# Patent

## Title
Fungal endophytes for improved crop yields and protection from pests

## Abstract

The invention provides a synthetic combination of a crop and at least one fungal endophyte, wherein the crop is a host plant of the endophyte. Provided are also methods and compositions for producing such synthetic combinations. The endophyte reproduces and enhances the agronomic characteristics of the crop. Methods for inoculating the host plant with the endophyte, for propagating the host-endophyte combination, and for detecting the presence of the endophyte and of its metabolites within a host plant are also described.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application Nos. 61/900,929 and 61/900,935, both filed Nov. 6, 2013, which are herein incorporated by reference in their entirety.


INCORPORATION OF SEQUENCE LISTING
The sequence listing that is contained in the file named TAMC027US_ST25.txt, which is 33 kilobytes as measured in Microsoft Windows operating system and was created on Nov. 6, 2014, is filed electronically herewith and incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to fungal endophytes of agricultural crops for improving yield and/or for protection from pests.
DESCRIPTION OF RELATED ART
Fungal endophytes are fungi that internally colonize plant tissues without causing evident damage or disease. Particular fungal endophytes, such as mycorrhiza, survive within various host plant tissues, often colonizing the intercellular spaces of host leaves, stems, flowers or roots. The symbiotic endophyte-host relationships can provide several fitness benefits to the host plant, such as enhancement of nutrition, and/or increased drought tolerance. Root-colonizing mycorrhizae survive on photosynthetic carbohydrates from the plant, and in return, aid in the solubilization and uptake of water and minerals to the host, which can lead to the promotion of seed germination and plant growth. Additionally, the association of a fungal endophyte with a host plant can provide tolerance to a variety of biotic and abiotic stresses. Host growth, fitness promotion and protection are thought to be achieved through multiple beneficial properties of the endophyte-host association. For instance, the endophytic organisms may produce growth-regulating substances to induce biomass production and alkaloids or other metabolites. Additionally, fungal endophytes may directly suppress or compete with disease-causing microbes, protecting the plant from potential pathogens.
SUMMARY OF THE INVENTION
In one aspect, the invention provides methods for improving a trait in an agricultural plant comprising contacting an agricultural seed of said plant with a formulation comprising a purified facultative fungal endophytes of at least one species, wherein the endophytes are capable of producing substances that are beneficial to plants or detrimental to pests or both, and wherein the endophytes are present in the formulation in an amount effective to modulate the colonization frequencies of the endophytes that are native to the agricultural plant grown from the seed compared to a reference seed that is planted in an agricultural environment, and to provide a benefit to the seeds or the agricultural plants grown from the seeds.
In another aspect, the invention provides methods for providing a benefit to an agricultural plant comprising treating said plant, the seed of said plant, or the rhizosphere of said plant or seed with a composition comprising purified facultative fungal endophytes and an agriculturally-acceptable carrier, wherein the endophyte is capable of at least one of: reducing pest reproduction, killing pests, and deterring pests, and wherein the endophyte is present in the composition in an amount effective to provide a benefit to the seeds or the agricultural plants derived from the seeds.
In yet another aspect, the invention provides methods for providing a benefit to an agricultural plant, comprising obtaining a synthetic combination of an agricultural plant seed and a purified facultative fungal endophyte, wherein the endophyte is capable of at least one of: reducing pest reproduction, killing pests, and deterring pests, and wherein the endophyte is present in the synthetic combination in an amount effective to provide a benefit to the seeds or the agricultural plants derived from the seeds.
In another embodiments, methods of producing a plant with a non-naturally occurring ratio of endophytes is provided, where the methods comprise contacting an agricultural seed of the plant with a formulation comprising facultative fungal endophytes of at least one species, wherein endophytes are present in the formulation in an amount effective to modulate the colonization frequencies of the endophytes that are native to the agricultural plant grown from the seed compared to a reference seed that is planted in an agricultural environment, wherein the plant with the non-naturally occurring ratio of endophytes has an improved trait as compared to a plant with a naturally-occurring ratio. In a further aspect, the facultative fungal endophytes are capable of producing substances that are beneficial to plants or detrimental to pests or both.
In another aspect, the invention provides methods for altering the systemic defensive pathway in a plant comprising contacting an agricultural seed of said plant with a formulation comprising a purified facultative fungal endophytes of at least one species, wherein the endophytes are capable of producing substances that are beneficial to plants or detrimental to pests or both, and wherein the endophyte is present in the synthetic combination in an amount effective to modulate the level of at least one phytohormone within an agricultural plant grown from the plant seed, and to provide a benefit to the seeds or the agricultural plants grown from the seeds. In a further aspect, the facultative fungal endophytes are capable of producing substances that are beneficial to plants or detrimental to pests or both.
In other embodiments, the invention provides methods of modulating the colonization frequencies of endophytes that are native to the agricultural plant grown from the seed compared to a reference seed that is planted in an agricultural environment, comprising contacting the seed of the agricultural plant with a formulation comprising facultative fungal endophytes of at least one species, and wherein endophytes are present in the formulation in an amount effective to modulate the colonization frequencies of native endophytes and to provide a benefit to the seeds or the agricultural plants grown from the seeds. In certain aspects, the native endophytes are of genus Alternaria. In a further aspect, the facultative fungal endophytes are capable of producing substances that are beneficial to plants or detrimental to pests or both.
In another aspect, the invention provides methods for altering the systemic defensive pathway in a plant comprising contacting an agricultural seed of said plant with a formulation comprising a purified facultative fungal endophytes of at least one species, and wherein the endophyte is present in the synthetic combination in an amount effective to modulate the level of at least one phytohormone within an agricultural plant grown from the plant seed, and to provide a benefit to the seeds or the agricultural plants grown from the seeds. In a further aspect, the facultative fungal endophytes are capable of producing substances that are beneficial to plants or detrimental to pests or both.
In yet another aspect, the invention provides methods of producing a plant with a network of fungal endophytes that comprises endophytes of the genus Alternaria, comprising (a) contacting the seed of an agricultural plant with a formulation comprising facultative fungal endophytes of at least one non-Alternaria species, wherein endophytes are present in the formulation in an amount effective to provide a benefit to the seeds or the agricultural plants grown from the seeds, and wherein the plant grown from the seed comprises endophytes of the genus Alternaria. In a further aspect, the facultative fungal endophytes are capable of producing substances that are beneficial to plants or detrimental to pests or both.
Also provided herein are synthetic combinations of an agricultural plant seed and a composition comprising purified entomopathogenic fungal endophytes of at least one species, wherein the endophytes are capable of (1) colonizing the agricultural plant grown from the plant seed (2) and at least one of: reducing pest reproduction, killing pests, and deterring pests, from within the agricultural plant; wherein the endophytes are not of species Beauveria bassiana, and wherein the endophyte is present in the synthetic combination in an amount effective to provide a benefit other than enhanced resistance to biotic stress to the seeds or the agricultural plants derived from the seeds when the seeds or plants are grown in an agricultural setting.
In yet another aspect, the invention provides synthetic combinations of an agricultural plant seed and a composition comprising purified facultative fungal endophytes of at least one species, wherein the endophyte is present in the synthetic combination in an amount effective to modulate the level of at least one phytohormone within an agricultural plant grown from the plant seed, and to provide a benefit to the seeds or the agricultural plants grown from the seeds. In a further aspect, the facultative fungal endophytes are capable of producing substances that are beneficial to plants or detrimental to pests or both.
In another embodiment, the invention provides synthetic combinations of an agricultural plant seed and a composition comprising purified facultative fungal endophytes of at least one species, wherein the facultative fungal endophytes are present in the synthetic combination in an amount effective to modulate the colonization frequencies of endophytes that are native to the agricultural plant grown from the seed compared to a reference seed that is planted in an agricultural environment, and to provide a benefit to the seeds or the agricultural plants grown from the seeds. In a further aspect, the facultative fungal endophytes are capable of producing substances that are beneficial to plants or detrimental to pests or both. In certain aspects, the facultative fungal endophytes are present in the synthetic combination in an amount effective to modulate the colonization frequencies of endophytes of genus Alternaria that are native to the agricultural plant grown from the seed compared to a reference seed that is planted in an agricultural environment.
In a further aspect for certain of these methods and synthetic combinations, the composition comprising purified facultative fungal endophytes also comprises an agriculturally acceptable carrier.
In a further aspect for certain of these methods and synthetic combinations, the facultative fungal endophyte may be a filamentous fungal endophyte. In other embodiments, the facultative endophyte may be spore-forming. In yet other embodiments, the facultative fungal endophyte may be a septate fungal endophyte. In yet other embodiments, the facultative fungal endophyte may be a dark septate fungal endophyte. In some embodiments, the facultative endophyte may be an entomopathogen. In some embodiments, the facultative fungal endophyte may belong to the phylum Ascomycota or Basidiomycota. In a further aspect, the facultative fungal endophyte may belong to subphylum Pezizomycotina, Agaricomycotina, or Ustilaginomycotina. In yet another aspect, facultative fungal endophyte may belong to class Sordariomycetes, Dothideomycetes, Agaricomycetes, Ustilaginomycetes, Orbiliomycetes, or Eurotiomycetes. In yet another aspect, the facultative fungal endophyte may belong to order Hypocreales, Pleosporales, Capnodiales, Sordariales, Polyporales, Diaporthales, Ustilaginales, Xylariales, Orbiliales, Trichosphaeriales, or Eurotiales.
In a further aspect, the facultative fungal endophyte may be a species from Table 1, namely Acremonium altematum, Alternaria altemata, Alternaria brassicae, Alternaria compacta, Alternaria dianthi, Alternaria longipes, Alternaria mali, Alternaria sesami, Alternaria solani, Alternaria sp., Alternaria tenuissima, Ascomycota sp., Bipolaris spicifera, Cercospora canescens, Cercospora capsici, Cercospora kikuchii, Cercospora zinnia, Chaetomium globosum, Chaetomium piluliferum, Chaetomium sp., Cladosporium cladosporioides, Cladosporium sp., Cladosporium uredinicola, Cochliobolus sp, Phanerochaete crassa, Phoma americana, Phoma subherbarum, Phomopsis liquidambari, Phomopsis sp., Pleospora sp., Pleosporaceae sp., Polyporales sp., Preussia africana, Preussia sp., Pseudozyma sp., Pyrenophora teres, Colletotrichumcapsici, Coniolariella gamsii, Coniothyrium aleuritis, Coniothyrium sp., Corynespora cassiicola, Diaporthe sp., Diatrype sp., Drechslerella dactyloides, Embellisia indefessa, Epicoccum nigrum, Epicoccum sp., Exserohilum rostratum, Fusarium chlamydosporum, Fusarium sp., Gibellulopsis nigrescens, Gnomoniopsis sp., Lewia infectoria, Mycosphaerella coffeicola, Mycosphaerellaceae sp., Nigrospora oryzae, Nigrospora sp., Nigrospora sphaerica, Paecilomyces sp., Penicillium citrinum, Retroconis sp., Rhizopycnis sp., Schizothecium inaequale, Stagonospora sp., Stemphylium lancipes, Thielavia hyrcaniae, Thielavia sp., Ulocladium chartarum, Verticillium sp., Beauveria bassiana, Aspergillus parasiticus, Lecanicillium lecanii, and Paecilomyces lilacinus. 
In a further aspect, the facultative fungal endophyte comprises a nucleic acid that is at least 97% identical, for example, at least 98% identical, at least 99% identical, at least 99.5% identical, or 100% identical to the nucleic acids provided in any of SEQ ID NO:7 through SEQ ID NO:77, for example those listed in Example 16.
In another aspect for certain of these methods is an additional step of packaging the contacted seeds in a container may be included. In certain aspects, the packaging material may be selected from a bag, box, bin, envelope, carton, or container, and may comprise a dessicant.
In a further aspect for certain of these methods and synthetic combinations, the benefit to the treated seed or plant grown from the treated seed is measured at the level of the population, as compared to a reference population of plants. In certain aspects, the facultative fungal endophyte may be providing a benefit to a crop comprising a plurality of agricultural plants produced from the seeds treated with the endophyte. In certain aspects, the present invention discloses a substantially uniform population of plants produced by growing the population of seeds described above. In one embodiment, at least 75%, at least 80%, at least 90%, at least 95% or more of the plants comprise in one or more tissues an effective amount of the endophyte or endophytes. In another embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, at least 80%, at least 90%, at least 95% or more of the plants comprise a microbe population that is substantially similar.
In a further aspect for certain of these methods and synthetic combinations, the plant is grown in an agricultural setting or environment, including a greenhouse. In one embodiment, the agricultural setting or environment comprises at least 100 plants. In another embodiment, the population occupies at least about 100 square feet of space, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% of the population comprises an effective amount of the microbe. In another embodiment, the population occupies at least about 100 square feet of space, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% of the population comprises the microbe in reproductive tissue. In still another embodiment, the population occupies at least about 100 square feet of space, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% of the population comprises at least 10 CFUs, 100 CFUs, 1,000 CFUs, 10,000 CFUs or more of the facultative fungal endophyte of the invention. In yet another embodiment, the population occupies at least about 100 square feet of space, wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% of the population comprises the facultative fungal endophyte of the invention.
In one embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or more of the seeds in the population, contains a viable endophyte or endophytes disposed on the surface of the seeds. In a particular embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or more of the seeds in the population contains at least 10 CFU, for example, at least 30 CFU, at least 100 CFU, at least 300 CFU, at least 1,000 CFU, at least 3,000 CFU, at least 10,000 CFU or more, of the endophyte or endophytes coated onto the surface of the seed.
In a further aspect for certain of these methods and synthetic combinations, the endophytes that are native to the agricultural plant and whose colonization frequencies or ratios are altered may belong to phylum Ascomycota or Basidiomycota. In yet another aspect, the endophytes that are native to the agricultural plant may be of class Leotiomycetes, Dothideomycetes, Eurotiomycetes, Saccharomycetes, Sordariomycetes, Agaricomycetes, Microbotryomycetes, Tremellomycetes. In yet another aspect, the native endophytes may belong to order Capnodiales, Pleosporales, Chaetothyriales, Eurotiales, Saccharomycetales, Diaporthales, Hypocreales, Ophiostomatales, Sordariales, Trichosphaeriales, Xylariales, Cantharellales, Corticiales, Polyporales, Russulates, Sporidiobolales, or Tremellales. In a further aspect, the native endophytes may belong to genus Davidiellaceae, Mycosphaerellaceae, Pleosporaceae, Didymellaceae, Sporormiaceae, Chaetothyriaceae, Trichocomaceae, Saccharomycetaceae, Gnomoniaceae, Cordycipitaceae, Nectriaceae, Hypocreaceae, Plectosphaerellaceae, Ophiostomataceae, Chaetomiaceae, Lasiosphaeriaceae, Trichosphaeriaceae, Ceratobasidiaceae, Corticiaceae, Coriolaceae, Peniophoraceae, Sporidiobolaceae, or Tremellaceae. In a further aspect, the endophytes that are native to the agricultural plant may be a species from Table 2, namely Cladosporium sp., Cladosporium cladosporioides, Davidiella sp., Cercospora sp., Cercospora beticola, Alternaria sp., Alternaria alternata, Alternaria citri, Alternaria tenuissima, Cochliobolus sp., Curvularia sp., Exserohilum sp., Lewia sp., Lewia infectoria, Pyrenophora sp., Pyrenophora tritici-repentis, Pleospora sp., Phoma americana, Preussia africana, Penicillium sp., Thermomyces sp., Thermomyces lanuginosus, Candida sp., Candida quercitrusa, Candida tropicalis, Cyberlindnera sp., Cyberlindnera jadinii, Kluyvemmyces sp., Kluyveromyces marxianus, Gnomoniopsis sp., Beauveria bassiana, Cordyceps sp., Cordyceps bassiana, Fusarium sp., Gibellulopsis nigrescens, Hypocrea sp., Hypocrea lixii, Hypocrea virens, Trichoderma sp., Trichoderma tomentosum, Verticillium sp., Ophiostoma sp., Ophiostoma dendifundum, Chaetomium sp., Chaetomium globosum, Thielavia hyrcaniae, Taifanglania sp., Taifanglania inflata, Schizothecium inaequale, Nigrospora sp., Rhizoctonia sp., Phanerochaete sp., Trametes sp., Trametes hirsuta, Trametes viliosa, Rhodotorula sp., Rhodotorula mucilaginosa, Cryptococcus sp, Cryptococcus skinneri, or Tremella sp.
