Enhanced nitrogen-fixing microorganisms for use in agriculture

Abstract

The present disclosure relates to genetically modified microorganisms of the genus Paenibacillus for improving the phenotype of plants, such as nitrogen utilization in non-leguminous plants. Comprising novel strains of microbial bodies, microbial consortia and agricultural compositions comprising the same. In addition, the present disclosure teaches methods of utilizing the microorganisms, microbial consortia, and agricultural compositions comprising the same in methods of conferring beneficial properties to target plant species. In particular aspects, the disclosure provides methods of enhancing desired plant traits in agronomically important species, such as fixation, utilization, regulation, absorption, acquisition, tolerance and / or processing of nitrogen in plants.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 376,601, filed on September 21, 2022, under 35 USC 119(e), which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically

[0004] An official copy of the sequence listing is submitted electronically as a WIPOST26-compliant XML sequence listing and is submitted simultaneously with this specification, the file name of the sequence listing is 22039_SeqListing.xml, created on August 30, 2023, and is 367,414 bytes in size. The sequence listing contained in this XML format file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to isolated and biologically pure microorganisms having applications, particularly in agriculture. The disclosed microorganisms can be used in their isolated and biologically pure states or can be formulated into agriculturally acceptable compositions. Methods for using isolated microorganisms or agriculturally acceptable compositions in agricultural applications are also disclosed. Background Art

[0006] According to the United Nations World Food Program, there are nearly 900 million undernourished people worldwide. The malnutrition epidemic is particularly acute in developing countries around the world, where one in six children is underweight. The lack of available food can be attributed to a variety of socioeconomic factors; however, regardless of the ultimate cause, the fact remains that there is a shortage of food available to feed a growing world population that is expected to reach 9 billion people by 2050. The United Nations estimates that agricultural production must increase by 70% to 100% in 2050 to feed the projected global population.

[0007] These alarming world population and malnutrition figures highlight the importance of agricultural efficiency and productivity in sustaining a growing worldwide population. The technological advances achieved through modern row crop agriculture, which have resulted in unprecedented crop yields, are impressive. However, despite advances made through technological innovations such as genetically engineered crops and new insecticidal and herbicidal compounds, improved crop performance is still needed to meet the needs of an exponentially increasing global population.

[0008] Scientists estimate that if the global agricultural "yield gap" (i.e., the difference between the best observed yield and what is achieved elsewhere) could be closed, then worldwide crop production could rise by 45% to 70%. In other words, if all farmers (regardless of their location in the world) could achieve the highest achievable yields expected for their respective regions, much of the shortfall in world food production could be addressed. However, addressing the question of how to achieve higher yields in a heterogeneous global landscape is difficult.

[0009] Typically, yield gaps are explained by insufficient water, substandard farming practices, insufficient fertilizers, and the inability to use herbicides and pesticides. However, a significant increase in the use of water, fertilizers, herbicides, and pesticides worldwide would not only be economically unfeasible for large parts of the world, but would also have adverse environmental consequences.

[0010] Therefore, it is simply not feasible to meet global agricultural output expectations by simply scaling up the current high-input agricultural systems used in most developed countries.

[0011] Therefore, there is a pressing need in the art for improved methods of enhancing crop performance and imparting beneficial traits to desirable plant species.

[0012] The technology described herein includes environmental nitrogen-fixing bacteria that have been genetically edited to increase the amount of atmospheric nitrogen fixed.

[0013] These gene-edited bacterial strains confer improved phenotypes to plants, such as crop plants, to increase the amount of nitrogen available to the plants and improve plant health and productivity. Summary of the invention

[0014] The present invention includes isolated and biologically pure microorganisms that have applications, particularly in agriculture. The disclosed microorganisms can be used in their isolated and biologically pure states or formulated into agriculturally acceptable compositions. Also provided are agriculturally beneficial microbial consortia (which comprise at least two members of the disclosed microorganisms) and methods of utilizing the consortia in agricultural applications. In some aspects, genome modification of microorganisms (individuals, consortia, and / or colonies) is contemplated to improve microbial traits and improve plants associated with microorganisms.

[0015] Successful editing of strains of the Gram-positive spore-forming bacterium Paenibacillus across multiple different species, across subgroups I and II of the genus, is presented herein. Several different edits, including deletions, truncations, and knockouts, were evaluated at several different loci within the Paenibacillus genome, including two GlnR binding sites upstream of the nif operon (sites I and II, respectively), and the locus of the CueR gene.

[0016] The present disclosure addresses the important question of how to improve plant performance, thereby closing the yield gap worldwide, while providing a means of imparting other beneficial traits to plant species. The novel edited strains of Paenibacillus described herein improve plant performance by enabling plants to increase and / or improve nitrogen utilization, fixation, uptake, acquisition, tolerance, distribution, regulation, processing, and / or any combination of the foregoing and / or any combination of the foregoing.

[0017] In some embodiments, the plant is a non-leguminous crop plant.

[0018] In some embodiments, the plant is a dicot. In some embodiments, the plant is a vegetable, herb, ornamental, or fruit plant. In some embodiments, the plant is selected from the group consisting of soybean, cotton, canola, rapeseed, kale, spinach, lettuce, carrot, potato, beet, radish, tomato, broccoli, cauliflower, pumpkin, mustard, berry, pepper, leafy greens, cowpea, cantaloupe, cucumber, basil, grape, and okra.

[0019] In some embodiments, the plant is a monocot. In some embodiments, the plant is a C3 monocot. In some embodiments, the plant is a C4 monocot. In some embodiments, the plant is selected from the group consisting of corn, wheat, rice, sorghum, sugar cane, onion, bamboo, palm, garlic, ginger, lily, daffodil, iris, orchid, bluebell, tulip, amaryllis, banana, plantain, ginger, turmeric, cardamom, asparagus, pineapple, sedge, rush, leek, forage grass, lawn grass, buckwheat, quinoa, chia and millet.

[0020] In some embodiments, the plant is a dicot. In some embodiments, the plant is selected from the group consisting of soybean, cotton, canola, rapeseed, legumes (e.g., peas, beans, lentils, peanuts), mint, lettuce, tomato, pepper, eggplant, dwarf melon, broccoli, carrot, cauliflower, potato. In some embodiments, the plant is an arbor, such as apple, peach, pear, cherry, almond, walnut, lemon, lime, orange. In some embodiments, the plant is a fruit, such as blackberry, strawberry, blueberry, raspberry.

[0021] The solution for improving crop performance and increasing yield provided by the present disclosure is not harmful to the earth's resources because it does not rely on increasing water consumption or increasing the input of synthetic chemicals in the system. Instead, the present disclosure utilizes microorganisms to impart beneficial traits to desired plants, including increased yield.

[0022] Thus, the present disclosure provides an environmentally sustainable solution that allows farmers to increase yields of important crops that does not rely on increasing the use of synthetic herbicides and pesticides.

[0023] In embodiments, the present disclosure provides efficient and broadly applicable agricultural platforms that utilize microorganisms and microbial consortia (multiple microorganisms, in some aspects, multiple microorganisms that improve the health or desired phenotype (such as agronomic traits) of the plant with which they are associated) that promote one or more desired plant characteristics.

[0024] Microorganisms disclosed herein improve the performance of plants (such as crop plants) by direct and indirect mechanisms. In some respects, microorganisms form a symbiotic relationship with plants. In some respects, microorganisms produce compounds (for example, metabolites, toxins, proteins, lipopeptides or other compositions) that give benefits to plants or plants can be used for improved properties. In some respects, microorganisms improve the solubility of one or more compositions (such as nutrients), thereby benefiting plants. In some respects, microorganisms give tolerance to exogenous substances (such as herbicides or pesticides) to plants. In some respects, microorganisms produce compositions that are harmful to plant pests (such as insects). In some respects, microorganisms fix nitrogen, thereby improving the nutritional status of plants. Other aspects other than the exemplary non-limiting aspects listed above are contemplated.

[0025] In some embodiments, a single microorganism is utilized. In some aspects, the single microorganism is isolated and purified. In some aspects, the single microorganism is a classified species of bacteria. In some aspects, the single microorganism is an identifiable strain of a classified species of bacteria. In some aspects, the single microorganism is a new, recently discovered strain of a classified species of bacteria.

[0026] In some aspects, the single microorganism (whether a taxonomically identifiable species or strain) is combined with one or more other microorganisms of a different species or strain. In certain aspects, the combination of two or more microorganisms forms a consortia or consortium. The terms consortia and consortium are used interchangeably.

[0027] In certain aspects, the present disclosure provides the development of highly functional microbial consortia that help promote the development and expression of desired phenotypic or genotypic plant traits. In some embodiments, the consortia of the present disclosure possess functional attributes that do not exist in nature when the individual microorganisms live alone. That is, in various embodiments, specific microbial species are combined into a consortium such that the microbial combination possesses functional attributes that any individual member of the consortium does not possess when considered alone.

[0028] In some embodiments, the functional attribute possessed by the microbial consortium is the ability to confer one or more beneficial characteristics to a plant species, such as: increased growth, increased yield, increased nutrient utilization (e.g., nitrogen, phosphate, etc.), increased nitrogen use efficiency, increased stress tolerance, increased drought tolerance, increased photosynthetic rate, increased water use efficiency, increased pathogen resistance, modification of plant architecture (which does not necessarily affect plant yield, but addresses plant function), etc. Beneficial characteristics of pest resistance and / or tolerance are also contemplated, including adverse effects on nematodes, insects or other pests.

[0029] In some embodiments, individual microorganisms do not possess the ability to impart these beneficial properties to plants when they exist in nature. Instead, in some embodiments, these microorganisms are manually combined into consortia by humans to form functional compositions that possess attributes and functional properties that do not exist in nature. In some embodiments, the consortia may include microorganisms that have been genetically edited, altered, or modified by modifying cellular composition (including DNA, RNA, protein, and / or combinations thereof) through techniques known to those of ordinary skill in the art.

[0030] However, in other embodiments, the present disclosure provides isolated and biologically pure individual microorganisms that are capable of conferring beneficial traits to desired plant species without the need to combine the microorganisms into a consortium.

[0031] In some embodiments, the microorganism is a strain of Paenibacillus that has been genetically modified to have increased nitrogen fixation capabilities.

[0032] Thus, the present disclosure provides environmentally sustainable solutions that allow farmers to increase the yield of important crops without relying on increased use of synthetic fertilizers, herbicides, and / or pesticides. For example, in one aspect, the present disclosure describes isolated microorganisms that have been genetically modified to increase the ability of the microorganism to fix nitrogen. In some embodiments, the endogenous nif gene of the isolated microorganism is genetically modified to increase the nitrogen fixation ability of the microorganism. In some embodiments, the endogenous glnR gene encoding the protein GlnR is genetically modified to increase the nitrogen fixation ability of the microorganism.

[0033] In some embodiments, the present disclosure relates to an isolated genetically modified microorganism, the isolated genetically modified microorganism comprising one or more genetic modifications selected from the following: genetic modification of an endogenous glnR gene encoding GlnR; and genetic modification of a regulatory sequence of an endogenous nif gene. The genetic modification of the endogenous glnR gene encoding GlnR in the genetically modified microorganism is characterized as providing a mutant glnR gene that produces a GlnR protein variant. In addition, the genetic modification of the regulatory sequence within the endogenous nif gene is characterized as providing improved binding affinity for GlnR compared to a non-genetically modified regulatory sequence. The one or more genetic modifications are characterized as providing improved nitrogen fixation activity to the genetically modified microorganism compared to a non-genetically modified strain of the microorganism.

[0034] In some embodiments, the genetically modified microorganism of separation described herein is characterized as having constitutive expression of nif genes, regardless of the local nitrogen concentration in the microorganism surrounding environment. For example, in some embodiments, the genetically modified microorganism of separation described herein is characterized as having constitutive expression of nif genes under nitrogen-limited conditions. In some embodiments, the genetically modified microorganism of separation described herein is characterized as having constitutive expression of nif genes under nitrogen-rich conditions.

[0035] The present disclosure also relates to a method for preparing an isolated genetically modified microorganism, wherein the method comprises genetically modifying an endogenous glnR gene encoding GlnR, genetically modifying a regulatory sequence within an endogenous nif gene, or a combination thereof; and isolating the microorganism. The step of genetically modifying the endogenous glnR gene encoding GlnR comprises editing the endogenous glnR gene to produce a mutant gene encoding a GlnR protein variant. The step of genetically modifying the regulatory sequence within the endogenous nif gene comprises replacing the regulatory sequence with a DNA sequence characterized as providing improved binding affinity for GlnR compared to the native regulatory sequence. In addition, the microorganism is characterized as having nitrogen fixation activity, wherein genetically modifying the endogenous glnR gene encoding GlnR, and / or genetically modifying the regulatory sequence within the endogenous nif gene, provides improved nitrogen fixation activity compared to a non-genetically modified strain of the microorganism.

[0036] The present disclosure also relates to agricultural compositions, which include one or more strains of the isolated genetically modified microorganisms disclosed herein and an agriculturally acceptable carrier. In some embodiments, the agricultural compositions include one or more additional agricultural beneficial agents (e.g., fertilizers, biofertilizers, bionematicides, biostimulants, synthetic insecticides and / or synthetic herbicides).

[0037] Also disclosed herein are methods of conferring one or more beneficial traits to plants, wherein the methods comprise applying an agriculturally effective amount of one or more isolated genetically modified microorganisms or agricultural compositions disclosed herein.

[0038] In some embodiments, the Paenibacillus strain is described in Table 1a, Table 1b, or Table 1c. In some embodiments, the Paenibacillus strain comprises a polynucleotide sequence at least 90% identical to any one or more of SEQ ID NOs. 1-123. In some embodiments, the Paenibacillus strain is a species selected from the group consisting of Paenibacillus aceris, Paenibacillus aestuarii, Paenibacillus agarexedens, Paenibacillus agaridevorans, Paenibacillus albidus, Paenibacillus alginolyticus, Paenibacillus alkaliterrae, Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus anaericanus, Paenibacillus antarcticus, Paenibacillus apiary, Paenibacillus spp. apiaries), Paenibacillus assamensis, Paenibacillus azotifigens, Paenibacillus baetica, Paenibacillus barcinonensis, Paenibacillus barengoltzii, Paenibacillus borealis, Paenibacillus caespitis, Paenibacillus camelliae, Paenibacillus castaneae, Paenibacillus catalpa, Paenibacillus cavernae, Paenibacillus cellulolyticuscellulosilyticu), Paenibacilluschartariuss, Paenibacillus chibensis, Paenibacillus chinjuensis, Paenibacillus chitinolyticus, Paenibacillus chondroitinus, Paenibacillus cineris, Paenibacillus cisolokensis, Paenibacillus contaminans, Paenibacillus cookii, Paenibacillus crassostreae, Paenibacillus cucumis, Paenibacillus galactolyticus curdlanolyticus), Paenibacillus daejeonensis, Paenibacillus darwinianus, Paenibacillus dongdonensis, Paenibacillus donghaensis, Paenibacillus doosanensis, Paenibacillus drentensis, Paenibacillus durus, Paenibacillus usedaphicus, Paenibacillus ehimensis, Paenibacillus elgii, Paenibacillus endophyticus, Paenibacillus etheri, Paenibacillus euphorbia favisporus), Paenibacillus filicis, Paenibacillus frigoriresistens, Paenibacillus gansuensisgansuensis), Paenibacillusginsengarvi, Paenibacillus glacialis, Paenibacillus glebae, Paenibacillus glucanolyticus, Paenibacillus glycanilyticus, Paenibacillusgraminis, Paenibacillusgranivorans, Paenibacillusharenae, Paenibacillushelianthin, Paenibacillushordei, Paenibacillushumicus, Paenibacillusshunanensis, Paenibacillusillinois, Paenibacillus illinoisensis、Paenibacillus jilunlii、Paenibacillus kobensis、Paenibacillus koleovorans、Paenibacillus konkukensis、Paenibacillus konsidensis、Paenibacillus kukuduoahitejonii、Paenibacillus lactis、Paenibacillus lautus、Paenibacillus liaoningensis、Paenibacillus lupini、Paenibacillus macquariensis、Paenibacillusmarchantiophytorum、Paenibacillus marquariensis, Paenibacillus mendelii, Paenibacillus mobilis, Paenibacillus motobuensis, Paenibacillus colloidusmucilaginosus), Paenibacillus naganoensis, Paenibacillus naganoensis, Paenibacillus nanensis, Paenibacillus nasutitemitis, Paenibacillus nebraskensis, Paenibacillus nicotianae, Paenibacillus nitroguajacolicus, Paenibacillus oceanisediminis, Paenibacillus odorifer, Paenibacillus oryzae, Paenibacillus oryzisoli, Paenibacillus ourofinensis, Paenibacillus fodder pabuli), Paenibacillus panacisoli, Paenibacillus panaciterrae, Paenibacillus pectinilyticus, Paenibacillus peoriae, Paenibacillus periandrae, Paenibacillus phoenicis, Paenibacillus phoenicis, Paenibacillus physcomitrellae, Paenibacillus pini, Paenibacillus pinisoli, Paenibacillus plakortidis, Paenibacillus pocheonensis, Paenibacillus polymyxa polymyxa), Paenibacillus polysaccharolyticus, Paenibacillus populi, Paenibacillus profundus, Paenibacillus Provenceprovencensis), Paenibacillus purispatii, Paenibacillus qinlingensis, Paenibacillus rhizoplanae, Paenibacillus rhizoryzae, Paenibacillus rhizosphaerae, Paenibacillus rigui, Paenibacillus sacheonensis, Paenibacillus segetis, Paenibacillus sepulcri, Paenibacillus shirakamiensis, Paenibacillus silagei, Paenibacillus silvae, Paenibacillus sinopodophylli, Paenibacillus solanacearum, Paenibacillus soli, Paenibacillus sonchi, Paenibacillus sputi, Paenibacillus stellifer, Paenibacillus susongensis, Paenibacillus taichungensis, Paenibacillus taihuensis, Paenibacillus taiwanensis, Paenibacillus taohuashanense, Paenibacillus starimensis, Paenibacillus telluris, Paenibacillus terrae, Paenibacillus terrigena, Paenibacillus hailandensis, Paenibacillus thalictra, Paenibacillus thermophilus, Paenibacillus Tianmushan tianmuensis), Paenibacillus tianmuensistimonensis), Paenibacillus tritici, Paenibacillus tundrae, Paenibacillus turicensis, Paenibacillus tylopili, Paenibacillus typhae, Paenibacillus suliginis, Paenibacillus validus, Paenibacillus vini, Paenibacillus vulneris, Paenibacillus wooponensis, Paenibacillus wynnii, Paenibacillus xanthanilyticus, Paenibacillus xinjiangensis, Paenibacillus xylan-eating xylanexedens), Paenibacillus xylanilyticus, Paenibacillus yonginensis, and Paenibacillus yunnanensis. In some embodiments, the Paenibacillus strain belongs to subgroup I. In some embodiments, the Paenibacillus strain belongs to subgroup II. In some embodiments, the Paenibacillus strain is selected from the group consisting of: NRRL deposit numbers: B-68102, B-68103, B-68104, B-68105, B-68106, B-68107, B-68108, B-68109, and B-68110.

[0039] Any strain disclosed herein can be further combined with one or more other microorganisms to form a microbial consortium. A microbial consortium can be any combination of one or more individual microorganisms. In certain embodiments, the microbial consortium comprises two microorganisms or three microorganisms or four microorganisms or five microorganisms or six microorganisms or seven microorganisms or eight microorganisms or nine microorganisms or 10 microorganisms or more than 10 microorganisms.

[0040] Another object of the present disclosure is to design a microbial consortium that is capable of performing a common multidimensional activity. In some aspects, the microorganisms that comprise the consortium act synergistically. In various aspects, the effect of the microbial consortium on a certain plant characteristic is greater than the effect that would be observed when any individual microbial member of the consortium is used singly. That is, in some aspects, the consortium exhibits a greater than additive effect on a desired plant characteristic compared to the effect that would be found when any individual member of the consortium is used alone.

[0041] In some aspects, the consortium leads to the establishment of other plant-microbe interactions, for example by serving as primary colonizers or founder populations that set the trajectory of future microbiome development.

[0042] In embodiments, the present disclosure relates to synergistic combinations (or mixtures) of microbial isolates.

[0043] In some aspects, the consortia taught herein provide a wide range of agricultural applications, including: increasing the yield of grains, fruits and flowers; increasing the growth of plant parts; increasing the ability to utilize nutrients (e.g., nitrogen, phosphates, etc.), increasing disease resistance; bio-insecticide effects, including increasing resistance to fungi, insects and nematodes; increasing survival in extreme climates; and improving other desired plant phenotypic characteristics. Obviously, these benefits to plants and / or adverse effects on target pests and / or pathogens can be obtained without any harmful side effects to the environment.

[0044] In some aspects, individual microorganisms of the present disclosure or consortia comprising the same can be combined into agriculturally acceptable compositions.

[0045] In some embodiments, the agricultural compositions of the present disclosure include, but are not limited to, wetting agents, extenders, defoamers, detergents, chelating agents, drift reducers, neutralizers, buffers, corrosion inhibitors, dyes, odorants, extenders, penetration aids, stickers, binders, dispersants, thickeners, stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, fertilizers, pesticides, nematicides, insecticides, herbicides, inert carriers, polymers, and the like.

[0046] In one embodiment of the present disclosure, microorganisms (including isolated single species or strains, consortia, or their compositions, such as metabolites) are supplied in the form of seed coatings or other seed applications. In embodiments, seed coatings can be applied to bare and untreated seeds. In other embodiments, seed coatings can be applied to previously treated seeds. Therefore, in some embodiments, the present disclosure teaches a method for treating seeds, which includes applying isolated bacterial strains or microbial consortia to seeds. In certain embodiments, isolated bacterial strains or microbial consortia are applied in the form of agricultural compositions comprising agriculturally acceptable carriers. In some embodiments, agricultural compositions can be formulated as: soil drenches, foliar sprays, immersion treatments, furrow treatments, soil conditioners, granules, broadcast treatments, post-harvest disease control treatments, or seed treatments. In some embodiments, agricultural compositions can be applied alone or in a rotary spraying procedure with other agricultural products. In some embodiments, agricultural compositions can be compatible with tank mixing. In some embodiments, agricultural compositions can be compatible with tank mixing with other agricultural products. In some embodiments, the agricultural compositions are compatible with equipment for ground, aerial, and irrigation applications.

[0047] In some embodiments, the applied microorganism may become endogenous and thus may be present in the treated growing plant and its progeny. In other embodiments, the microorganism may be applied simultaneously with the seed treatment in a co-treatment.

[0048] In one embodiment of the present disclosure, the microorganism is supplied in the form of a granule or plug or soil drench applied to a plant growth medium. In other embodiments, the microorganism is supplied in the form of a foliar application, such as a foliar spray or a liquid composition. The foliar spray or liquid application can be applied to a growing plant or a growth medium, such as soil.

[0049] In other embodiments, microorganisms (including separated single species or strains or consortia or their compositions, such as metabolites) are supplied in the form of fertilizers, pesticides or other amendments that can be applied to soil. In some embodiments, microorganisms are supplied in the form of fertilizers, pesticides or other amendments that are applied to soil before planting. In some embodiments, microorganisms are supplied in the form of fertilizers, pesticides or other amendments that are applied to soil while planting. In some embodiments, microorganisms are supplied in the form of fertilizers, pesticides or other amendments that are applied to soil after planting.

[0050] In other embodiments of the present disclosure, microorganisms (including isolated single species or strains or consortia) and / or compositions thereof (e.g., metabolites) are supplied in the form of a post-harvest disease control application.

[0051] In embodiments, the agricultural compositions of the present disclosure may be formulated as: (1) solutions; (2) wettable powders; (3) dusting powders; (4) soluble powders; (5) emulsion or suspension concentrates; (6) seed dressings, (7) tablets; (8) water-dispersible granules; (9) water-soluble granules (slow release or quick release); (10) microencapsulated granules or suspensions; (11) as irrigation components, and (12) components of fertilizers, pesticides and other compatible improvers, etc. In certain aspects, the composition may be diluted in an aqueous medium prior to conventional spray application. The compositions of the present disclosure may be applied to soil, plants, seeds, rhizosphere, rhizosheaths, or other areas where application of the microbial composition would be beneficial.

[0052] Another object of the present disclosure relates to agricultural compositions, which are formulated to provide high colony forming unit (CFU) bacterial colonies or consortia. In some aspects, agricultural compositions have adjuvants that provide relevant shelf life. In embodiments, the CFU concentration of the agricultural compositions taught is higher than the concentration at which the microorganisms will naturally occur outside the disclosed method. In another embodiment, the agricultural compositions include microbial cells at a concentration of 10^2-10^12 CFU / gram carrier or 10^5-10^9 CFU / gram carrier. In one aspect, microbial cells are directly applied to seeds as seed coatings at a concentration of 10^5-10^9 CFU. In other aspects, microbial cells are applied to the top of another sub-coating as seed outer coatings at a concentration of 10^5-10^9 CFU. In other aspects, microbial cells are applied to the top of another sub-coating with a concentration of 10^5-10^9 CFU together with another sub-treatment agent as a co-treatment agent at a ratio of 10^5-10^9 CFU.

[0053] In various aspects, the present disclosure relates to agricultural microbial preparations that promote plant growth. In various aspects, the present disclosure provides isolated microorganisms and consortia comprising them as taught to be formulated as agricultural biological inoculants. The taught biological inoculants can be applied to plants, seeds or soil, or combined with fertilizers, pesticides and other compatible modifiers. Suitable examples of preparing biological inoculants comprising isolated microorganisms can be found in U.S. Patent No. 7,097,830, which is incorporated herein by reference.

[0054] The disclosed microbial formulations can: reduce the need for nitrogen-containing fertilizers, solubilize minerals, provide biopesticide protection to plants, protect plants from pathogens (e.g., fungi, insects and nematodes), and make valuable nutrients such as nitrogen and / or phosphates available to plants, thereby reducing and eliminating the need to use chemical pesticides and chemical fertilizers.

[0055] In some embodiments, the isolated and biologically pure microorganisms of the present disclosure can be utilized in methods of conferring one or more beneficial characteristics or traits to a desired plant species.

[0056] In some embodiments, agriculturally acceptable compositions comprising an isolated and biologically pure microorganism of the present disclosure can be utilized in methods of imparting one or more beneficial characteristics or traits to a desired plant species.

[0057] In some embodiments, the consortia of the present disclosure may be utilized in methods of conferring one or more beneficial characteristics or traits to a desired plant species.

[0058] In some embodiments, agriculturally acceptable compositions comprising the consortia of the present disclosure may be utilized in methods of imparting one or more beneficial characteristics or traits to a desired plant species.

[0059] In some aspects, the isolated and biologically pure microorganisms of the present disclosure and / or the consortia of the present disclosure are derived from an accelerated microbial selection process ("AMS" process). The AMS process utilized in some aspects of the present disclosure is described, for example, in the following documents: (1) International Patent Application No. PCT / NZ2012 / 000041, published as International Publication No. WO 2012125050 A1 on September 20, 2012, and (2) International Patent Application No. PCT / NZ2013 / 000171, published as International Publication No. WO 2014046553 A1 on March 27, 2014, each of which is incorporated herein by reference in its entirety for all purposes.

[0060] However, in other embodiments, the microorganism of the present disclosure is not derived from an accelerated microorganism selection process. In some aspects, the microorganism utilized in the embodiments of the present disclosure is selected from the microorganism members present in the database. In specific aspects, the microorganism utilized in the embodiments of the present disclosure is selected from the microorganism present in the database based on the specific characteristics of the microorganism.

[0061] The present disclosure provides that plant components or plant parts can be effectively enhanced by coating the plant components or plant parts with an isolated microorganism or consortium of microorganisms in an amount not normally found on the plant components or plant parts.

[0062] Some embodiments described herein are methods for preparing agricultural seed compositions or seed coatings, comprising: contacting the surface of the seed with a preparation comprising a purified microbial colony, the purified microbial colony comprising at least one isolated microorganism that is heterologous to the seed or that rarely exists on the seed. Other embodiments require the preparation of an agricultural plant composition, comprising: contacting the surface of the plant with a preparation comprising a purified microbial colony, the purified microbial colony comprising at least one isolated microorganism that is heterologous to the plant. In other aspects, the preparation or microorganism is introduced into the interior of the seed, for example, into cotyledon or plumule other seed tissues.

[0063] In some aspects, applying the isolated microorganisms, microbial consortia, exudates, metabolites and / or agricultural compositions of the present disclosure to seeds or plants can modulate agronomically important traits. Agronomically important traits can be, for example, disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen stress resistance, improved nitrogen fixation, improved nutrient (e.g., phosphate, potassium, etc.) utilization, insect resistance, herbivore resistance, pathogen resistance, reduced pathogen levels (e.g., via secretion of metabolites that affect pathogen survival), increased yield, increased yield under water-limited conditions, enhanced health, increased vigor, improved growth, increased photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased bud length, increased root length, improved root configuration, increased seed weight, faster seed germination, altered seed carbohydrate composition, altered seed oil composition, number of pods, delayed aging, stay-green, and altered seed protein composition. In some aspects, at least 2, 3, 4 or more agronomically important traits are modulated. In some aspects, the modulation is a positive effect on one of the aforementioned agronomic traits.

[0064] In some aspects, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to modulate or alter plant characteristics, such as altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, increased nitrogen fixation, improved nitrogen utilization efficiency, improved root architecture, improved water use efficiency, increased biomass, reduced biomass, increased root length, reduced root length, increased seed weight, increased shoot length, reduced shoot length, yield relative to a reference plant. increase, increased yield under water-limiting conditions, kernel mass, kernel moisture content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutritional enhancement, pathogen resistance, insect resistance, improved photosynthetic capacity, salt tolerance, stay-green, improved vigour, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased pod length per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant and increased number of non-wilted leaves per plant, detectable modulation at the level of metabolites, detectable modulation at the level of transcripts, and detectable modulation at the level of the proteome.

[0065] In some embodiments, the agricultural formulations taught herein comprise at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

[0066] The methods described herein can include contacting a seed or plant with at least 100 CFU or spores, at least 300 CFU or spores, at least 1,000 CFU or spores, at least 3,000 CFU or spores, at least 10,000 CFU or spores, at least 30,000 CFU or spores, at least 100,000 CFU or spores, at least 300,000 CFU or spores, at least 1,000,000 CFU or spores, or more of a microorganism taught herein.

[0067] The methods described herein may include contacting a seed or plant with a composition comprising a metabolite produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 1 mg of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 10 mg of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 100 mg of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 1 g of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 10 g of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 100 g of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method comprises contacting a seed or plant with a composition comprising at least 1 kg of a metabolite produced by a single microorganism or a consortium of microorganisms disclosed herein. In some aspects, the method comprises contacting a seed or plant with a composition comprising greater than 1 kg of a metabolite produced by a single microorganism or a consortium of microorganisms disclosed herein.

[0068] In some embodiments of the methods described herein, the isolated microorganisms of the present disclosure are present in the formulation in an amount effectively detectable in and / or on a target tissue of an agricultural plant. For example, at least 100 CFU or spores, at least 300 CFU or spores, at least 1,000 CFU or spores, at least 3,000 CFU or spores, at least 10,000 CFU or spores, at least 30,000 CFU or spores, at least 100,000 CFU or spores, at least 300,000 CFU or spores, at least 1,000,000 CFU or spores, or more of the microorganisms are detected in and / or on a target tissue of a plant. Alternatively or additionally, the microorganisms of the present disclosure may be present in a formulation in an amount effective to increase the biomass and / or yield of a plant to which the formulation is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more, when compared to a reference agricultural plant to which the formulation of the present disclosure has not been applied. Alternatively or additionally, the microorganisms of the present disclosure may be present in a formulation in an amount effective to detectably modulate an agronomic trait of interest of a plant to which the formulation is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more when compared to a reference agricultural plant to which the formulation of the present disclosure has not been applied.

