of strain Curtobacterium salicis (WW7) consists of 18 scaffolds (N50 = 329,216 bp), is 3,489,963 bp in length, and has a G+C content of 71.35%, corresponding to approximately 84× coverage. Genome completeness of WW7 was calculated based on the presence of Actinobacteria lineage-specific single-copy marker genes using CheckM v1.0.8. In this respect, 99% completeness was achieved. Gene prediction of the draft assembly was performed using Prokka v1.11 (7). Of the 3363 predicted genes, 3286 were protein-coding genes, 53 were tRNAs, 12 were miscRNAs, 1 was tmRNA, and 11 were rRNAs. A total of 1,114 genes were assigned to Clusters of Orthologous Groups (COG), 1,082 were annotated with Enzyme Commission (EC) numbers, and 1,722 were assigned to KEGG Orthology (KO).

[0066] To classify WW7 to species level, two different strategies were used: (i) average nucleotide identity (ANI) analysis using the ANIm algorithm (Seemann T. (2014). Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068-2069. doi:10.1093 / bioinformatics / btu153) and (ii) calculation of intraspecific probabilities (Printra-species) using the genome-wide-based average nucleotide identity (gANI) strategy described in Varghese et al. (Richter M and Rossello-Mora R. (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA.106: 19126-19131). In this regard, the WW7 genome was compared with all publicly available Curtobacterium genome assemblies in the NCBI GenBank database: a total of 107 Curtobacterium genomes. Using the ANIm algorithm, we aligned all genomes to each other and used the ANI values ​​to create an adjacency matrix, which we converted to a similarity matrix (Figure 8A) and extracted clusters of closely related genomes using a cutoff value of 0.9 (corresponding to an ANI of 90%).WW7 was closely related (ANI > 90%) to Curtobacterium herbarum DSM 14013 (ASM1690733v1) and seven new Curtobacterium strains isolated from leaf soil in Southern California (NCBI BioProject Accession Number: PRJNA391502): Curtobacterium sp. MCPF17_052 (NCBI Assembly ID: ASM323408v1), Curtobacterium sp. MCPF17_047(NCBI Assembly ID :ASM323404v1), Curtobacterium sp. MCPF17_031(NCBI Assembly ID : ASM323403v1), Curtobacterium sp. MCPF17_011 (NCBI Assembly ID: ASM323414v1), Curtobacterium sp. MCPF17_001 (NCBI Assembly ID: ASM323461v1), Curtobacterium sp. MCLR17_032 (NCBI Assembly ID: ASM323479v1), Curtobacterium sp. MCBD17_030 (NCBI Assembly ID: ASM322425v1) - see Figure 8A. Additional WW7 strains were distantly related (84% < ANI) to two strains isolated from leaf soil in Massachusetts (MCBA15_007 (ASM186490v1), MCBA15_005 (ASM186485v1)) and to a strain of Curtobacterium pusillum isolated from maize roots (NCBI Assembly ID: ASM202564v1).

[0067] To further evaluate the relatedness of WW7 to Curtobacterium type strains, pairwise digital DNA-DNA hybridization values ​​(dDDH) were calculated for WW7 to determine its interspecies relationship with representative strains of the genus Curtobacterium (type strains). The pairwise dDDH values ​​between WW7 and Curtobacterium type strains were lower than 70%, indicating that WW7 is a representative of a novel Curtobacterium species, as shown in Figure 8B. See Kim MK, Kim YJ, Kim HB, Kim SY, Yi TH, Yang DC 2008. Curtobacterium ginsengisoli sp. nov., isolated from soil of a ginseng field. Int J Syst Evol Microbiol. 58(10):2393-7. Similarly, the Genomic Taxonomy Database (GTDB) identified WW7 as the sole member of a new species cluster, namely, Curtobacterium flaccumfaciens (https: / / gtdb.ecogenomic.org / species?id=Curtobacterium flaccumfaciens), further supporting that this strain does not belong to any known Curtobacterium species.

[0068] Phylogenetic distances were also calculated using 605 concatenated single-copy protein-coding genes assigned to clusters conserved in all GTDB Curtobacterium representatives according to the Anvi'o pan-genome pipeline. The resulting phylogenetic tree shows a clear separation of strain WW7 from other Curtobacterium species and identifies C. herbarum as the closest type strain. See Figure 8C. Using the Anvi'o pan-genome analysis pipeline, 605 single-copy core genes were identified and partition files were generated. Partition analysis was then performed using IQ-TREE to calculate the best-fitting substitution model for each single-copy core gene. Bootstrap values ​​were calculated based on 1000 iterations, and only nodes with bootstrap values ​​≥ 80% are displayed. Asterisks indicate the type strain of Curtobacterium according to the List of Prokaryotic names with Standing in Nomenclature (LPSN) database. Clavibacter michiganensis was used as the outgroup.

[0069] Example 1C Genome analysis of Rhizobium populi (PTD1) To assess the affiliation of strain PTD1 to the Rhizobium type strain, pairwise digital DNA-DNA hybridization values ​​(dDDH) were calculated for strain PTD1 to determine its interspecies relationship with the closest representative strain (type strain) of the Rhizobium genus. As shown in Figure 9, the pairwise dDDH values ​​between strain PTD1 and the Rhizobium type strain were less than 70%, indicating that strain PTD1 is a representative of a novel Rhizobium species.

[0070] Example 2A Two-strain product fermentation formulation under nitrogen-limited medium conditions. Cultivation of the two endophytic strains WW6 and WW7: Each strain was first plated on nitrogen limited medium (NLM) semi-solid medium. Three to four colonies can be picked and used to inoculate a 2-liter seed train using fresh sterile NLM medium. The 2-liter flasks can be cultured at about 25°C to about 30°C with constant agitation at about 200 rpm and about 500 rpm for 72 hours. After completion, the two strains can be pooled in one carboy 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 fermenter. The co-fermentation can be carried out for 3 days with an aeration rate of 20 PSI. The resulting colony forming units of the two strains when plated on tryptic soy broth agar (TSBA) are 2.2 × 10 for WW6. 8 CFU / mL ~ 1.23 × 10 9 CFU / mL, WW7 was 1.03 × 10 8 CFU / mL ~ 1.4 × 10 9 CFU / mL. This procedure can also be applied to other endophyte strain combinations such as any combination of WW5, WW6, WW7 and PTD1.

[0071] Example 2B The endophyte composition was prepared by mixing with two commonly used dry fertilizers and dry powder for combination use in agriculture. Two new compositions were prepared by adding WW6 and WW7 endophyte strains and sodium alginate (DuPont Nutrition USA, In) and dry fertilizer Triple Superphosphate 0-28-0 (OCP group) and separately Dolomite lime (Down To Earth, Inc). 3 mL of the mixture containing WW6+WW7+0.5% alginate was added separately to 5 g of dry fertilizer. The compositions were dried and stored at room temperature for 24 h. The colony forming unit (CFU / gram) counts of the two strains were then determined by plating on NLM semi-solid medium.

[0072] The results shown in Figure 10A indicate that the survival rates of both endophyte strains were slightly decreased when mixed with different fertilizers in the new compositions. These results indicate that the liquid endophyte composition can be used to create a new composition for application in commercial agriculture as a NUE endophyte-enriched agricultural fertilizer.

[0073] Additionally, a liquid fermentation composition containing WW6 and WW7 endophyte strains and sodium alginate (DuPont Nutrition USA, In) was used to create new compositions containing three different powdered dry carriers in combination with whey protein (Chemital tecnicas alimentarias), sodium bentonite (Specialty Minerals, Inc) and coconut core (W. Atlee Burpee & Co). 3ml of liquid composition containing a mixture of WW6+WW7+alginic acid 0.5% was added to 5g of different dry powdered carriers to create the compositions. The powdered carrier-endophyte compositions were dried separately at room temperature for 24 hours and then stored before being plated on NLM semi-solid medium to calculate colony forming units (CFU / gram).

[0074] The results are shown in Figure 10B. The viability of both endophyte strains was slightly decreased when mixed with different dry powder carriers, demonstrating that the endophyte composition can be used with different dry carriers for blending, coating and feeding in commercial agricultural practices.

[0075] Example 2C The viability of endophytic strains (WW6 and WW7) was assayed after freeze-drying and combining with different powder carriers along with Mycorrhizae powder.

[0076] Various powder carrier mixtures (maltodextrin sucrose, dextrose, whey) were assayed for compatibility with the WW6+WW7 FD powder mixture. The mixture ratios tested were: powder carrier 2.09g (~95wt%), freeze-dried powder WW6+WW7 0.11g (~5wt%), Mycorrhizae 0.0022g (~0.1wt%). The compatibility test results showed the following results as shown in Figure 11.

[0077] Compatibility tests performed with Mycorrhizae by mixing freeze-dried WW6+WW7 with four possible bulking agents (maltodextrin, sucrose, dextrose and whey) showed good results. The powder carrier that reduced the most CFU was the whey product, which only slightly inhibited WW6. Maltodextrin and dextrose slightly reduced the CFU of the WW6 strain. The other powder products did not reduce the CFU very much. Sucrose was the best powder carrier, not reducing the CFU of any of the strains in the assay.

[0078] The results of the examples demonstrate that the freeze-dried endophyte powder can be mixed with a variety of carriers and the freeze-dried inoculum can be diluted to low levels and used as a fertilizer coating or as a reconstituted powder for subsequent resuspension and various aqueous foliar applications.

[0079] Example 2D Short-term and long-term viability of endophytic strains (WW5, WW6, WW7, PTD1, and WP1) with powdered carrier biochar alone or in combination with biochar and carbohydrate molasses after 1–22 days and 2 weeks.

[0080] Biochar was treated with co-fermented WW5, WW6, WW7, PTD1 and WP1 inoculant compositions 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 2 weeks, dried biochar (0.1 g) was resuspended in 1 mL potassium phosphate buffer and assayed for endophyte strain viability, with the results expressed as CFU / ml. Data is shown in Figure 12.

[0081] The results demonstrate that 50 μL of co-fermented endophyte mix with 950 μL of 1 / 10× molasses per g of biochar is an effective ratio for the viability and stability of four of the five endophyte strains tested for at least two weeks on the biochar powder carrier.

[0082] Example 3 Endophytic strain WW7 solubilizes insoluble forms of phosphate The ability of endophytic strains to enhance endophytic metabolism, allowing them to utilize insoluble phosphorus (P) from soil or soil solution, mobilize P within the plant body, and enhance P uptake compared to other soil particles or internal metal ions. Heterologous grafting of endophytic strain WW7 has been shown to be effective in enhancing P uptake in the host plant. Heterologous WW7 appears to be able to solubilize various forms of P that are insoluble in the medium-solution mixture. Genomic analysis of WW7 suggested possible genetic mechanisms for the biosynthesis of Krebs cycle intermediates such as the organic oxyacids malate and citrate. In addition, endophytes have exudates that may help keep phosphate ligands free inside the plant by competing with other metals that may tightly bind phosphate making it insoluble again and unavailable for assimilation. Genomic data of WW7 was used to identify protein-coding genes involved in the reactions of interest. The predicted proteome of WW7 was functionally annotated through the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using the KofamKOALA genome jp tool (https: / / www.genome.jp / tools / kofamkoala / ). Using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database as a functional database, the proteome of WW7 was searched for enzyme reactions and pathways that catalyze the synthesis of malate and citrate, which are exuded from the roots and solubilize insoluble malate. 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 assimilation and conversion of aspartate and glutamate. In this regard, WW7 possesses a complete set of enzymes that catalyze the conversion of glutamate and aspartate to L-argininosuccinate, which is further converted to arginine, resulting in the release of a fumarate molecule for use in the citric acid cycle pathway (see Figure 13A). Glutamate is converted to 1 mole of NH 3からIt is the first amino acid product of the GS-GOGAT pathway that produces one mole of glutamate, and is the GOGAT pathway responsible for atmospheric nitrogen fixation in bacteria. Figure 13A shows the WW7 enzyme pathway involved in the synthesis of fumarate from glutamate and aspartate, with the enzymatic reactions catalyzed by the WW7 enzymes (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, 4.3.2.1).

[0083] Phosphate solubilization genes According to KEGG annotation, genes involved in inorganic and organic phosphate solubilization in other species were also detected in WW7 (see Figure 13B). The results revealed the presence of an acid phosphatase (AcPase) gene and genes involved in acetate and gluconate synthesis. AcPase has been shown in other species to be involved in solubilizing phosphate from phosphomonoesters, and acetate and gluconate have been shown in other species to be involved in solubilizing phosphate from inorganic forms.

[0084] The ability of endophytes to solubilize insoluble phosphorus (P) from soil and soil solution and exogenously produce a variety of mobilizing compounds is important for plants to more efficiently acquire needed P and other nutrients, such as potassium K, and micronutrient ions from the soil. These chelating compounds, i.e., acidic compounds that lower the pH, produced by endophytes appear to help plant roots better utilize these minerals from the soil and aid in their uptake and translocation from plant roots to shoots.

[0085] To demonstrate the ability of WW7 to solubilize insoluble phosphate through the secretion of exogenous compounds, an insoluble phosphate solubilization assay was performed in liquid medium using a phosphate-sensitive dye and microplate reader method to further confirm the biochemical ability of WW7 to solubilize different phosphate species. A physiological assay method was also developed and modified from a previous method by Varga et al., Endophyte-Promoted Phosphorus Solubilization in Populus, Frontiers in Plant Science, 11; 2020; 1585 (hereafter referred to as "Varga"). WW7 cells were grown in modified NBRIP (National Botanical Research Institute) broth without phosphate to remove residual phosphate inside. Five milliliters of NBRIP broth culture with or without phosphate was added to 10 mL of WW7 cell culture as previously described by Varga. The 10 mL tube was incubated at 25 °C and 220 rpm for 3 days. The 10 mL tubes were then removed from the shaker and left to stand for 90 minutes. One mL of culture was spun at 5,000 rpm for 5 minutes at 25°C, and the supernatant was used to measure solubilized phosphate. The absorbance of solubilized phosphate was measured at 650 nm (A650) using a phosphate colorimetric measurement kit (Sigma-Aldrich, MAK030). The supernatant of each sample was added to a 96-well plate at different dilutions. In addition, phosphate standards included in the kit were prepared to calculate the linear equation used to measure solubilized phosphate in each sample. Four to five technical replicates were used to determine statistical differences between samples.

