US008975489B2

(12) United States Patent (10) Patent No.: US 8,975.489 B2 Craven (45) Date of Patent: Mar. 10, 2015

(54) GRASS FUNGAL ENDOPHYTES AND USES (56) References Cited THEREOF U.S. PATENT DOCUMENTS

(75) Inventor: Kelly Craven, Ardmore, OK (US) 5,880,343 A ck 3, 1999 Hiruma et al...... 800,320 2010.0024.076 A1 1/2010 Craven (73) Assignee: The Samuel Roberts Noble Foundation, Ardmore, OK (US) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this Kawait (GenBank ACCSSO ion No.NO AB518683.1, firstSawaale availabl O li patent is extended or adjusted under 35 Dashtban et al (Int J Biochem Mol Biol 1(1):36-50, first available U.S.C. 154(b) by 218 days. online May 23, 2010).* Chen et al (Functional Plant Biology, 2004, 31, 235-245).* (21) Appl. No.: 13/306,841 Dien etal (Bioenerg. Res. (2009) 2:153-164).* y x- - - 9 USDA Technical Notes, Agronomy #35, NRCS-Iowa, published Jan. 2009.* (22) Filed: Nov. 29, 2011 Gardes et al. "ITS primers with enhanced specificity for Basidimycetes—application to the identification if mycorrhizae and (65) Prior Publication Data rusts.” Molec Ecol. 2: 113-118, 1993. Ghimire etal. “Biodiversity of fungalendophyte communities inhab US 2012/O144,533 A1 Jun. 7, 2012 iting Switchgrass (Panicum virgatum L.) growing in the native tallgrass prairie of northern Oklahoma.” Fungal Diversity. 47(1), 19-27, 2010. Related U.S. Application Data Khu et al., “QTL mappig of aluminum tolerance intetraploid alfalfa.” AV Joint Meeting of the 41 North American Alfalfa Improvement Con (60) Provisional application No. 61/419.242, filed on Dec. ference & 20' Trifolium Conference, Dallas, Texas, Jun. 2, 2008 2, 2010. (Abstract). Martin et al. "Fungal-specific PCR primers developed for analysis of (51) Int. Cl. the ITS region of environmental DNA extracts.” BMC Microbiol. AOIH 5/00 (2006.01) st Arsenal hizal associates of 39th Ko C Ericaceae.”elosse et al.,New SepinalesPhytologist, are 174(4):864-878, common mycorrhizal 2007. associates o ( .01) White et al. "Amplification and direct sequencing of fungal AOIN 65/44 (2009.01) ribosomal RNA genes for phylogenetics.” In: PCR Protocols: Aguide CI2N L/4 (2006.01) to methods and applications (eds. Innis MA, Gelfand DH. Sninsky J.J. A23K L/00 (2006.01) White TJ). Academic Press Inc., New York, 1990. AOIN 63/04 (2006.01) CIOL I/02 (2006.01) * cited by examiner CI2P 7/10 (2006.01) CI2R L/645 (2006.01) Primary Examiner — David T Fox (52) U.S. Cl. Assistant Examiner — Lee A Visone CPC ...... A23K I/009 (2013.01): A0IN 63/04 (74) Attorney, Agent, or Firm — Dentons US LLP (2013.01); A23K I/006 (2013.01); CIOL I/02 (2013.01); CIOL I/023 (2013.01); C12P 7/10 (57) ABSTRACT (2013.01); CI2R I/645 (2013.01); Y02E 50/16 The invention provides isolated fungal endophytes and Syn (2013.01); Y02E 50/30 (2013.01) thetic combinations thereof with host grass plants. Methods USPC ...... 800/320:435/254.1; 435/267; 435/161; for inoculating grass plant with the endophytes, for propagat 504/117: 800/278; 800/289: 800/320.1; 800/320.2: ing the grass-endophyte combinations, and for producing 8OO/32O3 feeds and biofuels from grass-endophyte combinations are (58) Field of Classification Search also provided. None See application file for complete search history. 31 Claims, No Drawings US 8,975,489 B2 1. 2 GRASS FUNGAL ENDOPHYTES AND USES may be selected from those provided in Table 2 or may be THEREOF defined as comprising a ribosomal DNA (rDNA) sequence comprising one of the sequences of SEQID NOs: 1-555. CROSS-REFERENCE TO RELATED Inafurther embodiment, the invention provides a synthetic APPLICATIONS 5 combination of a grass plant and a fungal endophyte provided herein. In one embodiment, the endophyte comprises a rDNA This application claims the priority of U.S. Provisional sequence selected from the group consisting of SEQID NOs: Application No. 61/419.242, filed Dec. 2, 2010, the entire 1-555. In certain aspects, the fungal endophyte may primarily colonize a root or stem tissue of the plant. In further aspects, disclosure of which is incorporated herein by reference. 10 the synthetic combination may comprise two or more differ STATEMENT REGARDING FEDERALLY ent fungal endophytes. For example, a grass plant may com SPONSORED RESEARCH ORDEVELOPMENT prise at least a first fungal endophyte that colonizes a root tissue and at least a first fungal endophyte that colonizes a This invention was made with government Support under stem tissue. In some aspects, a grass plant comprises two, grant number DE-AC05-00OR22725 awarded by the U.S. 15 three, four, five, six, or more different endophytes, such as Department of Energy. The United States government has bacterial endophytes or fungal endophytes, including one or certain rights in the invention. more of those provided in Table 2. In certain embodiments, a synthetic combination accord INCORPORATION OF SEQUENCE LISTING ing to the invention comprises an agronomically elite grass 2O plant and a fungal endophyte. For example, the grass plant The Sequence Listing, which is a part of the present dis may comprise one or more agronomically elite traits, such as closure, includes a computer readable 473 KB file entitled drought resistance, insect resistance, fungus resistance, virus “NBLE076US ST25.txt comprising nucleotide and/or resistance, bacteria resistance, cold tolerance, salt tolerance, amino acid sequences of the present invention Submitted via increased yield, enhanced nutrient use efficiency, increased EFS-Web. The subject matter of the Sequence Listing is 25 fermentable carbohydrate content, reduced lignin content, or incorporated herein by reference in its entirety. resistance to biotic or abiotic stress. In certain aspects, the grass plant may comprise a transgene. Such as a transgene that BACKGROUND OF THE INVENTION confers an agronomic trait. For example, a transgene may confer herbicide tolerance, drought resistance, insect resis 1. Field of the Invention 30 tance, fungus resistance, virus resistance, bacteria resistance, The invention relates to fungal endophytes of host plants, male sterility, cold tolerance, salt tolerance, increased yield, such as grass plants. In particular, the invention relates to enhanced nutrient use efficiency, increased fermentable car prairie grass endophytes and combinations of these endo bohydrate content, or reduced lignin content. Grass plants phytes with agronomically elite grass plants. that may be used in the synthetic endophyte combinations 2. Description of the Related Art 35 according to the invention include, but are not limited to, Endophytes are fungal or bacterial organisms that live Switchgrass (Panicum virgatum), wheat, durum wheat, tall within plants. Fungal endophytes, such as mycorrhiza, Sur wheatgrass, western wheatgrass, maize, rice, Sorghum, Vive within various host plant tissues, often colonizing the meadow fescue, tall fescue, cereal rye, Russian wild rye, oats, intercellular spaces of host leaves, stems, flowers or