WO2022256254A1 - Methods of selecting microbes for improvement of plant growth - Google Patents

Abstract

Provided are methods for selecting microbes for improvement of plant growth, including identifying microbes that confer early seedling vigor. Some aspects include enriching a microbe in a plant or seed. The microbe may be transmitted vertically through plant generations. The microbe may be isolated and used to enhance growth of other plants.

Description

METHODS OF SELECTING MICROBES FOR IMPROVEMENT OF PLANT GROWTH

CROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Application No.63/195, 363, filed June 1, 2021, which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] Agricultural products such as crops are critical. Improved crop growth and abundance is important in view of increasing population size and potential loss of suitable land. Microbes may have properties that may help improve crop growth. For example, microbes with anti fungal properties may be used to reduce crop lost. Additional microbes may exist that potentially provide additional benefits for crop growth.

SUMMARY

[0003] Disclosed herein, in some aspects, are methods comprising: (a) inoculating a microbial population with an endophyte obtained by: (i) screening a plant comprising the endophyte for speed of growth or germination, (ii) obtaining a seed from the plant comprising the endophyte, and (iii) obtaining the endophyte from the seed; (b) incubating the microbial population with the endophyte to produce a second microbial population comprising the endophyte; and (c) sequencing nucleic acids of the second microbial population, thereby obtaining nucleotide sequence information of a microbe that interacts with the endophyte to enhance growth of the endophyte. In some aspects, sequencing the nucleic acids of the second microbial population comprises obtaining operational taxonomic unit (OTU) abundances of the microbes of the second microbial population. Some aspects include identifying the microbe as enhancing the growth of the endophyte. Some aspects include identifying an increase in the growth of the endophyte in the presence of the microbe, relative to growth of the endophyte in the absence of the microbe or in the presence of another microbe. Some aspects include isolating the endophyte. Some aspects include isolating the microbe. Some aspects include inoculating a seed or crop with the microbe and the endophyte. In some aspects, the plant comprises a leafy green vegetable. In some aspects, the leafy green vegetable comprises lettuce. In some aspects, the plant comprises a legume such as soybean. Other plants may be used. In some aspects, the endophyte comprises a microbial endophyte. In some aspects, the microbial endophyte comprises a bacterium. In some aspects, the endophyte comprises a population of endophytes. [0004] Disclosed herein, in some aspects, are methods comprising: (a) growing a plurality of plant seedlings, and identifying one or more early emergers of the plurality of seedlings, the one or more early emergers comprising a seedling that (i) germinates earlier than an average germination time of the plurality of seedlings or (ii) grows faster than an average rate of growth of the plurality of seedlings; (b) growing a plurality of second generation seedlings from seeds of the one or more early emergers, and identifying a second generation early emerger of the second generation seedlings, the second generation early emerger comprising a seedling that (i) germinates earlier than an average germination time of the plurality of second generation seedlings or (ii) grows faster than an average rate of growth of the plurality of second generation seedlings, (c) obtaining or isolating an endophyte from the second generation early emerger; and (d) identifying a property of the endophyte that enhances plant germination or growth. In some aspects, the property comprises plant hormone production by the endophyte, or enhancement of plant hormone production by the plant. In some aspects, the plant hormone comprises ethylene, indole-3 -acetic acid (IAA), or giberellic acid. In some aspects, the property comprises amylase, protease, or lipase production by the endophyte, or enhancement of amylase, protease, or lipase production by the plant. In some aspects, the property comprises 1-aminocyclopropane-l- carboxylic acid (ACC) deaminase production. In some aspects, the property comprises increasing plant root surface area. In some aspects, the endophyte is vertically transmitted from the one or more early emergers of the plurality of plant seedlings. In some aspects, (a) comprises growing plant seedlings from seeds of plants of 1, 2, 3, 4 or more earlier generations of early emergers. In some aspects, growing comprises plant development through to seed set. An amount of time for such growing may be reduced by a method described herein. In some aspects, growing comprises growing indoors. Some aspects include identifying a microbe that enhances growth of the endophyte. Some aspects include isolating the microbe. Some aspects include inoculating a seed or crop with the microbe. Some aspects include inoculating a seed or crop with the endophyte. In some aspects, the plant comprises a leafy green vegetable. In some aspects, the leafy green vegetable comprises lettuce. In some aspects, the plant comprises a legume such as soybean. Other plants may be used. In some aspects, the endophyte comprises a microbial endophyte. In some aspects, the microbial endophyte comprises a bacterium. In some aspects, the endophyte comprises a population of endophytes.

[0005] Disclosed herein, in some aspects, are methods comprising: (a) growing plant seedlings under a stress condition, and identifying first early emergers of the seedlings, the first early emergers comprising seedlings that germinate or grow faster than other seedlings of the seedlings; (b) growing second generation seedlings from seeds of the early emergers under the stress condition, and identifying a second generation early emerger of the second generation seedlings, the second generation early emerger comprising a seedling that germinates or grows faster than other seedlings of the second generation seedlings, and comprising an endophyte vertically transmitted from one of the early emergers; and (c) obtaining or isolating the endophyte from the second generation early emerger. In some aspects, the stress condition comprises an environmental stress condition. In some aspects, the environmental stress condition comprises low temperature, low pH, high pH, or high salt. In some aspects, the stress condition comprises scarcity of a nutrient. In some aspects, the stress condition comprises a presence of a pathogen. In some aspects, the pathogen comprises a soil or foliar pathogen. In some aspects, the pathogen comprises a fungus. In some aspects, (a) comprises growing plant seedlings from seeds of plants of 1, 2, 3, 4 or more earlier generations of early emergers. In some aspects, growing comprises plant development through to seed set. An amount of time for such growing may be reduced by a method described herein. In some aspects, growing comprises growing indoors. Some aspects include identifying a microbe that enhances growth of the endophyte. Some aspects include isolating the microbe. Some aspects include inoculating a seed or crop with the microbe. Some aspects include inoculating a seed or crop with the endophyte. In some aspects, the plant comprises a leafy green vegetable. In some aspects, the leafy green vegetable comprises lettuce. In some aspects, the plant comprises a legume such as soybean. Other plants may be used. In some aspects, the endophyte comprises a microbial endophyte. In some aspects, the microbial endophyte comprises a bacterium. In some aspects, the endophyte comprises a population of endophytes.

INCORPORATION BY REFERENCE

[0006] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0008] FIG. 1 illustrates a schematic of examples methods described in the disclosure.

[0009] FIG. 2 illustrates a schematic of example methods described in the disclosure. DETAILED DESCRIPTION

[0010] There is a need in the art for accelerating germination and growth, or improving seedling root system architectures for indoor agriculture. Microbiome enrichment may accelerate germination or growth, or improve seedling root system architectures. For example, enrichment of enrichment of lettuce endophytes may promote early seedling vigor. Microbes may be used to inhibit the growth of pathogens that may kill or damage the plants and agriculture.

[0011] Endophytes and other microbes may live within plants and provide benefits to the plants. Endophytes may be present in the seeds or other parts of plants. Endophytes and associated microbes may provide benefits to seeds or plants such that the plant may comprise particular characteristics. For example, endophytes may improve the plant’s resistance to abiotic or biotic stresses, for example, drought, changes in the environments, or pathogens. Additionally, a plant or seed comprising a given endophyte may grow faster compared to a plant or seed without an endophyte or with a different endophyte. As such, identifying and isolating endophytes may be beneficial to improving plant and agricultural growth.

