US 20160286821A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0286821 A1 SWORD (43) Pub. Date: Oct. 6, 2016

(54) FUNGAL ENDOPHYTES FOR IMPROVED Publication Classification CROPYELDS AND PROTECTION FROM PESTS (51) Int. Cl. AOIN 63/04 (2006.01) (71) Applicant: THE TEXAS A & M UNIVERSITY (52) U.S. Cl. SYSTEM, College Station, TX (US) CPC ...... A0IN 63/04 (2013.01) (72) Inventor: Gregory A. SWORD, College Station, TX (US) (57) ABSTRACT (21) Appl. No.: 15/034,862 (22) PCT Filed: Nov. 6, 2014 The invention provides a synthetic combination of a crop and at least one fungal endophyte, wherein the crop is a host (86). PCT No.: PCT/US1.4/64411 plant of the endophyte. Provided are also methods and S 371 (c)(1), compositions for producing Such synthetic combinations. (2) Date: May 5, 2016 The endophyte reproduces and enhances the agronomic characteristics of the crop. Methods for inoculating the host Related U.S. Application Data plant with the endophyte, for propagating the host-endo (60) Provisional application No. 61/900,935, filed on Nov. phyte combination, and for detecting the presence of the 6, 2013, provisional application No. 61/900,929, filed endophyte and of its metabolites within a host plant are also on Nov. 6, 2013. described. Patent Application Publication Oct. 6, 2016 Sheet 1 of 25 US 2016/0286821 A1

FIG. 1

Colonization efficiencies Seedlings (cotyledons) 14 days after planting Tissues sampled: leaves, stems, roots

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control control Conidia concentration Conidia concentration Patent Application Publication Oct. 6, 2016 Sheet 3 of 25 US 2016/0286821 A1

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Endophytic Chaetonium globosun negatively affects root-knot nematode reproduction. Major reductions in galls and egg production independent of concentration.

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two replicate trials with very similar results insect trials ongoing Candidate for 2014 field trials Six more genetically-distinct C. globulosun isolates still to be tested Patent Application Publication Oct. 6, 2016 Sheet 4 of 25 US 2016/0286821 A1

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(both comparisons: P & 0.0001, Wilcoxon Signed Rank Test) Patent Application Publication Oct. 6, 2016 Sheet 7 of 25 US 2016/0286821 A1

FIGs. 7A, 7B

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2013 field trial - Boll retention s Aug. 9, 2013 P0.004 Fungicide seed treatments: Metalaxyl (systemic) Triadimenol (systemic) -- Trifloxystrobin o 2-(Thiocyanome e thylthio) o benzothioazole d o s insecticide seed . L treatents Thiodicarb limidacloprid Chloropyrifos

Commercial FM 1740B2F seeds planted May 21st Sprayed with endophyte spores at 5th true leaf stage Patent Application Publication Oct. 6, 2016 Sheet 10 of 25 US 2016/0286821 A1

FIG 10

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FIG 11

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25% higher yields in endophyte treated cotton plants. Patent Application Publication Oct. 6, 2016 Sheet 12 of 25 US 2016/0286821 A1

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US 2016/0286821 A1 Oct. 6, 2016

FUNGAL ENDOPHYTES FOR IMPROVED 0006. In another aspect, the invention provides methods CROPYELDS AND PROTECTION FROM for providing a benefit to an agricultural plant comprising PESTS treating said plant, the seed of said plant, or the rhizosphere of said plant or seed with a composition comprising purified CROSS REFERENCE TO RELATED facultative fungal endophytes and an agriculturally-accept APPLICATIONS able carrier, wherein the endophyte is capable of at least one 0001. This application claims priority to U.S. Provisional of reducing pest reproduction, killing pests, and deterring Patent Application Nos. 61/900,929 and 61/900,935, both pests, and wherein the endophyte is present in the compo filed Nov. 6, 2013, which are herein incorporated by refer sition in an amount effective to provide a benefit to the seeds ence in their entirety. or the agricultural plants derived from the seeds. 0007. In yet another aspect, the invention provides meth INCORPORATION OF SEQUENCE LISTING ods for providing a benefit to an agricultural plant, com prising obtaining a synthetic combination of an agricultural 0002 The sequence listing that is contained in the file plant seed and a purified facultative fungal endophyte, named TAMC027WO ST25.txt, which is 33 kilobytes as wherein the endophyte is capable of at least one of reducing measured in Microsoft Windows operating system and was pest reproduction, killing pests, and deterring pests, and created on Nov. 6, 2014, is filed electronically herewith and wherein the endophyte is present in the synthetic combina incorporated herein by reference. tion in an amount effective to provide a benefit to the seeds FIELD OF THE INVENTION or the agricultural plants derived from the seeds. 0008. In another embodiments, methods of producing a 0003. The present invention relates to fungal endophytes plant with a non-naturally occurring ratio of endophytes is of agricultural crops for improving yield and/or for protec provided, where the methods comprise contacting an agri tion from pests. cultural seed of the plant with a formulation comprising facultative fungal endophytes of at least one species, DESCRIPTION OF RELATED ART wherein endophytes are present in the formulation in an 0004 Fungal endophytes are fungi that internally colo amount effective to modulate the colonization frequencies of nize plant tissues without causing evident damage or dis the endophytes that are native to the agricultural plant grown ease. Particular fungal endophytes, such as mycorrhiza, from the seed compared to a reference seed that is planted Survive within various host plant tissues, often colonizing in an agricultural environment, wherein the plant with the the intercellular spaces of host leaves, stems, flowers or non-naturally occurring ratio of endophytes has an improved roots. The symbiotic endophyte-host relationships can pro trait as compared to a plant with a naturally-occurring ratio. vide several fitness benefits to the host plant, such as In a further aspect, the facultative fungal endophytes are enhancement of nutrition, and/or increased drought toler capable of producing Substances that are beneficial to plants ance. Root-colonizing mycorrhizae Survive on photosyn or detrimental to pests or both. thetic carbohydrates from the plant, and in return, aid in the 0009. In another aspect, the invention provides methods solubilization and uptake of water and minerals to the host, for altering the systemic defensive pathway in a plant which can lead to the promotion of seed germination and comprising contacting an agricultural seed of said plant with plant growth. Additionally, the association of a fungal endo a formulation comprising a purified facultative fungal endo phyte with a host plant can provide tolerance to a variety of phytes of at least one species, wherein the endophytes are biotic and abiotic stresses. Host growth, fitness promotion capable of producing Substances that are beneficial to plants and protection are thought to be achieved through multiple or detrimental to pests or both, and wherein the endophyte beneficial properties of the endophyte-host association. For is present in the synthetic combination in an amount effec instance, the endophytic organisms may produce growth tive to modulate the level of at least one phytohormone regulating Substances to induce biomass production and within an agricultural plant grown from the plant seed, and alkaloids or other metabolites. Additionally, fungal endo to provide a benefit to the seeds or the agricultural plants phytes may directly Suppress or compete with disease grown from the seeds. In a further aspect, the facultative causing microbes, protecting the plant from potential patho fungal endophytes are capable of producing Substances that genS. are beneficial to plants or detrimental to pests or both. 0010. In other embodiments, the invention provides SUMMARY OF THE INVENTION methods of modulating the colonization frequencies of 0005. In one aspect, the invention provides methods for endophytes that are native to the agricultural plant grown improving a trait in an agricultural plant comprising con from the seed compared to a reference seed that is planted tacting an agricultural seed of said plant with a formulation in an agricultural environment, comprising contacting the comprising a purified facultative fungal endophytes of at seed of the agricultural plant with a formulation comprising least one species, wherein the endophytes are capable of facultative fungal endophytes of at least one species, and producing Substances that are beneficial to plants or detri wherein endophytes are present in the formulation in an mental to pests or both, and wherein the endophytes are amount effective to modulate the colonization frequencies of present in the formulation in an amount effective to modu native endophytes and to provide a benefit to the seeds or the late the colonization frequencies of the endophytes that are agricultural plants grown from the seeds. In certain aspects, native to the agricultural plant grown from the seed com the native endophytes are of genus Alternaria. In a further pared to a reference seed that is planted in an agricultural aspect, the facultative fungal endophytes are capable of environment, and to provide a benefit to the seeds or the producing Substances that are beneficial to plants or detri agricultural plants grown from the seeds. mental to pests or both. US 2016/0286821 A1 Oct. 6, 2016

0011. In another aspect, the invention provides methods Zation frequencies of endophytes of genus Alternaria that for altering the systemic defensive pathway in a plant are native to the agricultural plant grown from the seed comprising contacting an agricultural seed of said plant with compared to a reference seed that is planted in an agricul a formulation comprising a purified facultative fungal endo tural environment. phytes of at least one species, and wherein the endophyte is 0016. In a further aspect for certain of these methods and present in the synthetic combination in an amount effective synthetic combinations, the composition comprising puri to modulate the level of at least one phytohormone within an fied facultative fungal endophytes also comprises an agri agricultural plant grown from the plant seed, and to provide culturally acceptable carrier. a benefit to the seeds or the agricultural plants grown from 0017. In a further aspect for certain of these methods and the seeds. In a further aspect, the facultative fungal endo synthetic combinations, the facultative fungal endophyte phytes are capable of producing Substances that are benefi may be a filamentous fungal endophyte. In other embodi cial to plants or detrimental to pests or both. ments, the facultative endophyte may be spore-forming. In 0012. In yet another aspect, the invention provides meth yet other embodiments, the facultative fungal endophyte ods of producing a plant with a network of fungal endo may be a septate fungal endophyte. In yet other embodi phytes that comprises endophytes of the genus Alternaria, ments, the facultative fungal endophyte may be a dark comprising (a) contacting the seed of an agricultural plant septate fungal endophyte. In some embodiments, the facul with a formulation comprising facultative fungal endophytes tative endophyte may be an entomopathogen. In some of at least one non-Alternaria species, wherein endophytes embodiments, the facultative fungal endophyte may belong are present in the formulation in an amount effective to to the phylum Ascomycota or Basidiomycota. In a further provide a benefit to the seeds or the agricultural plants grown aspect, the facultative fungal endophyte may belong to from the seeds, and wherein the plant grown from the seed Subphylum Pezizomycotina, Agaricomycotina, or Ustilag comprises endophytes of the genus Alternaria. In a further inomycotina. In yet another aspect, facultative fungal endo aspect, the facultative fungal endophytes are capable of phyte may belong to class Sordariomycetes, Dothideomy producing Substances that are beneficial to plants or detri cetes, Agaricomycetes, Ustilaginomycetes, Orbilliomycetes, mental to pests or both. or Eurotiomycetes. In yet another aspect, the facultative 0013 Also provided herein are synthetic combinations of fungal endophyte may belong to order Hypocreales, Pleo an agricultural plant seed and a composition comprising sporales, Capnodiales, Sordariales, Polyporales, Diaportha purified entomopathogenic fungal endophytes of at least one les, Ustilaginales, Xylariales, Orbiliales, Trichosphaeriales, species, wherein the endophytes are capable of (1) coloniz or Eurotiales. ing the agricultural plant grown from the plant seed (2) and 0018. In a further aspect, the facultative fungal endophyte at least one of reducing pest reproduction, killing pests, and may be a species from Table 1, namely Acremonium alter deterring pests, from within the agricultural plant; wherein natum, Alternaria alternata, Alternaria brassicae, Alter the endophytes are not of species Beauveria bassiana, and naria compacta, Alternaria dianthi, Alternaria longipes, wherein the endophyte is present in the synthetic combina Alternaria mali, Alternaria sesami, Alternaria Solani, Alter tion in an amount effective to provide a benefit other than naria sp., Alternaria tenuissima, Ascomycota sp., Bipolaris enhanced resistance to biotic stress to the seeds or the spicifera, Cercospora canescens, Cercospora capsici, Cer agricultural plants derived from the seeds when the seeds or cospora kikuchii, Cercospora Zinnia, Chaetonium globo plants are grown in an agricultural setting. sum, Chaetomium piluliferum, Chaetomium sp., Cladospo 0014. In yet another aspect, the invention provides syn rium cladosporioides, Cladosporium sp., Cladosporium thetic combinations of an agricultural plant seed and a uredlinicola, Cochliobolus sp., Phanerochaete crassa, composition comprising purified facultative fungal endo Phoma americana, Phoma subherbarum, Phomopsis liq phytes of at least one species, wherein the endophyte is uidambari, Phomopsis sp., Pleospora sp., Pleosporaceae present in the synthetic combination in an amount effective sp., Polyporales sp., Preussia africana, Preussia sp., to modulate the level of at least one phytohormone within an Pseudozyma sp., Pyrenophora teres, Colletotrichuncapsici, agricultural plant grown from the plant seed, and to provide Coniolariella gamsii, Coniothyrium aleuritis, Coniothyrium a benefit to the seeds or the agricultural plants grown from sp., Corynespora cassicola, Diaporthe sp., Diatrype sp., the seeds. In a further aspect, the facultative fungal endo Drechslerella dactyloides, Embellisia indefessa, Epicoccum phytes are capable of producing Substances that are benefi nigrum, Epicoccum sp., Exserohilum rostratum, Fusarium cial to plants or detrimental to pests or both. chlamydosporum, Fusarium sp., Gibellulopsis nigrescens, 0015. In another embodiment, the invention provides Gnomoniopsis sp., Lewia infectoria, Mycosphaerella coffei synthetic combinations of an agricultural plant seed and a cola, Mycosphaerellaceae sp., Nigrospora Oryzae, composition comprising purified facultative fungal endo Nigrospora sp., Nigrospora sphaerica, Paecilomyces sp., phytes of at least one species, wherein the facultative fungal Penicillium citrinum, Retroconis sp., Rhizopycnis sp., endophytes are present in the synthetic combination in an Schizothecium inaequale, Stagonospora sp., Stemphylium amount effective to modulate the colonization frequencies of lancipes, Thielavia hyrcaniae, Thielavia sp., Ulocladium endophytes that are native to the agricultural plant grown chartarum, Verticillium sp., Beauveria bassiana, Aspergillus from the seed compared to a reference seed that is planted parasiticus, Lecanicillium lecanii, and Paecilomyces lilaci in an agricultural environment, and to provide a benefit to S. the seeds or the agricultural plants grown from the seeds. In 0019. In a further aspect, the facultative fungal endophyte a further aspect, the facultative fungal endophytes are comprises a nucleic acid that is at least 97% identical, for capable of producing Substances that are beneficial to plants example, at least 98% identical, at least 99% identical, at or detrimental to pests or both. In certain aspects, the least 99.5% identical, or 100% identical to the nucleic acids facultative fungal endophytes are present in the synthetic provided in any of SEQ ID NO:7 through SEQ ID NO:77, combination in an amount effective to modulate the coloni for example those listed in Example 16. US 2016/0286821 A1 Oct. 6, 2016