In a further aspect for certain of these methods and synthetic combinations, the benefit provided by the facultative fungal endophyte to the agricultural plant is an improved agronomic property selected from the group consisting of increased biomass, increased tillering, increased root mass, increased flowering, increased yield, increased water use efficiency, reduction of yield loss, altered plant height, decreased time to emergence, increased seedling height, increased root length, increased chlorophyll levels, retention of developing flowers, retention of developing fruits, altered phytohormone levels, and enhanced resistance to environmental stress relative to a reference plant. In some aspects, the benefit provided is the alteration of levels of at least two phytohormones. In some aspects, the environmental stress is selected from the group consisting of drought stress, cold stress, heat stress, nutrient deficiency, salt toxicity, aluminum toxicity, grazing by herbivores, insect infestation, nematode infection, and fungal infection, bacterial infection and viral infection. In some aspects, the benefit to agricultural plants derived from the seed is increased yield in a population of said plants by about 5%, 10%, 15%, 20%, 30%, 40%, or 45% relative to a reference population of plants. In other aspects, the benefit to agricultural plants derived from the seed is a reduction of yield loss in a population of said plants by more than 40%, 30%, 20%, 10%, 5%, or 1% relative to a reference population of plants. In some aspects, treatment of seeds with facultative fungal endophytes may decrease thrip damage, decrease fleahopper damage, increase canopy temperature, increase drought tolerance, increase above ground biomass, and increase below ground biomass in the plants grown from the treated seeds.
In a further aspect for certain of these methods and synthetic combinations, the facultative fungal endophyte is present in the synthetic combination in an amount effective to obtain at least 50% colonization of the leaves, stems or roots of an agricultural plant grown from the seed.
In a further aspect for certain of these methods and synthetic combinations, the facultative fungal endophytes are capable of producing substances that are detrimental to pests. In certain aspects, the pest may be a nematode and/or an insect, for example, a root knot nematode, a aphid, a lygus bug, a stink bug, or combinations thereof.
In a further aspect for certain of these methods and synthetic combinations, the synthetic combination may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 facultative fungal endophytes. In one aspect, the invention provides a synthetic combination of a cotton plant or seed and a fungal endophyte comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 endophytes selected from those in Table 1, wherein the cotton or seed is a host of the endophyte.
In another aspect, a seed coating is provided comprising a fungal endophyte comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 endophytes from Table 1; and at least one sticker, wherein the fungal endophyte is in contact with the sticker. In certain aspects, the sticker may comprise, for example, alginic acid, carrageenan, dextrin, dextran, pelgel, polyethelene glycol, polyvinyl pyrrolidone, methyl cellulose, polyvinyl alcohol, gelatin, or combinations thereof. In certain aspects, the sticker may have a weight ratio between fungal endophyte and sticker of 1:1-10, 1:10-50, 1:50-100, 1:100-500, 1:500-1000, or 1:1000-5000. The seed coating may be a solid or fluid. In certain aspects, the seed coating is a powder. In certain aspects, the fungal endophyte may comprise fungal spores. In various aspects, the seed coating may comprise about 1, 2, 5, 10, 50, 102, 103, 104, 105, 106, 107, 108, or 109 or more colony forming units per gram or spores per gram.
In certain embodiments, compositions for foliar or soil application may comprise a fungal endophyte comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 endophytes from Table 1, and at least one carrier, surfactant or diluent. In certain aspects, the compositions may comprise may comprise about 1, 2, 5, 10, 50, 102, 103, 104, 105, 106, 107, 108, or 109 or more colony forming units per gram or spores per gram. In various aspects, the composition may comprise water, a detergent, Triton X, insecticides, fungicides, or combinations thereof, for example. In further embodiments, seed compositions comprise a plant seed and the above-described seed coating. In certain aspects, the plant seed comprises a cotton seed, a seed of an agronomically elite plant, a dicot plant seed, and/or a monocot plant seed. In certain aspects, the seed composition may be resistant to a pest comprising an insect and/or a nematode.
In yet another aspect, the invention provides methods for preventing pest infestation or increasing yield, which may comprise treating a plant, plant seed, or the rhizosphere of said plant or seed with the endophyte containing compositions described herein. In certain aspects, the method may also comprise identifying a plant or seed as in need of endophyte treatment. The pest may comprise, for example, a nematode and/or insect. In certain aspects, the pest may comprise a root knot nematode, a aphid, a lygus bug, a stink bug, or combinations thereof.
In still yet another aspect, methods for preventing pest infestation are provided comprising obtaining a seed described herein and planting the seed. The method may further comprise identifying a need of preventing pest infestation. In certain aspects, the pest may comprise a nematode and/or a insect; and/or the pest may comprise a root knot nematode, a aphid, a lygus bug, a stink bug, or combinations thereof.
In a further embodiment, a method for treating a pest infestation comprises identifying a plant suspected of being infected with a pest, applying an above-described composition to the plant, whereby an endophyte-treated plant is generated. In certain aspects, the pest may comprise a nematode and/or an insect; and/or the pest may comprise a root knot nematode, a aphid, a lygus bug, a stink bug, or combinations thereof.
In still yet another aspect, a method of manufacturing pest-resistant seeds is provided comprising providing a fungal endophyte composition comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 endophytes from Table 1, providing seeds; and combining the seeds with the endophyte composition, whereby pest-resistant seeds are generated. In certain aspects, the method increases the percentage of colonization with the endophyte of the plant developing from the seed.
In still yet another aspect, methods of increasing a yield of a crop or a reduction of loss are disclosed comprising providing a fungal endophyte composition comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 endophytes from Table 1; and applying the endophyte composition to a seed, plant or part thereof, whereby the yield of the crop increases. In certain aspects, the crop may be cotton, and the increase of yield may be at least about 2%, 3% 5%, 15%, 20%, or 25% relative to a crop to which no endophyte composition has been applied. In certain aspects, the increase of yield is about 2%-5%, 3%-5%, 5%-10%, 10%-15%, or greater than about 20%, 30%, or more relative to a crop to which no endophyte composition has been applied. In certain aspects, the crop is cotton and the increase of yield comprises reduced boll damage. In certain aspects, the reduction of loss comprises reduction of loss due to insect infestation or drought, and the loss is less than 50%, 40%, 30%, 20%, 10%, 5%, or 5% relative to a crop to which no endophyte composition has been applied.
Also described herein are commodity plant products comprising a plant or part of a plant (including a seed) and further comprising the facultative fungal endophyte described above that is present in a detectable level, for example, as detected by the presence of its nucleic acid by PCR. In another aspect, disclosed is a method of producing a commodity plant product, comprising obtaining a plant or plant tissue from the synthetic combination described above, and producing the commodity plant product therefrom. The commodity plant product can be produced from the seed, or the plant (or a part of the plant) grown from the seed. The commodity plant product can also be produced from the progeny of such plant or plant part. The commodity plant product can be is selected from the group consisting of grain, flour, starch, seed oil, syrup, meal, flour, oil, film, packaging, nutraceutical product, an animal feed, a fish fodder, a cereal product, a processed human-food product, a sugar or an alcohol and protein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: The colonization efficiencies demonstrate that endophytes can be manipulated in the field. Depicted are the mean+/−SE endophytic colonization frequencies of cotton seedlings under field conditions inoculated by seed treatments with different spore concentrations of either (left) Paecilomyces lilacinus or (right) Beauveria bassiana. 
FIG. 2: The endophytic fungus Paecilomyces lilacinus negatively affects root knot nematode (Meloidogyne incognita) reproduction when present as an endophyte in cotton. At high nematode inoculum levels (10,000 eggs), the endophyte reduced egg production in plants following treatment of seeds with solutions containing either 106 or 107 spores/ml when compared to untreated control seeds. At field inoculum levels (2000 eggs), the presence of the endophyte significantly reduced both galls and egg production at both seed treatment concentrations.
FIG. 3: Endophytic Chaetomium globosum negatively affects root-knot nematode reproduction. Negative effects of endophytic Chaetomium globosum on root-knot nematode gall formation and egg production following cotton seed soaking treatments in solutions of 0 (untreated controls), 106 and 108 spores/ml. Seedlings were inoculated with 1000 nematode eggs and grown in the greenhouse. Egg production by hatching nematodes that successfully infected the seedlings was quantified 60 days later.
FIGS. 4A, 4B: The effect of endophytic fungi on cotton aphids (Aphis gossypii) reproduction. FIG. 4A demonstrates that the presence of Beauveria bassiana in cotton negatively affects the reproduction of cotton aphids. FIG. 4B demonstrates that the presence of Paecilomyces lilacinus in cotton negatively affects the reproduction of cotton aphids.
FIG. 5: Effects of Chaetomium globosum on cotton aphids. Endophytic Chaetomium globosum in cotton negatively affects cotton aphid population growth rates as evidenced by reduced reproduction after 14 days on endophyte-colonized versus control plants. Cotton plants were grown from seeds treated by soaking in spore solutions of 0 (control), 106 (low) and 108 (high) spores/ml.
FIGS. 6A, 6B: The effect of the endophytic fungi Beauveria bassiana and Paecilomyces lilacinus on western tarnished plant bugs Lygus hesperus (Miridae). FIG. 6A demonstrates that Beauveria bassiana and Paecilomyces lilacinus negatively affect host plant selection of western tarnished plant bugs when present as an endophyte in cotton. FIG. 6B demonstrates that Beauveria bassiana and Paecilomyces lilacinus negatively affect host plant selection behavior of western tarnished plant bugs when present as an endophyte in cotton.
FIGS. 7A, 7B: The effect of the endophytic fungi Beauveria bassiana and Paecilomyces lilacinus on southern green stink bugs (Nezara viridula (Pentatomidae). FIG. 7A demonstrates that Beauveria bassiana and Paecilomyces lilacinus negatively affect host plant selection of southern green stink bugs when present as an endophyte in cotton. FIG. 7B demonstrates that Beauveria bassiana and Paecilomyces lilacinus negatively affect host plant selection behavior of southern green stink bugs when present as an endophyte in cotton.
FIG. 8: A reduction in cotton boll damage was observed during field trials. Relative to control plants, levels of insect-related boll damage were lower among plants that were treated by soaking seeds in spore solutions of Beauveria bassiana and Paecilomyces lilacinus at concentrations of 106 and 108 spore/ml.
FIG. 9: Foliar application of cotton in the field with spores of endophytic entomopathogenic fungi improves plant performance. Cotton (variety FM1740B2F) seeds treated with a variety of typical fungicide (Metalaxyl, Triadimenol, Trifloxystrobin, 2-(Thiocyanome-thylthio)benzothioazole) and insecticide (Thiodicarb, Imidacloprid, Chloropyrifos) seed treatments were planted and grown under field conditions. The plants were sprayed at the 5th true leaf stage with aqueous solutions of Beauveria bassiana and Paecilomyces fumosoroseus. Sucrose was included (1% wt/vol) as an additional nutritional resource for the fungi. Significantly higher first position boll (developing fruit) retention was observed in plants sprayed with Beauveria bassiana without sucrose and Paecilomyces fumosoroseus plus sucrose.
FIG. 10: Positive effects of fungal endophytes on cotton plant performance under field conditions. Box A (left) demonstrates an early season trend for higher square retention in the treated versus untreated plants. Box B (right) demonstrates that significantly more bolls were retained in the endophyte treatment groups later in the season, relative to control. This is demonstrated with both endophyte species used and with both seed treatment concentration employed (Repeated measures ANOVA: Time, P<0.001; Time*Endophyte, P=0.045, Endophyte, P=0.003).
FIG. 11: Positive effects of fungal endophytes on cotton yields under field conditions. The data demonstrate that endophyte treatments achieved 25% higher yields in treated cotton plants.
FIG. 12: Positive effects of fungal endophytes on sorghum (a) plant height and (b) total fresh biomass under growth chamber seedling assays. Data shown is average plant height (cm) and total fresh biomass (g) of n=10 independent replicates. Error bars represent±1 standard error. All three fungal endophytes improve both traits relative to the untreated control.
FIG. 13: The in-field modulation of the colonization of endogenous cotton endophytes in (a, b) stems and (c, d) roots when treated with fungal endophytes Paecilomyces lilacinus (a, c) and Beauveria bassiana (b, d). Data shown is a percentage change in colonization relative to the corresponding untreated control and plant tissue.
FIG. 14: Average percent difference in yield between endophyte treated and control cotton plants (n=6 replicate plots in a dryland field, College Station, Tex.) for 15 facultative fungal endophytes in the Phytogen (PHY 499WRF) cultivar.
FIG. 15: Aggregated average percent difference in yield between endophyte treated and control cotton plants (n=6 replicate plots in a dryland field, College Station, Tex.) for 15 facultative fungal endophytes and two cotton cultivars; Delta Pine (DP 0912B2RF) and Phytogen (PHY 499WRF). Bars represent a 95% confidence interval around the mean.
FIG. 16: Average percent difference in thrip damage (A) and fleahopper damage (B) between endophyte treated and control cotton plants. The thrip damage was assessed in the Delta Pine (DP 0912B2RF) cultivar (n=6 replicate plots in a dryland field, College Station, Tex.) for 15 facultative fungal endophytes. 12 out of the 15 facultative fungal endophytes tested showed a decrease in thrip damage relative to the untreated cotton plants. The fleahopper damage was assessed in cotton plants of the Phytogen (PHY 499WRF) cultivar (n=6 replicate plots in a dryland field, College Station, Tex.) for 15 facultative fungal endophytes. 6 out of the 15 facultative fungal endophytes tested showed an average decrease in fleahopper damage as compared to untreated cotton plants.
FIG. 17: Mid-season field-trait measured in June at the dryland trial of (A) root length and (B) belowground weight. Data presented is the average of n=10 independent replicates and error bars represent±one standard error.
FIG. 18: Mid-season field-trait measured in July at the dryland trial of canopy temperature (Celsius) for the (blue bars) Delta Pine and (green bars) Phyton cultivars. Data presented is the block-controlled average of n=10 independent replicates, relative to the control plot and error bars represent±one standard error.
FIG. 19: Mid-season field-trait measured in August at the dryland trial of NDVI for the (blue bars) Delta Pine and (green bars) Phyton cultivars. Data presented is the block-controlled average of n=10 independent replicates, relative to the control plot and error bars represent±one standard error.
FIG. 20: Mid-season field-trait measured in August at the dryland trial of first position square retention for the (blue bars) Delta Pine and (green bars) Phyton cultivars. Data presented is the block-controlled average of n=10 independent replicates, relative to the control plot and error bars represent±one standard error.
FIG. 21: Mid-season field-trait measured in August at the dryland trial of plant height (cm) for the (blue bars) Delta Pine and (green bars) Phyton cultivars. Data presented is the block-controlled average of n=10 independent replicates, relative to the control plot and error bars represent±one standard error.
FIG. 22: Mid-season field-trait measured in July at the dryland trial of plant height (cm) for the (blue bars) Delta Pine and (green bars) Phyton cultivars. Data presented is the block-controlled average of n=10 independent replicates, relative to the control plot and error bars represent±one standard error.
FIG. 23: Picture showing increased biomass in the plants treated with endophytes (right half of the image) compared to untreated control (left half of the image).
FIG. 24: Table showing the time to wilt following drought stress in days for plants grown from seeds treated with fungal endophytes and control.
FIG. 25: Table showing the time to death following drought stress in days for plants grown from seeds treated with fungal endophytes and control.