roots. bermudagrass, Kentucky bluegrass, big bluestem, little The symbiotic endophyte-host relationships can provide sev- 40 bluestem, Miscanthus sp., Miscanthusxgiganteus, blue eral fitness benefits to the host plant, Such as enhancement of grama, black grama, side-oat grama, johnsongrass, buffa nutrition, increased drought tolerance and/or chemical lograss, creeping bentgrass, or Sugarcane. In certain aspects, defense from potential herbivores and often enhanced biom the host plant is a forage grass host plant. In one embodiment, ass production. Root-colonizing mycorrhizae Survive on pho the host plant is Switchgrass (Panicum virgatum). tosynthetic carbohydrates from the plant, and in return, aid in 45 In some further embodiments, combinations of grass the solublization and uptake of water and minerals to the host, plants and fungal endophytes according to the invention dis which can lead to the promotion of seed germination and play increased biomass, enhanced drought tolerance, plant growth. Additionally, the association of a fungal endo increased nitrogen use efficiency, increased phosphorus uti phyte with a host plant often provides protection from patho lization, disease resistance, and/or increased vigor relative to gens or tolerance to a variety of biotic and abiotic stresses, 50 a host grass plant of the same genotype that lacks the endo Such as insect infestation, grazing, water or nutrient defi phyte, when grown under the same conditions. In another ciency, heat stress, Salt or aluminum toxicity, and freezing aspect, the endophyte protects the hostgrass plant from biotic temperatures. Host growth and fitness promotion and protec stresses such as insect infestation, nematode infestation, and tion are thought to be achieved through multiple beneficial herbivore grazing, and/or abiotic stresses, such as water defi properties of the endophyte-host association. For instance, 55 ciency, nutrient deficiency, heat stress, fungal infection, salt the endophytic organisms may produce growth-regulating toxicity, aluminum toxicity, heavy metal toxicity, and freez Substances to induce biomass production and alkaloids or ing temperatures. other metabolites that have anti-insect and anti-herbivore In certain embodiments, the host grass plant is artificially properties. Additionally, fungal endophytes may directly Sup inoculated with the endophyte. The endophyte-host combi press or compete with disease-causing microbes, protecting 60 nation may be achieved, for example, by introduction of the the plant from potential pathogens. endophyte to the host grass plant by a method selected from the group consisting of inoculation, infection, grafting, and SUMMARY OF THE INVENTION combinations thereof. In a yet another embodiment, the invention provides a seed In a first embodiment, the invention provides an isolated 65 comprising a grass plant embryo and a fungal endophyte fungal endophyte that has been initially identified from a (e.g., an endophyte comprising a rNA sequence selected prairie grass species. For example, the isolated endophyte from the group consisting of SEQID NOs: 1-555). In certain US 8,975,489 B2 3 4 aspects, a fungal endophyte according to the invention is mercial product therefrom. For example, a plant or plant part provided into or onto the exterior of the seed. In still yet described herein can be manufactured into a product Such as another aspect, the invention relates to a method for propa paper, paper pulp, ethanol, biodiesel, Silage, animal feed, or gating a hostgrass plant-fungal endophyte combination com fermentable biofuel feedstock. prising: a) obtaining a synthetic combination of a fungal In yet another embodiment, the invention provides a endophyte and a host grass plant, and b) vegetatively repro method of producing ethanol comprising: (a) obtaining a ducing the hostgrass plant tissue colonized by the endophyte. grass plant comprising a fungal endophyte according to the In still yet another embodiment, the invention provides a invention; (b) treating tissue from the plant to render carbo method for cultivating a hostgrass plant comprising: contact hydrates in the tissue fermentable; and (c) fermenting the ing the host grass plant or a seed (or other propagating mate 10 rial) that produces the plant with a fungal endophyte, Such carbohydrates to produce ethanol. that the endophyte colonizes the plant. In one aspect, coloni In yet another embodiment, the invention provides a zation of the hostgrass is achieved by a method selected from method for processing lignocellulosic biomass from a plant the group consisting of inoculation, infection, grafting, and or plant part described herein. In one embodiment, the combinations thereof. In another aspect, the host grass plant 15 method for processing lignocellulosic biomass from a has enhanced root growth, more tillers, enhanced total biom plant or plant part may comprise acid and/or enzymatic treat ass, or enhanced seed yield relative to a hostgrass plant of the ment(s). The enzymatic treatment(s) may comprise treatment same genotype that lacks the endophyte, when grown under with one or more cellulolytic enzymes, such as a cellulase. In the same conditions. In yet other aspects, the host grass plant another embodiment, the method comprises an acid treatment displays tolerance to stress as relative to a hostgrass plant of prior to or during a treatment to render carbohydrates in the the same genotype that lacks the endophyte, when grown plant fermentable. In yet another embodiment, no acid treat under the same conditions. The stress may be selected from ment is performed. the group consisting of a biotic stress, a pest stress, an insect The use of the word “a” or “an' when used in conjunction stress, an abiotic stress, and a water deficit stress. In one with the term “comprising in the claims and/or the specifi embodiment, the stress may be biotic stress caused by at least 25 cation may mean "one.” but it is also consistent with the one organism selected from the group consisting of a mam meaning of “one or more.” “at least one and “one or more malian or insect herbivore, or a microbial pathogen (e.g., than one.” nematode, fungus, bacteria, or virus). In a further aspect, the Other objects, features, and advantages of the present stress is abiotic stress selected from the group consisting of invention will become apparent from the following detailed water deficiency, nutrient deficiency, heat stress, Salt toxicity, 30 description. It should be understood, however, that the aluminum toxicity, heavy metal toxicity, and freezing tem detailed description and the specific examples, while indicat peratures. ing specific embodiments of the invention, are given by way In still yet another embodiment, the invention provides a of illustration only, since various changes and modifications method for cultivating a hostgrass plant comprising: contact within the spirit and scope of the invention