[0012] Growing plants or seedlings may include a propagation phase. In some cases, leafy green plants such as lettuce grown indoors may need a propagation phase. The propagation phase may include 1-2 days for germination and 9-12 days of growth to reach a maturity, size, or complexity before seedlings may be transferred to a farm such as an outdoor farm for additional growth. It may be commercially advantageous to speed up a propagation phase, for example to get through more crop cycles in any given time and enhance plant seedling production.

[0013] Disclosed herein, in some aspects, are methods that include microbial enrichment such as endophyte enrichment, or that include use of microbes such as endophytes. The systems and methods described herein may be used to select or identify endophytes and associated microbes. The endophytes may be present in the plant such as in the seed. A method may include growing a plurality of plants or plant seedlings. The plurality of plants or plant seedlings may be observed for a particular or given characteristic. For example, growth speed or time for emergence from a seed may be used to characterize the plants or plant seedlings. Some aspects include identifying one or more early emergers of the plurality of seedlings. In some aspects, the one or more early emergers include a seedling that germinates earlier than an average germination time of the plurality of seedlings. In some aspects, the one or more early emergers include a seedling that grows faster than an average rate of growth of the plurality of seedlings. [0014] In various aspects, the methods may include growing a plurality of second generation seedlings from seeds of an earlier plurality of seedling. For example, a plurality of seedlings derived from the seeds of one or more early emergers may be used to grow a second generation of seedlings. The second generation of seedlings may be observed for particular characteristics, and may be similar characteristics that were observed or selected for in an earlier generation of plants or seedlings. For example, the method may include identifying a second generation early emerger of the second generation seedlings. The second generation early emerger may include a seedling that germinates earlier than an average germination time of the plurality of second generation seedlings. The second generation early emerger may include a seedling that grows faster than an average rate of growth of the plurality of second generation seedlings. Some aspects include obtaining or isolating an endophyte from the second generation early emerger. The methods may be performed iteratively. For example, a first set of plants or seedling may be grown and observed. Plants or seedlings with a given, particular, or desired characteristic may be harvested or allowed to propagate. Seeds (or other plant matter) derived from the plants with a given, particular, or desired characteristic (e.g., the top ten fastest growing plants in a set) may then be used to generate a new set of plants and be monitored. This new set of plants may then be observed for characteristics and seeds (or other plant matter) from certain selected planted may be harvested and allowed to generate an additional set of plants. This process can be repeated multiple times to generate new plants. Harvesting, extracting, isolating, or other reaction (or process) such as those described elsewhere herein may be performed on any of the plants throughout the iterative process. For example, endophytes may be isolated from a nth, n+1, n+2, etc., generation and analyzed. The iterative process may be stopped once a desired or particular characteristic is observed in the plant. For example, a plant may be partially or minimally resistant to a pathogen. After multiple generation of iteratively obtaining seeds from plants that demonstrate resistance to a pathogen, the method may result in the generation of a plant that has improved resistance to the pathogen.

[0015] In various aspects, the endophyte is vertically transmitted from the plant, seed, or seedling, of an earlier generation. The endophyte may be vertically transmitted from the one or more plants that demonstrate a trait or characteristic of interest from a plurality of plant seedlings or an earlier generation. In some aspects, the endophyte is vertically transmitted from the one or more early emergers of the plurality of plant seedlings. In some aspects, an endophyte is obtained by (i) screening a plant comprising the endophyte for speed of growth or germination, (ii) obtaining a seed from the plant comprising the endophyte, and (iii) obtaining the endophyte from the seed. In some aspects, growing a plurality of plant seedlings includes growing plant seedlings from seeds of plants of 1, 2, 3, 4 or more earlier generations of early emergers.

[0016] In various aspects, plants and plant seedlings are grown in cultivation conditions.

The cultivation conditions may be a time, temperature, soil type, soil pH, salinity, or with the presence of abiotic stimuli or stress, or the presence of biotic stimuli or stress. In some embodiments, the growing comprises growing indoors for 10 to 14 days. In some aspects, growing indoors comprises 1 or 2 days of indoor growing to germinate. In some aspects, growing indoors comprises 9 to 12 days of indoor growing to reach sufficient maturity to be transplanted outdoors. In some embodiments, the growing comprises growing indoors for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 30 or more days. In some embodiments, the growing comprises growing indoors for no more than 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 30 or less days. In some aspects, growing comprises growing indoors. In some aspects, growing comprises growing outdoors. In some aspect, growing comprises growing indoors and growing outdoors. In some aspects, growing comprises plant development through to seed set. An amount of time for such growing may be reduced by a method described herein. The growing time may be influenced by the endophyte, such that the average growing time is less than the average growing time for a plant without the endophyte. The speed of growing may be used as a metric to identify plants with potential characteristic of interest. For example, the fastest growing plant may be analyzed, or have their associated endophytes analyzed or isolated.

[0017] In some aspects, growing comprises growing under a stress condition. In some aspects, the stress condition comprises an environmental stress condition. In some aspects, the environmental stress condition comprises low temperature, low pH, high pH, or high salt, low water availability, low mineral availability, the presence of organic or inorganic toxins, low nutrient availability, or poor air quality . For example, the temperature may be less than 20°C. For example, the temperature may be less than 15°C. For example, the temperature may be less than 10°C. For example, the temperature may be less than 5°C. In some cases, the pH is greater than 8. Some temperature examples include 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, irC, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C, or a range defined by any two of the aforementioned temperatures. In some cases, the pH is greater than 9. In some cases, the pH is greater than 10. In some cases, the pH is greater than 11. In some cases, the pH is less than 6. In some cases, the pH is less than 5. In some cases, the pH is less than 4. Some pH examples include a pH of 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,

4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,

6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3,

8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3,

10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0, or a range defined by any two of the aforementioned pH values. In some aspects, the stress condition comprises scarcity of a nutrient. For example, the stress condition may comprise a scarcity of a mineral, carbohydrate, polypeptide, lipid, phosphate, nitrogen, oxygen, carbon or other element or chemical.

[0018] In one embodiment, the stress condition is applied to the plant or the soil (or other growth medium that the plant in grown in). The stress condition may be applied at any time, for example, while the plant in still a seed, or when the plant is in the process of sprouting.

[0019] The plants may be grown and subjected to the selective pressure for any appropriate length of time before they are selected and harvested. By way of example only, the plants and any microorganisms associated with them may be selected and harvested at any time during the growth period of a plant, in one embodiment, any time after germination of the plant. In a preferred embodiment, the plants are grown or allowed to multiply for a period which allows one to distinguish between plants having desirable phenotypic features and those that do not.

[0020] In various aspects, multiple stress conditions may be applied to the plants. Two or more stress conditions may be applied simultaneously. Two or more stress conditions may be applied simultaneously.