DETAILED DESCRIPTION OF THE INVENTION
Endophytic fungi are ubiquitous in nature, infecting virtually all plants in both natural and agronomic ecosystems. Plants commonly harbor a diversity of fungi living within their tissues as asymptomatic endophytes that can provide protection from a range of biotic and abiotic stressors. The present disclosure describes certain fungal endophytes that can be pathogens, parasites or antagonists to plant pathogens, insects, and nematode pests, thereby providing health and performance benefits to crop plants. The symbiotic endophyte-host relationships can provide several general health and fitness benefits to the host plant, such as enhancement of nutrition, increased drought tolerance and/or chemical defense from potential herbivores and often enhanced biomass production. Root-colonizing mycorrhizae survive on photosynthetic carbohydrates from the plant, and in return, aid in the solubilization and uptake of water and minerals to the host, which can lead to the promotion of seed germination and plant growth. Additionally, the association of a fungal endophyte with a host plant often provides protection from pathogens or tolerance to a variety of biotic and abiotic stresses, such as insect infestation, grazing, water or nutrient deficiency, heat stress, salt or aluminum toxicity, and freezing temperatures. Host growth and fitness promotion and protection are thought to be achieved through multiple beneficial properties of the endophyte-host association.
These fungal endophytes provided in Table 1 were originally collected as fungal endophytes of cotton. These endophytic fungi can be inoculated to live within cotton using either seed, soil or foliar applications and exhibited surprisingly beneficial effects by providing protection from pest infestation. Pests can be nematode and/or insect pests. In addition, these endophytic fungi have an unexpected beneficial effect on cotton yield.
Described is the application of beneficial fungi to establish endophytically within crop plants to improve plant performance and yield while conferring protection against insect and nematode pests. In this regard, the present invention overcomes the limitations of the prior art such as the susceptibility of the fungi to degradation by UV light, desiccation or heat after exposure to the environment following application as an inundative soil or foliar biopesticide. Inoculation and endophytic establishment of the fungi within the plant protects the fungi from UV light, desiccation, and unfavorable temperatures, while harboring the fungi in the very plant tissues they are intended to protect. Introducing fungi to live endophytically within plants requires no genetic modification of the plant or microorganisms, and the fungi themselves can be a source for natural products. In various embodiments, the fungal inoculant can be formulated and applied, for example, as treatment of seeds, in furrow applications, before or during planting, or as foliar application after plant germination, and after inoculation, the fungal endophytes provide season-long protective effects and higher crop yields (approximately 25% higher). In certain embodiments, the increase of yield is about 5%, 10%, 15%, 20%, 30%, 40%, 45%, 50%, or greater than 50% relative to a crop to which no endophyte composition has been applied. In further embodiments, the increase of yield is the result of reduction of loss that comprises reduction of loss due to insect infestation or drought and the loss is less than 50%, 40%, 30%, 20%, 10%, 5%, or 5% relative to a crop to which no endophyte composition has been applied. In certain embodiments, the crop is cotton and the reduction of loss comprises reduced boll damage.
Thus, in one aspect, the invention provides a combination (also termed a “symbiotum”) of a host plant and an endophyte that allows for improved agronomic properties of host plants. The combination may be achieved by artificial inoculation, application, or other infection of a host plant or seeds thereof, such as a cotton plant or seed thereof, or host plant tissues, with a fungal endophyte strain of the present invention. Thus, a combination achieved by such an inoculation is termed a “synthetic” combination, synthetic composition, synthetic seed coating, and/or synthetic pest-resistant seed composition. The fungal endophyte may be present in intercellular spaces within plant tissue, such as the root. Its presence may also occur or may also be maintained within a plant or plant population by means of grafting or other inoculation methods such as treating seeds, plants or parts thereof with endophyte mycelia, or endophyte spores. In certain embodiments, the plant, part of the plant, roots, seed, or leaves are sterilized to remove microorganisms before applying the endophyte. In particular embodiments, seeds are sterilized to remove native endophytes before adding the endophyte compositions herein described. In certain aspects, the ability of the seed to germinate is not affected by the sterilization.
The invention also provides methods for detecting the presence of the fungal endophyte of the present invention within a host plant. This may be accomplished, for instance, by isolation of total DNA from tissues of a potential plant-endophyte combination, followed by PCR, or alternatively, Southern blotting, western blotting, or other methods known in the art, to detect the presence of specific nucleic or amino acid sequences associated with the presence of a fungal endophyte strain of the present invention. Alternatively, biochemical methods such as ELISA, HPLC, TLC, or fungal metabolite assays may be utilized to determine the presence of an endophyte strain of the present invention in a given sample of crop tissue. Additionally, methods for identification may include microscopic analysis, such as root staining, or culturing methods, such as grow out tests or other methods known in the art (Deshmukh et al. 2006). In particular embodiments, the roots of a potential grass plant-endophyte combination may be stained with fungal specific stains, such as WGA-Alexa 488, and microscopically assayed to determine fungal root associates.
In certain embodiments, the agronomic qualities may be selected from the group consisting of: increased biomass, increased tillering, increased root mass, increased flowering, increased seed yield, and enhanced resistance to biotic and/or abiotic stresses, each of these qualities being rated in comparison to otherwise identical plants grown under the same conditions, and differing only with respect to the presence or absence of a fungal endophyte. The synthetic combinations and methods of the present invention may be applied to respond to actual or anticipated stresses. Such stresses may include, for instance, drought (water deficit), cold, heat stress, nutrient deficiency, salt toxicity, aluminum toxicity, grazing by herbivores, insect infestation, nematode infection, and fungal, bacteria or viral infection, among others.
The present disclosure provides, in one embodiment, fungal endophytes selected from those in Table 1 that negatively affect the reproduction of insect herbivores feeding on leaves above ground (cotton aphids, Aphis gossypii) and plant parasitic nematodes attacking roots below ground (root knot nematodes, Meloidogyne incognita). In addition, improved plant performance and yields in colonized versus uncolonized control plants may be observed in field trials employing seed treatment with such endophytes. Plant growth enhancement and increased resistance to root knot nematodes was demonstrated in cotton, for example, employing Chaetomium globosum as an endophyte in greenhouse trials. In addition and as a further non-limiting illustrative example, using Beauveria bassiana as an endophyte in cotton, reductions in insect (cotton aphid) reproduction was demonstrated in both greenhouse and field trials. The endophytic presence of Paecilomyces lilacinus and Beauveria bassiana also had negative effects on the host selection behavior of key sucking bug pests (Lygus hesperus and Nezara viridula) that attack developing flowers and fruits in cotton. Furthermore, in field trials using Beauveria bassiana as an endophyte in cotton positive effects on plant performance and higher yields in endophyte colonized versus uncolonized control plants was demonstrated.
Metabolomic differences between the plants can be detected using methods known in the art. For example, a biological sample (whole tissue, exudate, phloem sap, xylem sap, root exudate, etc.) from the endophyte-associated and reference agricultural plants can be analyzed essentially as described in Fiehn et al., (2000) Nature Biotechnol., 18, 1157-1161, or Roessner et al., (2001) Plant Cell, 13, 11-29. Such metabolomic methods can be used to detect differences in levels in hormones, nutrients, secondary metabolites, root exudates, phloem sap content, xylem sap content, heavy metal content, and the like.
In another embodiment, the present invention contemplates methods of coating the seed of a plant with a plurality of endophytes, as well as seed compositions comprising a plurality of endophytes on and/or in the seed. The methods according to this embodiment can be performed in a manner similar to those described herein for single endophyte coating. In one example, multiple endophytes can be prepared in a single preparation that is coated onto the seed. The endophytes can be from a common origin (i.e., a same plant). Alternatively, the endophytes can be from different plants.
Where multiple endophytes are coated onto the seed, any or all of the endophytes may be capable of conferring a beneficial trait onto the host plant. In some cases, all of the endophytes are capable of conferring a beneficial trait onto the host plant. The trait conferred by each of the endophytes may be the same (e.g., both improve the host plant's tolerance to a particular biotic stress), or may be distinct (e.g., one improves the host plant's tolerance to drought, while another improves phosphate utilization). In other cases the conferred trait may be the result of interactions between the endophytes.
DEFINITIONS
In the description and tables herein, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
When a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. The singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof.
The term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and agriculturally acceptable carriers. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for applying the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
Biological control: the term “biological control” and its abbreviated form “biocontrol,” as used herein, is defined as control of a pest, pathogen, or insect or any other undesirable organism by the use of at least one endophyte.
A “composition” is intended to mean a combination of active agent and at least another compound, carrier or composition, inert (for example, a detectable agent or label or liquid carrier) or active, such as a pesticide.
As used herein, an “agricultural seed” is a seed used to grow plants in agriculture (an “agricultural plant”). The seed may be of a monocot or dicot plant, and is planted for the production of an agricultural product, for example grain, food, fiber, etc. As used herein, an agricultural seed is a seed that is prepared for planting, for example, in farms for growing. Agricultural seeds are distinguished from commodity seeds in that the former is not used to generate products, for example commodity plant products.
As used herein, a “commodity plant product” refers to any composition or product that is comprised of material derived from a plant, seed, plant cell, or plant part of the present invention. Commodity plant products may be sold to consumers and can be viable or nonviable. Nonviable commodity products include but are not limited to nonviable seeds and grains; processed seeds, seed parts, and plant parts; dehydrated plant tissue, frozen plant tissue, and processed plant tissue; seeds and plant parts processed for animal feed for terrestrial and/or aquatic animal consumption, oil, meal, flour, flakes, bran, fiber, and any other food for human or animal consumption; and biomasses and fuel products. Any such commodity plant product that is derived from the plants of the present invention may contain at least a detectable amount of the specific and unique DNA corresponding to the endophytes described herein. Any standard method of detection for polynucleotide molecules may be used, including methods of detection disclosed herein.
As used herein, the phrase “agronomically elite plants” refers to a genotype or cultivar with a phenotype adapted for commercial cultivation. Traits comprised by an agronomically elite plant may include biomass, carbohydrate, and/or seed yield; biotic or abiotic stress resistance, including drought resistance, insect resistance, fungus resistance, virus resistance, bacteria resistance, cold tolerance, and salt tolerance; improved standability, enhanced nutrient use efficiency, and reduced lignin content.
In certain embodiments, cotton agronomically elite plants include, for example, known cotton varieties AM 1550 B2RF, NG 1511 B2RF, NG 1511 B2RF, FM 1845LLB2, FM 1944GLB2, FM 1740B2F, PHY 499 WRF, PHY 375 WRF, PHY 367 WRF, PHY 339 WRF, PHY 575 WRF, DP 1252 B2RF, DP 1050 B2RF, DP 1137 B2RF, DP 1048 B2RF, and/or DP 1137 B2RF.
As used herein, the phrase “culture filtrate” refers to broth or media obtained from cultures inoculated with a strain of fungi and allowed to grow. The media is typically filtered to remove any suspended cells, leaving the nutrients, hormones, or other chemicals.
As used herein, the term “endophyte” refers to an organism capable of living within a plant or plant tissue. An endophyte may comprise a fungal organism that may confer an increase in yield, biomass, resistance, or fitness in its host plant. Fungal endophytes may occupy the intracellular or extracellular spaces of plant tissue, including the leaves, stems, flowers, or roots.
The phrase “pest resistance” refers to inhibiting or reducing attack from pests. Pest resistance provides at least some increase in pest resistance over that which is already possessed by the plant.
As used herein, the term “genotypes” refers to the genetic constitution of a cell or organism.
As used herein, the term “phenotype” refers to the detectable characteristics of a cell or organism, which characteristics are either the direct or indirect manifestation of gene expression.
As used herein, the phrase “host plant” refers to any plant that an endophytic fungi colonizes. In certain embodiments, the host plant comprises progeny of colonized plant.
As used herein, the phrase “increased yield” refers to an increase in biomass or seed weight, seed or fruit size, seed number per plant, seed number per unit area, bushels per acre, tons per acre, kilo per hectare, carbohydrate yield, or cotton yield. Such increased yield is relative to a plant or crop that has not been inoculated with the endophyte. In certain embodiments, the increase yield is relative to other commonly used pest treatments or other methods of addressing the biotic or abiotic stress.
As used herein, the phrase “biomass” means the total mass or weight (fresh or dry), at a given time, of a plant tissue, plant tissues, an entire plant, or population of plants, usually given as weight per unit area. The term may also refer to all the plants or species in the community (community biomass).
As used herein, “sticker” refers to compounds to enhance binding of spores to the seed surface. Non-limiting examples of such compounds are alginic acid, carrageenan, dextrin, dextran, pelgel, polyethelene glycol, polyvinyl pyrrolidone, methyl cellulose, polyvinyl alcohol, or gelatin.
As used herein, an “agriculturally acceptable” excipient or carrier is one that is suitable for use in agriculture without undue adverse side effects to the plants, the environment, or to humans or animals who consume the resulting agricultural products derived therefrom commensurate with a reasonable benefit/risk ratio.
As used herein, the term “synthetic” or the phrase “synthetic combination” refers to an artificial combination that includes mycelia and/or spores of a endophyte that is or leads to an endophytic fungal-host relationship (also termed a “symbiotum”) of a host plant and an endophyte. The synthetic combination may be achieved, for example, by artificial inoculation, application, or other infection of a host plant, host plant seeds, or host plant tissues with the endophyte. In addition, the combination of host plant and an endophyte may be achieved by inoculating the soil or growth media of the plant.
The present invention contemplates the use of “isolated” microbe. As used herein, an isolated microbe is a microbe that is isolated from its native environment, and carries with it an inference that the isolation was carried out by the hand of man. An isolated microbe is one that has been separated from at least some of the components with which it was previously associated (whether in nature or in an experimental setting) or occurs at a higher concentration, viability, or other functional aspect than occurring in its native environment. Therefore, an “isolated” microbe is partially or completely separated from any other substance(s) as it is found in nature or as it is cultured, propagated, stored or subsisted in naturally or non-naturally occurring environments. Specific examples of isolated microbes include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.
As used herein, a microbe is considered to be “native” to a plant or a portion of the plant, and is said to be “natively” present in the plant or a portion of plant, if that plant or portion of the plant contains the microbe, for example, in the absence of any contacting with the microbe preparation, or contains the microbe at much lower concentrations than the contacting with the microbe preparation would provide.
Some of the methods described herein allow the colonization of plant seeds by microbes. As used herein, a microbe is said to “colonize” a plant or seed when it can exist in a symbiotic or non-detrimental relationship with the plant in the plant environment, for example on, in close proximity to or inside a plant, including the seed.
A “population” of plants, as used herein, refers to a plurality of plants that were either grown from the seeds treated with the endophytes as described herein, or are progeny of a plant or group of plants that were subjected to the inoculation methods. The plants within a population are typically of the same species, and/or typically share a common genetic derivation.
EXAMPLES
Example 1
Creating Spore Suspensions and Treatment of Seeds
Cultivation of plants and endophytic fungi strains: The cotton seed variety used in particular embodiments was variety LA122 (available from All-Tex Seed, Inc., Levelland, Tex. 79336). Paecilomyces lilacinus and Chaetomium globosum were obtained from cotton plants as described (Ek-Ramos et al. 2013, PLoS ONE 8(6): e66049. doi:10.1371/journal.pone.0066049). Persons of ordinary skill in the art can obtain endophytes suitable for performing the various embodiments of the present invention by performing the procedures described therein. In short, plant samples were rinsed in tap water and surface sterilized by immersion in 70% ethanol for 5 min, 10% bleach solution for 3 min, and rinsed twice with autoclaved distilled water. Samples were blotted dry using autoclaved paper towels. Five individual surface sterilized leaves, squares and bolls (N=15 total samples) were randomly selected and imprinted onto fresh potato dextrose agar (PDA) and V8 media as a way to monitor surface sterilization efficiency. For endophyte isolation, leaves were cut in small fragments of approximately 1 cm2. Squares and bolls were cut in six pieces. Any fiber present was removed and cut into six smaller pieces. Leaf fragments were placed upside down on PDA and V8 medium plates in triplicate. Each plate contained 3 leaf fragments for a total of 9 fragments assayed per plant. For squares collected early in the season, 3 slices per square were plated on PDA and V8 media as with the leaf fragments. Because of similarity in size and location within a plant, when collected later in the season, squares and bolls from a given plant were plated together on petri dishes containing two square slices, two boll slices and two pieces of fiber. Antibiotics Penicillin G (100 Units/mL) and Streptomycin (100 μg/mL) (Sigma, St Louis, Mo., USA) were added to the media to suppress bacterial growth. All plates were incubated in the dark at room temperature for, in average, two weeks until growth of fungal endophyte hyphae from plant tissues was detected.
An inclusive combination of morphological and molecular fungal endophyte identification was employed for identification. Once fungal hyphae were detected growing from the plant material, samples were taken to obtain pure fungal isolates. For identification by PCR, genomic DNA was extracted from mycelium of each isolated fungal strain, following a chloroform:isoamyl alcohol 24:1 protocol and fungal specific primers were used to amplify the ITS (Internal Transcribed Spacer) region of nuclear ribosomal DNA. This region is the primary barcoding marker for fungi and includes the ITS1 and ITS2 regions, separated by the 5.8S ribosomal gene. In order to avoid introducing biases during PCR (taxonomy bias and introduction of mismatches), it has been suggested to amplify the ITS1 region only, therefore the primers ITS1 (5′ TCC GTA GGT GAA CCT GCG G 3′) (SEQ ID NO:5) and ITS2 (5′ GCT GCG TTC TTC ATC GAT GC 3′) (SEQ ID NO:6) were used to amplify and sequence the  ̃240 bp ITS1 region of each one of the isolated fungal strains. The resulting sequences were aligned as query sequences with the publicly available databases GenBank nucleotide, UNITE and PlutoF. The last two are specifically compiled and used for fungi identification. Table 1 provides a list of endophytes identified and useful in the present invention. All of these endophytes belong to phylum Ascomycota, subphylum Pezizomycotina, except for Phanerochaete crassa, which belongs to phylum Basidiomycota, subphylum Agaricomycotina, and Pseudozyma sp, which belongs to phylum Basidiomycota, subphylum Ustilaginomycotina. Table 1 shows the species/genus, family, order, subclass, class, and the SEQ ID NO corresponding to the  ̃240 bp ITS1 region for each one of the isolated fungal strains, except for Beauveria bassiana, Aspergillus parasiticus, Lecanicillium lecanii, and Paecilomyces lilacinus, where the sequences shown includes the ITS1, ITS2, 5.8S, 18S, and 28S sequences and were obtained from the UNITE database for GenBank numbers JF837090, JX857815, FJ643076, and EU553283, respectively.