will become ing the host grass plant or a seed thereof with a filtrate of a 35 apparent to those skilled in the art from this detailed descrip cultured fungal endophyte strain, wherein the plant has tion. enhanced root growth, more tillers, enhanced total biomass, or enhanced seed yield or germination relative to a host grass DETAILED DESCRIPTION OF THE INVENTION plant of the same genotype that lacks the filtrate, when grown under the same conditions. In one aspect, the host grass plant 40 Endophytic fungi are ubiquitous in nature, infecting virtu displays tolerance to stress relative to a hostgrass plant of the ally all plants in both natural and agronomic ecosystems. same genotype that lacks the endophyte, when grown under Provided herein are endophytic fungi initially identified from the same conditions, wherein the stress is selected from the Switchgrass and having utility for improving the phenotype of group consisting of a biotic stress, a pest stress, an insect a grass plant. Given the constraints involved in producing a stress, an abiotic stress, and a water deficit stress. 45 bioenergy crop, for example, these beneficial endophytes can In another embodiment, the invention relates to a method be used to maximize the Sustainability and minimize the for increasing the biomass of a plant or increasing the fer economic cost of growing biofuel crops, such as Switchgrass. mentable biomass of a plant comprising: contacting the host Novel combinations of fungalendophytes with grasses can be grass plant with an endophyte provided herein, such that the used to enhance agronomic characteristics of grass, Such as endophyte colonizes the plant, wherein the plant exhibits 50 nutrient use efficiency and stress tolerance, as well as to increased biomass relative to a host grass plant of the same increase yield. Combining grass species with fungal endo genotype that lacks the endophyte, when grown under the phytes thus represents a technique that can be used in parallel same conditions. with plant breeding and transgenic technologies to improve In a further embodiment, the invention provides a com yield from grass crops and reduce the cost of cellulosic bio modity product obtained from a plant comprising a synthetic 55 fuel production. combination of the invention, such as comprising an endo Thus, in one aspect, the invention provides a combination phyte with a rDNA sequence selected from the group consist (also termed a “symbiotum) of a host plant and an endophyte ing of SEQID NOs: 1-555. For example, a commodity prod that allows for improved agronomic properties of host plants. uct may be an animal feed, a biofuel (e.g., ethanol or The combination may be achieved by artificial inoculation, biodiesel), a paper or paper pulp, Silage, or a fermentable 60 application, or other infection of a host plant, such as a grass biofuel feedstock. In certain aspects, the invention provides plant, or host plant tissues, with a fungal endophyte Strain of an isolated nucleic acid molecule comprising a sequence the present invention. Thus, a combination achieved by Such selected from the group consisting of SEQID NOs: 1-555. an inoculation is termed a “synthetic' combination. The fun In still a further embodiment, there is provided a method gal endophyte may be present in intercellular spaces within for the manufacture of a commercial product comprising 65 plant tissue. Such as the root. Its presence may also occur or obtaining a grass plant or grass plant part comprising a fungal may also be maintained within a grass plant or plant popula endophyte according to the invention and producing a com tion by means of grafting or other inoculation methods. US 8,975,489 B2 5 6 These endophytes may also be introduced or maintained by presence of an endophyte strain of the present invention in a Such procedures, into various grasses, such as Switchgrass, given sample of grass plant tissue. Additionally, methods for wheat (Triticum aestivum), durum wheat (Triticum turgidium identification may include microscopic analysis, Such as root ssp. durum), tall wheatgrass (Thinopyrum ponticum), western staining, or culturing methods, such as grow out tests or other wheatgrass (Pascopyrum Smithii), maize (Zea mays), rice 5 methods known in the art (Deshmukh et al. 2006). In particu (Oyrza sativa), Sorghum (Sorghum bicolor), meadow fescue lar embodiments, the roots of a potential grass plant-endo (Festuca pratensis), tall fescue (Festuca arundinacea), cereal phyte combination may be stained with fungal specific stains, rye (Secale cereale), Russian wild rye (Psathyrostachys jun such as WGA-Alexa 488, and microscopically assayed to cea), oats (Avena sativa), bermudagrass (Cynodon dactylon), determine fungal root associates, as described below. Kentucky bluegrass (Poa pratensis), big bluestem (Andro 10 pogon gerardii), little bluestem (Schizachyrium scoparium), DEFINITIONS blue grama (Bouteloua gracilis), black grama (Bouteloua eriopoda), side-oat grama (Bouteloua curtipendula), Agronomically elite plants: Refers to a genotype or cultivar johnsongrass (Sorghum halepense), buffalograss (Buchloe with a phenotype adapted for commercial cultivation. Traits dactyloides), and creeping bentgrass (Agrostis stolonifera). 15 comprised by an agronomically elite plant may include bio In one embodiment, the host plant is defined as a monocot. In mass, carbohydrate, and/or seed yield; biotic or abiotic stress an additional embodiment, the host plant is a forage grass host resistance, including drought resistance, insect resistance, plant or a cereal. In a particular embodiment, the host plant is fungus resistance, virus resistance, bacteria resistance, cold a grass host plant such as Switchgrass (Panicum virgatum). tolerance, and salt tolerance; improved standability, Endophytes for use according to the invention include any enhanced nutrient use efficiency, and reduced lignin content. of those provided in Table 2 below. For example, the endo Biofuel crop species: A plant that may be used to provide phyte may be an endophyte of the Hypocreales order, such as biomass for production of lignocellulosic-derived ethanol. the clavicipitaceous, seed-borne Neotyphodium endophytes, Examples of Such plants include Switchgrass (Panicum vir Fusarium spp. or Acremonium spp. Thus, a combination gatum), giant reed (Arundo donax), reed canarygrass according to the invention may comprise a grass plantandone 25 (Phalaris arundinacea), MiscanthusXgiganteus, Miscanthus of the Acremonium strictum endophytes represented Table 2. sp., sericea lespedeza (Lespedeza cuneata), corn, Sugarcane, In certain embodiments, the agronomic qualities for Sorghum, millet, ryegrass (Lolium multiflorum, Lolium sp.), improvement may be selected from the group consisting of timothy, Kochia (Kochia scoparia), Sunn hemp, kenaf, bahia increased biomass, increased tillering, increased root mass, grass, bermudagrass, dallisgrass, pangolagrass, big