0020. In another aspect for certain of these methods is an mycetes, Dothideomycetes, Eurotiomycetes, Saccharomy additional step of packaging the contacted seeds in a con cetes, Sordariomycetes, Agaricomycetes, Microbotryomy tainer may be included. In certain aspects, the packaging cetes, Tremellomycetes. In yet another aspect, the native material may be selected from a bag, box, bin, envelope, endophytes may belong to order Capnodiales, Pleosporales, carton, or container, and may comprise a dessicant. Chaetothyriales, Eurotiales, Saccharomycetales, Diaportha 0021. In a further aspect for certain of these methods and les, Hypocreales, Ophiostomatales, Sordariales, Trichospha synthetic combinations, the benefit to the treated seed or eriales, Xylariales, Cantharellales, Corticiales, Polyporales, plant grown from the treated seed is measured at the level of Russulales, Sporidiobolales, or Tremellales. In a further the population, as compared to a reference population of aspect, the native endophytes may belong to genus Davi plants. In certain aspects, the facultative fungal endophyte diellaceae, Mycosphaerellaceae, Pleosporaceae, Didymel may be providing a benefit to a crop comprising a plurality laceae, Sporormiaceae, Chaetothyriaceae, Trichocomaceae, of agricultural plants produced from the seeds treated with Saccharomycetaceae, Gnomoniaceae, Cordycipitaceae, the endophyte. In certain aspects, the present invention Nectriaceae, Hypocreaceae, Plectosphaerellaceae, Ophios discloses a Substantially uniform population of plants pro tomataceae, Chaetomiaceae, Lasiosphaeriaceae, Trichos duced by growing the population of seeds described above. phaeriaceae, Ceratobasidiaceae, Corticiaceae, Corio In one embodiment, at least 75%, at least 80%, at least 90%, laceae, Peniophoraceae, Sporidiobolaceae, O at least 95% or more of the plants comprise in one or more Tremellaceae. In a further aspect, the endophytes that are tissues an effective amount of the endophyte or endophytes. native to the agricultural plant may be a species from Table In another embodiment, at least 10%, 20%, 30%, 40%, 50%, 2, namely Cladosporium sp., Cladosporium cladospori 60%, 70%, 75%, at least 80%, at least 90%, at least 95% or Oides, Davidiella sp., Cercospora sp., Cercospora beticola, more of the plants comprise a microbe population that is Alternaria sp., Alternaria alternata, Alternaria citri, Alter Substantially similar. naria tenuissima, Cochliobolus sp., Curvularia sp., Exsero 0022. In a further aspect for certain of these methods and hilum sp., Lewia sp., Lewia infectoria, Pyrenophora sp., synthetic combinations, the plant is grown in an agricultural Pyrenophora tritici-repentis, Pleospora sp., Phoma ameri setting or environment, including a greenhouse. In one cana, Preussia africana, Penicillium sp., Thermomyces sp., embodiment, the agricultural setting or environment com Thermomyces lanuginosus, Candida sp., Candida quercit prises at least 100 plants. In another embodiment, the rusa, Candida tropicalis, Cyberlindnera sp., Cyberlindnera population occupies at least about 100 square feet of space, jadinii, Kluyveromyces sp., Kluyveromyces marxianus, Gino wherein at least about 10%, 20%, 30%, 40%, 50%, 60%, moniopsis sp., Beauveria bassiana, Cordyceps sp., 70%, 80%, 90% or more than 90% of the population Cordyceps bassiana, Fusarium sp., Gibellulopsis nigre comprises an effective amount of the microbe. In another scens, Hypocrea sp., Hypocrea lixii, Hypocrea virens, embodiment, the population occupies at least about 100 Trichoderma sp., Trichoderma tomentosum, Verticillium sp., square feet of space, wherein at least about 10%, 20%, 30%, Ophiostoma sp., Ophiostoma dendiflundum, Chaetomium 40%, 50%, 60%, 70%, 80%, 90% or more than 90% of the sp., Chaetomium globosum, Thielavia hyrcaniae, Taifangla population comprises the microbe in reproductive tissue. In nia sp., Taifanglania inflata, Schizothecium inaequale, still another embodiment, the population occupies at least Nigrospora sp., Rhizoctonia sp., Phanerochaete sp., Tram about 100 square feet of space, wherein at least about 10%, etes sp., Trametes hirsuta, Trametes villosa, Rhodotorula 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than sp., Rhodotorula mucilaginosa, Cryptococcus sp. Crypto 90% of the population comprises at least 10 CFUs, 100 coccus Skinneri, or Tremella sp. CFUs, 1,000 CFUs, 10,000 CFUs or more of the facultative 0025. In a further aspect for certain of these methods and fungal endophyte of the invention. In yet another embodi synthetic combinations, the benefit provided by the faculta ment, the population occupies at least about 100 square feet tive fungal endophyte to the agricultural plant is an of space, wherein at least about 10%, 20%, 30%, 40%, 50%, improved agronomic property selected from the group con 60%, 70%, 80%, 90% or more than 90% of the population sisting of increased increased biomass, increased tittering, comprises the facultative fungal endophyte of the invention. increased root mass, increased flowering, increased yield, 0023. In one embodiment, at least 10%, at least 20%, at increased water use efficiency, reduction of yield loss, least 30%, at least 40%, at least 50%, at least 60%, at least altered plant height, decreased time to emergence, increased 70%, at least 75%, at least 80%, at least 90%, at least 95% seedling height, increased root length, increased chlorophyll or more of the seeds in the population, contains a viable levels, retention of developing flowers, retention of devel endophyte or endophytes disposed on the Surface of the oping fruits, altered phytohormone levels, and enhanced seeds. In a particular embodiment, at least 10%, at least resistance to environmental stress relative to a reference 20%, at least 30%, at least 40%, at least 50%, at least 60%, plant. In some aspects, the benefit provided is the alteration at least 70%, at least 75%, at least 80%, at least 90%, at least of levels of at least two phytohormones. In some aspects, the 95% or more of the seeds in the population contains at least environmental stress is selected from the group consisting of 10 CFU, for example, at least 30 CFU, at least 100 CFU, at drought stress, cold stress, heat stress, nutrient deficiency, least 300 CFU, at least 1,000 CFU, at least 3,000 CFU, at salt toxicity, aluminum toxicity, grazing by herbivores, least 10,000 CFU or more, of the endophyte or endophytes insect infestation, nematode infection, and fungal infection, coated onto the surface of the seed. bacterial infection and viral infection. In some aspects, the 0024. In a further aspect for certain of these methods and benefit to agricultural plants derived from the seed is synthetic combinations, the endophytes that are native to the increased yield in a population of said plants by about 5%, agricultural plant and whose colonization frequencies or 10%, 15%, 20%, 30%, 40%, or 45% relative to a reference ratios are altered may belong to phylum Ascomycota or population of plants. In other aspects, the benefit to agricul Basidiomycota. In yet another aspect, the endophytes that tural plants derived from the seed is a reduction of yield loss are native to the agricultural plant may be of class Leotio in a population of said plants by more than 40%, 30%, 20%, US 2016/0286821 A1 Oct. 6, 2016

10%. 5%, or 1% relative to a reference population of plants. positions described herein. In certain aspects, the method In some aspects, treatment of seeds with facultative fungal may also comprise identifying a plant or seed as in need of endophytes may decrease thrip damage, decrease fleahopper endophyte treatment. The pest may comprise, for example, damage, increase canopy temperature, increase drought tol a nematode and/or insect. In certain aspects, the pest may erance, increase above ground biomass, and increase below comprise a root knot nematode, a aphid, alygus bug, a Stink ground biomass in the plants grown from the treated seeds. bug, or combinations thereof. 0026. In a further aspect for certain of these methods and 0032. In still yet another aspect, methods for preventing synthetic combinations, the facultative fungal endophyte is pest infestation are provided comprising obtaining a seed present in the synthetic combination in an amount effective described herein and planting the seed. The method may to obtain at least 50% colonization of the leaves, stems or further comprise identifying a need of preventing pest roots of an agricultural plant grown from the seed. infestation. In certain aspects, the pest may comprise a 0027. In a further aspect for certain of these methods and nematode and/or a insect; and/or the pest may comprise a synthetic combinations, the facultative fungal endophytes root knot nematode, a aphid, a lygus bug, a stink bug, or are capable of producing Substances that are detrimental to combinations thereof. pests. In certain aspects, the pest may be a nematode and/or 0033. In a further embodiment, a method for treating a an insect, for example, a root knot nematode, a aphid, a pest infestation comprises identifying a plant Suspected of lygus bug, a stink bug, or combinations thereof. being infected with a pest, applying an above-described 0028. In a further aspect for certain of these methods and composition to the plant, whereby an endophyte-treated synthetic combinations, the synthetic combination may plant is generated. In certain aspects, the pest may comprise comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, a nematode and/or an insect; and/or the pest may comprise 15, 16, 17, 18, 19, or 20 facultative fungal endophytes. In a root knot nematode, a aphid, a lygus bug, a Stink bug, or one aspect, the invention provides a synthetic combination combinations thereof. of a cotton plant or seed and a fungal endophyte comprising 0034. In still yet another aspect, a method of manufac at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, turing pest-resistant seeds is provided comprising providing 18, 19, or 20 endophytes selected from those in Table 1, a fungal endophyte composition comprising at least 1, 2, 3, wherein the cotton or seed is a host of the endophyte. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 0029. In another aspect, a seed coating is provided com endophytes from Table 1, providing seeds; and combining prising a fungal endophyte comprising at least 1, 2, 3, 4, 5, the seeds with the endophyte composition, whereby pest 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 resistant seeds are generated. In certain aspects, the method endophytes from Table 1; and at least one sticker, wherein increases the percentage of colonization with the endophyte the fungal endophyte is in contact with the Sticker. In certain of the plant developing from the seed. aspects, the Sticker may comprise, for example, alginic acid, 0035. In still yet another aspect, methods of increasing a carrageenan, dextrin, dextran, pelgel, polyethelene glycol, yield of a crop or a reduction of loss are disclosed compris polyvinyl pyrrolidone, methyl cellulose, polyvinyl alcohol, ing providing a fungal endophyte composition comprising at gelatin, or combinations thereof. In certain aspects, the least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, Sticker may have a weight ratio between fungal endophyte 18, 19, or 20 endophytes from Table 1; and applying the and sticker of 1:1-10, 1:10-50, 1:50-100, 1:100-500, 1:500 endophyte composition to a seed, plant or part thereof, 1000, or 1:1000-5000. The seed coating may be a solid or whereby the yield of the crop increases. In certain aspects, fluid. In certain aspects, the seed coating is a powder. In the crop may be cotton, and the increase of yield may be at certain aspects, the fungal endophyte may comprise fungal least about 2%. 3% 5%, 15%, 20%, or 25% relative to a crop spores. In various aspects, the seed coating may comprise to which no endophyte composition has been applied. In about 1, 2, 5, 10, 50, 10, 10, 10, 10, 10, 107, 10, or 10 certain aspects, the increase of yield is about 2%-5%. or more colony forming units per gram or spores per gram. 3%-5%. 5%-10%, 10%-15%, or greater than about 20%, 0030. In certain embodiments, compositions for foliar or 30%, or more relative to a crop to which no endophyte soil application may comprise a fungal endophyte compris composition has been applied. In certain aspects, the crop is ing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, cotton and the increase of yield comprises reduced boll 17, 18, 19, or 20 endophytes from Table 1, and at least one damage. In certain aspects, the reduction of loss comprises carrier, Surfactant or diluent. In certain aspects, the compo reduction of loss due to insect infestation or drought, and the sitions may comprise may comprise about 1, 2, 5, 10, 50. loss is less than 50%, 40%, 30%, 20%, 10%, 5%, or 5% 10, 10, 10, 10, 10, 107, 10, or 10 or more colony relative to a crop to which no endophyte composition has forming units per gram or spores per gram. In various been applied. aspects, the composition may comprise water, a detergent, 0036) Also described herein are commodity plant prod Triton X, insecticides, fungicides, or combinations thereof, ucts comprising a plant or part of a plant (including a seed) for example. In further embodiments, seed compositions and further comprising the facultative fungal endophyte comprise a plant seed and the above-described seed coating. described above that is present in a detectable level, for In certain aspects, the plant seed comprises a cotton seed, a example, as detected by the presence of its nucleic acid by seed of an agronomically elite plant, a dicot plant seed, PCR. In another aspect, disclosed is a method of producing and/or a monocot plant seed. In certain aspects, the seed a commodity plant product, comprising obtaining a plant or composition may be resistant to a pest comprising an insect plant tissue from the synthetic combination described above, and/or a nematode. and producing the commodity plant product therefrom. The 0031. In yet another aspect, the invention provides meth commodity plant product can be produced from the seed, or ods for preventing pest infestation or increasing yield, which the plant (or a part of the plant) grown from the seed. The may comprise treating a plant, plant seed, or the rhizosphere commodity plant product can also be produced from the of said plant or seed with the endophyte containing com progeny of Such plant or plant part. The commodity plant US 2016/0286821 A1 Oct. 6, 2016 product can be is selected from the group consisting of grain, lomyces lilacinus negatively affect host plant selection flour, starch, seed oil, syrup, meal, flour, oil, film, packaging, behavior of Southern green Stink bugs when present as an nutraceutical product, an animal feed, a fish fodder, a cereal endophyte in cotton. product, a processed human-food product, a Sugar or an 0044 FIG. 8: A reduction in cotton boll damage was alcohol and protein. observed during field trials. Relative to control plants, levels of insect-related boll damage were lower among plants that BRIEF DESCRIPTION OF THE DRAWINGS were treated by Soaking seeds in spore solutions of Beau veria bassiana and Paecilomyces lilacinus at concentrations 0037 FIG. 1: The colonization efficiencies demonstrate of 10° and 10 spore/ml. that endophytes can be manipulated in the field. Depicted are 004.5 FIG.9: Foliar application of cotton in the field with the mean +/tSE endophytic colonization frequencies of spores of endophytic entomopathogenic fungi improves cotton seedlings under field conditions inoculated by seed plant performance. Cotton (variety FM1740B2F) seeds treatments with different spore concentrations of either (left) treated with a variety of typical fungicide (Metalaxyl, Tri Paecilomyces lilacinus or (right) Beauveria bassiana. adimenol, Trifloxystrobin, 2-(Thiocyanome-thylthio) benzo 0038 FIG. 2: The endophytic fungus Paecilomyces thioazole) and insecticide (Thiodicarb. Imidacloprid, Chlo lilacinus negatively affects root knot nematode (Meloid ropyrifos) seed treatments were planted and grown under ogyne incognita) reproduction when present as an endophyte field conditions. The plants were sprayed at the 5th true leaf in cotton. At high nematode inoculum levels (10,000 eggs), stage with aqueous solutions of Beauveria bassiana and the endophyte reduced egg production in plants following Paecilomyces filmosoroseus. Sucrose was included (1% treatment of seeds with solutions containing either 10° or wit/vol) as an additional nutritional resource for the fungi. 107 spores/ml when compared to untreated control seeds. At Significantly higher first position boll (developing fruit) field inoculum levels (2000 eggs), the presence of the retention was observed in plants sprayed with Beauveria endophyte significantly reduced both galls and egg produc bassiana without Sucrose and Paecilomyces fumosoroseus tion at both seed treatment concentrations. plus Sucrose. 0039 FIG. 3: Endophytic Chaetomium globosum nega 0046 FIGS. 10A, 10B: Positive effects of fungal endo tively affects root-knot nematode reproduction. Negative phytes on cotton plant performance under field conditions. effects of endophytic Chaetomium globosum on root-knot FIG. 10A demonstrates an early season trend for higher nematode gall formation and egg production following square retention in the treated versus untreated plants. FIG. cotton seed soaking treatments in solutions of 0 (untreated 10B demonstrates that significantly more bolls were retained controls), 10° and 10 spores/ml. Seedlings were inoculated in the endophyte treatment groups later in the season, with 1000 nematode eggs and grown in the greenhouse. Egg relative to control. This is demonstrated with both endophyte production by hatching nematodes that Successfully infected species used and with both seed treatment concentration the seedlings was quantified 60 days later. employed (Repeated measures ANOVA: Time, P-0.001; 0040 FIGS. 4A, 4B: The effect of endophytic fungi on Time Endophyte, P=0.045, Endophyte, P=0.003). cotton aphids (Aphis gossypii) reproduction. FIG. 4A dem 0047 FIG. 11: Positive effects of fungal endophytes on onstrates that the presence of Beauveria bassiana in cotton cotton yields under field conditions. The data demonstrate negatively affects the reproduction of cotton aphids. FIG. 4B that endophyte treatments achieved 25% higher yields in demonstrates that the presence of Paecilomyces lilacinus in treated cotton plants. cotton negatively affects the reproduction of cotton aphids. 0048 FIG. 12: Positive effects of fungal endophytes on 0041 FIG. 5: Effects of Chaetomium globosum on cotton Sorghum (a) plant height and (b) total fresh biomass under aphids. Endophytic Chaetonium globosum in cotton nega growth chamber seedling assays. Data shown is average tively affects cotton aphid population growth rates as evi plant height (cm) and total fresh biomass (g) of n=10 denced by reduced reproduction after 14 days on endophyte independent replicates. Error bars represent +1 standard colonized versus control plants. Cotton plants were grown error. All three fungal endophytes improve both traits rela from seeds treated by soaking in spore solutions of 0 tive to the untreated control. (control), 10' (low) and 108 (high) spores/ml. 0049 FIG. 13: The in-field modulation of the coloniza 0042 FIGS. 6A, 6B: The effect of the endophytic fungi tion of endogenous cotton endophytes in (a,b) stems and (c. Beauveria bassiana and Paecilomyces lilacinus on western d) roots when treated with fungal endophytes Paecilomyces tarnished plant bugs Lygus hesperus (Miridae). FIG. 6A lilacinus (a,c) and Beauveria bassiana (b. d). Data shown demonstrates that Beauveria bassiana and Paecilomyces is a percentage change in colonization relative to the corre lilacinus negatively affect host plant selection of western sponding untreated control and plant tissue. tarnished plant bugs when present as an endophyte in cotton. 0050 FIG. 14: Average percent difference in yield FIG. 6B demonstrates that Beauveria bassiana and Paeci between endophyte treated and control cotton plants (n=6 lomyces lilacinus negatively affect host plant selection replicate plots in a dryland field, College Station, Tex.) for behavior of western tarnished plant bugs when present as an 15 facultative fungal endophytes and two cotton cultivars; endophyte in cotton. Delta Pine (DP 0912B2RF) and Phytogen (PHY 499WRF). 0043 FIGS. 7A, 7B: The effect of the endophytic fungi On Delta Pine, 11 out of 15 and on Phytogen 14 out of 15 Beauveria bassiana and Paecilomyces lilacinus on Southern facultative fungal endophytes tested showed an increase in green stink bugs (Nezara viridula (Pentatomidae). FIG. 7A yield relative to untreated cotton plants. demonstrates that Beauveria bassiana and Paecilomyces 0051 FIG. 15: Aggregated average percent difference in lilacinus negatively affect host plant selection of Southern yield between endophyte treated and control cotton plants green Stink bugs when present as an endophyte in cotton. (n=6 replicate plots in a dryland field, College Station, Tex.) FIG. 7B demonstrates that Beauveria bassiana and Paeci for 15 facultative fungal endophytes and two cotton culti US 2016/0286821 A1 Oct. 6, 2016