TABLE 1







endophytes identified and useful in the present invention












Genus/Species
Family
Order
Subclass
Class
SEQ ID NO.















Acremonium
Incertaesedis
Hypocreales
Hypocreomycetidae
Sordariomycetes
7


altematum







Alternaria
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
8


altemata







Alternaria
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
9


brassicae







Alternaria
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
10


compacta







Alternaria dianthi
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
11


Alternaria
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
12


longipes







Alternaria mall
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
13


Alternaria sesami
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
14


Alternaria solani
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
15


Alternaria sp.
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
16


Alternaria
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
17


tenuissima







Bipolaris
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
18


spicifera







Cercospora
Mycosphaerellaceae 
Capnodiales
Dothideomycetidae
Dothideomycetes
19


canescens







Cercospora
Mycosphaerellaceae 
Capnodiales
Dothideomycetidae
Dothideomycetes
20


capsici







Cercospora
Mycosphaerellaceae 
Capnodiales
Dothideomycetidae
Dothideomycetes
21


kikuchii







Cercospora
Mycosphaerellaceae 
Capnodiales
Dothideomycetidae
Dothideomycetes
22


zinnia







Chaetomium
Chaetomiaceae
Sordariales
Sordariomycetidae
Sordariomycetes
23


globosum







Chaetomium
Chaetomiaceae
Sordariales
Sordariomycetidae
Sordariomycetes
24


piluliferum







Chaetomium sp.
Chaetomiaceae
Sordariales
Sordariomycetidae
Sordariomycetes
25


Cladosporium
Cladosporiaceae
Capnodiales
Dothideomycetidae
Dothideomycetes
26


cladosporioides







Cladosporium sp.
Cladosporiaceae
Capnodiales
Dothideomycetidae
Dothideomycetes
27


Cladosporium
Cladosporiaceae
Capnodiales
Dothideomycetidae
Dothideomycetes
28


uredinicola







Cochliobolus sp
Pleosporaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
29


Phanerochaete
Phanerochaetaceae
Polyporales
Incertae sedis
Agaricomycetes
30


crassa







Phoma
Incertae sedis
Pleosporales
Pleosporomycetidae 
Dothideomycetes
31


americana







Phoma
Incertae sedis
Pleosporales
Pleosporomycetidae 
Dothideomycetes
32


subherbarum







Phomopsis
Diaporthaceae
Diaporthales
Sordariomycetidae
Sordariomycetes
33


liquidambari







Phomopsis sp.
Diaporthaceae
Diaporthales
Sordariomycetidae
Sordariomycetes
34


Pleospora sp.
Pleosporaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
35


Pleosporaceae
Pleosporaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
36


sp.







Preussia africana
Sporormiaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
37


Preussia sp.
Sporormiaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
38


Pseudozyma sp.
Ustilaginaceae
Ustilaginales
Ustilaginomycetidae
Ustilaginomycetes
39


Pyrenophora
Pleosporaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
40


teres







Colletotrichum
Glomerellaceae
Incertae sedis
Sordariomycetidae
Sordariomycetes
41


capsici







Coniolariella
Incertae sedis
Xylariales
Xylariomycetidae
Sordariomycetes
42


gamsii







Coniothyrium
Coniothyriaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
43


aleuritis







Coniothyrium sp.
Coniothyriaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
44


Corynespora
Corynesporascaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
45


cassiicola







Diaporthe sp.
Diaporthaceae
Diaporthales
Sordariomycetidae
Sordariomycetes
46


Diatrype sp.
Diatrypaceae
Xylariales
Xylariomycetidae
Sordariomycetes
47


Drechslerella
Orbiliaceae
Orbiliales
Orbiliomycetidae
Orbiliomycetes
48


dactyloides







Embellisia
Pleosporaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
49


indefessa







Epicoccum
Pleosporaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
50


nigrum







Epicoccum sp.
Pleosporaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
51


Exserohilum
Pleosporaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
52


rostraturn







Fusarium
Nectriaceae
Hypocreales
Hypocreomycetidae
Sordariomycetes
53


chlamydosporum







Fusarium sp.
Nectriaceae
Hypocreales
Hypocreomycetidae
Sordariomycetes
54


Gibellulopsis
Plectosphaerellaceae
Incertae sedis
Hypocreomycetidae
Sordariomycetes
55


nigrescens







Gnomoniopsis sp.
Glomerellaceae
Incertae sedis
Hypocreomycetidae
Sordariomycetes
56


Lewia infectoria
Pleosporaceae
Pleosporales
Pleosporomycetidae
Dothideomycetes
57


Mycosphaerella
Mycosphaerellaceae
Capnodiales
Dothideomycetidae
Dothideomycetes
58


coffeicola







Mycosphaerellac
Mycosphaerellaceae
Capnodiales
Dothideomycetidae
Dothideomycetes
59


eae sp.







Nigrospora
Incertae sedis
Trichosphaeriales
Incertae sedis
Sordariomycetes
60


oryzae







Nigrospora sp.
Incertae sedis
Trichosphaeriales
Incertae sedis
Sordariomycetes
61


Nigrospora
Incertae sedis
Trichosphaeriales
Incertae sedis
Sordariomycetes
62


sphaerica







Paecilomyces sp.
Trichocomaceae
Eurotiales
Eurotiomycetidae
Eurotiomycetes
63


Penicillium
Trichocomaceae
Eurotiales
Eurotiomycetidae
Eurotiomycetes
64


citrinum







Retroconis sp.
Incertae sedis
Incertae sedis
Incertae sedis
Incertae sedis
65


Rhizopycnis sp.
Incertae sedis
Incertae sedis
Incertae sedis
Dothideomycetes
66


Schizothecium
Lasiosphaeriaceae
Sordariales
Sordariomycetidae
Sordariomycetes
67


inaequale







Stagonospora sp.
Phaeosphaeriaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
68


Stemphylium
Pleosporaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
69


lancipes







Thielavia
Chaetomiaceae
Sordariales
Sordariomycetidae
Sordariomycetes
70


hyrcaniae







Thielavia sp.
Chaetomiaceae
Sordariales
Sordariomycetidae
Sordariomycetes
71


Ulocladium
Pleosporaceae
Pleosporales
Pleosporomycetidae 
Dothideomycetes
72


chartarum







Verticillium sp.
Plectosphaerellaceae
Incertae sedis
Hypocreomycetidae
Sordariomycetes
73