[0086] The results shown in Figure 13C indicate that the growth and molecular activity of the endophyte strain WW7 resulted in a statistically significant increase (p < 0.01) in phosphate solubilization for both insoluble suspended aluminum and tricalcium phosphates, but not for iron phosphate. The WW7 strain increased the rate of phosphate solubilization from insoluble aluminum phosphate by an average of 29% and from insoluble tricalcium phosphate by an average of 100%. These results indicate substantial mobilization of insoluble phosphate by WW7.

[0087] WW7 may mix with other heterotrophic microorganisms to mobilize P, and the transformation of insoluble phosphate by WW7 and the potential uptake of soluble P from soil and rock by the host plant may be combined with enhanced nitrogen acquisition (e.g., from the atmosphere). These mechanisms combined may significantly improve the metabolic performance of the treated host plants, enhancing their biomass, stress tolerance, and other advantageous traits.

[0088] Example 4 Genomic and biochemical iron siderophore production assay Endophytic strains can 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. Endophytic strains may also improve the host plant's ability to acquire the necessary iron and other metals and micronutrient ions from soil, especially positively charged divalent cations. These iron-siderophore chelating compounds made by some bacterial and yeast endophytes may help plants to better compete with the high cation exchange capacity of soil clay particles and aid in the uptake and translocation of metal cations from plant roots to shoots. To confirm the ability of strain WW7, we performed a genome-wide analysis of the WW7 genes. It was shown that WW7 has the genetic equipment required to make iron siderophores that can transport or scavenge iron ions. InterProscan results show the presence of a gene cluster efeUOB involved in the salvage of ferrous and ferric iron from exogenous heme, as well as three genes encoding NADPH-dependent ferric reductases. See Figure 14A. The latter catalyzes the reduction of iron complexed with various siderophores, such as ferric tricatecholate and ferric dicitrate, resulting in the release of the bound ferrous iron. AntiSmash analysis also revealed that the NADPH-dependent ferric siderophore reductase (Ga0372474_197) of WW7 was clustered with a gene encoding a non-ribosomal peptide synthetase-like protein (Ga0372474_207), an enzyme typically found in biosynthetic gene clusters (BGCs) involved in the synthesis of ferric siderophores.

[0089] Assay of iron siderophore production in all four endophytic strains Iron siderophore production ability of endophytic strains WW5, WW6, WW7 and PTD1. Three of the tested endophytic strains were confirmed to produce iron siderophore. The four strains were assayed using the microbial growth solid agar plate-based Fe-siderophore CAS medium. The agar plates were plated with chromazurol as a color indicator and chloramphenicol as a colorimetric indicator. FeCl Prepared using the CAS agar plate preparation method containing 3. Endophytic strains were spread onto individual plates.

[0090] Iron solubilization was evidenced by the presence of a discolored zone on the CAS test plate, which appears as a clear white color surrounding the streak of bacterial growth on the plate. The zone was measured and quantified using image analysis software (e.g., ImageJ, a publicly available image analysis program from the National Institutes of Health (http: / / rsb.info.nih.gov / ij / )). The ability of the strains to solubilize iron was photographed, measured, and compared. See Figure 14B.

[0091] The in vitro results in Figures 14B-14D show that WW7 produced extracellular compounds that scavenge insoluble iron. WW7 showed the greatest iron solubilization activity among all strains. WW5 and WW6 also showed significant iron solubilization. However, the PTD1 Rhizobium populi strain showed little iron solubilization activity, and insoluble iron was observed very close to the colony streaks. The results demonstrate the production of iron siderophores by WW5, WW6, and WW7. The results show iron solubilization by each of the WW5, WW6, and WW7 strains, suggesting that WW7 produced the most iron siderophores, followed by WW5 and WW6. Endophytic strains that produce these compounds can aid in the solubilization and mobilization of iron in the host plant. The iron solubilization activity of each strain is quantified in Figure 14C and graphed in Figure 14D.

[0092] Example 5 Gas chromatographic analysis and identification Bacterial identification was performed by gas chromatography analysis of fatty acid methyl esters (GC-FAME). GC-FAME analysis yields highly specific FAME ID chemical identification chromatograms for each endophyte strain. This allows the isolated microorganisms to be individually tracked and each one positively identified based on their unique fatty acid methyl ester signatures. The unique chromatograms for each strain are shown in Figure 15.

[0093] How to use The formulations of the present invention may be advantageously applied to plants by several means, including, but not limited to, spraying, dredging, coating, soaking, injecting, in-furrow, or any combination thereof. The compositions of the present invention may be applied to leaves, roots, thallus, shoots, flowers, plant cells, plant tissues, seeds (e.g., as a coating or by treating the seeds by spraying or soaking, etc.), pre-emergence (before the seedlings emerge above ground or appear), grains, fruits, tubers, spores, cuttings, slips, meristems, plant cells, nuts, or embryos. In some examples, the compositions may be applied as part of a soak to the roots and / or other tissues of the host plant, as a seed coating, as a coating applied to the leaves and / or other elements of the host plant, as a powder on the surface of the leaves and / or other elements of the host plant, as a spray on the leaves and / or other elements of the host plant, as part of a drip on the soil and / or roots of the host plant, as dry alginate beads that encapsulate the endophyte and deliver it to the roots, or as other suitable methods or inoculations.

[0094] The compositions of the present invention are effective in improving the metabolism (e.g., nutrient uptake, carbon uptake, growth, etc.) of the host plant. Thus, the compositions and methods of the present invention can be of significant economic advantage due to the improved growth characteristics, which can lead to increased harvestable crop yields and more robust plants. Exemplary methods are described below.

[0095] Example 6 PCR analysis of endophyte colonization of host plants following root dip inoculation The ability of the endophytic strains to heterologously colonize host crop plants was tested using PCR techniques. The results revealed that the endophyte was present inside surface-sterilized plant tissues. Successful colonization was demonstrated by in-plant PCR in agriculturally important wheat, rice and barley seeds inoculated with the WW6 (Pseudomonas siliginis) endophytic strain via seed treatment.

[0096] After the seeds were treated as follows, four sets of plants were cultivated: an appropriate number of seeds were placed in a seed germination box of 11 cm x 11 cm, and 20 mL of inoculum solution (co-fermented endophyte WW6 + WW7, both in NLM medium) was added. 107 CFU / ml). Seeds were allowed to germinate for 5 days and then transplanted into 3.5 inch pots containing a mixture of washed play sand, vermiculite and perlite. Plants were grown in a growth room at 25°C under sodium halide lights with a 14 hour light / 10 hour dark cycle. Additionally, the plants were watered and fertilized 2-3 times a week in trays with Hoagland's solution containing reduced nitrogen at 25 ppm. Plants were harvested individually 14 days after transplanting. Plants were then removed from the soil and processed. PureLink TM DNA was isolated from the treated samples using the Microbiome DNA Purification Kit DNA isolation kit (Thermo Fisher Scientific).

[0097] A 20 microliter PCR reaction was set up using 2X HotStart PCR Master Mix (MCLAB), 1uL of template DNA, and 1uM of primers specific to the gene of WW6 for the hypothetical protein. 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 a termination at 72°C for 5 minutes. 10uL of each PCR reaction was loaded onto a 1.2% agarose gel and run at 120V through a DNA QS710 Electrophoresis (IBISCI). Figure 16 shows that WW6 is present in all three host plant species tested (wheat, rice, and barley) inoculated pre-emergence and absent from the controls for each of the three tested plants. Bands in lanes 2 (wheat treated with WW6), 4 (rice treated with WW6), and 6 (barley treated with WW6) indicate the presence of genes specific to WW6. Thus, it was revealed that the WW6 endophyte strain can effectively colonize several host plants.

[0098] Example 7 PCR analysis of endophyte colonization of host plants after seed coating The ability of endophytic strains to heterologously colonize host crop plants when applied as seed coatings was tested using PCR techniques. Shoots and roots of winter wheat (Triticum aestivum) and broccoli (Brassica oleracea) plants treated with WW6 (Pseudomonas siliginis) were evaluated for colonization and incorporation of the WW6 endophytic strain into plant tissues. After seed treatment with WW6, plants were grown as follows: WW6 was fermented according to the method disclosed herein, blended with 0.5% sodium alginate (Scogin™ LDH) and used to coat raw wheat seeds. The same seed coating agent without the endophytic strain was applied to control plants. The coated seeds were air-dried at room temperature and stored for one month after coating treatment.

[0099] An appropriate number of seeds were placed in 11cm x 11cm germination boxes. Seeds were allowed to germinate for 5 days and then transplanted into 3.5 inch pots containing a mixture of washed play sand, vermiculite and perlite. The plants were grown in a growth room at 25°C under sodium halide light with a 14 hour light / 10 hour dark cycle. Additionally, the plants were watered and fertilized with a reduced nitrogen Hoagland solution at 25 ppm in trays 2-3 times a week. 14 days after transplanting, the seedlings were removed from the soil and processed. The root and shoot tissues of the plants were harvested. PureLink TM DNA was isolated from the treated samples using the Microbiome DNA Purification Kit DNA isolation kit (Thermo Fisher Scientific).

[0100] PCR was performed as described in Example 5 above. Figures 17A and 17B are electrophoretic gel data of DNA encoding an unknown hypothetical protein present only in WW6. The gels demonstrate the presence of WW6-specific DNA in the shoot and root tissues of winter wheat and broccoli host plants grown from treated seeds, but not in the control shoot and root tissues. Figure 17A is electrophoretic gel data demonstrating the presence of WW6 in the shoot 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 generated DNA bands indicative of the specific presence of the WW6 strain, but not in the control shoot and root tissues. Thus, it is evident that the WW6 endophyte strain can effectively colonize the root and shoot tissues of wheat host plants following seed inoculation.

[0101] Figure 17B shows electrophoretic PCR gel data showing the presence of WW6 in the root tissue of seedlings inoculated with WW6 seed treatment after surface sterilization of the root tissue. Multiple treatment groups with WW6 endophyte strain: treatment 1 with WW6 liquid fermentation, treatment 2 with broccoli seeds treated with liquid fermentation of 0.5% sodium alginate (DuPont's Scogin™ LDH) mixed with seed coating material, and treatment 3 with WW6 liquid fermentation of 1% sodium alginate mixed with seed coating material. Broccoli seeds were subjected to the three treatments. Plant growth, DNA isolation, and PCR were performed as described in Example 5 above.

[0102] Figure 17B shows electrophoretic gel data showing the presence of WW6 in the shoot and root tissues of winter wheat, rice, soybean, broccoli, and corn host plants grown from treated seeds, but not in the shoot and root tissues of controls, due to the presence of genome-specific PCR primers designed to an unknown hypothetical protein that generated a unique DNA band indicative of the presence of the WW6 strain. Thus, it was revealed that the WW6 endophyte strain can effectively colonize the root and shoot tissues of several host plants after seed inoculation.

[0103] Example 8 Analysis of endophyte colonization of host plants following foliar application The field performance of spinach (Spinacia oleracea) foliarly sprayed with WW6 and WW7 endophytes was tested, and the ability of the endophytic strains to heterologously colonize the host plant was also measured.

[0104] A foliar inoculant composition containing freeze-dried powders of co-fermented WW6 and WW7 suspended in water was sprayed on spinach plants according to commercial rates together with 10-5-3 CaO liquid fertilizer formulation Greenstim TM (Masso, SA Agro Department, concentrated glycine betaine extracted from beetroot, 12% total nitrogen). Spinach was grown in a fully conventional hydroponics culture with 550 L (715 kg, 18 kg / day) of 10-5-3 3% CAO fertilizer applied daily to each plot. WW6+WW7 endophyte concentration was 20 g / L, with a foliar application rate of 1 L / ha, applied at the stage of 4 true leaves. The experimental field was divided into 3 blocks of 6 beds each. Sampling was performed in the center of the 4 central beds in each block. Canopy coverage was measured using a software tool (Canopeo) that analyzes and measures canopy coverage from photographs. TM ) 35 days after application. At the same time, leaves were harvested for nutrient analysis, surface sterilization, and quantification of in-plant endophytes. Plant coverage results for the plot bed data showed that endophyte foliar treatments increased spinach leaf canopy coverage by 30.92%, a statistically significant increase (p<0.05), compared to commercial fertilizer alone, and by 58%, a statistically significant increase (p<0.05), compared to the control treatment. See Figure 18A.

[0105] To measure and quantify the presence of endophytes in spinach plants, leaves and roots were harvested 35 days after the foliar inoculation treatment, washed and surface-sterilized to detect strains WW6 and WW7 in the plant. All samples were weighed and photographed, and CFUs were calculated in relation to the sample weight and leaf or root surface area. Leaf and root ends were placed separately in plastic bags and surface-sterilized in a laminar flow hood. Surface washing with distilled water was performed to remove dust, soil and other contaminants. Samples were then placed in a sterile flask, 70% ETOH alcohol was added, and the flask was shaken (150 rpm) for 2 min. After shaking, the alcohol solution was removed and a 1% solution of sodium hypochlorite bleach was added. The mixture was shaken for another 2 min. The sodium hypochlorite solution was then also removed and the samples were washed in sterile water by manual shaking three times for 1 min.

[0106] Leaves and roots were crushed in 50 ml of 0.9% saline. The extract was placed in a sterile tube and left for 1 hour to allow the endophytes to be released from the aboveground tissues. The extract was serially diluted several times using sterile water. 100 μl of each dilution was spread onto potato dextrose agar (PDA) plates and actinomycete isolation agar medium with glycerol.

[0107] Agar plates were incubated until bacterial growth was visible. Bacterial concentrations were calculated as CFU / g and CFU / g of the analyzed material. cm We obtained the result shown in 2.