vars; Delta Pine (DP 0912B2RF) and Phytogen (PHY DETAILED DESCRIPTION OF THE 499WRF). Bars represent a 95% confidence interval around INVENTION the mean. 0062 Endophytic fungi are ubiquitous in nature, infect 0052 FIG. 16: Average percent difference in thrip dam ing virtually all plants in both natural and agronomic eco age (A) and fleahopper damage (B) between endophyte systems. Plants commonly harbor a diversity of fungi living treated and control cotton plants. The thrip damage was within their tissues as asymptomatic endophytes that can assessed in the Delta Pine (DP 0912B2RF) cultivar (n=6 provide protection from a range of biotic and abiotic stress replicate plots in a dryland field, College Station, Tex.) for ors. The present disclosure describes certain fungal endo 15 facultative fungal endophytes. 12 out of the 15 facultative phytes that can be pathogens, parasites or antagonists to fungal endophytes tested showed a decrease in thrip damage plant pathogens, insects, and nematode pests, thereby pro relative to the untreated cotton plants. The fleahopper dam viding health and performance benefits to crop plants. The age was assessed in cotton plants of the Phytogen (PHY symbiotic endophyte-host relationships can provide several 499WRF) cultivar (n=6 replicate plots in a dryland field, general health and fitness benefits to the host plant, such as College Station, Tex.) for 15 facultative fungal endophytes. enhancement of nutrition, increased drought tolerance and/ 6 out of the 15 facultative fungal endophytes tested showed or chemical defense from potential herbivores and often an average decrease in fleahopper damage as compared to enhanced biomass production. Root-colonizing mycorrhizae untreated cotton plants. Survive on photosynthetic carbohydrates from the plant, and in return, aid in the solubilization and uptake of water and 0053 FIG. 17: Mid-season field-trait measured in June at minerals to the host, which can lead to the promotion of seed the dryland trial of (A) root length and (B) belowground germination and plant growth. Additionally, the association weight. Data presented is the average of n=10 independent of a fungal endophyte with a host plant often provides replicates and error bars represent tone standard error. protection from pathogens or tolerance to a variety of biotic 0054 FIG. 18: Mid-season field-trait measured in July at and abiotic stresses. Such as insect infestation, grazing, the dryland trial of canopy temperature (Celsius) for the water or nutrient deficiency, heat stress, salt or aluminum (blue bars) Delta Pine and (green bars) Phyton cultivars. toxicity, and freezing temperatures. Host growth and fitness Data presented is the block-controlled average of n=10 promotion and protection are thought to be achieved through independent replicates, relative to the control plot and error multiple beneficial properties of the endophyte-host asso bars represent tone standard error. ciation. 0055 FIG. 19: Mid-season field-trait measured in August 0063. These fungal endophytes provided in Table 1 were at the dryland trial of NDVI for the (blue bars) Delta Pine originally collected as fungal endophytes of cotton. These and (green bars) Phyton cultivars. Data presented is the endophytic fungi can be inoculated to live within cotton block-controlled average of n=10 independent replicates, using either seed, soil or foliar applications and exhibited relative to the control plot and error bars represent tone Surprisingly beneficial effects by providing protection from standard error. pest infestation. Pests can be nematode and/or insect pests. In addition, these endophytic fungi have an unexpected 0056 FIG. 20: Mid-season field-trait measured in August beneficial effect on cotton yield. at the dryland trial of first position square retention for the 0064. Described is the application of beneficial fungi to (blue bars) Delta Pine and (green bars) Phyton cultivars. establish endophytically within crop plants to improve plant Data presented is the block-controlled average of n=10 performance and yield while conferring protection against independent replicates, relative to the control plot and error insect and nematode pests. In this regard, the present inven bars represent tone standard error. tion overcomes the limitations of the prior art such as the 0057 FIG. 21: Mid-season field-trait measured in August susceptibility of the fungi to degradation by UV light, at the dryland trial of plant height (cm) for the (blue bars) desiccation or heat after exposure to the environment fol Delta Pine and (green bars) Phyton cultivars. Data presented lowing application as an inundative soil or foliar biopesti is the block-controlled average of n=10 independent repli cide. Inoculation and endophytic establishment of the fungi cates, relative to the control plot and error bars represent within the plant protects the fungi from UV light, desicca tone standard error. tion, and unfavorable temperatures, while harboring the fungi in the very plant tissues they are intended to protect. 0058 FIG. 22: Mid-season field-trait measured in July at Introducing fungi to live endophytically within plants the dryland trial of plant height (cm) for the (blue bars) Delta requires no genetic modification of the plant or microorgan Pine and (green bars) Phyton cultivars. Data presented is the isms, and the fungi themselves can be a source for natural block-controlled average of n=10 independent replicates, products. In various embodiments, the fungal inoculant can relative to the control plot and error bars represent tone be formulated and applied, for example, as treatment of standard error. seeds, in furrow applications, before or during planting, or 0059 FIG. 23: Picture showing increased biomass in the as foliar application after plant germination, and after inocu plants treated with endophytes (right half of the image) lation, the fungal endophytes provide season-long protective compared to untreated control (left half of the image). effects and higher crop yields (approximately 25% higher). In certain embodiments, the increase of yield is about 5%, 0060 FIG. 24: Table showing the time to wilt following 10%, 15%, 20%, 30%, 40%, 45%, 50%, or greater than 50% drought stress in days for plants grown from seeds treated relative to a crop to which no endophyte composition has with fungal endophytes and control. been applied. In further embodiments, the increase of yield 0061 FIG. 25: Table showing the time to death following is the result of reduction of loss that comprises reduction of drought stress in days for plants grown from seeds treated loss due to insect infestation or drought and the loss is less with fungal endophytes and control. than 50%, 40%, 30%, 20%, 10%, 5%, or 5% relative to a US 2016/0286821 A1 Oct. 6, 2016 crop to which no endophyte composition has been applied. ing on leaves above ground (cotton aphids, Aphis gossypii) In certain embodiments, the crop is cotton and the reduction and plant parasitic nematodes attacking roots below ground of loss comprises reduced boll damage. (root knot nematodes, Meloidogyne incognita). In addition, 0065. Thus, in one aspect, the invention provides a com improved plant performance and yields in colonized versus bination (also termed a “symbiotum) of a host plant and an uncolonized control plants may be observed in field trials endophyte that allows for improved agronomic properties of employing seed treatment with Such endophytes. Plant host plants. The combination may be achieved by artificial growth enhancement and increased resistance to root knot inoculation, application, or other infection of a host plant or nematodes was demonstrated in cotton, for example, seeds thereof. Such as a cotton plant or seed thereof, or host employing Chaetomium globosum as an endophyte in green plant tissues, with a fungal endophyte strain of the present house trials. In addition and as a further non-limiting illus invention. Thus, a combination achieved by Such an inocu trative example, using Beauveria bassiana as an endophyte lation is termed a 'synthetic' combination, synthetic com in cotton, reductions in insect (cotton aphid) reproduction position, synthetic seed coating, synthetic pest-resistant seed was demonstrated in both greenhouse and field trials. The composition. The fungal endophyte may be present in inter endophytic presence of Paecilomyces lilacinus and Beau cellular spaces within plant tissue. Such as the root. Its veria bassiana also had negative effects on the host selection presence may also occur or may also be maintained within behavior of key Sucking bug pests (Lygus hesperus and a plant or plant population by means of grafting or other Nezara viridula) that attack developing flowers and fruits in inoculation methods such as treating seeds, plants or parts cotton. Furthermore, in field trials using Beauveria bassiana thereof with endophyte mycelia, or endophyte spores. In as an endophyte in cotton positive effects on plant perfor certain embodiments, the plant, part of the plant, roots, seed, mance and higher yields in endophyte colonized versus or leaves are sterilized to remove microorganisms before uncolonized control plants was demonstrated. applying the endophyte. In particular embodiments, seeds 0069 Metabolomic differences between the plants can be are sterilized to remove native endophytes before adding the detected using methods known in the art. For example, a endophyte compositions herein described. In certain aspects, biological sample (whole tissue, exudate, phloem sap, xylem the ability of the seed to germinate is not affected by the sap, root exudate, etc.) from the endophyte-associated and sterilization. reference agricultural plants can be analyzed essentially as 0066. The invention also provides methods for detecting described in Fiehn et al., (2000) Nature Biotechnol., 18, the presence of the fungal endophyte of the present inven 1157-1161, or Roessner et al., (2001) Plant Cell, 13, 11-29. tion within a host plant. This may be accomplished, for Such metabolomic methods can be used to detect differences instance, by isolation of total DNA from tissues of a poten in levels in hormones, nutrients, secondary metabolites, root tial plant-endophyte combination, followed by PCR, or exudates, phloem sap content, Xylem sap content, heavy alternatively, Southern blotting, western blotting, or other metal content, and the like. methods known in the art, to detect the presence of specific 0070. In another embodiment, the present invention con nucleic or amino acid sequences associated with the pres templates methods of coating the seed of a plant with a ence of a fungal endophyte strain of the present invention. plurality of endophytes, as well as seed compositions com Alternatively, biochemical methods such as ELISA, HPLC, prising a plurality of endophytes on and/or in the seed. The TLC, or fungal metabolite assays may be utilized to deter methods according to this embodiment can be performed in mine the presence of an endophyte strain of the present a manner similar to those described herein for single endo invention in a given sample of crop tissue. Additionally, phyte coating. In one example, multiple endophytes can be methods for identification may include microscopic analy prepared in a single preparation that is coated onto the seed. sis, such as root staining, or culturing methods, such as grow The endophytes can be from a common origin (i.e., a same out tests or other methods known in the art (Deshmukh et al. plant). Alternatively, the endophytes can be from different 2006). In particular embodiments, the roots of a potential plants. grass plant-endophyte combination may be stained with 0071. Where multiple endophytes are coated onto the fungal specific stains, such as WGA-Alexa 488, and micro seed, any or all of the endophytes may be capable of scopically assayed to determine fungal root associates. conferring a beneficial trait onto the host plant. In some 0067. In certain embodiments, the agronomic qualities cases, all of the endophytes are capable of conferring a may be selected from the group consisting of increased beneficial trait onto the host plant. The trait conferred by biomass, increased tittering, increased root mass, increased each of the endophytes may be the same (e.g., both improve flowering, increased seed yield, and enhanced resistance to the host plants tolerance to a particular biotic stress), or may biotic and/or abiotic stresses, each of these qualities being be distinct (e.g., one improves the host plants tolerance to rated in comparison to otherwise identical plants grown drought, while another improves phosphate utilization). In under the same conditions, and differing only with respect to other cases the conferred trait may be the result of interac the presence or absence of a fungal endophyte. The synthetic tions between the endophytes. combinations and methods of the present invention may be 0072 Definitions applied to respond to actual or anticipated stresses. Such 0073. In the description and tables herein, a number of stresses may include, for instance, drought (water deficit), terms are used. In order to provide a clear and consistent cold, heat stress, nutrient deficiency, salt toxicity, aluminum understanding of the specification and claims, the following toxicity, grazing by herbivores, insect infestation, nematode definitions are provided. Unless otherwise noted, terms are infection, and fungal, bacteria or viral infection, among to be understood according to conventional usage by those others. of ordinary skill in the relevant art. 0068. The present disclosure provides, in one embodi 0074. When a term is provided in the singular, the inven ment, fungal endophytes selected from those in Table 1 that tors also contemplate aspects of the invention described by negatively affect the reproduction of insect herbivores feed the plural of that term. The singular form “a,” “an,” and US 2016/0286821 A1 Oct. 6, 2016