Beauveria
Cordycipitaceae
Hypocreales
Hypocreomycetidae
Sordariomycetes
74


bassiana







Aspergillus
Trichocomaceae
Eurotiales
Eurotiomycetidae
Eurotiomycetes
75


parasiticus







Lecanicillium
Cordycipitaceae
Hypocreales
Hypocreomycetidae
Sordariomycetes
76


lecanii







Paecilomyces
Trichocomaceae
Eurotiales
Eurotiomycetidae
Eurotiomycetes
77


lilacinus









TABLE 1 List of Endophytes:
Acremonium alternatum, Alternaria alternata, Alternaria brassicae, Alternaria compacta, Alternaria dianthi, Alternaria longipes, Alternaria mali, Alternaria sesami, Alternaria solani, Alternaria sp., Alternaria tenuissima, Ascomycota sp., Bipolaris spicifera, Cercospora canescens, Cercospora capsici, Cercospora kikuchii, Cercospora zinnia, Chaetomium globosum, Chaetomium piluliferum, Chaetomium sp., Cladosporium cladosporioides, Cladosporium sp., Cladosporium uredinicola, Cochliobolus sp, Phanerochaete crassa, Phoma americana, Phoma subherbarum, Phomopsis liquidambari, Phomopsis sp., Pleospora sp., Pleosporaceae sp., Polyporales sp., Preussia africana, Preussia sp., Pseudozyma sp., Pyrenophora teres, Colletotrichumcapsici, Coniolariella gamsii, Coniothyrium aleuritis, Coniothyrium sp., Corynespora cassiicola, Diaporthe sp., Diatrype sp., Drechslerella dactyloides, Embellisia indefessa, Epicoccum nigrum, Epicoccum sp., Exserohilum rostratum, Fusarium chlamydosporum, Fusarium sp., Gibellulopsis nigrescens, Gnomoniopsis sp., Lewia infectoria, Mycosphaerella coffeicola, Mycosphaerellaceae sp., Nigrospora oryzae, Nigrospora sp., Nigrospora sphaerica, Paecilomyces sp., Penicillium citrinum, Retroconis sp., Rhizopycnis sp., Schizothecium inaequale, Stagonospora sp., Stemphylium lancipes, Thielavia hyrcaniae, Thielavia sp., Ulocladium chartarum, Verticillium sp., Beauveria bassiana, Aspergillus parasiticus, Lecanicillium lecanii, Paecilomyces lilacinus. 
Beauveria bassiana was cultured from a commercially obtained strain (available from Botanigard). Beauveria bassiana, Paecilomyces lilacinus, and Chaetomium globosum were cultured on potato dextrose agar media (PDA). Stock spore concentration solutions of each fungi were made by adding 10 ml of sterile water to the fungi plates and scraping them free of the agar with a sterile scalpel. The resulting mycelia and spores obtained were then filtered into a sterile beaker utilizing a cheese cloth to filter out the mycelia, thereby creating stock solutions. A haemocytometer was used to measure and calculate spore concentrations of the stock solutions. The desired concentrations were created by dilution, and seeds were placed into spore suspensions with the desired spore concentrations. In various embodiments, the final treatment concentrations can be about 102, 103, 104, 105, 106, 107, 108, or 109 spores/ml which can be reached by serial dilutions in sterile water or in an appropriate solution or buffer.
For seed inoculation, the seeds were surface sterilized prior to soaking them in spore suspensions with the desired concentration by immersion the seeds in 70% ethanol for 3 minutes with constant shaking followed by incubation in 2% NaOCl for 3 minutes; followed by three washes in sterile water. The third sterile water wash was plated onto potato dextrose agar media (PDA) to confirm that surface sterilization was effective. Seeds were then soaked for 24 hours in beakers containing spore suspensions with two different concentrations of fungi. Control group seeds were treated with sterile water only. Spore concentrations for Beauveria bassiana were zero (control), 1×106 (treatment 1) and 1×109 (treatment 2) and for Paecilomyces lilacinus or Chaetomium globosum were zero (control), 1×106 (treatment 1) and 1×107 (treatment 2). These beakers were incubated for 24 hours at 32° C. in a culture chamber until next day for planting (24 hr).
Soaked seeds were planted in L22 mix soil (Borlaug Institute, Texas A&M). All plants were grown in a laboratory greenhouse at  ̃28° C. with a natural light photoperiod. There was no fertilization of the plants, and watering was done consistently across all treatments as needed.
Direct seed inoculation: In particular embodiments, individual seeds and the surrounding soil can be directly inoculated with the spore solution (102-103, 103-104, 104-105, 106-107, or 107-108 spores/ml) at planting before covering the seed with soil.
In various embodiments, any seed or plant treatments that are suitable for application of biological agents to seeds or plants and known to persons having ordinary skill in the art can be employed.
Example 2
Application of Endophyte Spores as a Dry Powder Composition
In addition to application of a spore solution for seed treatment, the endophytes or endophyte spores can also be applied as dry powder or using a sicker such as methyl cellulose for seed treatment. In certain embodiments, the concentration may be at least 105, 106, 107, 108, 109, or higher colony forming units or spores/g dry weight.
In certain embodiments, endophytes can be grown in fungi cultivation media in a fermenter. Endophytic mycelial fragments or spores can be collected, dried and ground. A sticker such as caboxymethyl cellulose may also be added to the ground endophytic material.
In certain embodiments the weight ratio between endophytic material and sticker may be between 1:10-50, 1:50-100, 1:100-500, or 1:500-1000 to obtain the seed coating or seed inoculation material. This seed inoculation material can be applied to seeds. In various embodiments, the weight ratio between seed inoculation material and seed may be 1:10-50, 1:50-100, 1:100-500, 1:500-1000, or 1:1000-5000.
Example 3
Soil (in Furrow) Endophyte Treatments
Soil drench (in furrow) application may be performed by applying an endophyte composition to the surface of the soil and/or seed during planting. In particular embodiments, the endophyte composition may comprise an endophyte suspension or an endophyte dry powder formulation. In various embodiments the endophyte may comprise mycelia and/or spores. In particular embodiments, the soil drench application may comprise applying the endophyte composition to the surface of the soil directly above each seed. In certain embodiments, the endophyte composition may comprise 0.01-0.1, 0.1-1, or 1-10 ml endophyte suspension, which may be a endophyte spore suspension.
Soil inoculation: In certain embodiments, seeds can be planted into inoculated soil. The inoculum can be obtained by multiplying the endophyte on fungal growth media. The fungal growth media can be potato dextrose agar media (PDA). In other embodiments the fungal growth media can be as wheat grain. In a non-limiting example, 100 g of wheat grain can be washed and soaked overnight in sterile water. Excess water can be drained, seeds dried on paper towel, packed in a 500 ml conical flask and autoclaved at 15 psi for 1 h. One milliliter of the endophytic fungal spore suspension (107 spores/ml) can be inoculated to the flask, and the cultures can be incubated at 25° C. for 2 weeks. To avoid clumping, the flasks can be shaken vigorously to separate the grain and break the mycelial mat. Approximately 5 g of inoculum can be placed in soil at planting. In certain embodiments, the inoculum can be placed in the soil at the same time or within 1 month of planting the seeds. In certain embodiments, the seeds may comprise sterilized seeds.
Example 4
Foliar Endophyte Treatments
Plants were inoculated via foliar application at the third true leaf stage by spraying the surface of fully expanded leaves to run-off with a spore suspension (108 spores/ml) using a hand-held plastic sprayer (1 L). In certain embodiments, endophyte spore suspensions were made in water. In certain embodiments, the water was supplemented with a detergent. In a particular non-limiting example, the spore suspension contained 0.02% Triton X 100 as a detergent.
Foliar endophyte treatment may be performed using any suitable method known to a person having ordinary skill in the art. In particular, foliar endophyte treatment may be performed using a sprayer by directly spraying leaves with an endophyte suspension, which may be a endophyte spore suspension.
FIG. 9 demonstrates that foliar application of cotton in the field with spores of endophytic entomopathogenic fungi improved plant performance. Cotton (variety FM1740B2F) seeds were treated with a variety of typical fungicide (Metalaxyl, Triadimenol, Trifloxystrobin, 2-(Thiocyanome-thylthio)benzothioazole) and insecticide (Thiodicarb, Imidacloprid, Chloropyrifos), and seed treatments were planted and grown under field conditions. The plants were sprayed at the 5th true leaf stage with aqueous solutions of Beauveria bassiana and Paecilomyces fumosoroseus. Sucrose was included (1% wt/vol) as an additional nutritional resource for the fungi. Significantly higher first position boll (developing fruit) retention was observed in plants sprayed with Beauveria bassiana without sucrose and P. fumosoroseus plus sucrose.
Example 5
Confirmation of Plant Colonization by Endophytic Fungi
Plants were individually placed in plastic bags, which were labeled with plant number, treatment, and final aphid number, and stored in 4° C. until the next day for endophyte confirmation. Half of each plant was utilized for plating on PDA agar and the other half was freeze-dried for to conduct diagnostic PCR assays for endophyte confirmation. The surface sterilization protocol and plating of third sterile water wash on PDA to test for surface contamination was conducted as described above. For diagnostic PCR assays, plant tissue was freeze-dried and DNA was extracted utilizing the CTAB protocol (Doyle & Doyle, 1987, Phytochemistry Bulletin 19:11-15). The oligonucleotide primer sequences synthesized were based upon a NCBI BLAST search corresponding to the laboratory culture sequence results isolated (Ek-Ramos et al., 2013). Sense and antisense oligonucleotide sequences for Beauveria bassiana were: 5′-CGGCGGACTCGCCCCAGCCCG-3′ (SEQ ID NO:1) and 5′-CCGCGTCGGGGTTCCGGTGCG-3′ (SEQ ID NO:2) respectively. The oligonucleotides used to amplify Paecelomyces lilacinus were: 5′ CTCAGTTGCCTCGGCGGGAA 3′ (SEQ ID NO:3) and 5′ GTGCAACTCAGAGAAGAAATTCCG 3′ (SEQ ID NO:4).
The PCR protocol consisted of a denaturation step at 95° C. for 5 min, followed by alignment of oligonucleotides at 56° C. for 2 min and an extension step of 7 min at 72° C. with a total of 35 cycles. The PCR products were visualized in a 2% agarose gel containing 1% ethidium bromide. Electrophoresis was performed at 70 volts for 30 min.
Example 6
Endophytic Fungi can be Manipulated in the Field
A field trial using isolates of Paecilomyces lilacinus and Beauveria bassiana was conducted during the summer. A randomized block design with five replicate plots that were planted with seeds that were inoculated by soaking for 9 hr in three different aqueous spore concentrations (0, 106, or 108 spores/ml) of the candidate endophyte (such as Paecilomyces lilacinus or Beauveria bassiana). Each plot consisted of four 15.24 m (40 ft) rows, each separated by 101.6 cm (40 in).
Colonization efficiency: At the first true leaf stage, four plants from each plot for a total of 20 plants per treatment were randomly sampled and tested for colonization by each of the candidate endophytes. Colonization frequencies were determined by incubating surface sterilized root, stem and leaf fragments on PDA media and observing for fungal growth. Colonization frequencies are reported as the number of plants per treatment group with at least one positively colonized plant fragment.
The high endophytic colonization frequency of seedlings by Paecilomyces lilacinus or Beauveria bassiana demonstrates that the presence of specific endophytes can be manipulated under field planting conditions (FIG. 1).
Example 7
Cotton Aphid Reproduction Test
A colony of A. gossypii was reared on cotton in cages in a greenhouse kept at approximately 28° C. with natural light photoperiod. Second instar nymphs were placed directly onto endophyte-treated cotton plants and control plants. Ten plants were utilized per treatment group and ten aphids were placed per plant. After plants were inoculated with the aphids, the plants were placed in individual plastic 45×20 cm cups and sealed with no-see-um mesh (Eastex products, NJ) to avoid aphid movement from plant to plant. In one embodiment, the plants used were 13 days old, approximately in the first true leaf stage, and aphids were left to reproduce for seven days under greenhouse conditions. In another embodiment, aphids were left to reproduce for 14 days on plants initially 20 days old at the beginning of the experiment, approximately in the third true leaf stage. At the end of each embodiment, aphid numbers were counted and recorded per individual plant. The presence of Beauveria bassiana or Paecilomyces lilacinus as an endophyte in cotton significantly reduced the reproduction of cotton aphids on endophyte treated plants versus untreated control plants (FIG. 4A, 4B, and FIG. 5)
Example 8
Fungal Endophytes Reduce Nematode Reproduction
Plants were germinated from treated and untreated control seeds in an environment chamber and then transplanted to soil in pots 11 days after planting. Two replicate seedlings per treatment were sampled to examine the endophyte colonization efficiency by surface sterilization and plating on PDA agar. Nematode treatment group seedlings were treated with either 2,000 or 10,000 eggs/plant at day six after transplanting. Plants were harvested and processed 6 weeks after nematode inoculation. The numbers of galls per gram of root tissue and total egg numbers in the population for each plant were quantified to compare nematode performance between endophyte-treated and untreated (control) plants.
FIGS. 2 and 3 demonstrate that the endophytic fungi Paecilomyces lilacinus and Chaetomium globosum negatively affected root knot nematode (Meloidogyne incognita) reproduction when present as an endophyte in cotton. At high nematode inoculum levels (10,000 eggs), Paecilomyces lilacinus reduced egg production in plants following treatment of seeds with solutions containing either 106 or 107 spores/ml when compared to untreated control seeds. At field inoculum levels (2000 eggs), the presence of Paecilomyces lilacinus significantly reduced both galls and egg production at both seed treatment concentrations. Endophytic Chaetomium globosum negatively affects root-knot nematode reproduction. Negative effects of endophytic Chaetomium globosum on root-knot nematode gall formation and egg production were demonstrated following cotton seed soaking treatments in solutions of 0 (untreated controls), 106 and 108 spores/ml.
Example 9
Effect of Fungal Endophytes on Insects
Endophyte-treated and control plants were grown from non-transgenic cotton seeds (Gossypium hirsutum)(variety LA122, AllTex Seed Co.). Seeds were soaked for 24 hours in beakers containing 108 spores/ml solutions of the fungi utilized plus sterile water-only as a control. The beakers were placed in a 32° C. culture chamber overnight (approx. 9 h) until planting the next day. The plants were grown under both greenhouse and field conditions. Greenhouse plants were first germinated in seedling trays and then transferred to 30 cm pots. Field grown plants were concurrently planted and grown.
Behavioral assays: No-choice and choice behavioral assays were conducted to compare the response of western tarnished plant bugs (L. hesperus) and green stink bugs (N. viridula) to squares and bolls from endophyte-treated and untreated plants. The assays were conducted at 30° C. in 10 cm diameter petri dishes with a thin layer of 2% agar on the bottom to provide moisture for the squares (L. hesperus assays) and bolls (N. viridula assays) from experimental plants offered to the insects during the observations. For no-choice assays, a single square or boll was inserted by the base into the agar in the center of the dish. A single young adult (1-7 days post molt) insect was placed in each dish and covered with the top. A total of 30 insects were observed in each trial with N=10 insects each in the Beauveria bassiana, Paecilomyces lilacinus and control treatment groups. The L. hesperus no-choice trials were replicated four times (N=40 per treatment) with squares from greenhouse grown plants used in all but one trial. The N. viridula no-choice trials were replicated three times (N=20 per treatment) with bolls from greenhouse grown plants used in one trial.
Choice tests were conducted under the similar conditions using the same arenas, but with two equal sized squares (L. hesperus) or bolls (N. viridula) placed 4 cm apart in the center of the petri dish. The two squares or bolls per arena were from an untreated control plant and either a Beauveria bassiana or Paecilomyces lilacinus treated plant. A total of 20 insects were observed in each trial, with N=10 each in the Beauveria bassiana vs. control and Paecilomyces lilacinus vs. control treatment groups. The L. hesperus and N. viridula choice trials were both replicated twice (N=20 per treatment) with squares from field-grown plants in all trials.
Insects were observed for 6 hours per trial using a point sampling procedure for both the no-choice and choice assays. Preliminary observations indicated that the insects of both species were more active at the beginning of the assay, thus staged sampling schedule was adopted with observations recorded at 5 minute intervals early in the assay (0-60 min), 15 minute intervals in the middle (61-180 min) and 30 minute intervals late (181-360 min) in the assay. At each sampling interval, the insects were recorded as either off the square/boll or feeding or roosting upon the square/boll.
Data analysis: In the no-choice assays, the proportion of insects observed either feeding or resting upon cotton squares (L. hesperus) or bolls (N. viridula) was compared between treatment groups at each observation point across the duration of the assay using the Wilcoxon Signed Ranks Test. To test for variation in responses over time, for each individual the proportion of observations either feeding or upon the plant sample was calculated for early (0-60 min), middle (61-180 min) and late (181-360 min) periods of the assay and compared across treatment groups using a repeated measures analysis of variance (ANOVA) with the endophyte treatment group as the main factor and time as the repeat effect. The observed frequency of individuals failing to make contact with squares or bolls from endophyte-treated plants was compared to the expected frequency of individuals failing to do so based on the control group using a X2 test. Among the insects that did make contact with either a square or boll, the time to first contact (latency) was compared among treatment groups using a one-way ANOVA. All analyses including tests of normality and homogeneity of variances were conducted in SPSS 21 (SPSS Inc.).
Results of the L. hesperus no-choice assays: Over the duration of the assay, a significantly higher proportion of L. hesperus individuals over time was observed in contact with and feeding upon squares from untreated control plants relative to those from either of the Beauveria bassiana or Paecilomyces lilacinus endophyte treatment groups (Wilcoxon Signed Ranks test, P<0.0001 for both comparisons) (FIG. 6A). Repeated measures ANOVA indicated a significant effect of time (F1,116=86.175; P<0.001) with a higher proportion of insects contacting the square as the assay progressed (FIG. 6B). There was also a significant effect of endophyte treatment (F2,116=4.929; P=0.009) with no significant time X endophyte treatment interaction (F2,116=1.015; P=0.366). Of the 40 insects in each treatment group, 12.5% of the control group failed to make contact with the square over the course of the assay, while a significantly higher 35% and 32.5% the Beauveria bassiana and Paecilomyces lilacinus treatment group insect respectively failed to make contact (X2 test, P<0.0001). Among the insects that did make contact with a square, there was significant difference in the latency to first contact among the treatment groups (F2,85=7.225; P<0.0001) with the control group exhibiting a shorter latency to contact than either the Beauveria bassiana (posthoc LSD test; P=0.001) or Paecilomyces lilacinus endophyte treatment groups (posthoc LSD test; P=0.006 (FIG. 6A).
Results of the L. hesperus choice assays: In simultaneous choice tests, L. hesperus individuals selected squares from untreated control plants more often than those from endophyte-treated plants. Response ratios were significantly greater than 0.5 over the duration of the assays, indicating that the insects non-randomly selected bolls from control plants over bolls from plants endophytically colonized by either (A) Beauveria bassiana (P<0.0001; Wilcoxon Signed Ranks test) or (B) Paecilomyces lilacinus (P<0.0001; Wilcoxon Signed Ranks test)(FIG. 6B).
Results of the N. viridula no-choice assays: Over the duration of the assay, a significantly higher proportion of N. viridula individuals over time was observed in contact with and feeding upon bolls from untreated control plants relative to those from either of the Beauveria bassiana or Paecilomyces lilacinus endophyte treatment groups (Wilcoxon Signed Ranks test, P<0.0001 for both comparisons)(FIG. 7A). Repeated measures ANOVA indicated a significant effect of time (F1,116=86.175; P<0.001) with a higher proportion of insects contacting the square as the assay progressed (FIG. 1), There was also a significant effect of endophyte treatment (F2,116=4.929; P=0.009) with no significant time X endophyte treatment interaction (F2,116=1.015; P=0.366). Of the 40 insects in each treatment group, 12.5% of the control group failed to make contact with the square over the course of the assay, while a significantly higher 35% and 32.5% the Beauveria bassiana and Paecilomyces lilacinus treatment group insect respectively failed to make contact (X2 test, P<0.0001). Among the insects that did make contact with a square, there was significant difference in the latency to first contact among the treatment groups (F2,85=7.225; P<0.0001) with the control group exhibiting a shorter latency to contact than either the Beauveria bassiana (posthoc LSD test; P=0.001) or Paecilomyces lilacinus endophyte treatment groups (posthoc LSD test; P=0.006 (FIG. 7B).
Example 10
More Bolls are Retained after Endophyte Treatment
During the field trial, cotton phenology and development was quantified using a plant mapping and information system developed specifically for cotton to track fruit development and retention by the plant as a means of monitoring plant development and stress (COTMANTM, Cotton Inc.). One measure of cotton stress is the retention of developing flowers (squares) and fruits (bolls) in the first fruiting position on branches. First position squares and bolls were measured on 5 plants per row in two rows in each of the five replicate plots (N=10 plants per plot) for each treatment group.
FIG. 10 demonstrates that early in the growing season as flowers begin to develop, a trend for higher square retention in the endophyte-treated plants relative to controls was observed. This trend continued later in the season as evidenced by significantly higher boll retention among the endophyte treatment groups relative to the untreated control plants.
FIG. 8 demonstrates reduction in cotton boll damage during field trials. Relative to control plants, levels of insect-related boll damage were lower among plants that were treated by soaking seeds in spore solutions of Beauveria bassiana and Paecilomyces lilacinus at concentrations of 106 and 108 spore/ml. Positive effects of fungal endophytes on cotton plant performance under field conditions.
Example 11
Endophyte Treatment Increases Yield
At the end of the field trial employing endophyte treatment and treatment plants, plots were machine harvested with a 1-row picker. Surprisingly, the final yields at harvest were significantly higher than expected (25% higher than the untreated controls). Unexpectedly, treatment with Paecilomyces lilacinus or Beauveria bassiana resulted in higher yields than untreated control plants with regardless of the initial seed treatment concentration. (FIG. 11)
Example 12
Endophyte Treatment of Sorghum Increased Growth in the Greenhouse
The effect of the described microbial compositions on sorghum was tested in a seedling assay. Sorghum bicolor seeds were surface sterilized using ethanol and bleach as described in Example 1 for cotton. Three strains (B. bassiana, P. fumosoroseus, and P. lilacinus) were prepared as conidia suspensions at 107 conidia/ml, and coated on the sorghum seeds as described in Example 1. Control seeds were soaked in sterile water instead of a conidia suspension. Planted seeds were held in constant growth chamber conditions for two weeks at a replication of 10. At the end of two weeks, the plants were removed from the growth chamber and the plant height and biomass were measured. FIG. 12A shows the increase in plant height when applied with the described microbial composition relative to the control (p<0.05). FIG. 12B shows the increase in plant biomass in plants grown from seed that were treated with the described microbial composition relative to the control (p<0.05).
Example 13
Treatment with Fungal Endophytes Modulates the Colonization Frequencies of Native Endophytes
To determine whether endophyte seed treatments could alter the microbiome of the plant grown from the seed, cotton seeds were treated with spore suspensions of Paecilomyces lilacinus or Beauveria bassiana. Plants were grown in the field as part of a field trial planted and maintained under standard agricultural practices. Endophytic fungi were isolated on PDA media separately from surface-sterilized above-ground stem/leaf and below-ground root tissue to assess changes in the microbial community. The comparison shown in FIG. 13 is relative to the fungal endophyte communities in untreated control plants. The results show that these treatments can alter the colonization rates of native fungal endophytes.
Fungal endophyte treatments may alter the colonization frequencies of any of the fungal endophytes naturally present in plants. To determine what other native endophytes may be affected by seed treatments with fungal endophytes, the identity of cotton fungal endophytes isolated from plants of two commercial cotton varieties, CG3787B2RF and PHY499WRF, were assessed. The samples were obtained during a variety trial near Lubbock, Tex., USA identified as Lubbock-RACE. One single healthy leaf was collected from each of nine individual plants sampled per variety across multiple replicate plots arranged in a randomized block design to control for spatial variation in the field. To identify the fungal endophyte species, whole genomic DNA was extracted and the ribosomal DNA internal transcribed spacer (ITS) region was amplified as a barcode for 454 pyrosequencing using ITS1F forward and ITS2 reverse universal fusion primers. The fungal endophytes identified in this experiment, along with those shown in FIG. 13, are listed in Table 2.