[0108] The data in Figure 18B demonstrate the presence of endophytes (WW6 and WW7) in the leaves and roots of surface-sterilized spinach host plants inoculated with the foliar inoculant composition. This endophyte strain was not present in spinach plants that received only the Greenstim fertilizer treatment (see detailed discussion above). Thirty-five days after treatment with the foliar inoculant composition containing WW6 and WW7 endophytes, WW6 and WW7 endophytes were detected in both the leaves and roots of the host plants. No endophytes were present, and no endophytes were detected in the standard treated control plants. Endophyte concentrations, expressed as CFU / g, were higher in the leaves than in the roots, but the difference was small and the concentrations in the leaves and roots were considered to be the same in both parts. CFU / g cm 2Surface area was calculated from visual analysis and measurements using Adobe Photoshop software.

[0109] The above data show that the endophyte successfully colonized and improved foliar growth and biomass of treated host spinach plants 35 days after foliar application, resulting in improved establishment, growth, and soil cover of spinach host plants, thereby increasing productivity, demonstrating the efficacy of the composition and foliar application method.

[0110] Example 9 Effect of single endophytic strain seed treatments on total nutrient accumulation Corn seeds (Zea mays) were treated with a seed inoculant composition containing one heterologous endophyte strain selected from WW5, WW6, and WW7, and compared to a control corn seed treated with an inoculant composition without endophytes. An appropriate amount of corn seeds was placed in the bottom of a large gallon-sized zip-lock bag and sealed. Three groups of corn seeds were each treated with a specific endophyte culture (WW5, WW6, and WW7). Cultures of WW5, WW6, and WW7 were grown under NLM medium + sodium alginate (0.5% w / v) conditions (endophyte-containing). ~ The cultures were prepared at 107 CFU / ml and stored refrigerated. The cultures were removed from refrigeration, mixed thoroughly, and then carefully pipetted onto the seeds in a sterile laminar flow hood at a ratio of 3.4 mL / lb in 1 mL drops. After each addition, the bag and seeds were manually inverted and carefully massaged. After the full 3.4 mL / lb was added, the seeds were massaged, shaken, tumbled, and left for 2-3 minutes until all corn seeds in the bag were visibly wet. The bag was then opened to air dry in a sterile laminar flow hood. After drying, the seeds were stored at room temperature for 3 weeks, then planted in a pot of a mixture of washed play sand, vermiculite, and perlite, placed in 1-gallon felt Smart Pots, and stored for 25-48 hours. ℃、 The lighting cycle was 14 h light / 10 h dark and the mice were exposed to sodium halide light (luminous flux 710 μmol m-2 s Plants were germinated in a growth chamber under 100% CO2 (-1). After germination, the trays were watered and fertilized with Hoagland's solution adjusted to 50 ppm reduced nitrogen 2-3 times a week as needed to maintain the soil moist to slightly dry. Controls were grown under the same conditions without pre-treatment before planting. Plants were individually harvested on day 24, dried, and weighed. Tissues were sent for inductively coupled plasma mass spectrometry (ICP-MS) to measure tissue ion content. Nutrient accumulation in shoot biomass was calculated by multiplying the total shoot dry weight by the shoot concentration in each sample.

[0111] As shown in Figure 19, corn plants inoculated with endophytic strains WW5, WW6, or WW7 accumulated significantly higher levels of macro- and micromineral nutrients across the entire key mineral ion profile, as measured by percent change in total nutrient content of shoot biomass, when grown in Hoagland's drop out N nutrient solution supplemented with 50 ppm bioavailable nitrogen, compared to untreated control plants.

[0112] The data in Figure 19 show that the heterologous endophyte successfully improved both macro- and micronutrient uptake and uptake in host corn plants grown from seeds treated under reduced nitrogen. The results demonstrated that the heterologous endophyte was effective in improving the physiological performance of the foreign host plants and providing atmospheric nitrogen. The endophyte seed treatment composition increased corn shoot nitrogen by: WW5 47%, WW6 45%, and WW7 29%.

[0113] Example 10 Endophyte screen assay of crop plant yield when grown under conditions of limited bioavailable forms of nitrogen and phosphorus. The WW5, WW6, WW7 and PTD1 endophyte strains were further screened in a greenhouse pot experiment in which plants were inoculated with either endophyte strains encapsulated in alginate beads, either alone or in a mixture of all four strains. Alginate beads containing endophytes encapsulated in calcium alginate were placed next to the seeds, one per seed, and the pots were watered evenly by controlled drip irrigation to allow the seeds to germinate. The potting medium contained nitrate <13 ppm, ammonia nitrogen <6 ppm and phosphate <11 ppm.

[0114] The results in Figure 20 show that the four selected endophytes showed positive responses in a wide variety of crop plants increasing yields under bioavailable nitrogen and phosphorus limited conditions when using commercially available agronomically appropriate commonly used seeds.

[0115] Example 11 Effects of combined endophytic strain (WW6+WW7) seed treatments on total nutrient accumulation and shoot biomass Canola seeds (Brassica napus) treated with a seed inoculant composition co-fermented with WW6 and WW7 heterologous endophyte strains were grown and compared to control canola plant seeds treated with an inoculant composition without endophytes. Treated seeds were grown as follows: an appropriate amount of canola seeds were commercially treated with 500 mL of mixed co-fermented WW6 and WW7 strains per ton of seeds along with 500 mL of 1% alginate with Integral pro (BASF) and prebiotic UBS 016 (Unium Bioscience Ltd.) according to the manufacturer's instructions to aid in endophyte survival, colony growth and colonization of the host plant. The prebiotics include a microbial nutrition package, plant biostimulants, osmotic protectants, buffers and seed lubricants. Endophyte survival was confirmed by resuspension of seeds in 0.2 M phosphoric acid and plating on NLM semi-solid medium at appropriate dilutions. Control canola seeds were also treated with the same crop protection package. They were then stored for one month under normal seed storage conditions (4°C–15°C) and commercially planted in autumn at Coxwold, Lincolnshire, UK, using a seed drill in a large-scale CRO field trial.

[0116] Shoots were harvested in mid-spring at the beginning of growth and sent for agronomic mineral nutrition analysis. Nutrient accumulation in shoot biomass was calculated by multiplying the total shoot weight by the shoot ion concentration. As shown in Figure 21, canola co-fermented with strains WW6 and WW7 accumulated significantly higher concentrations of macro- and micronutrients.

[0117] The above data indicate that the co-fermented heterologous endophytes WW6 and WW7 successfully improved the uptake and incorporation of macro- and micronutrients in canola plants grown from treated seeds. The results demonstrate the effectiveness of co-fermented heterologous endophytes for improving the physiological performance of non-foreign host plants.

[0118] Example 12 Effect of WW6+WW7 composite strains on total nutrient accumulation and biomass Winter wheat seeds (Triticum aestivum) treated with a seed inoculant composition co-fermented with WW6 and WW7 heterologous endophyte strains were grown and compared to control winter wheat seeds treated with an inoculant composition without endophytes. The inoculant composition was combined with a prebiotic composition UBS 016 from Unium Bioscience Ltd. to aid in endophyte survival, colony growth and colonization of the host plant. Treated seeds were grown as follows: an appropriate amount of wheat seeds was commercially treated with 500 mL of the mixed co-ferment, 500 mL of 1% alginate and 10% UBS 016 in 1000 mL of water per tonne of seeds. A crop protection package consisting of fludioxonil and sedaxane was also added to prevent seed-borne diseases. Syngenta AG's Vibrance Duo® was used as the crop protection package. Vibrance Duo® was applied at a rate of 2 liters per tonne.

[0119] Control seeds of the same cultivars received the same crop protection package and survival of the endophyte on the seeds was confirmed by spiking the seeds in a 0.2 M phosphate resuspension and plating at appropriate dilutions on NLM semi-solid medium. Seeds were then stored for one month under normal industry conditions (4°C–15°C) and a large-scale CRO field trial was conducted in conventional cultivation in Caxwold, Lincolnshire, UK in autumn using a seed drill.

[0120] In late spring, 5 months after planting, vegetative shoots were harvested and sent for agronomic mineral nutrition analysis. Nutrient accumulation in shoot biomass was calculated by multiplying the total shoot weight by the shoot ion concentration. As shown in Figure 22, winter wheat inoculated with co-fermented WW6+WWW7 accumulated significantly more macro- and micronutrients, and the increase in accumulation was expressed as the Total Nutrient Content of Shoot Biomass, expressed as the percentage change from the control.

[0121] The above data indicate that the co-fermented heterologous endophytes WW6 and WW7 successfully improved the uptake and incorporation of macro- and micronutrients in winter wheat grown from inoculated seeds. The results demonstrate the effectiveness of co-fermented heterologous endophytes for improving the physiological performance of non-foreign host plants.

[0122] Example 13 Effect of WW6 and WW7 combination on total nutrient accumulation Shoot biomass 107Spring oat seeds (Avena sativa var Elyann and SO1) treated with seed inoculant compositions containing WW6 and WW7 ferments co-fermented with CFU / ml of heterologous endophyte strains were grown and compared to control seeds treated with inoculant compositions without endophyte strains. Treated seeds were grown as follows: an appropriate amount of oat seeds was commercially treated with 500 mL of the mixed co-ferment and 500 mL of 1% alginic acid and 10% prebiotic composition UBS 016 (Unium Bioscience Ltd.) and seed disease protectant Redigo (Bayer) in water at 1000 mL per ton of seeds, according to the manufacturer's instructions. Survival of endophytes on seeds was confirmed by resuspending seeds in 0.2 M phosphoric acid and plating on NLM semi-solid medium at appropriate dilutions. Control seeds of the same variety received the same prebiotic treatment but no endophyte spray. The oat seeds were then stored for one month under normal seed storage conditions and commercially planted in May in Suffolk, UK, in a large-scale replicated CRO field trial conducted with conventional fertiliser rates.

[0123] In mid-spring, five months after planting, early vegetative shoots were harvested and sent for agronomic mineral nutrition analysis. Nutrient accumulation in shoot biomass was calculated by multiplying the total shoot weight by the shoot ion concentration. As shown in Figure 23, spring oats inoculated with WW6 + WW7 co-ferment accumulated significantly higher concentrations of macro- and micronutrients.

[0124] The above data indicate that the co-fermented heterologous endophytes WW6 and WW7 successfully improved the uptake and incorporation of macro- and micronutrients in spring oat plants grown from inoculated seeds. The results demonstrate the effectiveness of co-fermented heterologous endophytes for improving the physiological performance of non-foreign host plants.

[0125] Example 14 Effect of the complex WW5+WW6+WW7+PTD1 on nutrient concentrations Oryza sativa hybrid XP753 seeds were pretreated with two fungicides, GA3 (gibberellic acid), dyes, and a mobile zinc micronutrient coating, and then overlaid with a seed inoculant composition containing the co-fermented WW5+WW6+WW7+PTD1 heterologous endophyte strains. 500 mL of endophyte fermentation broth and 1% alginic acid were added to 2,205 lbs of rice seeds. Testing of seed coat quality of the commercially treated seeds confirmed the survival of WW5+WW6+WW7+PTD1 endophyte strains, expressed as colony forming units (CFU) per seed, as follows: WW5 2.0 x WW6+WW7+PTD1 endophyte strains. 106 ;WW6 8.0 x 105 ;WW7 3.6 x 106 ; PTD1 1.2 x 106 Control seeds were treated with a pre-treatment rather than the seed inoculant composition. Seeds were then stored for 2 months under normal seed storage conditions and commercially planted in spring 2020 by a large-scale grower in Clay County, Arkansas, USA. Paddy fields were fertilized with 400 pounds of urea per acre, which is equivalent to 184 pounds of nitrogen per acre. Shoots including leaves were pooled at mid-growth stage (early bud break) and again separately at late growth stage (late bud break), air-dried and sent for agronomic mineral content analysis. As can be seen in Figure 24, plants inoculated with (WW5+WW6+WW7+PTD1) accumulated higher levels of plant-associated macro- and micro-nutrients, and these differences are expressed as the percentage change in leaf nutrient concentration from the control.

[0126] Example 15 Effects of seed treatments (WW5, WW6, WW7 and PTD1) on crop yield under reduced and normal nitrogen fertilizer application in arable land. Broccoli seeds were first commercially treated with a mixture of endophyte ferment and 1% alginate and sprayed at different rates using two different crop protection packages and an industry leading conventional method. Endophyte application rates consisted of 10mL, 50mL and 100mL of ferment mixed with the commercial slurry and sprayed per kg of broccoli seeds. Control seeds of the same variety were treated with the same crop protection package minus the endophyte. The endophyte viability was then measured to confirm the presence of each microorganism. The microbial mix demonstrated the survival of the strains (WW5, WW6, WW7, PTD1) on the seeds after drying. Ten seeds that had been washed with 10mL of water and had their seed coats removed were used to count the number of seed coats. Dilution plating results showed that the bacterial titers, provided as colony forming units / seed (CFU / seed) for each strain, were still viable and were successfully dehydrated, with the survival of the strains that had been dormant on the seeds afterwards being evident (Figure 25A).

[0127] Broccoli seeds were then grown under normal industry conditions (dark packaging). 25The plants were stored at 40°C or below for 2 months and commercially planted in the fall in a large replicated CRO field trial at a commercial production farm in Salinas, California, using nitrogen fertilization rates that were 25% lower than the conventional and commercial rates. Calcium ammonium nitrate (17-0-0) was applied for the entire season at 12 gal / acre twice and 5 gal / acre twice. The 25% reduced nitrogen treatment reduced the nitrogen rate by 25% at each application. In addition, all treatments received 0-0-6-3% Ca fertilizer a total of eight times during the season. Irrigation was managed at the farm manager's discretion according to commercial agricultural standards. After one month of growth, there was a significant difference in plant size between the treatments that received 100% nitrogen and those that received 25% less nitrogen. No disease or insect symptoms were observed in any of the treatments during the trial. At harvest, there was still a noticeable difference in leaves between the treatments that received 100% N and 25% less N. All treatments were then measured for uniformity and commercial quality at harvest. When selecting commercial broccoli heads, growers considered a variety of criteria, including head size (diameter in inches), head smoothness, dark green color, and firmness. The largest mean head diameter at harvest was observed at two endophyte treatment doses, 100 mL and 50 mL per kg, for both seed crop protection packages, with 25% N reduced fertilizer and regular 100% fertilizer, as shown in Figure 25B for 75% N and 100% N, respectively.