bluestem, increased flowering, increased seed yield, and enhanced 30 indiangrass, fescue (Festuca sp.), Dactylis sp., Brachypodium resistance to biotic and/or abiotic stresses, each of these quali distachyon, Smooth bromegrass, orchardgrass, Kentucky ties being rated in relation to plants of the same genotype bluegrass, and fonio (Digitaria sp.), among others. grown under the same conditions, and differing only with Biomass: The total mass or weight, at a given time, of a respect to the presence or absence of a fungal endophyte. The plant or population of plants, usually given as weight per unit stresses may include, for instance, drought (water deficit), 35 area. The term may also refer to all the plants or species in the cold, heat stress, nutrient deficiency, salt toxicity, aluminum community (community biomass). toxicity, heavy metal toxicity, grazing by herbivores, insect Culture filtrate: Broth or media obtained from cultures infestation, nematode infection, and fungal infection, among inoculated with a strain of fungi and allowed to grow. The others. In a particular embodiment, the enhanced resistance is media is typically filtered to remove any Suspended cells, provided by the endophyte and protects the host plant from 40 leaving the nutrients, hormones, or other chemicals. Subsequent infection by other fungal diseases, such as root Endophyte: An organism capable of living within a plant rot, powdery mildew, Fusarium blight, Pythium blight, leaf cell. An endophyte may refer to a fungal organism that may spot, rust, and Snow mold, among others. This resistance may confer an increase in yield, biomass, resistance, or fitness in allow for improved biomass or seed yield relative, for its host plant. Fungal endophytes may occupy the intracellu instance, to grass plants not colonized by an endophyte. In 45 lar or extracellular spaces of plant tissue, including the leaves, another embodiment, the invention may be defined as a grass stems, flowers, or roots. plant seed in combination with an endophyte strain or coated Genotype: The genetic constitution of a cell or organism. with a fungal endophyte Strain of the present invention. Host plant: Any plant which an endophytic fungicolonizes. The invention also relates to methods for protecting grass Increased yield: An increase in biomass or seed weight, plants from biotic or abiotic stress, by means of introducing 50 seed size, seed number per plant, seed number per unit area, an endophyte strain of the present invention into a grass plant, bushels per acre, tons per acre, kilo per hectare, or carbohy and propagating the plant-endophyte combination by vegeta drate yield. tive means. Vegetative propagation of the plant allows for Phenotype: The detectable characteristics of a cell or propagation of the combination, since fungal propagules organism, which characteristics are the manifestation of gene (e.g., mycelia, conidia, and chlamydospores) are present in or 55 expression. on plant tissue, or may infect the plant tissue. Regeneration: The process of growing a plant from a plant The invention also provides methods for detecting the pres cell (e.g., plant protoplast, callus, or explant). ence of a fungal endophyte of the present invention within a Synthetic combination: A combination (also termed a host plant. This may be accomplished, for instance, by isola “symbiotum) of a host plant and an endophyte. The combi tion of total DNA from tissues of a potential plant-endophyte 60 nation may beachieved, for example, by artificial inoculation, combination, followed by PCR, or alternatively, Southern application, or other infection of a host plant, such as a grass blotting, western blotting, or other methods known in the art, plant, or host plant tissues with an endophyte. to detect the presence of specific nucleic or amino acid Transgene: A segment of DNA which has been incorpo sequences associated with the presence of a fungal endophyte rated into a host genome or is capable of autonomous repli strain of the present invention (Selosse et al. 2007). Alterna 65 cation in a host cell and is capable of causing the expression tively, biochemical methods such as ELISA, HPLC, TLC, or of one or more coding sequences. Exemplary transgenes will fungal metabolite assays may be utilized to determine the provide the host cell, or plants regenerated therefrom, with a US 8,975,489 B2 7 8 novel phenotype relative to the corresponding non-trans TABLE 1 formed cell or plant. Transgenes may be directly introduced into a plant by genetic transformation, or may be inherited Sample collection sites. from a plant of any previous generation which was trans Location Coordinates formed with the DNA segment. Transgenic plant: A plant or progeny plant of any Subse D Lat. Lon. quent generation derived therefrom, wherein the DNA of the E1 36.38.38 96.23.47 plant or progeny thereof contains an introduced exogenous E 36.38.39 96.23.50 DNA segment not naturally present in a non-transgenic plant E 36.38.39 96.23.47 10 E 36.38.40 96.23.52 of the same strain. The transgenic plant may additionally E 36.41.39 96.20.02 contain sequences which are native to the plant being trans E 36.4140 96.20.01 E 36.4140 96.20.02 formed, but wherein the “exogenous gene has been altered in E 36.4141 96.20.00 order to alter the level or pattern of expression of the gene, for E 36.4.4.11 96.11.13 example, by use of one or more heterologous regulatory or 15 E10 36.4.4.12 96.11.12 other elements. E11 36.4.4.13 96.11.11 E12 36.44.16 96.11.12 E13 36.4431 96.21.50 EXAMPLES E14 36.44.32 96.21.50 E15 36.44.32 96.21.49 E16 36.44.33 96.21.49 The following examples are included to demonstrate pre E17 36.45.10 96.21.47 ferred embodiments of the invention. It should be appreciated E18 36.45.10 96.21.46 by those of skill in the art that the techniques disclosed in the E19 36.45.10 96.21.46 examples which follow represent techniques discovered by E20 36.45.10 96.21.47 E21 36.45.23 96.22.56 the inventors to function well in the practice of the invention, E22 36.45.24 96.22.56 and thus can be considered to constitute preferred modes for 25 E23 36.45.24 96.22.56 its practice. However, those of skill in the art should, in light E24 36.45.25 96.22.56 E25 36.45.45 96.10.27 of the present disclosure, appreciate that many changes can be E26 36.45.45 96.10.29 made in the specific embodiments which are disclosed and E27 36.45.45 96.10.26 still obtain a like or similar result without departing from the E28 36.45.47 96.10.26 concept, spirit and scope of the invention. More specifically, 30 E29 36.45.48 96.10.26 E30 36.46.08 96.23.24 it will be apparent that certain agents which are both chemi E31 36.46.08 96.2326 cally and physiologically related may be substituted for the E32 36.46.08 96.2326 agents described herein while the same or similar results E33 36.46.09 96.23.24 would be achieved. All such similar substitutes and modifi E34 36.47.10 96.23.49 35 E35 36.47.10 96.23.49 cations