TABLE 2







Native fungal endophytes that may be altered by seed treatments with other fungal endophytes











Phylum
Class
Order
Family
Genus species





Ascomycota







Leotiomycetes






Leotiomycetes


Geomyces auratus



Dothideomycetes
Botryosphaeriales
Botryosphaeriaceae
Macrophomina sp.



Dothideomycetes
Capnodiales
Davidiellaceae




Dothideomycetes
Capnodiales
Davidiellaceae
Cladosporium sp.



Dothideomycetes
Capnodiales
Davidiellaceae
Cladosporium






cladosporioides



Dothideomycetes
Capnodiales
Davidiellaceae
Davidiella sp.



Dothideomycetes
Capnodiales
Mycosphaerellaceae
Cercospora sp.



Dothideomycetes
Capnodiales
Mycosphaerellaceae
Cercospora beticola



Dothideomycetes
Pleosporales





Dothideomycetes
Pleosporales
Pleosporaceae




Dothideomycetes
Pleosporales
Pleosporaceae
Alternaria sp.



Dothideomycetes
Pleosporales
Pleosporaceae
Alternaria alternata



Dothideomycetes
Pleosporales
Pleosporaceae
Alternaria citri



Dothideomycetes
Pleosporales
Pleosporaceae
Alternaria porri



Dothideomycetes
Pleosporales
Pleosporaceae
Alternaria tenuissima



Dothideomycetes
Pleosporales
Pleosporaceae
Cochliobolus sp.



Dothideomycetes
Pleosporales
Pleosporaceae
Curvularia sp.



Dothideomycetes
Pleosporales
Pleosporaceae
Epicoccum sp.



Dothideomycetes
Pleosporales
Pleosporaceae
Exserohilum sp.



Dothideomycetes
Pleosporales
Pleosporaceae
Lewia sp.



Dothideomycetes
Pleosporales
Pleosporaceae
Lewia infectoria



Dothideomycetes
Pleosporales
Pleosporaceae
Pyrenophora sp.



Dothideomycetes
Pleosporales
Pleosporaceae
Pyrenophora tritici-






repentis



Dothideomycetes
Pleosporales
Pleosporaceae
Pleospora sp.



Dothideomycetes
Pleosporales
Didymellaceae
Phoma americana



Dothideomycetes
Pleosporales
Sporormiaceae
Preussia africana



Eurotiomycetes
Chaetothyriales





Eurotiomycetes
Chaetothyriales
Chaetothyriaceae




Eurotiomycetes
Eurotiales
Trichocomaceae




Eurotiomycetes
Eurotiales
Trichocomaceae
Aspergillus sp.



Eurotiomycetes
Eurotiales
Trichocomaceae
Penicillium sp.



Eurotiomycetes
Eurotiales
Trichocomaceae
Thermomyces sp.



Eurotiomycetes
Eurotiales
Trichocomaceae
Thermomyces






lanuginosus



Saccharomycetes
Saccharomycetales





Saccharomycetes
Saccharomycetales
Saccharomycetaceae




Saccharomycetes
Saccharomycetales
Saccharomycetaceae
Candida sp.



Saccharomycetes
Saccharomycetales
Saccharomycetaceae
Candida quercitrusa



Saccharomycetes
Saccharomycetales
Saccharomycetaceae
Candida tropicalis



Saccharomycetes
Saccharomycetales
Saccharomycetaceae
Cyberlindnera sp.



Saccharomycetes
Saccharomycetales
Saccharomycetaceae
Cyberlindnera jadinii



Saccharomycetes
Saccharomycetales
Saccharomycetaceae
Kluyveromyces sp.



Saccharomycetes
Saccharomycetales
Saccharomycetaceae
Kluyveromyces






marxianus



Sordariomycetes






Sordariomycetes
Diaporthales
Gnomoniaceae
Gnomoniopsis sp.



Sordariomycetes
Hypocreales
Cordycipitaceae
Beauveria bassiana



Sordariomycetes
Hypocreales
Cordycipitaceae
Cordyceps sp.



Sordariomycetes
Hypocreales
Cordycipitaceae
Cordyceps bassiana



Sordariomycetes
Hypocreales
Nectriaceae




Sordariomycetes
Hypocreales
Nectriaceae
Fusarium sp.



Sordariomycetes
Hypocreales
Hypocreaceae




Sordariomycetes
Hypocreales
Hypocreaceae
Gibellulopsis






nigrescens



Sordariomycetes
Hypocreales
Hypocreaceae
Hypocrea sp.



Sordariomycetes
Hypocreales
Hypocreaceae
Hypocrea lixii



Sordariomycetes
Hypocreales
Hypocreaceae
Hypocrea virens



Sordariomycetes
Hypocreales
Hypocreaceae
Trichoderma sp.



Sordariomycetes
Hypocreales
Hypocreaceae
Trichoderma






tomentosum



Sordariomycetes
Hypocreales
Plectosphaerellaceae 
Verticillium sp.



Sordariomycetes
Ophiostomatales
Ophiostomataceae




Sordariomycetes
Ophiostomatales
Ophiostomataceae
Ophiostoma sp.



Sordariomycetes
Ophiostomatales
Ophiostomataceae
Ophiostoma






dendifundum



Sordariomycetes
Sordariales
Chaetomiaceae
Chaetomium sp.



Sordariomycetes
Sordariales
Chaetomiaceae
Chaetomium






globosum



Sordariomycetes
Sordariales
Chaetomiaceae
Thielavia hyrcaniae



Sordariomycetes
Sordariales
Chaetomiaceae
Taifanglania sp.



Sordariomycetes
Sordariales
Chaetomiaceae
Taifanglania inflata



Sordariomycetes
Sordariales
Lasiosphaeriaceae
Schizothecium






inaequale



Sordariomycetes
Trichosphaeriales
Trichosphaeriaceae
Nigrospora sp.



Sordariomycetes
Xylariales
Amphisphaeriaceae
Truncatella angustata


Basidiomycota
Agaricomycetes
Cantharellales
Ceratobasidiaceae
Rhizoctonia sp.



Agaricomycetes
Corticiales
Corticiaceae




Agaricomycetes
Corticiales
Corticiaceae
Phanerochaete sp



Agaricomycetes
Polyporales
Coriolaceae




Agaricomycetes
Polyporales
Coriolaceae
Trametes sp.



Agaricomycetes
Polyporales
Coriolaceae
Trametes hirsuta



Agaricomycetes
Polyporales
Coriolaceae
Trametes villosa



Agaricomycetes
Russulales
Peniophoraceae




Microbotryomycetes
Sporidiobolales





Microbotryomycetes
Sporidiobolales 
Sporidiobolaceae 
Rhodotorula sp.



Microbotryomycetes
Sporidiobolales
Sporidiobolaceae
Rhodotorula






mucilaginosa



Tremellomycetes






Tremellomycetes
Tremellales





Tremellomycetes
Tremellales
Tremellaceae
Cryptococcus sp



Tremellomycetes
Tremellales
Tremellaceae
Cryptococcus






skinneri



Tremellomycetes
Tremellales
Tremellaceae
Tremella sp.









Example 14
Fungal Endophyte Seed Treatment Leads to Modulation of Phytohormone Levels in Plants Grown from the Seed
To determine whether fungal endophyte seed treatment affects phytohormone levels in plants grown from the seed, tissue was harvested from the root or third true leaf of cotton plants inoculated with either endophytic Beauveria bassiana or Paecilomyces lilacinus. The experiment was done with three endophyte treatments (uncolonized control, B. bassiana or P. lilacinus) and, for Beauveria bassiana, two herbivory treatments (no aphids, or aphid herbivory for either 1, 4, 8, 24 or 48 hours). Phytohormone levels for abscisic acid (ABA), tuberonic acid (12-OH-JA, an oxidation product of JA-Ile) (TA), ascorbic acid (AA), 12-Oxophytodienoic acid (a JA precursor) (OPDA), JA isoleucine (JA-Ile), and salicylic acid (SA) were assessed by LC-MS in leaf and root tissues separately. All phytohormone level comparisons were made versus plants in the uncolonized control group with significance at P<0.05. Phytohormone levels in plants grown from seed treated with Beauveria bassiana are shown in Table 3, and phytohormone levels in plants grown from seed treated with Paecilomyces lilacinus are shown in Table 4.