[0128] The highest mean head weights at harvest were observed at the two highest endophyte treatment rates of 100 mL and 50 mL per kg seed for both crop protection packages, both with reduced 25% N fertilizer and with regular 100% fertilizer, as shown in Figure 25C for 75% N and 100% N, respectively.

[0129] Example 16 Efficacy of individual seed treatments of WW6 and WW7 under reduced nitrogen fertilizer (32 ppm N) in Hoagland leaf litter indoor pot trials. WW6 and WW7 endophytes were applied alone to nitrogen-limited Brassica plants under controlled conditions to test their early effects on shoot growth. Endophyte strains WW6 and WW7 were applied in fermentation broth to broccoli seeds germinated and grown in artificial soilless medium within a commercial seed coating process using both clay and dip coat. The control was the commercial coating without the endophyte ferment mixture. Eight seeds per treatment were planted 1 / 2 inch deep in 2 inches of mineral medium (1 / 3 play sand, 1 / 3 perlite, 1 / 3 vermiculite) in 10" x 20" plastic growing trays. Trays were watered on Mondays, Wednesdays, and Fridays for the duration of the experiment with Hoagland's N drop out solution adjusted to 32 ppm total nitrogen with 70% reduction in optimal nitrogen, pH 7. The study period was 27 days, with 710μL of fermentation for 14 hours per day in an indoor growing chamber. mol / m2s1 under greenhouse lighting at an ambient temperature of 25°C and humidity of 50%. Seed coat viability and dilution plating were measured to determine the survival of endophyte compositions and crop protection agents after dehydration on commercially coated seeds. See Figure 26A.

[0130] At harvest (27 days old), the total fresh weight of seedlings was determined for all treatments and the results are reported below. The WW7 treatment (RD12378) showed a significant 30% increase in seedling weight compared to the control. The WW6 treatment (RD12381) showed a significant 47% increase over the control. See Figure 26B.

[0131] Example 17 Efficacy of WW5, WW6, WW7 and the co-fermented mixture when applied as seed treatment under reduced nitrogen fertilizer in a controlled environment growth chamber. The effect of WW5, WW6, and WW7 endophytes on shoot growth was tested when applied individually to nitrogen-limited corn seeds in a controlled environment. Endophyte strain formulations were applied in combination with 1% w / v sodium alginate (Scogin LDH) to germinated corn seeds and grown in artificial soilless medium in 1-gallon felt smart pots. Eight seeds per treatment were planted 1 / 2 inch deep in mineral medium (pure water washed; 1 / 3 play sand, 1 / 3 perlite, 1 / 3 vermiculite). Pots were watered on Mondays, Wednesdays, and Fridays throughout the experimental period with Hoagland's N drop out solution adjusted to contain 50 ppm total nitrogen. Two control groups were treated with modified Hoagland's solution and contained no endophyte. The study was performed in a greenhouse growth chamber at 710 μmol / m2. s 1. Under the conditions of 25℃ temperature and 50% humidity, 14 hours of greenhouse lighting per day was irradiated. 24 days after heading, the plants were harvested, washed, and placed in individual paper bags for 45 ℃で The shoots were dried for one month. The dry weight of the shoots was then measured and recorded. The results are shown in Figure 27. WW6 +102% p > 0.05, WW5 84% p > 0.05, WW7 49% p > 0.01.

[0132] Example 18 Effects of WW6 and WW7 field seed treatments on winter wheat. Winter wheat seeds were co-fermented with WW6+WW7 endophyte inoculant. The co-fermented WW6+WW7 endophyte mixture was fermented in low nitrogen medium and then freeze-dried to powder. Five grams of the freeze-dried fermentate and five grams of dried sodium alginate were mixed in one liter of water. This mixture was then mixed with a commercial prebiotic composition UBS 016 (manufactured by Unium Bioscience Ltd.) at a ratio of about 3:1 to about 5:1 to the prebiotic composition. This combination was finally sprayed as seed slurry at a rate of about 4 L to about 6 L per ton of winter wheat seeds. The control group was administered prebiotics without the fermentate. Seeds were commercially planted in the field in early November and the trial was conducted until the usual harvest in the middle of the summer of the following year. The treatment group showed a 10% increase in crop yield as shown in Figure 28A.

[0133] Additionally, nitrogen accumulation was measured in winter wheat shoots from February to June. Plants were harvested at each time point, 1 metre square, dried and subjected to total nitrogen determination by Kjeldahl method. A consistent increase in total nitrogen per hectare of wheat shoots was measured and is depicted in the results shown in Figure 28B which shows a +30% increase in total nitrogen accumulation Kg of wheat shoots / Ha measured at the last sampling in June. This result supports the conclusion that diazotrophy is N The impact of the two fixing endophyte strains on this wheat cultivar in the field was clearly demonstrated: endophyte treatments increased total plant shoot nitrogen per hectare throughout the growing season, with a 100% increase in shoot nitrogen accumulation in early May and a 30% increase in shoot nitrogen accumulation per hectare in early June.

[0134] Example 19 Carbon accumulation in plants treated with endophytic strains. The ability of WW5, WW6, WW7 and PTD1 endophytic bacterial strains to increase total plant carbon accumulation when grown under field conditions was evaluated using fast-growing Populus trees planted in field soil in the Lower Mississippi Alluvial Valley. Trees were treated with approximately 20 calcium alginate beads containing encapsulated endophytes applied to the base of Populus cuttings at the time of planting. Field replicate blocks were planted (3 × 5 trees) and there were five replicate blocks in the field. Dormant hybrid poplar cuttings with root lengths of 22.86 cm obtained from Greenwood Resources (Portland, OR, USA) were treated with Admire® Pro (Bayer, Whippany, NJ, USA).

[0135] For carbon sampling, leaf samples were returned to the lab and dried in an oven at 60 °C before being finely ground and placed in tin capsules. Samples were analyzed using an ECS 4010 CHNS-O analyzer (Costech Analytical Technologies Inc.).

[0136] As a result, as shown in Figure 29, endophyte inoculation increased the total carbon content of the plants by 71.01%, with a p-value = 0.063. The total carbon content was calculated by multiplying the total biomass dry weight of n=12 treated trees and n=12 control trees by the carbon percentage by dry weight. The results shown in Figure 29 indicate a significant increase in carbon accumulation in the treatment group compared to the control group.

[0137] Example 20 Compatibility with crop protection chemicals commonly used in seed treatments Endophyte strains WW5, WW6, WW7 and PTD1 were tested for their viability when combined with various commonly used seed crop protection chemicals. The survival rate of endophyte strains was evaluated when added to five different seed crop protection chemical solutions: Beret Gold® (Syngenta), Laxylstar® (Bayer CropScience), Redigro Pro® (Bayer CropScience), Vibrance Duo® (Syngenta) and Latitude® (Bayer CropScience). The solution mixes were prepared according to the manufacture specifications. Five to six minutes after making the mixtures, the colony forming units (CFU / ml) of the strains were measured by exhaustive dilution and plating on NLM semi-solid medium. The results shown in Figure 30A show that all strains can survive in the five mixed solutions.

[0138] The survival rate of WW5, WW6, WW7 and PTD1 was evaluated when applied to seeds of different crops with different seed crop protection chemical active ingredients: Mefenoxam, Fludioxonil, Azoxystrobin, Sedaxane, Thiabendazole Thiram, Metalaxyl, Hymexazol, Penthiopyrad, Poncho Beta and Thiamethoxam. The solution mixes were prepared according to the manufacturing specifications and the different strains were added to the solutions before application to the seeds with a commercial seed treater. The colony forming units (CFU / ml) of the strains that survived on the seeds were evaluated by adding the seeds to a 0.2M phosphoric acid resuspension and plating appropriate dilutions on NLM semi-solid medium. The results shown in Figure 30B indicate that the composition of endophytic strains is tolerant and survives when mixed with different commercial products and tolerates the temperature and dryness conditions found in commercial seed treatment processes.

[0139] Example 21 Compatibility of fertilizers, micronutrients and herbicides Commonly used after sowing and as foliar sprays. The ability of the endophytic strains to be added to various commonly used tank mix solutions and applied as seeding and foliar sprays was evaluated based on the survival of the strains over various time periods ranging from 3 hours to 1 month. The survival of WW5 and WP1 was evaluated in ammonium polyphosphate (10-34-0), a liquid starter fertilizer for post-seeding fertilizer applications, and a micronutrient fertilizer (4% ammoniacal nitrogen, 3% soluble nitrogen, 9.0% chelated zinc). The volume was reduced from 5 gal / acre to 50 ml / acre for the experiment. The mix composition consisted of 32 fl oz / acre of micronutrients, 5 gal / acre of 10-34-0 fertilizer, 16 fl oz / acre of WW5 strain inoculant, and water. Three hours after making the mix, colony forming units (CFU / ml) of the two strains were measured by plating on NLM semi-solid medium. The results shown in Figure 31A indicate that both WW5 and WP1 survived when 10-34-0 was included in the aqueous in-seed fertilizer + endophyte tank mix.

[0140] Based on these results, a tank mix of 3 gal / acre of 10-34-0 fertilizer and 16 fl oz / acre of WW5 + WP1 was evaluated in a replicated block field study applied as a pre-plant tank mix to growing corn. The tank mix was dripped onto the planted seed. The fields were either not fertilized with nitrogen or had 180 lbs / acre of nitrogen applied.

[0141] The results shown in Figure 31B indicate that when WW5 + WP1 lines were combined with 10-34-0 fertilizer and corn was grown under conventional full-rate NPK fertilizer in Wisconsin, Midwest USA, grain yield increased by +12% at commercial harvest.

[0142] Additionally, mineral nutrient content was measured in maize leaves during the V9 growth stage. Figure 31C shows that total nitrogen, potassium, and phosphate (NPK) were consistently increased when WW5 and WP1 were sown in the fertilizer tank mix at planting. Importantly, the increase in potassium (K) in leaves from blocks that did not receive nitrogen was significant at +9.5%, demonstrating enhanced shoot uptake and assimilation of K in addition to N and P.

[0143] Example 22 Compatibility of fertilizers, micronutrients and herbicides Commonly used after sowing and as foliar sprays. The WW6 and WW7 strains were used to test the effectiveness of endophyte fertilizers and the compatibility of micronutrients. The WW6 and WW7 strains were evaluated for their long-term viability when mixed with 6-22-6-4 (a common liquid starter fertilizer often used for post-seeding fertilizer applications). The test solution mixture volume was reduced from 5 gal / acre to 50 ml to take into account the size of the experimental container. The composition contents were 5 gal / acre of 6-22-6-4 fertilizer and 40 fl oz / acre of WW6+WW7 microbial composition. The colony forming units (CFU / ml) of the two strains were significantly increased from 1 week to 5 weeks on NLM semi-solid medium. The results in Figure 32 show that the viability of both strains was slightly reduced when 6-22-6-4 was present in the mixed solution.

[0144] Example 23 Endophyte compositions tested for compatibility with foliar herbicides The viability of W6 and WW7 was evaluated in a composition containing glyphosate, a broad-spectrum systemic herbicide used in foliar sprays, and / or adjuvants (modified vegetable oil, polyoxyethylene sorbitan fatty esters, vegetable oil, soybean oil ethoxylated) and / or micronutrient composition (sulfur 3.6%, boron 0.1%, manganese 3.0%, zinc 4.0%). The mixture solution was scaled down from 10 gal / acre to 50 ml for experimental purposes. The mixture contained a proprietary blend of the following products: 32 fl oz / acre of micronutrients, 16 fl oz / acre of adjuvants, 24 fl oz / acre of glyphosate, and 16 fl oz / acre of WW6+WW7 microbial fermentation composition. 24 hours after the mixture was made, the colony forming units (CFU / ml) of the two strains (WW6+WW7) were measured by plating on NLM semi-solid medium. The results shown in Figure 33A show that the viability of both strains did not decrease in the different compositions.

[0145] Field trials were conducted to demonstrate the effectiveness of WW6 and WW7 when applied as foliar sprays on corn with a glyphosate herbicide and adjuvant. A tank mix was prepared containing 32 oz / acre of WW6 and WW7 inoculant, 32 oz / acre of Cornerstone 5 Plus (Winfield United's glyphosate herbicide), 32 oz / acre of Masterlock (Winfield United's adjuvant), and 10 gal / acre of water. Field studies included 27.5 ft x 5 ft. high yielding and low yielding corn varieties. Three plots were treated with the tank mix and three with the same ingredients except for the endophyte strain. The results shown in Figure 33B indicate that the corn yield of the low-yielding variety increased by 10.6 bu / acre, while the high-yielding variety increased by 11.3 bu / acre with the WW6 + WW7 endophyte strains compared to the control without endophyte treatment.

[0146] Example 24 Enhancement effect of WW5, WW6, WW7 and PTD1 on plant resistance to flooding and saturated soil Waterlogging of agricultural soils is a major problem in food production systems in the United States and worldwide, often resulting in large-scale plant mortality and crop losses. To test the ability of endophytes to confer flooding tolerance to crops, beet (Beta vulgaris) was treated with a commercial seed treatment method both with and without application of heterologous endophytes (WW5, WW6, WW7, PTD1). Control strains were seed coated, and experimental seeds were seed coated in combination with co-fermenting endophyte strains WW5, WW6, WW7, and PTD1. The coated seeds were then washed and the survival of the endophyte strains was tested in the experimental groups. Ten seeds were vortexed and washed with 10 mL of potassium phosphate (KP) buffer to remove and dissolve the coating, and then diluted and plated on NLM agar medium to measure bacterial counts. In the assay, endophyte survival was evident, and the titers are shown in CFU / seed in Figure 34A.