apparent to those skilled in the art are deemed to be E36 36.47.10 96.23.48 within the spirit, scope and concept of the invention as defined E37 36.47.11 96.23.49 by the appended claims. E38 36.47.56 96.24.48 E39 36.47.56 96.24.48 E40 36.47.57 96.24.48 Example 1 E41 36.47.57 96.24.48 40 E42 36.48.39 96.26.03 Isolation of Wild Grass Endophytes E43 36.48.39 96.26.03 E44 36.48.39 96.26.02 E45 36.48.40 96.26.02 Plant samples were collected from Alfalfa, Grant, Kay, and E46 36.48.48 96.26.22 Osage counties of Northern Oklahoma. The sampling points E47 36.48.48 96.26.23 were located between the GPS coordinates of 36°38'38" to 45 E48 36.48.48 96.26.23 36°48'48" N latitude and 96°10'26" to 08°16'11" W longitude W1 36.48.44 97.18.15 W2 36.48.44 97.18.15 (Table 1). This region has a Sub-humid continental climate, W3 36.48.44 97.18.13 with a mean annual temperature of 15° C. and a growing W4 36.48.40 97.27.14 season of 177 to 220 days. Mean annual precipitation ranges W5 36.48.40 97.27.14 from 76 to 112 cm, of which more than 70% usually falls in 50 W6 36.48.37 97.31.34 W7 36.48.37 97.31.37 April through October (USDA 1985; USDA 2007a; USDA W8 36.48.37 97.31.40 2007b; USDA 2008). Although all four counties studied are W9 36.4.842 97.4041 part of the North American tallgrass prairie, Sampling sites W10 36.484.1 98.01.19 from Alfalfa, Grant, and Kay counties were from quite diverse W11 36.48.46 97.4041 55 W12 36.484.1 98.01.19 habitats (e.g., grassland, pasture, lake shores, salt plains, or W13 36.4.842 98.01.18 marshy land). Conversely, those from Osage county were W14 36.484.1 98.01.15 predominantly composed of natural grassland from the W15 36.48.38 98.0459 Tallgrass Prairie Preserve (TGPP). Since 1989, the Nature W16 36.48.36 98.OS.OO W17 36.48.36 98.05:01 Conservancy has owned and managed the TGPP by recreat W18 36.45.01 98.09:42 ing a semi-natural grazing and disturbance regime with bison 60 W19 36.45.04 98.09.44 herds and a stochastic fire regime (Hamilton 1996). In addi W2O 36.45.04 98.0745 tion to habitat differences, Alfalfa, Grant, and the western part W21 36.45.06 98.07.43 W22 36.45.06 98.07.44 of Kay counties receive significantly lower average annual W23 36.41.04 98.12.09 precipitation than Osage county (82 cm versus 112 cm). Sam W24 36.41.04 98.12.10 pling sites from Alfalfa, Grant, and Kay counties are hence 65 W25 36.40.51 98.14.07 forth referred to as the west part and those from Osage county W26 36.40.52 98.14.08 are referred to as the east part of the study area. US 8,975,489 B2 9 10 TABLE 1-continued maintained in the greenhouse. Root and shoot samples were cut into 3-4 cm pieces prior to rigorous Surface sterilization Sample collection sites. (95% ethanol for 30s, 70% ethanol for 5 min followed by 3% sodium hypochlorite for 25-30 min). Surface sterilized tissue Location Coordinates were rinsed three times with sterile water, blot dried, cut into small pieces (1-1.5 cm) and plated on PDA plates amended ID Lat. Lon. with 100 ppm ampicillin sodium salt, 50 ppm chlorampheni W27 36.40.52 98.14.08 col, and 50 ppm streptomycin sulfate. Plates were incubated W28 36.42.35 98.16.10 W29 36.42.34 98.16.11 in the dark for up to two months at 24° C. and examined W30 36.42.34 98.16.11 10 regularly for emerging fungal colonies. Emerging fungal W31 36.42.28 98.16.10 colonies were passed through two rounds of Subculture prior W32 36.42.25 98.16.10 to preparing agar Slants for long-term storage and collecting W33 36.42.22 98.16.08 W34 36.42.23 98.16.11 fungal materials for DNA extraction. W35 36.42.22 98.16.08 15 Example 2 Plant samples were collected during early vegetative, full Characterization of Isolated Endophytes reproductive, and senescence stages of switchgrass growth in the months of April, July, and October of 2009, respectively. Fungal material for DNA extraction was harvested from 1 Each sampling consisted of up to 83 whole plant samples (5 to to 2 week-old cultures grown on potato dextrose agar (PDA) 10 tillers per sample) from different parts of the tallgrass by cutting an agar block of 1.5 cm. Agar blocks were placed prairie that included at least 35 samples each from the east and in a 1.5 ml micro-tube with a single 4.5 mm stainless steel west. GPS coordinates were recorded for each sampling site bead. These micro-tubes were arranged in a rack and covered in the April sampling, and the same general coordinates with an AirPore(R) filter, stored at -80° C. overnight and (within the same field) were used for Subsequent samplings. lyophilized for 24 h. The DNA was extracted from lyo More than 210 total plant samples were collected and all 25 philized tissue using QIAGEN MagAttract.R 96 DNA Plant samples were processed for shoot and root inhabiting endo Core Kit according to the manufacturers instructions. phytic fungal communities. Twenty-four representative soil The internal transcribed spacer (ITS) regions of fungal samples from these GPS locations, 12 each from the east and ribosomal DNA (rDNA) are highly variable in sequence, and west parts of prairie were collected and analyzed for pH, thus of great importance in distinguishing fungal species organic matter, phosphorus, potassium, calcium, magnesium, 30 (White et al. 1990). The fungal-specific primers ITS 1F and and sodium content. ITS4, amplifying the highly variable ITS 1 and ITS2 Collected plants with approximately 25-35 cm of both sequences Surrounding the 5.8S-coding sequences, were used above- and below-ground tissues were transported to the in this study. These primer sets have been used widely laboratory on ice and processed within 24 h of collection. (Gardes & Bruns 1993; Martin & Rygiewicz, 2005) and are Each plant sample was divided into two parts. The roots from 35 thus well represented in the NCBI nucleotide database. PCR the first half were harvested immediately for endophyte iso primers were used to sequence the purified PCR products as lation, while the second half was cutback at 10-12 cm above described previously (Puckette et al. 2009). Gene sequences ground level, transplanted into 3.8 L containers with Metro were manually inspected, edited, and appended into contigs mix 350 and maintained in the greenhouse for six weeks prior using DNA sequence assembly software Sequencher R ver to endophyte isolation from shoot tissues. This was done to 40 sion 4.9 (Gene Code Corporation, Ann Arbor, Michigan). minimize excessive saprophytic fungal contamination. Test sequences were compared to the NCBI fungal database Processing of the root tissues involved thorough rinsing of and the top three hits with >99% sequence similarities were multiple roots (5-10/plant), with tap water to remove excess used to determine the identity of test isolates to the deepest soil. The basal portion of shoots was collected from the plants possible taxonomic resolution. TABLE 2 Identification of fungal endophytes by rBNA sequence. SEQ Plant ID NO Isolate ID Location* Part Initial ID (Accession No.)