TABLE 3







Phytohormone levels in plants grown from seed treated with Beauveria bassiana












Herbivory
Phytohormone
Tissue
Upregulated/downregulated
Tissue
Upregulated/downregulated





Yes
ABA
Leaves
Down at 8 hours of feeding
Roots
Upregulated at 48 hrs of feeding


No


Not significant

Upregulated


Yes
TA
Leaves
Not significant
Roots
Upreguated at 48 hrs of feeding


No


Not significant

Not significant


Yes
AA
Leaves
Down at 4 hrs up at 24 hrs
Roots
Up at 8 hrs down at 48 hrs


No


Not significant

Upregulated


Yes
OPDA
Leaves
Not significant
Roots
Up at 4 hrs and 8 hrs


No


Not significant

Upregulated


Yes
JA-Ile
Leaves
Up at 48 hrs
Roots
Up at 48 hrs


No


Not significant

Upregulated


Yes
SA
Leaves
Up at l hr, 8 hr, 24 and 48 hr
Roots
Down at 4 hr the rest n.s


No


Not significant

Not significant
















TABLE 4





Phytohormone levels in plants grown from seed treated with Paecilomyces lilacinus




















Yes
ABA
Leaves
Down at 48 hrs
Roots
Up at 1 hr and 8 hrs


Yes
TA
Leaves
down at 4 and 8 hrs
Roots
up at 4 hrs


Yes
AA
Leaves
down at 4 and 8 hrs
Roots
up at 4 hrs


Yes
OPDA
Leaves
down at 4 and 8 hrs
Roots
Up at 4 and 48 hrs, down at 24 hrs


Yes
JA-Ile
Leaves
Down at 8 and 48 hrs
Roots
Up at 4 and 24 hrs


Yes
SA
Leaves
Up at 1 and 4 hr, down at 8 hrs
Roots
Up at 1, down at 8 hrs









Example 15
Fungal Endophyte Seed Treatments Alter Traits in Certain Cotton Cultivars in Field Trials
The 2014 field trials were executed in a similar fashion as described in Example 6. A field trial using isolates of listed below was conducted during the summer. Each plot consisted of four 15.24 m (40 ft) rows, each separated by 101.6 cm (40 in), and there were 6 replicate plots per treatment. Yield from plots treated with the described microbial compositions was compared relative to the untreated control plots. For thrips, this damage assessment was on a scale of 0-5; 0=no damage, 1=noticeable feeding scars, but no stunting, 2=noticeable feeding and 25% stunting, 3=feeding with blackened leaf terminals and 50% stunting, 4=severe feeding and 75% stunting, and 5=severe feeding and 90% stunting. For fleahoppers, the number of insects per plant were quantified and reported as an average for each plot. FIG. 14 shows the yield improvement of crops when treated with the described microbial compositions, for Delta Pine and Phytogen cultivars, respectively. FIG. 15 shows the aggregated yield improvement of the microbes across the two cultivars. Bars represent 95% confidence intervals. FIG. 16A shows the beneficial effect of 12 out of 15 microbial compositions tested on thrip damage in the Delta Pine cultivar. In the Phytogen cultivar, only 2 out of the 15 microbial compositions tested showed a benefit by reducing thrip damage. FIG. 16B shows the beneficial effect of reducing fleahopper damage in the Phytogen cultivar, where 6 out of the 15 facultative fungal endophytes tested showed an average decrease in fleahopper damage as compared to untreated cotton plants. In the Delta Pine cultivar, only one microbial composition showed a beneficial effect on fleahopper damage.
A number of other mid-season plant traits were also assessed in the field to determine the effect of the described fungal endophyte compositions. FIG. 17A shows the beneficial increase of the described microbial compositions on mid-season mean root length. FIG. 17B shows the beneficial increase of the described fungal endophyte compositions on mid-season belowground weight. FIG. 18 shows the beneficial increase of the described fungal endophyte compositions on mid-season canopy temperature for both Delta Pine and Phyton cultivars. FIG. 19 shows the beneficial increase of the described fungal endophyte compositions on mid-season NDVI (Normalized Difference Vegetation Index) for both Delta Pine and Phytogen cultivars. NDVI is a measure of chlorophyll content. FIG. 20 shows the beneficial increase of the described fungal endophyte compositions on mid-season first-position square retention for both Delta Pine and Phytogen cultivars. FIG. 21 and FIG. 22 show the modulation (up in July and down in August) of mid-season plant height when treated with the described fungal endophyte compositions for both Delta Pine and Phytogen cultivars. FIG. 23 shows increased biomass in the plants treated with endophytes (right half of the image) compared to untreated control (left half of the image).
In FIGS. 15 through 22, TAM505 is Acremonium sp., TAM32 is Epicoccum nigrum, TAM534 is Cladosporium urdinicola, TAM244 is Cladosporium sp., TAM514 is Cladosporium urdinicola, TAM474 is Cladosporium cladosporoides, TAM554 is Chaetomium globosum, TAM15 is Exserohilum sp., TAM488 is Epicoccum nigrum, TAM452 is Cladosporium urdinicola, TAM490 is Paecilomyces lilacinus, TAMBB is Beauveria bassiana, TAM105 is Cochliobolus sp., TAM189 is Bipolaris sp., and TAM47 is Epicoccum nigrum. 
Example 16
Fungal Endophyte Seed Treatments Provide Drought Tolerance in Cotton Cultivars in Greenhouse Trials
Cotton plants were germinated from endophyte-treated and untreated control seeds in the greenhouse. All seeds watered for 7 days or until cotyledon stage using pre-determined soil saturation volume of water per plant. At 7 DAP, water was withheld from water stressed plants while controls continued to be watered. Time to wilt and time to death were measured at a max of 21 DAP. The data in FIG. 24 shows the mean time to wilt, and the data in FIG. 25 shows the mean time to death. Endophyte treatment increased the survival of plants subjected to drought stress in both the Delta Pine (DTP) and the Phytogen (PHY) cultivars. In FIGS. 24 and 25, endophyte number 194 is Epicoccum nigrum, 249 is Cladosporium cladosporioides, 355 is Chaetomium globusum, 46 is Epicoccum sp., 463 is Cladosporium sp., 534 is Cladosporium uredinicola, 554 is Chaetomium globosum, 58 is Epicoccum nigrum, and control is no endophyte treatment.
Example 17
Identification of Fungal Endophytes with at Least 97% Identity to Those in Table 1
All known fungal endophytes with 97% identity to SEQ ID NO:7 through SEQ ID NO:77 were identified and are listed here by accession number: FJ425672,AY526296,JQ760047,UDB014465,KC662098,HQ649874,JQ764783,EU881906,K F251285,JQ862870,AB019364,AB594796,JF773666,JN034678,KC343142,EU707899,AB62 7855,GU138704,JN695549,DQ279491,HM776417,AB361643,DQ782839,AF222826,EU682 199,DQ782833,EU054429,FJ025275,AY354239,AF222828,GU721921,GU721920,DQ09371 5,AJ309335,FR774125,JQ747741,EF042603,KC968942,HE584924,AY740158,FJ645268,H Q692590,GQ203786,AY233867,HE579398,AB777497,KF435523,DQ420778,JQ649365,AJ2 71430,GQ996183,EF070423,FJ172277,AF483612,JX675127,EF070420,EF070421,AB74159 7,JN225408,DQ019364,KF251279,EF194151,EU977196,JX981477,EU686115,JX021531,FJ 527863,AJ302451,AJ302455,JN975370,EU754952,AF284388,KF296855,AF502785,JX3172 07,AF502781,DQ278915,EU686867,KC179120,HM991270,AF284384,DQ632670,JQ75980 6,JQ747685,EU885302,GU721781,EF434047,EF505854,JQ666587,JQ619887,GQ919270,K F531831,AB627854,DQ914679,DQ914681,HQ599592,DQ279490,DQ660336,JX069862,AB 607957,HE820869,FJ859345,JX966567,GU910230,AB627850,JX144030,DQ914723,HM59 5556,KC771473,DQ849310,EU179868,KF312152,JN890447,JX042854,EU554174,JN19851 8,HM992813,JQ845947,KF251310,JQ758707,AM930536,KF296912,JN865204,JN943512,G Q921743,EU245000,EU977304,EU144787,HE579322,HE579402,GU910171,HE792919,KC 960885,DQ485941,JN604449,HQ607913,AF502620,DQ468027,JX944132,JN207338,JQ922 240,JN207336,JX559559,JN207330,JN207333,HE820882,JX969625,HQ339994,JF744950,H E584937,JN120351,JX298885,DQ872671,AJ877102,JQ081564,DQ019391,AF071342,EF10 4180,JQ759755,GU827492,JN418769,GU324757,JX984750,JX256420,KF436271,JX205162,JN712450,KF435911,GU367905,JX416919,KC315933,JQ736648,AY904051,AF404126,FJ4 66722,HE584965,JN890282,HE584966,HQ166312,KC305124,HE977536,KC305128,AY907 040,JF710504,AF483609,AJ302460,AJ302461,AJ302462,AY969615,EU685981,U75615,AJ 302468,FJ210503,GU237860,JX960591,JX143632,HM044649,EU164404,HE584824,HQ116 406,DQ156342,JX416911,U75617,GU721359,KC427041,EU254839,JX262800,KC179307,HQ107993,KF361474,GU721420,HM053659,EF619702,EU686156,HE820839,HQ634617,G U721810,AB277211,AJ302417,KC315945,JQ002571,AM237457,AF009805,JX489795,EU6 80554,KC507199,FJ236723,HQ692618,JN846717,JX944160,JQ585672,KF435573,EU52059 0,HM581946,DQ250382,JX243908,KC343184,KC485454,GQ479695,GU237760,KF147147,EF619849,GU237767,GU237766,AB818997,AF502847,EU683672,KF225801,KC965743,AJ 488254,DQ825983,JN031007,DQ825985,KF028765,AB818999,HQ238268,EU685984,KC96 6180,HE998711,HQ533007,AM113729,KF251637,FN394692,KF435172,JN207307,JQ8143 05,HM770988,KC145175,AB511813,EU552102,AJ309344,EU645686,JQ936328,JN038492,DQ875349,EU977228,JQ814357,KF040480,JX317350,DQ401548,DQ318195,DQ318194,G U721776,KF193449,AF178544,AM262354,AB540567,AY627787,HE792907,HE579333,EU 445372,AF362069,GU973687,HM053663,AB374284,DQ062977,GU237797,JQ760783,EF0 29240,HM751829,FM200445,AY953383,AY233922,JF742784,HM626650,FN610871,JX15 5902,JX006065,AB566289,AF163078,AY344976,AB566287,AF282089,AY251441,AF3956 93,JQ761899,AJ315835,HQ187633,KC287233,AJ315831,HE820745,JN418779,M13906,JQ7 61896,AJ315838,AY536373,HQ328035,JX838793,JQ758986,HQ166357,JN163855,KC9655 95,JN545789,JN545788,GU944558,HE579247,KF296900,EF377335,KC965954,GU269703,AB095511,EF419913,DQ993641,AB325678,HQ223035,AY513945,FJ197013,FR799277,H M071900,JN207293,FJ025268,JQ758966,GU138733,GU138730,DQ267595,GQ919269,JF77 0450,GU138734,DQ279488,DQ279486,DQ242472,EU164804,EF104177,GU366726,KF212 243,DQ923534,GU079598,JX987761,JX984765,AY585343,JQ769260,GU721919,DQ92353 8,EU686756,EU040222,U75626,GU004264,EU686753,JQ765651,JX270629,JN943408,EF0 42604,AJ271588,HE579386,GQ479556,JQ759962,JX317413,EU516867,DQ780361,JQ9056 44,HQ649792,JQ247355,FN386296,AY004778,DQ102374,KF251383,GU237835,DQ38364 2,FN868479,GU237814,KC343032,JN943394,HQ450001,KC800573,AB217793,GQ851883,EU330630,JF309198,AY489281,GU325687,JX399008,AB164703,EF159407,AJ302429,UD B008141,UDB008140,FM200496,AJ302426,AJ302422,AJ302423,JQ683725,KF193481,JQ6 83727,HE792931,AB220252,FJ013057,DQ286207,JQ759811,JF414842,JX088707,JN41575 4,AY787715,JX559577,KC776206,GU166440,KC460867,FJ515595,KF056850,DQ118964,KC806227,KC631802,EU823315,AY528970,HQ116401,JX317516,KF251313,KC800565,A F502705,AF502810,JQ747697,AY527407,EU680518,AJ621773,AB374285,HQ832827,GU1 74316,DQ974750,JN198507,JF749806,JQ782739,HQ023202,AY616234,KC965315,AB7437 81,EU554161,KC507201,HM036624,EF464164,JX391942,AB743995,FJ415474,AY647237,KC965503,AB540553,HQ377280,JX898571,JN969419,DQ166962,HM123519,GU237881,A B683953,AY681487,EU498738,EU687037,AB540550,EF394866,AY853245,EU680532,HQ 450006,AM292674,KF435452,AF502638,JN890354,JX256427,JF773646,KC916704,FJ3470 31,JN572154,AF443850,AY273300,JQ247392,JQ247393,HQ316569,GU324760,AB120858,JF440978,HQ115719,JF440976,DQ124120,HQ022342,AF333138,AB255293,GQ999456,DQ 286209,HE820785,AF451751,JN038479,JQ044421,JQ044422,KC968911,KC492447,FM172 902,AF437754,HM030631,HM595545,AY510424,JX414184,HQ184179,AB588822,JQ8138 16,JQ813817,FJ025255,AY745019,EU668292,HM216214,AF427105,EU479799,JQ769257,HM484866,EU301059,EU564808,AY265329,HQ701737,KC677889,AY907030,GU721349,AY304513,GU062277,AY907037,HM484859,AB576865,JX090109,UDB004179,JN692542,JQ327868,AY756490,JN890185,JX042994,FJ613832,AF009815,HQ332534,AF009816,EU6 86781,DQ520639,KC247154,HE820841,HE820847,JN717228,JX944174,GU721348,AB444 657,KF435560,JQ585546,JQ775577,UDB004443,JF744968,KF192823,JN102440,AM50405 8,JX164074,GU907781,HQ889707,FJ612980,KF251355,AF502854,AF350291,HQ649989,G U966521,FJ481149,AY916491,AB444663,FR799197,KC691458,HE820786,JN802324,AF14 9926,AY372686,AY233908,HQ631033,UDB004677,KF251596,EU479757,GU079602,KC6 91456,DQ420883,DQ914680,DQ914683,KC305134,JN207313,AB512307,JN807326,GQ395 365,JN207256,FJ425678,AB000932,JN207252,KF293814,GU138728,AY160210,UDB01500 6,KC565735,FJ524302,AF404127,EU272486,JF796251,JF439458,AY304511,KC592278,JX 143583,JF440977,EU686925,JX982370,EU687082,JX966607,GU222370,JN687988,JN0067 71,JX436806,JQ936201,KF481950,AF178551,KC181937,JX144778,DQ790541,JF796076,J X898576,JX418352,AF097902,FJ411320,AF309617,FR863589,HM469970,AF163069,KF58 2795,AB566293,HE820790,GQ267191,JX130356,JN049828,HM060596,KF436001,GQ9192 83,HQ832834,JN049822,EU041786,AB594789,HE579259,HE584944,GU004268,GU237770,GQ921765,HE579253,KC305158,AF043599,GQ267190,AY344968,JN601031,JN969420,G U328624,AB540507,AM691002,JN102384,EU480019,JN545815,DQ993651,JX130360,JX3 98990,AY969704,KF251559,AF395695,HQ449993,U94714,KF435968,JX966550,AB85976 2,JF749808,U94713,GU981750,AF177152,FJ430599,JQ647433,GU981756,GU981757,EF10 4164,JN802311,GQ266146,HQ445083,JX155909,KF436256,DQ318204,GU078649,JN8901 15,DQ386141,GQ999487,EU686744,FJ426983,UDB013022,FN435799,EF600976,HM59601 2,JF825143,AM711381,EU816668,AJ972833,JQ905735,AF004686,EU266103,EU266107,H Q166334,EF679384,UDB004580,AM691001,JX399012,KC460880,JX982437,AB482221,A M292048,KF251253,AF350308,JF502446,JQ905803,KC179320,KF251393,GU053815,DQ3 23686,DQ323681,KC343119,HE820747,KF251529,DQ676536,U17215,DQ278919,EU4899 50,FR668016,GU903287,AJ302439,AJ302438,AJ302435,AJ302434,JN807325,AB741584,K C790941,DQ394387,FJ403513,GQ461566,KF193491,KC305164,AF502895,GU237707,EU9 77520,AB247177,AB482220,AY929321,GU004278,AB247171,GU461294,GU461295,JX12 3570,AY684241,EU686968,JX944143,JN871718,JQ796813,HQ829122,KF435590,KC80623 1,JX414183,GU944858,AF502733,JN662314,HQ022970,AY510418,KC623569,KC216145,KF129059,DQ279515,KF251526,JN192379,JN192376,HM140630,DQ006928,AF011289,E U089663,FJ825373,DQ307292,JN890424,KF155521,AB670714,GQ927271,AB670717,AB6 70711,AB670713,KF435279,GU053814,KF435375,JX414188,AF033422,GU225946,EU520 610,JF773645,KC595884,KC965570,DQ812921,EU885299,DQ078757,FJ612618,KF018920,JX077035,EU686911,JX270567,HE579352,EU885297,FJ418185,DQ914724,HQ608112,HQ 450016,GU174399,JN890327,HM999913,GU079580,HE584936,JQ765675,GU726947,JQ76 5670,HM588120,AY969986,JN120335,JQ247384,HQ891112,JQ769297,JN207242,EU00288 8,EU479803,AY365468,AF163083,DQ534482,KC146356,KF436052,AF416460,JX537970,J X156018,AY907035,GQ241278,AF409972,JQ388941,FR668022,EU687151,DQ468026,AY 251418,AB508842,AB508840,DQ233665,GU721949,AJ302444,JF927155,AJ302442,GU721 976,AJ302440,KC790931,UDB004433,GU328539,EU479791,HQ649905,KC797566,JQ7539 68,GU721449,HQ701742,AY613410,GU062246,AY907045,HM991267,DQ979608,JQ7818 40,GU721442,EU426553,DQ980024,HQ634638,AF222836,GU222372,AY969338,EF10415 8,AY431101,JQ081415,FJ649318,AY152583,JN943058,EU885294,HQ231255,FJ179477,EU 304350,KC005785,FR799224,EF070422,HQ533789,AJ289870,KF025952,HQ611347,DQ48 5934,KC989106,JQ081921,HE820871,AF404125,JN603182,KF436170,HQ832964,DQ1850 74,KC216108,JN102460,GU553324,DQ318207,HQ589260,AB819001,AY699669,EU81250 1,AB819004,HQ436065,KC013976,KF251204,KF435307,AF249905,EF029217,EF029216,F J708614,EF029198,JQ517314,GU199416,HM180398,EU479748,GU721599,DQ185081,EF1 04175,JX021528,KF251430,AY611071,AY329221,JN207241,HM235963,JN890375,JF5060 92,KF193461,KF453551,HM123501,HM051074,AB255269,HQ904082,KF193500,FN56203 8,GU721911,EF417805,KF193504,KF028766,HE579312,EF433991,KF144910,KF144911,F M200450,AF163090,AB444665,AB444664,HQ649964,AB444666,AB444661,AY528998,D Q525492,KC870889,EF543844,GU073125,AY684240,JN163853,EU680538,AF395694,KC1 79102,KC778197,JN102425,DQ520638,EU244997,GU994552,DQ279527,KC179418,EF495 164,AY999117,JX860441,JQ793663,DQ836775,EU479964,AY772736,AJ875343,KC01397 2,AJ875346,AY208785,HE614864,HF570009,KF435344,KC148376,EF641857,JX625368,A B512308,KC305146,AY266384,KC662096,HE579269,GU004277,GU004276,EF504668,EU 687114,GU004272,GU004271,EU516731,KC213751,JN102394,HQ654776,JQ862729,EU68 7052,JX868653,FJ172294,JX130355,HE584891,FJ427063,GQ996174,FN252438,AJ633598,JX398987,EU245009,HM069466,FJ859344,JN942165,FJ785433,EF504592,HQ449989,HQ4 49988,JN120346,JX868648,EF600969,HQ529711,JN383815,KF003112,JN890192,GU98174 8,EU715654,EF535663,GU328634,UDB004320,GQ999475,FR731421,GU322457,EF550969,GU322450,FJ477838,KC305130,AJ247519,JQ026214,AJ972825,KC305135,EU520614,EU 338415,JQ747670,EU040241,HE584979,KF477240,HM162095,AB746179,KC963934,AY9 06949,JN975339,EU520120,HM071902,JX399005,EU828350,JX399006,EF070418,FJ02523 1,EF070415,JN859327,JQ517292,JX399009,KF297004,JN618372,AY233888,EU784271,A M292673,EU514295,GQ921804,GU595027,HM008727,GU174426,JN673038,AF442801,E U686126,JF440982,EU754960,GQ154505,GU055711,FJ175159,KC354573,DQ993639,JQ62 1881,JN102454,AY177233,FJ013071,AY566992,GQ120971,EF408555,JX317505,AF52490 5,FJ887922,AF264905,AF264906,HM997113,EF619857,KC537805,KC537804,FJ887928,A B255303,HQ671302,FJ210537,FN386267,HQ649813,GU083033,KF251334,GU721297,KC1 81926,DQ832329,JQ781696,KF251233,KF251234,GQ505688,AJ437294,AJ437295,EF6793 63,HE820831,FN868450,GU174305,AY428866,AY956759,JQ759940,DQ489291,AJ271418,AY157952,EU784408,FJ427055,EF419900,FN813731,FJ427059,KF435462,JQ860113,KF20 9290,JF439437,KC565714,FJ228189,AF377282,JQ814364,HM991266,EF458676,AY76204 6,JN048884,HQ896484,HE579345,AB444659,EU076958,HQ402674,AF540504,AM922204,EU479758,JN943840,JN943841,FJ427025,KC584194,AF502754,FJ418192,KC343004,AB5 24806,AJ877224,DQ394377,FJ427028,AF282090,GQ927270,EU178738,DQ059579,EF5356 99,KF040479,AF163085,JX256429,AY999125,KF477238,KC513506,GQ999534,GU237837,EU002898,HM164732,AF443193,AJ315828,AJ315829,AY586560,JX868722,EU686847,D Q875350,DQ421277,AM176740,JX280875,AM691003,KF302463,GQ921786,KC965801,A M691004,EF452446,EU040235,KC662103,KC662102,AY251073,DQ993637,AY489282,FJ 151434,JQ936199,EF505495,JN163856,JN659510,EF452449,EF504607.1,GQ516009.1,GQ5 08761.1,KC800847.1,JX187590.1,GQ508832.1,KC800841.1,KC800840.1,EF504876.1,HQ54 0685.1,EF505180.1,AY842353.1,GU014821.1,FJ761203.1,GQ510033.1,EF504642.1,GU014 822.1,AY998786.1,AB581046.1,EF452470.1,FJ907534.1,EF504721.1,Y08744.2,FJ757587.1,GU014820.1,AF400896.1,KC800831.1,EF505804.1,EF505121.1,JX187587.1,KC800858.1,G Q866210.1,GQ522120.1,Y10748.1,EF504853.1,EF452471.1,KJ834329.1,AB581446.1,JX187 588.1,AF163061.1,AB632670.1,Y08746.1,EF505082.1,JX187589.1,EF504723.1,AF400889.1,KC800835.1, and EF505282.1.
Example 18
Endophytes and Combination Thereof
The protocols as described in Examples 1-16 are used in connection with the endophytes of Table 1 to confirm beneficial properties on plant health, such as yield and/or past resistance, for example. In particular, endophytes from Table 1 are employed in a synthetic combination with a plant as described herein with crop plants, such as cotton. Any single or combination of endophytes listed in Table 1 can also be used in this manner, employing for example seed coatings or foliar, soil, or rhizosphere applications. A seed composition may comprise seeds and any combination of endophytes listed in Table 1. Endophytes listed in Table 1 or combinations thereof are thus employed in methods for preventing pest infestation, increased yield, treating a pest infestation, manufacturing pest-resistant seeds; or increasing a yield or reducing loss of a crop according to the methods of Examples 1-15.