[0147] Twenty-four endophyte-treated seeds and 24 control seeds were planted in individual cells. Seeds were germinated in standard commercial transplanting medium, kept constantly moist in fully saturated soil. The medium was moistened daily to saturation and then allowed to stand for 25 min. ℃の Plants were grown under natural sunlight conditions. Once germinated, the plants were constantly overwatered and exposed to flood-like conditions. After this flooding exposure, the plants were evaluated for flood stress symptoms, germination and overall growth effects. Plants inoculated with endophytic strains showed faster germination and establishment, less flood damage and better growth under continuously flooded saturated soil conditions compared to the control, as shown in Figure 34B.

[0148] The above data show that the co-fermented heterologous endophytes WW5+WW6+WW7+PTD1 improved the response of beet plants to biotic stress (flooding) due to over-wetting in saturated medium, enhanced 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 enhance the physiological performance of exotic host crop plants.

[0149] Example 25 Effect of seed treatment with multiple endophytic strains (WW6+WW7) on the cold tolerance of plants. Two sets of broad bean (Vicia faba) plants were prepared: a control group treated with a commercial seed treatment and an experimental group treated with the commercial seed treatment plus the co-fermented heterologous endophyte strains WW6, WW7 and the prebiotic UBS 016 (by Unium Bioscience Ltd.). The co-fermented WW6+WW7 endophyte mixture was fermented in low nitrogen medium and then freeze-dried to powder. Five grams of the freeze-dried fermentate and five grams of dried sodium alginate were mixed in 500 ml of water. This mixture was then mixed with 500 ml of prebiotic UBS 016 and the endophyte sodium alginate mixture and prebiotic composition in a 1:1 ratio. It was then incorporated into a commercial seed treatment slurry at a ratio of about 4 L to about 6 L per ton of broad bean seeds. The control group of seeds were treated with the prebiotic only. The seeds were germinated in standard potting medium and grown in a greenhouse in 6-inch pots with daytime minimum temperatures ranging from 45°F to 55°F. °F, maximum temperature between 65°F and 70°F Grown under natural daylight conditions at temperatures ranging from 100°F to 120°C. After germination, fertilize with a 90-day supply of slow-release complete fertilizer (Osmocote 15-9-12). Six weeks after germination, both sets were grown at 34°F. The plants were exposed to a cold shock treatment of 100°F for 6 hours. After this exposure, the plants were photographed and evaluated for cold stress symptoms and damage. As shown in Figure 35, the experimental plants inoculated with WW6+WW7 had less wilting and cold damage and more severe wilting than the control plants. Furthermore, the endophyte-treated plants fully recovered from the cold stress, whereas the non-endophyte-treated plants did not fully recover and showed signs of chlorosis and necrosis.

[0150] The above data indicate that the co-fermented heterologous endophytes WW6+WW7 successfully improved the response of broad bean plants to biotic stress (cold shock) compared to the control. This result demonstrates the effectiveness of co-fermented heterologous endophytes for improving the physiological performance of non-foreign host plants.

[0151] Example 26 Effect of individual and combined endophytic strains on plant resistance to boron- and chloride-rich sodic / saline soils. Sodic and saline soils limit plant growth, often resulting in plant death and reduced yields. The United States Department of Agriculture estimates that 40% of once arable farmland is no longer usable for agriculture due to high salinity of soils. The endophyte strains of the present invention were tested for their ability to increase crop plant growth after seed treatment in sodic and saline soils. Control and experimental groups using heritage broccoli (Brassica oleracea) seeds were prepared in the following manner. The two experimental groups were coated using a commercial seed treatment method that included a combination of polymer dip coat and WW7 (group 1) or endophyte strains WW5 + WW6 + WW7 + PTD1 + WP1, while the control group was treated with polymer dip coat and broccoli protection package. Seeds were then assayed for overall survival of the endophyte in the commercial seed coat, as shown in Figure 37A.

[0152] Seeds from the two endophyte treatments (groups 1 and 2) and the control were then grown in a commercial transplant greenhouse in Santa Monica, California, and planted in the field trial. A site was selected for the field trial by the United States Department of Agriculture (USDA) that is characterized by high cationic / saline soils and high boron and chloride concentrations. The site is Five Points, California. A soil is considered sodic when its exchangeable sodium percentage (ESP) is greater than 6%, and a soil is considered highly sodic when its ESP is greater than 15%. The ESP value indicates the percentage of the soil's cation exchange capacity (CEC) that is accounted for by sodium. The chemical profile of the poor quality soil is as follows. As shown in Figure 36B, the soil used in the trial had an ESP of 12.9. The soil also contained 25 ppm of boron. The soil also had a very high chloride concentration of 68 ppm. These characteristics indicate that the soil used in the trial was very poor.

[0153] Three identical 72-inch beds were created using a rotor tiller in a homogenous high salinity field (RRR west) and two drip tape irrigation lines were carefully installed in each bed. Bed size was 100 feet long and 6 feet wide in two rows (2 feet from the edge of each bed and 2 feet between rows). 290 plants each were planted from the two endophyte treatments and the untreated control seed. Photographs of the beds taken at harvest, 91 days after transplanting, are shown in Figure 36C. The salt tolerance of the endophyte-enhanced plants (labeled WW7 and Phase A in the figure) can be easily visualized in Group 2 (Phase A).

[0154] Seven week old broccoli seedlings were harvested and weighed 91 days after transplanting. Fresh weight of plants in group 2 showed a statistically significant increase of 13.24% (p<0.05) compared to the control group. Figure 36D shows the graph from the broccoli field trial. After harvesting, the plants were dried in a drying oven. Total dry weight of group 2 (Phase A) was increased by 47.06% (p<0.05) and total dry weight of group 1 (WW7) was increased by 16.81% (p<0.05) compared to the control group. See Figure 36E.

[0155] The data show that (1) the heterologous endophyte co-fermented WW5+WW6+WW7+PTD1+WP1 successfully improved the fresh and dry weight of broccoli compared to the control, and (2) the endophyte strains improved the dry weight of broccoli compared to the control under biotic stress conditions (sodic / saline soil conditions). This result demonstrates the effectiveness of heterologous endophytes to enhance the physiological performance of exotic host plants.

[0156] Example 27 Combined effects of different endophytic strains on enhancing and recovering plant tolerance under drought conditions Drought stress has adverse effects on crops, plants, grasses and trees, often leading to plant death and crop loss. The occurrence of drought is ever increasing, and drought induces many physiological and molecular biological changes in plants. The internal processes that allow plants to tolerate drought stress include scavenging of reactive oxygen species (ROS), osmoregulation (OA), stomatal closure and synthesis of protective molecules such as inducible dehydrins. Plant recovery after drought stress involves a series of steps that occur over time, which may be aided or promoted by beneficial endophytes.

[0157] To demonstrate the effect of endophytes in increasing tolerance to desiccation, a fermentation mixture containing endophyte strains WW5+WW6+WW7+PTD1 and fungal yeast endophyte strain WP1 was mixed and named "Phase A Mix". The Phase A Mix fermentation was then applied to tall fescue seeds (Festuca arundinacea, a pasture grass used in livestock farming) as a commercial seed treatment incorporating a polymer with talc powder as a carrier to dry the seeds and form a natural hard coating like a husk. There were two treatment groups: Group 1 was treated with 0.5 L of fermentation liquid per ton of seeds; Group 2, treated with 1.0 L (0.5 L fermentation broth + 0.5 L 2% alginate) per ton of seeds;

[0158] As a control, a commercial seed treatment containing no endophyte was used. These treatments were assayed and the survival of the endophyte was confirmed on the seeds shown in 37A.

[0159] The control and experimental groups 1 and 2 were planted at the same density in three flats containing low carbon growing medium consisting of washed play sand, perlite and vermiculite. They were sown three times a week for four weeks with an amended low nitrogen Hoagland nutrient solution containing 65 ppm nitrogen. The grass was grown for 30 min at 40°C. ℃で After growth, the plants were subjected to drought stress without irrigation for 14 days to allow the growth medium to dry out completely. After drought stress treatment was discontinued, watering of the flats was resumed to give the turf a chance to recover. Each group was then harvested and weighed. There was a statistically significant increase in dry weight of 42% and 67% for groups 1 and 2, respectively (p<0.05). See graph in Figure 37B for total weight results.

[0160] Taken together, our data demonstrated that the heterologous endophyte co-fermented with WW5+WW6+WW7+PTD1+WP1 successfully improved the fresh weight of fescue under biotic stress conditions (drought conditions) compared to the control. This result demonstrates the effectiveness of heterologous endophytes to enhance the physiological performance of exotic host plants.

[0161] Example 28 Endophytes increase seedling germination, seedling emergence, and seedling biomass weight. A series of tests were conducted to examine the effect of spraying endophytic strains on seed germination, seedling emergence from seed coats and soil, and seedling biomass weight increase. In the first experiment, romaine seeds were treated with 10 treatment groups and a control group, as identified in Figure 38. Each treatment was applied to romaine seeds with clayey seed coats and a limited nitrogen endophyte ferment mixture in 1 w / v% alginic acid solution. The control did not contain endophyte ferment. The control seeds were coated with commercially available methods such as clay coat or seed coat polymer. The seeds were germinated on square petri dishes containing seed germination paper moistened with deionized sterile water. The seeds were then germinated and the seedlings were grown under fluorescent light. The results showed that the inoculated seedlings were larger and grew better under nitrogen-limited conditions. This result strongly suggests that the inoculated plants were able to fix atmospheric nitrogen and utilize the nutrients available from the seedling germination paper better than the control. In relation to nitrogen fixation, the strains that seemed to fix the most nitrogen and grow best under nitrogen-free bacterial medium conditions were also observed to have the greatest lettuce seedling weights in the following order: WW6 > WW5 > WW6 / WW7 > endophyte mix (WW5, WW6, WW7, PTD1 + WP1) > PTD1 > WW7.

[0162] Example 29 An experiment was conducted to examine the effect of endophytic strains on seedling biomass weight in broccoli seeds. The experiment included four treatment groups (WW7 strain only and a mix of WW5, WW6, and WW7 with PTD1) and a control group, as shown in Figure 39. Each treatment was applied to broccoli seeds with clayey seed coats and contained a limited nitrogen endophyte ferment mix in 1% w / v alginate solution. The control did not contain endophyte ferment. Seeds, including control seeds, were coated with commercially available methods such as clay coat or seed coat polymer. Seeds were germinated on square petri dishes lined with seed germination paper moistened with deionized sterile water.

[0163] Germination was performed on a square petri dish covered with seed germination paper moistened with 14ml of deionized sterile water per 4x5" seed germination container. The weight of the seedlings was then measured after 14 days of nutrient-free growth under fluorescent lights. The results showed that the inoculated seedlings were larger and grew well under limited conditions. These inoculated plants were able to fix atmospheric nitrogen and utilize the nutrients obtained from the seedling germination paper better than the control, which resulted in the maximum seedling weight, as shown in Figure 39.

[0164] Example 30 An experiment was conducted to examine the effect of applying endophytic strains to barley seeds on seedling emergence. The experiment included treatment groups treated with co-ferment WW6+WW7 and a control group. Each treatment consisted of a mixture of limited nitrogen endophyte ferment in 1 w / v% alginic acid solution applied to barley seeds with clayey seed coats. The control group did not receive endophyte ferment. The control seeds were coated with commercial seed treatments containing polymers and crop protection agents, including clay coat and seed coat polymers. Seeds were germinated on square petri dishes lined with seed germination paper moistened with sterile deionized water. The seedlings were then allowed to germinate nutrient-free under white light for 4 days and were first photographed. The results showed that the inoculated seedlings visibly grew larger, as shown in Figure 40A, and continued to grow over time, as shown in Figure 40B after 4 days.

[0165] Example 31 An experiment was conducted to examine the effect of endophytic strains on seedling emergence in broccoli seeds. The experiment included a group treated with WW7 fermentation liquid, a group treated with a four-strain mix (I4WP), and a control group. Each treatment consisted of broccoli seeds with clayey seed coats treated with a mixture of limited nitrogen endophyte fermentates in a 1 w / v% alginate solution. The control seeds were treated without endophyte fermentates. Seeds, including the control seeds, were coated with commercial seed treatments containing clay coat, polymers containing seed coat polymers, and crop protection agents.

[0166] Broccoli seedlings from the treatment group and the control were planted in individual cells. Seeds were germinated in standard commercially available transplant potting medium. The medium was watered daily and incubated for 25 min. ℃の The plants were cultured under natural daylight conditions for 15 days. After that, the germination of the plants was evaluated. In the endophyte-treated group, the germination rate increased after 15 days. The plants inoculated with the endophyte strain germinated (see Figure 41) and established quickly, and grew well.

[0167] Example 32 An experiment was carried out in which the endophytic strains were applied to seeds of sugar beet varieties (C578 and M5) that had been treated with a seed inoculant composition. The experiment included a group treated with WW7 fermentation broth, a group treated with a four-strain mix (I4WP) and a control group. The treated seeds were sprayed with 50 mL of commercial fermentation broth per kg of seeds together with the crop protection agents Thiram, Metalaxyl, Hymexazol, Penthiopyrad and Poncho Beta (Clothianidin). Control seeds of the same varieties were treated with the same preparation without the fermentation. The seeds were then stored for one month under normal industry conditions and commercially planted.

[0168] Beet seedling emergence after sewing was measured over time. The endophyte seed treatment composition improved plant emergence at 29 days on average for beet cultivars WW5, WW6, WW7, and PTD1, all four of which survived (I4WP), compared to the control. In this study, emergence of 200 plants was treated as equivalent to 75% establishment of sugar beets, and the endophyte improved average emergence at 13, 16, and 29 days after planting. 200 beet plants emerged (13 more plants) in the four cultivars (I4WP) compared to 187 plants emerged at 29 days for the C578 control, and 177 beet plants emerged (16 more plants) in the treatment group treated with the I4WP mixture compared to 161 plants emerged at 29 days for the M5 control (Figures 42A and 42B).