“the include plural references unless the context clearly PHY 375 WRF, PHY 367 WRF, PHY 339 WRF, PHY 575 dictates otherwise. For example, the term “a cell includes WRF, DP 1252 B2RF, DP 1050 B2RF, DP 1137 B2RF, DP one or more cells, including mixtures thereof. 1048 B2RF, and/or DP 1137 B2RF. 0075. The term “comprising is intended to mean that the I0082. As used herein, the phrase “culture filtrate” refers compositions and methods include the recited elements, but to broth or media obtained from cultures inoculated with a not excluding others. "Consisting essentially of when used strain of fungi and allowed to grow. The media is typically to define compositions and methods, shall mean excluding filtered to remove any Suspended cells, leaving the nutrients, other elements of any essential significance to the combi hormones, or other chemicals. nation. Thus, a composition consisting essentially of the I0083. As used herein, the term “endophyte' refers to an elements as defined herein would not exclude trace contami organism capable of living within a plant or plant tissue. An nants from the isolation and purification method and agri endophyte may comprise a fungal organism that may confer culturally acceptable carriers. “Consisting of shall mean an increase in yield, biomass, resistance, or fitness in its host excluding more than trace elements of other ingredients and plant. Fungal endophytes may occupy the intracellular or Substantial method steps for applying the compositions of extracellular spaces of plant tissue, including the leaves, this invention. Embodiments defined by each of these tran stems, flowers, or roots. sition terms are within the scope of this invention. I0084. The phrase "pest resistance' refers to inhibiting or 0076 Biological control: the term “biological control reducing attack from pests. Pest resistance provides at least and its abbreviated form “biocontrol,” as used herein, is Some increase in pest resistance over that which is already defined as control of a pest, pathogen, or insect or any other possessed by the plant. undesirable organism by the use of at least one endophyte. I0085. As used herein, the term “genotypes” refers to the 0077. A “composition' is intended to mean a combina genetic constitution of a cell or organism. tion of active agent and at least another compound, carrier I0086. As used herein, the term "phenotype” refers to the or composition, inert (for example, a detectable agent or detectable characteristics of a cell or organism, which char label or liquid carrier) or active, such as a pesticide. acteristics are either the direct or indirect manifestation of 0078. As used herein, an "agricultural seed' is a seed gene expression. used to grow plants in agriculture (an "agricultural plant'). The seed may be of a monocot or dicot plant, and is planted I0087 As used herein, the phrase “host plant” refers to for the production of an agricultural product, for example any plant that an endophytic fungi colonizes. In certain grain, food, fiber, etc. As used herein, an agricultural seed is embodiments, the host plant comprises progeny of colonized a seed that is prepared for planting, for example, in farms for plant. growing. Agricultural seeds are distinguished from com I0088 As used herein, the phrase “increased yield’ refers modity seeds in that the former is not used to generate to an increase in biomass or seed weight, seed or fruit size, products, for example commodity plant products. seed number per plant, seed number per unit area, bushels 0079. As used herein, a “commodity plant product” refers per acre, tons per acre, kilo per hectare, carbohydrate yield, to any composition or product that is comprised of material or cotton yield. Such increased yield is relative to a plant or derived from a plant, seed, plant cell, or plant part of the crop that has not been inoculated with the endophyte. In present invention. Commodity plant products may be sold to certain embodiments, the increase yield is relative to other consumers and can be viable or nonviable. Nonviable com commonly used pest treatments or other methods of address modity products include but are not limited to nonviable ing the biotic or abiotic stress. seeds and grains; processed seeds, seed parts, and plant I0089. As used herein, the phrase “biomass” means the parts; dehydrated plant tissue, frozen plant tissue, and pro total mass or weight (fresh or dry), at a given time, of a plant cessed plant tissue; seeds and plant parts processed for tissue, plant tissues, an entire plant, or population of plants, animal feed for terrestrial and/or aquatic animal consump usually given as weight per unit area. The term may also tion, oil, meal, flour, flakes, bran, fiber, and any other food refer to all the plants or species in the community (commu for human or animal consumption; and biomasses and fuel nity biomass). products. Any Such commodity plant product that is derived 0090. As used herein, “sticker refers to compounds to from the plants of the present invention may contain at least enhance binding of spores to the seed surface. Non-limiting a detectable amount of the specific and unique DNA corre examples of Such compounds are alginic acid, carrageenan, sponding to the endophytes described herein. Any standard dextrin, dextran, pelgel, polyethelene glycol, polyvinyl pyr method of detection for polynucleotide molecules may be rolidone, methyl cellulose, polyvinyl alcohol, or gelatin. used, including methods of detection disclosed herein. 0091. As used herein, an "agriculturally acceptable' 0080. As used herein, the phrase "agronomically elite excipient or carrier is one that is suitable for use in agricul plants' refers to a genotype or cultivar with a phenotype ture without undue adverse side effects to the plants, the adapted for commercial cultivation. Traits comprised by an environment, or to humans or animals who consume the agronomically elite plant may include biomass, carbohy resulting agricultural products derived therefrom commen drate, and/or seed yield; biotic or abiotic stress resistance, Surate with a reasonable benefit/risk ratio. including drought resistance, insect resistance, fungus resis 0092. As used herein, the term “synthetic' or the phrase tance, virus resistance, bacteria resistance, cold tolerance, “synthetic combination” refers to an artificial combination and salt tolerance; improved Standability, enhanced nutrient that includes mycelia and/or spores of a endophyte that is or use efficiency, and reduced lignin content. leads to an endophytic fungal-host relationship (also termed 0081. In certain embodiments, cotton agronomically elite a “symbiotum) of a host plant and an endophyte. The plants include, for example, known cotton varieties AM synthetic combination may be achieved, for example, by 1550 B2RF, NG 1511 B2RF, NG 1511 B2RF, FM artificial inoculation, application, or other infection of a host 1845LLB2, FM 1944GLB2, FM 1740B2F, PHY 499 WRF, plant, host plant seeds, or host plant tissues with the endo US 2016/0286821 A1 Oct. 6, 2016

phyte. In addition, the combination of host plant and an lation, leaves were cut in Small fragments of approximately endophyte may be achieved by inoculating the soil or 1 cm. Squares and bolls were cut in six pieces. Any fiber growth media of the plant. present was removed and cut into six smaller pieces. Leaf 0093. The present invention contemplates, in one fragments were placed upside down on PDA and V8 embodiment, the use of an "isolated microbe. As used medium plates in triplicate. Each plate contained 3 leaf herein, an isolated microbe is a microbe that is isolated from fragments for a total of 9 fragments assayed per plant. For its native environment, and carries with it an inference that squares collected early in the season, 3 slices per square the isolation was carried out by the hand of man. An isolated were plated on PDA and V8 media as with the leaf frag microbe is one that has been separated from at least some of ments. Because of similarity in size and location within a the components with which it was previously associated plant, when collected later in the season, squares and bols (whether in nature or in an experimental setting). from a given plant were plated together on petri dishes 0094. As used herein, a microbe is considered to be containing two square slices, two boll slices and two pieces “native' to a plant or a portion of the plant, and is said to be of fiber. Antibiotics Penicillin G (100 Units/mL) and Strep “natively present in the plant or a portion of plant, if that tomycin (100 ug/mL) (Sigma, St Louis, Mo., USA) were plant or portion of the plant contains the microbe, for added to the media to Suppress bacterial growth. All plates example, in the absence of any contacting with the microbe were incubated in the dark at room temperature for, in preparation. average, two weeks until growth of fungal endophyte 0095 Some of the methods described herein allow the hyphae from plant tissues was detected. colonization of plant seeds by microbes. As used herein, a 0098. An inclusive combination of morphological and microbe is said to “colonize' a plant or seed when it can molecular fungal endophyte identification was employed for exist in a symbiotic or non-detrimental relationship with the identification. Once fungal hyphae were detected growing plant in the plant environment, for example on and/or inside from the plant material, Samples were taken to obtain pure a plant, including the seed. fungal isolates. For identification by PCR, genomic DNA 0096. A "population of plants, as used herein, refers to was extracted from mycelium of each isolated fungal strain, a plurality of plants that were either grown from the seeds following a chloroform:isoamyl alcohol 24:1 protocol and coated with the endophytes as described herein, or are fungal specific primers were used to amplify the ITS (Inter progeny of a plant or group of plants that were subjected to nal Transcribed Spacer) region of nuclear ribosomal DNA. the inoculation methods. The plants within a population will This region is the primary barcoding marker for fungi and typically be of the same species, and will also typically share includes the ITS1 and ITS2 regions, separated by the 5.8S a common genetic derivation ribosomal gene. In order to avoid introducing biases during PCR (taxonomy bias and introduction of mismatches), it has EXAMPLES been suggested to amplify the ITS1 region only, therefore the primers ITS1 (5' TCC GTAGGT GAA CCT GCG G 3') Example 1 (SEQ ID NO:5) and ITS2 (5 GCT GCG TTC TTC ATC GAT GC 3') (SEQ ID NO:6) were used to amplify and Creating Spore Suspensions and Treatment of sequence the ~240 by ITS1 region of each one of the isolated Seeds fungal Strains. The resulting sequences were aligned as 0097 Cultivation of plants and endophytic fungi strains: query sequences with the publicly available databases Gen The cotton seed variety used in particular embodiments was Bank nucleotide, UNITE and PlutoF. The last two are variety LA 122 (available from All-Tex Seed, Inc., Level specifically compiled and used for fungi identification. Table land, Tex. 79336). Paecilomyces lilacinus and Chaetomium 1 provides a list of endophytes identified and useful in the globosum were obtained from cotton plants as described present invention. All of these endophytes belong to phylum (Ek-Ramos et al. 2013, PLoS ONE 8(6): e66049. doi:10. Ascomycota, Subphylum Pezizomycotina, except for Pha 1371/journal.pone.0066049). Persons of ordinary skill in the nerochaete crassa, which belongs to phylum Basidiomy art can obtain endophytes Suitable for performing the vari cota, Subphylum Agaricomycotina, and Pseudozyma sp. ous embodiments of the present invention by performing the which belongs to phylum Basidiomycota, Subphylum Usti procedures described therein. In short, plant samples were laginomycotina. Table 1 shows the species/genus, family, rinsed in tap water and Surface sterilized by immersion in order, subclass, class, and the SEQID NO corresponding to 70% ethanol for 5 min, 10% bleach solution for 3 min, and the -240 by ITS1 region for each one of the isolated fungal rinsed twice with autoclaved distilled water. Samples were strains, except for Beauveria bassiana, Aspergillus parasiti blotted dry using autoclaved paper towels. Five individual cus, Lecanicillium lecanii, and Paecilomyces lilacinus, surface sterilized leaves, squares and bols (N=15 total where the sequences shown includes the ITS1, ITS2, 5.8S, samples) were randomly selected and imprinted onto fresh 18S, and 28S sequences and were obtained from the UNITE potato dextrose agar (PDA) and V8 media as a way to database for GenBank numbers JF837090, JX857815, monitor surface sterilization efficiency. For endophyte iso 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 alternatin US 2016/0286821 A1 Oct. 6, 2016 10