[0021] In some aspects, the stress condition comprises a presence of a pathogen or other organism that is detrimental to plant survival and growth including fungi, bacteria, viruses, or nematodes. In some aspects, the pathogen comprises a soil or foliar pathogen. In some aspects, the pathogen comprises a fungus. The fungal pathogen may be a fungal pathogen of the genus Fusarium. The fungal pathogen may be a Fusarium oxysporum. The fungal pathogen can be a fungal pathogen in the genus Albugo, Alternaria, Aphanomyces, Armillaria, Aspergillus,

Botrytis, Botrydiplodia, Botrytinia, Bremia, Cercospora, Cercosporella, Cladosporium, Colletotrichum, Cordana, Corynespora, Cylindrocarpon, Daktulosphaira, Didymella, Elsinoe, Erysiphe, Eutypa, Fusarium, Ganoderma, Guignardia, Gymnoconia, Helminthosporium, Leptosphaeria, Leveillula, Macrophomina, Microsphaera, Monolinia, Mycosphaerella,

Oidopsis, Passalora, Peronospora, Phomopsis, Phytophthora, Peronospora, Phoma, Plasmodiophora, Plasmopara, Podosphaera, Polyscytalum, Pseudocercospora, Puccinia, Pucciniastrum, Pythium, Ralstonia, Ramularia, Rhizoctonia, Rhizopus, Septoria, Sclerotinia, Sclerotium, Sphaerotheca, Sphaceloma, Spongospora, Stemphylium, Synchytrium, Thielaviopsis, Uncinula, Uromyces, or Verticillium. The fungal pathogen can be Albugo Candida, Albugo occidentalis, Alternaria alternata, Alternaria cucumerina, Alternaria dauci, Alternaria solani Alternaria tenuis, Alternaria tenuissima, Alternaria tomatophila,, Aphanomyces euteiches, Aphanomyces raphani, Armillaria mellea, Botrydia theobromae, Botrytis cinerea, Botrytinia fuckeliana, Bremia lactuca, Cercospora beticola, Cercosporella rubi, Cladosporium herbarum, Colletotrichum acutatum, Colletotrichum gloeosporioides, Colletotrichum lindemuthianum, Colletotrichum musae, Colletotrichum spaethanium, Cordana musae, Corynespora cassiicola, Daktulosphaira vitifoliae, Didymella bryoniae, Elsinoe ampelina, Elsinoe mangiferae, Elsinoe veneta, Erysiphe cichoracearum, Erysiphe necator, Eutypa lata, Fusarium germinareum, Fusarium oxysporum, Fusarium solani, Ganoderma boninense, Guignardia bidwellii, Gymnoconia peckiana, Helminthosporium solani, Leptosphaeria coniothyrium, Leptosphaeria maculans, Leveillula taurica, Macrophomina phaseolina, Microsphaera alni, Monilinia fructicola, Monilinia vaccinii-corymbosi, Mycosphaerella angulate, Mycosphaerella brassicicola, Mycosphaerella fragariae, Mycosphaerella fijiensis, Oidopsis taurica, Passalora fulva, Peronospora sparse, Peronospora farinosa, Phoma exigua, Phomopsis obscurans, Phomopsis vaccinia, Phomopsis viticola, Phytophthora capsica, Phytophthora erythroseptica, Phytophthora infestans, Phytophthora parasitica, Plasmopara viticola, Plasmodiophora brassicae, Podosphaera macularis, Polyscytalum pustulans, Pseudocercospora vitis, Puccinia allii, Puccinia sorghi, Pucciniastrum vaccinia, Pythium debaryanum, Pythium sulcatum,

Pythium ultimum, Ralstonia solanacearum, Ramularia tulasneii, Rhizoctonia solani, Rhizopus arrhizus, Rhizopus stoloniferz, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotium cepivorum, Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum, Septoria apiicola, Septoria lactucae, Septoria lycopersici, Septoria petroelini, Sphaceloma perseae, Sphaerotheca macularis, Spongospora subterrannea, Stemphylium vesicarium, Synchytrium endobioticum, Thielaviopsis basicola, Uncinula necator, Uromyces appendiculatus, Uromyces betae, Verticillium albo-atrum, Verticillium dahliae, Verticillium theobromae, or a combination thereof. [0022] In various aspects, the plants and seed may be observed based at least on a resistance to a stress condition. For example, the plants or seed may be observed to show some resistance to a fungal infection. A plant may show fewer symptoms of fungal infection as compared to an average plant. A plant that shows fewer symptoms of fungal infection may comprise an endophyte or associated microbes that are conferring a partial or full fungal resistance. By observing and selecting (or harvesting seeds or other plant material from) plants that show fewer symptoms, additional generations of plants may be grown that have fewer symptoms of fungal infections. Endophytes may be extracted from the plants.

[0023] Plants, seedlings, or seeds may be observed and selected based at least on any observable characteristic or phenotype of the plant, including for example growth rate, height, weight, color, leaf shape, leaf health, leaf structure, leaf size, stem size, root size, taste, smell, changes in the production of one or more molecules by the plant, such as metabolites, polypeptides, carbohydrates, lipids, or hormones.. Plants may be selected based at least on resistance (including partial or nominally improved resistance) to a stress condition. Plants may also be observed and selected based at least on speed of seed germination; quantity of biomass produced, increased root, leaf, stem, or seedling growth, or other characteristics associated with crop yield.

[0024] Some aspects include inoculating a seed or crop with the endophyte. In some aspects, the endophyte comprises a microbial endophyte. In some aspects, the microbial endophyte comprises a bacterium. In some aspects, the endophyte comprises a population of endophytes. The endophyte be selected and identified by using the methods of selecting for plants as described elsewhere herein. The endophyte may be selected and identified based at least on its association with a plant. A plant, or seedling may demonstrate characteristics or phenotype which may be associated with an endophyte. The endophyte may then be extracted or isolated as potentially able to confer the plant with the characteristic observed. The endophyte may provide beneficial properties to the seed or crop.

[0025] In various aspects, the plants, seedlings or seeds may be observed or monitored. The plurality of plants or plant seedlings may be observed for a particular or given characteristic. For example, growth speed or time for emergence from a seed may be used to characterize the plants or plant seedlings. Some aspects include identifying one or more early emergers of the plurality of seedlings. In some aspects, the one or more early emergers include a seedling that germinates earlier than an average germination time of the plurality of seedlings. In some aspects, the one or more early emergers include a seedling that grows faster than an average rate of growth of the plurality of seedlings. The plants may be monitored and measured based on various parameters related to growth of the plants, such as growth rate or percentages of seed that sprout, or weight of plant. The plant may be monitors based on vigor index. Vigor index may be calculated wherein Vigor index (VI) =(RL+SL)xGP, where RL is root length (cm), SL is shoot length (cm) and GP is germination percentage. Vigor index may be calculated based on dry plant matter production of a seedling.

[0026] The plant may include any plant. In some aspects, the plant comprises a leafy green vegetable. In some aspects, the leafy green vegetable comprises lettuce. In some aspects, the plant comprises a legume. An example of a legume includes a soybean.

[0027] Some aspects include identifying a property of the endophyte that enhances plant germination or growth. For example, the endophyte may increase root surface area to allow for improved water uptake. The mechanism identified may be the increased root surface area or may be the underlying chemical mechanism that is generating the increase root surface area. For example, the property may be expression of a polypeptide, or the generation of a metabolite or signaling molecule. In some aspects, the property comprises plant hormone production by the endophyte, or enhancement of plant hormone production by the plant. In some aspects, the plant hormone comprises ethylene, indole-3 -acetic acid (IAA), or giberellic acid. In some aspects, the property comprises amylase, protease, or lipase production by the endophyte, or enhancement of amylase, protease, or lipase production by the plant. In some aspects, the property comprises 1- aminocyclopropane-1 -carboxylic acid (ACC) deaminase production. In some aspects, the property comprises increasing plant root surface area.

[0028] Some aspects include identifying a microbe that enhances growth of the endophyte. For example, a microbe may interact directly with the endophyte The microbe may directly interact with the endophyte a produce a multicellular structure. The microbe may directly interact with the endophyte and facilitate interaction of the endophyte and the plant. The microbe may produce a metabolite. The metabolite may be consumed by the endophyte or may be otherwise used by the endophyte. The microbe may produce an enzyme or an enzyme substrate. The enzyme or substrate may allow for the generation of a metabolite or a nutrient, or may generate an environment for improved endophyte or plant growth. For example, the microbe may generate or remove oxygen, or acidify or de-acidify the environment, which may improve endophyte growth. The microbe may produce a hormone or other signaling molecule that may improve endophyte growth or function. The hormone or signaling molecule may also affect the plant growth.