[0169] Example 33 An experiment was carried out in which the endophytic strains were applied to wheat cultivar seeds treated with a seed inoculant composition. The experiment included a seed treatment group with co-fermented WW6 / WW7 fermentate and a control group. Seeds in the treatment group were sprayed with 500 mL of fermentate and 500 mL of 1% alginic acid per tonne of seed together with the prebiotic UBS 016 according to the manufacturer's instructions. Control seeds of the same cultivar were treated with the same preparation without the fermentate. Seeds were then stored for one month under normal industry conditions and commercially planted.

[0170] Heading of wheat seedlings was measured over time in a CRO field trial. The treatments improved wheat seedling growth after heading over time, randomly harvested from appropriate field plots. Final seedling fresh weights after 15 days were 1.054g leaf and 0.781g root for the WW6 / WW7 combo, compared to 0.95g leaf and 0.44g root for the control. Fresh weights of new plants pulled on each day are shown below in Figures 43A and 43B.

[0171] Example 34 Effect of WW6 or WW7 seed treatments on grain yield in the field. Spring wheat seeds were treated with WW6 or WW7 endophyte NLM ferment. Prior to seed treatment, the ferment was freeze-dried to a powder. Five grams of freeze-dried ferment along with 5 grams of dry sodium alginate per liter of water were added to prepare a seed treatment slurry. Six liters of seed slurry were used to treat one ton of spring wheat seed cultivar Tibalt. The results shown in Figure 44 show 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 the non-endophyte treated control.

[0172] Example 35 Efficacy of WW6 or WW7 applied as a seed treatment to increase resistance to plant diseases of the host plants treated with the inoculant composition. Field trials were conducted in Lincolnshire, UK. Wheat (var. Skyfall) seeds were treated with a labelled rate of Vibrance Duo® (Syngenta, Basel, Switzerland), a conventional crop protection package, plus WW6+WW7 endophyte LNM fermentate freeze-dried powder (5 grams combined with 5 grams of dried sodium alginate in 1 L of H2O2). Oに Suspension) was added along with 100ml diluted to 1 litre of commercial prebiotic UBS 016 (Unitium Biosciences, Brindle, Howden, UK). The final seed treatment slurry volume was 4L and applied to 1 tonne of wheat seed. Seed was sown in autumn 2020 at a rate of 200Kg / ha. The untreated plot received only the commercial crop protection agent seed treatment.

[0173] The experiment involved layering two nitrogen treatments; commercial 220Kg nitrogen and a lower 180Kg / ha nitrogen (supplied as ammonium nitrate). In both cases 50 units were supplied at planting and the remainder sprayed the following spring. Each treatment consisted of 10 x 25 m blocks with 4 replicates. Industry standard plant protection foliar fungicides were applied following the UK programme recommended by the Farmers Weekly Journal (Guide to tackling Septoria disease in wheat; https: / / www.fwi.co.uk / arable / crop-management / disease-management / a-guide-to-tackling-septoria-in-wheat).

[0174] Septoria disease of wheat is the most devastating disease of wheat in Northern Europe. In these trials, Septoria was assessed on flag leaves and leaf 2 throughout the season. Figure 45A shows that wheat plants treated with the endophyte seed treatment were significantly less infected than the conventionally treated control. Figure 45B shows the visual and qualitative difference in the health of leaf 2 samples from endophyte treated plants and leaf 2 samples from control plants.

[0175] Example 36 Compatibility of endophytic herbicides commonly used during agricultural foliar applications to control weeds in a wide range of monocotyledonous and dicotyledonous crops. Three liquid foliar herbicides, Enlistone (Corteva Agriscience, LLC), Impact (AMVAC Chemical Corporation), and Callisto (Syngenta Crop Protection, LLC), were evaluated for compatibility in tank mixes with the WW6 and WW7 strains. The mixture solution was scaled down from 20 gallons / acre to 10 ml for experimental purposes. WW6 + WW7 endophyte ferment composition was combined at 32 fl oz / acre (per strain): Enlistone (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) at 16 fl oz / acre, Impact (Topramezone 29.7% w / v, inactive ingredients 70.3% w / v), Callisto 3 fl oz / acre (ethylene glycol < 15% w / v, other ingredients > 45% w / v, mesotrione 40% w / v) or water. Four hours after making the mixture, colony forming units (CFU / ml) of the two strains were measured by plating on NLM semi-solid medium (Figure 46).

[0176] The results shown in Figure 46 indicate that there was no significant decrease in the viability of both endophyte strains in the different solution mixtures over a long period of time, allowing the use of these endophyte nutrient use efficiency, biomass and stress tolerance enhancing formulations in combination with foliar applications of commercial agricultural herbicides.

[0177] Example 37 Examples of fertilizer and biostimulant compatibility for common agricultural foliar applications on a wide range of monocotyledonous and dicotyledonous crops. Two foliar fertilizers or biostimulants, Isabion (Syngenta Agro AG) and Megafol (Syngenta Crop Protection AG), were tested for compatibility with the WW6 and WW7 strain compositions as foliar tank mixes. For experimental purposes, the tank mix was scaled down from 400 liters / ha to 10 ml. The WW6+WW7 microbial composition was mixed at 2.4 liters / ha with the following product combinations: Isabion 6 liters / ha, Megafol 3 liters / ha, or water. The colony forming units (CFU / ml) of the two strains were evaluated 4 and 24 hours after making the mixture by plating on NLM semi-solid medium. See Figure 47.

[0178] The results shown in Figure 47 indicate that there was no significant decrease in the viability of both strains in the different solution mixes over a long period of time, indicating that this endophyte composition can be used in commercial agricultural fertilizer and foliar application of biostimulants.

[0179] Example 38 Examples of adaptation of stable freeze-dried powder endophytes to commonly used foliar herbicides in various field crops and monocotyledonous cereals. For strains WW6 and WW7, the freeze-dried compositions were reconstituted in water as aqueous solutions and mixed separately with various standard herbicide tank mixes commonly used in cereal crops for compatibility testing and evaluation. Compatibility with Azimut (Comercial Quimica Masso, SA), Guadana (Comercial Quimica Masso, SA) and Tower (Comercial Quimica Masso, SA) products was all tested individually. Mixtures were scaled down from 400 litres / ha to 10ml for experimental purposes. The microbial preparations WW6 + WW7 were mixed at 0.25% (10 g of lyophilisate mixed in 1 L), separately with the following preparations: 0.13% Azimut (Florasulam 5 g / L (0,5% w / v) + Aminopyralid (potassium salt) 10 g / L (1 % w / v) + 2,4-D (Ester-2-ethylhexyl) 180 g / L (18% w / v)), 0.15% Guadana (Flufenacet 40% w / v (400 g / l) (32.4% w / w) + Diflufenican 20% w / v (200 g / l) (16.2% w / w)), 0.50% Towr (Diflufenican 4% + Chlortoluron 25% + Pendimethalin 30% (SC)) or with water as control. Four hours after the mixture was made and stored, the colony forming units (CFU / ml) of the two strains were measured by plating on NLM semi-solid medium. See Figure 48.

[0180] The results shown in Figure 48 showed no significant decrease in the viability of both strains in different product mixes over a long period of time, indicating that this endophyte composition can be used in nutrient utilization efficiency, biomass and stress tolerance enhancing product formulations in combination with commercial agricultural foliar herbicide applications.

[0181] Example 39 Ability to promote atmospheric nitrogen fixation after treatment with endophyte-inoculating seed coat suspensions. A test was carried out to demonstrate the ability of different endophytes to fix atmospheric nitrogen in crop plants germinated from hybrid maize seeds coated with inoculum using seed treatment suspensions mixed with 0.5% w / v nutritional additive sodium alginate carbohydrate suspension. Incubation of endophyte inoculum as strains WW5, WW6 and PTD1 showed that the cultures were at least 1.0 E The adjusted fermentation inoculum was used until a titer of 8 cells / mL was reached, and then combined with carbohydrates as a nutritional additive. Colony morphology of the appropriate species and strains was genetically confirmed using specific primers for colony PCR. The endophyte solution seed treatment composition was mixed with widely used chemical seed treatments, including the fungicides fludioxonil and mefenoxam, and the neonicotinoid insecticide thiamethoxam. The endophyte inoculum seed treatment was sprayed on corn seeds at a rate of 2.4 mL / 1800 kernels and added to the chemical seed treatments according to the manufacturer's recommended rates and application procedures for corn seeds. The treated seeds were then dried and viable colony counts were determined on nitrogen-limited medium NLM plus agar by seed coat washing and bacterial count dilution with KP buffer. As a result, the microbial survival rates on seeds one month after the mixed slurry seed coat application were as follows: WW6-400 CFU / seed, WW5-40 CFU / seed, and PTD1-40 CFU / seed.

[0182] Germinated seeds were potted in 2-gallon pots containing 2.5 kg of perlite-containing field soil and nutrient-rich soil containing 1,200 ppm total nitrogen plus soluble nitrogen ( NO 3+ NH 3) Plants were grown in 10 ppm soil without fertilizer for 4-5 weeks until V6 stage. Plants were grown in a greenhouse. 15N Biological nitrogen fixation (BNF) was analyzed using isotope dilution. The proportional dependence of inoculated maize plants on atmospheric and soil nitrogen was consistent with the natural nitrogen content of the inoculated plant biomass. 15N content and that of adjacent reference non-inoculated plants growing on soil nitrogen only. 15N The total N in maize shoot tissue was estimated by comparing the N content in the shoot tissue and the total N content in the shoot tissue. 15N のIsotope concentrations were measured at the V6 growth stage, 4–5 weeks after planting, using an Elementar EA Vario Pyrocube to measure total N, followed by IRMS using an Elementar IRMS GeoVisION, an isotope ratio mass spectrometer (IRMS). 15N を To quantify the percentage of nitrogen derived from air (NDFA%) in plant shoots, we used IRMS. 15N NDFA was calculated by subtracting isotopic nitrogen from total nitrogen concentration and reporting the difference as a percentage of the total nitrogen pool.

[0183] The test results are shown in Figure 49 and demonstrate that by seed treatment with endophyte seed treatment inoculant, the major proportion of total nitrogen in corn sprouts was derived from air as follows; PTD1 42% NDFA, WW5 36% NDFA and WW6 69% NDFA. This result clearly indicates that corn endophyte seed treatment can be stabilized after drying and used to inoculate seeds and subsequent crop plants sprouted from the seeds enhanced biological nitrogen fixation at the V6 plant growth stage of corn plants.

[0184] Example 40 Ability of liquid endophytes to promote atmospheric nitrogen fixation following direct treatment of wheat roots. The endophyte liquid inoculum, which was co-cultured with the WW5, WW6, and PTD1 strains and the synergistic mixture of WW6+WW7 strains, was cultured in the crop plant to demonstrate the atmospheric nitrogen fixation ability of different endophytes after inoculation into the soil or rhizosphere. The culture suspensions were maintained at least 1.0 ENitrogen-limited NLM medium supplemented with nutrient additives was used in parallel to reach a titer of 8 cells / mL. The nutrient-supplemented inoculum was then analyzed for viable cells and colony morphology. Specific primers for colony PCR were used to confirm the presence of WW5, WW6, PTD1 and the co-cultured mixture of WW6+WWW7 in the respective inoculum. The endophyte suspension was inoculated onto the roots of two-week-old wheat plants. 1 mL of the culture suspension was used per plant and sprayed on the roots of the plants after transplanting from the potting soil. The nutrient-supplemented inoculum was supplemented with 1,200 ppm total nitrogen and 10 ppm soluble nitrogen ( NO 3+ NH 3) was added to the field soil, but no fertilizer was added, and the plants were transplanted into 1-gallon greenhouse pots filled with 1.5 kg of field soil amended with perlite. Approximately 4-5 weeks after transplanting, the plants were grown in the greenhouse until the junction growth stage. The tissues were then harvested and biological nitrogen fixation (BNF) was measured as air-derived nitrogen using an Elementar EA Vario Pyrocube for total nitrogen analysis. 15Nは The measurements were made using an Elementar IRMS GeoVisION, an isotope ratio mass spectrometer (IRMS).

[0185] The results are shown in Figure 50 and indicate that seed treatment with endophyte seed treatment inoculants resulted in a substantial percentage of air-derived nitrogen in wheat sprouts as follows: 43% NDFA for PTD1, 51% NDFA for WW5, 38% NDFA for WW6 and 47% NDFA for the synergistic combination of WW6+WW7. This result clearly indicates that inoculation of wheat roots with endophytes, both with single strains and with co-fermented nutrient additives, enhances the biological nitrogen fixation capacity and carries over to the main growth stages of wheat plants.

[0186] Example 41 Efficacy of the endophytic synergistic combination WW6+WW7 against single strains when applied as a freeze-dried reconstituted seed treatment formulation on barley. The aim of this study was to determine whether a synergistic mixed consortium of endophytes could increase the total plant biomass (shoot + root) of barley more than a single strain used alone. Dry weight biomass was measured after 26 days of growth in a controlled environment with reduced nitrogen. A seed treatment slurry of bacterial inoculum, freeze-dried, stored and resuspended at the original growth solution moisture content, was used as the endophyte seed inoculation medium (500 mL resuspended endophyte freeze-dried culture 1.1% w / v + sterile sodium alginate solution 2% w / v) and a sterile control nitrogen-limited medium (NLM) with final alginate 2% w / v was added alone without endophyte addition. All treatments were sprayed at a rate of 1 L per tonne of seed. Spring barley was treated with three seed formulation solutions using endophyte strains WW6, WW7 and co-fermented WW6+WW7 and dried overnight in a laminar flow cabinet. Four pots were planted with one plant per pot, with n=4 plants per treatment. Control plants received Hoagland's nitrogen dropout solution at 65 ppm N (100%) and reduced at 32 ppm N (50%). Experimental treatments inoculated with endophyte received reduced levels of 32 ppm N (50%). Hoagland's nutrient was injected into the trays on Mondays, Wednesdays and Fridays and drained ½ hour later. Plants were grown under artificial lighting with a 12-hour light / 12-hour dark cycle. 25 After artificial insemination, the plants were grown for 26 days in a plant growth incubator at 37 °C, then harvested and placed in paper bags. 45 It was dried at ℃ for 48 days.