TABLE 1-continued endophytes identified and useful in the present invention Genus Species Family Order Subclass Class SEQ ID NO. Alternaria Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 8 alternata Alternaria Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 9 brassicae Alternaria Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 10 compacta Alternaria dianthi Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 11 Alternaria (OSO3C(86. (OSO8ES eosporomycetidae Dothideomycetes 12 longipes Alternaria mai (OSO3C(86. (OSO8ES Pleosporomycetidae Dothideomycetes 13 Alternaria Sesami (OSO3C(86. (OSO8ES Pleosporomycetidae Dothideomycetes 14 Alternaria Soiani (OSO3C(86. (OSO8ES Pleosporomycetidae Dothideomycetes 15 Alternaria sp. (OSO3C(86. (OSO8ES Pleosporomycetidae Dothideomycetes 16 Alternaria (OSO3C(86. (OSO8ES Pleosporomycetidae Dothideomycetes 17 tentissina Bipolaris Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 18 spicifera Cercospora Mycosphaerellaceae Capnodiales Dothideomycetidae Dothideomycetes 19 C(i.e.SC&S Cercospora Mycosphaerellaceae Capnodiales Dothideomycetidae Dothideomycetes 2O capsici Cercospora Mycosphaerellaceae Capnodiales Dothideomycetidae Dothideomycetes 21 kikuchi Cercospora Mycosphaerellaceae Capnodiales Dothideomycetidae Dothideomycetes 22 zinnia Chaetonium Chaetomiaceae Sordariales Sordariomycetidae Sordariomycetes 23 globosum Chaetonium 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 tiredlinicola Cochliobolus sp Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 29 Phanerochaete Phanerochaetaceae Polyporales Incertae sedis Agaricomycetes 30 CFSS Phoma ncertae sedis Pleosporales Pleosporomycetidae Dothideomycetes 31 americana Phoma ncertae sedis Pleosporales Pleosporomycetidae Dothideomycetes 32 subherharum 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 Pseudozyna sp. Ustilaginaceae Ustilaginales Ustilaginomycetidae Ustilaginomycetes 39 Pyrenophora Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 40 iefeS Colleiotrichtim Glomerellaceae ncertae sedis Sordariomycetidae Sordariomycetes 41 capsici Conioiarielia ncertae sedis Xylariales Xylariomycetidae Sordariomycetes 42 gamsii Coniothyrium Coniothyriaceae Pleosporales Pleosporomycetidae Dothideomycetes 43 a lettritis Coniothyrium sp. Coniothyriaceae Pleosporales Pleosporomycetidae Dothideomycetes 44 Corynespora Corynesporascaceae Pleosporales Pleosporomycetidae Dothideomycetes 45 cassicola Diaporihe sp. Diaporthaceae Diaporthales Sordariomycetidae Sordariomycetes 46 Diatrype sp. Diatrypaceae Xylariales Xylariomycetidae Sordariomycetes 47 Drechsierelia Orbiliaceae Orbilliales Orbilliomycetidae Orbilliomycetes 48 dactyloides Embellisia Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 49 indefessa Epicocci in Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 50 nigrum Epicoccim sp. Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 51 Exserohilum Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 52 iOSirciitii? US 2016/0286821 A1 Oct. 6, 2016 11

TABLE 1-continued endophytes identified and useful in the present invention Genus Species Family Order Subclass Class SEQ ID NO. Fusarium Nectriaceae Hypocreales Hypocreomycetidae Sordariomycetes 53 chlamydosportin Fusarium sp. Nectriaceae Hypocreales Hypocreomycetidae Sordariomycetes S4 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 Mycosphaereliaceae Mycosphaerellaceae Capnodiales Dothideomycetidae Dothideomycetes 59 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 Penicilium Trichocomaceae Eurotiales Eurotiomycetidae Eurotiomycetes 64 citrintin Retroconis sp. Incertae sedis Incertae sedis ncertae sedis Incertae sedis 65 Rhizopycnis sp. Incertae sedis Incertae sedis ncertae 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 Uiociadium Pleosporaceae Pleosporales Pleosporomycetidae Dothideomycetes 72 chartarum Verticilium sp. Plectosphaerellaceae Incertae sedis Hypocreomycetidae Sordariomycetes 73 Beativeria Cordycipitaceae Hypocreales Hypocreomycetidae Sordariomycetes 74 bassiana Aspergilius Trichocomaceae Eurotiales Eurotiomycetidae Eurotiomycetes 75 parasiticits Lecaniciiium Cordycipitaceae Hypocreales Hypocreomycetidae Sordariomycetes 76 iecani Paecilomyces Trichocomaceae Eurotiales Eurotiomycetidae Eurotiomycetes 77 iiacinus

0099 TABLE 1 List of endophytes: sp., Ulocladium chartarum, Verticillium sp., Beauveria 0100 Acremonium alternatum, Alternaria alternata, bassiana, Aspergillus parasiticus, Lecanicillium lecanii, Alternaria brassicae, Alternaria compacta, Alternaria dian Paecilomyces lilacinus. thi, Alternaria longipes, Alternaria mali, Alternaria sesami, 0101 Beauveria bassiana was cultured from a commer Alternaria Solani, Alternaria sp., Alternaria tenuissima, cially obtained strain (available from Botanigard). Beau Ascomycota sp., Bipolaris spicifera, Cercospora canescens, veria bassiana, Paecilomyces lilacinus, and Chaetomium Cercospora capsici, Cercospora kikuchii, Cercospora Zin globosum were cultured on potato dextrose agar media nia, Chaetomium globosum, Chaetomium piluliferum, (PDA). Stock spore concentration solutions of each fungi Chaetomium sp., Cladosporium cladosporioides, Cladospo were made by adding 10 ml of sterile water to the fungi rium sp., Cladosporium uredinicola, Cochliobolus sp., Pha plates and scraping them free of the agar with a sterile nerochaete crassa, Phoma americana, Phoma subherba Scalpel. The resulting mycelia and spores obtained were then rum, Phomopsis liquidambari, Phomopsis sp., Pleospora filtered into a sterile beaker utilizing a cheese cloth to filter sp., Pleosporaceae sp., Polyporales sp., Preussia africana, out the mycelia, thereby creating Stock solutions. A haemo Preussia sp., Pseudozyma sp., Pyrenophora teres, Col cytometer was used to measure and calculate spore concen letotrichuncapsici, Coniolariella gamsii, Coniothyrium trations of the stock solutions. The desired concentrations aleuritis, Coniothyrium sp., Corynespora cassicola, Dia were created by dilution, and seeds were placed into spore porthe sp., Diatrype sp., Drechslerella dactyloides, Embel Suspensions with the desired spore concentrations. In vari lisia indefessa, Epicoccum nigrum, Epicoccum sp., Exsero ous embodiments, the final treatment concentrations can be hilum rostratum, Fusarium chlamydosporum, Fusarium sp., about 10, 10, 10, 10, 10, 107, 10, or 10° spores/ml Gibellulopsis nigrescens, Gnomoniopsis sp., Lewia infecto which can be reached by serial dilutions in sterile water or ria, Mycosphaerella coffeicola, Mycosphaerellaceae sp., in an appropriate solution or buffer. Nigrospora Oryzae, Nigrospora sp., Nigrospora sphaerica, 0102 For seed inoculation, the seeds were surface ster Paecilomyces sp., Penicillium citrinum, Retroconis sp., ilized prior to soaking them in spore suspensions with the Rhizopycnis sp., Schizothecium inaequale, Stagonospora desired concentration by immersion the seeds in 70% etha sp., Stemphylium lancipes, Thielavia hyrcaniae, Thielavia nol for 3 minutes with constant shaking followed by incu US 2016/0286821 A1 Oct. 6, 2016

bation in 2% NaOCl for 3 minutes; followed by three washes sition to the surface of the soil directly above each seed. In in sterile water. The third sterile water wash was plated onto certain embodiments, the endophyte composition may com potato dextrose agar media (PDA) to confirm that surface prise 0.01-0.1, 0.1-1, or 1-10 ml endophyte suspension, sterilization was effective. Seeds were then soaked for 24 which may be a endophyte spore Suspension. hours in beakers containing spore Suspensions with two 0110 Soil inoculation: In certain embodiments, seeds can different concentrations of fungi. Control group seeds were be planted into inoculated soil. The inoculum can be treated with sterile water only. Spore concentrations for obtained by multiplying the endophyte on fungal growth Beauveria bassiana were Zero (control), 1x10' (treatment 1) media. The fungal growth media can be potato dextrose agar and 1x10 (treatment 2) and for Paecilomyces lilacinus or media (PDA). In other embodiments the fungal growth Chaetomium globosum were zero (control), 1x10' (treat media can be as wheat grain. In a non-limiting example, 100 ment 1) and 1x107 (treatment 2). These beakers were incu g of wheat grain can be washed and soaked overnight in bated for 24 hours at 32° C. in a culture chamber until next sterile water. Excess water can be drained, seeds dried on day for planting (24 hr). paper towel, packed in a 500 ml conical flask and autoclaved 0103 Soaked seeds were planted in L22 mix soil (Bor at 15 psi for 1 h. One milliliter of the endophytic fungal laug Institute, Texas A&M). All plants were grown in a spore suspension (10 spores/ml) can be inoculated to the laboratory greenhouse at -28°C. with a natural light pho flask, and the cultures can be incubated at 25° C. for 2 toperiod. There was no fertilization of the plants, and weeks. To avoid clumping, the flasks can be shaken vigor watering was done consistently across all treatments as ously to separate the grain and break the mycelial mat. needed. Approximately 5 g of inoculum can be placed in soil at 0104 Direct seed inoculation: In particular embodi planting. In certain embodiments, the inoculum can be ments, individual seeds and the Surrounding soil can be placed in the soil at the same time or within 1 month of directly inoculated with the spore solution (10°-10, 10 planting the seeds. In certain embodiments, the seeds may 10, 10-10, 10°-107, or 107-10 spores/ml) at planting comprise sterilized seeds. before covering the seed with soil. 0105. In various embodiments, any seed or plant treat Example 4 ments that are suitable for application of biological agents to seeds or plants and known to persons having ordinary skill Foliar Endophyte Treatments in the art can be employed. 0111 Plants were inoculated via foliar application at the third true leaf stage by spraying the surface of fully Example 2 expanded leaves to run-off with a spore suspension (10 Application of Endophyte Spores as a Dry Powder spores/ml) using a hand-held plastic sprayer (1 L). In certain Composition embodiments, endophyte spore Suspensions were made in water. In certain embodiments, the water was Supplemented 0106. In addition to application of a spore solution for with a detergent. In a particular non-limiting example, the seed treatment, the endophytes or endophyte spores can also spore suspension contained 0.02% Triton X 100 as a deter be applied as dry powder or using a sicker Such as methyl gent. cellulose for seed treatment. In certain embodiments, the 0112 Foliar endophyte treatment may be performed concentration may be at least 10, 10, 107, 10, 10, or using any suitable method known to a person having ordi higher colony forming units or spores/g dry weight. nary skill in the art. In particular, foliar endophyte treatment 0107. In certain embodiments, endophytes can be grown may be performed using a sprayer by directly spraying in fungi cultivation media in a fermenter. Endophytic myce leaves with an endophyte Suspension, which may be a lial fragments or spores can be collected, dried and ground. endophyte spore Suspension. A Sticker Such as caboxymethyl cellulose may also be added 0113 FIG. 9 demonstrates that foliar application of cot to the ground endophytic material. ton in the field with spores of endophytic entomopathogenic 0108. In certain embodiments the weight ratio between fungi improved plant performance. Cotton (variety endophytic material and sticker may be between 1:10-50, FM1740B2F) seeds were treated with a variety of typical 1:50-100, 1:100-500, or 1:500-1000 to obtain the seed fungicide (Metalaxyl, Triadimenol, Trifloxystrobin, 2-(Thio coating or seed inoculation material. This seed inoculation cyanome-thylthio) benzothioazole) and insecticide (Thiodi material can be applied to seeds. In various embodiments, carb. Imidacloprid, Chloropyrifos), and seed treatments the weight ratio between seed inoculation material and seed were planted and grown under field conditions. The plants may be 1:10-50, 1:50-100, 1:100-500, 1:500-1000, or were sprayed at the 5th true leaf stage with aqueous solu 1:1OOO-5OOO. tions of Beauveria bassiana and Paecilomyces fumosoro seus. Sucrose was included (1% wt/vol) as an additional Example 3 nutritional resource for the fungi. Significantly higher first position boll (developing fruit) retention was observed in Soil (in Furrow) Endophyte Treatments plants sprayed with Beauveria bassiana without Sucrose and 0109 Soil drench (in furrow) application may be per P. fimosoroseus plus Sucrose. formed by applying an endophyte composition to the Surface of the Soil and/or seed during planting. In particular embodi Example 5 ments, the endophyte composition may comprise an endo phyte Suspension or an endophyte dry powder formulation. Confirmation of Plant Colonization by Endophytic In various embodiments the endophyte may comprise myce Fungi lia and/or spores. In particular embodiments, the soil drench 0114 Plants were individually placed in plastic bags, application may comprise applying the endophyte compo which were labelled with plant number, treatment, and final US 2016/0286821 A1 Oct. 6, 2016