[0069] In some embodiments of the methods described herein, one or more metabolites isolated from the microorganisms or consortia of the present disclosure are present in the formulation in an amount that can be effectively detected within and / or on the target tissue of the agricultural plant. For example, metabolites of at least 1 mg, at least 10 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 400 mg, at least 600 mg, at least 800 mg, at least 1 g or more are detected in and / or on the target tissue of the plant. Alternatively or additionally, metabolites isolated from the microorganisms and consortia of the present disclosure can be present in the formulation in an amount that effectively increases the biomass and / or yield of the plant to which the formulation is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more when compared to a reference agricultural plant to which the formulation of the present disclosure is not applied. Alternatively or additionally, metabolites isolated from the microorganisms and consortia of the present disclosure may be present in a formulation in an amount effective to detectably modulate an agronomic trait of interest in a plant to which the formulation of the present disclosure is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more when compared to a reference agricultural plant to which the formulation of the present disclosure has not been applied.

[0070] In some embodiments, the agricultural composition taught herein is shelf stable. In some aspects, the microorganism taught herein is freeze dried. In some aspects, the microorganism taught herein is spray dried. In some aspects, the microorganism taught herein is placed in a liquid formulation. In some aspects, the microorganism taught herein is present on a particle.

[0071] Also described herein are a plurality of isolated microorganisms enclosed within an article selected from the group consisting of a bottle, a jar, an ampoule, a package, a vessel, a bag, a box, a storage box, an envelope, a carton, a container, a silo, a shipping container, a carriage, and a case.

[0072] In some aspects, the selected plant species is combined with the disclosed microorganisms (operational taxonomic units (OTUs), strains, or compositions comprising any of the foregoing) to result in increased crop yields and the generation of their products. Therefore, in one aspect, the present disclosure provides a synthetic combination of a seed of a first plant and a microbial preparation coated on the surface of the seed of the first plant, such that the microorganism is present on the surface of the seed at a higher level than that present on the surface of an uncoated reference seed. In another aspect, the present disclosure provides a synthetic combination of a portion of a first plant and a microbial preparation coated on the surface of the portion of the first plant, such that the microorganism is present on the surface of the portion of the first plant at a higher level than that present on the surface of an uncoated reference plant portion. The above methods can be used alone or simultaneously with plant breeding and transgenic techniques.

[0073] In some embodiments, the Paenibacillus strain is described in Table 1a, Table 1b, or Table 1c. In some embodiments, the Paenibacillus strain comprises a polynucleotide sequence at least 90% identical to any one or more of SEQ ID NOs. 1-123. In some embodiments, the Paenibacillus strain is a species selected from the group consisting of Paenibacillus aceris, Paenibacillus aestuarii, Paenibacillus agarexedens, Paenibacillus agaridevorans, Paenibacillus albidus, Paenibacillus alginolyticus, Paenibacillus alkaliterrae, Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus anaericanus, Paenibacillus antarcticus, Paenibacillus apiary, Paenibacillus spp. apiaries), Paenibacillus assamensis, Paenibacillus azotifigens, Paenibacillus baetica, Paenibacillus barcinonensis, Paenibacillus barengoltzii, Paenibacillus borealis, Paenibacillus caespitis, Paenibacillus camelliae, Paenibacillus castaneae, Paenibacillus catalpa, Paenibacillus cavernae, Paenibacillus cellulolyticuscellulosilyticu), Paenibacilluschartariuss, Paenibacillus chibensis, Paenibacillus chinjuensis, Paenibacillus chitinolyticus, Paenibacillus chondroitinus, Paenibacillus cineris, Paenibacillus cisolokensis, Paenibacillus contaminans, Paenibacillus cookii, Paenibacillus crassostreae, Paenibacillus cucumis, Paenibacillus galactolyticus curdlanolyticus), Paenibacillus daejeonensis, Paenibacillus darwinianus, Paenibacillus dongdonensis, Paenibacillus donghaensis, Paenibacillus doosanensis, Paenibacillus drentensis, Paenibacillus durus, Paenibacillus usedaphicus, Paenibacillus ehimensis, Paenibacillus elgii, Paenibacillus endophyticus, Paenibacillus etheri, Paenibacillus euphorbia favisporus), Paenibacillus filicis, Paenibacillus frigoriresistens, Paenibacillus gansuensisgansuensis), Paenibacillusginsengarvi, Paenibacillus glacialis, Paenibacillus glebae, Paenibacillus glucanolyticus, Paenibacillus glycanilyticus, Paenibacillusgraminis, Paenibacillusgranivorans, Paenibacillusharenae, Paenibacillushelianthin, Paenibacillushordei, Paenibacillushumicus, Paenibacillusshunanensis, Paenibacillusillinois, Paenibacillus illinoisensis、Paenibacillus jilunlii、Paenibacillus kobensis、Paenibacillus koleovorans、Paenibacillus konkukensis、Paenibacillus konsidensis、Paenibacillus kukuduoahitejonii、Paenibacillus lactis、Paenibacillus lautus、Paenibacillus liaoningensis、Paenibacillus lupini、Paenibacillus macquariensis、Paenibacillusmarchantiophytorum、Paenibacillus marquariensis, Paenibacillus mendelii, Paenibacillus mobilis, Paenibacillus motobuensis, Paenibacillus colloidusmucilaginosus), Paenibacillus naganoensis, Paenibacillus naganoensis, Paenibacillus nanensis, Paenibacillus nasutitemitis, Paenibacillus nebraskensis, Paenibacillus nicotianae, Paenibacillus nitroguajacolicus, Paenibacillus oceanisediminis, Paenibacillus odorifer, Paenibacillus oryzae, Paenibacillus oryzisoli, Paenibacillus ourofinensis, Paenibacillus fodder pabuli), Paenibacillus panacisoli, Paenibacillus panaciterrae, Paenibacillus pectinilyticus, Paenibacillus peoriae, Paenibacillus periandrae, Paenibacillus phoenicis, Paenibacillus phoenicis, Paenibacillus physcomitrellae, Paenibacillus pini, Paenibacillus pinisoli, Paenibacillus plakortidis, Paenibacillus pocheonensis, Paenibacillus polymyxa polymyxa), Paenibacillus polysaccharolyticus, Paenibacillus populi, Paenibacillus profundus, Paenibacillus Provenceprovencensis), Paenibacillus purispatii, Paenibacillus qinlingensis, Paenibacillus rhizoplanae, Paenibacillus rhizoryzae, Paenibacillus rhizosphaerae, Paenibacillus rigui, Paenibacillus sacheonensis, Paenibacillus segetis, Paenibacillus sepulcri, Paenibacillus shirakamiensis, Paenibacillus silagei, Paenibacillus silvae, Paenibacillus sinopodophylli, Paenibacillus solanacearum, Paenibacillus soli, Paenibacillus sonchi, Paenibacillus sputi, Paenibacillus stellifer, Paenibacillus susongensis, Paenibacillus taichungensis, Paenibacillus taihuensis, Paenibacillus taiwanensis, Paenibacillus taohuashanense, Paenibacillus starimensis, Paenibacillus telluris, Paenibacillus terrae, Paenibacillus terrigena, Paenibacillus hailandensis, Paenibacillus thalictra, Paenibacillus thermophilus, Paenibacillus Tianmushan tianmuensis), Paenibacillus tianmuensistimonensis), Paenibacillus tritici, Paenibacillus tundrae, Paenibacillus turicensis, Paenibacillus tylopili, Paenibacillus typhae, Paenibacillus suliginis, Paenibacillus validus, Paenibacillus vini, Paenibacillus vulneris, Paenibacillus wooponensis, Paenibacillus wynnii, Paenibacillus xanthanilyticus, Paenibacillus xinjiangensis, Paenibacillus xylan-eating xylanexedens), Paenibacillus xylanilyticus, Paenibacillus yonginensis, and Paenibacillus yunnanensis. In some embodiments, the Paenibacillus strain belongs to subgroup I. In some embodiments, the Paenibacillus strain belongs to subgroup II. In some embodiments, the Paenibacillus strain is selected from the group consisting of: NRRL deposit numbers: B-68102, B-68103, B-68104, B-68105, B-68106, B-68107, B-68108, B-68109, and B-68110.

[0074] In some embodiments, the isolated bacterial strain has a morphology and physiological characteristics substantially similar to the isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain has a genetic characteristic substantially similar to the isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain is a naturally occurring or artificial mutant of the isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain is a bacterial strain that has been genetically edited, changed or modified. In some embodiments, the isolated bacterial strain of the present disclosure is located in a substantially pure culture. In some embodiments, the isolated bacterial strain of the present disclosure is located in a pure culture. In some embodiments, the isolated bacterial strain of the present disclosure is located in a cell fraction, an extract or a supernatant.

[0075] In some embodiments, progeny and / or mutants of the isolated bacterial strains of the present disclosure are contemplated. In some embodiments, progeny, mutants and / or genetically modified forms of the isolated bacterial strains of the present disclosure are contemplated.

[0076] In some embodiments, cell-free or inactivated preparations of isolated bacterial strains of the present disclosure, or mutants of the isolated bacterial strains are contemplated. In some embodiments, cell-free or inactivated preparations of isolated bacterial strains of the present disclosure, or mutants of the isolated bacterial strains, or genetically edited, altered, or modified variants are contemplated. In some embodiments, metabolites produced by isolated bacterial strains of the present disclosure, or mutants of the isolated bacterial strains are contemplated. In some embodiments, metabolites produced by isolated bacterial strains of the present disclosure, or mutants of the isolated bacterial strains, or genetically modified variants are contemplated.

[0077] In some embodiments, the agricultural composition comprises an isolated bacterial strain and an agriculturally acceptable carrier. The isolated bacterial strain may be present in the composition at 1×10^2 to 1×10^12 CFU / gram. The agricultural composition may be formulated as a seed coating.

[0078] In some embodiments, the method of conferring at least one beneficial trait to a plant species comprises applying an isolated bacterial strain to a plant or a growth medium in which the plant is located. In some embodiments, the method of conferring at least one beneficial trait to a plant species comprises applying an agricultural composition of the present disclosure to the plant or a growth medium in which the plant is located.

[0079] In some embodiments, the plant is a non-leguminous crop plant. In some embodiments, the plant is a monocot. In some embodiments, the plant is a C3 monocot. In some embodiments, the plant is a C4 monocot. In some embodiments, the plant is selected from the group consisting of corn, wheat, rice, sorghum, sugarcane, onion, bamboo, palm, garlic, ginger, lily, daffodil, iris, orchid, bluebell, tulip, amaryllis, banana, plantain, ginger, turmeric, cardamom, asparagus, pineapple, sedge, rush, leek, forage grass, buckwheat, quinoa, chia and millet.

[0080] In some embodiments, the present disclosure teaches a method of growing a plant having at least one beneficial trait. In some embodiments, the method comprises applying an isolated bacterial strain or microbial consortium to a seed of a plant; sowing or planting the seed; and growing the plant. In certain embodiments, the isolated bacterial strain or microbial consortium is applied in the form of an agricultural composition that also comprises an agriculturally acceptable carrier.

[0081] In some embodiments, the microbial consortium has a morphology and physiological properties substantially similar to the microbial consortium of the present disclosure. In some embodiments, the microbial consortium has genetic properties substantially similar to the microbial consortium of the present disclosure. In some embodiments, the microbial consortium is located in a substantially pure culture. In some embodiments, subsequent generations of any microorganism of the microbial consortium are envisioned. In some embodiments, mutants of any microorganism of the microbial consortium are envisioned. In some embodiments, variants of any microorganism of the microbial consortium are envisioned that are genetically edited, altered or modified. In some embodiments, a cell-free or inactivated preparation of a microbial consortium or a mutant of any microorganism in the microbial consortium or a variant that is genetically edited, altered or modified is envisioned. In some embodiments, metabolites produced by a mutant of any microorganism in the microbial consortium or a variant that is genetically edited, altered or modified is envisioned.

[0082] In some embodiments, the agricultural composition comprises a microbial consortium and an agriculturally acceptable carrier. The microbial consortium of the agricultural composition may be present in the composition at 1×10^3 to 1×10^12 bacterial cells / gram. In some embodiments, the agricultural composition is formulated as a seed coating. In some embodiments, a method of imparting at least one beneficial trait to a plant species comprises applying a microbial consortium to the plant or a growth medium in which the plant is located. In some embodiments, a method of imparting at least one beneficial trait to a plant species comprises applying an agricultural composition to a plant or a growth medium in which the plant is located.

[0083] In any of the methods, the microorganism can comprise a 16S rRNA nucleic acid sequence having at least 97% sequence identity to a 16S rRNA nucleic acid sequence of a bacterium selected from the organisms provided in Table 1a, Table 1b, and / or Table 1c.

[0084] Description of Sequence Listing

[0085] The present disclosure may be more fully understood through the following detailed description and sequence listing, which constitute a part of this application. Also contemplated are the sequences, strains, and edits described in PCT / US2022 / 021213, filed March 21, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0086] The sequence description and accompanying sequence listing comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 CFR §§ 1.821 and 1.825.

[0087] Descriptions of the parental lines, edited lines and sequences disclosed herein are given in Table 1a, Table 1b and Table 1c.

[0088] Table 1a: Wild-type strains of Paenibacillus, taxa and sources

[0089] Species names are determined from 16S rRNA. *Note: strain identifiers may further include an optional prefix (e.g., "CM" or "PM"), as indicated in the table. For example, strain 8619 may optionally be synonymously referred to as CM8619.

[0090]

[0091]

[0092] Table 1b: Edited strains of Paenibacillus

[0093] *Note: The strain identifier may further include an optional prefix, as shown in the table. For example, the parental strain (PM)53593 with editing type D would be "(PE)53953-G3", and the parental strain and edited strain have the prefixes "PM" (see Table 1a) and "PE" (or "CM" and "CE"), respectively, which are optional additional names.

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] Table 1c: Sequence

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] The microorganisms described in this application are deposited with the Agricultural Research Type Culture Collection (NRRL), an international depository located at 1815 North University Street, Peoria, IL 61604, USA.

[0108] These deposits were made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made in accordance with and meet the standards set forth in 37 CFR §§1.801-1.809 and Manual of Patent Examining Procedure §§2402-2411.05.

[0109] Bacillus strain ID 17899-G13A was deposited with the NRRL as B-68110 on March 11, 2022.

[0110] Bacillus polymyxa strain ID 55083-G14 was deposited with the NRRL on March 11, 2022 as B-68106.

[0111] Bacillus polymyxa strain ID 68890-G12 was deposited with the NRRL as B-68103 on March 11, 2022.

[0112] Bacillus polymyxa strain ID 77155-G3 was deposited with the NRRL on March 11, 2022 as B-68105.

[0113] Bacillus polymyxa strain ID 77155-G46 was deposited with the NRRL on March 11, 2022 as B-68102.

[0114] Bacillus polymyxa strain ID 8619-G25 was deposited with the NRRL on March 11, 2022 as B-68109.

[0115] Bacillus polymyxa strain ID 8619-G50 was deposited with the NRRL on March 11, 2022 as B-68108.

[0116] Bacillus polymyxa strain ID 8619-G53 was deposited with the NRRL on March 11, 2022 as B-68107.

[0117] Bacillus polymyxa strain ID 8619-G88 was deposited with the NRRL on March 11, 2022 as B-68104. DETAILED DESCRIPTION

[0118] Although the following terms are considered to be well understood by those of ordinary skill in the art, they are set forth to facilitate explanation of the presently disclosed subject matter.

[0119] The term "a" or "an" refers to one or more of the entity, i.e., multiple referents. Thus, the terms "a" or "an", "one (kind) or more (kinds)" and "at least one (kind)" are used interchangeably herein. In addition, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element.

[0120] As used herein, the term "microorganism" or "microorganism" should be interpreted in a broad sense. These terms are used interchangeably and include, but are not limited to, two prokaryotic domains (i.e., bacteria and archaea) as well as eukaryotic fungi and protists. In some embodiments, the present disclosure relates to the "microorganisms" of Table 1a, Table 1b, and Table 1c, or the "microorganisms" of various other tables or paragraphs present in the present disclosure. This characterization can refer not only to the classified bacterial genus identified in the table, but also to the classified species identified, as well as various novel and newly identified bacterial strains in the table.

[0121] As used herein, the term "microorganism" or "microorganism" refers to any species or taxon of a microorganism, including but not limited to archaea, bacteria, microalgae, fungi (including mold and yeast species), mycoplasmas, microspores, nanobacteria, oomycetes, and protozoa. In some embodiments, a microorganism or microorganism encompasses a single cell (e.g., a unicellular microorganism) or more than one cell (e.g., a multicellular microorganism). Thus, a "microorganism population" may refer to multiple cells of a single microorganism, wherein the cells share a common genetic derivation.

[0122] As used herein, the term "bacteria" generally refers to any prokaryotic organism, and may relate to organisms from the Eubacteria (bacteria), Archaea (archaea) or both. In some cases, bacterial genus or other taxonomic classifications may be in classification flux, have been reassigned for other reasons (evolutionary domains such as but not limited to whole genome sequencing), and / or may be variable based on methodology, and it should be understood that such naming variability is within the scope of any claimed taxonomy. For example, some species of Erwinia (Erwinia) have been described in the literature as belonging to Pantoea (Pantoea) (Zhang, Y. and Qiu, S., Examining phylogenetic relationships of Erwinia and Pantoeaspecies using whole genome sequence data.Antonie van Leeuwenhoek 108, 1037–1046 (2015)).

[0123] The term "16S" refers to the DNA sequence of the 16S ribosomal RNA (rRNA) sequence of bacteria. 16S rRNA gene sequencing is an established method for studying the phylogeny and taxonomy of bacteria.

[00166] As used herein, the term "fungus" generally refers to any organism from the kingdom Fungi. Historically, fungi have been taxonomically classified based on morphological manifestations. Since the mid-nineteenth century, it has been recognized that some fungi have a polymorphic life cycle, and different nomenclature names are used for different forms of the same fungi. In 1981, the Sydney Congress of the International Mycological Association established the rule that fungi are named asexual, sexual or holotype according to their state (Taylor, JW One Fungus = One Name: DNA and fungal nomenclature twenty years after PCR. IMA Fungus 2, 113–120 (2011)). With the development of genome sequencing, it is clear that taxonomic classification based on molecular phylogenetics does not match the nomenclature based on morphology (Shenoy, BD, Jeewon, R. and Hyde, KD (2007). Impact of DNA sequence-data on the taxonomy of anamorphic fungi. Fungal Diversity 26: 1-54). Therefore, in 2011, the International Botanical Congress passed a resolution approving the International Code of Nomenclature for Algae, Fungi and Plants (Melbourne Code) (2012), where the result was the designation of "one fungus = one name" (Hawksworth, DL Managing and coping with names of pleomorphic fungi in a period of transition. IMA Fungus 3, 15–24 (2012)).

[0124] The term "internal transcribed spacer" ("ITS") refers to the spacer DNA (non-coding DNA) between the small subunit ribosomal RNA (rRNA) and large subunit (LSU) rRNA genes in the corresponding transcribed regions in the chromosome or in the polycistronic rRNA precursor transcript. ITS gene sequencing is an established method for studying the phylogeny and taxonomy of fungi. In some cases, the "large subunit" ("LSU") sequence is used to identify fungi. LSU gene sequencing is an established method for studying the phylogeny and taxonomy of fungi. Some fungal microorganisms of the present invention can be described by ITS sequences, and some fungal microorganisms can be described by LSU sequences. It should be understood that both are equally descriptive and accurate for determining taxonomy.

[0125] The term "microbial consortia" or "microbial consortium" refers to a subset of a microbial community of individual microbial species, or strains of species, that can be described as performing a common function, or can be described as participating in or causing or being associated with an identifiable parameter or plant phenotypic trait. The community can include one or more species, or strains of species, of microorganisms. In some cases, these microorganisms coexist in a symbiotic manner within the community.

[0126] The term "microbial community" means a group of microorganisms comprising two or more species or strains. Unlike a microbial consortium, a microbial community does not necessarily perform a common function or participate in or cause or correlate with an identifiable parameter or plant phenotypic trait.

[0127] The term "accelerated microbial selection" or "AMS" is used interchangeably with the term "directed microbial selection" or "DMS" and refers to an iterative selection method that is used in some embodiments of the present disclosure to obtain the claimed microbial species or consortia of such species.

[0128] As used herein, "isolate," "isolated," "isolated microorganism," and similar terms are intended to mean that one or more microorganisms have been separated from at least one material with which they were associated in a particular environment (e.g., soil, water, plant tissue).

[0129] Thus, an "isolated microorganism" does not exist in its naturally occurring environment; rather, the microorganism is removed from its natural environment and placed in a non-naturally occurring state by the various techniques described herein. Thus, an isolated strain can exist, for example, in the form of a biologically pure culture or spores (or other forms of strains) associated with an agricultural carrier.

[0130] In certain aspects of the present disclosure, an isolated microorganism is present in the form of an isolated and biologically pure culture. It will be understood by those skilled in the art that an isolated and biologically pure culture of a particular microorganism means that the culture is substantially free of (within scientific reason) other living organisms and contains only the individual microorganism in question. The culture may contain varying concentrations of the microorganism. The present disclosure indicates that an isolated and biologically pure microorganism is generally "certainly distinct from less pure or impure material." See, for example, In re Bergstrom, 427F.2d 1394, (CCPA 1970) (discussing purified prostaglandins), also see In re Bergy, 596F.2d 952 (CCPA 1979) (discussing purified microorganisms), also see Parke-Davis & Co. v HK Mulford & Co., 189F.95 (SDNY 1911) (Learned Hand, discussing purified epinephrine), partially maintained, partially revoked, 196F.496 (2d Cir. 1912), each of which is incorporated herein by reference. In addition, in some aspects, the present disclosure provides certain quantitative measurements of concentrations or purity limits that will be found in isolated and biologically pure microbial cultures. In certain embodiments, the presence of these purity values ​​is another attribute that distinguishes microorganisms disclosed in the present invention from those microorganisms that exist in a natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing the purity limits of vitamin B12 produced by microorganisms), which is incorporated herein by reference.

[0131] As used herein, "individual isolate" should be considered to mean a composition or culture that primarily contains a single genus, species, or strain of a microorganism, after separation from one or more other microorganisms. The phrase should not be considered to indicate the degree of separation or purification of a microorganism. However, an "individual isolate" may contain essentially only one genus, species, or strain of a microorganism.

[0132] With respect to a microorganism, the term "modified" or "edited" (and its corresponding terms "modification", "editing", etc.) means that the microorganism has been changed by non-natural means compared to the native state when found. In this case, "modified" or "edited" is synonymous with "engineered" and indicates that artificial participation has been involved in creating the modification. In some cases, the modification includes changes in polynucleotides within the microorganism (e.g., in its genome). The modification can include the deletion, insertion, substitution and / or chemical alteration of at least one nucleotide, including any multiple of the foregoing items and / or a combination of the foregoing items, and can cause a change in the phenotype of the edited organism (e.g., upregulation of a specific pathway, downregulation of a specific pathway, knockout of a gene or protein function) and / or a change in the phenotype of another heterologous organism associated or becoming associated with the microorganism.

[0133] As used herein, the term "growth medium" is any medium suitable for supporting plant growth. As examples, the medium can be natural or artificial, including but not limited to: soil, potting mix, bark, vermiculite, hydroponic solutions alone and applied to a solid plant support system, and tissue culture gel. It should be understood that the medium can be used alone or in combination with one or more other mediums. It can also be used with or without the addition of exogenous nutrients and physical support systems for roots and leaves.

[0134] In one embodiment, the growth medium is a naturally occurring medium, such as soil, sand, mud, clay, humus, topsoil, rock or water. In another embodiment, the growth medium is artificial. Such artificial growth medium can be constructed to simulate the conditions of a naturally occurring medium; however, this is not required. The artificial growth medium can be made of one or more of any number and combination of materials, including sand, minerals, glass, rock, water, metals, salts, nutrients, water. In one embodiment, the growth medium is sterile. In another embodiment, the growth medium is not sterile.

[0135] The culture medium may be modified or enriched with additional compounds or components, such as components that may facilitate the interaction and / or selection of a particular population of microorganisms with the plant and with each other. For example, antibiotics (such as penicillin) or sterilizing agents (e.g., quaternary ammonium salts and oxidizing agents) may be present, and / or physical conditions (such as salinity, plant nutrients (e.g., organic and inorganic minerals (such as phosphorus, nitrogen salts, ammonia, potassium and micronutrients, such as cobalt and magnesium), pH and / or temperature) may be modified.

[0136] The term "plant" generally includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds and their offspring. Plant cells include, but are not limited to, cells from seeds, suspension cultures, plumules, meristem zones, calli, leaves, roots, buds, gametophytes, sporophytes, pollen and microspores. As used herein, the term "plant components" refers to plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, complete plant callus, plant mass and plant cells in plant or plant parts such as plumules, pollen, ovules, seeds, leaves, flowers, branches, fruits, grains, spikes, cobs, outer skins, stalks, roots, root tips, anthers, etc. and their own parts. Offspring, variants and mutants of regenerated plants are also included within the scope of the present invention, provided that these parts comprise the polynucleotides introduced.

[0137] "Plant component" is intended to refer to whole plants or plant components, which may include differentiated and / or undifferentiated tissues, such as, but not limited to, plant tissues, parts, and cell types. In one embodiment, plant component is one of the following: whole plants, seedlings, meristems, basic tissues, vascular tissues, cortical tissues, seeds, leaves, roots, buds, stems, flowers, fruits, runners, bulbs, tubers, corms, high buds, buds, bracts, tumor tissues, and various forms of cells and cultures (e.g., unicellular, protoplasts, embryos, callus). The term "plant organ" refers to plant tissues or tissue groups that constitute plant parts that are different in morphology and function. As used herein, "plant part" is synonymous with "part" of a plant, and refers to any part of a plant, and may include different tissues and / or organs, and may be used interchangeably with the term "tissue" throughout.

[0138] Similarly, "plant propagation components" are intended to refer generally to any part of a plant that is capable of starting other plants via sexual or asexual propagation of the plant, such as, but not limited to, seeds, seedlings, roots, buds, cuttings, scions, grafts, runners, bulbs, tubers, corms, high buds, or bracts. Plant components may be located in the plant or in plant organs, tissue cultures, or cell cultures. Plant "vegetative components" are intended to refer to any non-reproductive part of a plant associated with growth and / or development, such as, but not limited to, roots, stems, leaves.

[0139] "Progeny" includes any subsequent generation of an organism produced by sexual or asexual reproduction.

[0140] "Grain" is intended to mean mature seeds produced by commercial growers for purposes other than growing or reproducing the species.

[0141] The term "monocotyledonous" or "monocotyledonous plant" refers to the subclass of angiosperms, also known as the "monocots," whose seeds typically contain only one embryonic leaf or cotyledon. The term includes reference to whole plants, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0142] The term "dicotyledonous" or "dicotyledonous plant" refers to the subclass of angiosperms, also known as the "dicots," whose seeds typically contain two embryonic leaves or cotyledons. The term includes reference to whole plants, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0143] As used herein, the term "cultivar" refers to a plant variety, line or race that has been produced by horticultural or agronomic techniques and that is not normally found in wild-type populations.

[0144] As used herein, "improved" should be interpreted broadly to encompass improvements in the properties of a plant compared to a control plant or compared to a known average quantity associated with the property in question. For example, "improved" plant biomass associated with the application of the beneficial microorganisms or consortia of the present disclosure can be demonstrated by comparing the biomass of a plant treated with the microorganisms taught herein with the biomass of an untreated control plant. Alternatively, the biomass of a plant treated with the microorganisms taught herein can be compared with the average biomass typically achieved by a given plant (as indicated in scientific or agricultural publications known to those skilled in the art). In the present disclosure, "improved" does not necessarily require that the data be statistically significant (e.g., p<0.05); rather, any quantifiable difference indicating that one value (e.g., average treatment value) is different from another value (e.g., average control value) can be raised to the extent of "improved".

[0145] As used herein, "inhibit and repress" and similar terms should not be construed as requiring complete inhibition or repression, although this may be desired in some embodiments.

[0146] As used herein, the term "genotype" refers to the genetic constitution of an individual cell, cell culture, tissue, organism (eg, plant), or population of organisms.

[0147] The compositions and methods herein can provide plants with improved "agronomic traits" or "agronomically important traits" or "agronomically interesting traits" which may include, but are not limited to, disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen use efficiency, improved nitrogen fixation, insect resistance, herbivore resistance, pathogen resistance, increased yield, enhanced health, improved vigor, improved growth, increased photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, metabolite modulation, proteome modulation, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition, as compared to an isoline plant that does not contain the modifications derived from the methods or compositions herein.

[0148] "Agronomic potential" is intended to mean the ability of a plant component to express a phenotype (preferably an improved agronomic trait) at some point in its life cycle or to transmit the phenotype to another plant component with which it is associated in the same plant.

[0149] As used herein, the term "molecular marker", "marker" or "genetic marker" refers to an indicator used in a method for observing differences in nucleic acid sequence characteristics. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence characterization amplified regions (SCARs), enzyme-cut amplified polymorphic sequences (CAPS) markers or isozyme markers or a combination of markers described herein (which define specific genes and chromosome positions). The positioning of molecular markers near alleles is a procedure that can be performed by an ordinary person with experience in molecular biotechnology.

[0150] As used herein, the term "trait" refers to a characteristic or phenotype. For example, in the context of some embodiments of the present disclosure, crop yields relate to the amount of marketable biomass (e.g., fruit, fiber, grain) produced by plants. Desired traits may also include other plant characteristics, including but not limited to: water use efficiency, nutrient use efficiency, productivity, mechanical harvestability, fruit maturity, shelf life, insect resistance / disease resistance, early plant maturity, stress tolerance, etc. Traits can be dominant or recessive, or inherited in a partially or incompletely dominant manner. Traits may be monogenic (i.e., determined by a single locus) or polygenic (i.e., determined by more than one locus) or may also be produced by the interaction of one or more genes with the environment.

[0151] As used herein, the term "phenotype" refers to an observable characteristic of an individual cell, cell culture, organism (eg, plant), or population of organisms that results from the interaction between the genetic makeup (ie, genotype) of the individual and the environment.

[0152] As used herein, a "synthetic nucleotide sequence" or "synthetic polynucleotide sequence" is a nucleotide sequence that is not known to occur in nature or that does not occur naturally. Generally, such a synthetic nucleotide sequence will contain at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence.

[0153] As used herein, the term "nucleic acid" refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length or their analogs. The term refers to the primary structure of the molecule, and therefore includes double-stranded and single-stranded DNA, and double-stranded and single-stranded RNA. It also includes modified nucleic acids, such as methylated and / or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, etc. The terms "nucleic acid" and "nucleotide sequence" are used interchangeably.