[0029] Some aspects include isolating the microbe. One or more plants may be harvested and tissues or other plant matter derived from the plant may be obtained. For example, the seed, stems, roots, leaves, flowers, or other tissues from the plants may be harvested. The plants may be analyzed such to identify the presence of a microbe in the tissues. The plant matter may also be sterilized or otherwise processed to remove contaminating microbes. For example, the microbe may be an endophyte present in the plant seed. Contaminating microbes may be present on the outside of the seed that are not associated with or confer the benefits associated with the endophyte. By sterilizing the outside of the seed, the contaminating microbes may be removed and the endophyte may be later extracted. The extraction of endophyte may comprise excision of plant material.

[0030] The microbe may be isolated via culturing, plating or other methods. For example, plant matter comprising the microbes may be added to liquid solutions to allow the microbes to be suspended in liquid suspension. The liquid suspension may then be added to liquid media or agar or other solid media. The microbes may be cultured in a liquid media and diluted. The microbe may be plated at a dilution such that individual microbial colonies may be isolated and then subsequently cultured individually. For example, the plurality of microbes can be subjected to serial dilutions such that a colony of a particular microbe can be isolated. The serial dilutions can each be cultured in liquid, semi-solid, or solid media. On a semi-solid or solid media such as an agar plate, the plurality of microbes can form colonies. The colonies can be well dispersed so that a colony can contain a single strain or species of microbe. Isolation of a particular microbe can also be performed using physical separation methods such a centrifugation. For example, a plurality of microbes may be cultured in liquid media and centrifuged in order to isolate the microbes from the culture. A particular microbe may also be isolated using a particular growth condition. For example, a particular microbe may have higher viability when compared to another microbe when cultured in anaerobic conditions. A particular microbe may have a high viability compared to another microbe when cultured in a media rich in a particular nutrient. [0031] Isolated microbes may be screened to identify particular characteristics associated with the microbe. For example, the microbes may be plated on an solid media and allowed to interact with a fungal pathogen. The microbe and fungal pathogen may be observed to identify anti-pathogenic activity in the microbe. The isolated microbe may be applied to a plant or seedling to confer any beneficial characteristics to the plant or seedling.

[0032] Some aspects include inoculating a seed or crop with a microbe, such as an endophyte. Inoculation may be performed by directly adding the microbe to a seed or crop at any time during the life cycle of the plant. For example, the seed may be dipped in a liquid culture comprising a microbe and the planted in soil or other suitable growth environment The microbe may be added to the soil prior to planting of a seed. The microbe may be inoculated by contacting the root, stem, or leaves of the plant. The microbe may be added prior to or after germination of the plant. The plant, seedling, or seed may be inoculated with multiple microbes. For example, the plant, seedling, or seed may be inoculated with the endophyte as well as other microbes that provide a synergistic benefit to the plant, seed, or seedling. The plant, seed, or seedling may be inoculated with any microbe or microbial population as generated using the methods described elsewhere herein.

[0033] Some aspects include incubating a microbial population with an endophyte to produce a second microbial population comprising the endophyte. The second microbial population may comprise microbes that interact with the endophyte. The second microbial population may comprise microbes that enhance the growth of endophytes or enhance the growth of the plant. The second microbial population may comprise an enriched amount of a microbe as compared the original microbial population. Some aspects include sequencing nucleic acids of the second microbial population. Some aspects include obtaining nucleotide sequence information of a microbe that interacts with the endophyte to enhance growth of the endophyte. In some aspects, sequencing the nucleic acids of the second microbial population comprises obtaining operational taxonomic unit (OTU) abundances of the microbes of the second microbial population. Some aspects include identifying the microbe as enhancing the growth of the endophyte. Some aspects include identifying an increase in the growth of the endophyte in the presence of the microbe, relative to growth of the endophyte in the absence of the microbe or in the presence of another microbe.

[0034] Upon culturing a microbial population for a duration of time, the overall percentage representation of each microbe in the subset may change from the percentage at the start of culturing. For example, microbes which remain viable among other microbes after different periods of culturing may indicate a symbiotic relationship or interaction between the microbes of the culture and these microbes may form a microbial consortium. The microbial consortia can be tested for efficacy of producing a characteristic or phenotype in a manner similar to methods used for identifying the efficacy of microbes as described elsewhere herein.

[0035] Methods of identifying and/or selecting for a endophyte composition can comprise culturing the endophyte in isolation or with a plurality of other microbes along with a stress condition such as a fungal pathogen. For example, the endophyte can be cultured with a fungal pathogen to identify efficacy of the endophyte to inhibit growth of the fungal pathogen. The efficacy of the endophyte to inhibit the growth of the fungal pathogen can be determined by the observing the growth parameters of the fungal pathogen. For example, the lack of living fungal pathogen close to the endophyte on a growth media may be used determine a high efficacy of inhibition. The optical density of a liquid media containing the endophyte and the fungal pathogen may be used to identify an efficacy of the endophyte. Efficacy may be observed for microbial populations similarly as to for an isolated microbe and may be used to identify compositions with synergistic microbes.

[0036] The endophyte may be affected by other microbes. The microbes can behave synergistically when cultured together such that the properties conferred to the plant (such as resistance to a pathogen) are improved when cultured (or inoculated) together compared to when separate. For example, the endophyte may have increased viability when cultured with another microbe. The endophyte may have increased proliferation when cultured with another microbe. The endophyte may use chemicals or metabolites produced by another microbe. The endophyte may interact directly with another microbe. For example, the endophyte and another microbe may form biofilms or a multicellular structure. The endophyte may produce and/or secrete an increased amount of the secondary metabolite when cultured with another microbe. For example, the endophyte may produce an intermediate metabolite, which in turn is processed by another microbe resulting in the secondary metabolite. Methods disclosed elsewhere herein can be used to identify microbes which may benefit from culturing with another microbe, as well as identify endophyte compositions comprising a first microbe and a second microbe wherein the second microbe is not identical to the first microbe.

[0037] Methods for identifying or selecting microbial compositions can be used to generate microbial populations with particular or specific characteristics. For example, methods as disclosed in U.S. Patent Publication No. 20180127796 can be used for identifying or selecting for microbial consortia, including microbes and microbial consortia that may interact with the endophyte. For example, a plurality of microbes including the endophyte can be grown together. The aforementioned patent publication is incorporated herein by reference in its entirety. In some cases, the method can comprise diluting a sample to form plurality of dilution, wherein a dilution in the plurality of dilutions comprises a subset of the plurality of microbes. The dilutions may allow for the generation of a plurality of subsets in which different microbes of the plurality of microbes are allowed to interact. The subset of the plurality of microbes can be subjected to culturing such that the microbes may proliferate. The subsets can be subjected to sequencing reactions such that sequences of the microbes can be obtained. From the sequencing reaction, the species, strain, or other taxonomic information can be obtained. The subsets can be subjected to varying culturing times such can be subjected to sequencing reactions at various times to monitor the presences and/or relative abundance of a particular species, strain or other taxonomic category. By observing the changes in the presence and/or relative abundance of a particular species, strain or other taxonomic category, the interaction between multiple microbes, such as interactions with endophytes and other microbes, can be determined. For example, a first microbe may have a higher relative abundance when cultured with a second microbe when compared to a relative abundance when not cultured with the second microbe. In this example, the first microbe may interact with the second microbe such that the first microbe’s overall viability is increased. The plurality of dilutions can each be subjected to sequencing reactions such that the microbes of each dilution can be identified, and can allow for a multiplexed, high throughput approach.