aphid number, and stored in 4° C. until the next day for 45x20 cm cups and sealed with no-see-um mesh (Eastex endophyte confirmation. Half of each plant was utilized for products, NJ) to avoid aphid movement from plant to plant. plating on PDA agar and the other half was freeze-dried for In one embodiment, the plants used were 13 days old, to conduct diagnostic PCR assays for endophyte confirma approximately in the first true leaf stage, and aphids were tion. The surface sterilization protocol and plating of third left to reproduce for seven days under greenhouse condi sterile water wash on PDA to test for surface contamination tions. In another embodiment, aphids were left to reproduce was conducted as described above. For diagnostic PCR for 14 days on plants initially 20 days old at the beginning assays, plant tissue was freeze-dried and DNA was extracted of the experiment, approximately in the third true leaf stage. utilizing the CTAB protocol (Doyle & Doyle, 1987, Phyto At the end of each embodiment, aphid numbers were chemistry Bulletin 19:11-15). The oligonucleotide primer counted and recorded per individual plant. The presence of sequences synthesized were based upon a NCBI BLAST Beauveria bassiana or Paecilomyces lilacinus as an endo search corresponding to the laboratory culture sequence phyte in cotton significantly reduced the reproduction of results isolated (Ek-Ramos et al., 2013). Sense and antisense cotton aphids on endophyte treated plants versus untreated oligonucleotide sequences for Beauveria bassiana were: control plants (FIGS. 4A, 4B, and FIG. 5) 5'-CGGCGGACTCGCCCCAGCCCG-3' (SEQ ID NO:1) and 5'-CCGCGTCGGGGTTCCGGTGCG-3' (SEQ ID Example 8 NO:2) respectively. The oligonucleotides used to amplify Paecelomyces lilacinus were: 5' CTCAGTTGCCTCG Fungal Endophytes Reduce Nematode GCGGGAA 3 (SEQID NO:3) and 5' GTGCAACTCAGA Reproduction GAAGAAATTCCG 3' (SEQ ID NO:4). I0120 Plants were germinated from treated and untreated 0115 The PCR protocol consisted of a denaturation step control seeds in an environment chamber and then trans at 95° C. for 5 min, followed by alignment of oligonucle planted to soil in pots 11 days after planting. Two replicate otides at 56°C. for 2 min and an extension step of 7 min at seedlings per treatment were sampled to examine the endo 72° C. with a total of 35 cycles. The PCR products were phyte colonization efficiency by Surface sterilization and visualized in a 2% agarose gel containing 1% ethidium plating on PDA agar. Nematode treatment group seedlings bromide. Electrophoresis was performed at 70 volts for 30 were treated with either 2,000 or 10,000 eggs/plant at day six 1. after transplanting. Plants were harvested and processed 6 weeks after nematode inoculation. The numbers of galls per Example 6 gram of root tissue and total egg numbers in the population for each plant were quantified to compare nematode perfor Endophytic Fungi can be Manipulated in the Field mance between endophyte-treated and untreated (control) 0116. A field trial using isolates of Paecilomyces lilacinus plants. and Beauveria bassiana was conducted during the Summer. I0121 FIGS. 2 and 3 demonstrate that the endophytic A randomized block design with five replicate plots that fungi Paecilomyces lilacinus and Chaetomium globosum were planted with seeds that were inoculated by soaking for negatively affected root knot nematode (Meloidogyne incog 9 hr in three different aqueous spore concentrations (0, 10. nita) reproduction when present as an endophyte in cotton. or 10 spores/ml) of the candidate endophyte (such as At high nematode inoculum levels (10,000 eggs), Paecilo Paecilomyces lilacinus or Beauveria bassiana). Each plot myces lilacinus reduced egg production in plants following consisted of four 15.24 m (40 ft) rows, each separated by treatment of seeds with solutions containing either 10° or 101.6 cm (40 in). 107 spores/ml when compared to untreated control seeds. At 0117 Colonization efficiency: At the first true leaf stage, field inoculum levels (2000 eggs), the presence of Paecilo four plants from each plot for a total of 20 plants per myces lilacinus significantly reduced both galls and egg treatment were randomly sampled and tested for coloniza production at both seed treatment concentrations. Endo tion by each of the candidate endophytes. Colonization phytic Chaetomium globosum negatively affects root-knot frequencies were determined by incubating Surface steril nematode reproduction. Negative effects of endophytic ized root, stem and leaf fragments on PDA media and Chaetomium globosum on root-knot nematode gall forma observing for fungal growth. Colonization frequencies are tion and egg production were demonstrated following cotton reported as the number of plants per treatment group with at seed soaking treatments in Solutions of 0 (untreated con least one positively colonized plant fragment. trols), 10° and 10 spores/ml. 0118. The high endophytic colonization frequency of seedlings by Paecilomyces lilacinus or Beauveria bassiana Example 9 demonstrates that the presence of specific endophytes can be manipulated under field planting conditions (FIG. 1). Effect of Fungal Endophytes on Insects 0.122 Endophyte-treated and control plants were grown Example 7 from non-transgenic cotton seeds (Gossypium hirsutum) (variety LA1 22, AllTex Seed Co.). Seeds were soaked for Cotton Aphid Reproduction Test 24 hours in beakers containing 10 spores/ml solutions of the 0119) A colony of A. gossypii was reared on cotton in fungi utilized plus sterile water-only as a control. The cages in a greenhouse kept at approximately 28°C. with beakers were placed in a 32° C. culture chamber overnight natural light photoperiod. Second instar nymphs were placed (approx. 9h) until planting the next day. The plants were directly onto endophyte-treated cotton plants and control grown under both greenhouse and field conditions. Green plants. Ten plants were utilized per treatment group and ten house plants were first germinated in seedling trays and then aphids were placed per plant. After plants were inoculated transferred to 30 cm pots. Field grown plants were concur with the aphids, the plants were placed in individual plastic rently planted and grown. US 2016/0286821 A1 Oct. 6, 2016

0123. Behavioral assays: No-choice and choice behav ANOVA. All analyses including tests of normality and ioral assays were conducted to compare the response of homogeneity of variances were conducted in SPSS 21 western tarnished plant bugs (L. hesperus) and green Stink (SPSS Inc.). bugs (N. viridula) to squares and bolls from endophyte I0127. Results of the L. hesperus no-choice assays: Over treated and untreated plants. The assays were conducted at the duration of the assay, a significantly higher proportion of 30°C. in 10 cm diameter petri dishes with a thin layer of 2% L. hesperus individuals over time was observed in contact agar on the bottom to provide moisture for the squares (L. with and feeding upon Squares from untreated control plants hesperus assays) and bolls (N. viridula assays) from experi relative to those from either of the Beauveria bassiana or mental plants offered to the insects during the observations. Paecilomyces lilacinus endophyte treatment groups (Wil For no-choice assays, a single Square or boll was inserted by coxon Signed Ranks test, P-0.0001 for both comparisons) the base into the agar in the center of the dish. A single (FIG. 6A). Repeated measures ANOVA indicated a signifi young adult (1-7 days post molt) insect was placed in each cant effect of time (F-86.175: P-0.001) with a higher dish and covered with the top. A total of 30 insects were proportion of insects contacting the square as the assay observed in each trial with N=10 insects each in the Beau progressed (FIG. 6B). There was also a significant effect of veria bassiana, Paecilomyces lilacinus and control treat endophyte treatment (F-4.929; P=0.009) with no sig ment groups. The L. hesperus no-choice trials were repli nificant time X endophyte treatment interaction (F-1. cated four times (N=40 per treatment) with squares from 015; P=0.366). Of the 40 insects in each treatment group, greenhouse grown plants used in all but one trial. The N. 12.5% of the control group failed to make contact with the viridula no-choice trials were replicated three times (N=20 square over the course of the assay, while a significantly per treatment) with bolls from greenhouse grown plants used higher 35% and 32.5% the Beauveria bassiana and Paeci in one trial. lomyces lilacinus treatment group insect respectively failed 0124 Choice tests were conducted under the similar to make contact (X2 test, P-0.0001). Among the insects that conditions using the same arenas, but with two equal sized did make contact with a square, there was significant dif squares (L. hesperus) or bolls (N. viridula) placed 4 cm apart ference in the latency to first contact among the treatment in the center of the petri dish. The two squares or bolls per groups (Fass=7.225; P-0.0001) with the control group arena were from an untreated control plant and either a exhibiting a shorter latency to contact than either the Beau Beauveria bassiana or Paecilomyces lilacinus treated plant. veria bassiana (posthoc LSD test; P=0.001) or Paecilomy A total of 20 insects were observed in each trial, with N=10 ces lilacinus endophyte treatment groups (posthoc LSD test; each in the Beauveria bassiana VS. control and Paecilomy P=0.006 (FIG. 6A). ces lilacinus VS. control treatment groups. The L. hesperus I0128 Results of the L. hesperus choice assays: In simul and N. viridula choice trials were both replicated twice taneous choice tests, L. hesperus individuals selected (N=20 per treatment) with squares from field-grown plants squares from untreated control plants more often than those in all trials. from endophyte-treated plants. Response ratios were signifi 0.125 Insects were observed for 6 hours per trial using a cantly greater than 0.5 over the duration of the assays, point sampling procedure for both the no-choice and choice indicating that the insects non-randomly selected bolls from assays. Preliminary observations indicated that the insects of control plants over bolls from plants endophytically colo both species were more active at the beginning of the assay, nized by either (A) Beauveria bassiana (P<0.0001; Wil thus staged sampling schedule was adopted with observa coxon Signed Ranks test) or (B) Paecilomyces lilacinus tions recorded at 5 minute intervals early in the assay (0-60 (P<0.0001; Wilcoxon Signed Ranks test) (FIG. 6B). min), 15 minute intervals in the middle (61-180 min) and 30 I0129 Results of the N. viridula no-choice assays: Over minute intervals late (181-360 min) in the assay. At each the duration of the assay, a significantly higher proportion of sampling interval, the insects were recorded as either off the N. viridula individuals over time was observed in contact square/boll or feeding or roosting upon the square/boll. with and feeding upon bolls from untreated control plants 0126 Data analysis: In the no-choice assays, the propor relative to those from either of the Beauveria bassiana or tion of insects observed either feeding or resting upon cotton Paecilomyces lilacinus endophyte treatment groups (Wil squares (L. hesperus) or bolls (N. viridula) was compared coxon Signed Ranks test, P-0.0001 for both comparisons) between treatment groups at each observation point across (FIG. 7A). Repeated measures ANOVA indicated a signifi the duration of the assay using the Wilcoxon Signed Ranks cant effect of time (F-86.175: P-0.001) with a higher Test. To test for variation in responses over time, for each proportion of insects contacting the square as the assay individual the proportion of observations either feeding or progressed (FIG. 1), There was also a significant effect of upon the plant sample was calculated for early (0-60 min), endophyte treatment (F 4929; P=0.009) with no sig middle (61-180 min) and late (181-360 min) periods of the nificant time X endophyte treatment interaction (F-1. assay and compared across treatment groups using a 015; P=0.366). Of the 40 insects in each treatment group, repeated measures analysis of variance (ANOVA) with the 12.5% of the control group failed to make contact with the endophyte treatment group as the main factor and time as the square over the course of the assay, while a significantly repeat effect. The observed frequency of individuals failing higher 35% and 32.5% the Beauveria bassiana and Paeci to make contact with squares or bolls from endophyte lomyces lilacinus treatment group insect respectively failed treated plants was compared to the expected frequency of to make contact (X2 test, P-0.0001). Among the insects that individuals failing to do so based on the control group using did make contact with a square, there was significant dif a X2 test. Among the insects that did make contact with ference in the latency to first contact among the treatment either a square or boll, the time to first contact (latency) was groups (Fass=7.225; PK0.0001) with the control group compared among treatment groups using a one-way exhibiting a shorter latency to contact than either the Beau US 2016/0286821 A1 Oct. 6, 2016

veria bassiana (posthoc LSD test; P=0.001) or Paecilomy seeds were surface sterilized using ethanol and bleach as ces lilacinus endophyte treatment groups (posthoc LSD test; described in Example 1 for cotton. Three strains (B. bassi P=0.006 (FIG. 7B). ana, P. fumosoroseus, and P lilacinus) were prepared as conidia suspensions at 107 conidia/ml, and coated on the Example 10 Sorghum seeds as described in Example 1. Control seeds were soaked in sterile water instead of a conidia Suspension. More Bolls are Retained after Endophyte Treatment Planted seeds were held in constant growth chamber con 0130. During the field trial, cotton phenology and devel ditions for two weeks at a replication of 10. At the end of two opment was quantified using a plant mapping and informa weeks, the plants were removed from the growth chamber tion system developed specifically for cotton to track fruit and the plant height and biomass were measured. FIG. 12A development and retention by the plant as a means of shows the increase in plant height when applied with the monitoring plant development and stress (COTMANTM, described microbial composition relative to the control Cotton Inc.). One measure of cotton stress is the retention of (p<0.05). FIG. 12B shows the increase in plant biomass in developing flowers (squares) and fruits (bols) in the first plants grown from seed that were treated with the described fruiting position on branches. First position squares and microbial composition relative to the control (p<0.05). 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 Example 13 treatment group. Treatment with Fungal Endophytes Modulates the 0131 FIG. 10 demonstrates that early in the growing season as flowers begin to develop, a trend for higher square Colonization Frequencies of Native Endophytes retention in the endophyte-treated plants relative to controls I0135) To determine whether endophyte seed treatments was observed. This trend continued later in the season as could alter the microbiome of the plant grown from the seed, evidenced by significantly higher boll retention among the cotton seeds were treated with spore suspensions of Paeci endophyte treatment groups relative to the untreated control lomyces lilacinus or Beauveria bassiana. Plants were grown plants. in the field as part of a field trial planted and maintained 0132 FIG. 8 demonstrates reduction in cotton boll dam understandard agricultural practices. Endophytic fungi were age during field trials. Relative to control plants, levels of isolated on PDA media separately from surface-sterilized insect-related boll damage were lower among plants that above-ground stem/leaf and below-ground root tissue to were treated by soaking seeds in spore solutions of Beau assess changes in the microbial community. The comparison veria bassiana and Paecilomyces lilacinus at concentrations shown in FIG. 13 is relative to the fungal endophyte of 10° and 10 spore/ml. Positive effects of fungal endo communities in untreated control plants. The results show phytes on cotton plant performance under field conditions. that these treatments can alter the colonization rates of native fungal endophytes. Example 11 0.136 Fungal endophyte treatments may alter the coloni Zation frequencies of any of the fungal endophytes naturally Endophyte Treatment Increases Yield present in plants. To determine what other native endophytes may be affected by seed treatments with fungal endophytes, 0133. At the end of the field trial employing endophyte the identity of cotton fungal endophytes isolated from plants treatment and treatment plants, plots were machine har of two commercial cotton varieties, CG3787B2RF and vested with a 1-row picker. Surprisingly, the final yields at PHY499WRF, were assessed. The samples were obtained harvest were significantly higher than expected (25% higher during a variety trial near Lubbock, Tex., USA identified as than the untreated controls). Unexpectedly, treatment with Lubbock-RACE. One single healthy leaf was collected from Paecilomyces lilacinus or Beauveria bassiana resulted in each of nine individual plants sampled per variety across higher yields than untreated control plants with regardless of multiple replicate plots arranged in a randomized block the initial seed treatment concentration. (FIG. 11) design to control for spatial variation in the field. To identify Example 12 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 pyrose Endophyte Treatment of Sorghum Increased quencing using ITS1F forward and ITS2 reverse universal Growth in the GH fusion primers. The fungal endophytes identified in this 0134. The effect of the described microbial compositions experiment, along with those shown in FIG. 13, are listed in on Sorghum was tested in a seedling assay. Sorghum bicolor 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 at traits Dothideomycetes Botryosphaeriales Botryosphaeriaceae Macrophonina sp. Dothideomycetes Capnodiales Davidiellaceae US 2016/0286821 A1 Oct. 6, 2016 16