[0154] As used herein, the term "gene" refers to any DNA fragment associated with a biological function. Therefore, a gene includes, but is not limited to, a coding sequence and / or a regulatory sequence required for its expression. A gene may also include unexpressed DNA fragments that, for example, form recognition sequences for other proteins. A gene may be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0155] As used herein, the term "homolog" or "homologue" or "ortholog" is known in the art and refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms "homology", "homolog", "substantially similar" and "substantially corresponding" are used interchangeably herein. They refer to nucleic acid fragments in which changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the present disclosure, such as the deletion or insertion of one or more nucleotides that do not substantially change the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. Therefore, it should be understood that the present disclosure encompasses more than specific exemplary sequences, as will be understood by those skilled in the art. These terms describe the relationship between a gene found in a species, subspecies, variant, cultivar or strain and a corresponding or equivalent gene in another species, subspecies, variant, cultivar or strain. For the purposes of the present disclosure, homologous sequences are compared. "Homologous sequences" or "homologues" or "orthologs" are considered, believed or known to be functionally related. Functional relationships can be indicated in any of a variety of ways, including but not limited to: (a) degree of sequence identity and / or (b) identical or similar biological functions. Preferably, both (a) and (b) are indicated. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (ed. by FM Ausubel et al., 1987) Supplement 30, Section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, UK), ALIGN Plus (Scientific and Educational Software, Pennsylvania), and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), which uses default parameters.

[0156] As used herein, the term "nucleotide changes" refers to, for example, nucleotide substitutions, deletions, insertions, chemical alterations, or any of the foregoing, as is well understood in the art.

[0157] As used herein, the term "protein modification" refers to, for example, amino acid substitutions, amino acid modifications, deletions and / or insertions, as are well understood in the art.

[0158] As used herein, the term "at least a portion" or "fragment" of a nucleic acid or polypeptide means a portion having the minimum size characteristic of such a sequence, or any larger fragment of a full-length molecule (up to and including the full-length molecule). Fragments of the polynucleotides of the present disclosure may encode biologically active portions of gene regulatory elements. The biologically active portion of a gene regulatory element may be prepared by isolating a portion of one of the polynucleotides of the present disclosure that contains the gene regulatory element and evaluating the activity as described herein. Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, etc., up to a full-length polypeptide. The length of the portion to be used will depend on the specific application. The portion of a nucleic acid that can be used as a hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides. The portion of a polypeptide that can be used as an epitope may be as short as 4 amino acids. The portion of a polypeptide that functions as a full-length polypeptide will generally be longer than 4 amino acids.

[0159] As used herein, the term "primer" refers to an oligonucleotide that can anneal to an amplification target and allow a DNA polymerase to connect, thereby acting as a starting point for DNA synthesis under conditions that induce the synthesis of primer extension products (i.e., in the presence of nucleotides and reagents for polymerization such as DNA polymerase and at a suitable temperature and pH). (Amplification) primers are preferably single-stranded to obtain maximum amplification efficiency. Preferably, primers are oligodeoxyribonucleotides. Primers must be long enough to initiate the synthesis of extension products in the presence of reagents for polymerization. The exact length of a primer will depend on many factors, including the composition of temperature and primers (A / T vs. G / C content). A pair of bidirectional primers is composed of a forward primer and a reverse primer as commonly used in the field of DNA amplification (such as PCR amplification).

[0160] The term "stringency" or "stringent hybridization conditions" refers to hybridization conditions that affect the stability of the hybrid, such as temperature, salt concentration, pH, formamide concentration, etc. These conditions are optimized empirically to maximize the specific binding of the primer or probe to its target nucleic acid sequence and minimize non-specific binding. The term used includes reference to such conditions under which the probe or primer will hybridize to its target sequence to a detectably greater extent than other sequences (e.g., at least 2 times greater than the background). Stringent conditions are sequence-dependent and will vary in different situations. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. Tm is the temperature (under a defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a fully matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+ ions, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes or primers (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. Exemplary low stringency conditions or "conditions of reduced stringency" include hybridization at 37°C with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS and washing in 2×SSC at 40°C. Exemplary high stringency conditions include hybridization at 37°C in 50% formamide, 1 M NaCl, 1% SDS, and washing in 0.1×SSC at 60°C. Hybridization procedures are well known in the art and are described, for example, by Ausubel et al., 1998 and Sambrook et al., 2001. In some embodiments, stringent conditions are hybridization at 45°C in 0.25 M Na2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5% to 20% sodium dodecyl sulfate (such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%), followed by washing in 5×SSC containing 0.1% (w / v) sodium dodecyl sulfate at 55°C to 65°C.

[0161] In some embodiments, the cell or organism has at least one heterologous trait. As used herein, the term "heterologous trait" refers to a phenotype conferred to a cell or organism by an exogenous molecule or other organism (e.g., a microorganism), a DNA fragment, a heterologous polynucleotide, or a heterologous nucleic acid.

[0162] Various changes in phenotype are of interest in the present disclosure, including, but not limited to, modifying fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, increasing the yield of a plant with economically important traits (e.g., grain yield, forage yield, etc.), etc. These results can be achieved by providing expression of a heterologous product or increased expression of an endogenous product in a plant using the methods and compositions of the present disclosure.

[0163] A "synthetic combination" may include a combination of plants and microorganisms of the present disclosure. The combination may be achieved, for example, by coating the surface of a seed of a plant (such as an agricultural plant) or a host plant tissue (root, stem, leaf, etc.) with a microorganism of the present disclosure. In addition, a "synthetic combination" may include a combination of microorganisms of various strains or species. A synthetic combination has at least one variable that can distinguish the combination from any combination existing in nature. The variable may be, in particular, the concentration of microorganisms on seeds or plant tissues that would not naturally occur, or a combination of microorganisms and plants that would not naturally occur, or a combination of microorganisms or strains that would not naturally co-exist. In each of these cases, the synthetic combination shows traces of artificiality and has structural and / or functional attributes that do not exist when the individual elements in the combination are considered separately.

[0164] In some embodiments, a microorganism may be "endogenous" to a seed or plant. As used herein, a microorganism is considered "endogenous" to a plant or seed if it originates from a plant sample from which it is derived. That is, a situation in which the microorganism is found associated with the plant in nature. In embodiments in which endogenous microorganisms are applied to plants, the endogenous microorganisms are applied in amounts that differ from the levels found on the plant in nature. Therefore, if a microorganism endogenous to a given plant is present on the plant at a level that is not present in nature, the microorganism may still form a synthetic combination with the plant.

[0165] In some embodiments, a composition (such as a microorganism) may be "heterologous" (also referred to as "exogenous") to another composition (such as a seed or a plant), and in some aspects, is referred to herein as a "heterologous composition". As used herein, a microorganism is considered to be "heterologous" to a plant or seed if the microorganism is not derived from the plant sample from which it is derived. That is, a situation in which the microorganism is not found associated with the plant in nature. For example, a microorganism that is normally associated with the leaf tissue of a maize plant is considered to be exogenous to the leaf tissue of another maize plant that does not contain the microorganism in nature. In another example, a microorganism that is normally associated with a maize plant is considered to be exogenous to a wheat plant that does not contain the microorganism in nature.

[0166] A composition is "heterogeneously placed" when mechanically or manually applied, artificially inoculated, associated or placed on or in a plant component, seedling, plant, or on or in a plant growth medium or on or in a treatment formulation such that the treatment agent is present on or in the plant component, seedling, plant, plant growth medium or formulation in a manner not found in nature prior to application of the treatment agent, e.g., in that plant variety, at that stage of plant development, in that plant tissue, in that abundance, or in that growth environment (e.g., drought). In some embodiments, this manner is contemplated as being selected from the group consisting of: presence of microorganisms; presence of microorganisms in different cell numbers, concentrations, or amounts; presence of microorganisms in different plant components, tissues, cell types, or other physical locations in or on a plant; presence of microorganisms at different time periods, such as during the developmental period of a plant or plant component, time of day, time of season, and combinations thereof. In some embodiments, "placed heterogenously" means that the microorganism is applied to a tissue or cell type of a plant component that is different from the location where the microorganism naturally occurs. In some embodiments, "heterogeneously placed" means that a microorganism is applied to a certain developmental stage of a plant component, seedling or plant, and the microorganism is not associated in nature at this developmental stage, but may be associated at other stages. For example, if a microorganism is usually found in the flowering stage of a plant and not found in other stages, the microorganism applied at the seedling stage can be considered to be heterogenously placed. In some embodiments, if a microorganism is usually found in the root tissue of a plant component and not found in leaf tissue, and the microorganism is applied to the leaf, the microorganism is heterogenously placed. In another non-limiting example, if the microorganism is present in the mesophyll layer of leaf tissue in nature but is applied to the epithelial layer, the microorganism will be considered to be heterogenously placed. In some embodiments, "heterogeneously placed" means that a natural plant component, seedling or plant does not contain a detectable level of microorganisms in the same plant component, seedling or plant. In some embodiments, "being placed heterologously " means that compared with the concentration, quantity or amount present in described plant component, seedling or plant in nature, microorganism is applied to this plant component, seedling or plant with larger concentration, quantity or amount.For example, when compared with the concentration existing before placing microorganism with at least 1.5 times, between 1.5 times and 2 times, between 2 times, between 2 times and 3 times, between 3 times, between 3 times and 5 times, between 5 times, between 5 times and 7 times, between 7 times, between 7 times and 10 times, between 10 times or even more than 10 times quantity, amount or concentration exist, described microorganism is placed heterologously.In another non-limiting example, the microorganism present in cypress (cupressaceous tree) tissue in nature will be regarded as heterologous for the tissue of maize, wheat, cotton, soybean plant.In another example, a microorganism that occurs in nature in leaf tissue of a maize, spring wheat, cotton, or soybean plant is considered heterologous to leaf tissue of another maize, spring wheat, cotton, or soybean plant that does not contain the microorganism in nature or contains a different amount of the microorganism.

[0167] A microorganism may also be "heterologously placed" on a given plant tissue. This means that the microorganism is placed on a plant tissue on which it is not found in nature. For example, if a given microorganism only occurs naturally on the roots of a given plant, the microorganism may be exogenously applied to the plant's above-ground tissue and would thereby be "heterologously placed" on the plant tissue. Thus, a microorganism is considered to be heterologously placed when applied to a plant that does not naturally occur with the microorganism or does not naturally have the microorganism present in the amount applied.

[0168] The compositions and methods herein can provide a "modulated," "agronomic trait," or "agronomically important trait" to a host plant, which can include, but are not limited to, the following: altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, and altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, increased nitrogen fixation, improved nitrogen utilization efficiency, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, compared to isotype plants grown from seeds without the seed treatment formulation , increased yield, increased yield under water-limited conditions, grain quality, grain moisture content, metal tolerance, number of ears, number of grains per ear, number of pods, nutritional enhancement, pathogen resistance, insect resistance, increased photosynthetic capacity, salt tolerance, stay-green, increased vigor, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased pod length per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, and increased number of non-wilted leaves per plant, detectable modulation of metabolite levels, detectable modulation of transcript levels, and detectable modulation of the proteome. The term "modulated" is intended to refer to changes in agronomic traits that are altered by the presence of microorganisms, exudates, broths, metabolites, etc. In various aspects, the modulation provides for the conferment of beneficial traits.

[0169] Microorganisms and microorganisms

[0170] As used herein, the term "microorganism" should be interpreted in a broad sense. It includes, but is not limited to, prokaryotic bacteria and archaea as well as eukaryotic fungi and protists.

[0171] In certain embodiments, the microorganism is an endophyte, or an epiphyte, or a microorganism that resides in the rhizosphere or rhizosheath of a plant. That is, the microorganism can be found in soil material attached to the roots of a plant or in the area immediately adjacent to the roots of a plant.

[0172] In one embodiment, the microorganism is an endophyte. Endophytes can benefit the host plant by preventing pathogenic organisms from colonizing it. Endophytes create a "barrier effect" to widespread colonization of plant tissues, where local endophytes outcompete and prevent pathogenic organisms from taking hold. Endophytes can also produce chemicals that inhibit the growth of competitors, including pathogenic organisms.

[0173] In certain embodiments, the microorganism is not culturable. This should be taken to mean that the microorganism is not known to be culturable or is difficult to culture using methods known to those skilled in the art.

[0174] The microbial organisms of the present disclosure may be collected or obtained from any source, or contained within and / or associated with material collected from any source.

[0175] In one embodiment, the microorganism or combination of microorganisms can provide a possible or predicted benefit to the plant. For example, the microorganism can be predicted to: increase nitrogen fixation; release phosphate from soil organic matter; release phosphate from inorganic forms of phosphate (e.g., rock phosphate); "fix carbon" in root microspheres; live in the rhizosphere of the plant, thereby helping the plant absorb nutrients from the surrounding soil and then provide these nutrients more easily to the plant; increase the number of nodules on the plant roots, thereby increasing the number of symbiotic nitrogen fixers (e.g., Rhizobium species) per plant and the amount of nitrogen fixed by the plant; trigger plant defense responses, such as ISR (induced systemic resistance) or SAR (systemic acquired resistance), which help plants resist invasion and spread of pathogenic microorganisms; compete with microorganisms that are harmful to plant growth or health through antagonism or competitive use of resources (such as nutrients or space); change the color of one or more parts of the plant, or change the chemical condition of the plant, its smell, taste, or one or more other characteristics.

[0176] Microorganisms of the present disclosure can be separated in basically pure or mixed cultures. They can be concentrated, diluted or provided at the natural concentrations present in the source material. For example, microorganisms from salt deposits can be separated for use in the present disclosure in the following manner: the deposits are suspended in fresh water and the deposits are dropped to the bottom. Water containing most of the microorganisms can be removed by decantation after a suitable settling time period and directly applied to plant growth medium, or concentrated by filtration or centrifugation, diluted to a suitable concentration and applied to plant growth medium with most of the salt removed. As another example, microorganisms from mineralization or toxic sources can be similarly processed to recycle microorganisms to apply to plant growth materials, thereby minimizing the possibility of damaging plants.

[0177] In some embodiments, mixed populations of microorganisms are used in the methods of the present disclosure.

[0178] Genome modification of microorganisms

[0179] In some embodiments, a microorganism can have its genome altered in a manner to provide an improved trait of interest, such as improved nitrogen fixation in a non-leguminous crop.

[0180] Various methods are known in the art for modifying polynucleotides in cells (including but not limited to any polynucleotide sequence contained in the cell, including genome, chromosome and plasmid DNA). In short, according to the needs of the practitioner, a single-strand break or a double-strand break is introduced into the target polynucleotide (the object of modification), and the result can be the insertion of at least one nucleotide, the deletion of at least one nucleotide, the substitution of at least one nucleotide, or any combination of the foregoing.

[0181] Single-strand breaks or double-strand breaks (SSBs or DSBs) can be accomplished by any of a variety of methods, including the use of chemicals or radiation, as a result of a homologous recombination process, by the introduction of specific or nonspecific nucleases, or by any combination of the foregoing methods.

[0182] Enzymes that affect polynucleotide cleavage are known in the art and may include, without limitation, restriction endonucleases, meganucleases, TALENs, zinc fingers, or Cas endonucleases.

[0183] In some aspects, the present disclosure relates to an isolated genetically modified microorganism having improved nitrogen fixation activity compared to a non-genetically modified variant of the same species or strain of microorganism.

[0184] Glutamine (Gln) is a universal nitrogen signal in all free-living nitrogen-fixing organisms (see, e.g., Wang et al., PLOS Genetics, 2018). Gram-negative bacteria, such as Klebsiella and Pseudomonas, have clear nitrogen pathways and easier and more predictable gene delivery and expression for genome-modified strains. In the Gram-negative organism Klebsiella, NifL is a negative regulator of the nif operon. When intracellular glutamine is high (nitrogen excess), NifL forms a repressor complex to inactivate nif operon expression. In the Gram-negative organism Azospirillum, NifA activates transcription of the nif operon. Glutamine regulates the expression of nifA through ntrB phosphorylation of ntrC. Nitrogenase is inactivated after transcription.

[0185] In contrast, Gram-positive bacteria, such as the Paenibacillus described herein, are more difficult to transform and have less studied nitrogen fixation pathways.

[0186] Therefore, for Gram-positive bacteria like Paenibacillus, successful cell modifications leading to greater nitrogen fixation capacity are not only surprising, but also highly desirable in agricultural biotechnology. Due to the spore-forming ability of Paenibacillus, the commercial potential of products comprising gene-edited Paenibacillus strains that improve nitrogen fixation in crop plants is increased.

[0187] In Gram-positive bacteria, the nif operon controls the nitrogen fixation pathway via GlnR. Binding of GlnR to site I activates Nif expression, whereas binding of GlnR to site II represses Nif expression. Therefore, the gene target for improving nitrogen fixation in Paenibacillus is the Nif activator / repressor GlnR and its binding site.

[0188] In the genus Paenibacillus, there are two different subgroups, subgroup I and subgroup II, each containing a different operon composition. Subgroup I Paenibacillus, such as Paenibacillus polymyxa, contains in this order: nifB, nifH, nifD, nifK, nifE, nifN, nifZ, hesA, nifV. Subgroup II Paenibacillus, such as Paenibacillus graminearum, contains in this order: nifB, nifH, nifD, nifK, nifE, nifV, nifZ, orf1, hesA, nifV.

[0189] In some embodiments, the Paenibacillus microorganism has a genetic modification to the nif gene, which encodes an enzyme responsible for the microbial nitrogen fixation activity. In other embodiments, the isolated microorganism has a genetic modification to glnR, which encodes a protein, GlnR, which is involved in sensing local ammonia concentrations and regulating the expression of the nif gene. In certain embodiments, the isolated microorganism has genetic modifications to both the nif and glnR genes.

[0190] In some embodiments, the present disclosure relates to an isolated genetically modified microorganism, which includes one or more genetic modifications selected from the following: genetic modification of an endogenous glnR gene encoding GlnR; and genetic modification of the upstream (5') regulatory region of an endogenous nif gene or a GlnR binding region, wherein: the genetic modification of the endogenous glnR gene encoding GlnR is characterized as providing a mutant glnR gene that produces a GlnR protein variant; the genetic modification of the 5' regulatory region sequence is characterized as providing improved binding affinity for GlnR compared to a non-genetically modified 5' regulatory region sequence; and the one or more genetic modifications are characterized as providing improved nitrogen fixation activity to the microorganism compared to a non-genetically modified strain of the microorganism.

[0191] In some embodiments, the genetic modification of the endogenous glnR gene is characterized as providing a mutant glnR gene that produces a GlnR protein variant. In some embodiments, the genetic modification of the glnR gene produces a mutant gene that can be transcribed and translated to produce a modified version of the GlnR protein. In other words, in some embodiments, the isolated microorganisms described herein are not modified to knock out or delete the glnR gene, or otherwise prevent the production of the GlnR protein. In certain embodiments, the GlnR variant protein retains one or more functions of the unmodified endogenous wild-type GlnR protein. For example, in some embodiments, the variant protein is still able to recognize and bind to the recognition element or 5' regulatory region sequence within the nif gene. In certain embodiments, the GlnR variant protein is still able to regulate the expression of the nif gene, particularly with respect to activating or upregulating the expression of the nif gene.

[0192] Although the present disclosure excludes isolated microorganisms that have been genetically modified to prevent the production of GlnR protein, for example by knocking out or deleting the glnR gene, the present disclosure encompasses genetically modified microorganisms in which the glnR gene is deleted and replaced with a recombinant glnR gene. In some embodiments, the recombinant glnR gene can be derived from a different microbial species or strain than the microorganism into which the recombinant gene is incorporated. In some embodiments, the recombinant glnR gene can be a non-natural gene or a synthetic gene. In some embodiments, the recombinant glnR gene can encode a variant of the GlnR protein that more effectively regulates the expression of nif, for example, by binding with higher affinity to the GlnR recognition element in the 5' regulatory region of nif, or by forming a complex with other proteins required for nif transcription with higher affinity.

[0193] In some embodiments, the genetic modification of the endogenous glnR gene includes truncation, meaning that the GlnR protein variant encoded by the gene lacks one or more amino acid residues compared to the protein encoded by the native gene or endogenous gene. One of ordinary skill in the art will recognize that genetic modification to produce a truncated mutant gene encoding GlnR can be achieved via a variety of molecular biological methods known in the art. For example, in some embodiments, a truncated mutant gene encoding a GlnR protein can be prepared by inserting a stop codon in a gene upstream of an endogenous stop codon in a native glnR gene. Alternatively, in some embodiments, a truncated mutant glnR gene can be prepared by genetically editing an endogenous glnR gene to delete a DNA sequence encoding an amino acid that will be excluded from the truncated variant of the GlnR protein.

[0194] In some embodiments, the truncation modification of the glnR gene comprises a truncation comprising a deletion of a portion of the endogenous glnR gene, the portion comprising the C-terminal domain of the GlnR protein. In certain embodiments, the deletion of a portion of the C-terminal domain of the GlnR protein is the result of a deletion of a portion of the endogenous gene encoding the GlnR that specifically encodes the C-terminal domain. In certain embodiments, the deletion of a portion of the C-terminal domain of the GlnR protein is the result of inserting a stop codon immediately upstream of the portion of the endogenous gene encoding the GlnR that specifically encodes the C-terminal domain. In certain embodiments, the modified portion of the endogenous glnR gene encoding the GlnR in the isolated microorganism comprises a portion encoding approximately the last 25 C-terminal amino acids of the GlnR protein.

[0195] Without being bound by theory, the C-terminal domain of the GlnR protein is associated with the perception of local ammonia concentrations, for example, via the formation of a complex with the protein glutamine synthetase. Under conditions of elevated local ammonia concentrations, GlnR binds to glutamine synthetase and forms a complex, ultimately repressing the transcription of the nif gene, thereby reducing the nitrogen fixation activity of the microorganism. Therefore, by truncating the GlnR protein to delete the C-terminal domain, the variant protein cannot form a complex with glutamine synthetase, which means that the ability of the variant to negatively regulate the expression of the nif gene is reduced. Therefore, microorganisms with C-terminal truncated variants of the GlnR protein are able to maintain the expression of the nif gene, even under conditions of high local concentrations of ammonia in the environment surrounding the microorganism.

[0196] The present disclosure also contemplates genetic modification of an endogenous glnR gene to produce a mutant glnR gene encoding a GlnR variant protein characterized as having improved binding affinity to a recognition element or 5' regulatory region sequence in an endogenous nif gene compared to a wild-type GlnR protein. In some embodiments, a mutant glnR gene comprising a truncation of a portion of a glnR gene encoding a C-terminal domain of a GlnR protein produces a GlnR variant protein having improved binding affinity to a recognition element or 5' regulatory region sequence in an endogenous nif gene compared to a wild-type GlnR protein. In other embodiments, the mutant glnR gene may include a mutation to a portion of a glnR gene encoding a DNA binding domain of GlnR. In certain embodiments, mutations in the portion of the glnR gene encoding the DNA binding domain of GlnR produce a mutant glnR gene encoding a GlnR protein variant having improved binding affinity for a recognition element or 5' regulatory region sequence in an endogenous nif gene, for example, by altering specific amino acid residues required for the interaction of the DNA binding domain with nucleotides in the recognition element or 5' regulatory region sequence of the nif gene.

[0197] The endogenous gene encoding nif contains at least two 5' regulatory region sequences that can be recognized and bound by GlnR proteins. The first 5' regulatory region sequence is located upstream of the transcription start site of the gene's 5' regulatory region. Without being bound by theory, the binding of GlnR to the first 5' regulatory region sequence is associated with the activation or upregulation of expression of the nif gene. Therefore, compared with non-genetically modified microorganisms of the same species or strain, genetic modification of the first 5' regulatory region sequence (characterized as providing improved binding affinity for GlnR) provides enhanced or upregulated expression of the nif gene, thereby improving the nitrogen fixation activity of the isolated microorganism. The second 5' regulatory region sequence is located downstream of the transcription start site. Without being bound by theory, the binding of GlnR to the second 5' regulatory region sequence is associated with the repression or downregulation of nif gene expression.

[0198] In some embodiments, the isolated microorganism comprises a genetic modification of a 5' regulatory region sequence within an endogenous nif gene, wherein the 5' regulatory region sequence is located upstream of the transcription start site of the nif gene.

[0199] In some embodiments, the 5' regulatory region sequence located upstream of the transcription start site of the nif gene includes the following sequence:

[0200] 5'–X1-N-X2-X3-X4-A-X5-X6-X3-X3-AN-X7-TNA-X8-X1-X5–3'[SEQ ID NO:19]

[0201] Wherein: each X1 is independently selected from A, G and T; X2 is selected from C and G;

[0202] each X3 is independently selected from A and T; X4 is selected from C and T; each X5 is selected from G and T; X6 is selected from A, C and G; X7 is selected from A, C and T; X8 is selected from A and C; and each N is selected from A, C, G and T.

[0203] In some embodiments, the 5' regulatory region sequence located upstream of the transcription start site of the nif gene includes a sequence selected from at least one of the following:

[0204] 5'-ACGATATATTACTTGACGT-3'[SEQ ID NO:20]'5'-GTGATATCTTACCTAACGT-3'[SEQ ID NO:21]; 5'-AAGTTATGTAAGTTAACAT-3'[SEQ ID NO:22]; NO:23]; 5'-ATCATATATTAGTTGAATG-3' [SEQ ID NO: 24]; 5'-AAGTTAGCTAAGCTAACAT-3' [SEQ ID NO: 25]; 5'-ACGATATATTACTTGACGT-3' [SEQ ID NO: 26]; 5'-ACGATATATTACTTGACGT-3' [SEQ ID NO:27];5'-AGGTTATATAAACTAACAT-3'[SEQ ID NO:28];5'-AGGTTATATAAACTCACAT-3'[SEQ ID NO:29]; 5'-AGGTTATATAAACTAACAT-3'[SEQ ID NO:30]; 5'-AGGTTATATAAACTAACAT-3'[SEQ ID NO:31]; 5'-ATGTTATCTAATATTACAT-3'[SEQ ID NO:32]; 5'-TGGTCAGGAAAGTTAACAT-3'[SEQ ID NO:33]; 5'-AAGTTATATAAGTTAACAT-3'[SEQ ID NO:34]. In some embodiments, the 5' regulatory region sequence located upstream of the transcription start site of the nif gene includes the sequence 5'-CGATATATTACTTGACG-3'[SEQ ID NO:35].

[0205] In some embodiments, the isolated genetically modified microorganism described herein further comprises genetic modification of a second 5' regulatory region sequence within an endogenous nif gene. In some embodiments, the isolated genetically modified microorganism described herein further comprises genetic modification of a second 5' regulatory region sequence within an endogenous nif gene, wherein the second 5' regulatory region sequence is located downstream of the transcription start site in the endogenous nif gene.

[0206] In some embodiments, the natural second 5' regulatory region sequence in the endogenous nif gene can be recognized and bound by the GlnR protein. In some embodiments, the genetic modification of the second 5' regulatory region sequence in the endogenous nif gene is characterized as reducing the negative regulation or repression of GlnR on the expression of the endogenous nif gene. In some embodiments, the genetic modification of the second 5' regulatory region sequence in the endogenous nif gene includes the genetic modification of the 5' regulatory region sequence that produces a reduced affinity for the GlnR protein relative to the natural second 5' regulatory region sequence. In some embodiments, the genetic modification of the second 5' regulatory region sequence in the endogenous nif gene includes the genetic modification of the 5' regulatory region sequence that produces a 5' regulatory region sequence that cannot be recognized or bound by the GlnR protein. For example, in certain embodiments, the genetic modification of the second 5' regulatory region sequence in the endogenous nif gene includes deleting or knocking out the second 5' regulatory region sequence from the endogenous nif gene. In certain other embodiments, the genetic modification of the second 5' regulatory region sequence in the endogenous nif gene includes replacing the second 5' regulatory region sequence in the endogenous nif gene with a DNA sequence characterized as being unrecognizable by GlnR. The DNA sequence characterized as being unrecognizable by GlnR is not particularly limited, including any sequence characterized as having no affinity for the GlnR protein or significantly reduced affinity for the GlnR protein compared to the native second 5' regulatory region sequence.

[0207] In some embodiments, the second 5' regulatory region sequence within the endogenous nif gene comprises the following sequence:

[0208]

[0209] wherein: each Y1 is independently selected from A, G and T; Y2 is selected from A, C and T; each Y3 is independently selected from A and T; Y4 is selected from A and G; Y5 is selected from C and T; and each N is independently selected from A, C, G and T.

[0210] In some embodiments, the second 5' regulatory region sequence within the endogenous nif gene includes a sequence selected from at least one of the following: 5'-ATGTAAGGGAATATAACGT-3' [SEQ ID NO:37]; 5'-ATGTAAGGTAATTTAACGT-3' [SEQ ID NO:38]; 5'-GTGTTATGAAATATAACAT-3' [SEQ ID NO:39]; 5'-GTGTTAACTAAAATTACAT-3' [SEQ ID NO:40]; 5'-AAGTGAGGAAACATAACGT-3' [SEQ ID NO:41]; 5'-GGGTCAGGAAACATAACAT-3' [SEQ ID NO:42]; 5'-ATGTAAGGTAATATAACGT-3' [SEQ ID NO:43]; 5'-GTGTAAGGAAATATAACGT-3' [SEQ ID NO:44]; 5'-GTGTTAACTAAAATTACAT-3' [SEQ ID NO:45] NO:45]; 5'-GTGTTAACTAAAATTACAT-3'[SEQ ID NO:46]; 5'-GTGTTAGATAAAATTACAT-3'[SEQ ID NO:47]; 5'-GTGTTAACTAAAATTACAT-3'[SEQ ID NO:48]; 5'-TTGTTAGTAAATATAACAC-3'[SEQ ID NO:49]; 5'-TTGTTAGGAAACATAACAC-3'[SEQ ID NO:50]; 5'-ATGTTATGAAATATAACAT-3'[SEQ ID NO:51]. In some embodiments, the second 5' regulatory region sequence within the endogenous nif gene comprises the sequence 5'-TGTAAGGGAATATAACG-3'[SEQ ID NO:37].

[0211] In some embodiments, the isolated genetically modified microorganisms disclosed herein include a genetic modification of an endogenous glnR gene encoding GlnR and a genetic modification of a 5' regulatory region sequence within an endogenous nif gene.

[0212] In some embodiments, the genetic modification of the 5' regulatory region sequence in the endogenous nif gene includes replacing the 5' regulatory region sequence with a DNA sequence characterized as providing improved binding affinity to GlnR compared to the 5' regulatory region sequence. Those of ordinary skill in the art will recognize that the replacement of the 5' regulatory region sequence in the endogenous nif gene can be achieved by a variety of molecular biological methods known in the art. For example, in some embodiments, the 5' regulatory region sequence in the endogenous nif gene is replaced via site-directed mutagenesis or related processes to mutate specific nucleotides in the 5' regulatory region sequence to produce a DNA sequence characterized as providing improved binding affinity to GlnR. In other embodiments, the 5' regulatory region sequence in the endogenous nif gene is replaced via a gene editing method to excise the natural 5' regulatory region sequence and insert a recombinant DNA sequence characterized as providing improved binding affinity to GlnR.

[0213] In some embodiments, the DNA sequence characterized as providing improved binding affinity to GlnR is derived from a second 5' regulatory region sequence in an endogenous nif gene. In other words, the 5' regulatory region sequence in an endogenous nif gene can be replaced by a second 5' regulatory region sequence from an endogenous nif gene at a replacement site in the gene. For example, in some embodiments, the DNA sequence characterized as providing improved binding affinity to GlnR is derived from a second 5' regulatory region sequence in an endogenous nif gene, wherein the second 5' regulatory region sequence is located downstream of the transcription start site in the endogenous nif gene. In certain embodiments, the genetically modified microorganism of the separation of the present invention includes genetic modification of a 5' regulatory region sequence in an endogenous nif gene, wherein the 5' regulatory region sequence is located upstream of the transcription start site of the endogenous nif gene, and the genetic modification of the 5' regulatory region sequence includes replacing the 5' regulatory region sequence with a DNA sequence derived from a second 5' regulatory region sequence in an endogenous nif gene. In certain embodiments, the isolated genetically modified microorganism of the present invention includes a genetic modification of a 5' regulatory region sequence within an endogenous nif gene, wherein the 5' regulatory region sequence is located upstream of the transcription start site of the endogenous nif gene, and the genetic modification of the 5' regulatory region sequence includes replacing the 5' regulatory region sequence with a DNA sequence derived from a second 5' regulatory region sequence within the endogenous nif gene, wherein the second 5' regulatory region sequence within the endogenous nif gene is located downstream of the transcription start site within the endogenous nif gene.