## Claims

1. A method for improving a trait in a cotton plant, the method comprising:
contacting a seed of said cotton plant with a formulation comprising purified filamentous, spore-forming, facultative fungal endophytes of at least one species,
wherein the facultative fungal endophytes are Dothideomycetes capable of producing substances that are beneficial to plants or detrimental to pests or both,
and are present in the formulation in an amount effective to decrease colonization frequencies of endophytes of genus Alternaria native to the cotton plant and to provide a benefit to the cotton plant compared to a cotton plant grown from a seed untreated with the Dothideomycetes facultative fungal endophytes,
wherein the benefit is selected from the group consisting of reduced nematode reproduction, reduced insect infestation, increased boll retention, increased yield, reduction of yield loss, increased growth, modulated phytohormone, enhanced resistance to drought stress, enhanced resistance to cold stress, enhanced resistance to heat stress, enhanced resistance to nutrient deficiency, enhanced resistance to salt toxicity, enhanced resistance to aluminum toxicity, enhanced resistance to grazing by herbivores, enhanced resistance to nematode infection, enhanced resistance to fungal infection, enhanced resistance to bacterial infection, enhanced resistance to viral infection and pest reduction.

2. The method of claim 1, wherein the facultative fungal endophyte is native to the cotton plant.

3. The method of claim 1, wherein the benefit is reduced nematode production.

4. The method of claim 1, wherein the benefit is reduced insect infestation.

5. The method of claim 1, wherein the benefit is increased boll retention.

6. The method of claim 1, wherein the benefit is increased yield by about 5%.

7. The method of claim 1, wherein the benefit is a reduction of yield loss by more than 5%.

8. The method of claim 1, wherein the benefit is enhanced resistance to drought stress.

9. The method of claim 1, wherein the formulation contains at least 100 (10^2) spores/ml or 100,000 (10^5) spores/g dry weight of the facultative fungal endophytes.

10. The method of claim 1, wherein the benefit is pest reduction.

11. The method of claim 10, wherein the pest is selected from the group consisting of a root knot nematode, an aphid, a lygus bug, a stink bug, or combinations thereof.

12. The method of claim 1, wherein the formulation comprises at least 2 species of facultative fungal endophytes.

13. The method of claim 1, wherein the formulation contains at least 1000 (10^3) spores/ml or 1,000,000 (10^6) spores/g dry weight of the facultative fungal endophytes.

14. The method of claim 1, wherein the formulation contains at least 1,000,000 (10^6) spores/ml of the facultative fungal endophytes.

15. The method of claim 6, wherein yield is increased by about 10%.

16. The method of claim 7, wherein yield loss is decreased by about 10%.

17. The method of claim 1, wherein the formulation contains at least 100 (10^2) spores/ml or 100,000 (10^5) spores/g dry weight of the facultative fungal endophytes and yield is increased by about 5%.

18. A synthetic combination of a cotton seed and purified filamentous, spore-forming facultative fungal endophytes of at least one species,
wherein the facultative fungal endophytes are Dothideomycetes capable of producing substances that are beneficial to plants or detrimental to pests or both
and are present in the synthetic combination in an amount effective to decrease the colonization frequencies of genus Alternaria native to the cotton plant grown from the seed and to provide a benefit to the cotton plant compared to a cotton plant grown from a seed untreated with the Dothideomycetes facultative fungal endophytes
wherein the benefit is selected from the group consisting of reduced nematode reproduction, reduced insect infestation, increased boll retention, increased yield, reduction of yield loss, increased growth, modulated phytohormone, enhanced resistance to drought stress, enhanced resistance to cold stress, enhanced resistance to heat stress, enhanced resistance to nutrient deficiency, enhanced resistance to salt toxicity, enhanced resistance to aluminum toxicity, enhanced resistance to grazing by herbivores, enhanced resistance to nematode infection, enhanced resistance to fungal infection, enhanced resistance to bacterial infection, enhanced resistance to viral infection and pest reduction.

19. The synthetic combination of claim 18, wherein the facultative fungal endophytes are in spore form.

20. The synthetic combination of claim 18, wherein the benefit is reduced nematode production.

21. The synthetic combination of claim 18, wherein the benefit is enhanced resistance to drought stress.

22. The synthetic combination of claim 18, wherein the benefit is reduced insect infestation.

23. The synthetic combination of claim 18, comprising at least 2 species of facultative endophytes.

24. The synthetic combination of claim 18, wherein the benefit is increased yield by about 5%.

25. The synthetic combination of claim 18, wherein the benefit is a reduction of yield loss by more than 5%.

26. The synthetic combination of claim 18, wherein the facultative fungal endophyte is native to the cotton plant grown from the seed.

27. The synthetic combination of claim 18, wherein the facultative fungal endophytes are present at a concentration of at least 100 (10^2) spores/seed on the surface of the seed.

28. The synthetic combination of claim 18, wherein the facultative fungal endophytes are present at a concentration of at least 1,000 (10^3) spores/seed on the surface of the seed.

29. The synthetic combination of claim 18, wherein the facultative fungal endophytes are present at a concentration of at least 10,000 (10^4) spores/seed on the surface of the seed.

30. The synthetic combination of claim 24, wherein yield is increased by about 10%.

31. The synthetic combination of claim 25, wherein yield loss is reduced by about 10%.

32. The synthetic combination of claim 18, wherein the benefit is increased boll retention.

33. The synthetic combination of claim 18, wherein the benefit is pest reduction.

34. The synthetic combination of claim 33, wherein the pest is selected from the group consisting of a root knot nematode, an aphid, a lygus bug, and a stink bug.

35. The synthetic combination of claim 18, wherein the facultative fungal endophytes are present at a concentration of at least 100 (10^2) spores/seed on the surface of the seed and the yield is increased by about 5%.