TABLE 2-continued Native fungal endophytes that may be altered by seed treatments with other fungal endophytes Phylum Class Order Family Genus species Dothideomycetes Capnodiales Davidiellaceae Cladosporium sp. Dothideomycetes Capnodiales Davidiellaceae Cladosporium cladosporioides Dothideomycetes Capnodiales Davidiellaceae Davidiella sp. Dothideomycetes Capnodiales Mycosphaerellaceae Cercospora sp. Dothideomycetes Capnodiales Mycosphaerellaceae Cercospora beiicola 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 tentissina Dothideomycetes Pleosporales Pleosporaceae Cochliobolus sp. Dothideomycetes Pleosporales Pleosporaceae Curvularia sp. Dothideomycetes Pleosporales Pleosporaceae Epicoccim 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 Aspergilius sp. Eurotiomycetes Eurotiales Trichocomaceae Penicillium sp. Eurotiomycetes Eurotiales Trichocomaceae Thermomyces sp. Eurotiomycetes Eurotiales Trichocomaceae Thermomyces lanuginostis Saccharomycetes Saccharomycetales Saccharomycetes Saccharomycetales Saccharomycetaceae Saccharomycetes Saccharomycetales Saccharomycetaceae Candida sp. Saccharomycetes Saccharomycetales Saccharomycetaceae Candida quiercitritsa Saccharomycetes Saccharomycetales Saccharomycetaceae Candida tropicalis Saccharomycetes Saccharomycetales Saccharomycetaceae Cyberlindnera sp. Saccharomycetes Saccharomycetales Saccharomycetaceae Cyberlindneraliadinii Saccharomycetes Saccharomycetales Saccharomycetaceae Kluyveromyces sp. Saccharomycetes Saccharomycetales Saccharomycetaceae Kluyveromyces marxiantis Sordariomycetes Sordariomycetes Diaporthales Gnomoniaceae Gnomoniopsis sp. Sordariomycetes Hypocreatles Cordycipitaceae Beativeria bassiana Sordariomycetes Hypocreatles Cordycipitaceae Cordyceps sp. Sordariomycetes Hypocreatles Cordycipitaceae Cordyceps bassiana Sordariomycetes Hypocreatles Nectriaceae Sordariomycetes Hypocreatles Nectriaceae Fusarium sp. Sordariomycetes Hypocreatles Hypocreaceae Sordariomycetes Hypocreatles Hypocreaceae Gibellulopsis nigrescens Sordariomycetes Hypocreatles Hypocreaceae Hypocreasp. Sordariomycetes Hypocreatles Hypocreaceae Hypocrea ixi Sordariomycetes Hypocreatles Hypocreaceae Hypocrea virens Sordariomycetes Hypocreatles Hypocreaceae Trichoderma sp. Sordariomycetes Hypocreatles Hypocreaceae Trichoderma iOileriiOSiii? Sordariomycetes Hypocreatles Plectosphaerellaceae Verticilium sp. Sordariomycetes Ophiostomatales Ophiostomataceae Sordariomycetes Ophiostomatales Ophiostomataceae Ophiostoma sp. Sordariomycetes Ophiostomatales Ophiostomataceae Ophiostoma dendiflundum Sordariomycetes Sordariales Chaetomiaceae Chaetomium sp. Sordariomycetes Sordariales Chaetomiaceae Chaetonium globosum Sordariomycetes Sordariales Chaetomiaceae Thielavia hyrcaniae Sordariomycetes Sordariales Chaetomiaceae Taifangiania sp. Sordariomycetes Sordariales Chaetomiaceae Taifangiania inflata Sordariomycetes Sordariales Lasiosphaeriaceae Schizothecium inaequale US 2016/0286821 A1 Oct. 6, 2016 17

TABLE 2-continued Native fungal endophytes that may be altered by seed treatments with other fungal endophytes Phylum Class Order Family Genus species Sordariomycetes Trichosphaeriales Trichosphaeriaceae Nigrospora sp. Sordariomycetes Xylariales Amphisphaeriaceae Truncatelia anguistata 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 viliosa Agaricomycetes Russulales Peniophoraceae Microbotryomycetes Sporidiobolales Microbotryomycetes Sporidiobolales Sporidiobolaceae Rhodotorula sp. Microbotryomycetes Sporidiobolales Sporidiobolaceae Rhodotoria mucilaginosa Tremellomycetes Tremellomycetes Tremellales Tremellomycetes Tremellales Tremellaceae Cryptococcus sp Tremellomycetes Tremellales Tremellaceae Cryptococcus Skinneri Tremellomycetes Tremellales Tremellaceae Tremella sp.

Example 14 herbivory treatments (no aphids, or aphid herbivory for either 1, 4, 8, 24 or 48 hours). Phytohormone levels for Fungal Endophyte Seed Treatment Leads to abscisic acid (ABA), tuberonic acid (12-OH-JA, an oxida Modulation of Phytohormone Levels in Plants tion product of JA-Ile) (TA), ascorbic acid (AA), 12-Oxo Grown from the Seed phytodienoic acid (a JA precursor) (OPDA), JA isoleucine (JA-Ile), and salicylic acid (SA) were assessed by LC-MS in 0.137 To determine whether fungal endophyte seed treat leaf and root tissues separately. All phytohormone level ment affects phytohormone levels in plants grown from the comparisons were made versus plants in the uncolonized seed, tissue was harvested from the root or third true leaf of control group with significance at PK0.05. Phytohormone cotton plants inoculated with either endophytic Beauveria levels in plants grown from seed treated with Beauveria bassiana or Paecilomyces lilacinus. The experiment was bassiana are shown in Table 3, and phytohormone levels in done with three endophyte treatments (uncolonized control, plants grown from seed treated with Paecilomyces lilacinus B. bassiana or P lilacinus) and, for Beauveria bassiana, two 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 (8WCS Down at 8 hours of feeding Roots Upregulated at 48 hrs of feeding No Not significan Upregulated Yes TA (8WCS Not significan Roots Upregulated at 48 hrs of feeding No Not significan Not significant Yes AA (8WCS Down at 4 hrs up at 24 hrs Roots Op at 8 hrs down at 48 hrs No Not significan Upregulated Yes OPDA (8WCS Not significan Roots Op at 4 hrs and 8 hrs No Not significan Upregulated Yes JA-Ile (8WCS Up at 48 hrs Roots Op at 48 hrs No Not significan Upregulated Yes SA (8WCS Up at 1 hr., 8 hr, 24 and 48 hr Roots Down at 4 hr the rest n.s No Not significan Not significant US 2016/0286821 A1 Oct. 6, 2016

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 compositions for both Delta Pine and Phytogen cultivars. FIG. 23 shows increased biomass in the Fungal Endophyte Seed Treatments Alter Traits in plants treated with endophytes (right half of the image) Certain Cotton Cultivars in Field Trials compared to untreated control (left half of the image). 0140. In FIGS. 15 through 22, TAM505 is Acremonium 0.138. The 2014 field trials were executed in a similar sp., TAM32 is Epicoccum nigrum, TAM534 is Cladospo fashion as described in Example 6. A field trial using isolates rium urdinicola, TAM244 is Cladosporium sp., TAM514 is of listed below was conducted during the summer Each plot Cladosporium urdinicola, TAM474 is Cladosporium cla consisted of four 15.24 m (40 ft) rows, each separated by dosporoides, TAM554 is Chaetomium globosum, TAM15 is 101.6 cm (40 in), and there were 6 replicate plots per Exserohilum sp., TAM488 is Epicoccum nigrum, TAM452 is treatment. Yield from plots treated with the described micro Cladosporium urdinicola, TAM490 is Paecilomyces lilaci bial compositions was compared relative to the untreated nus, TAMBB is Beauveria bassiana, TAM 105 is Cochliobo control plots. For thrips, this damage assessment was on a lus sp., TAM189 is Bipolaris sp., and TAM47 is Epicoccum scale of 0-5; 0 no damage, 1-noticeable feeding scars, but nigrum. no stunting, 2-noticeable feeding and 25% stunting, 3-feed ing with blackened leaf terminals and 50% stunting, Example 16 4-severe feeding and 75% stunting, and 5-severe feeding and 90% stunting. For fleahoppers, the number of insects per Fungal Endophyte Seed Treatments Provide plant were quantified and reported as an average for each Drought Tolerance in Cotton Cultivars in plot. FIG. 14 shows the yield improvement of crops when Greenhouse Trials treated with the described microbial compositions, for Delta Pine and Phytogen cultivars, respectively. FIG. 15 shows the 0141 Cotton plants were germinated from endophyte aggregated yield improvement of the microbes across the treated and untreated control seeds in the greenhouse. All two cultivars. Bars represent 95% confidence intervals. FIG. seeds watered for 7 days or until cotyledon stage using 16A shows the beneficial effect of 12 out of 15 microbial pre-determined soil saturation volume of water per plant. At compositions tested on thrip damage in the Delta Pine 7 DAP, water was withheld from water stressed plants while cultivar. In the Phytogen cultivar, only 2 out of the 15 controls continued to be watered. Time to wilt and time to microbial compositions tested showed a benefit by reducing death were measured at a max of 21 DAP. The data in FIG. thrip damage. FIG. 16B shows the beneficial effect of 24 shows the mean time to wilt, and the data in FIG. 25 reducing fleahopper damage in the Phytogen cultivar, where shows the mean time to death. Endophyte treatment 6 out of the 15 facultative fungal endophytes tested showed increased the Survival of plants Subjected to drought stress in an average decrease in fleahopper damage as compared to both the Delta Pine (DTP) and the Phytogen (PHY) culti untreated cotton plants. In the Delta Pine cultivar, only one vars. In FIGS. 24 and 25, endophyte number 194 is Epic microbial composition showed a beneficial effect on flea Occum nigrum, 249 is Cladosporium cladosporioides, 355 is hopper damage. Chaetomium globusum, 46 is Epicoccum sp., 463 is Cla 0.139. A number of other mid-season plant traits were also dosporium sp., 534 is Cladosporium uredinicola, 554 is assessed in the field to determine the effect of the described Chaetomium globosum, 58 is Epicoccum nigrum, and con fungal endophyte compositions. FIG. 17A shows the ben trol is no endophyte treatment. eficial increase of the described microbial compositions on Example 17 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 ben Identification of Fungal Endophytes with at Least eficial increase of the described fungal endophyte compo 97% Identity to Those in Table 1 sitions on mid-season canopy temperature for both Delta 0.142 All known fungal endophytes with 97% identity to Pine and Phyton cultivars. FIG. 19 shows the beneficial SEQ ID NO:7 through SEQ ID NO:77 were identified and increase of the described fungal endophyte compositions on are listed here by accession number: FJ425672.AY526296, mid-season NDVI (Normalized Difference Vegetation JQ760047.UDB014465, KC662098.HQ649874.JQ764783, Index) for both Delta Pine and Phytogen cultivars. NDVI is EU881906.K F251285.JQ862870, AB019364AB594796, a measure of chlorophyll content. FIG. 20 shows the ben JF773666,JN034678, KC343142, EU707899, AB62 7855, eficial increase of the described fungal endophyte compo GU138704,JN695549, DQ279491, HM776417, AB361643, sitions on mid-season first-position Square retention for both DQ782839, AF222826,EU682 199,DQ782833, EU054429, Delta Pine and Phytogen cultivars. FIG. 21 and FIG. 22 FJO25275,AY354239, AF222828,GU721921,GU721920, show the modulation (up in July and down in August) of DQ0937 15. AJ309335,FR774125.JQ747741,EF042603, mid-season plant height when treated with the described KC968942, HE584924AY740158.FJ645268.HQ6925.90,

US 2016/0286821 A1 Oct. 6, 2016 beneficial properties on plant health, such as yield and/or composition may comprise seeds and any combination of past resistance, for example. In particular, endophytes from endophytes listed in Table 1. Endophytes listed in Table 1 or Table 1 are employed in a synthetic combination with a plant combinations thereof are thus employed in methods for as described herein with crop plants, such as cotton. Any preventing pest infestation, increased yield, treating a pest single or combination of endophytes listed in Table 1 can infestation, manufacturing pest-resistant seeds; or increasing also be used in this manner, employing for example seed a yield or reducing loss of a crop according to the methods coatings or foliar, soil, or rhizosphere applications. A seed of Examples 1-15.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 77

<21 Os SEQ ID NO 1 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide

<4 OOs SEQUENCE: 1

cggcggactic goccCaggcc g 21

<21 Os SEQ ID NO 2 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide

<4 OOs SEQUENCE: 2

21

<21 Os SEQ ID NO 3 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide

<4 OOs SEQUENCE: 3 Ctcagttgcc toggcgggaa

<21 Os SEQ ID NO 4 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide

<4 OOs SEQUENCE: 4

gtgcaactica gagaagaaat tcc.g 24

<21 Os SEQ ID NO 5 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide

<4 OOs SEQUENCE: 5

toctaggtg aacctgcg 18

<21 Os SEQ ID NO 6 &211s LENGTH: 2O &212s. TYPE: DNA

US 2016/0286821 A1 Oct. 6, 2016 23

- Continued

ORGANISM: Alternaria dilanthi

< 4 OOs SEQUENCE: 11

Cctic.cggact ggcticgagga ggttggcaac gaccacctica agc.cggaaag ttggtcaaac 6 O tcggtcattt agaggaagta aaagt cqtaa caatttct co gtaggtgaac Ctgcggaggg 12 O at Cattacac agg tatgaag gcgggctgga atct ct cqgg gttacagcct tctgaatta 18O ttcaccc.gtg tottttgcgt acttcttgtt toctdggtgg gttcgcc cac caccagg acc 24 O aaccataaac ctitttgtaat togcaatcago gtcagtaiaca aaataataat tacaact 297

SEQ ID NO 12 LENGTH: 249 TYPE: DNA ORGANISM: Alternaria longipes

< 4 OOs SEQUENCE: 12 tggtaattac aaaatgaagc gggctggacc tict cqgggitt acagcctgct gaatt attca 6 O c ccttgtc.tt ttgcgtactt Cttgtttcct tdgtgggttc gcc caccact aggacaaaca 12 O taaaccttitt gtaattgcaa totagogt cag taacaaatta ataattacaa ctittcaacaa 18O cggat.ct ctt ggttctggca t catgaaga acgcagdaaa ttaatgc.cgg Ctggaacgc.c 24 O tctgggata 249