[0214] In other embodiments, the DNA sequence characterized as providing improved binding affinity to GlnR is a non-natural DNA sequence, meaning that the DNA sequence is known not to exist naturally in endogenous nif genes as a binding site for GlnR. Non-natural DNA sequences can be designed de novo using techniques and methods known in the art, such as binding assays designed to test the interaction of GlnR with specific DNA sequences. For example, binding assays based on techniques including, but not limited to, fluorescence polarization and surface plasmon resonance can be used to determine the affinity of GlnR for specific DNA sequences. Thus, a large number of variable DNA sequences can be screened for GlnR binding affinity, and those sequences that exhibit the greatest affinity can be incorporated into the 5' regulatory region of the endogenous nif gene to provide regulated regulation of the nif gene by GlnR.

[0215] In some embodiments, the DNA sequence characterized as providing improved binding affinity to GlnR is recognized and bound by GlnR with a dissociation constant or Kd of about 1 / 2 to about 1 / 25 of the dissociation constant of a complex of GlnR and a native 5' regulatory region sequence. In other embodiments, the DNA sequence characterized as providing improved binding affinity to GlnR is recognized and bound by GlnR with a dissociation constant or Kd of about 1 / 1.1 to about 1 / 25 of the dissociation constant of a complex of GlnR and a native 5' regulatory region sequence. In some embodiments, a DNA sequence characterized as providing improved binding affinity for GlnR is recognized and bound by GlnR with a dissociation constant that is about 1 / 2, about 1 / 3, about 1 / 4, about 1 / 5, about 1 / 6, about 1 / 7, about 1 / 8, about 1 / 9, about 1 / 10, about 1 / 11, about 1 / 12, about 1 / 13, about 1 / 14, about 1 / 15, about 1 / 16, about 1 / 17, about 1 / 18, about 1 / 19, about 1 / 20, about 1 / 21, about 1 / 22, about 1 / 23, about 1 / 24, or about 1 / 25 of the dissociation constant of a complex of GlnR and a native 5' regulatory region sequence. In certain embodiments, a DNA sequence characterized as providing improved binding affinity for GInR is recognized and bound by GInR with a dissociation constant that is about 1 / 17 the dissociation constant of a complex of GInR and a native 5' regulatory region sequence.

[0216] In some embodiments, the isolated genetically modified microorganism described herein comprises a genetic modification of an endogenous glnR gene encoding GlnR and a genetic modification of a 5' regulatory region sequence within an endogenous nif gene, wherein the genetic modification of the endogenous glnR gene comprises truncation of a portion of the gene encoding the C-terminal domain of GlnR, and the genetic modification of the 5' regulatory region sequence within the nif gene comprises replacement of the 5' regulatory region sequence upstream of the transcription start site with a DNA sequence that is recognized and bound by GlnR with higher binding affinity than the native 5' regulatory region sequence.

[0217] In some embodiments, the isolated genetically modified microorganism described herein comprises a genetic modification of an endogenous glnR gene encoding GlnR and a genetic modification of a 5' regulatory region sequence within an endogenous nif gene, wherein the genetic modification of the endogenous glnR gene comprises truncation of a portion of the gene encoding the C-terminal domain of GlnR, and the genetic modification of the 5' regulatory region sequence within the nif gene comprises knocking out or deleting the 5' regulatory region sequence downstream of the transcription start site.

[0218] In some embodiments, the isolated genetically modified microorganism described herein comprises genetic modification of an endogenous glnR gene encoding GlnR and genetic modification of a 5' regulatory region sequence within an endogenous nif gene, wherein the genetic modification of the endogenous glnR gene comprises truncation of a portion of the gene encoding the C-terminal domain of GlnR, the genetic modification of the 5' regulatory region sequence within the nif gene comprises replacement of the 5' regulatory region sequence upstream of the transcription start site with a DNA sequence that is recognized by GlnR and bound with higher binding affinity than the native 5' regulatory region sequence, and the genetic modification of the 5' regulatory region sequence within the nif gene also comprises knocking out or deleting the 5' regulatory region sequence downstream of the transcription start site.

[0219] In some embodiments, the isolated genetically modified microorganism described herein includes genetic modification of a 5' regulatory region sequence within an endogenous nif gene, wherein the genetic modification of the 5' regulatory region sequence within the nif gene includes replacing the 5' regulatory region sequence upstream of the transcription start site with a DNA sequence that is recognized by GlnR and bound with higher binding affinity than the native 5' regulatory region sequence, and the genetic modification of the 5' regulatory region sequence within the nif gene also includes knocking out or deleting the 5' regulatory region sequence downstream of the transcription start site.

[0220] In some embodiments, the genetically modified microorganism of separation of the present disclosure is characterized as having the constitutive expression of nif genes. In some embodiments, the genetically modified microorganism of separation of the present disclosure is characterized as having the constitutive expression of nif genes, and has nothing to do with local nitrogen or ammonia concentrations. For example, in certain embodiments, the genetically modified microorganism of separation is characterized as having the constitutive expression of nif genes under nitrogen-limited conditions. In certain embodiments, the genetically modified microorganism of separation is characterized as having the constitutive expression of nif genes under nitrogen-rich conditions. In certain embodiments, the genetically modified microorganism of separation is characterized as having the constitutive expression of nif genes under nitrogen-limited conditions or nitrogen-rich conditions.

[0221] The present disclosure also encompasses isolated genetically modified microorganisms, wherein the microorganism lacks endogenous nif and / or glnR genes, and the microorganism is genetically edited to incorporate exogenous nif and / or glnR genes. As described herein, the exogenous nif and / or glnR genes incorporated into the isolated microorganism may include one or more genetic modifications to the nif and / or glnR genes. For example, the exogenous glnR gene incorporated into the microorganism may encode a GlnR variant protein (the variant protein includes a truncation of the C-terminal domain of the protein), and / or be characterized as being able to bind to the recognition element or 5' regulatory region sequence within the nif gene with enhanced affinity. The exogenous nif gene incorporated into the isolated microorganism may include one or more recognition elements or 5' regulatory region sequences that can be recognized by GlnR and bound with enhanced or weakened affinity. In addition to exogenous nif and / or glnR genes, auxiliary genes known in the art to improve nitrogen fixation ability may also be incorporated into microorganisms lacking endogenous nif and / or glnR genes. Compared with the incorporation of exogenous nif and glnR genes alone, the further incorporation of these auxiliary genes can enhance the nitrogen fixation ability of the microorganism to a greater extent.

[0222] The present invention also contemplates genetic modification of the endogenous cueR gene. CueR belongs to the helix-turn-helix transcriptional regulatory factor family (Mer family HTH regulatory factor), similar to GlnR. CueR and GlnR have the same pfam domain.

[0223] Both GlnR and glutamine synthetase (GS, encoded by glnA within the glnRA operon) are required to repress nif expression under conditions of nitrogen excess. Under conditions of glutamine excess, a feedback-inhibitory form of glutamine synthetase (GS), encoded by glnA within the glnRA operon, interacts directly with the C-terminal domain of GlnR, thereby controlling GlnR activity. In addition, overexpression of glnR or deletion of glnA or mutation of GlnR binding site II results in constitutive nif expression in the absence or presence of high (100 mM) concentrations of ammonia. (Wang, 2018).

[0224] Also provided are strains and modifications of the NrgA locus. In Paenibacillus, the nrgA gene is the major ammonium transporter. Nitrogen fixation is tightly regulated and is repressed by excess ammonium levels within the cell. To reduce the amount of ammonium available to repress nitrogenase expression, nrgA is knocked out by seamlessly removing the entire open reading frame from the first nucleotide to the last nucleotide, leaving the immediately surrounding sequence intact, but without leaving any gene sequence.

[0225] Provided herein are isolated, edited (genetically modified) strains of various Paenibacillus species having one or more modifications of one or more endogenous loci that confer improved nitrogen fixation capabilities to the microorganism.

[0226] In some embodiments, the isolated genetically modified microorganism is characterized as a Gram-positive bacterial species or strain. In some embodiments, the isolated genetically modified microorganism is characterized as a spore-forming Gram-positive bacterial species or strain.

[0227] Microbial consortium

[0228] In some aspects, the disclosure provides a microbial consortium comprising a combination of at least any two microorganisms, wherein one microorganism is a Paenibacillus strain described in Table 1a, Table 1b, or Table 1c. In some embodiments, the Paenibacillus strain comprises a polynucleotide sequence having at least 90% identity to any one or more of SEQ ID NOs. 1-123. In some embodiments, the Paenibacillus strain is a species selected from the group consisting of Paenibacillus aceris, Paenibacillus aestuarii, Paenibacillus agarexedens, Paenibacillus agaridevorans, Paenibacillus albidus, Paenibacillus alginolyticus, Paenibacillus alkaliterrae, Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus anaericanus, Paenibacillus antarcticus, Paenibacillus apiary, Paenibacillus spp. apiaries), Paenibacillus assamensis, Paenibacillus azotifigens, Paenibacillus baetica, Paenibacillus barcinonensis, Paenibacillus barengoltzii, Paenibacillus borealis, Paenibacillus caespitis, Paenibacillus camelliae, Paenibacillus castaneae, Paenibacillus catalpa, Paenibacillus cavernae, Paenibacillus cellulolyticuscellulosilyticu), Paenibacilluschartariuss, Paenibacillus chibensis, Paenibacillus chinjuensis, Paenibacillus chitinolyticus, Paenibacillus chondroitinus, Paenibacillus cineris, Paenibacillus cisolokensis, Paenibacillus contaminans, Paenibacillus cookii, Paenibacillus crassostreae, Paenibacillus cucumis, Paenibacillus galactolyticus curdlanolyticus), Paenibacillus daejeonensis, Paenibacillus darwinianus, Paenibacillus dongdonensis, Paenibacillus donghaensis, Paenibacillus doosanensis, Paenibacillus drentensis, Paenibacillus durus, Paenibacillus usedaphicus, Paenibacillus ehimensis, Paenibacillus elgii, Paenibacillus endophyticus, Paenibacillus etheri, Paenibacillus euphorbia favisporus), Paenibacillus filicis, Paenibacillus frigoriresistens, Paenibacillus gansuensisgansuensis), Paenibacillusginsengarvi, Paenibacillus glacialis, Paenibacillus glebae, Paenibacillus glucanolyticus, Paenibacillus glycanilyticus, Paenibacillusgraminis, Paenibacillusgranivorans, Paenibacillusharenae, Paenibacillushelianthin, Paenibacillushordei, Paenibacillushumicus, Paenibacillusshunanensis, Paenibacillusillinois, Paenibacillus illinoisensis、Paenibacillus jilunlii、Paenibacillus kobensis、Paenibacillus koleovorans、Paenibacillus konkukensis、Paenibacillus konsidensis、Paenibacillus kukuduoahitejonii、Paenibacillus lactis、Paenibacillus lautus、Paenibacillus liaoningensis、Paenibacillus lupini、Paenibacillus macquariensis、Paenibacillusmarchantiophytorum、Paenibacillus marquariensis, Paenibacillus mendelii, Paenibacillus mobilis, Paenibacillus motobuensis, Paenibacillus colloidusmucilaginosus), Paenibacillus naganoensis, Paenibacillus naganoensis, Paenibacillus nanensis, Paenibacillus nasutitemitis, Paenibacillus nebraskensis, Paenibacillus nicotianae, Paenibacillus nitroguajacolicus, Paenibacillus oceanisediminis, Paenibacillus odorifer, Paenibacillus oryzae, Paenibacillus oryzisoli, Paenibacillus ourofinensis, Paenibacillus fodder pabuli), Paenibacillus panacisoli, Paenibacillus panaciterrae, Paenibacillus pectinilyticus, Paenibacillus peoriae, Paenibacillus periandrae, Paenibacillus phoenicis, Paenibacillus phoenicis, Paenibacillus physcomitrellae, Paenibacillus pini, Paenibacillus pinisoli, Paenibacillus plakortidis, Paenibacillus pocheonensis, Paenibacillus polymyxa polymyxa), Paenibacillus polysaccharolyticus, Paenibacillus populi, Paenibacillus profundus, Paenibacillus Provenceprovencensis), Paenibacillus purispatii, Paenibacillus qinlingensis, Paenibacillus rhizoplanae, Paenibacillus rhizoryzae, Paenibacillus rhizosphaerae, Paenibacillus rigui, Paenibacillus sacheonensis, Paenibacillus segetis, Paenibacillus sepulcri, Paenibacillus shirakamiensis, Paenibacillus silagei, Paenibacillus silvae, Paenibacillus sinopodophylli, Paenibacillus solanacearum, Paenibacillus soli, Paenibacillus sonchi, Paenibacillus sputi, Paenibacillus stellifer, Paenibacillus susongensis, Paenibacillus taichungensis, Paenibacillus taihuensis, Paenibacillus taiwanensis, Paenibacillus taohuashanense, Paenibacillus starimensis, Paenibacillus telluris, Paenibacillus terrae, Paenibacillus terrigena, Paenibacillus hailandensis, Paenibacillus thalictra, Paenibacillus thermophilus, Paenibacillus Tianmushan tianmuensis), Paenibacillus tianmuensistimonensis), Paenibacillus tritici, Paenibacillus tundrae, Paenibacillus turicensis, Paenibacillus tylopili, Paenibacillus typhae, Paenibacillus suliginis, Paenibacillus validus, Paenibacillus vini, Paenibacillus vulneris, Paenibacillus wooponensis, Paenibacillus wynnii, Paenibacillus xanthanilyticus, Paenibacillus xinjiangensis, Paenibacillus xylan-eating xylanexedens), Paenibacillus xylanilyticus, Paenibacillus yonginensis, and Paenibacillus yunnanensis. In some embodiments, the Paenibacillus strain belongs to subgroup I. In some embodiments, the Paenibacillus strain belongs to subgroup II. In some embodiments, the Paenibacillus strain is selected from the group consisting of: NRRL deposit numbers: B-68102, B-68103, B-68104, B-68105, B-68106, B-68107, B-68108, B-68109, and B-68110.

[0229] In certain embodiments, the consortium of the present disclosure comprises two microorganisms or three microorganisms or four microorganisms or five microorganisms or six microorganisms or seven microorganisms or eight microorganisms or nine microorganisms or ten or more microorganisms. The microorganisms of the consortium are different microbial species, or different strains of microbial species.

[0230] Compositions produced by microorganisms

[0231] In some cases, the microorganisms of the present disclosure can produce one or more compounds and / or have one or more activities, such as one or more of the following: production of a metabolite, production of a plant hormone (such as an auxin), production of acetoin, production of an antimicrobial compound, production of a siderophore, production of a polyketide, production of a phenazine, production of a cellulase, production of a pectinase, production of a chitinase, production of a glucanase, production of a xylanase or a protease or an organic acid or a lipopeptide or a polynucleotide or a polypeptide, nitrogen fixation, mineral phosphate solubilization, or any combination and / or multiple of the foregoing.

[0232] For example, the microorganisms of the present disclosure can produce a plant hormone selected from the group consisting of auxins, cytokinins, gibberellins, ethylene, brassinolide, and abscisic acid.

[0233] Thus, "metabolites produced by a microorganism of the present disclosure" is intended to reflect any molecule (small molecule, vitamin, mineral, protein, nucleic acid, lipid, fat, carbohydrate, etc.) produced by a microorganism. In general, the precise mechanism of action by which a microorganism of the present disclosure confers a beneficial trait to a given plant species is unknown. It is speculated that in some cases, a microorganism produces a metabolite that is beneficial to a plant. Thus, in some aspects, a cell-free or inactivated preparation of a microorganism is beneficial to a plant, as the microorganism does not have to be alive to confer a beneficial trait to a given plant species, as long as the preparation contains a metabolite produced by the microorganism and that is beneficial to the plant.

[0234] In one embodiment, the microorganisms of the present disclosure can produce auxins (e.g., indole-3-acetic acid (IAA)). The production of auxins can be analyzed. Many of the microorganisms described herein are capable of producing the plant hormone auxin indole-3-acetic acid (IAA) when grown in culture. Auxins play a key role in altering the physiology of plants, including the extent of root growth.

[0235] Thus, in one embodiment, the microorganisms of the present disclosure are present as a population disposed on the surface or within the tissue of a given plant species. The microorganisms can produce a composition, such as a metabolite, in an amount effective to cause a detectable increase in the amount of the composition found on or within the plant when compared to a reference plant not treated with the microorganisms of the present disclosure or a cell-free or inactive preparation. The composition produced by the microbial population can be beneficial to the plant species.

[0236] Such microbial produced compositions may be present in the cell culture broth or culture medium in which the microorganism is grown, or may encompass exudates produced by the microorganism. As used herein, "exudate" refers to one or more compositions secreted by or extracted from one or more microbial cells. As used herein, "broth" refers to the common composition of the cell culture medium after the microbial cells are placed in the culture medium. The composition of the broth may change over time, changing during different stages of microbial growth and / or development. Broth and / or exudates may improve the traits of the plant with which it becomes associated.

[0237] Microbial-induced traits in plants

[0238] The present disclosure utilizes microorganisms to impart beneficial properties (or beneficial traits) to desired plant species, such as agronomic species of interest. In the present disclosure, the terms "beneficial properties," "beneficial traits," or "traits of interest" are used interchangeably and refer to the modulation of a desired plant phenotype or genetic trait of interest by the application of a microorganism or microbial consortium as described herein. As previously noted, in some aspects, it may be desirable that the metabolites produced by a given microorganism are ultimately responsible for the modulation or imparting of a beneficial trait to a given plant.

[0239] There are many beneficial traits that can be modulated by applying the microorganisms of the present disclosure. For example, the microorganisms can have the ability to impart one or more beneficial characteristics to plant species, such as: increased growth, increased yield, increased nitrogen use efficiency, increased stress tolerance, increased drought tolerance, increased photosynthetic rate, increased water use efficiency, increased pathogen resistance, modification of plant architecture (which does not necessarily affect plant yield, but addresses plant function, causing the plant to increase production of metabolites of interest), etc.

[0240] In various aspects, the microorganisms taught herein provide a wide range of agricultural applications, including: increasing grain, fruit and flower yields; increasing the growth of plant parts; increasing the ability to utilize nutrients (e.g., nitrogen, phosphates, etc.); increasing disease resistance; bio-insecticide effects (including increasing resistance to fungi, insects and / or nematodes); increasing survival in extreme climates; and improving other desired plant phenotypic characteristics.

[0241] In some aspects, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to modulate or alter plant characteristics, such as altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, increased nitrogen fixation, improved nitrogen utilization efficiency, improved nutrient (e.g., phosphate, potassium, etc.) utilization efficiency, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, yield under water-limiting conditions, etc., relative to a reference plant. The invention also provides for the invention of the invention a method for regulating the growth of plants and crops, for example, by regulating the growth of seeds, for example, by regulating the growth of seeds, for example, by regulating the growth of crops, for example, by regulating the growth of plants, for example, by regulating the growth of crops, for example, by regulating the growth of plants, for example, by regulating the growth of crops, for example, by regulating the growth of plants, for example, by regulating the growth of crops, for example, by regulating the growth of plants, for example, by regulating the growth of crops, for example, by regulating the growth of plants

[0242] In some aspects, the isolated microorganisms, consortia and / or agricultural compositions of the present disclosure can be applied to plants to regulate specific plant characteristics in a negative manner. For example, in some aspects, the microorganisms of the present disclosure can reduce phenotypic traits of interest because such functionality may be desirable in some applications. For example, the microorganisms of the present disclosure may have the ability to reduce root growth or reduce root length. Or the microorganisms may have the ability to reduce bud growth or reduce plant growth rate because these adjustments of plant traits may be desirable in certain applications.

[0243] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure may be applied to plants to confer nematode stress tolerance to the plants.

[0244] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure may be applied to plants to provide biological stimulation (biostimulant effect) to the plants. In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure may be applied to plants to provide disease resistance to the plants.

[0245] Agricultural composition

[0246] In some embodiments, the microorganisms of the present disclosure are combined with agricultural compositions. Agricultural compositions generally refer to such organic and inorganic compounds, which may include compositions that promote the cultivation of microorganisms and / or plant components; compositions that participate in the preparation of microorganisms to be applied to plant components (such as but not limited to: wetting agents, extenders (also known as "compatibilizers"), defoamers, detergents, chelating agents, drift reducers, neutralizers and buffers, corrosion inhibitors, dyes, odorants, extenders (also known as "spreaders"), penetration aids (also known as "penetrants"), adhesives (also known as "adhesives" or "binders"), dispersants, thickeners (also known as "thickeners"), stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, etc.); compositions that participate in imparting protection to plant components or plants (such as but not limited to: insecticides, nematicides, fungicides, bactericides, herbicides, etc.); and other compositions that may be of interest for specific applications.

[0247] In some embodiments, the agricultural composition of the present disclosure is solid. When using solid compositions, it may be desirable to include one or more carrier materials and isolated active microorganisms or consortia. In some embodiments, the present disclosure teaches the use of carriers, which include but are not limited to: mineral soils such as silicon dioxide, silica gel, silicates, talc, kaolin, activated clay, limestone, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, plant-derived products such as grain flour, bark powder, wood powder and husk powder, cellulose powder, attapulgite, montmorillonite, mica, vermiculite, synthetic silica and synthetic calcium silicate, or compositions of these substances.

[0248] Growth composition

[0249] In some embodiments, compositions that promote growth and development are provided to microorganisms and / or plant components. Exemplary compositions include liquids (such as broth, culture media) and / or solids (such as soil, nutrients). Various organic or inorganic compounds may be added to the growth composition alone or in combination with plant components to benefit the health of the microorganisms, such as, but not limited to: amino acids, vitamins, minerals, carbohydrates, monosaccharides, lipids.

[0250] Preparation composition

[0251] One or more compositions other than the microorganism or microorganism-produced composition may be combined for various application, stability, activity and / or storage reasons. The additional composition may be referred to as a "formulation component".

[0252] In some embodiments, the agricultural compositions disclosed herein may be formulated as a liquid, solid, gas, or gel.

[0253] Thus, in some embodiments, the present disclosure teaches that the agricultural compositions disclosed herein may include compounds or salts such as monoethanolamine salts, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium dihydrogen phosphate, diammonium monophosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate, or ammonium carbamate.

[0254] In some embodiments, the present disclosure teaches that agricultural compositions may include binders such as: polyvinyl pyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethyl cellulose, starch, vinyl pyrrolidone / vinyl acetate copolymers and polyvinyl acetate, or combinations thereof; lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or combinations thereof; defoamers such as silicone emulsions, long chain alcohols, phosphate esters, acetylene glycols, fatty acids or organofluorine compounds, and complexing agents such as: salts of ethylenediaminetetraacetic acid (EDTA), salts of trinitrilotriacetic acid or salts of polyphosphoric acid, or combinations thereof.

[0255] In some embodiments, agricultural compositions include surfactants. In some embodiments, surfactants are added to liquid agricultural compositions. In other embodiments, surfactants are added to solid formulations, especially those designed to be diluted with a carrier before application. Therefore, in some embodiments, agricultural compositions include surfactants. Surfactants are sometimes used alone or with other additives (such as minerals or vegetable oils) as adjuvants for spray tank mixtures to improve the biological performance of microorganisms to targets. The type of surfactant used for bioenhancers generally depends on the properties and mode of action of the microorganism. The characteristics of surfactants can be anionic, cationic or nonionic, and can be used as emulsifiers, wetting agents, suspending agents or for other purposes. In some embodiments, surfactants are nonionic surfactants, such as: alkyl ethoxylates, linear fatty alcohol ethoxylates and fatty amine ethoxylates. Surfactants commonly used in the formulation art and which may also be used in the formulation of the present invention are described in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, NJ, 1998, and Encyclopedia of Surfactants, Vols. I-III, Chemical Publishing Co., New York, 1980-81. In some embodiments, the present disclosure teaches the use of surfactants, which include alkali metal, alkaline earth metal or ammonium salts of aromatic sulfonic acids (e.g., ligninsulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid and dibutylnaphthalenesulfonic acid) and fatty acids of arylsulfonates, alkyl ethers, dodecyl ethers, fatty alcohol sulfates and fatty alcohol glycol ether sulfates; condensates of sulfonated naphthalene and its derivatives with formaldehyde; condensates of naphthalene or naphthalenesulfonic acid with phenol and formaldehyde; condensates of phenol or phenolsulfonic acid with formaldehyde; condensates of phenol with formaldehyde and sodium sulfite; polyoxyethylene octylphenyl ether; ethoxylated isooctylphenol, ethoxylated octylphenol or ethoxylated nonylphenol; tributylphenyl polyglycol ether; alkylaryl polyether alcohol; isotridecyl alcohol; ethoxylated castor oil; ethoxylated triarylphenol; salts of phosphorylated triarylphenol ethoxylates; dodecanol polyglycol ether acetate; sorbitan esters; lignin-sulfite waste liquor or methylcellulose, or combinations of these substances.

[0256] In some embodiments, the present disclosure teaches other suitable surfactants, including salts of alkyl sulfates, such as diethanolammonium dodecyl sulfate; alkylaryl sulfonates, such as calcium dodecylbenzene sulfonate; alkylphenol-alkylene oxide addition products, such as nonylphenol-C18 ethoxylate; ethanol-alkylene oxide addition products, such as tridecyl alcohol-C16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalene-sulfonates, such as sodium dibutylnaphthalene sulfonate; dialkyl esters of sulfosuccinates, such as di(2-ethylhexyl)sulfonate; succinate; sorbitol esters, such as sorbitol oleate; quaternary amines, such as dodecyltrimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of monoalkyl and dialkyl phosphates; vegetable oils, such as soybean oil, rapeseed / canola oil, olive oil, castor oil, sunflower oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil, etc.; and esters of the above vegetable oils, especially methyl esters.

[0257] In some embodiments, the agricultural composition comprises a wetting agent. A wetting agent is a substance that, when added to a liquid, increases the spreading or penetration ability of a liquid by reducing the interfacial tension between the liquid and the surface on which the liquid is spread. Wetting agents are used in agricultural chemical formulations for two main functions: to increase the wettability of powders in water during processing and manufacturing to prepare soluble liquid concentrates or suspension concentrates; and to shorten the wetting time of wettable powders and increase the penetration rate of water into water-dispersible granules during mixing of the product with water in a spray tank or other container. In some embodiments, examples of wetting agents used in agricultural compositions of the present disclosure (including wettable powders, suspension concentrates, and water-dispersible granule formulations) are: sodium lauryl sulfate; sodium dioctyl sulfosuccinate; alkylphenol ethoxylates; and fatty alcohol ethoxylates.

[0258] In some embodiments, agricultural compositions of the present disclosure include dispersants. Dispersants are substances that are adsorbed on the surface of particles and help to maintain the dispersed state of particles and prevent them from re-aggregating. In some embodiments, dispersants are added to agricultural compositions of the present disclosure to promote dispersion and suspension during manufacture, and ensure that particles are redispersed in water in a spray tank. In some embodiments, dispersants are used in wettable powders, suspension concentrates, and water-dispersible granules. The surfactant used as a dispersant has the ability to be strongly adsorbed on the surface of particles and provide a charged or spatial barrier that prevents particles from re-aggregating. In some embodiments, the most commonly used surfactants are anionic, nonionic, or a mixture of these two types.

[0259] In some embodiments, for wettable powder formulations, the most commonly used dispersant is sodium lignin sulfonate. In some embodiments, suspension concentrates use polyelectrolytes (such as sodium naphthalene sulfonate formaldehyde condensates) to provide excellent adsorption and stabilization. In some embodiments, tristyrylphenol ethoxylate phosphate is also used. In some embodiments, such as alkyl aryl ethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionic surfactants as dispersants for suspension concentrates.

[0260] In some embodiments, the agricultural compositions of the present disclosure include polymeric surfactants. In some embodiments, polymeric surfactants have extremely long hydrophobic "backbones" and a large number of ethylene oxide chains, which form the "teeth" of a "comb-like" surfactant. In some embodiments, these high molecular weight polymers can provide excellent long-term stability of suspension concentrates because the hydrophobic backbone has many points of anchoring to the surface of the particles. In some embodiments, examples of dispersants used in the agricultural compositions of the present disclosure are: sodium lignin sulfonate; sodium naphthalene sulfonate formaldehyde condensate; tristyrylphenol ethoxylate phosphate; fatty alcohol ethoxylate; alkyl ethoxylate; EO-PO block copolymer; and graft copolymer.

[0261] In some embodiments, agricultural compositions of the present disclosure include emulsifiers. Emulsifiers are substances that stabilize a suspension of droplets of a liquid phase in another liquid phase. In the absence of emulsifiers, the two liquids will separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends include alkylphenols or fatty alcohols with 12 or more ethylene oxide units and oil-soluble calcium salts of dodecylbenzene sulfonic acid. Hydrophile-lipophile balance ("HLB") values ​​within the range of 8 to 18 will generally provide good stable emulsions. In some embodiments, emulsion stability can sometimes be improved by adding a small amount of EO-PO block copolymer surfactants.

[0262] In some embodiments, the agricultural compositions of the present disclosure include a solubilizing agent. A solubilizing agent is a surfactant that will form micelles in water at a concentration exceeding the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials within the hydrophobic portion of the micelle. The types of surfactants commonly used for dissolution are nonionic surfactants: sorbitan monooleate; sorbitan monooleate ethoxylate; and methyl oleate.

[0263] In some embodiments, the agricultural composition of the present disclosure comprises an organic solvent. Organic solvents are mainly used in the preparation of emulsifiable concentrates, ULV preparations, and to a lesser extent in granular preparations. Sometimes a mixture of solvents is used. In some embodiments, the present disclosure teaches the use of solvents, and the solvents include aliphatic paraffin oils, such as kerosene or refined paraffin. In other embodiments, the present disclosure teaches the use of aromatic solvents, such as the higher molecular weight fractions of xylene and C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons can be used as cosolvents to prevent insecticide crystallization when the preparation is emulsified in water. Alcohol is sometimes used as a cosolvent to improve dissolving power.

[0264] In some embodiments, the agricultural composition comprises a gelling agent. Thickeners or gelling agents are mainly used in the formulation of suspension concentrates, emulsions and suspoemulsions to change the rheology or fluidity of the liquid and prevent the separation and sedimentation of dispersed particles or droplets. Thickeners, gelling agents and anti-settling agents generally belong to two categories, namely water-insoluble particles and water-soluble polymers. It is possible to use clay and silica to produce suspension concentrate formulations. In some embodiments, the agricultural composition comprises one or more thickeners, including but not limited to: montmorillonite, such as bentonite; magnesium aluminum silicate; and attapulgite. In some embodiments, the present disclosure teaches the use of polysaccharides as thickeners. The most commonly used types of polysaccharides are natural extracts of seeds and seaweed or synthetic derivatives of cellulose. Some embodiments utilize xanthan gum and some embodiments utilize cellulose. In some embodiments, the present disclosure teaches the use of thickeners, including but not limited to: guar gum; locust bean gum; carrageenan; alginates; methylcellulose; sodium carboxymethylcellulose (SCMC); hydroxyethylcellulose (HEC). In some embodiments, the present disclosure teaches the use of other types of anti-settling agents, such as modified starches, polyacrylates, polyvinyl alcohols, and polyethylene oxides. Another good anti-settling agent is xanthan gum.