[0038] In some embodiments, the method comprises: diluting a sample to form a plurality of dilutions of the sample (e.g., via dispersal or chance), wherein the sample comprises a plurality of microorganisms including the endophyte; cultivating (or enriching) the plurality of dilutions of the sample in a first cultivation condition (also referred to as environmental filtering); determining taxonomic information of taxa in the plurality of dilutions of the sample cultivated in the first cultivation condition (e.g., using gene amplicon sequencing, such as gene amplicon sequencing of 16S rRNA, 12S rRNA, 18S rRNA, 28S rRNA, 13S rRNA and 23S rRNA, internal transcribed spacer (ITS), ITS1, ITS2, cytochrome oxidase I (COI), or cytochrome b), wherein the taxonomic information comprises the abundance of each taxon of the taxa in the plurality of dilutions of the sample cultivated in the first cultivation condition; and determining, based on the taxonomic information of the taxa in the plurality of dilutions of the sample cultivated in the first cultivation condition, interactions (e.g., biotic interactions) of the plurality of taxa of microorganisms in the sample in the first cultivation condition. In some embodiments, the method comprises designing a microbial community with the property of interest. The cultivation conditions may be conditions comprising stress conditions as described elsewhere herein.

[0039] In some embodiments, diluting the sample to form plurality of dilutions of the sample comprises: diluting the sample serially to form a plurality of serial dilutions of the sample. Organisms in the plurality of serial dilutions of the sample can be due to dispersal or chance. The plurality of serial dilutions can be different in different implementations. In some embodiments, the plurality of serial dilutions of the sample can comprise, or about, 1:10, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000, 1:10000000, 1:100000000, 1:1000000000, or a number or a range between any two of these values, dilutions of the sample. In some embodiments, the plurality of serial dilutions of the sample can comprise at least, or at most, 1:10, 1 : 100, 1 : 1000, 1:10000, 1:100000, 1:1000000, 1:10000000, 1:100000000, or 1:1000000000 dilutions of the sample. For example, a sample can be diluted 10 times into a 1 : 10 dilution of the sample using, for example, a buffer. The 1:10 dilution of the sample can be diluted 10 times into a 1 : 100 dilution of the sample. The plurality of serial dilutions can comprise the 1:10 dilution of the sample, 1:100 dilution of the sample, and other dilutions of the sample similarly prepared. As another example, a sample can be diluted 10 times into a 1 : 10 dilution of the sample using, for example, a buffer. The sample can be diluted 100 times into a 1 : 100 dilution of the sample. The plurality of serial dilutions can comprise the 1:10 dilution of the sample, 1:100 dilution of the sample, and other dilutions of the sample similarly prepared.

[0040] The plurality of serial dilutions of the sample can comprise dilutions of a number of orders of magnitudes of the sample. In some embodiments, the plurality of serial dilutions of the sample comprises, or about, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, or a number or range between any two of these values, folds dilutions of the sample. In some embodiments, the plurality of serial dilutions of the sample comprises at least, or at most, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8,

4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1,

7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4,

9.5, 9.6, 9.7, 9.8, 9.9, or 10 folds dilutions of the sample.

[0041] In some embodiments, each dilution is cultivated in replicates and tested. In some embodiments, the method is multiplexed. For example, the number of combinations of cultivation conditions, dilutions, and replicates can be, or be about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 100000, 1000000, 10000000, 100000000, 1000000000, or a number or a range between any two of these values. As another example, the number of combinations of cultivation conditions, dilutions, and replicates for each dilution tested can be at least, or at most, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000. For example, if the number of cultivation condition is 2, the number of dilutions is 5 (e.g., 1:10,

1 : 100, 1 : 1000, 1 : 10000, and 1 : 100000), and the number of replicates for each dilution cultivated and tested is 100000, then the number of combinations of cultivation conditions, dilutions, and replicates is 1000000 (2 x 5 x 10000). As another example, if the number of cultivation condition is 10, the number of dilutions is 5 (e.g., 1:10, 1:100, 1:1000, 1:10000, and 1:100000), and the number of replicates for each dilution cultivated and tested is 100000, then the number of combinations of cultivation conditions, dilutions, and replicates is 2500000 (5 x 5 x 10000). [0042] In some embodiments, determining the taxonomic information of the plurality of dilutions of the sample cultivated in the first cultivation condition comprises: determining the taxonomic information of the plurality of dilutions of the sample cultivated in the first cultivation condition using 16S rRNA gene amplicon sequencing. Determining the taxonomic information of the taxa in the plurality of dilutions of the sample cultivated in the first cultivation condition can comprise: determining one or more errors in the taxonomic information of the taxa in the dilutions; and removing at least one of the one or more errors in the taxonomic information of the taxa dilutions. The one or more errors in the taxonomic information of the taxa can be a result of a barcode sequencing error or a contamination of a reagent used in determining the taxonomic information of the taxa in the dilutions.

[0043] To determine the relative abundance of each OTU in microbial compositions, cultivable organism pool can be predicted from MPN estimates. Using the initial estimated abundances, a number of communities (e.g., 10000) can be simulated using a null model of community assembly. The taxonomic information of the communities simulated can be compared with the taxonomic information of the plurality of dilutions of the sample cultivated. The number of communities simulated can be different in different implementations. In some embodiments, the number of communities simulated can be, or about, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 1000000, 10000000, 100000000, 100000000, or a number or a range between any two of these values. In some embodiments, the number of communities simulated can be at least, or at most, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 1000000, 10000000, 100000000, or 100000000. Final relative abundances can be simulated from initial estimated abundances simulated by assuming no net positive or negative interactions, all growth rates are identical, and detection is unbiased. The number of communities simulated can be related to the number of combinations of cultivation conditions, dilutions, and replicates of each dilution cultivated. In some embodiments, the number of communities simulated can be, or about, 0.0000000001,

0.000000001, 0.00000001, 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000, 100000, 1000000, 10000000, 100000000, 1000000000 times, or a number or a range between any two of these values, the number of combinations of cultivation conditions, dilutions, and replicates of each dilution cultivated. In some embodiments, the number of communities simulated can be at least, or at most, 0.0000000001, 0.000000001, 0.00000001, 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000, 100000, 1000000, 10000000, 100000000, or

1000000000 times the number of combinations of cultivation

[0044] The method can include inoculating a sample of microorganisms (e.g., the endophyte and associated microbes) into microwells of one or more microwell plates. The number of microwells per microwell plate can be different in different implementations. In some embodiments, a microwell plate can include, or about, 96, 384, 1536, 2000, 3000, 4000, 5000,

6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, or a number or a range between any two of these values, microwells. In some embodiments, a microwell plate can include at least, or at most, 96, 384, 1536, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,

90000, or 100000 microwells. The method can comprise systematically manipulating bacterial diversity by subsampling a single “regional” species pool at several dilutions in order to create many “local” communities that varied in their membership. [0045] In various aspects, the methods may comprise determining interactions of microbial populations and endophytes. The determining the interactions may comprises determining a pair of taxa or microbes that positively or negatively interact with each other. The pair of taxa or microbes negatively interacts with each other if one taxon of the pair of the taxa inhibits growth or maintenance of the other taxon of the pair of taxa. In some embodiments, determining the interactions of the plurality of taxa of microorganisms comprises: determining, based on a null model of community assembly and the taxonomic information of the taxa in the plurality of dilutions of the sample cultivated in the first cultivation condition, taxa that occur together significantly non-randomly in the plurality of dilutions of the sample cultivated in a first cultivation condition. Determining the taxa that occur together significantly non-randomly in the plurality of samples cultivated in the first cultivation condition can comprise: determining co occurrence probabilities of microbes of a plurality of sample cultivated in a first cultivation condition.