1 April.09 S OO 13ES1 W 13 Shoot Fusarium ngamai (X94174) 2 April.09 S OO 18ES1 E18 Shoot Emericellopsis terricola (FJ430737) 3 April.09 S OO 18ES2 E18 Shoot Cladosporium colombiae (FJ936159) 4 April.09 S OO 19ES2 E18 Shoot Uncultured Ascomycete sp. (EU489902) 5 April 09 S OO 1WS1 W14 Shoot Phoma glomerata (AY183371) 6 April.09 S OO 20WS3 W20 Shoot Acremonium sp. (AM901 698) 7 April.09 S OO 21WS1 W21 Shoot Stachybotrys elegans (AF081481) 8 April O9 S OO 21WS2 W21 Shoot Myrothecium melanosporum (FJ235086) 9 April 09 S OO 22WS1 W22 Shoot Gibberella sp. (FJ466715) 10 April.09 S OO 22WS2 W22 Shoot Gibberella sp. (FJ466715) 11 April.09 S OO 22WS3 W22 Shoot Ascomycete sp. (AY243057) 12 April.09 S OO 23WS1 W223 Shoot Emericellopsis terricola (FJ430737) 13 April.09 S OO 24WS3 W24 Shoot Biomectria rossmaniae (AM944351) 14 April.09 S OO 26WS1 W26 Shoot F. Oxysportin f sp. vaSinfectum (AF322074) 15 April.09 S OO 26WS2 W26 Shoot Emericellopsis terricola (FJ430737) 16 April.09 S OO 26WS.5 W27 Shoot F. Oxysportin f sp. vaSinfectum (AF322074) US 8,975,489 B2 11 12 TABLE 2-continued Identification of fungal endophytes by rDNA sequence. SEQ ant ID NO Isolate ID Location art Initial ID (Accession No.)

17 April 09 S hoot 27WS3 W27 OO Uncultured endophytic fungus (EF505542) 18 April.09 S hoot 28ES1 E28 S OO Uncultured endophytic fungus (EF505542) 19 April 09 S hoot 28WS2 OO Myrothecium melanosporum (FJ235086) 20 April O9 S hoot 29ES1 OO Hypocrea ixi (EF392760) 21 April.09 S hoot 29ES2 OO Hypocrea ixi (EF392760) 22 April O9 S hoot 29ES3 OO Hypocrea ixi (EF392760) 23 April O9 S hoot 29WS1 OO Buergenerula spariinae (AF422960) 24 April.09 S hoot 29WS2 OO Gibberella acuminata (U85533) 25 April.09 S hoot 2WS1 OO Emericeliopsis terricola (FJ430737) 26 April O9 S hoot 2WS2 OO Ascomycete sp. (AJ279460) 27 April O9 S hoot 3OWS2 OO Emericeliopsis terricola (FJ430737) 28 April O9 S hoot 32ES1 OO Fusarium proliferatum (AF291061) 29 April.09 S hoot 32WS OO Stachybotry's elegans (AF081481) 30 April O9 S hoot 34ES2 OO Eutypa scoparia (AF373064) 31 April O9 S hoot 34ES3 OO Sordariomycete sp. (EU680539) 32 April O9 S hoot 35WS1 OO Uncultured soil fungus (EU479884) 33 April.09 S hoot 35WS4 OO Myrothecium melanosporum (FJ235086) 34 April O9 S hoot 35WS5 OO Leptosphaeria avenariaf sp. triticea (U77357) 35 April O9 S hoot 35WS6 OO Acremonium sp. (AM901 698) 36 April.09 S hoot 3WS1 OO Penicilium citreonigrum (EU497942) 37 April O9 S hoot 44ES1 OO Fungal endophyte sp. (EU977213) 38 April O9 S hoot 46ES1 OO Fusarium proliferatum (AF291061) 39 April O9 S hoot 46ES2 OO Emericeliopsis terricola (FJ430737) 40 April.09 S hoot 4ES1 OO Emericeliopsis terricola (FJ430737) 41 April.09 S hoot SWS2 OO Acremonium strictum (EU497953) 42 April.09 S hoot 6ES2 OO Emericeliopsis minima (U57675) 43 April.09 S hoot 6WS2 OO Alternaria mali (AY154683) 44 April.09 S hoot 8ES1 OO Bipolaris heveae (AB179834) 45 April 09 S hoot 9ES1 OO Stachybotry's bishyi (AF081480) 46 April.09 S hoot 9WS1 OO Dothideomycete sp. (EU680559) 47 uly09 S OO OO Acremonium strictum (GU219467) 48 uly09 S OO OO Pleosporaceae sp. (EU330624) 49 uly09 S OO OO Alternaria sp. (FJ210481) 50 uly09 S OO OO Fusarium moniiformae (EU364864) 51 uly09 S OO OO Fusarium moniiformae (AB369908) 52 uly09 S OO OO Sporisorium everhartii (AY740159) 53 uly09 S OO OO Alternaria sp. (FJO37742, GQ389617) S4 uly09 S OO OO Fusarium proliferatum (AF291061) 55 uly09 S OO OO Fusarium ngamai (U34568) 56 uly09 S OO OO Periconia macrospinosa (FJ536208) 57 uly09 S OO OO Codinaeopsis sp. (EF488392) 58 uly09 S OO OO Sporisorium everhartii (AY740159) 59 uly09 S OO OO Fusarium proliferatum (AF291061) 60 uly09 S OO OO Fusarium moniiformae (AB369908) 61 uly09 S OO OO Fusarium proliferatum (EU888923) 62 uly09 S OO OO Fusarium moniiformae (EU364864) 63 uly09 S OO OO Acremonium strictum (EU497953) 64 uly09 S OO OO Myrothecium verrucaria (AY303603) 65 uly09 S OO OO Fusarium subglutinans (GQ167234) 66 uly09 S OO OO Leptosphaeria bicolor (AF455415) 67 uly09 S OO OO Acremonium sp. (AM901 698) 68 uly09 S OO OO Acremonium strictum (U57671) 69 uly09 S OO OO Periconia macrospinosa (FJ536208) 70 uly09 S OO OO Fusarium pseudograminearlin (DQ4598710) 71 uly09 S OO 22WS2 W22 OO Acremonium sp. (AM924149) 72 uly09 S OO 23ES2 E23 OO Fusarium ngamai (U34568) 73 uly09 S OO 25ES1 E25 OO Gibberella sp. (FJOO8984) 74 uly09 S OO 25ES2 E25 OO Colietotrichum graminicola (GQ221855) 75 uly09 S OO 25WS3 W25 OO Sporisorium everhartii (AY740159) 76 uly09 S OO 26ES1 E26 OO Monographeila sp. (FJ228195) 77 uly09 S OO 26ES2 E26 OO Myrothecium verrucaria (AY303603) 78 uly09 S OO 27ES1 E27 OO Alternaria arborescens (AY154706) 79 uly09 S OO 27ES3 E27 OO Alternaria alternata (FJ872066) 8O uly09 S OO 27ES4 E27 OO Parasarcopodium ceratocarvi (AY344479) 81 uly09 S OO OO Acremonium sp. (AM924149) 82 uly09 S OO OO Acremonium strictum (EU497953) 83 uly09 S OO OO Sporisorium everhartii (AY740159) 84 uly09 S OO OO Fusarium ngamai (U34568) 85 uly09 S OO OO Acremonium sp. (AM924149) 86 uly09 S OO OO Alternaria mali (AY154683) US 8,975,489 B2 13 14 TABLE 2-continued dentification of fungal endophytes by rDNA sequence. SEQ Plant ID NO solate ID Location Part Initial ID (Accession No.)