[0265] In some embodiments, the presence of surfactants (which reduce interfacial tension) can cause water-based formulations to foam during the mixing operation in the preparation and application by spray cans. Therefore, in some embodiments, in order to reduce the foaming tendency, defoamers are usually added during the preparation stage or before filling into the bottle / spray can. Generally speaking, there are two types of defoamers, i.e. silicones and non-silicone defoamers. Silicones are usually aqueous emulsions of dimethylpolysiloxane, while non-silicone defoamers are water-insoluble oils, such as octanol and nonanol, or silicon dioxide. In both cases, the function of the defoamer is to transfer the surfactant from the air-water interface.

[0266] In some embodiments, the agricultural composition comprises a preservative.

[0267] In some embodiments, the agricultural composition can be formulated as a soil drench, foliar spray, drench treatment, in-furrow treatment, soil conditioner, granules, broadcast treatment, post-harvest disease control treatment, or seed treatment. In some embodiments, the agricultural composition can be applied alone or in combination with other agricultural products in a rotary spray procedure.

[0268] In some embodiments, the agricultural composition is compatible with tank mixing. In some embodiments, the agricultural composition is compatible with tank mixing with other agricultural products. In some embodiments, the agricultural composition is compatible with equipment for ground, aerial, and irrigation applications.

[0269] In some embodiments, the agricultural composition may be applied to genetically modified seeds or plants.

[0270] Protective composition

[0271] In addition, the individual microorganisms or microorganism consortia or microbial communities developed according to the disclosed method can be combined with known active agents available in the agricultural field, such as: pesticides, herbicides, bactericides, fungicides, insecticides, virucides, acaricides, nematicides, scabicides, plant growth regulators, rodenticides, anti-algae agents, biological control or beneficial agents. In addition, the microorganisms, microorganism consortia or microbial communities developed according to the disclosed method can be combined with known fertilizers. Such combinations can show synergistic properties. In addition, the individual microorganisms or microorganism consortia or microbial communities developed according to the disclosed method can be combined with inert ingredients. In addition, in some aspects, the disclosed microorganisms are combined with bioactive agents.

[0272] In some embodiments, a single microorganism or microbial consortium or microbial community developed according to the disclosed methods can be combined with a biopesticide that acts as a herbicide, bactericide, fungicide, insecticide, viricide, acaricide, nematicide, scabicide, rodenticide, and / or anti-algae agent. Such biopesticides can be, but are not limited to, macrobiological organisms (e.g., beneficial nematodes, etc.), microbial organisms (e.g., Serenade, Bt, etc.), plant extracts (e.g., Timorex Gold, etc.), biochemicals (e.g., insect pheromones, etc.), and / or minerals and oils (e.g., canola oil).

[0273] Pesticides and biopesticides

[0274] In some embodiments, the agricultural compositions of the present disclosure comprise a pesticide used in combination with the taught microorganisms. In some embodiments, the agricultural compositions of the present disclosure comprise a biopesticide used in combination with the taught microorganisms.

[0275] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with known pesticides in the agricultural field, such as: pesticides that act as herbicides, bactericides, fungicides, insecticides, virucides, miticides, nematicides, scabicides, rodenticides and / or anti-algae agents.

[0276] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with known biopesticides in the agricultural field, such as: biopesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, miticides, nematicides, scabicides, rodenticides and / or anti-algae agents.

[0277] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients, including: macrobiological organisms (e.g., beneficial nematodes, etc.), microbial organisms (e.g., Serenade, Bt, etc.), plant extracts (e.g., Timorex Gold, etc.), biochemicals (e.g., insect pheromones, etc.), and / or minerals and oils (e.g., canola oil).

[0278] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with a herbicide selected from the group consisting of: acetamides selected from the group consisting of: acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, flufenacet, benzathine, isopropylamine, pyraclostrobin, naproxen, pethoxydim, pretilachlor, cypermethrin and dimethenamid; amino acid derivatives selected from the group consisting of: bialaphos, glufosinate and glufosinate; aryloxyphenoxypropionates selected from the group consisting of: clodinafop-butyl, cyhalofop-butyl, oxazolidinone, fluazifop-butyl, fluazifop-butyl, oxadiazol-butyl, quinazolin-butyl, quinazolin-butyl and quinazolin-butyl; quinazolin-butyl, quinazolin-butyl, quinazolin-butyl, quinazolin-butyl and quinazolin-butyl; quat and paraquat; (thio) carbamates selected from the group consisting of: cypermethrin, butadiene, carbamate, betamethrin, piperocarb, EPTC, cypermethrin, cypermethrin, cypermethrin, betamethrin, betamethrin, cypermethrin, cypermethrin and wild wheat cypermethrin; cyclohexanedione selected from the group consisting of: butadiene, clethodim, cypermethrin, cypermethrin, sethoxydim, pyraclostrobin and oxamethoxam; dinitroanilines selected from the group consisting of: fluazifop, ethylbutene, oryzalin, pendimethalin, aminofluralin and trifluralin; diphenyl ethers selected from the group consisting of: acifluorfen, benfibrate, cypermethrin, difluanid, chlorpyrifos, fomesulfuron, lactofen and oxyfluorfen ; Hydroxybenzonitriles selected from the group consisting of bromoxynil, dichlobenil and ioxynil; Imidazolinones selected from the group consisting of imazapic, imazapic, methyl imazapic, imazapic, imazapic and imazapic; Phenoxyacetic acids selected from the group consisting of chloranil, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-DB, dichlorprop, MCPA, MCPA-thioethyl, MCPB and 2-methyl-4-chloropropionic acid; Pyrazines selected from the group consisting of herbicide, flupyridazine, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl and fluazifop-butyl; Pyridines selected from the group consisting of clomiphene carboxylic acid, clopyralid, difluanid, fluthiopyr, flupyralid, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl , fluazifop and thiabendazole; a sulfonylurea selected from the group consisting of acylsulfuron-methyl, tetrazosulfuron-methyl, benzylsulfuron-methyl, chlorimuron-methyl, chlorsulfuron-methyl, ethoxysulfuron-methyl, flazasulfuron-methyl, flutosulfuron-methyl, flutosulfuron-methyl, foramsulfuron-methyl, chlorpyrisulfuron-methyl, pyrazosulfuron-methyl, iodosulfuron-methyl, mesosulfuron-methyl, metsulfuron-methyl, nicosulfuron, epoxysulfuron-methyl, primisulfuron-methyl, prosulfuron, pyrazosulfuron-methyl, sulfonesulfuron-methyl, sulfosulfuron-methyl, thifensulfuron-methyl, etherbensulfuron-methyl, bensulfuron-methyl, trifloxysulfuron, trifloxysulfuron, trifloxysulfuron and 14 (2-chloro-6-propyl-imidazo[1,2-b]pyridazin-3-yl)sulfonyl)-3-(4,6-dimethoxy-pyrimidin-2-yl)urea;A triazine selected from the group consisting of: ametryn, atrazine, cypermethrin, isopentanol, ethidium, hexazinone, metamitron, metribuzin, promethazine, simazine, terbuthion, benzamid and triazinon; a urea compound selected from the group consisting of: chlorotoluron, cypermethrin, diuron, flumethuron, isoproturon, linuron, methylbenzenesulfuron and tetrathion; an acetolactate synthase inhibitor selected from the group consisting of: sodium salt of bispyribac, chlorpyrifos, bispyribac, bispyribac, fluazifop, sulfamethoxam, sulfamethoxam, sulfamethoxam, penoxsulam, propoxysulfone, propylsulfuron ... oxazolidinone, pyrimidine oxime, pyrimidine oxime, pyrimidine oxime, pyriproxyfen, pyrimidine oxime, sulfamethoxazole and sulfamethoxazole; and a compound selected from the group consisting of: amitriptyline, aminotriazole, thiamethoxam, fluazifop, chloranil, bencarbazone, benfluresate, pyraclostrobin, bentazon, bicyclosulfuron, herbicide, bromobutyric acid, fluazifop-butyl, chloranil, fenpyroxil ... monoceras), grass algae, ethoxyfuroxan, ethoxybencloram, tetrazoline, flufenac, fluazifop, flufenac, fluchloridone, furochloridone, indole, isoxaflutamide, isoxaflutamide, cypermethrin, propanil, propargyl, quinclorac, clomiphenecarb, mesotrione, methylarsonic acid, oxadiazine, oxadiazine, oxadiazine, cyclopentadiazine, pinoxaden, bispyribac, pyraclostrobin, pyrasuloftole, benzaclostrobin, pyrazol, pyrazol, quinoline, sulfaquinoxaline, terbacil, furansulfuron, cypermethrin, thiamethoxam, 4-hydroxy-3-[2-(2-methoxy-ethoxymethyl)-6-trifluoromethyl-pyridine-3-carbonyl]-dimethoate Cyclo[3.2.1]oct-3-en-2-one, (3-[2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydro-2H-pyrimidin-1-yl)-phenoxy]-pyridin-2-yloxy)-acetic acid ethyl ester, 6-amino-5-chloro-2-cyclopropyl-pyrimidine-4-carboxylic acid methyl ester, 6-chloro-3-(2-cyclopropyl-6-methyl-phenoxy)-pyridazin-4-ol, 4-amino-3-chloro-6-(4-chloro-phenyl)-5-fluoro-pyridine-2-carboxylic acid, 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxy-phenyl)-pyridine-2-carboxylic acid methyl ester and 4-amino-3-chloro-6-(4-chloro-3-dimethylamino-2-fluoro-phenyl)-pyridine-2-carboxylic acid methyl ester. ;

[0279] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with an insecticide selected from the group consisting of: an organo(thio)phosphate selected from the group consisting of acephate, methyl pyraclostrobin, azinphos-methyl, chlorpyrifos, methyl chlorpyrifos, chlorfenvinphos, diazinon, dichlorvos, dicrotophos, dimethoate, ethion, ethion, fenitrothion, fenthion, isoxazophos, malathion, methamidophos, methidathion, methyl parathion, mefenphos, monocrotophos, sulfone, paraoxon, parathion, fenthion, phosphamidon ... Phosphorus, triazophos and trichlorfon; carbamates selected from the group consisting of: aldicarb, bensulfuron, carbaryl, carbofuran, carbofuran, fenoxycarb, furamicarb, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, thiodicarb and trichlorfon; pyrethroids selected from the group consisting of: allethrin, bifenthrin, cypermethrin, flucythrin, phenothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, zeta-cypermethrin, deltamethrin, cyper ... Tefluthrin, tetramethrin, tralomethrin, transfluthrin, profluthrin and transfluthrin; an insect growth regulator selected from the group consisting of: a) a chitin synthesis inhibitor, wherein the chitin synthesis inhibitor is a benzoyl urea selected from the group consisting of: chlorpyrifos, cyramazin, diflubenzuron, flufenoxuron, flufenoxuron, fluflumuron, lufenuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron; buprofezin, benzathine, hexathiazox, etoxazole and clofentezin; b) an ecdysone antagonist selected from the group consisting of: chlorfenapyr, methoxyfenopyr, fenbufenozide and azadirachtin; c) a juvenile hormone analog selected from the group consisting of: pyriproxyfen, methoprene and phenoxyprogesterone. or d) a lipid biosynthesis inhibitor selected from the group consisting of spirodiclofen, spiromesifen and spirotetramat; a nicotinic receptor agonist / antagonist compound selected from the group consisting of clothianidin, dinotefuran, imidacloprid, thiamethoxam, nitenpyram, acetamiprid, thiamethoxam and 1-(2-chloro-thiazol-5-ylmethyl)-2-nitrylimino-3,5-dimethyl-[1,3,5]triazine; a GABA antagonist compound selected from the group consisting of endosulfan, ethiprole, fipronil, flupyrazone, pyrazine fipronil, pyraclostrobin and 5-amino-1-(2,6-dichloro-4-methyl-phenyl)-4-sulfonylaminoacyl-1H-pyrazole-3-thiocarboxamide;A macrolide insecticide selected from the group consisting of avermectin, emamectin, mibemycin, lepidomectin, spinosad and ethyl spinosad; a mitochondrial electron transport inhibitor (METI) I scabicide selected from the group consisting of fenazaquin, pyraclostrobin, tebufenpyrad, tolfenpyrad and pyrimidine; a METI selected from the group consisting of II and III compounds: acequinoxaline, fluacyprim and hydrazone; chlorfenapyr; oxidative phosphorylation inhibitors selected from the group consisting of tricyclam, diafenthiuron, fenbutatin and propargite; cryomazine; piperonyl butoxide; sodium channel blockers selected from the group consisting of indoxacarb and metaflumizone; and compounds selected from the group consisting of benclothiaz, bifenazate, batan, flonicamid, pyridalyl, pymetrozine, sulfur, flubendiamide, chlorfenapyr, cyantraniliprole (HGW86), cypermethrin, flufenapyr, fluazifop, flufenapyr, sulfamethoxam, imicyafos, bistrifluan and fluquintocet. ;

[0280] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial consortia disclosed herein with known pesticides in the agricultural field, such as: pesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, miticides, nematicides, scabicides, rodenticides and / or anti-algae agents.

[0281] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial consortia disclosed herein with known biopesticides in the agricultural field, such as: biopesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, miticides, nematicides, scabicides, rodenticides and / or anti-algae agents.

[0282] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a pesticide, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a pesticide, a synergistic effect on a plant phenotypic trait of interest is observed.

[0283] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biopesticide, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biopesticide, a synergistic effect on a plant phenotypic trait of interest is observed.

[0284] The synergistic effect obtained by the methods taught can be quantified according to the Colby formula (i.e., (E) = X + Y - (X * Y / 100)). See Colby, RS, "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations," 1967 Weeds, Vol. 15, pp. 20-22, which is incorporated herein by reference in its entirety. Thus, the term "synergistic" is intended to be an amount that, when present, increases the desired effect beyond the additive amount.

[0285] The isolated microorganisms and consortia of the present disclosure can enhance the efficacy of agriculturally active pesticide compounds as well as agriculturally adjunct pesticide compounds in a synergistic manner.

[0286] The isolated microorganisms and consortia of the present disclosure can enhance the efficacy of agriculturally active biopesticide compounds as well as agriculturally auxiliary biopesticide compounds in a synergistic manner.

[0287] Plant growth regulators and biostimulants

[0288] In some embodiments, the agricultural compositions of the present disclosure include plant growth regulators and / or biostimulants used in combination with the taught microorganisms.

[0289] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with known plant growth regulators in the agricultural field, such as: auxins, gibberellins, cytokinins, ethylene generators, growth inhibitors, and growth retardants.

[0290] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients, including: cyprodinil, butralin, alcohol, chlormequat, cytokinin, butyric acid, ethepohon, furamidol, gibberellic acid, gibberellic acid mixture, indole-3-butyric acid (IBA), maleic hydrazide, mefludide, mepiquat, mepiquat pentaborate, naphthaleneacetic acid (NAA), 1-naphthaleneacetamide (NAD), n-decanol, placlobutrazol, prohexadione-calcium, trinexapac-ethyl, uniconazole, salicylic acid, abscisic acid, ethylene, brassinolide, jasmonate, polyamines, nitric oxide, strigolactones or karrikins, etc.

[0291] In some embodiments, individual microorganisms or consortia or communities of microorganisms developed according to the disclosed methods can be combined with seed inoculants known in the agricultural arts, such as: In some embodiments, a Bradyrhizobium inoculum is used in combination with any single microorganism or microbial consortium disclosed herein. In certain aspects, when one of the aforementioned inoculants (e.g., or Bradyrhizobium) with a microorganism or a consortium of microorganisms as taught herein, a synergistic effect was observed.

[0292] In some embodiments, an agricultural composition of the present disclosure comprises a plant growth regulator comprising: kinetin, gibberellic acid, and indolebutyric acid and copper, manganese, and zinc.

[0293] In some embodiments, the present disclosure teaches agricultural compositions comprising one or more commercially available plant growth regulators, including but not limited to:

[0294] and

[0295] In some embodiments, the present invention teaches the synergistic use of the disclosed microorganisms or microbial consortia with plant growth regulators and / or stimulants, such as plant hormones or chemicals that affect the production or destruction of plant growth regulators.

[0296] In some embodiments, the present invention teaches plant hormones which may include: auxins (e.g., indoleacetic acid, IAA), gibberellins, cytokinins (e.g., kinetin), abscisic acid, ethylene (and its production, as regulated by ACC synthetase and destroyed by ACC deaminase).

[0297] In some embodiments, a single microorganism or a consortium of microorganisms or a community of microorganisms developed according to the disclosed methods can be combined with a biostimulant. Such biostimulants can be, but are not limited to, microbial organisms, plant extracts, algae, acids, biochar, and the like.

[0298] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with fertilizers, which can be organic (e.g., manure, blood meal, fish meal, etc.), nitrogen-based (e.g., nitrates, ammonium, urea, etc.), phosphate and potash fertilizers. Such fertilizers may also contain micronutrients, including but not limited to sulfur, iron, zinc, etc.

[0299] In some embodiments, the present invention teaches additional plant growth promoting chemicals that can act synergistically with the microorganisms and microbial consortia disclosed herein, such as: humic acid, fulvic acid, amino acids, polyphenols, and protein hydrolysates.

[0300] Thus, in some embodiments, the present disclosure provides for combining the taught microorganisms with In addition, the present disclosure provides for applying the taught microorganisms with The combination can be applied to any crop and using any method or application rate.

[0301] In some embodiments, the present disclosure teaches agricultural compositions having biostimulants.

[0302] As used herein, the term "biostimulant" refers to any substance used to stimulate the growth of microorganisms that may be present in soil or other plant growth media.

[0303] The level of microorganisms in the soil or growth medium is directly related to plant health. Microorganisms feed on biodegradable carbon sources, and therefore plant health is also related to the amount of organic matter in the soil. While fertilizers provide nutrients to feed and grow plants, in some embodiments, biostimulants provide biodegradable carbon (e.g., molasses, carbohydrates (e.g., sugars)) to feed and grow microorganisms. Unless otherwise expressly stated, a biostimulant may comprise a single ingredient, or a combination of several different ingredients that are capable of enhancing microbial activity or plant growth and development due to the effects of one or more of the ingredients, acting alone or in combination.

[0304] In some embodiments, biostimulants are compounds that produce non-nutritional plant growth responses. In some embodiments, many important benefits of biostimulants are based on their ability to influence hormonal activity. Hormones in plants (phytohormones) are chemical messengers that regulate normal plant development and responses to the environment. Root and shoot growth, as well as other growth responses, are regulated by phytohormones. In some embodiments, compounds in biostimulants can change the hormonal status of plants and have a significant impact on their growth and health. Therefore, in some embodiments, the present disclosure teaches kelp, humic acid, fulvic acid, and vitamin B as common components of biostimulants. In some embodiments, the biostimulants of the present disclosure enhance antioxidant activity, thereby enhancing the defense system of the plant. In some embodiments, vitamin C, vitamin E, and amino acids (such as glycine) are antioxidants contained in biostimulants.

[0305] In other embodiments, biostimulants can be used to stimulate the growth of microorganisms present in soil or other plant growth media. Previous studies have shown that when certain biostimulants comprising specific organic seed extracts (e.g., soybeans) are used in combination with a microbial inoculum, the biostimulants are able to stimulate the growth of microorganisms contained in the microbial inoculum. Thus, in some embodiments, the present disclosure teaches one or more biostimulants that, when used with a microbial inoculum, are able to enhance the population of native microorganisms and inoculum microorganisms. For a review of some popular uses of biostimulants, see Calvo et al., 2014, Plant Soil 383:3-41.

[0306] Combinations of plant parts, microorganisms and agricultural compositions

[0307] In some embodiments, the present disclosure teaches that a single microorganism or a consortium of microorganisms or a community of microorganisms or any combination of the foregoing (e.g., a microorganism comprising any one or more of the edited Paenibacillus strains described herein) can be applied to plant parts (optionally in combination with any agricultural composition) to improve plant phenotype.

[0308] Isolated microorganisms or communities or consortia (often interchangeably referred to as "microbes" or "microbes") can be applied to heterologous plant components to form a synthetic combination. A microorganism is considered heterologous to a plant component if it is not normally associated with the plant component in nature or, when present, is applied in an amount different from that found in nature. In some embodiments, a microorganism may be present in nature in one part of a plant and not in another part, and introduction of the microorganism into the other part of the plant is considered a heterologous association.

[0309] It is further contemplated that the microorganism, isolated or combined with a plant or plant component, can be further associated with one or more agricultural compositions, such as those described above.

[0310] Synthetic combinations of microorganisms and plant components, microorganisms and agricultural compositions, and microorganisms and plant components and agricultural compositions (often referred to as "synthetic compositions," ie, compositions comprising components not normally found associated in nature) are contemplated.

[0311] Plant component processing

[0312] In some embodiments, the present disclosure also relates to the discovery that treating plant parts with a combination of one or more of the disclosed microorganisms or agricultural compositions prior to sowing or planting can enhance desirable plant traits, such as plant growth, plant health, and / or plant insect resistance.

[0313] Thus, in some embodiments, the present disclosure teaches the use of one or more of a microorganism or microbial consortium as a plant component treatment agent. A plant component treatment agent can be a plant component coating applied directly to an untreated and "naked" plant component. However, a plant component treatment agent can be a plant component outer coating applied to a plant component that has been coated with one or more previous plant component coatings or plant component treatment agents. Previous plant component treatment agents can contain one or more active compounds (chemical or biological) and one or more inert ingredients.

[0314] The term "plant component treatment" generally refers to the application of materials to plant components prior to or during planting in the soil. Plant component treatments with the microorganisms and other agricultural compositions of the present disclosure have the advantage of delivering the treatment to areas where plant components are planted shortly before plant component germination and plant component emergence.

[0315] In other embodiments, the present disclosure also teaches that the use of plant component treatments can minimize the amount of microorganisms or agricultural compositions required to successfully treat plants and further limit worker exposure to microorganisms and compositions compared to application techniques such as spraying above the soil or over emerging plant components.

[0316] In addition, in some embodiments, the present disclosure teaches that the microorganisms disclosed herein are important for enhancing the early stages of plant life (e.g., within the first thirty days after emergence of plant components). Thus, in some embodiments, delivering the microorganisms and / or compositions of the present disclosure in the form of plant component treatments can place them in the area of ​​action at a time that is important for microbial activity.

[0317] In some embodiments, the microbial composition of the present disclosure is formulated as a plant component treatment agent. In some embodiments, it is envisioned that by using a treatment agent application equipment that is specifically designed and manufactured to accurately, safely and effectively apply plant component treatment products to plant components, one or more layers of microorganisms and / or agricultural compositions disclosed herein can be substantially uniformly applied to plant components using conventional mixing, spraying methods or their combinations. This type of equipment uses various types of coating techniques, such as a rotary coater, a drum coater, a fluidized bed technology, a fountain bed, a rotary spray or their combinations. Liquid plant component treatment agents (such as liquid plant component treatment agents of the present disclosure) can be applied by a spin "atomizer" disk or a spray nozzle, which evenly distributes the plant component treatment agent on the plant component when moving in a spray mode. In various aspects, the plant component is then mixed or tumbled for a period of time to realize additional treatment agent distribution and drying.

[0318] Prior to coating with the microbial composition, the plant components may be primed or unprimed to improve uniformity of germination and emergence. In an alternative embodiment, the dry powder formulation may be provided on the moving plant components by metering and allowed to mix until fully distributed.

[0319] In some embodiments, at least a portion of the surface area of ​​the plant component is coated with a microbial composition according to the present disclosure. In some embodiments, the plant component coating comprising the microbial composition is applied directly to the naked plant component. In some embodiments, the plant component outer coating comprising the microbial composition is applied to the plant component on which the plant component coating has been applied. In some aspects, the plant component may have a plant component coating comprising, for example, clothianidin and / or Bacillus firmus-I-1582, on top of which the present composition will be applied in the form of a plant component outer coating. In some aspects, the microbial composition taught is applied in the form of a plant component outer coating to a plant component that has been treated with PONCHO TM VOTiVO TM In some aspects, the plant component may have a plant component coating, which includes, for example, metalaxyl and / or clothianidin and / or Bacillus firmus-I-1582, on top of which the composition of the present invention is applied in the form of a plant component coating. In some aspects, the microbial composition taught is applied in the form of a plant component coating to a plant component that has been treated with ACCELERON TM Processed plant components.

[0320] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 10^2 to 10^12, 10^2 to 10^11, 10^2 to 10^10, 10^2 to 10^9, 10^2 to 10^8, 10^2 to 10^7, 10^2 to 10^6, 10^2 to 10^5, 10^2 to 10^4, or 10^2 to 10^3 per plant component.

[0321] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 10^3 to 10^12, 10^3 to 10^11, 10^3 to 10^10, 10^3 to 10^9, 10^3 to 10^8, 10^3 to 10^7, 10^3 to 10^6, 10^3 to 10^5, or 10^3 to 10^4 per plant component.

[0322] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 10^4 to 10^12, 10^4 to 10^11, 10^4 to 10^10, 10^4 to 10^9, 10^4 to 10^8, 10^4 to 10^7, 10^4 to 10^6, or 10^4 to 10^5 per plant component.

[0323] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 10^5 to 10^12, 10^5 to 10^11, 10^5 to 10^10, 10^5 to 10^9, 10^5 to 10^8, 10^5 to 10^7, or 10^5 to 10^6 per plant component.

[0324] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 10^5 to 10^9 per plant component.

[0325] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or microbial cell concentration of at least about 1×10^3 or 1×10^4 or 1×10^5 or 1×10^6 or 1×10^7 or 1×10^8 or 1×10^9 per plant component.

[0326] In some embodiments, the amount of one or more of the microorganisms and / or agricultural compositions applied to plant components depends on the final formulation, and the size or type of the plant or plant components utilized. In some embodiments, one or more of the microorganisms are present with about 2% w / w to about 80% w / w of the total formulation. In some embodiments, by weight, one or more of the microorganisms employed in the composition are about 5% w / w to about 65% w / w of the total formulation, or 10% w / w to about 60% w / w.

[0327] In some embodiments, the plant components may also have more spores or microbial cells per plant component, for example, about 10^2, 10^3, 10^4, 10^5, 10^6, 10^7, 10^8, 10^9, 10^10, 10^11, 10^12, 10^13, 10^14, 10^15, 10^16, or 10^17 spores or cells per plant component.

[0328] In some embodiments, the thickness of the coating of the plant component of the present disclosure can be at most 10μm, 20μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 110μm, 120μm, 130μm, 140μm, 150μm, 160μm, 170μm, 180μm, 190μm, 200μm, 210μm, 220μm, 230μm, 240μm, 250μm, 260μm, 270μm, 280μm, 290μm, 300μm, 310μm, 320μm, 330μm, 340μm, 350μm, 360μm, 370μm, 380μm, 390μm, 400μm, 410μm, 420μm, 430μm, 440μm, 450μm, 460μm, 470μm, 480μm, 490μm, 500μm, 510μm, 520μm, 530μm, 540μm, 550μm, 560μm, 570μm, 580μm, 590μm, 600μm, 610μm, 620μm, 630μm, 640μm, 650μm, 660μm, 670μm, 680μm, 690μm, 700μm, 710μm, 720μm, 730μm, 740μm, 750μm, 760μm, 770μm, 780μm, 790μm, 800μm, 810μm, 820μm, 830μm, 840μm, 850μm, 860μm, 870μm, 880μm, 890μm, 900μm, 910μm, 920μm, 930μm, 940μm, 950μm, 960μm, 970μm, 980μm, 990μm, 1000μm, 1010μm, 1020μm, 1030μm, 1040μm, 1050μm, 1060μm, 1070μm, 1080μm, 1090μm, 1100μm, 1110μm, 1120μm, 1130μm, 1140μm, 1150μm, 1160μm, 1170μm, 1180μm, 1190μm, 1200μm, 1210μm, 1220μm, 1230μm, 1240μm, 1250μm, 1260μm, 1270μm, 1280μm, 1290μm, 1300μm, 1310μm, 1320μm, 1330μm, 1340μm, 1350μm, 1360μm, 1370μm, 1380μm, 1390μm, 1400μm, 1410μm, 1420μm, 1430μm, 1440μm, 1450μm, 1460μm, 1470μm, 1480μm, 1490μm, 1500μm, 1510μm, 1520μm, 1530μm, 1540μm,1550μm, 1560μm, 1570μm, 1580μm, 1590μm, 1600μm, 1610μm, 1620μm, 1630μm, 1640μm, 1650μm, 1660μm, 1670μm, 1680μm, 1690μm, 1700μm, 1710μm, 1720μm ,1730μm,1740μm,1750μm,1760μm,1770μm,1780μm,1790μm,1800μm,1810μm,1820μm,1830μm,1840μm,1850μm,1860μm,1870μm,1880μm,1890μm,1900μm m, 1910μm, 1920μm, 1930μm, 1940μm, 1950μm, 1960μm, 1970μm, 1980μm, 1990μm, 2000μm, 2010μm, 2020μm, 2030μm, 2040μm, 2050μm, 2060μm, 2070μm, 2080 μm, 2090μm, 2100μm, 2110μm, 2120μm, 2130μm, 2140μm, 2150μm, 2160μm, 2170μm, 2180μm, 2190μm, 2200μm, 2210μm, 2220μm, 2230μm, 2240μm, 2250μm, 226 0μm, 2270μm, 2280μm, 2290μm, 2300μm, 2310μm, 2320μm, 2330μm, 2340μm, 2350μm, 2360μm, 2370μm, 2380μm, 2390μm, 2400μm, 2410μm, 2420μm, 2430μm, 24 40μm, 2450μm, 2460μm, 2470μm, 2480μm, 2490μm, 2500μm, 2510μm, 2520μm, 2530μm, 2540μm, 2550μm, 2560μm, 2570μm, 2580μm, 2590μm, 2600μm, 2610μm, 2 620μm, 2630μm, 2640μm, 2650μm, 2660μm, 2670μm, 2680μm, 2690μm, 2700μm, 2710μm, 2720μm, 2730μm, 2740μm, 2750μm, 2760μm, 2770μm, 2780μm, 2790μm, 2800μm, 2810μm, 2820μm, 2830μm, 2840μm, 2850μm, 2860μm, 2870μm, 2880μm, 2890μm, 2900μm, 2910μm, 2920μm, 2930μm, 2940μm, 2950μm, 2960μm, 2970μm,2980μm, 2990μm or 3000μm.

[0329] In some embodiments, the thickness of the plant component coating of the present disclosure can be 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

[0330] In some embodiments, the coating of the plant components of the present disclosure can be at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24% by weight of the uncoated plant component. , 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37% , 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5% or 50%.

[0331] In some embodiments, microbial spores and / or cells may be freely applied to the plant component or they may be formulated in a liquid or solid composition prior to being applied to the plant component. For example, a solid composition comprising microorganisms may be prepared by mixing a solid carrier with a suspension of spores until the solid carrier is impregnated with the spore or cell suspension. The mixture may then be dried to obtain the desired particles.

[0332] In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the present disclosure further contain functional agents, such as activated carbon, nutrients (fertilizers), and other agents capable of improving germination and quality of the product or a combination thereof.

[0333] Plant component coating methods and compositions known in the art may be particularly useful when they are modified by adding one of the embodiments of the present disclosure. Such coating methods and their application equipment are disclosed, for example, in U.S. Pat. Nos. 5,916,029, 5,918,413, 5,554,445, 5,389,399, 4,759,945, 4,465,017, and U.S. Patent Application No. 13 / 260,310, each of which is incorporated herein by reference.

[0334] Plant part coating compositions are disclosed, for example, in U.S. Pat. Nos. 5,939,356, 5,876,739, 5,849,320, 5,791,084, 5,661,103, 5,580,544, 5,328,942, 4,735,015, 4,634,587, 4,372,080, 4,339,456, and 4,245,432, each of which is incorporated herein by reference.