[0046] In some embodiments, the method comprises: determining, based on the interactions of the plurality of microorganisms with the endophyte, one or more microorganisms that contribute to a property of interest, such as a synergistic interaction with the endophyte such to improve on a trait or characteristic of the plant. The trait or characteristic of the plant can comprise performing a specific metabolic function, a molecular of interest, a molecular of interest, a perturbation, or any combination thereof, such as those described elsewhere herein. For example, the trait or characteristic of the plant may be pathogen resistance.

[0047] In some embodiments, the method can be used to determine the specific microbial taxa, within a complex consortium of mixed taxa, that are interacting with each other within an environment of interest. By determining pairs of microbes that positively or negatively interacting within a microbial community in a given cultivation condition, the methods, systems and compositions disclosed herein enable the design and management of microbial compositions that may confer benefits to plants.

[0048] In some embodiments, a microbial community comprising the endophyte is inoculated into a large number of separate enrichment cultures and cultivated under conditions appropriate to detect interactions of interests so that each enrichment culture represents a small fraction of the original community complexity. Then DNA is extracted and sequence or taxonomic information is acquired from each culture. Presence / absence data on each taxon or microbe is used to determine taxa or microbes that occur together in significantly non-random patterns across all enrichment cultures. Compared to a bottom-up, one-by-one comparison of several species of interest, this top-down approach quickly queries potential interactions among assemblages of co-occurring microorganisms.

[0049] In various aspects, nucleic acid sequencing is performed. The sequencing may be performed on any nucleic acid derived from the plants or seedlings. The sequencing may be performed on microbes or endophyte that are associated with the plants or seedlings. The sequencing may comprising obtain taxonomic information, such as an operational taxonomic unit (OTU). Taxonomic information can be determined using sequencing a variety of sequences for example, sequencing of 16S rRNA, 12S rRNA, 18S rRNA, 28S rRNA, 13S rRNA and 23S rRNA, internal transcribed spacer (ITS), ITS1, ITS2, cytochrome oxidase I (COI), cytochrome b, or any combination thereof. The method can comprise counting the reads matched to each organism to determine relative abundances of microorganisms in a given reaction, plant, or seed. [0050] The nucleic acids may be generated from the microbes or endophytes. The microbes and endophytes may be isolated from the plants prior to sequencing. The microbes or endophytes may be lysed. Nucleic acids may be isolated, extracted, purified or otherwise separated from other cellular components of the lysed cells.

[0051] A method may include determining a microbial interaction. Some such methods may include any aspect of the following: diluting a sample comprising a plurality of taxa of microorganisms to form a plurality of dilutions of the sample; cultivating a first subset of the plurality of dilutions of the sample in a first cultivation condition to generate a first plurality of cultivated dilutions, wherein the first subset of the plurality of dilutions comprises a first dilution and a second dilution with an identical inoculum density, wherein the first dilution comprises a first taxon and a second taxon, wherein the second dilution comprises the first taxon and not the second taxon, and wherein an abundance of the first taxon in a first cultivated dilution of the first plurality of cultivated dilutions cultivated from the first dilution is different from an abundance of the first taxon in a second cultivated dilution of the first plurality of cultivated dilutions cultivated from the second dilution; subjecting the first plurality of cultivated dilutions of the sample to sequencing to generate taxonomic information of taxa in the first plurality of cultivated dilutions, wherein the taxonomic information comprises abundances of at least one taxon of the taxa in the first plurality of cultivated dilutions; and processing the taxonomic information of the taxa in the first plurality of cultivated dilutions to identify a non-random occurrence of the first taxon in the presence or absence of the second taxon, thereby determining an interaction within the plurality of taxa of microorganisms in the sample in the first cultivation condition. [0052] Methods of selecting microbial population may be performed using screening based at least of physical traits of plants. Endophytes and associated microbes may be present in the seeds or other parts of plants. Endophytes and associated microbes may provide benefits to seeds or plants such that the plant may comprise particular characteristics. For example, a plant or seed comprising a given endophyte may grow faster compared to a plant or seed without an endophyte or with a different endophyte. As such particular endophytes may be screened or selected based on the plant phenotype.

[0053] The endophyte may be formulated as a endophyte composition for applying to plants. The endophyte composition may comprise additional microbes, such as those that are synergistic with the endophyte . endophyte composition can be formulated as a liquid formulation or a dry formulation. The liquid formulation can be a flowable or aqueous suspension. The liquid formulation can comprise the endophyte or a secondary metabolite thereof suspended in water, oil, or a combination thereof (an emulsion). A dry formulation can be a wettable powder, a dry flake, a dust, or a granule. A wettable powder can be applied to the plant, the seed, the flower, or the produce thereof as a suspension. A dust can be applied to the plant, the seed, or the produce thereof dry, such as to seeds or foliage. A granule can be applied dry or can be mixed with water to create a suspension. The endophyte or a secondary metabolite thereof can be formulated as a microencapsulation, wherein the endophyte or a secondary metabolite thereof has a protective inert layer. The protective inert layer can comprise any suitable polymer.

[0054] The endophyte composition can further comprise an additional compound. The additional compound can be a carrier, a surfactant, a wetting agent, a penetrant, an emulsifier, a spreader, a sticker, a stabilizer, a nutrient, a binder, a desiccant, a thickener, a dispersant, a UV protectant, or a combination thereof. The carrier can be a liquid carrier, a mineral carrier, or an organic carrier. Examples of a liquid carrier include, but are not limited to, vegetable oil or water. Examples of a mineral carrier include, but are not limited to, kaolinite clay or diatomaceous earth. Examples of an organic carrier include, but are not limited to, grain flour. The surfactant can be an anionic surfactant, a cationic surfactant, an amphoteric surfactant, or a nonionic surfactant. The surfactant can be Tween 20 or Tween 80 The wetting agent can comprise a polyoxyethylene ester, an ethoxy sulfate, or a derivative thereof. In some cases a wetting agent is mixed with a nonionic surfactant. A penetrant can comprise a hydrocarbon. A spreader can comprise a fatty acid, a latex, an aliphatic alcohol, a crop oil (e.g. cottonseed), or an inorganic oil. A sticker can comprise emulsified polyethylene, a polymerized resin, a fatty acid, a petroleum distillate, or pregelantinized com flour. The oil can be coconut oil, palm oil, castor oil, or lanolin. The stabilizer can be lactose or sodium benzoate. The nutrient can be molasses or peptone. The binder can be gum arabic or carboxymethylcellulose. The desiccant can be silica gel or an anhydrous salt. A thickener can comprise a polyacrylamide, a polyethylene polymer, a polysaccharide, xanthan gum, or a vegetable oil. The dispersant can be microcrystalline cellulose. The UV protectant can be oxybenzone, blankophor BBH, or lignin. The endophyte composition may comprise dipicolinic acid.

[0055] The endophyte can comprise an effective amount of isolated and purified microbes isolated and purified from a liquid culture. The endophyte from the liquid culture can be air- dried, freeze-dried, spray-dried, or fluidized bed-dried to produce a dry formulation. The dry formulation can be reconstituted in a liquid to produce a liquid formulation.

[0056] The endophyte composition can be formulated such that the endophyte can replicate once they are applied/or delivered to the target habitat (e.g. the soil, the plant, the seed, and/or the produce).