[0187] The results shown in Figure 51 show the synergistic effect of the combined treatment WW6+WWW7 which increased the total dry weight biomass of barley the most under 50% nitrogen reduction, significantly increasing the total biomass by 75%, while WW6 alone increased the total biomass by a non-significant 17%, and WW7 alone increased the total dry weight biomass by a non-significant 17%.

[0188] Example 42 Increased grain yield biomass with synergistic application of stacked endophytic strains as inoculated seed treatments in commercial field spring wheat. To test the ability of specific endophyte combinations to confer synergistic benefits to cereal crops, spring wheat cultivars (Sy Ingmar) were commercially treated with WW6+WW7 coferment and WW5+WW6+WW7 coferment using a seed treatment method. No other seed treatments were performed. Seeds were soaked in the prepared solutions (>1.0 mL). E6 Wheat seeds were coated with a seed treatment slurry of 1-0.5% w / v (CFU / mL NLM ferment + carbohydrate solution) at a rate of 0.23 mL per pound of wheat seeds using a seed treatment device. Controls were not inoculated with endophyte ferment. Wheat was planted in late May in Berthold, ND, USA, and harvested in September after 117 days of field growth. Field size was 5ft x 30ft, and soil was Williams silt loam. Wheat was planted in 4 rows per plot, with 10 seeds per row evenly spaced. A randomized complete block design was used with 4 replicates per treatment, with 1.5 million seeds sown per acre. Grain yield data was adjusted for 14% moisture. Fertilizer was applied at planting with a blend of 10-34-0 at 15 gal / ac and 28-0-0 at 44.5 gal / ac. Soil tests were conducted on soil from 0-24 inches deep and showed 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, Zn: 1.11 ppm.

[0189] The results of the study are shown in Figure 52. The two-stalk treatment (WW6+WW7) increased the average yield by 0.7 bu / ac, while the three-stalk stacking treatment showed a synergistic effect, increasing the average yield by an additional 3.33 bu / ac over the control, which was statistically significant at p<0.1.

[0190] Example 43 Synergistic effects of stacking endophytic strains to increase biomass yield from inoculated seed treatments of commercial field romaine lettuce. To test the ability of specific endophyte combinations to provide yield benefits to lettuce crops, a romaine variety (River Road CVS) was commercially treated with single bacterial endophyte strains and strain combinations (strains 2, 3, and 4) in which strains WW5, WW6, WW7, and PTD1 were co-cultured with the endophytic yeast WP1. All fermentations were mixed into a treatment slurry with the following formulation: CFU / mL > 1.0. E 6 cells (NLM ferment) + 1% w / v carbohydrate solution. Treatment slurry was also mixed into clay seed coats using a seed treatment device and spread on seeds using standard commercial techniques. Endophyte solutions were applied at the following rates: 10 mL of endophyte inoculum per 1 / 3 pound of romaine seed. For co-fermentates containing two endophyte strains, 5 mL of each strain was applied. For co-fermentates containing five endophyte strains, 2 mL of each strain was co-applied. Standard commercial field planting conditions included an 80" bed with 142,000 seeds planted on approximately one acre near Spreckels, California.

[0191] As shown in Figure 53A and 53B, single strain treatments improved yield. The combined treatments of WW5, WW6, WW7, PTD1 and A. WP1 significantly increased shoot biomass weight by 113% after commercial field growth.

[0192] Example 44 Efficacy of the endophytic synergistic combination WW6 + WW7 against single strains when mixed with a prebiotic carrier composition in the form of a compatible biostimulant and then applied as a seed treatment to canola. A study was conducted to determine whether a mixed synergistic consortium of endophytes, when mixed with a prebiotic plant microbial booster, could increase the total biomass (shoot + root) of canola plants more than when the prebiotic plant microbial booster was used alone. Plants were grown in a controlled environment with reduced nitrogen and fresh weight biomass was measured after 21 days. The bacterial inoculum was freeze-dried, stored, and then resuspended at the moisture content of the original growth solution and incorporated into a formulated seed treatment slurry. This slurry was used to prepare an endophyte seed inoculum with the following formulation: 500 mL of endophyte freeze-dried culture resuspended at 1.1% w / v, 500 mL of prebiotic plan and microbial biostimulant, and 500 mL of H2O. Oで Sterile 4% w / v sodium alginate solution. Sterile control nitrogen-limited medium (NLM) containing prebiotic plan and microbial biostimulant and 4% w / v sterile sodium alginate. Treatments were applied at a rate of 1 litre per tonne of seed. Seeds (untreated spring canola ‘Atomic TT’) were treated with three seed preparations using endophytic strains WW6, WW7 and WW6+WW7. After application, the seed was dried overnight in a laminar flow cabinet. Three pots were prepared with one plant per pot, and n=3 plants per treatment were planted and grown. Control plants were treated with Hoagland’s nitrogen dropout solution at 65 ppm N (100%) and 32 ppm N (50%). Experimental plants were treated with 32 ppm N (50%). Nutrients were injected into the trays on Mondays, Wednesdays and Fridays and replenished after the trays were drained. Plants were grown under artificial lighting with a 12-hour light / 12-hour dark cycle. 25 After artificial insemination, the plants were grown in a plant growth incubator at 37 °C for 21 days and then harvested.

[0193] As shown in Figure 54, the combined synergistic mixed treatment of WW6 + WW7 + prebiotics significantly increased the total biomass weight by 82% and increased the total biomass the most, whereas WW6 + prebiotics increased the total biomass by 20% and WW7 increased the total biomass by 55%.

[0194] Example 45 Use of three synergistic endophytic strains as foliar sprays on maize shoots reduces fertilizer requirements and increases harvested grain yield. A trial was conducted to determine the effectiveness of foliar applications of a novel endophyte combination (WW5+WW6+WW7) to improve nitrogen fixation in a corn hybrid variety (Channel 113 day213-19VT2PRIB). The corn variety was first commercially treated with the seed chemical Accelon according to the manufacturer's method. Corn was planted in early May in Mead, Nebraska, USA. The soil was Tomek silt loam with pre-sowing nutrient levels of 11.1 ppm P, 344 ppm K, 7.4 ppm S, pH 5.8, 4.1% OM, and CEC 17.6. Either the control or experimental treatments containing endophyte strains WW5, WW6, and WW7 were foliar-applied to V6 by mist spray using a pressurized sprayer. Field size was 10 ft x 40 ft with four rows of corn planted per field. Split block design with six replicates per treatment. Fertilization consisted of soil nitrate at 17 lbs / acre, UAN 32-0-0 liquid nitrogen at 75 lbs / acre before planting, plus an additional 37.5 lbs / acre for a total of 130 lbs / acre of nitrogen. Nitrogen application to treatments was 75% of standard nitrogen application to planted areas. A secondary control received 170 lbs of nitrogen (100% of standard nitrogen application). Herbicides were also applied: PreAculon + Roundup in May immediately after planting. 13 The two center rows were harvested in late October after 163 days in the field, and the grains were weighed and statistical analysis was performed. The results are summarized in Figure 55.

[0195] At the 75% N fertilization rate, results show that the synergistic effect of foliar sprays of the three strains of endophyte at V6 significantly increased corn average grain yield by +44.5 bu / ac (p=0.03). Foliar sprays of endophyte at V6 resulted in an increase in average yield compared to the full 100% N control treatment of 35 bu / ac. Example 46 By stacking two endophytic strains and applying them synergistically in crop fields, crop yields were increased with a 75% reduction in nitrogen. A trial was conducted to determine the effect of a specific endophyte combination, WW5+WP1, applied as a seed solution composition to improve nitrogen fixation inoculum and yield in a corn hybrid variety (Channel 113 day213-19VT2PRIB). The corn variety was first commercially treated with the seed chemical Accelon according to the manufacturer's method. Corn was planted in early May in Mead, Nebraska, USA. The soil was Tomek silt loam with pre-seeding nutrient levels of 11.1 ppm P, 344 ppm K, 7.4 ppm S, pH 5.8, 4.1% OM, and CEC 17.6. At corn planting, the WW5+WP1 endophyte solution was applied as an overlay over the seeds in the furrow using a dribble tube. Field size was 10 ft x 40 ft, with four rows of corn planted per field. Split block design with six replicates per treatment. Fertilization consisted of soil nitrate at 17 lbs / acre, UAN 32-0-0 liquid nitrogen at 75 lbs / acre before planting, plus an additional 37.5 lbs / acre for a total of 130 lbs / acre of nitrogen. Nitrogen application to treatments was 75% of standard nitrogen application to planted areas. A secondary control received 170 lbs of nitrogen (100% of standard nitrogen application). Herbicides were also applied: PreAculon + Roundup in May immediately after planting. 13 The two center rows were harvested in late October after 163 days in the field, and the grains were weighed and statistical analysis was performed. The results are summarized in Figure 56.

[0196] Results at a 75% N fertilizer rate show that furrow application of two endophytes at planting increased corn average grain yield by +30.2 bu / ac (p=0.07). Endophyte inoculation at planting resulted in an increase in average yield compared to the full 100% N control treatment of 20 bu / ac. Example 47 Stacking of endophytic strains for synergistic application using liquid root sprays of transplants prior to planting in the field to increase strawberry biomass yield. A trial was conducted on an organic strawberry farm in Salinas, California to determine the effectiveness of a specific endophyte combination, WW5+WP1, applied as a liquid root spray to improve biomass yield of Albion strawberries. Various endophyte inoculants were applied in a thin mist until the roots were covered. Treatments included WW5 alone, WW6 alone, WW7 alone, PTD1 alone, and a mix of WW5, WW6, WW7, and PTD1. The control group contained no endophyte strains. Treatments were applied and planted using standard methods in early November 2016. Strawberries were planted in beds spaced 25-30 cm apart in two rows (200-240 plants per row), with three beds per treatment spaced 120 cm apart. Strawberries were fertilized using standard methods under the guidance of a registered CCA and were harvested in May 2017 after 28 weeks of growth. 20 It was harvested on the day.

[0197] As shown in Figure 57, the treatment with a mixture of four strains, WW5, WW6, WW7 and PTD1, increased fruit yield measured by fresh weight by 25% over the control. The single strain inoculation treatments showed less increase compared to the control.

[0198] Example 48 Combination of endophytic strains in hard partially hydrated beads as dry granular carriers for synergistic application to increase tomato transplant biomass. A trial was conducted to examine the effect of WW5 alone, the combination of WW6 and WW7, and the combination of four endophyte strains WW5, WW6, WW7, and PTD1 on increasing biomass yield in tomato. The fermentation suspension was blended into a sodium alginate slurry and dripped into a 100 mM calcium chloride water bath where the cation exchange reaction took place, creating fully hydrated but hard calcium alginate beads, which were then dried to a final bead size of 2 mm and a moisture content of approximately 4% to approximately 6%. Quality 47 (Q47) hybrid tomato seeds were placed on or adjacent to a single bead containing the endophyte treatment; the control was a bead treatment without the endophyte strain. They were then germinated and grown in 125-cell transplant planter trays with a commercial organic-rich transplant potting mix based on peat and perlite, with a standard commercial greenhouse fertilizer rate and fertilizer rate. 25 Plants were grown for 3 weeks under normal lighting at 20°C. After 21 days, 8 replicates per treatment were weighed and the total plant biomass dry weight was collected. The survival rate of Q47 tomato plants inoculated with the mixed fungus was 100%, while the germination rate of the uninoculated control plants was 88%. Figure 58 shows the endophyte enhanced biomass (shoot + root).

[0199] The results clearly showed a synergistic effect on growth in tomato plants inoculated with alginate beads containing a combination of four endophyte strains WW5, WW6, WW7 and PTD1 compared to the control. The endophyte mix resulted in a statistically significant 35% increase in total plant weight (shoots and roots). The single endophyte treatments WW5 and the combination of WW6 and WW7 resulted in smaller increases.

[0200] Example 49 Combinations of endophytic strains in rigid partially hydrated beads as dry granular carriers for synergistic inoculation of loose-leaf lettuce grown under conditions deficient in bioavailable N and P. A trial was conducted to determine the effectiveness of an endophytic strain in dehydrated calcium alginate beads in reducing nitrate and phosphate application rates and increasing edible yields of loose-leaf lettuce (Lactuca sativa, cv. Refugio). The experiment was starved of bioavailable N and P and used 9 kg of Terra Green, 0.66 kg of peat moss, 40 g of 8-3-5 organic fertilizer, and well-drained organic-rich soil containing 12 ppm nitrate, 5 ppm ammonia, 11 ppm phosphate, 328 ppm potassium, 690 ppm sulfate, SAR 2.71, pH 7.28, EC 2.89 dS / m, TEC 18.76 meq / 100 g, and 1831 ppm total nitrogen. In a greenhouse trial, plants were harvested 106 days after planting. Each seed was planted in a pint-sized starter pot, one bead per seed placed 1 / 2 inch into the growing medium, and watered. The alginate beads were applied adjacent or adjacent to the germinated seeds in the soil. After 3 weeks, the plants were carefully transplanted, with all roots removed with a hand trowel and the surrounding soil still intact. The roots, beads, and soil were placed in equal sized holes and placed into 2 gallon felt Smart Pots. All plants were automatically watered with the same amount of water every 12 hours using a controlled drip system. Low evening sunlight was supplemented with greenhouse lighting (high pressure sodium halide lamps) to allow for a 12 hour growth cycle from 4:15pm to 7:15pm. Lettuce plants were inoculated with beads containing WW7 or PTD1 alone or a mixture of four bacterial endophytes (WW5+WW6+WW7+PTD1). Controls received alginate beads without endophytes. Each treatment had 6 replicates with n=6 pots.

[0201] Figure 59 shows the analysis results of fresh shoot biomass. The treatment with the mixture of four endophytes performed best, with an average shoot weight of 4.23g per plant, a 191% increase compared to the uninoculated control. WW7 beads produced an average yield of 3.96g per plant, with an average shoot weight increase of 173%. Inoculation with PTD1 beads increased shoot biomass by an average of 2.74g per plant, with an average yield increase of 89%.