[0335] In some embodiments, a variety of additives can be added to the plant component treatment formulation comprising the composition of the present invention. A binder can be added, and the binder includes a binder consisting of a natural or synthetic adhesive polymer that has no phytotoxic effect on the applied plant component. The binder can be selected from polyvinyl acetate; polyvinyl acetate copolymer; ethylene-vinyl acetate (EVA) copolymer; polyvinyl alcohol; polyvinyl alcohol copolymer; cellulose, including ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose and carboxymethyl cellulose; polyvinyl pyrrolidone; polysaccharides, including starch, modified starch, dextrin, maltodextrin, alginate and deacetylated chitosan; fat; oil; protein, including gelatin and zein; gum arabic; shellac; vinylidene chloride and vinylidene chloride copolymer; calcium lignin sulfonate; acrylic acid copolymer; polyvinyl acrylate; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methacrylamide monomer; and polychloroprene.

[0336] Any of a variety of colorants may be employed, including organic chromophores, which are classified as nitroso; nitro; azo, including monoazo, disazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. Other additives that may be added include trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.

[0337] Polymers or other dust control agents may be applied to retain the treatment on the surfaces of plant components.

[0338] In some embodiments, in addition to microbial cells or spores, the coating may also include an adhesive layer. The adhesive should be non-toxic, biodegradable and adhesive. Examples of such materials include, but are not limited to, polyvinyl acetate; polyvinyl acetate copolymers; polyvinyl alcohol; polyvinyl alcohol copolymers; cellulose, such as methylcellulose, hydroxymethylcellulose and hydroxymethylpropylcellulose; dextrin; alginate; sugar; molasses; polyvinyl pyrrolidone; polysaccharides; proteins; fats; oils; gum arabic; gelatin; syrups; and starch. More examples can be found, for example, in U.S. Pat. No. 7,213,367, which is incorporated herein by reference.

[0339] Various additives may also be included in the plant component treatment formulation, such as adhesives, dispersants, surfactants and nutrients and buffering components. Other conventional plant component treatment additives include, but are not limited to, coating agents, wetting agents, buffers and polysaccharides. At least one agriculturally acceptable carrier, such as water, solids or dry powders, may be added to the plant component treatment formulation. Dry powders may be derived from a variety of materials, such as calcium carbonate, gypsum, vermiculite, talc, humus, activated carbon and a variety of phosphorus compounds.

[0340] In some embodiments, the plant component coating composition may include at least one filler, which is an organic or inorganic, natural or synthetic component with which the active ingredients are combined to facilitate its application on the plant component. In various aspects, the filler is an inert solid such as clay, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (e.g., ammonium salts), natural soil minerals (such as kaolin, clay, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earth) or synthetic minerals (such as silica, alumina or silicates, especially aluminum silicate or magnesium silicate).

[0341] In some embodiments, the plant component treatment formulation may also include one or more of the following ingredients: other insecticides, including compounds that work only below the ground; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadone, and isomers of each of these materials, etc.; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxy groups, ureas, and benzoic acid; herbicide safeners , such as benzoxazines, benzhydryl derivatives, N,N-diallyl dichloroacetamide, various dihaloacetyl, oxazolidinyl and thiazolidinyl compounds, acetone, naphthalic anhydride compounds and oxime derivatives; chemical fertilizers; biological fertilizers; and biological control agents, such as other naturally occurring or recombinant bacteria and fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. These ingredients can be added as a separate layer on the plant component, or alternatively can be added as part of the plant component coating composition of the present disclosure.

[0342] In some embodiments, the formulations used in the present disclosure for treating plant parts may be in the form of suspensions; emulsions; slurries of particles in an aqueous medium (e.g., water); wettable powders; wettable granules (dry flowable); and dry granules. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation may be from about 0.5% to about 99% by weight (w / w), or 5% to 40% by weight, or otherwise formulated by one skilled in the art.

[0343] As mentioned above, other conventional inactive or inert ingredients may be incorporated into the formulation. Such inert ingredients include, but are not limited to, conventional adhesives; dispersants, such as methylcellulose, for example, acting as a combined dispersant / adhesive for plant component treatment agents; polyvinyl alcohol; lecithin, polymeric dispersants (e.g., polyvinyl pyrrolidone / vinyl acetate); thickeners (e.g., clay thickeners for increasing viscosity and reducing sedimentation of particle suspensions); emulsion stabilizers; surfactants; antifreeze compounds (e.g., urea), dyes, colorants, etc. Additional inert ingredients that may be used in the present disclosure may be found in McCutcheon's, Vol. 1, "Emulsifiers and Detergents," MC Publishing Company, Glen Rock, NJ, USA, 1996, which is incorporated herein by reference.

[0344] The plant component coating formulations of the present disclosure can be applied to the plant component by a variety of methods, including but not limited to: mixing in a container (e.g., a bottle or bag), mechanical application, tumbling, spraying, and immersion. A variety of active or inert materials can be used to contact the plant component with the microbial composition according to the present disclosure.

[0345] In some embodiments, the amount of microorganism or agricultural composition used to treat the plant component will vary depending on the type of plant component and the type of active ingredient, but treatment will include contacting the plant component with an agriculturally effective amount of a composition of the invention.

[0346] As discussed above, an effective amount means an amount of a composition of the invention sufficient to affect beneficial or desired results. An effective amount can be administered over the course of one or more administrations.

[0347] In some embodiments, in addition to the coating layer, the plant parts may be treated with one or more of the following ingredients: other pesticides, including fungicides and herbicides; herbicide safeners; fertilizers and / or biological control agents. These ingredients may be added as a separate layer, or alternatively may be added in the coating layer.

[0348] In some embodiments, the plant component coating formulations of the present disclosure can be applied to the plant component using a variety of techniques and machines, such as fluidized bed technology, roller mill methods, drum electrostatic plant component processors, and drum coaters. Other methods (such as spouted beds) may also be useful. The plant component can be pre-sized before coating. After coating, the plant component is typically dried and then transferred to a sizing machine for sizing. Such procedures are known in the art.

[0349] In some embodiments, the plant parts treated with microorganisms can also be wrapped with a film outer coating to protect the coating. Such outer coatings are known in the art and can be applied using fluidized bed and drum film coating techniques.

[0350] In other embodiments of the present disclosure, can cause by using solid matrix to be introduced on the plant component according to the composition of the present disclosure.For example, a certain amount of the present composition can be mixed with solid matrix material, and then can place plant component and make it contact with solid matrix material for a period of time to allow composition to be introduced into plant component.Then, can optionally plant component be separated from solid matrix material and store or use, or can directly store or plant the mixture that solid matrix material adds plant component.Solid matrix material that can be used for the present disclosure comprises polyacrylamide, starch, clay, silicon dioxide, aluminum oxide, soil, sand, polyurea, polyacrylate or any other material that can absorb or adsorb the present composition for a period of time and the composition is released in the plant component or is released on the plant component.It is very useful to ensure that the present composition and solid matrix material are compatible with each other.For example, solid matrix material should be selected so that it can be released at a reasonable rate (for example in the time period of a few minutes, a few hours or a few days).

[0351] In some embodiments, the present disclosure teaches that individual microorganisms or consortia of microorganisms or communities of microorganisms developed according to the disclosed methods can be combined with any plant biostimulant.

[0352] In some embodiments, the present disclosure teaches agricultural compositions comprising one or more commercially available biostimulants, including but not limited to: Diehard TM Diehard TM Fe, Diehard TM Soluble Kelp, Diehard TM Humate SP, Foliar Plus TM 、Plant Plus TM 、Accomplish Soil Builder TM 、Nutri Life、SoilSolution TM 、Seed Coat TM 、PercPlus TM 、Plant Thrust TM , and wait.

[0353] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with an active chemical agent, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with an active chemical agent, a synergistic effect on a plant phenotypic trait of interest is observed.

[0354] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a fertilizer, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a fertilizer, a synergistic effect on a plant phenotypic trait of interest is observed.

[0355] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a plant growth regulator, an additive effect on a plant phenotypic trait of interest is observed. In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a plant growth regulator, a synergistic effect is observed. In some aspects, combining a microorganism of the present disclosure with a plant growth regulator Combine and observe synergistic effects on one or more phenotypic traits of interest.

[0356] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biostimulant, an additive effect on a plant phenotypic trait of interest is observed. In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biostimulant, a synergistic effect is observed.

[0357] The synergistic effect obtained by the methods taught can be quantified according to the Colby formula (i.e., (E) = X + Y - (X * Y / 100)). See Colby, RS, "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations," 1967 Weeds, Vol. 15, pp. 20-22, which is incorporated herein by reference in its entirety. Thus, the term "synergistic" is intended to be an amount that, when present, increases the desired effect beyond the additive amount.

[0358] The isolated microorganisms and consortia of the present disclosure can enhance the efficacy of agriculturally active compounds as well as agricultural adjuvant compounds in a synergistic manner.

[0359] In other embodiments, a synergistic effect is observed when a microorganism or consortium of microorganisms identified according to the taught methods is combined with a fertilizer.

[0360] In addition, in certain embodiments, the present disclosure utilizes synergistic interactions to define microbial consortia. That is, in certain aspects, the present disclosure combines certain isolated microbial species that act synergistically together into a consortium, which imparts beneficial traits to plants, or is associated with enhancing beneficial plant traits.

[0361] Agricultural compositions developed according to the present disclosure can be formulated with certain adjuvants to increase the activity of known active agricultural compounds. This has the advantage that the amount of active ingredient in the formulation can be reduced while maintaining the efficacy of the active compound, thus keeping the cost as low as possible and complying with any official regulations. In individual cases, it is also possible to broaden the scope of action of the active compound, because plants (which are not successfully treated with a specific active ingredient without addition) can actually be successfully treated by adding certain adjuvants and the disclosed microbial isolates and consortia. In addition, when environmental conditions are unfavorable, the performance of the active substance can be increased in individual cases by suitable formulations.

[0362] Such auxiliary agents that can be used in agricultural compositions can be adjuvants. Usually, adjuvants are in the form of surface-active or salt compounds. According to their mode of action, they can be roughly classified as regulators, activators, fertilizers, pH buffers, etc. Regulators affect the wetting, adhesion and spreading characteristics of the preparation. Activators destroy the waxy surface layer of plants and improve the penetration of active ingredients into the surface layer (short-term (several minutes) and long-term (several hours)). Fertilizers (such as ammonium sulfate, ammonium nitrate or urea) improve the absorption and solubility of active ingredients and can reduce the antagonistic behavior of active ingredients. pH buffers are routinely used to adjust the preparation to the optimal pH.

[0363] In some embodiments, the plant component is a plant reproductive component (e.g., a seed, tuber, bulb, and / or bud). In some embodiments, the plant component is other than a plant reproductive component (e.g., a leaf, stem, and / or root). In some embodiments, a plurality of plant components are associated with a microorganism as described herein.

[0364] In some embodiments, a plant or plant component is associated with one or more microorganisms described herein via an indirect method, such as, but not limited to, treatment of a growth medium in which the plant or plant component is placed.

[0365] For additional embodiments of the agricultural compositions of the present disclosure, see "Chemistry and Technology of Agrochemical Formulations," DA Knowles, ed., copyright 1998, Kluwer Academic Publishers, which is hereby incorporated by reference.

[0366] Botanical and Agronomic Benefits

[0367] A wide variety of plants, including those cultivated agriculturally, can benefit from the application of microorganisms, such as those described herein, including single microorganisms, consortia, and / or compositions produced therefrom, or including any of the foregoing. Many various plants, including mosses and lichens and algae, can be used in the methods of the present disclosure. In embodiments, plants have economic, social or environmental value. For example, plants can include those plants used as food crops, fiber crops, oil crops, for forestry, for pulp and paper industry, as raw materials for biofuel production, and as ornamental plants.

[0368] The genetically modified microorganisms disclosed herein can be used to improve nitrogen fixation in plants. In some embodiments, such plants include those that lack natural nitrogen fixing symbionts (e.g., non-leguminous crops), such as, but not limited to, wheat, maize (corn), rice, and vegetables. In some embodiments, such plants include those that would benefit from additional nitrogen fixation.

[0369] Application method

[0370] The microorganisms may be applied to plants, seedlings, cuttings, propagules, etc. and / or to a growth medium containing the plants using any suitable technique known in the art.

[0371] However, as an example, the microorganisms, consortia, or compositions comprising and / or produced therefrom may be applied to plants, seedlings, cuttings, propagules, etc. by spraying, coating, spreading, or any other method known in the art.

[0372] In another embodiment, the isolated microorganism, consortium or composition comprising the same may be applied directly to plant seeds prior to sowing.

[0373] In another embodiment, the isolated microorganism, consortium or composition comprising the same may be applied directly to plant seeds in the form of a seed coating.

[0374] In one embodiment of the present disclosure, the isolated microorganism, consortium or composition comprising the same is supplied in the form of granules or a plug or soil drench for application to a plant growth medium.

[0375] In other embodiments, the isolated microorganism, consortium, or composition comprising the same is provided in the form of a foliar application, such as a foliar spray or a liquid composition. The foliar spray or liquid application can be applied to growing plants or a growing medium, such as soil.

[0376] In some embodiments, the isolated microorganism, consortium, or composition comprising the same is provided in a form selected from the group consisting of a soil drench, a foliar spray, a drench treatment, an in-furrow treatment, a soil conditioner, a granule, a broadcast treatment, a post-harvest disease control treatment, or a seed treatment. In some embodiments, the agricultural composition may be applied alone or in a rotary spray procedure.

[0377] In some embodiments, the isolated microorganisms, consortia, or compositions comprising the same are compatible with tank mixing. In some embodiments, the agricultural compositions are compatible with tank mixing with other agricultural products. In some embodiments, the agricultural compositions are compatible with equipment for ground, aerial, and irrigation applications.

[0378] In another embodiment, the isolated microorganism, consortium or composition comprising the same may be formulated into granules and applied next to the seeds during planting. Or the granules may be applied after planting. Or the granules may be applied before planting.

[0379] In some embodiments, the isolated microorganism, consortium or composition comprising the same is applied to plants or growth media in the form of topical application and / or drench application to improve crop growth, yield and quality. Topical application can be carried out by using a dry mixture or a powder or dusting composition or can be a liquid-based formulation.

[0380] In embodiments, the isolated microorganisms, consortia, or compositions comprising the same may be formulated as: (1) solutions; (2) wettable powders; (3) dusting powders; (4) soluble powders; (5) emulsions or suspension concentrates; (6) seed dressings or seed coatings, (7) tablets; (8) water-dispersible granules; (9) water-soluble granules (slow release or quick release); (10) microencapsulated granules or suspensions; (11) as irrigation components, and (12) as components of fertilizers, pesticides, and other compatible improvers, etc. In certain aspects, the composition may be diluted in an aqueous medium prior to conventional spray application. The compositions of the present disclosure may be applied to soil, plants, seeds, rhizosphere, rhizosheath, or other areas that would be conducive to the application of the microbial composition. In addition, impact methods may be used as a means for introducing endogenous microorganisms.

[0381] In various aspects, the composition is applied to the leaves of the plant. The composition can be applied to the leaves of the plant in the form of an emulsion or suspension concentrate, a liquid solution or a foliar spray. The application of the composition can be carried out in a laboratory, a growth chamber, a greenhouse or a field.

[0382] In another embodiment, the microorganisms can be inoculated into the plant by pruning the roots or stems and exposing the plant surface to the microorganisms by spraying, dipping or otherwise applying a liquid microorganism suspension or a gel or powder.

[0383] In another embodiment, the microorganisms can be injected directly into the foliage or root tissue, or otherwise inoculated directly into or onto the foliage or root prunings, or inoculated into isolated embryos or radicles or coleoptiles. These inoculated plants can then be further exposed to growth media containing additional microorganisms; however, this is not required.

[0384] In other embodiments, particularly in the case where the microorganism is not culturable, the microorganism can be transferred to the plant by any one or combination of the following means: grafting, insertion into an explant, suction, electroporation, wounding, root pruning, inducing stomatal opening, or any physical, chemical or biological treatment that provides an opportunity for the microorganism to enter the plant cells or intercellular spaces. Those skilled in the art will readily appreciate the many alternative techniques that can be used.

[0385] In one embodiment, the microorganism infiltrates a part of a plant such as a root, stem, leaf, and / or reproductive plant part (becoming endophytic), and / or grows on the surface of a root, stem, leaf, and / or reproductive plant part (becoming epiphytic), and / or grows in the rhizosphere of the plant. In one embodiment, the microorganism forms a symbiotic relationship with the plant.

[0386] In some exemplary non-limiting embodiments, aspects of the invention are described below.

[0387] Aspect 1: A synthetic composition comprising a plant component and a Paenibacillus bacterium heterologously placed in the plant component, wherein the Paenibacillus bacterium comprises an edit in one or more loci of its genome; wherein the edit is a deletion of at least one nucleotide, an insertion of at least one nucleotide, and / or a substitution of at least one nucleotide at or near one or more of the following genomic loci: nifH, glnR, nifD, GlnR binding site I, GlnR binding site II, cueR, orf1, nrgA, glnA, nif operator a promoter region, a nif cluster component promoter, or any combination or multiple edits at any one or more of the genomic loci; wherein the Paenibacillus bacterium exhibits an improved phenotype compared to a Paenibacillus bacterium not comprising the edits, wherein the improved phenotype is selected from the group consisting of: increased acetylene reduction ability, improved nitrogen fixation ability, improved biofilm formation, increased culture turbidity, greater nitrogen fixation tolerance to oxygen levels, improved nitrogen fixation under high nitrogen conditions, improved nitrogen fixation under low nitrogen conditions, and any multiple of the foregoing and / or combinations of the foregoing.

[0388] Aspect 2: A synthetic composition as described in Aspect 1, wherein the editing is selected from the group consisting of: CueRC25 truncation, GlnR site II inactivation, CueR knockout, Nif PLH77 promoter insertion, Nif PLH77d promoter insertion, GlnR site II duplication and inactivation, GlnR C25 frameshift truncation and glnA SNP, NifH knockout, Orf1 knockout, GlnR C25 truncation, GlnR site II duplication, NrgA knockout, Suf PLH77 promoter exchange, Suf PLH77d promoter exchange, GlnR binding site II truncation, GlnR binding site II truncation and duplication, and any multiple of the foregoing items and / or combinations of the foregoing items, and any multiple of the foregoing items and / or combinations of the foregoing items.

[0389] Aspect 3: The synthetic composition of aspect 1, wherein the Paenibacillus bacterium comprises a sequence selected from the group consisting of SEQ ID NOs: 1-123.

[0390] Aspect 4: The synthetic composition of aspect 1, wherein the bacillus bacterium is a species selected from the group consisting of: Paenibacillus polymyxa, Paenibacillus tritici, Paenibacillus albus, Paenibacillus anaerobic, Paenibacillus nitrogen-fixing, Paenibacillus boraye, Paenibacillus tokai, Paenibacillus ehime, Paenibacillus graminearum, Paenibacillus lijilunshi, Paenibacillus heterotatissimus, Paenibacillus ginseng land, Paenibacillus phoenix, Paenibacillus sacheon, Paenibacillus rhizosphere, Paenibacillus silage, Paenibacillus peach blossom mountain, Paenibacillus thermophilus, Paenibacillus cattail, Paenibacillus tenacious, and Paenibacillus wensleyi.

[0391] Aspect 5: The synthetic composition according to aspect 1, wherein the Paenibacillus bacteria belongs to subgroup I.

[0392] Aspect 6: The synthetic composition according to aspect 1, wherein the Paenibacillus bacterium belongs to subgroup II.

[0393] Aspect 7: The synthetic composition of aspect 1, wherein the Paenibacillus bacterium is selected from the group consisting of: NRRL deposit numbers: B-68102, B-68103, B-68104, B-68105, B-68106, B-68107, B-68108, B-68109 and B-68110.

[0394] Aspect 8: The synthetic composition according to aspect 1, further comprising a formulation component and / or an agricultural composition.

[0395] Aspect 9: The synthetic composition of Aspect 1, wherein the Paenibacillus bacteria is present at a concentration of at least about 10^2 CFU / mL in a liquid formulation, or at a concentration of at least about 10^2 CFU / gram in a non-liquid formulation.

[0396] Aspect 10: The synthetic composition of Aspect 1, further comprising at least one additional microorganism.

[0397] Aspect 11: The synthetic composition of Aspect 1, wherein the plant component is a seed.

[0398] Aspect 12: The synthetic composition of Aspect 1, wherein the plant component is a seed comprising a transgene.

[0399] Aspect 13: The synthetic composition of Aspect 1, wherein the plant component is a leaf.

[0400] Aspect 14: The synthetic composition of Aspect 1, wherein the plant component is a root.

[0401] Aspect 15: The synthetic composition of Aspect 1, wherein the plant component is a whole plant.

[0402] Aspect 16: The synthetic composition of Aspect 1, wherein the plant component is a plant propagation component.

[0403] Aspect 17: The synthetic composition of aspect 1, wherein the formulation components are selected from the group consisting of: compounds that improve the stability of the microorganism, preservatives, carriers, surfactants, anti-complexing agents, and any combination thereof.

[0404] Aspect 18: The synthetic composition of aspect 1, wherein the agricultural composition comprises a fungicide, a nematicide, a bactericide, an insecticide, a herbicide, a micronutrient, a macronutrient, nitrogen, phosphorus, potassium, or any multiple of the foregoing and / or a combination of the foregoing.

[0405] Aspect 19: A plurality of synthetic compositions according to Aspect 1, wherein the synthetic composition is substantially enclosed within an article selected from the group consisting of a tube, a bottle, a jar, an ampoule, a package, a container, a bag, a box, a storage box, an envelope, a carton, a container, a silo, a shipping container, a carriage, and a case.

[0406] Aspect 20: A plurality of synthetic compositions according to aspect 19, wherein the synthetic compositions are at a temperature below zero degrees Celsius.

[0407] Aspect 21: The synthetic composition of Aspect 1, wherein the plant part is obtained from a monocotyledonous plant.

[0408] Aspect 22: The synthetic composition of aspect 21, wherein the monocot is a C3 monocot.

[0409] Aspect 23: The synthetic composition of aspect 21, wherein the monocot is a C4 monocot.

[0410] Aspect 24: The synthetic composition of Aspect 1, wherein the plant part is obtained from a dicotyledonous plant.

[0411] Aspect 25: The synthetic composition of Aspect 1, wherein the agricultural composition comprises a growth medium.

[0412] Aspect 26: The synthetic composition of Aspect 25, wherein the growth medium comprises soil.

[0413] Aspect 27: A plurality of synthetic compositions according to aspect 1, wherein the plurality of synthetic compositions are placed in the soil in a regular pattern with substantially equal spacing between each of the synthetic compositions.

[0414] Aspect 28: A method of improving the health, yield and / or vigor of a plant, the method comprising: (a) associating a component of the plant with a Paenibacillus bacterium comprising an edit in one or more loci of its genome; wherein the edit is a deletion of at least one nucleotide, an insertion of at least one nucleotide and / or a substitution of at least one nucleotide at or near one or more of the following genomic loci: nifH, glnR, nifD, GlnR binding site I, GlnR binding site II, cueR, orf1, nrgA, glnA, nif operon promoter region, nif cluster component promoter, or any combination or multiple edits at any one or more of the genomic loci; (b) placing the component of the crop plant in a medium that supports plant growth; (c) growing a plant from the component of the crop plant; (d) evaluating one or more characteristics of the plant, wherein at least one of the characteristics is improved compared to the same characteristic of a plant not obtained from the component associated with the Paenibacillus bacterium of (a).

[0415] Aspect 29: A method as described in Aspect 28, wherein the one or more characteristics of (d) include improved nitrogen fixation, increased biomass, increased leaf area, increased plant height, increased root area, increased shoot nitrogen composition, increased greenness, increased NDVI, increased NPCI, increased PSRI, increased CCI, increased yield, and any combination of the foregoing.

[0416] Aspect 30: The method of Aspect 28, further comprising at least one additional microorganism.

[0417] Aspect 31: The method of Aspect 28, wherein associating the component of the crop plant with the Paenibacillus bacterium comprising an edit in one or more loci of its genome is achieved by a method selected from the group consisting of: in-furrow application, soil sprinkle application, side application, and any combination of the foregoing.

[0418] Aspect 32: The method of Aspect 28, wherein associating the plant component with the Paenibacillus bacterium comprising an edit in one or more loci of its genome is achieved by coating the crop plant component with a liquid formulation of the bacterium.

[0419] Aspect 33: The method of Aspect 28, wherein associating the plant component with the Paenibacillus bacterium comprising an edit in one or more loci of its genome is achieved by coating the crop plant component with a substantially non-liquid formulation of the bacterium.

[0420] Aspect 34: The method of Aspect 28, wherein the plant component is a seed.

[0421] Aspect 35: The method of Aspect 28, wherein the plant component is a leaf.

[0422] Aspect 36: The method of Aspect 28, wherein the plant component is a root.

[0423] Aspect 37: The method of aspect 28, wherein the plant component is a whole plant.

[0424] Aspect 38: A modified Paenibacillus bacterium, wherein the Paenibacillus bacterium comprises an edit in one or more loci of its genome, wherein the edit is a deletion of at least one nucleotide, an insertion of at least one nucleotide, and / or a substitution of at least one nucleotide at or near one or more of the following genomic loci: nifH, glnR, nifD, GlnR binding site I, GlnR binding site II, cueR, orf1, nrgA, glnA, nif operator promoter region, nif cluster component promoter, or any combination or multiple edits at any one or more of the genomic loci.

[0425] Aspect 39: The modified Paenibacillus bacterium of Aspect 38, wherein the Paenibacillus bacterium exhibits an improved phenotype compared to a Paenibacillus bacterium not comprising the edit, wherein the improved phenotype is selected from the group consisting of increased acetylene reduction ability, improved biofilm formation, increased culture turbidity, greater tolerance to nitrogen fixation to oxygen levels, and any combination of the foregoing.

[0426] Aspect 40: A modified Paenibacillus bacterium as described in Aspect 38, wherein the editing is selected from the group consisting of: CueR C25 truncation, GlnR site II inactivation, CueR knockout, Nif PLH77 promoter insertion, NifPLH77d promoter insertion, GlnR site II duplication and inactivation, GlnR C25 frameshift truncation and glnA SNP, NifH knockout, Orf1 knockout, GlnRC25 truncation, GlnR site II duplication, NrgA knockout, Suf PLH77 promoter exchange, SufPLH77d promoter exchange, GlnR binding site II truncation, GlnR binding site II truncation and duplication, and any multiple of the foregoing items and / or combinations of the foregoing items.

[0427] Aspect 41: The modified Paenibacillus bacterium of aspect 38, wherein the Paenibacillus bacterium comprises a sequence selected from the group consisting of SEQ ID NOs: 1-123.

[0428] Aspect 42: The modified Paenibacillus bacterium of aspect 38, wherein the Paenibacillus bacterium is a species selected from the group consisting of Paenibacillus polymyxa, Paenibacillus tritici, Paenibacillus albus, Paenibacillus anaerobic, Paenibacillus nitrogen-fixing, Paenibacillus boraye, Paenibacillus tokai, Paenibacillus eihime, Paenibacillus graminearum, Paenibacillus lijilun, Paenibacillus heterotatis, Paenibacillus ginseng land, Paenibacillus phoenix, Paenibacillus sacheon, Paenibacillus rhizosphere, Paenibacillus silage, Paenibacillus peach blossom mountain, Paenibacillus thermophilus, Paenibacillus cattail, Paenibacillus tenacious, and Paenibacillus wensleyi.

[0429] Aspect 43: The modified Paenibacillus bacterium of Aspect 38, wherein the Paenibacillus bacterium belongs to subgroup I.

[0430] Aspect 44: The modified Paenibacillus bacterium of Aspect 38, wherein the Paenibacillus bacterium belongs to subgroup II.

[0431] Aspect 45: A substantially pure composition comprising the modified Paenibacillus bacterium of aspect 38.

[0432] Aspect 46: A bacterial culture comprising the modified Paenibacillus bacterium of Aspect 38.

[0433] Aspect 47: A fermentation culture comprising the modified Paenibacillus bacterium of aspect 38.

[0434] Aspect 48: An agricultural composition comprising the modified Paenibacillus bacterium of aspect 38 and an agriculturally acceptable carrier.

[0435] Aspect 49: The agricultural composition of aspect 48, further comprising a plant or a plant component, wherein the modified Paenibacillus bacterium is present in the agricultural composition in an amount effective for producing an improved phenotype in the plant.

[0436] Aspect 50: The agricultural composition of aspect 48, wherein the improved phenotype is an increase in health, yield and / or vigor of the plant.

[0437] Although the present invention is particularly shown and described in conjunction with preferred embodiments and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes may be made to the form and details thereof without departing from the spirit and scope of the present invention. Various changes, modifications and improvements (including certain changes, modifications, substitutions and improvements) of the present disclosure that are easily conceivable to those skilled in the art are also a part of the present disclosure. For example, although the following specific examples may use specific plants to illustrate the methods and embodiments described herein, the principles in these examples may be applied to any plant. Therefore, it should be understood that the scope of the present invention is covered by the embodiments described herein, rather than being covered by only the specific embodiments illustrated below.

[0438] All cited patents and publications mentioned in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each cited patent and publication was individually and specifically indicated to be incorporated by reference.

[0439] Example

[0440] The methods and compositions presented herein (based on the use of the disclosed isolated microorganisms, communities, consortia and / or compositions comprising and / or produced by microorganisms or consortia or communities) improve one or more characteristics of a plant (e.g., nitrogen fixation in agricultural crops).

[0441] The abbreviation "uL" means "microliter" and "ug" means "microgram".

[0442] Exemplary protocols are described herein in detail; however, it is to be understood that alternative approaches and / or variations may be employed.

[0443] Example 1: Microbial culture, sequencing and target selection

[0444] Paenibacillus strains 8619, 17899, 17911, 53072, 53953, 54805, 55083, 55136, 55146, 55470, 60721, 63764, 68890, 68892, 70995, 77155, 77357, 77359, 6219, 71001, 103408, 106159, 106276, and genetically modified strains of the foregoing strains are grown in culture to obtain adequate cell growth.

[0445] Subsamples of each line were then aseptically transferred to nitrogen-free growth medium and incubated under microaerobic conditions for 72 hours.

[0446] The isolates of interest were grown to mid-log phase in R2A medium. DNA was extracted with Qiagen Powersoil DNA extraction kit, and sequencing libraries were constructed with iGenomix RipTide kit according to the manufacturer's instructions. Sequencing was performed on Illumina HiSeq with PE150. The original Illumina reads were adjusted to Q15 with Trimmomatic v38 (Bolger AM, Lohse M, and Usadel B. (2014). Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics, btu170) and assembled with default parameters using SPAdes (Prjibelski A, Antipov D, Meleshko D, Lapidus A, and Korobeynikov A. (2020) Using SPAdes denovo assembler. Curr. Protoc. Bioinform. 70, e102). The assembled contigs were analyzed for purity using BinSantity 0.5.4 (Graham ED, Heidelberg JF and Tully BJ. (2017) BinSanity: unsupervised clustering of environmental microbial assemblies using coverage and affinity propagation. Peer J 5: e3035), with a contamination cutoff of <5%. The maximum bin was extracted and annotated with Prokka 1.8 (Seemann T. (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30 (14): 2068-9). The sequences of 16S rDNA were identified by Prokka 1.8 and extracted directly from the .ffn file.Taxonomy was assigned using GTDB-tk with default parameters against the April 2021 database (Pierre-Alain Chaumeil, Aaron J Mussig, Philip Hugenholtz, Donovan H Parks, GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database, Bioinformatics, Volume 36, Issue 6, March 15, 2020, Pages 1925–1927).