[0057] The endophyte composition can have a shelf life of at least one week, one month, six months, at least one year, at least two years, at least three years, at least four years, or at least five years. The shelf life can indicate the length of time the endophyte composition maintains at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of its anti -fungal properties. The endophyte composition can be stored at room temperate, at or below 4°C, at or below 0°C, or at or below -20°C.

[0058] The endophyte composition can comprise spores. Spore-containing compositions can be applied by methods described herein. Spore-containing compositions can extend the shelf life of the endophyte composition. Spore-containing compositions can survive low pH or low temperatures of a target habitat. For example, spore-containing compositions may be applied to the soil at a colder temperature (for example, below 10 °C) and can have anti -fungal properties for a seed planted at a higher temperature (for example, 20 °C). The spores may become vegetative cells, allowing them any advantages of vegetative cells.

[0059] The endophyte composition can comprise vegetative cells. Vegetative cell- containing compositions can be applied by methods described herein. Vegetative cells may proliferate and increase efficacy of the composition. For example, vegetative cells in the endophyte composition may proliferate after application increasing the surface area the plant that is exposed to the endophyte composition. In another example, vegetative cells in the endophyte composition may proliferate after application increasing the amount of the time the endophyte composition survives and thus extending the time the endophyte composition has efficacy. The vegetative cells may proliferate and compete for nutrients with a fungal pathogen. The vegetative cells may actively produce one or more secondary metabolites with anti-fungal properties. The vegetative cells may become spores, allowing them any advantages of spores. [0060] The endophyte composition can have anti-fungal activity, such as prevention of growth of a fungal pathogen or reduction of growth of a fungal pathogen on a plant, a seed, or a produce thereof. The endophyte composition can prevent growth of a fungal pathogen on the plant, seed, or produce thereof for at least 1, at least 2, at least 3, at least 4, or at least 5 days. The endophyte composition can prevent growth of a fungal pathogen on the plant, seed, or produce thereof for at least 1, at least 2, at least 3, at least 4, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days. The endophyte composition can prevent growth of a fungal pathogen on the plant, seed, or produce thereof for over 10 days.

[0061] The endophyte composition can reduce growth of the fungal pathogen on the plant, seed, or produce thereof relative to growth of the fungal pathogen on a control that is a plant, a seed, flower, or a produce thereof not exposed to the endophyte composition. The control can be a plant, a seed, or a produce thereof to which no anti-fungal agent has been applied or can be a plant, a seed, flower, or produce thereof to which a commercially available anti-fungal agent has been applied. Examples of commercially available anti-fungal agents include, but are not limited to, Bacillus subtilis strain QST713 (Serenade®), Bacillus subtilis strain GB02 (Kodiak®), Bacillus subtilis strain MBI 600 (Subtilex®), Bacillus pumilus strain GB34 (YieldShield), Bacillus licheniformis strain SB3086 (EcoGuard®). The endophyte composition can reduce growth of a fungal pathogen on the plant, seed, or produce thereof for at least 1, at least 2, at least 3, at least 4, or at least 5 days. The endophyte composition can reduce growth of a fungal pathogen on the plant, seed, or produce thereof for at least 1, at least 2, at least 3, at least 4, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.

The endophyte composition can reduce growth of a fungal pathogen on the plant, seed, or produce thereof for over 10 days. The endophyte composition can reduce growth of the fungal pathogen of at least 25% relative to growth of the fungal pathogen on the control. The endophyte composition can reduce growth of the fungal pathogen of at least 60% relative to growth of the fungal pathogen on the control. The endophyte composition can reduce growth of the fungal pathogen of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60 % 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more relative to growth of the fungal pathogen on the control.

[0062] Methods of conferring a phenotype, characteristic, or trait to a plant, a seed, or a produce thereof can comprise applying to the plant, the seed, flower, or the produce, before it has been harvested, an endophyte composition comprising at least one microbe described herein or one or more secondary metabolites thereof and a carrier. Harvesting the produce can refer to the removal of the edible portion of the plant from the remainder of the plant, or can refer to removal of the entire plant with subsequent removal of the edible portion later.

[0063] Applying the endophyte composition prior to harvest can comprise dusting, injecting, spraying, or brushing the plant, the seed, or the produce thereof with the endophyte composition. Applying the endophyte composition can comprise adding the endophyte composition to a drip line, an irrigation system, a chemigation system, a spray, or a dip. In some cases, the endophyte composition is applied to the root of the plant, the seed of the plant, the foliage of the plant, the soil surrounding the plant or the edible portion of the plant which is also referred to herein as the produce of the plant

[0064] The method can further comprise applying to the plant a fertilizer, an herbicide, a pesticide, other biocontrols, or a combination thereof. In some instances, the fertilizer, herbicide, pesticide, other biocontrols or combination thereof is applied before, after, or simultaneous with the endophyte composition.

[0065] Method of conferring a phenotype, characteristic, or trait to a plant, a seed, or a produce thereof, can comprise applying to the seed a endophyte composition comprising at least one microbe described herein or a secondary metabolite thereof and a carrier. Applying the endophyte composition to the seed of the plant can occur before planting, during planting, or after planting prior to germination. For example, the endophyte composition can be applied to the surface of the seed prior to planting. In some cases, a seed treatment occurring before planting can comprise addition of a colorant or dye, a carrier, a binder, a sticker, an anti-foam agent, a lubricant, a nutrient, or a combination thereof to the endophyte composition.

[0066] Methods of conferring a phenotype, characteristic, or trait to a plant, a seed, or a produce thereof, can comprise applying to the soil an endophyte composition comprising at least one microbe described herein or a secondary metabolite thereof and a carrier. The endophyte composition can be applied to the soil before, after, or during planting the soil with a seed, or before transfer of the plant to a new site. In one example, a soil amendment is added to the soil prior to planting, wherein the soil amendment results in improved growth of a plant, and wherein the soil amendment comprises the endophyte composition. In some cases, the soil amendment further comprises a fertilizer.

[0067] Method of conferring a phenotype, characteristic, or trait to a plant, a seed, or a produce thereof, can comprise applying to the root a endophyte composition comprising the endophyte described herein or a secondary metabolite thereof and a carrier. The endophyte composition can be directly applied to the root. One example of a direct application to the root of the plant can comprise dipping the root in a solution that includes the endophyte composition. The endophyte composition can be applied to the root indirectly. One example of an indirect application to the root of the plant can comprise spraying the endophyte composition near the base of the plant, wherein the endophyte composition permeates the soil to reach the roots.

EXAMPLES

Example 1: Enrichment for endophytes

[0068] Microbiomes may be enriched in plants. For example, endophytes that promote early seedling vigor may be enriched in leafy green plants such as lettuce. Any of the following steps may be performed in a process of enrichment: Trays of a plants (e.g. lettuce) are planted using indoor seeds and replicate indoor conditions. Measurements of speed of germination such as vigor index or co-efficient of germination are obtained. For example these measures may be obtained for of the population as a whole, or for the top ten seedlings, for each generation. The measurements may be used to monitor any improvements in germination speed or vigor over generations. Some examples of measures of speed of germination are included in Mia et al, “Effects of rhizobia and plant growth promoting bacteria inoculation on germination and seedling vigor of lowland rice,” African Journal of Biotechnology Vol. 11 (16), pp. 3758-3765, 23 February, 2012. The earliest emergers (e.g. top 10 earliest emergers) based on the one or more measurements of speed of germination (e.g. <14 days) are identified and harvested. The earliest emergers are allowed to generate seed and the seeds are harvested or otherwise obtained. The seeds of the earliest emergers are planted to obtain a second generation. The previous steps are repeated until a desired increase in emergence speed is achieved. For example, steps 1-4 or 1- 5 may be repeated 1, 2, 3, 4, or 5 more times. A desired increase in emergence speed may be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater increase in the one or more measurements of speed of germination. Endophytes are isolated from seeds of earliest emergers and endophytes can be extracted from earliest emergers from any or all generations. The isolated endophytes are then inoculated onto sterile seeds. The inoculated seeds may be compared with uninoculated control seeds to test for an improvement in germination and vigor by, for example, obtaining one or more measurements of speed of germination in seedlings of the inoculated seeds compared to the control seeds.