[0202] Example 50 Reconstitution of endophytic strains in freeze-dried powder combined with foliar application to jalapeno peppers in the field. To determine the effectiveness of endophyte inoculation from a freeze-dried endophyte mixture on the growth and biomass of jalapeno (cv. RPP7042). Approximately 420 jalapeno plants were foliar sprayed at flowering in early spring. Five endophyte strains (WW5, WW6, WW7, PTD1, WP1) were used, resuspended from freeze-dried powder. A mixture of the five resuspended endophyte strains was also prepared. One gram of freeze-dried endophyte was added per liter of purified water, and the freeze-dried endophyte was rehydrated and then placed in a foliar sprayer. Jalapeno seeds were planted in May to early June. Harvest occurred mid-September to early October. The reconstituted mixture was applied foliarly along each 50-foot treatment block. Each treatment section was separated by a 50-foot control section, with three treatments per row. 50 feet = approximately 75 plants. A series of growth and biomass analyses were conducted at harvest two months later.

[0203] As shown in Figure 60A, all inoculation treatments using lyophilized powder reconstituted at first flowering increased the average unripe pepper yield per plant compared to the control, except for the treatment with WP1. The mixture of five strains (Phase A) performed best and was found to have a synergistic effect. Phase A increased the total unripe pepper yield by 44%, favoring RPP7042 jalapeno.

[0204] Additionally, the ability of each treatment to increase the total number of peppers was measured, and the results are shown in Figure 60B. Phase A inoculation also showed the best results, with an initial increase of 66% in the average number of peppers per plant over the control, except for WP1. The mixtures of Phase A, PTD1, and WW7 were all statistically significant, indicating that the freeze-dried endophyte has an early positive effect on jalapenos.

[0205] The total yield of ripe peppers was also measured 3 months after the first inoculation and 4 months after planting, as shown in Figure 60C. The results showed that endophyte inoculation with reconstituted freeze-dried powder applied as a foliar spray at first flowering can increase the average yield per plant of jalapeno peppers compared to the control. Phase A inoculation increased the biomass yield of peppers more than the other treatments. All bacterial endophyte strains tested increased yield relative to the control strain, with Phase A showing an increased synergistic effect. This result supports the early positive impact of freeze-dried endophyte in solution and sprayed on flowers and leaves on the total yield of jalapeno peppers.

[0206] Example 51 Effects of different endophyte seed treatment compositions using WW5, WW6, WW7, PTD1 and WP1 on leaf chlorophyll. The synergistic endophyte mixture was freeze-dried and resuspended in water and used to treat canola seeds to determine whether the resuspended endophyte fermentate could increase chlorophyll in canola leaves after 36 days when grown under reduced nitrogen conditions. The endophyte seed inoculation medium consisted of endophyte freeze-dried powder suspended in 500 mL of water at a concentration of 1.1% w / v and mixed with 500 mL of 4% sterile aqueous sodium alginate solution and sprayed at a rate of 1 L per ton of seeds. Endophyte inoculation compositions were prepared for the following strain-strain combinations: WP1, GWW6+WWW7 and WW5+WWW6+WWW7+WP1+PTD1). The seed variety used in the study was spring canola "Atomic TT". Seeds were treated with the endophyte treatment compositions prepared: wp1, ww6+ww7, ww5+ww6+ww7+wp1+ptd1. Controls were treated with endophyte-free NLM only. After seed treatment, all groups were dried overnight at room temperature in a laminar flow cabinet. Five treatment groups were planted with 4 pots, 2 plants per pot (n=8 plants / treatment). Control plants were irrigated with Hoagland's nitrogen dropout solution at 65 ppm N (100% N) and 32 ppm N (50% N). Experimental groups were treated with 32 ppm N (50% N). Nutrients were flooded into the trays 3 days a week (Monday, Wednesday, Friday) and drained off after 30 minutes. Plants were grown under artificial lighting with a 12-hour light / 12-hour dark cycle for 25-48 hours. ℃の After artificial insemination, the plants were grown in a plant growth incubator for 36 days and then harvested.

[0207] The results showed that the endophyte compositions used as seed treatments on canola showed a synergistic effect on leaf chlorophyll, with the two strains WW6+WW7 increasing leaf chlorophyll by 14.3%, p<0.1, whereas the synergistic five-strain endophyte consortium composition increased leaf chlorophyll by 21.0%, p<0.05. See Figure 61.

[0208] Example 52 Synergistic application of stacked endophytic strains in alginate beads to increase chlorophyll in strawberry leaves. A trial was conducted to determine the efficacy of an endophyte inoculant applied as a composition to increase chlorophyll in the leaves of Albion strawberries. The trial was conducted at an organic strawberry farm in Salinas, California. Strawberry seedlings were transplanted onto five 2 mm calcium alginate beads that were placed in holes prior to planting. The calcium alginate beads were prepared in one of the following treatments: endophyte inoculation with WW5 alone, endophyte inoculation with WW6 alone, endophyte inoculation with WW7 alone, endophyte inoculation with PTD1 alone, endophyte inoculation with WW5, WW6, WW7, and PTD1, or a control without any endophyte strains. Planting was performed in early November 2016 using standard methods. Strawberries were planted in beds spaced 25–30 cm apart in two rows (200–240 plants / row), with three beds for each treatment spaced 120 cm apart. The strawberries were fertilized in a standard manner under the guidance of a registered CCA and were harvested in May 2017 after 28 weeks of growth. 20 It was harvested on the day. As shown in Figure 62, the treatment group that combined all four strains WW5, WW6, WW7 and PTD1 provided greater chlorophyll and showed the best result with a significant increase in chlorophyll of 5.5% p<0.05.

[0209] Example 53 Effect of different endophyte seed treatment compositions using WW6+WWW7 versus commercial key biological and biostimulants on total chlorophyll in winter wheat leaves. An experiment was conducted to determine whether a freeze-dried and resuspended endophyte composition applied to seeds could increase chlorophyll in the leaves of treated host plants. Endophytes were applied to winter wheat seeds AWC13. A freeze-dried endophyte seed inoculation composition was prepared by suspending freeze-dried WW6+WW7 endophyte strains in 500 mL water at a concentration of 1.1% w / v and mixing the suspension with 500 mL of 4% sterile aqueous sodium alginate solution. A control was prepared with 500 mL of sodium alginate solution and water without endophyte. Treatment composition was applied at a rate of 1 L per ton of seeds. Winter wheat was grown under optimal nitrogen in a field trial and harvested during the growing season. Chlorophyll was increased by 1 cm Extracted with acetone from leaf sections sized 2. The results in Figure 63 showed an average increase of 6% over the untreated control.

[0210] Example 54 Enhanced composition of glutamic acid / glutamine Glx in maize leaves after endophyte-inoculated seed coat treatment. The seed coat containing the heterologous endophytic composition is then incubated with atmospheric nitrogen. ガスをTests were conducted to determine whether WW6 fermentation can be used to fix and generate ammonium, which can then be converted to the primary amino acid end products glutamate and glutamine in the host plant via the GOGAT GS and GDH ammonium assimilation pathways. A seed treatment was prepared containing WW6 fermentation and sodium alginate 0.5% w / v. The seed treatment was applied to maize seeds, which were then dried and stored for one month. Treated seeds and control untreated seeds were potted in one gallon pots containing 1.5 kg sand, vermiculite and perlite and allowed to germinate and grow for three weeks. During the growth period, the seeds and resulting plants were watered with Hoagland's Nitrogen Dropout nutrient solution supplemented with 50 ppm nitrogen. The plants were grown in a greenhouse, harvested, freeze-dried and analyzed for common amino acids using a Shimadzu HPLC with post-column ninhydrin derivatization at AAA labs Inc USA. From the results shown in Figure 64, the glutamic acid and glutamine amino acid contents in the leaves of corn seeds inoculated with WW6 were significantly higher (p=0.01) than those of the control. Furthermore, the genome of WW6 was analyzed to check for the presence of genes encoding glutamine synthetase GS, and a total of six copies of the GS enzyme gene were found adjacent to other genes related to nitrogen assimilation, quorum sensing, and motility. These results suggest that atmospheric N2 Further supporting other fixation-related findings, N concentrations in WW6-treated maize shoots were increased. These results also provide a mechanistic basis for the effectiveness of WW6 as a biological inoculant to increase nitrogen assimilation into the amino acids glutamate and glutamine directly from the atmosphere in crop plants. The foregoing description of specific embodiments of the present invention has been presented for purposes of illustration and description, and many modifications and variations are possible in light of the above teachings. The embodiments have been chosen and described to best explain the principles of the invention and its practical application, so as to enable others skilled in the art to best utilize the invention and its various embodiments, with various modifications suited to the particular uses contemplated.

Claims

1. A plant inoculant composition for treating a host plant, comprising at least one heterologous endophyte strain selected for its ability to fix atmospheric nitrogen, and at least one nutrient additive activating to enhance the survival and colonization of the at least one heterologous endophyte strain in a host plant, wherein the at least one heterologous endophyte strain comprises at least one of the strains deposited with the ARS Culture Collection under accession numbers NRRL B-68081, NRRL B-68078, NRRL B-68080, and NRRL B-68079.

2. The aforementioned at least one heterologous endophyte strain contains exogenous ammonium (NH4). 4 ＋ The composition according to claim 1, selected for its ability to produce ).

3. The composition according to claim 1, wherein the at least one heterologous endophyte strain increases the solubilization of multiple forms of insoluble phosphorus in liquid bacterial culture.

4. The composition according to claim 1, wherein the at least one heterologous endophyte strain has a genetic mechanism for producing Fe siderophores.

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

6. The composition according to claim 1, wherein the at least one heterologous endophyte strain increases the acquisition of essential macronutrients and micronutrients from soil and air without substantially disrupting the phytonutrient stoichiometry of the host plant.

7. The composition according to claim 1, wherein the at least one heterologous endophyte strain is resistant to biocides, plant hormones, plant elicitors, mineral micronutrients, or mineral macronutrients.

8. The composition according to claim 1, wherein the nutritional additive comprises at least one of an organic or synthetic biostimulant, a fatty acid, a carbohydrate, an amino acid, a mineral nutrient, and a chemical prebiotic for combined seed treatment, combined foliar application, or combined infall application.

9. The composition according to claim 1, comprising a plurality of heterologous endophyte strains selected from the group consisting of strains deposited with the ARS Culture Collection under accession numbers NRRL B-68081, NRRL B-68078, NRRL B-68080 and NRRL B-68079.

10. The composition according to claim 5, wherein the plurality of heterologous endophyte strains comprises at least two, at least three, or at least four strains independently selected from the group of strains defined in claim 9.

11. The composition according to claim 1, further comprising at least one additional microbial species selected from Rhizobium species, Mycorrhizae species and endogenous yeast strains.

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

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

14. The composition according to claim 1, wherein the composition comprises a carrier composition that enables the application of the plant inoculant composition to seeds.

15. The composition according to claim 1, wherein the at least one heterologous endophyte strain is freeze-dried into a fluid powder before being incorporated into the inoculant composition.

16. The composition according to claim 1, wherein the at least one heterologous endophyte strain is encapsulated in microbeads or large beads incorporated into the inoculant composition.

17. The composition according to claim 16, wherein the inoculant composition is a suspension composition.

18. The composition according to claim 1, wherein the inoculant composition comprises a liquid composition that can be applied as a foliar spray.

19. A plant inoculant composition for heterologous application to host plants, comprising at least one heterologous endophyte strain selected from strains deposited in the ARS Culture Collection under accession numbers NRRL B-68081, NRRL B-68078, NRRL B-68080, and NRRL B-68079.

20. The composition according to claim 19, wherein the at least one heterologous endophyte strain is selected for its ability to fix atmospheric nitrogen and grow in nitrogen-restricted or nitrogen-free growth media.

21. The aforementioned at least one heterologous endophyte strain is found to be exogenous ammonium (NH₃) in nitrogen-restricted growth media and nitrogen-free growth media. 4 ＋ The composition according to claim 19, selected for its ability to produce ).

22. The composition according to claim 19, wherein the at least one heterologous endophyte strain increases the solubilization of multiple forms of insoluble phosphorus in liquid bacterial culture.

23. The composition according to claim 19, wherein the inoculant composition comprises a prebiotic composition that is activatable to enhance colonization of the host plant by the at least one heterologous endophyte bacterial strain, the prebiotic composition comprising at least one of a microbial nutrient package, a plant biostimulant, a systemic protective agent, a buffer, and a seed lubricant.

24. The composition according to claim 19, comprising a plurality of heterologous endophyte strains selected from the group consisting of strains deposited with the ARS Culture Collection under accession numbers NRRL B-68081, NRRL B-68078, NRRL B-68080 and NRRL B-68079.

25. The composition according to claim 24, wherein the plurality of heterologous endophyte strains comprises at least two, at least three, or at least four strains independently selected from the group of strains defined in claim 24.

26. The composition according to claim 19, wherein the inoculant composition further comprises at least one of the Rhizobium species, the Mycorrhizae species, and an endogenous yeast strain.

27. ​​The composition according to claim 24, wherein at least two of the plurality of heterologous endophyte strains are co-fermented.

28. A method for promoting nutrient uptake in a host plant, comprising applying the composition defined in claim 1 or 19 to the leaves or seeds of the host plant, or to the growing place of the plant, or to the place where the seeds are sown.

29. A method for increasing biomass in a host plant, comprising applying the composition defined in claim 1 or 19 to the leaves or seeds of the host plant, or to the growing place of the plant, or to the place where the seeds are sown.

30. A plant treated with a composition defined in claim 1 or 19, characterized in that at least one heterologous endophyte strain from the composition is incorporated into the plant tissue.

31. a. A step of obtaining a plant inoculant by fermenting at least one heterologous endophyte strain from among the strains deposited with the ARS Culture Collection under accession numbers NRRL B-68081, NRRL B-68078, NRRL B-68080 and NRRL B-68079; and b. A step of mixing the carrier and the plant inoculant. A method for producing a plant inoculant composition for strengthening a host plant, which includes the above.

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