[0447] According to the method of Xie et al. (2014), the selection of Paenibacillus strains was characterized as subgroup I or subgroup II (Comparative Genomic Analysis of N 2-Fixing and Non-N 2-Fixing Paenibacillus spp.: Organization, Evolution and Expression of the Nitrogen Fixation Genes. 10 (3)). Although the nif gene cluster consisting of nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV is highly conserved in 15 N2-fixing Paenibacillus strains, there are some variations in the DNA sequence of the nif cluster, and these clusters can be divided into two subgroups: subgroup I and subgroup II. The 9 genes nifB, HDK, ENX, hesAnifV of the nif gene cluster in subgroup I are continuous, while there is an ORF of 261-561 BP between nifX and hesA in subgroup II, and its predicted product is unknown. The species Paenibacillus polymyxa and Paenibacillus tritici of the genus Paenibacillus are examples of subgroup I. The species Paenibacillus albus, Paenibacillus anaericus, Paenibacillus azotigens, Paenibacillus borealis, Paenibacillus donghaensis, Paenibacillus ehimensis, Paenibacillus graminis, Paenibacillus jilunlii, Paenibacillus odorifer, Paenibacillus panacis isoli), P. phoenicis, P. pocheonensis, P. rhizoplanae, P. silage, P. taohuashanense, P. thermophilus, P. typhae, P. durus, and P. wynnii are examples of subgroup II.

[0448] Editing targets of various polynucleotides in the Paenibacillus genome were selected to increase nitrogen fixation in the absence of exogenously applied nitrogen, in the presence of minimal added nitrogen (e.g., ammonium), and in the presence of added nitrogen. Several approaches were developed, including shutting down negative regulation of the nif operon in the presence of ambient nitrogen, and promoting transcription under all conditions.

[0449] NiF

[0450] The nitrogenase complex consists of two conserved proteins: the MoFe protein, composed of subunits encoded by the nifD and nifK genes; and the Fe protein encoded by the nifH gene. The nitrogenase iron protein gene nifH is one of the oldest functional genes extant in the history of gene evolution. The nucleotide sequence of the coding region of the nifHDK gene is highly conserved in all nitrogen-fixing organisms. However, the copy number and arrangement of nifH, nifD and nifK are different in different nitrogen-fixing bacteria. Due to the nature of NifH, the entire coding region (ATG to stop codon) was removed by homologous recombination to generate a knockout to test the function. The resulting edit stitches the chromosomes together, minus the removed sequence and without the inserted nucleotides.

[0451] GqV

[0452] In Paenibacillus bacteria, the nif operon controls the nitrogen fixation pathway via GlnR; transcription of the nif operon genes is regulated by ammonium and oxygen. A knockout of GlnR (removal of the entire coding region ATG to M156 by homologous recombination, leaving a stop codon in the resulting sequence) and a C25 truncation (removal of the DNA encoding the last 25 amino acids of glnR) were generated to assess the effect on nitrogen fixation.

[0453] GlnR binding sites I and II

[0454] Binding of GlnR to site I activates Nif expression, while binding of GlnR to site II represses Nif expression. In addition, GlnR has a higher affinity for binding to site II. Several methods have been explored to increase expression of the nif operon to increase nitrogen fixation: inactivation of site I (replacing the last six nucleic acids of the native GlnR binding site I sequence), deletion of a portion of site II, inactivation of site II (replacing the last six nucleic acids of the native GlnR binding site II sequence), duplication of site II sequence and insertion into site I (e.g., replacing site I), and combinations of edits comprising any one or more of the foregoing.

[0455] CueR

[0456] This gene was misidentified as GlnR in 8619 and surprisingly gave a promising increase in activity. The misidentification was due to the lack of the GlnR ORF in the original Illumina genomic sequence of 8619 used to design the editing cassette. Structurally similar to GlnR, CueR was chosen as the closest match to the protein sequence of GlnR. Like GlnR, it is also an HTH-type transcriptional regulator. It is also located directly upstream of the nrgA (ammonium transporter) gene, sharing the same bidirectional transporter. It is possible that it is a novel transcriptional regulator.

[0457] In many bacterial species, CueR is generally considered to be a regulator of the Cue copper efflux system, activating gene expression in response to high levels of intracellular copper. In our P. polymyxa, the CueR ORF shares a bidirectional promoter with the ammonium transporter NrgA. CueR is a GlnR-like HTH-type transcriptional regulator, and its proximity to NrgA in P. polymyxa indicates that it may also play a role in the transcription of ammonium transporters. If this is the case, removal of CueR would misregulate the expression of NrgA relative to nitrogen levels, resulting in reduced ammonium transport into the cell. The reduction in ammonium levels in the cell involves the cell's utilization of atmospheric nitrogen through the expression of nitrogenase.

[0458] Locus CM8619_hybrid_04839 was extracted from the CM8619_hybrid_CE assembly and the properties of the MerR family HTH regulatory genes were analyzed. All orthologous Paenibacillus species Y412MC10 MerR family HTH regulatory genes were taken from the KEGG ortholog (KO) database. A BLAST database was constructed with MerR orthologous genes and a bidirectional BLAST search was performed on the putative MerR gene glnaRnt using blastp. The top hit was a hit with 62% identity to CueR. The Paenibacillus strain Y412MC10 assembly was taken from NCBI and the gene landscape of the CueR region matched that of CM8619, with nrgA immediately upstream and the zinc-cutting metalloproteinase located downstream.

[0459] orf1

[0460] In the native nif string of Paenibacillus heterosporus CM17899, the orf1 gene overlaps with the downstream sequence of nifX for the first 17 nucleotides of orf1. For orf1 knockout, these nucleotides are retained, and the gene sequence from nucleotide A18 to the last nucleotide G567 and including nucleotide A18 is seamlessly deleted. This deletion results in a severely truncated orf1 expression product with a seven amino acid sequence (including a short frameshift).

[0461] GlnA

[0462] Glutamine synthetase (GS), encoded by glnA within the glnRA operon, directly interacts with the C-terminal domain of GlnR, thereby controlling GlnR activity.

[0463] Example 2a: Editing of Paenibacillus strains

[0464] Editing of Paenibacillus microorganisms to produce engineered strains that confer plant improvement characteristics associated therewith can be achieved by nucleotide insertion, nucleotide deletion, nucleotide substitution, and / or any combination or multiples of the foregoing. The net effect can be one of: upregulation, downregulation, knockout (function of the target polynucleotide and / or its encoded RNA or protein), and / or any combination or multiples of the foregoing. Particularly contemplated herein are genomic targets in Paenibacillus, including cueR, GlnR binding site I upstream of the nif operon, GlnR binding site II upstream of the nif operon, Orf1, and glnR.

[0465] The successful editing and association with plants to confer plant improvement benefits was surprising and unexpected given the known challenges of working with Gram-positive bacteria such as Paenibacillus, as compared to more susceptible Gram-negative bacteria such as Klebsiella.

[0466] Editing of bacterial strains can be accomplished using any method known in the art. In some cases, the methods described herein are used. In some cases, previously described methods are used, see, for example, WO2023039463A1 "Blind Editing of Polynucleotide Sequences" published on March 16, 2023, which is incorporated herein by reference in its entirety.

[0467] The Gram-positive gene editing vector pMiniMad2 was obtained from the Bacillus Genetic Collection and modified by inserting the TraJ transfer origin originally from vector pKVM4 between the SalI and BamHI restriction sites, generating the mobilizable Gram-positive gene editing vector pMMmob.

[0468] Assembly of editing vector

[0469] Polymerase chain reaction (PCR) was performed using appropriate primers and Q5 high-fidelity polymerase to amplify upstream and downstream homology arms with appropriate Gibson assembly overhangs from purified genomic DNA from the target strain. PCR products were electrophoresed on agarose gels to confirm the appropriate size. Bands were cut from the gels and purified.

[0470] Digest the backbone vector pMMmob with restriction enzymes EcoRI and BamHI at 37°C for at least 30 minutes in Cutsmart buffer. Run the digest on an agarose gel to confirm the appropriate size. Purify the digested backbone from the gel.

[0471] Gibson assembly was performed by combining approximately 100 ng of digested pMMmob with the insert at a 1:3:3 backbone:insert:insert molar ratio in a 10 ul volume and then adding 10 ul of 2xGibson reagent. The reaction was incubated at 50C for 60 minutes and then used for transformation into E. coli DH5α.

[0472] 1-5ul Gibson assembly mixture is added to 50ul freshly thawed chemical competent Escherichia coli DH5α cells and finger vortexed. The cell-plasmid mixture is incubated on ice for 30 minutes, heat-shocked at 42°C for 30 seconds, then moved back on ice and kept for another 5 minutes. 1mL SOC (super optimal decomposition) culture medium is added, and the cells are recovered by incubation at 37°C with 200RPM shaking for 60-90 minutes. The dilution plate of the recovered culture is inoculated on the LB agar plate supplemented with 100ng / uL ampicillin and incubated overnight at 37°C.

[0473] The region of the plasmid containing the assembled insert was amplified from several recovered colonies via colony PCR using GoTaq polymerase, and the PCR products were electrophoresed on agarose gels to confirm the expected size of the product. The appropriately sized PCR products were sent for Sanger sequencing to confirm correct assembly and the absence of any off-target mutations in the editing cassette.

[0474] Colonies confirmed to carry the correct plasmid were inoculated into LB broth supplemented with 100 ng / uL ampicillin and grown overnight at 37C with shaking at 200 RPM. Plasmids were purified from overnight cultures and transformed into the conjugation donor strain E. coli BW29472 via electroporation.

[0475] 1ul purified plasmid was combined with 50ul freshly thawed Escherichia coli BW29472 electrocompetent cells and incubated on ice for 5 minutes. The cell-plasmid mixture was transferred to a precooled 1mm electroporation cuvette on ice. Using an electroporator, 1800V, 25uF, 200Ω of charge was applied to the cuvette, and the sample was immediately resuspended in 1mL SOC culture medium supplemented with 0.3mM 2,6-diaminopimelate (DAP). The resuspended cells were reclaimed at 37°C with 200RPM shaking incubation for 60-90 minutes. The dilution plate of the reclaimed culture was inoculated on an LB agar plate supplemented with 100ng / uL ampicillin and 0.3mM 2,6-diaminopimelate and incubated overnight at 37°C. The reclaimed transformant was used as the donor strain for conjugation.

[0476] join

[0477] The recipient strain was inoculated into 5 mL of Tryptic Soy Broth (TSB) medium in a 50 mL conical tube and grown overnight at 30C and 200 RPM shaking. The donor E. coli BW29427 carrying the plasmid to be mobilized was inoculated into 5 mL of LB medium supplemented with 100 ug / uL ampicillin and 0.3 mM 2,6-diaminopimelate (DAP) and grown overnight at 37C and 200 RPM shaking.

[0478] 1 ml aliquots of overnight donor and recipient cultures were centrifuged, washed in sterile water, combined and spotted onto the surface of LB agar plates supplemented with 0.3 mM DAP for conjugative mating. The mating plates were incubated overnight at 25°C, a permissive temperature for pMMmob replication in Gram-positive recipient strains.

[0479] The mating mixture was resuspended by adding 1 ml of sterile water on top of the spot and stirring with a sterile L-applicator. The resuspension was collected in a microcentrifuge tube, washed, and resuspended in 100 ul of sterile water. The concentrated cells were spread on TSA plates supplemented with MLS (25 ug / ml lincomycin, 1 ug / ml erythromycin) but without the addition of DAP and incubated at 25°C for 48-72 hours until metazygote colonies appeared.

[0480] Plasmid integration

[0481] The recovered transfer conjugants were inoculated into 5 mL TSB medium supplemented with MLS and grown at 25°C with shaking at 200 RMP for 48 hours or until turbid. Dilutions of the culture were plated on TSA + MLS plates and incubated overnight at 37°C (the restrictive temperature for plasmid replication) until integrated colonies appeared.

[0482] Plasmid excision

[0483] The integrated colony was inoculated into 5 mL TSB medium supplemented with MLS and incubated overnight at 37°C and 200 RPM shaking. 5 ul of the overnight culture was diluted into 5 mL of fresh TSB medium without antibiotics and grown overnight at 25°C and 200 RPM shaking. 5 ul of the overnight culture was subcultured into 5 mL of fresh TSB qt25°C and 200 RPM shaking was repeated twice for a total of three subcultures. Dilutions of the third round of overnight culture were plated onto R2A plates lacking antibiotics and incubated overnight at 30°C.

[0484] Excision of the plasmid was confirmed by picking individual colonies from the R2A plates and re-plating them in a grid format on agar plates with and without MLS. Both plates were incubated at 30C for 24-48 hours until colonies appeared. We confirmed that colonies that grew when plated on plates without MLS, but not on plates with MLS, had excised and lost the plasmid.

[0485] Confirmation of Editor

[0486] The edited region was amplified from the putative edited lines via colony PCR and the presence of the correct edit was confirmed by band size when electrophoresed on agarose gel (when possible) and / or by sanger sequencing. The absence of the plasmid backbone was confirmed by PCR assay for the MLS resistance cassette. Colonies that produced bands for the MLS cassette were confirmed to be not the correct edit.

[0487] Analyze the sequence results to distinguish colonies excised as wild type from correctly edited colonies and check the sequences for off-target mutations.

[0488] Colonies sequenced as correctly edited without off-target mutations were given modified strain names, added to the modified strain library, and made available to the project team for bioassay analysis.

[0489] The following edits were delivered to one or more parental lines and made available for in vitro and in planta evaluation.

[0490] Example 2b: Paenibacillus strains

[0491] Inactivation of GlnR binding site II

[0492] This replacement of nucleotides bound by GlnR during repression of nif expression with nucleotides that do not bind GlnR results in increased nitrogenase activity. This editing prevents the nif pathway from being repressed in response to excess nitrogen levels and is akin to removing an off switch.

[0493] The editing cassette was constructed by inserting the sequence "ATCGAT" between the natural genomic sequence from the seventh nucleotide to the last nucleotide of the GlnR binding site II and including approximately 1000 base pairs upstream of the seventh nucleotide and the natural genomic sequence from the last base pair of the GlnR binding site II and excluding approximately 1000 base pairs downstream of the last base pair.

[0494] GlnR binding site II replication

[0495] Such replacement of the native site I sequence with the native site II sequence is intended to increase nif expression, thereby increasing the binding affinity of GlnR to the region responsible for activating nif expression. Under all conditions, GlnR has a higher binding affinity for the site II sequence. Since the activation and repression activities of GlnR appear to be dependent on the position of binding, having an increased binding affinity to the position responsible for activation leads to increased nif expression.

[0496] The editing box is constructed by inserting the native GlnR binding site II sequence between the native genomic sequence approximately 1000 base pairs upstream of the first (and not including the first) nucleotide of GlnR binding site I and the native genomic sequence approximately 1000 base pairs upstream of the last (and not including the last) base pair of GlnR binding site I.

[0497] GlnR knockout

[0498] This edit is expected to result in basal levels of nif expression in both limiting and excess conditions. We initially saw unexpected activity in a line with this edit, but later determined that this line was incorrectly modified and was in fact a CueR knockout. If activity is seen in the correct GlnR knockout line, it could be due to the following reasons.

[0499] "GlnR is the main known regulator of nif expression in Paenibacillus, and its interaction with glutamine synthase is how the cell regulates nif expression based on nitrogen levels in the environment. By removing this regulator, nif expression becomes unregulated relative to environmental nitrogen levels, and instead may be driven by an unknown transcription factor that is not regulated by nitrogen levels. This could lead to an increase in nif expression, particularly under conditions of excess nitrogen."

[0500] The editing cassette was constructed by assembling the native genomic sequence up to and including the start codon of the glnR open reading frame approximately 700 base pairs upstream of the start codon and the native genomic sequence up to and including the stop codon of the glnR open reading frame 700 base pairs downstream of the stop codon.

[0501] GlnR C25 truncation

[0502] The alpha helical C25 region of GlnR folds back into the dimerization site, causing GlnR to exist primarily as a monomer in the cell. It is not until the feedback-inhibited glutamine synthase (FBI-GS) interacts with the GlnR monomer under conditions of excess nitrogen that the C-terminal region folds to promote stable dimerization. The GlnR dimer is then able to bind tightly to binding site II to tightly repress nif expression. By removing the C-terminal 25 amino acids, this regulatory interaction is disrupted, preventing the stable dimerization of GlnR and the binding from being controlled by nitrogen levels. This will allow for increased nif expression under conditions of excess nitrogen.

[0503] The editing cassette was constructed by assembling the native genomic sequence of the codon at the C-terminal twenty-five positions of the glnR open reading frame, excluding the codon approximately 500-1300 (depending on the strain) base pairs upstream of the codon, and the native genomic sequence of the stop codon of the glnR open reading frame, including the 500-900 (depending on the strain) base pairs downstream of the stop codon.

[0504] CueR knockout

[0505] The editing cassette was constructed by assembling the native genomic sequence up to and including the start codon of the cueR open reading frame approximately 1000 base pairs upstream of the start codon and the native genomic sequence up to and including the stop codon 1000 base pairs downstream of the stop codon of the cueR open reading frame.

[0506] CueR C25 truncated

[0507] The editing cassette was constructed by assembling the native genomic sequence up to and including the codon twenty-five positions from the C-terminus of the cueR open reading frame approximately 1000 base pairs upstream of the codon and the native genomic sequence up to and including the stop codon of the cueR open reading frame 1000 base pairs downstream of the stop codon.

[0508] Orf1 knockout

[0509] This is expected to demonstrate reduced nif activity due to reduced oxygen tolerance. We doubt that this edit will demonstrate to us the potential value of inserting this gene into a strain that lacks it. However, in the case where this edit showed increased nif expression, it could be due to the following reasons.

[0510] orf1 is an open reading frame found in the nif cluster of some Paenibacillus strains (called subclass II), but not in other strains (called subclass I). It is predicted to function at high oxygen levels. Its absence in many high-performing strains may indicate that it is unnecessary and that, in most cases, nitrogen assimilation is more efficient in its absence. This may be by removing the metabolic burden of expression of this ORF, or by excess activity of the expression product itself.

[0511] The editing cassette was constructed by assembling the native genomic sequence up to and including the stop codon of the nifX open reading frame approximately 1000 base pairs upstream of the stop codon with the native genomic sequence up to and including the stop codon of the Orf1 open reading frame excluding 1000 base pairs downstream of the stop codon.

[0512] In the native nif string of Paenibacillus heterosporus CM17899, the orf1 gene overlaps with the downstream sequence of nifX for the first 17 nucleotides of orf1. For orf1 knockout, these nucleotides are retained, and the gene sequence from nucleotide A18 to the last nucleotide G567 and including nucleotide A18 is seamlessly deleted. This deletion results in a severely truncated orf1 expression product with a seven amino acid sequence (including a short frameshift).

[0513] The editing cassette was assembled using standard molecular cloning techniques and Gibson assembly, where homology arms of the native Paenibacillus heterosporus CM17899 sequence, including the last nucleotide of the nifX gene and approximately 1000 nucleic acids upstream of the last nucleotide and the last nucleotide of orf1 and excluding approximately 1000 nucleic acids downstream of the last nucleotide, were seamlessly assembled into a scarless homologous recombination vector. The resulting editing vector was delivered to CM17899, and the resulting transformants were cultured in a manner sufficient to induce integration of the entire plasmid, followed by excision of unwanted genetic material, leaving only the desired edit. Sanger sequencing of the edited region confirmed the presence of the desired modification.

[0514] GlnR binding site II inactivation and replication

[0515] These edits are expected to act synergistically by preventing GlnR dimers from binding to the site II "off switch" and enhancing the "on switch" by increasing its binding efficiency. Together, these edits ensure that GlnR can only act as a strong positive activator of transcription of nif operon genes.

[0516] The editing cassette was constructed by inserting the native GlnR binding site II sequence between a genomic sequence approximately 1000 base pairs upstream of and excluding the first nucleotide of GlnR binding site I in a strain previously edited with GlnR binding site II inactivation, and a genomic sequence approximately 1000 base pairs downstream of and excluding the last base pair of GlnR binding site I in a strain previously edited with GlnR binding site II inactivation.

[0517] Inactivation of GlnR binding site II, truncation of CueR C25

[0518] These editors employ different approaches to prevent downregulation of nif expression, by preventing transcriptional repression through GlnR binding to site II and by potentially downregulating NrgA expression and thereby preventing downregulation triggered by ammonium accumulation.

[0519] Inactivation of GlnR binding site II, truncation of GlnR C25

[0520] The two edits work synergistically to increase the formation of GlnR dimers by removing the autoinhibitory regions of GlnR monomers and preventing these dimers from tightly binding site II. The more abundant dimers can only bind site I, leading to increased nif expression under all conditions.

[0521] Duplication of GlnR binding site II, truncation of GlnR C25

[0522] These two edits work synergistically to increase the formation of GlnR dimers by removing the autoinhibitory regions of GlnR monomers and increasing the binding affinity of these dimers to activation sites. More abundant dimers would have increased affinity for binding to activation sites, leading to increased nif expression, at least under nitrogen-limited conditions.

[0523] Orf1 knockout, GlnR C25 truncation

[0524] The edits take different approaches to improving nitrogen fixation. Orf1 knockout removes excess enzymes from the process, thereby increasing efficiency, while GlnR C25 truncation increases the capacity of available GlnR dimers while decoupling their dimerization levels from intracellular nitrogen levels.

[0525] Inactivation of GlnR binding site II and CueR knockout

[0526] These editors employ different approaches to prevent downregulation of nif expression, by preventing transcriptional repression through GlnR binding to site II and by potentially downregulating NrgA expression and thereby preventing downregulation triggered by ammonium accumulation.

[0527] GlnR binding site II duplication, CueR knockout

[0528] These edits employ different approaches to prevent downregulation of nif expression, by increasing the binding affinity of the GlnR dimer to the activation site and by potentially downregulating NrgA expression and thereby preventing the accumulation of ammonium that triggers downregulation.

[0529] GlnRC25 frameshift truncation and glnASNP

[0530] A sequence of 76 nucleotides between nucleotides A332 and T409 of the glnR gene, excluding these two nucleotides, was deleted, resulting in the breakage of the natural stop codon and a frameshift mutation. The frameshift mutation resulted in the addition of 13 amino acids to the GlnR protein before reaching the stop mutation. At the same time, a single nucleotide in the adjacent glnA gene was changed. Nucleotide C164 became A, resulting in the mutation of serine at amino acid position 55 of the GlnA protein to tyrosine.

[0531] To design an editing cassette that can delete the last 25 amino acids, primers were designed to amplify nucleotide arms of G333 and including approximately 500 nucleotides upstream of G333 and T409 and including approximately 500 nucleotides downstream of T409 of glnR in Paenibacillus heterosporus CM17899, so that 75 nucleotides were deleted from the glnR gene and 25 amino acids were deleted from the GlnR protein, but the natural stop codon was retained. An error occurred in the primer design, resulting in an upstream homology region extending from and including A332. This error resulted in a 76 nucleotide deletion in the editing cassette, destroying the natural stop codon and causing a frameshift mutation.

[0532] During the construction of the editing cassette, a single nucleotide polymorphism (SNP) was introduced during the polymerase chain reaction (PCR) to amplify the downstream homology arm in the glnA gene. The SNP may be introduced by random errors of the Q5 high-fidelity polymerase used in the PCR reaction, or it may be introduced by a SNP present in a copy of the polymyxa CM17899 genomic DNA used in the reaction. This nucleotide change is non-directional and is not based on known variations in the GlnA gene of species of the genus Paenibacillus. During the integration and excision of the plasmid editing into polymyxa CM17899, the off-target modification remains in the resulting homology region. These edits unexpectedly provide positive results for plants associated with the edited microorganisms.

[0533] NrgA knockout

[0534] In Paenibacillus, the nrgA gene is the major ammonium transporter. Nitrogen fixation is tightly regulated and is repressed by excess intracellular ammonium levels. To reduce the amount of available ammonium that would repress nitrogenase expression, nrgA was knocked out by seamlessly removing the entire open reading frame from the first to the last nucleotide, leaving the immediately surrounding sequence intact, but without leaving any gene sequence.

[0535] The editing cassette was assembled using standard molecular cloning techniques and Gibson assembly, where homology arms of the native Paenibacillus polymyxa CM8619 sequence, including the first nucleotide of the nrgA gene and excluding approximately 1000 nucleic acids upstream of the first nucleotide and the last nucleotide of nrgA and excluding approximately 1000 nucleic acids downstream of the last nucleotide, were seamlessly assembled into a scarless homologous recombination vector. The resulting editing vector was delivered to CM8619, and the resulting transformants were cultured in a manner sufficient to induce integration of the entire plasmid, followed by excision of unwanted genetic material, leaving only the desired edit. Sanger sequencing of the edited region confirmed the presence of the desired modification.

[0536] GlnR binding site II truncation

[0537] This loss of nucleotides bound by GlnR during repression of nif expression caused an increase in nitrogenase activity. This edit prevented the nif pathway from being repressed in response to excess nitrogen levels and was akin to removing an off switch.

[0538] The editing cassette was constructed by seamlessly assembling the native genomic sequence from the seventh nucleotide to the last nucleotide of the GlnR binding site II and including approximately 1000 base pairs upstream of the seventh nucleotide with the native genomic sequence from the last base pair of the GlnR binding site II and excluding approximately 1000 base pairs downstream of the last base pair.

[0539] GlnR binding site II truncation and duplication

[0540] These edits are expected to act synergistically by preventing GlnR dimers from binding to the site II "off switch" and enhancing the "on switch" by increasing its binding efficiency. Together, these edits ensure that GlnR can only act as a strong positive activator of transcription of nif operon genes.

[0541] The editing cassette was constructed by inserting the native GlnR binding site II sequence between the first nucleotide of GlnR binding site I and approximately 1000 base pairs upstream of the genomic sequence of the strain previously edited with GlnR binding site II truncation, and between the last base pair of GlnR binding site I and approximately 1000 base pairs downstream of the genomic sequence of the strain previously edited with GlnR binding site II truncation.

[0542] GlnR C25 frameshift truncation

[0543] The α-helical C25 region of GlnR folds back to the dimerization site, causing GlnR to exist primarily as a monomer in the cell. It is not until the feedback-inhibited glutamine synthetase (FBI-GS) interacts with the GlnR monomer under conditions of excess nitrogen that the C-terminal region is folded to promote dimerization. The GlnR dimer is then able to bind tightly to the binding site II to tightly repress nif expression. By deleting 76 nucleic acids upstream of the stop codon, the α-helix of the C-terminal 25 amino acids is deleted and a frameshift mutation is induced, resulting in the translation of 13 random amino acids. This removes the native C-terminal structure, disrupts the native regulatory interaction, and prevents the stable dimerization of GlnR and the binding from being controlled by nitrogen levels. This will allow increased nif expression under conditions of excess nitrogen.

[0544] The editing cassette was constructed by assembling the native genomic sequence at 331 nucleic acid positions from the start of the glnR open reading frame and excluding approximately 500 base pairs upstream of the nucleic acid and the native genomic sequence at 407 nucleic acid positions from the start of the glnR open reading frame and excluding 500 base pairs downstream of the nucleic acid.

[0545] GlnA SNP

[0546] The glutamine synthetase encoded by glnA is a key component of the regulatory apparatus of the Paenibacillus nif cluster. Single nucleotide polymorphisms (SNPs) introduced into the sequence may have an impact on the regulatory function of the protein, resulting in a net increase in nitrogen fixation activity. The GlnA SNP described herein is a replacement of the nucleic acid cytosine at position 164 of the open reading frame by an adenosine residue.

[0547] The editing cassette was constructed by assembling the native genomic sequence approximately 500-100 base pairs upstream of the SNP site and approximately 500-1000 base pairs downstream of the SNP site, and performing adenosine residue substitutions therebetween.

[0548] GlnR binding site II truncation

[0549] This loss of nucleotides bound by GlnR during repression of nif expression caused an increase in nitrogenase activity. This edit prevented the nif pathway from being repressed in response to excess nitrogen levels and was akin to removing an off switch.

[0550] The editing cassette was constructed by assembling the native genomic sequence from the seventh nucleotide to the last nucleotide of the GlnR binding site II and including approximately 1000 base pairs upstream of the seventh nucleotide with the native genomic sequence from the last base pair of the GlnR binding site II and excluding approximately 1000 base pairs downstream of the last base pair.

[0551] Promoter swapping and insertion

[0552] Paenibacillus polymyxa constitutive promoter

[0553] PLH-77 and PLH-77-d are constitutive promoters isolated from Paenibacillus polymyxa SC2-M1, a rhizosphere isolate of pepper plants from Guizhou, China. The promoter pLH77 was identified and reported by Li et al. (2019), and the replicating form pLH77-d was also described.

[0554] Nif promoter insertion

[0555] Promoters PLH-77 and PLH-77-d were operably linked to the nif cluster of Paenibacillus polymyxa CM8619 by inserting the promoter sequence upstream of the putative native ribosome binding site (RBS) but without removing any of the native promoter sequences.

[0556] The editing cassette was assembled using standard molecular cloning techniques and Gibson assembly, where homology arms of the native Paenibacillus polymyxa CM8619 sequence containing approximately 1000 nucleic acids upstream and downstream of the target promoter site were assembled into a scarless homologous recombination vector, and the synthetic promoter sequence was seamlessly assembled between them. The resulting editing vector was delivered to CM8619, and the resulting transformants were cultured in a manner sufficient to induce integration of the entire plasmid, followed by excision of unwanted genetic material, leaving only the desired edit. Sanger sequencing of the edited region confirmed the presence of the desired modification.

[0557] Suf promoter exchange

[0558] Promoters PLH-77 and PLH-77-d can be operably linked to the sufCDSUB cluster of Paenibacillus polymyxa CM8619 by inserting the promoter sequence upstream of the putative native ribosome binding site (RBS) and removing some or all of the native promoter sequence or leaving the native promoter sequence intact.

[0559] The editing cassette can be assembled using standard molecular cloning techniques and Gibson assembly, wherein the homology arms of the sequence of the natural polymyxa bacillus CM8619 or another target strain containing approximately 600 nucleic acids upstream of the natural promoter sequence and downstream of the natural promoter sequence are assembled into a scarless homologous recombination vector, and the synthetic promoter sequence is seamlessly assembled between them. The replacement cassette can be designed to retain the integrity of some or all of the natural promoter sequences. The resulting editing vector can be delivered to CM8619 or another target strain, and the resulting transformant can be cultured in a manner sufficient to induce integration of the entire plasmid, and then the unwanted genetic material is excised, leaving only the desired editing. Sanger sequencing of the editing region can confirm the presence of the desired modification.

[0560] Example 3: Cloning

[0561] The cloning vector was assembled by introducing the editing cassette (described above) into the pMMmob backbone. pMMmob [oriBsTstraJ ecol1mls amp] is a derivative of the plasmid pMiniMAD2 obtained from the Bacillus Genetic Collection. pMMmob was digested with restriction enzymes BamH1 and EcoR1, electrophoresed on a 10% agarose gel, and purified.

[0562] Using proofreading polymerase, upstream homology and downstream homology regions were amplified from genomic DNA extracts via PCR, and primers were designed to attach flanking sequences for subsequent Gibson assembly. For constructs with sequences added between the flanking homology arms, sequence introduction was achieved by inclusion in the primer flanking sequences. The PCR products were electrophoresed on 10% agarose gel and purified.