[0069] Optionally, the endophytes may be screened for synergists. The endophyte may be added into a microbial population and monitored such to identify synergistic microbes. An example of a synergist is a microbe that enhances growth of an endophyte. For example, the endophyte may be added to microbial population and the be allowed to propagate. The relative abundance of microbes in the population may allow for the detection of microbial interactions and potential synergists. In cases where synergists are found, at least some seeds inoculated with endophytes may also be inoculated with the synergists and the plants may be monitored [0070] Optionally, isolated endophytes may be screened to obtain information on a mode of action by which they enhance speed of germination. Some examples of modes of action may include production of: metabolites, signaling molecules, hormones, polypeptides or other molecules such as ethylene, amylase, protease, lipase, IAA, giberellic Acid ACC deaminase. Additionally, modes of action may include analysis of phenotypic or physical characteristics such as increased root surface area, improved water retention or uptake.

Example 2: Pathogen resistance

[0071] A plurality of lettuce trays are planted in soil infested with Fusarium oxysporum. The plants are monitor during growth for any Fusarium Wilt symptoms. The top ten plants that show the fewest symptoms of Fusarium Wilt are allowed to generate seeds. The seeds from the top ten plants showing the fewest symptom of fusarium wilt and planted to generate a second generation. The steps are repeated to generate new offspring from the plants that show the all offspring are disease resistant. Endophytes are isolated from the seeds by surface sterilizing seed and using standard microbiology methods. Microbes are screened for the ability to inhibit the growth of Fusarium oxysporum using a standard in vitro inhibition assay measuring zone of clearing on agar plates. The microbe(s) responsible for disease suppression are then identified. Lettuce seed treated with the microbe(s) identified are then plant in soil infested with Fusarium oxysporum. The plants are observed to determine if the plant are asymptomatic and allowed to produce seeds. Endophytes are isolated from seed as previously done and the presence of microbe previously identified is observed. The example show that the microbe may be vertically transmitted to their seed and offspring and allow for the breeding of plants with beneficial qualities that have been conferred by the endophytes.

[0072] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed is:

1. A method, comprising:

(a) inoculating a microbial population with an endophyte obtained by:

(i) screening a plant comprising the endophyte for speed of growth or germination,

(ii) obtaining a seed from the plant comprising the endophyte, and

(iii) obtaining the endophyte from the seed;

(b) incubating the microbial population with the endophyte to produce a second microbial population comprising the endophyte; and

(c) sequencing nucleic acids of the second microbial population, thereby obtaining nucleotide sequence information of a microbe that interacts with the endophyte to enhance growth of the endophyte.

2. The method of claim 1, wherein sequencing the nucleic acids of the second microbial population comprises obtaining operational taxonomic unit (OTU) abundances of the microbes of the second microbial population.

3. The method of claim 1 or 2, further comprising identifying the microbe as enhancing the growth of the endophyte.

4. The method of any one of claims 1-3, further comprising identifying an increase in the growth of the endophyte in the presence of the microbe, relative to growth of the endophyte in the absence of the microbe or in the presence of another microbe.

5. The method of any one of claims 1-4, further comprising isolating the endophyte.

6. The method of any one of claims 1-5, further comprising isolating the microbe.

7. The method of any one of claims 1-6, further comprising inoculating a seed or crop with the microbe and the endophyte.

8. A method, comprising:

(a) growing a plurality of plant seedlings, and identifying one or more early emergers of the plurality of seedlings, the one or more early emergers comprising a seedling that (i) germinates earlier than an average germination time of the plurality of seedlings or (ii) grows faster than an average rate of growth of the plurality of seedlings;

(b) growing a plurality of second generation seedlings from seeds of the one or more early emergers, and identifying a second generation early emerger of the second generation seedlings, the second generation early emerger comprising a seedling that (i) germinates earlier than an average germination time of the plurality of second generation seedlings or (ii) grows faster than an average rate of growth of the plurality of second generation seedlings,

(c) obtaining or isolating an endophyte from the second generation early emerger; and

(d) identifying a property of the endophyte that enhances plant germination or growth.

9. The method of claim 8, wherein the property comprises plant hormone production by the endophyte, or enhancement of plant hormone production by the plant.

10. The method of claim 9, wherein the plant hormone comprises ethylene, indole-3- acetic acid (IAA), or giberellic acid.

11. The method of claim 8, wherein the property comprises amylase, protease, or lipase production by the endophyte, or enhancement of amylase, protease, or lipase production by the plant.

12. The method of claim 8, wherein the property comprises 1-aminocyclopropane-l- carboxylic acid (ACC) deaminase production.

13. The method of claim 8, wherein the property comprises increasing plant root surface area.

14. The method of claim 8, wherein the endophyte is vertically transmitted from the one or more early emergers of the plurality of plant seedlings.

15. A method, compri sing :

(a) growing plant seedlings under a stress condition, and identifying first early emergers of the seedlings, the first early emergers comprising seedlings that germinate or grow faster than other seedlings of the seedlings;

(b) growing second generation seedlings from seeds of the early emergers under the stress condition, and identifying a second generation early emerger of the second generation seedlings, the second generation early emerger comprising a seedling that germinates or grows faster than other seedlings of the second generation seedlings, and comprising an endophyte vertically transmitted from one of the early emergers; and

(c) obtaining or isolating the endophyte from the second generation early emerger.

16. The method of claim 15, wherein the stress condition comprises an environmental stress condition.

17. The method of claim 16, wherein the environmental stress condition comprises low temperature, low pH, high pH, or high salt.

18. The method of claim 15, wherein the stress condition comprises scarcity of a nutrient.

19. The method of claim 15, wherein the stress condition comprises a presence of a pathogen.

20. The method of claim 19, wherein the pathogen comprises a soil or foliar pathogen.

21. The method of claim 19 or 20, wherein the pathogen comprises a fungus.

22. The method of any one of claims 8-21, wherein (a) comprises growing plant seedlings from seeds of plants of 1, 2, 3, 4 or more earlier generations of early emergers.

23. The method of any one of claims 8-25, wherein growing comprises growing indoors.

24. The method of any one of claims 8-23, further comprising identifying a microbe that enhances growth of the endophyte.

25. The method of claim 24, further comprising isolating the microbe.

26. The method of claim 24 or 25, further comprising inoculating a seed or crop with the microbe.

27. The method of any one of claims 8-26, further comprising inoculating a seed or crop with the endophyte.

28. The method of claim 27, wherein inoculating a seed or crop with the endophyte results in the seed or crop growing faster than without the inoculation.

29. The method of any one of claims 1-28, wherein the plant comprises a leafy green vegetable.

30. The method of claim 29, wherein the leafy green vegetable comprises lettuce.

31. The method of any one of claims 1-30, wherein the plant comprises a legume.

32. The method of any one of claims 1-31, wherein the endophyte comprises a microbial endophyte.

33. The method of claim 32, wherein the microbial endophyte comprises a bacterium.

34. The method of any one of claims 1-33, wherein the endophyte comprises a population of endophytes.