SEQ ID NO 13 LENGTH 227 TYPE: DNA ORGANISM: Alternaria mali

< 4 OOs SEQUENCE: 13 atcgtggagg tdaggact at tacacatatg aaggcgggct ggalacct ct c gggittacag 6 O ccttgctgaa ttatt caccc ttgtc.ttittg cqtacttctt gtttccttgg togggttcgc.c 12 O caccac tagg acaaacataa accttttgta attgcaatca gcgtcagtaa caaattaata 18O attacaactt to cacaacgg gat ct cittgg gttctgg cat cqctago 227

SEQ ID NO 14 LENGTH: 210 TYPE: DNA ORGANISM: Alternaria Sesami

< 4 OOs SEQUENCE: 14 aggcgggctg gCacct ct cq gggtggc.cag ccttgctgaa ttatt CC acc C9tgtcttitt 6 O gcqtacttct tdtttic ctitg gtgggct cqc ccaccacaag gacca accca taalacct titt 12 O tgtaatggca at cagogt ca gtaacaatgt aataattaca actittcaa.ca acggatctot 18O tggttctggc atcgatgaag aacgcagcaa. 21 O

SEO ID NO 15 LENGTH: 256 TYPE: DNA ORGANISM: Alternaria Solani

< 4 OOs SEQUENCE: 15 atgtgt catg gtatgaggcg ggctggacct Ctcggggitta cagccttgct gaatt attca 6 O c ccttgtc.tt ttgcgtactt Cttgtttcct tdgtgggttc gcc caccact aggacaaaca 12 O

US 2016/0286821 A1 Oct. 6, 2016 38

- Continued

<4 OO > SEQUENCE: 76 ccggcgt.ccg gacggcct cq ggc.ccggacc Caggcggc.cg ccggaga cct 6 O ctaaactctg tattat cago attittctgaa ggcaaaacaa atgaatcaaa 12 O actittcaa.ca acggaacctic ttgggttt cq ggcatcgatg aagaacgcag caaatgcga 18O taagtaatgt gaattgcaga att cagtgaa t catcgaatc tittgaacgca cattgcgc.cc 24 O gccago attc tgg.cgggCat gcc titt.cga gcqt catttic aac cct cqac titc cctittgg 3OO ggaaatcc.gc gttggggaaa cggcagcata cc.cgc Cinggc CCC galaatgg gagtggcggc 360 ccggtc.ccgc ng.cgaccott Ctgcgtaagt aat CCaactic ggc accggala CCCCnacgtg 42O gccaccccing taaaaCaCCC aact tcc.gaa 451

<210s, SEQ ID NO 77 &211s LENGTH: 549 212. TYPE : DNA <213> ORGANISM: Paecillomyces lilacinus

<4 OO > SEQUENCE: 77 ggagggat.ca ttaccgagtt tacaactic Co. aaaccc.cctg tgaacttata ccattactgt 6 O tgct tcggcg ggittattgcc CC9gggalagg at agggtgcc gcgaggtgcc Ctgc.ccgc.cc 12 O cc.ccggaaac aggcgc.ccgc cggaggactic aaact ctdta ttttittcttgttt tagtgta 18O tact atctga gtaaaaaa.ca atataatgaa toaaaactitt caacaacgga t ct cittggitt 24 O Ctggcatcga tgaagaacgc agcgaaatgc gataagtaat gtgaattgca gaatt cagtg 3OO aatcatcgaa t ctittgaacg cacattgcgc cc.gc.cagtat tctggcgggc atgcctgttc 360 gagggit catt toalaccCt. Ca agc cc ctittg gacttggtgt tggggaccgg C9atggaCaa 42O actgtc.ctitt cgcc.gc.cccc taaatgacitt ggcggCCtcg tcqcggc cct cotctg.cgta 48O gtag cacaca cct cqcaa.ca ggagc.ccggc gaatggccac tgcc.gtaaaa ccc.cccaact 54 O tttitt caga 549

1. A synthetic combination of a cotton seed or plant and 7. The seed coating of claim 3, wherein the seed coating a fungal endophyte comprising at least one endophyte from is a fluid. Table 1, wherein cotton is a host plant of the endophyte. 8. The seed coating of claim 3, wherein the seed coating 2. The synthetic combination of claim 1, comprising at is a powder. 9. The seed coating of claim 3, wherein the fungal least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, endophyte comprises fungal spores. 18, 19, or 20 endophytes. 10. The seed coating of claim 3, comprising about 10, 3. A seed coating, comprising 10, 10, 10, 10, 107, 10, or 10 colony forming units per a) a fungal endophyte comprising at least one endophyte gram or Spores per gram. from Table 1; and 11. A composition for foliar or soil application, compris b) at least one coating material, wherein the fungal 1ng endophyte is in contact with the coating material. a) at least one endophyte from Table 1; and b) at least one carrier, Surfactant or diluent. 4. The seed coating of claim 3, comprising at least 1, 2, 12. The composition of claim 11, comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 endophytes. 20 endophytes. 5. The seed coating of claim 3, wherein the coating 13. The composition of claim 11, comprising about 10, material comprises alginic acid, carrageenan, dextrin, dex 10, 10, 10, 10, 107, 10, or 10 colony forming units per tran, pelgel, polyethelene glycol, polyvinyl pyrrolidone, gram or Spores per gram. methyl cellulose, polyvinyl alcohol, gelatin, or combinations 14. The composition of claim 11, wherein the composition thereof. comprises water, a detergent, Triton X, insecticides, fungi 6. The seed coating of claim 3, wherein the weight ratio cides, or combinations thereof. between fungal endophyte and coating material is 1:1-10, 15. A seed composition comprising a plant seed and the 1:10-50, 1:50-100, 1:100-500, 1:500-1000, or 1:1000-5000. seed coating of claim 11. US 2016/0286821 A1 Oct. 6, 2016 39

16. The seed composition of claim 15, wherein the plant a) providing an endophyte composition comprising at seed comprises a cotton seed. least one endophyte from Table 1; and 17. The seed composition of claim 15, wherein the plant b) applying the endophyte composition to a seed, plant or seed comprise a seed of an agronomically elite plant. part thereof, whereby the yield of the crop increases. 18. The seed composition of claim 15, wherein the plant 37. The method of claim 36, wherein the endophyte seed comprises a dicot plant seed. composition comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 19. The seed composition of claim 15, wherein the plant 12, 13, 14, 15, 16, 17, 18, 19, or 20 endophytes. seed comprises a monocot plant seed. 38. The method of claim 36, wherein the crop is cotton, 20. The seed composition of claim 15, wherein the seed and the increase of yield is at least about 5%, 15%, 20%, or composition is resistant to a pest comprising an insect and/or 25% relative to a crop to which no endophyte composition a nematode. has been applied. 21. A method for preventing pest infestation or increased 39. The method of claim 36, wherein the increase of yield yield, comprising treating a plant, plant seed, or the rhizo is about 5%, 10%, 15%, 20%, 30%, 40%, or 45% relative to sphere of said plant or seed with the composition of claim a crop to which no endophyte composition has been applied. 11. 40. The method of claim 36, wherein the crop is cotton 22. The method of claim 21, further comprising identi and the reduction of loss comprises reduced boll damage. fying said plant as in need of endophyte treatment. 41. The method of claim 36, wherein the reduction of loss 23. The method of claim 21, wherein the seed is suspected comprises reduction of loss due to insect infestation or of needing pest treatment, and the pest comprises a nema drought and the loss is less than 40%, 30%, 20%, 10%, 5%, tode and/or insect. or 5% relative to a crop to which no endophyte composition 24. The method of claim 21, wherein the seed is suspected has been applied. of needing pest treatment, and the pest comprises a root knot 42. A method for improving a trait in an agricultural plant, nematode, a aphid, alygus bug, a Stink bug, or combinations the method comprising: thereof. a) contacting an agricultural seed of said plant with a 25. A method for preventing pest infestation, comprising formulation comprising purified filamentous, spore a) obtaining a pest-resistant seed composition according forming, facultative fungal endophytes of at least one to claim 15; and species, wherein the endophytes are capable of produc b) planting the pest-resistant seed composition. ing Substances that are beneficial to plants or detrimen 26. The method of claim 25, further comprising identi tal to pests or both, and wherein the endophytes are fying a need of preventing pest infestation. present in the formulation in an amount effective to 27. The method of claim 25, wherein the pest comprises modulate the colonization frequencies of the endo a nematode and/or a insect. phytes that are native to the agricultural plant grown 28. The method of claim 25, wherein the pest comprises from the seed compared to a reference seed that is a root knot nematode, a aphid, a lygus bug, a Stink bug, or planted in an agricultural environment, and to provide combinations thereof. a benefit to the seeds or the agricultural plants grown 29. A method for treating a pest infestation, comprising from the seeds. a) identifying a plant Suspected of being infected with a 43. The method of claim 42, further comprising packag pest; ing the contacted seeds in a container. b) applying the composition of claim 11 to the plant, 44. The method of claim 42, further comprising growing whereby an endophyte-treated plant is generated. a population of said agricultural plants, wherein the benefit is measured at the level of the population, as compared to a 30. The method of claim 29, wherein the pest comprises reference population of plants. a nematode and/or an insect. 45. The method of claim 42, wherein the endophytes that 31. The method of claim 29, wherein the pest comprises are native to the agricultural plant are of genus Alternaria. a root knot nematode, a aphid, a lygus bug, a Stink bug, or 46. The method of claim 42, wherein the benefit is an combinations thereof. improved agronomic property selected from the group con 32. A method of manufacturing pest-resistant seeds, com sisting of increased biomass, increased tillering, increased prising root mass, increased flowering, increased yield, increased a) providing an endophyte composition comprising at water use efficiency, reduction of yield loss, altered plant least one endophyte from Table 1: height, decreased time to emergence, increased seedling b) providing seeds; and height, increased root length, increased chlorophyll levels, c) combining the seeds with the endophyte composition, retention of developing flowers, retention of developing whereby pest-resistant seeds are generated. fruits, altered phytohormone levels, and enhanced resistance 33. The method of claim 32, wherein the endophyte to environmental stress relative to a reference plant. composition comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 47. The method of claim 46, wherein the benefit to 12, 13, 14, 15, 16, 17, 18, 19, or 20 endophytes. agricultural plants derived from the seed is increased yield 34. The method of claim 32, further comprising sterilizing in a population of said plants by about 5%, 10%, 15%, 20%, the seeds to remove microorganisms prior to combining the 30%, 40%, or 45% relative to a reference population of seeds with the endophyte composition. plants. 35. The method of claim 32, wherein the method increases 48. The synthetic combination of claim 46, wherein the the percentage of colonization with the endophyte of the benefit to agricultural plants derived from the seed is a plant developing from the seed. reduction of yield loss in a population of said plants by more 36. A method of increasing a yield or a reduction of loss than 40%, 30%, 20%, 10%, 5%, or 1% relative to a reference of a crop, comprising population of plants. US 2016/0286821 A1 Oct. 6, 2016 40

49. The method of claim 46, wherein the environmental 56. The synthetic combination of claim 54, wherein the stress is selected from the group consisting of drought stress, benefit is an improved agronomic property selected from the cold stress, heat stress, nutrient deficiency, salt toxicity, group consisting of increased biomass, increased tillering, aluminum toxicity, grazing by herbivores, insect infestation, increased root mass, increased flowering, increased yield, nematode infection, and fungal infection, bacterial infection increased water use efficiency, reduction of yield loss, and viral infection. altered plant height, decreased time to emergence, increased 50. The method of claim 42, wherein the endophyte is seedling height, increased root length, increased chlorophyll present in the synthetic combination in an amount effective levels, retention of developing flowers, retention of devel to obtain at least 50% colonization of the leaves, stems or oping fruits, altered phytohormone levels, and enhanced roots of an agricultural plant grown from the seed. resistance to environmental stress relative to a reference 51. The method of claim 42, wherein the pest comprises plant. a nematode and/or an insect. 57. The synthetic combination of claim 54, wherein the 52. The method of claim 42, wherein the pest comprises environmental stress is selected from the group consisting of a root knot nematode, a aphid, a lygus bug, a Stink bug, or drought stress, cold stress, heat stress, nutrient deficiency, combinations thereof. salt toxicity, aluminum toxicity, grazing by herbivores, 53. The method of claim 42, wherein the synthetic com insect infestation, nematode infection, and fungal infection, bination comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, bacterial infection and viral infection. 13, 14, 15, 16, 17, 18, 19, or 20 facultative fungal endo phytes. 58. The synthetic combination of claim 54, wherein the 54. A synthetic combination of an agricultural plant seed endophyte is present in the synthetic combination in an and a composition comprising purified filamentous, spore amount effective to obtain at least 50% colonization of the forming facultative fungal endophytes of at least one spe leaves, stems or roots of an agricultural plant grown from the cies, wherein the endophytes are capable of producing seed. substances that are beneficial to plants or detrimental to 59. The synthetic combination of claim 13, comprising at pests or both, and wherein the facultative fungal endophytes least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, are present in the synthetic combination in an amount 18, 19, or 20 strains of endophytes. effective to modulate the colonization frequencies of endo 60. The synthetic combination of claim 56, wherein the phytes that are native to the agricultural plant grown from benefit to agricultural plants derived from the seed is the seed compared to a reference seed that is planted in an increased yield in a population of said plants by about 5%, agricultural environment, and to provide a benefit to the 10%, 15%, 20%, 30%, 40%, or 45% relative to a reference seeds or the agricultural plants grown from the seeds. population of plants. 55. The synthetic combination of claim 54, wherein the facultative fungal endophytes are present in the synthetic 61. The synthetic combination of claim 56, wherein the combination in an amount effective to modulate the coloni benefit to agricultural plants derived from the seed is a Zation frequencies of endophytes of genus Alternaria that reduction of yield loss in a population of said plants by more are native to the agricultural plant grown from the seed than 40%, 30%, 20%, 10%, 5%, or 1% relative to a reference compared to a reference seed that is planted in an agricul population of plants. tural environment.