[0563] The backbone and insert were combined at a 1:3 backbone to insert molar ratio, combined with Gibson assembly reagent, and incubated at 50°C for 60 minutes for plasmid assembly. The Gibson assembly mixture was then transformed into chemically competent E. coli DH5α. Transformants were recovered on LB+100ug / uL ampicillin plates.

[0564] Correct assembly of the plasmid was confirmed by restriction digest analysis and PCR of the inserted region. The editing cassette was sequenced using Sanger sequencing to confirm the absence of off-target mutations.

[0565] Confirmed plasmids were extracted from overnight DH5α cultures and transformed into electrocompetent E. coli BW29472 via electroporation and recovered onto LB + 100 ug / uL ampicillin + 300 uM diaminopimelic acid plates for conjugation into the host strain.

[0566] Example 4: Gene Editing of Paenibacillus Species

[0567] Scarless homologous recombination

[0568] This is a general protocol for gene editing in Paenibacillus using a temperature-sensitive scarless homologous recombination plasmid and was used for the editing described herein. This protocol was developed for editing CM8619 and is broadly applicable to other Paenibacillus isolates with modifications. This protocol requires pre-assembly of one or more editing vectors designed for the desired editing using the pMMmob backbone in a host E. coli donor strain and one or more Paenibacillus recipient strains with confirmed susceptibility to the relevant antibiotic resistance marker.

[0569] join

[0570] The desired recipient strain is grown overnight in an appropriate growth medium. The donor strain is grown overnight in an appropriate growth medium supplemented with the relevant antibiotic marker to maintain the mobile plasmid. Aliquots of the overnight cultures are washed, combined, and plated on agar medium suitable for the growth of both strains. These plates are incubated overnight at a permissive temperature for plasmid replication in the recipient strain.

[0571] The mating mixture is recovered, washed, and replated onto agar plates supplemented with the appropriate antibiotic marker for selection of transconjugant recipient strains. The plates are incubated overnight at a permissive temperature for plasmid replication until transconjugant colonies appear.

[0572] Integration

[0573] Transconjugant colonies are grown overnight in liquid culture in the presence of a selective marker at a permissive temperature for plasmid replication. Dilutions of the liquid culture are plated on agar plates supplemented with a selective antibiotic and incubated overnight at a restrictive temperature for plasmid replication. Colonies recovered under these conditions are assumed to have integrated the editing plasmid by homologous recombination.

[0574] resection

[0575] Integrating colonies were inoculated into liquid cultures, grown until turbid at the permissive temperature for plasmid replication, then subcultured into fresh medium lacking antibiotics and grown again overnight at the permissive temperature. This serial subculture was repeated 2-3 more times, and dilutions of the final subculture were plated on agar plates lacking antibiotics.

[0576] Recovered colonies were assayed for plasmid loss by re-plating onto media with and without antibiotics. Colonies that grew in the absence of antibiotics but not in the presence of antibiotics were confirmed to have excised and lost the plasmid and were identified as putative edited lines.

[0577] confirm

[0578] By amplifying the editing region via polymerase chain reaction (PCR), putatively edited strains were screened to determine whether the edit was successfully delivered, or whether the strain reverted to wild type. PCR products were analyzed by gel electrophoresis and Sanger sequencing to confirm product size and sequence suitable for editing. Colonies with successfully delivered edits were confirmed by sanger sequencing to confirm that no off-target mutations were added to the editing region, and lack of growth on medium containing antibiotics to confirm the absence of the plasmid backbone.

[0579] Strains that pass all validation steps are assigned modified strain names, added to the modified isolate library, and provided to the bioassay panel for testing.

[0580] Other methods

[0581] Alternatively, any other method known in the art may be used to achieve any one or more polynucleotide editing described herein, such as, but not limited to: targeting and / or homing nucleases, restriction endonucleases, zinc finger nucleases, meganucleases, Cas endonucleases, TAL effector nucleases, guide nucleases, random site mutations, blind editing, chemical mutagenesis, or radiation mutagenesis. Typically, double-strand breaks are generated at or near the target site to be edited, and double-strand breaks are repaired by intracellular processes such as non-homologous end joining, homologous recombination, or homology-directed repair. The net effect may be any one or more of the following: insertion of at least one nucleotide, deletion of at least one nucleotide, substitution of at least one nucleotide, chemical alteration of at least one nucleotide. In some aspects, random, non-targeted, opportunistic, or other editing of the target polynucleotide (polynucleotide to be edited) may be achieved, such as by radiation mutagenesis, chemical mutagenesis, blind editing (see, for example, WO2023039463A1 published on March 16, 2023, which is incorporated herein by reference in its entirety) or any other method known in the art. For the purposes of the editing described herein, any technique desired by the practitioner may be used to achieve the end result.

[0582] Example 5: Identification and storage of microorganisms

[0583] Sequencing preparation and long-term storage for microbial identification can be performed by:

[0584] Day 1:

[0585] Use a 10 uL sterile pipette tip to transfer the colonies from the plates to flasks containing the appropriate liquid growth medium. Place the isolates on a shaker at room temperature and incubate for 2 days.

[0586] Day 3:

[0587] After 2 days on the shaking table, the tube can be turbid. All samples are analyzed by PCR. Vortex each tube, collect 50uL sample from each vortexed tube, and distribute it into 96-well plates. Use a multichannel pipette to distribute 15uL of the 50uL sample into a new 96-well plate. A 96-well plate containing 35uL of each sample will be used for phenotyping, and a 96-well plate containing 15uL of each sample will be used for PCR analysis. 27F / 1492R primers are commonly used for 16S PCR analysis because they obtain better results than PB36 / 38. The plate should be accompanied by appropriate negative controls and these negative controls are analyzed by PCR. The plate is analyzed by PCR using an Eppendorf thermal cycler. Once PCR is completed, the gel is run using standard gel electrophoresis technology. This is important because most isolates have grown to a sufficient degree, and ideally, they should be stored for a long time on the 3rd day. Use PCR and gel electrophoresis analysis to confirm that these isolates contain bacteria, rather than other microorganisms. For isolates that did not pass PCR or had clarified broth, vortex the tube and streak it on a petri dish using an inoculating loop. Check after a few days to see if anything has grown or if the tube is contaminated. For isolates that passed PCR, 600uL of 50% glycerol was dispensed into a 2ml screw cap tube and 1200uL of bacterial culture was added so that the broth was stored in 20% glycerol. The glycerol stock was stored at -80°C and images of the gels of the PCR samples were recorded.

[0588] Day 4:

[0589] Check the growth of the culture dishes of the streaked isolates that failed PCR. (During this time, the 2ml broth tube will remain on the shaker.) Once there is growth on the plate and the colonies appear to have been successfully isolated, 600uL of the broth-glycerol mixture is dispensed into small tubes and the two tubes are placed into their corresponding -80 boxes. The isolate may fail the PCR check for any of the following reasons: these primers may not be effective for all bacteria, the isolate is actually a fungus, the isolate has strong adherence and is therefore not homogenized in the broth, the isolate produces too much EPS and therefore needs to be diluted before the PCR setup, or the isolate grows very slowly. Over the next few days, continue to check the plate to confirm that only a single bacterial species is isolated. If contamination is observed, a new isolate is prepared. The viability of the prepared glycerol stock solution should be verified.

[0590] Example 6: Preparation of microorganisms

[0591] Microorganisms identified according to previous embodiments can be formulated with additional components so as to be applied via methods such as, but not limited to, seed treatment, root irrigation, root washing, seedling soaking, foliage application, soil inoculum, furrow application, lateral application, soil pretreatment, wound inoculation, drip irrigation, vector-mediated via pollinators, injection, osmotic initiation, hydroponics, aquaponics, aeroponics. Preparations containing microorganisms are prepared in the form of liquid, solid or gaseous preparations for agricultural applications. Application to plants is achieved, for example, in the form of powders for surface deposition on plant leaves, sprays for whole plants or selected plant components, a portion of drip irrigation fluids for soil or roots, or coatings for plant components before planting. Such embodiments are intended to be illustrative and do not limit the scope of the present invention.

[0592] The components of the culture medium used for the exemplary microorganism preparation are shown in Table 2 below. All contents are added and 50% of the final water volume is required, and the solution is stirred at high temperature until dissolved. After all contents have dissolved, the solution is fixed to the final required volume using sterile RO water. 4x formulations are usually used for field trial preparation.

[0593] Table 2a: Exemplary media components and concentrations of microbial preparations

[0594]

[0595] Table 2b: Exemplary media components for microbial preparations

[0596]

[0597]

[0598] Table 2c: Exemplary media components for microbial preparations

[0599] Components CAS# Tryptone 91079-40-2 Soybean Peptone 91079-46-8 NaCl 7647-14-5 K2HPO4 7758-11-4 glucose 50-99-7

[0600] Table 2d: Exemplary media components for microbial preparations

[0601] Components CAS# Trehalose 6138-23-4 Isomalt 64519-82-0 Xanthan gum 11138-66-2

[0602] The procedure for mixing the TIX formulation is as follows: measure all dry ingredients into a 50 ml tube. Vortex the ingredients thoroughly to ensure that the xanthan gum is "separated" from the other carbon sources. Add about half of the total sterile RO water to the mixture and vortex. Use the long end of an L-shaped applicator to break up large lumps as much as possible. Heat some sterile RO water in a microwave to warm water bath temperature (45-50°C). Add the remaining sterile RO water to the mixture and vortex. Repeat step 4 as needed and vortex until a clear solution with no lumps is obtained. Spin down the foam formed during mixing by "quick centrifuging" the centrifuge tube for 5 to 10 seconds. Remember to balance the formulation (TIX) tube. Let the formulation cool to room temperature. 1. Mix in the microbial consortium. Vortex to ensure homogeneity. Ideally, a concentration of 10^9 CFU / ml of microorganisms is added to the formulation.

[0603] The formulations are applied to plants or plant parts for testing in field trials.

[0604] Example 7: Application of microorganisms to plant parts and cultivation thereof

[0605] A microbial composition (comprising a single strain of one or more isolated microorganisms, a consortium, a population, a combination, or any combination of the foregoing) is prepared according to the previous examples. The microbial composition comprises one or more microorganisms, optionally in combination with one or more additional microorganisms disclosed herein.

[0606] Microbial compositions for administration

[0607] In some methods, the microbial composition is dried and applied directly to the plant parts.

[0608] In some methods, the microbial composition is suspended in a liquid formulation for application to the plant parts.

[0609] In some methods, the microbial composition is combined with another composition, such as but not limited to: a carrier, a wetting agent, a stabilizer, a salt. In some methods, the other composition comprises a molecule that brings additional agricultural beneficial results to the plant to which the microbial composition is applied. The other composition includes, for example, but not limited to: herbicides, fungicides, bactericides, insecticides, insecticides, nematicides, biostimulants.

[0610] Application type

[0611] The microbial composition is applied to the plant components at a time during development appropriate for the desired result, for example: in a formulation applied in soil drench / in-furrow before planting; as a seed or other propagation component treatment; as a propagation component application after planting; as an in-furrow, drip irrigation or drench application after planting; as a direct application to plant components (e.g., roots, leaves, stems); as an application to harvested plant components (e.g., fruit or grain). Combinations of application types are also tested.

[0612] Application method

[0613] The microbial composition is applied to (inoculated) plants or plant parts or plant products (before planting, after planting, before harvesting or after harvesting). This can be accomplished, for example, by applying the agricultural composition to a hopper or spreader or tank containing the microbial composition and configured to spread the microbial composition.

[0614] The seed coating of the microbial composition is applied to one or more seeds of a crop plant. After application of the isolated microorganisms in the form of a seed coating, the seeds are planted and cultivated according to established customary practices for the crop.

[0615] Alternatively, the microbial composition is applied to the soil to benefit plants present in the soil. Methods of soil application include in-furrow treatment, drench and drip irrigation application.

[0616] Alternatively, the microbial composition is applied to the surface of the plant or plant part after germination.

[0617] Alternatively, the microbial composition is applied to material obtained from the plant after harvesting.

[0618] A control plot of plants was also planted to which the isolated microorganism was not applied.The plants associated with the microbial composition exhibit improved traits of interest.

[0619] The method of administration can be performed according to any protocol known in the art.

[0620] The plant parts, plants or growth medium (eg, soil) may further be inoculated with the disease or pest, depending on the purpose of the test.

[0621] An exemplary, non-limiting protocol for watering a tomato plant is given below:

[0622] 1. Ten days after planting, carefully separate the plants so that there are 6 replicates per treatment group. The plants are delicate and the leaves can be easily torn off. Make sure the size and general appearance of the plants are as uniform as possible (the goal of thinning is to continue to maintain a uniform plant population). If there are not enough plants per replicate, transplant. See step 3 for guidelines on transplanting.

[0623] 2. Begin thinning the pots so that there is one plant per pot. Remove smaller plants, i.e., plants that are unhealthy or misshapen in some way. If there are 2 or more healthy plants per pot, transplant the extra plants to another pot. Use leftover soil pre-treated from the initial planting or from pots where the seeds did not germinate.

[0624] 3. Transplanting: If some pots do not germinate, fill them with plants from another container. To do this, simply dig out the extra plants with a scoopula (try to dig out as much root mass as possible without disturbing other plants) and place in the hole dug in the empty pot. Press lightly with your fingers to compact the soil around the plants.

[0625] 4. Space the pots into 6 rows of pots (1 row of pots per treatment group), which will require 4 RL98 trays. Once completed, take a look at all treatment groups and consider doing some pot swaps to ensure that some treatment groups do not have all tall plants and other treatment groups have all tall plants.

[0626] 5. Change gloves if necessary. Label each pot with the Avery labels you prepared. Treatments should be labeled in rows of 6 replicates, i.e. 1-1, 1-2, 1-3 to 1-6, etc. This will make it easier to find all the replicates for each treatment.

[0627] 6. Two weeks after planting (approximately 4 days after thinning and marking), obtain treatments from the Microbiology team; place trays of prepared plants on the table. Collect combitips, repeaters, and RO water. (Note: plants should be lightly watered on the day of treatment)

[0628] 7. Mix the microbial solution by inverting the tube / container (microbial treatment) 2 to 3 times or gently shaking. Set the dispensing tip to dispense 2 ml. Collect the treatment fluid into the dispensing tip and dispense the first step back into the tube. Make sure the treatment you have corresponds to the row of plants to be treated. Once confirmed, gently dispense 2 ml of the treatment onto the surface soil of each pot, close to the stem but avoiding direct contact with the stem and leaves.

[0629] 8. Dispose of the dispensing tip and repeat step 6 for all treatments. For the inoculated controls (IC or InoCon) and the untreated controls (UTC), RO water was applied instead of the treatments.

[0630] Once all treatments were applied, plants were returned to the growth chamber for processing (optional inoculation), growth, and evaluation.

[0631] Visualization of microorganisms associated with plant components

[0632] Individual microorganisms can be labeled with fluorescent proteins according to methods known in the art.Microscopic image analysis confirmed that the microorganisms disclosed herein were found associated with various plant tissues.

[0633] Example 8: In vitro testing

[0634] Wild-type and edited strains were evaluated for root colonization, acetylene reduction activity, biofilm formation, turbidity (OD at 600 nm), oxygen tolerance, and gene expression. Strain IDs are given in the form of <parental strain #-edit #>. Unless otherwise stated, protocols were performed using methods known in the art.

[0635] NF11 medium can be used. The formula of NF11 includes: autoclavable reagents (KH2PO4 5g / L 36.7mM; K2HPO4 5g / L 26.1mM; NaCl 1g / L 17.1mM; yeast extract 0.1g / L; monosodium glutamate (MSG) 0.86g / L5.9mM), filter sterilized reagents (glucose 10g / L; MgSO4–7H2O 0.2g / L 0.8mM; CaCl2–2H2O 23mg / L 156μM; trace elements: FeSO4 5.4mg / L 35μM, MnSO4–H2O 22.7mg / L 132μM, ZnSO4 0.0785mg / L0.48mM, CuSO4 0.001mg / L 6.2nM, NaMoO4 0.004mg / L 19nM, NaNH4PHO4-4H2O 0mM / 5mM).

[0636] ARA and GC-FID of Gram-positive strains

[0637] Ensure all equipment and materials are sterile. Before autoclaving, seal equipment containers with foil wrap so they can be opened in the anaerobic chamber transfer box and aseptically transferred into the chamber. Seal bottles with foil before sterilization. A loose "seal" is needed to allow for gas exchange in the transfer box.

[0638] 1. Streak the isolate from -80C and incubate at 30C or 25C until colonies are observed.

[0639] 2. Spread one plate per isolate and incubate at 25C or 30C until a lawn is observed.

[0640] 3. Harvest plates and equilibrate to approximately OD600 of 0.3 for each isolate to normalize the inoculum.

[0641] 4. Prepare the anaerobic chamber by cleaning surfaces and by sealing the equipment - ensuring that the container allows for gas exchange.

[0642] 5. Add 30 mL of NF11 to 70 mL vials – 3 replicates / isolate.

[0643] 6. Add 150uL of inoculum / vial and balance to OD600 of 0.3 with sterile water.

[0644] 7. Pass the vials through the anaerobic chamber and seal under anaerobic conditions - include an empty vial (with foil "cap") to add the anaerobic indicator for QC purposes.

[0645] 8. Place the vial at 30C, 200 rpm for 5 hours.

[0646] 9. After working in the fume hood for 5 hours, remove 10% (4 ml) of the headspace from each vial and replace with the same volume of acetylene gas.

[0647] 10. Incubate at 30C, 200 rpm for 48 hours.

[0648] 11. After 48 hours, take a 1 mL headspace sample and place in a GC collection tube.

[0649] 12. Run the sample in the GC using the instrumental method for ethylene analysis "Split 4" and measure the acetylene peak and the ethylene peak.

[0650] 13. Quantify the amount of gas by peak area.

[0651] 14. Take the OD600 reading of 200ul of culture and TVC of the culture.

[0652] 15. Analyze the ethylene gas as the percentage of acetylene converted to ethylene. This yields an estimate of the overall conversion.

[0653] The volume of gas (ethylene) produced can be quantified using calibration points in Chromeleon or by calculation from the % peak area. Acetylene + Ethylene Peak Area % must = 100%. From the known amount of acetylene added, the ethylene produced can be determined in mL. 1M gas = 24 dm3 or 24,000 ml. Therefore 1 mM gas = 24 ml.

[0654] To calculate how many mM ethylene was produced, divide the amount by 24: mM ET = ml / 24

[0655] To calculate the rate: mM / hour / CFU, mM needs to be calculated as described above.

[0656] Rate = Total ethylene mM / (time (hours) x total CFU)

[0657] ARA and oxygen tolerance test program

[0658] Ensure all equipment and materials are sterile. Before autoclaving, seal equipment containers with foil wrap so they can be opened in the anaerobic chamber transfer box and aseptically transferred into the chamber. Seal bottles with foil before sterilization. A loose "seal" is needed to allow for gas exchange in the transfer box.

[0659] 1. Streak the isolate from -80C and incubate at 30C or 25C until colonies are observed.

[0660] 2. Spread one plate per isolate and incubate at 25C or 30C until a lawn is observed.

[0661] 3. Harvest plates and equilibrate to approximately OD600 of 0.3 for each isolate to normalize the inoculum.

[0662] 4. Prepare the anaerobic chamber by cleaning surfaces and by sealing the equipment - ensuring that the container allows for gas exchange.

[0663] 5. Place the NF11 medium in the a...

Claims

1. A synthetic composition comprising a plant component and a Paenibacillus bacterium heterologously placed to the plant component, wherein the Paenibacillus bacterium comprises an edit in one or more loci of its genome; wherein the edit is a deletion of at least one nucleotide, an insertion of at least one nucleotide, and / or a substitution of at least one nucleotide at or near one or more of the following genomic loci: nifH, glnR, nifD, GlnR binding site I, GlnR binding site II, cueR, orf1, nrgA, glnA, nif operon promoter region, nif cluster component promoter, or any combination or multiple edits at any one or more of said genomic loci; wherein the Paenibacillus bacterium exhibits an improved phenotype compared to a Paenibacillus bacterium not comprising the edit, wherein the improved phenotype is selected from the group consisting of increased acetylene reduction ability, improved nitrogen fixation ability, improved biofilm formation, increased culture turbidity, greater tolerance to nitrogen fixation under oxygen levels, improved nitrogen fixation under high nitrogen conditions, improved nitrogen fixation under low nitrogen conditions, and any multiple of the foregoing and / or combinations of the foregoing.

2. The synthetic composition of claim 1, wherein the edit is selected from the group consisting of: CueR C25 truncation, CueR C25 truncation + GlnR site II inactivation, CueR knockout, CueR knockout + GlnR site II duplication and inactivation, CueR knockout + GlnR site II duplication and truncation, CueR knockout + GlnR site II inactivation, CueR knockout + GlnR site II truncation, CueR knockout + nif PLH77 promoter insertion, CueR knockout + nif PLH77 promoter insertion + NrgA knockout, CueR knockout + NrgA knockout, CueR KO + GlnR site II duplication, GlnA SNP, GlnR C25 frameshift truncation and GlnA SNP, GlnR C25 frameshift truncation and GlnA SNP + nif knockout, GlnR C25 frameshift truncation, GlnR C25 frameshift truncation + Orf1KO, GlnR C25 truncation, GlnR C25 truncation and GlnA SNP, GlnR C25 truncation + GlnR site II duplication, GlnR C25 truncation + GlnR site II duplication and inactivation, GlnR C25 truncation + GlnR site II inactivation, GlnR C25 truncation + nifH knockout, GlnR C25 truncation + Orf1 knockout, GlnR knockout, GlnR site II duplication, GlnR site II duplication and inactivation, GlnR site II duplication and inactivation + GlnR C25 truncation, GlnR site II duplication and inactivation + NrgA knockout, GlnR site II duplication and truncation, GlnR site II inactivation, GlnR site II inactivation and duplication + nifH knockout, GlnR site II inactivation + GlnR C25 truncation, GlnR site II inactivation + NrgA knockout, GlnR site II truncation, GlnR site II truncation + GlnR C25 truncation, nif PLH77 promoter insertion, nifPLH77 promoter insertion + CueR KO, nif PLH77 promoter insertion + GlnR C25 truncation, nif PLH77 promoter insertion + NrgA knockout, nif PLH77d promoter insertion + CueR KO, nif PLH77d promoter insertion, nif PLH77d promoter insertion + GlnR C25 truncation, nifH knockout, NrgA knockout, NrgA knockout + GlnR C25 truncation, NrgA knockout + GlnR site II truncation, Orf1 knockout, Orf1 knockout + GlnR C25 frameshift truncation, Orf1 knockout + GlnR C25 truncation, NrgA knockout + NifPLH77d promoter insertion, Suf promoter exchange, and any multiple of the foregoing items and / or combinations of the foregoing items.

3. The synthetic composition of claim 1, wherein the Paenibacillus bacterium comprises a sequence selected from the group consisting of SEQ ID NOs: 1-123.

4. The synthetic composition of claim 1, further comprising a formulation component and / or an agricultural composition.

5. The synthetic composition of claim 1, wherein the Paenibacillus bacteria is present at a concentration of at least about 10^2 CFU / mL in a liquid formulation, or at a concentration of at least about 10^2 CFU / gram in a non-liquid formulation.

6. The synthetic composition of claim 1, further comprising at least one additional microorganism.

7. The synthetic composition of claim 1, wherein the plant component is a seed.

8. The synthetic composition of claim 1, wherein the plant component is a seed comprising a transgene.

9. The synthetic composition of claim 1, wherein the plant component is obtained from vegetative tissue.

10. The synthetic composition of claim 1, wherein the plant component is a plant propagation component.

11. The synthetic composition of claim 1, wherein the plant component is a whole plant.

12. The synthetic composition of claim 1, wherein the formulation components are selected from the group consisting of: compounds that improve the stability of the microorganism, preservatives, carriers, surfactants, anti-complexing agents, and any multiple items and / or combinations thereof.

13. The synthetic composition of claim 1, wherein the agricultural composition comprises a fungicide, a nematicide, a bactericide, an insecticide, a herbicide, a micronutrient, a macronutrient, nitrogen, phosphorus, potassium, or any multiple of the foregoing and / or a combination of the foregoing.

14. A plurality of synthetic compositions as described in claim 1, wherein the synthetic composition is substantially enclosed within an article selected from the group consisting of: a tube, a bottle, a jar, an ampoule, a package, a container, a bag, a box, a storage box, an envelope, a carton, a container, a silo, a shipping container, a carriage, and a case.

15. A plurality of synthetic compositions as claimed in claim 14, wherein the synthetic compositions are at a temperature below zero degrees Celsius.

16. The synthetic composition of claim 1, wherein the plant parts are obtained from a monocotyledonous plant.

17. The synthetic composition of claim 16, wherein the monocot is a C3 monocot.

18. The synthetic composition of claim 16, wherein the monocot is a C4 monocot.

19. The synthetic composition of claim 1, wherein the plant parts are obtained from a dicotyledonous plant.

20. The synthetic composition of claim 1, wherein the agricultural composition comprises a growth medium.

21. The synthetic composition of claim 20, wherein the growth medium comprises soil.

22. A plurality of synthetic compositions as recited in claim 1, wherein said plurality of synthetic compositions are placed in said soil in a regular pattern with substantially equal spacing between each of said synthetic compositions.

23. A plant obtained, grown or derived from the synthetic composition of claim 1, wherein said plant comprises said Paenibacillus bacterium.

24. The plant of claim 23, wherein the plant exhibits improved agronomically important traits compared to plants not obtained, grown or derived from the synthetic composition of claim 1.

25. The plant of claim 24, wherein the improved agronomically important trait is yield, plant health and / or plant vigor.

26. A method of improving the health, yield and / or vigor of a plant, the method comprising: (a) associating a component of the plant with a Paenibacillus bacterium comprising an edit in one or more loci of its genome; wherein the edit is a deletion of at least one nucleotide, an insertion of at least one nucleotide, and / or a substitution of at least one nucleotide at or near one or more of the following genomic loci: nifH, glnR, nifD, GlnR binding site I, GlnR binding site II, cueR, Orf1, nrgA, glnA, the nif operon promoter region, the nif cluster component promoter, or any combination or multiple edits at any one or more of said genomic loci; (b) placing the component parts of the crop plant in a medium that supports plant growth; (c) growing plants from said component parts of said crop plants; (d) evaluating one or more characteristics of the plant, wherein at least one of the characteristics is improved compared to the same characteristic of a plant not obtained from a component associated with the Paenibacillus bacterium of (a).

27. The method of claim 26, wherein the one or more characteristics of (d) comprise improved nitrogen fixation, increased biomass, increased leaf area, increased plant height, increased root area, increased shoot nitrogen composition, increased greenness, increased NDVI, increased NPCI, increased PSRI, increased CCI, increased yield, and any combination of the foregoing.

28. The method of claim 26, further comprising at least one additional microorganism.

29. The method of claim 26, wherein associating the components of the crop plant with the Paenibacillus bacteria comprising an edit in one or more loci of its genome is accomplished by a method selected from the group consisting of in-furrow application, soil drench application, side-drip application, and any combination of the foregoing.

30. The method of claim 26, wherein associating the crop plant component with the Paenibacillus bacterium comprising an edit in one or more loci of its genome is accomplished by coating the crop plant component with a liquid formulation of the bacterium.

31. The method of claim 26, wherein associating the crop plant component with the Paenibacillus bacteria comprising an edit in one or more loci of its genome is accomplished by coating the crop plant component with a substantially non-liquid formulation of the bacteria.

32. The method of claim 26, wherein the plant component is a plant propagation component.

33. The method of claim 26, wherein the plant part is a leaf.

34. The method of claim 26, wherein the plant component is a whole plant.

35. A modified Paenibacillus bacterium, wherein the Paenibacillus bacterium comprises an edit in one or more loci of its genome, wherein the edit is a deletion of at least one nucleotide, an insertion of at least one nucleotide, and / or a substitution of at least one nucleotide at or near one or more of the following genomic loci: nifH, glnR, nifD, GlnR binding site I, GlnR binding site II, cueR, Orf1, nrgA, glnA, nif operon promoter region, nif cluster component promoter, or any combination or multiple edits at any one or more of the genomic loci.

36. The modified Paenibacillus bacterium of claim 35, wherein the Paenibacillus bacterium exhibits an improved phenotype compared to a Paenibacillus bacterium not comprising the edit, wherein the improved phenotype is selected from the group consisting of increased acetylene reduction ability, improved biofilm formation, increased culture turbidity, greater tolerance to nitrogen fixation to oxygen levels, and any combination of the foregoing.

37. The modified Paenibacillus bacterium of claim 35, wherein the editing is selected from the group consisting of CueR C25 truncation, CueR C25 truncation + GlnR site II inactivation, CueR knockout, CueR knockout + GlnR site II duplication and inactivation, CueR knockout + GlnR site II duplication and truncation, CueR knockout + GlnR site II inactivation, CueR knockout + GlnR site II truncation, CueR knockout + nif PLH77 promoter insertion, CueR knockout + nif PLH77 promoter insertion + NrgA knockout, CueR knockout + NrgA knockout, CueR KO + GlnR site II duplication, GlnA SNP, GlnR C25 frameshift truncation and GlnASNP, GlnR C25 frameshift truncation and GlnA SNP + nifH knockout, GlnR C25 frameshift truncation, GlnR C25 frameshift truncation + Orf1 KO, GlnR C25 truncation, GlnR C25 truncation and GlnA SNP, GlnR C25 truncation + GlnR site II duplication, GlnRC25 truncation + GlnR site II duplication and inactivation, GlnR C25 truncation + GlnR site II inactivation, GlnR C25 truncation + nifH knockout, GlnR C25 truncation + Orf1 knockout, GlnR knockout, GlnR site II duplication, GlnR site II duplication and inactivation, GlnR site II duplication and inactivation + GlnR C25 truncation, GlnR site II duplication and inactivation + NrgA knockout, GlnR site II duplication and truncation, GlnR site II inactivation, GlnR site II inactivation and duplication + nifH knockout, GlnR site II inactivation + GlnR C25 truncation, GlnR site II inactivation + NrgA knockout, GlnR site II truncation, GlnR site II truncation + GlnR C25 truncation, nif PLH77 promoter insertion, nif PLH77 promoter insertion + CueR KO, nifPLH77 promoter insertion + GlnR C25 truncation, nifPLH77 promoter insertion + NrgA knockout, nif PLH77d promoter insertion + CueR KO, nif PLH77d promoter insertion, nifPLH77d promoter insertion + GlnR C25 truncation, nifH knockout, NrgA knockout, NrgA knockout + GlnR C25 truncation, NrgA knockout + GlnR site II truncation, Orf1 knockout, Orf1 knockout + GlnR C25 frameshift truncation, Orf1 knockout + GlnR C25 truncation, and NrgA knockout + Nif PLH77d promoter insertion, Suf promoter exchange, and any multiple of the foregoing and / or combinations of the foregoing.

38. The modified Paenibacillus bacterium of claim 35, wherein the Paenibacillus bacterium comprises a sequence selected from the group consisting of SEQ ID NOs: 1-123.

39. A substantially pure composition comprising the modified Paenibacillus bacterium of claim 35.

40. A bacterial culture comprising the modified Paenibacillus bacterium of claim 35.

41. A fermentation culture comprising the modified Paenibacillus bacterium of claim 35.

42. An agricultural composition comprising the modified Paenibacillus bacterium of claim 35 and an agriculturally acceptable carrier.

43. The agricultural composition of claim 42, further comprising a plant or plant component, wherein the modified Paenibacillus bacterium is present in the agricultural composition in an amount effective to produce an improved phenotype in the plant.

44. The agricultural composition of claim 43, wherein the improved phenotype is increased health, yield and / or vigor of the plant.

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