87 y09 Shoot 3OES2 E30 OO Gibberella sp. (AM901682) 88 y09 Shoot 3OES3 E30 OO Fusarium proliferatum (AF291061) 89 y09 Shoot 3OES4 E30 OO Alternaria mali (AY154683) 90 y09 Shoot 31ES1 E31 OO Pseudozyma flocculosa (DQ411535) 91 y09 Shoot 32WS1 W32 OO Fusarium ngamai (U34568) 92 y09 Shoot 33WS1 W33 OO Fusarium ngamai (U34568) 93 y09 Shoot 33WS2 W33 OO Nigrospora oryzae (EU272488) 94 y09 Shoot 33WS3 W33 OO Fusarium ngamai (U34568) 95 y09 Shoot 33WS4 W33 OO Acremonium strictum (EU497953) 96 y09 Shoot 33WS5 W33 OO Fusarium proliferatum (AF291061) 97 y09 Shoot 33WS6 W33 OO Sporisorium everhartii (AY740159) 98 y09 Shoot 33WS7 W33 OO Uncultured root-associated fungus (EU144817) 99 l y09 Shoot 34WS1 W34 S OO Uncultured endophytic fungus (EF505.485) OO y09 Shoot 34WS3 OO Gibberella sp. (FJOO8984) O1 y09 Shoot 34WS4 OO Acremonium strictum (EU497953) O2 y09 Shoot 35ES1 OO Fusarium proliferatum (AF291061) O3 y09 Shoot 35ES2 OO Fusarium ngamai (X94174) O4 y09 Shoot 35ES3 OO Acremonium sp. (AM901 698) 05 y09 Shoot 35WS1 OO Gibberella sp. (AM901682) O6 y09 Shoot 35WS2 OO Gibberella sp. (FJOO8984) O7 y09 Shoot 35WS3 OO Gibberella sp. (AM901682) O8 y09 Shoot 35WS4 OO Gibberella sp. (AM901682) 09 y09 Shoot 35WS5 OO Fusarium proliferatum (EU272509) 10 y09 Shoot 35WS6 OO Alternaria alternata (AY433814) 11 y09 Shoot 36ES1 OO Sporisorium everhartii (AY740159) 12 y09 Shoot 42ES2 OO Sordariomycete sp. (EU680539) 13 y09 Shoot 45ES1 OO Fusarium proliferatum (AF291061) 14 y09 Shoot 45ES2 OO Fusarium moniiformae (AB369908) 15 y09 Shoot 48ES1 OO Fusarium ngamai (U34568) 16 y09 Shoot 48ES2 OO Fusarium proliferatum (AF291061) 17 y09 Shoot 48ES3 OO Fusarium ngamai (U34568) 18 y09 Shoot 4ES1 OO Exserohilum rostratum (GQ169762) 19 y09 Shoot 5ES1 OO Fusarium proliferatum (AF291061) 2O y09 Shoot 6WS1 OO Fusarium moniiformae (EU364864) 21 y09 Shoot 6WS2 OO Fusarium moniiformae (AB369908) 22 y09 Shoot 7ES1 OO Bipolaris oryzae (DQ3.00203) 23 y09 Shoot 7ES2 OO Gibberella sp. (FJOO8984) 24 y09 Shoot 8ES1 OO Colietotrichum graminicola (EU400146) 25 y09 Shoot 8ES3 OO Sporisorium everhartii (AY740159) 26 y09 Shoot 8ES4 OO Sporisorium everhartii (AY740159) 27 y09 Shoot 9ES2 OO Fusarium moniiformae (AB369908) 28 y09 Shoot 9ES3 OO Sporisorium everhartii (AY740159) 29 uly09 Shoot 9ES4 OO Sporisorium everhartii (AY740159) 30 Oct09 Shoot 10ES2 OO Sordariomycete sp. (EU680539) 31 Oct09 Shoot 10ES3 OO Sordariomycete sp. (EU680539) 32 Oct09 Shoot 28ES1 OO Fusarium ngamai (X94174) 33 Oct09 Shoot 3OES1 OO Fusarium acuminatum (GQ505462) 34 Oct09 Shoot 37ES1 OO Fusarium ngamai (X94174) 35 Oct09 Shoot 37ES2 OO Fusarium proliferatum (AF291061) 36 Oct09 Shoot 48ES1 OO Leaf litter ascomycetes (AF502815) 37 Oct09 Shoot 10WS1 OO Acremonium strictum (U57671) 38 Oct09 Shoot 14WS1 OO Fusarium ngamai (X94174) 39 Oct09 Shoot 16WS1 OO Fusarium ngamai (X94174) 40 Oct09 Shoot 16WS2 OO Fusarium ngamai (X94174) 41 Oct09 Shoot 24WS1 OO Stachybotry's elegans (AF081481) 42 Oct09 Shoot 24WS2 OO Stachybotry's elegans (AF081481) 43 Oct09 Shoot 24WS3 OO Stachybotry's elegans (AF081481) 44 April O9 Root 10WR1 Roo Periconia macrospinosa (FJ536207) 45 April.09 Root 10WR2 Roo Codinaeopsis sp. (EF488392) 46 April O9 Root 11WR1 Roo Fusarium ngamai (X94174) 47 April.09 Root 12ER1 Roo Gaeumannomyces incrustans (U17214) 48 April.09 Root 12ER2 Roo Periconia macrospinosa (FJ536207) 49 April.09 Root 12ER3 Roo Fusarium sp. (EF453116) 50 April.09 Root 12ER-A Roo Gaeumannomyces incrustans (U17214) 51 April O9 Root 12WR1 Roo Fusarium annulatum (AY213654) 52 April O9 Root 13ER1 Roo Fusarium ngamai (X94174) 53 April.09 Root 14ER2 Roo Periconia macrospinosa (FJ536207) S4 April O9 Root 15ER1 Roo Gaeumannomyces incrustans (U17214) 55 April.09 Root 16ER1 Roo Coprinus auricomus (FM163186) 56 April O9 Root 16ER2 Roo Coprinus auricomus (FM163186) 57 April.09 Root 16ER-A Roo Amyloathelia crassiuscula (DQ144610) 58 April O9 Root 16WR1 Roo Alternaria longissima (EU030349) 59 April O9 Root 18WR2 Roo Kabatiella microsticta (EU167608) US 8,975,489 B2 15 TABLE 2-continued dentification of fungal endophytes by rDNA sequence. SEQ Plan ID NO Isolate ID Location* Part Initial ID (Accession No.) 60 April.09 Root 19ER1 E19 Root Fusarium annulatum (AY213654) 61 April.09 Root 19ER2 E19 Root Fusarium ngamai (X94174) 62 April.09 Root 19ER3 E19 Root Fusarium ngamai (X94174) 63 April.09 Root 19WR1 W19 Root Alternaria mali (AY154683) 64 April.09 Root 19WR-A W19 Root Fusarium moniiformae (EU364865) 65 April.09 Root 1ER E1 Root Periconia macrospinosa (FJ536207) 66 April.09 Root 1ER3 E1 Root Uncultured soil fungus (EU480242) 67 April.09 Root 20ER1 E20 Root Gaeumannomyces incrustans (U17214) 68 April.09 Root 20ER2 E20 Root Fusarium ngamai (X94174) 69 April.09 Root 20WR2 W20 Root Eutpella sp. (FJ172283) 70 April.09 Root 21ER1 E21 Root Periconia macrospinosa (FJ536207) 71 April 09 Root 22WR1 W22 Root Fusarium ngamai (X94174) 72 April 09 Root 22WR2 W22 Root Fusarium ngamai (X94174) 73 April 09 Root 22WR3 W22 Root Fusarium sp. (AY143085) 74 April.09 Root 24WR2 W24 Root Fungal endophyte (FJ449944) 75 April 09 Root 26ER1 E26 Root Fusarium ngamai (X94174) 76 April 09 Root 26WR1 W26 Root Alternaria mali (AY154683) 77 April 09 Root 26WR2 W26 Root Fusarium ngamai (X94174) 78 April.09 Root 27ER1 E27 Root Fusarium ngamai (X94174) 79 April.09 Root 27WR1 W27 Root Microdochium sp. (FJ536210) 80 April.09 Root 29WR1 W29 Root Fusarium ngamai (X94174) 81 April.09 Root 29WR2 W29 Root Fusarium ngamai (X94174) 82 April.09 Root 29WR3 W29 Root Fusarium ngamai (X94174) 83 April.09 Root 2ER E2 Root Periconia macrospinosa (FJ536207) 84 April.09 Root 2WR1 W2 Root Uncultured Ascomycete sp. (EU358786) 85 April.09 Root 2WR2 W2 Root Uncultured Ascomycete sp. (EU358786) 86 April.09 Root 2WR3 W2 Root Uncultured Ascomycete sp. (EU003079) 87 April.09 Root 2WR4 W2 Root Uncultured Ascomycete sp. (EF154350) 88 April.09 Root 3OER1 E30 Root Fusarium ngamai (X94174) 89 April.09 Root 31ER2 E31 Root Fusarium ngamai (X94174) 90 April 09 Root 31ER3 E31 Root Fusarium sp. (GQ505756) 91 April 09 Root 31ER4 E31 Root Fusarium n-gamai (X94174) 92 April 09 Root 31WR1 W31 Root Fusarium ngamai (X94174) 93 April.09 Root 31WR2 W31 Root Fusarium ngamai (X94174) 94 April 09 Root 32WR1 W32 Root Gaeumannomyces incrustans (U17214) 95 April 09 Root 33ER1 E33 Root Periconia macrospinosa (FJ536207) 96 April 09 Root 33ER2 E33 Root Microdochium sp. (FJ536210) 97 April.09 Root 33ER3 E33 Root Periconia macrospinosa (FJ536207) 98 April 09 Root 33ER4 E33 Root Anthostomella brabei (EU552098) 99 April 09 Root 33WR1 W33 Root Gaeumannomyces incrustans (U17214) 200 April O9 Root 34ER1 E34 Root Gaeumannomyces incrustans (U17214) 201 April.09 Root 34ER2 E34 Root Periconia macrospinosa (FJ536207) 202 April O9 Root 37ER1 E37 Root Fusarium ngamai (X94174) 203 April O9 Root 37ER2 E37 Root Fusarium ngamai (X94174) 204 April O9 Root 37ER3 E37 Root Periconia macrospinosa (FJ536207) 205 April.09 Root 3ER1 E Root Halorosellinia sp. (EU715636) 206 April O9 Root 3ER2 E Root Fusarium sp. (EU750677) 207 April O9 Root 3ER3 E Root Halorosellinia sp. (EU715636) 208 April O9 Root 3ER-A E Root Uncultured root-associated fungus (EU144759) 209 April O9 Root 3WR1 W3 Root Penicilium verruculosum (AF510496) 210 April O9 Root 3WR2 W3 Root Fusarium ngamai (X94174) 211 April O9 Root 3WR3 W3 Root Gibberella sp. (GQ389619) 212 April O9 Root 3WR4 W3 Root Fusarium moniiformae (EU364865) 213 April O9 Root 41ER1 E41 Root Periconia macrospinosa (FJ536207) 214 April.09 Root 41ER2 E41 Root Periconia macrospinosa (FJ536207) 215 April.09 Root 41ER2- E41 Root Periconia macrospinosa (FJ536207) Green 216 April O9 Root 41ER3- E41 Root Periconia macrospinosa (FJ536207) Ash 217 April.09 Root 41ER-A E41 Root Periconia macrospinosa (FJ536207) 218 April O9 Root 42ER1 E42 Root Fusarium ngamai (X94174) 219 April O9 Root 44ER1 E44 Root Fusarium ngamai (X94174) 220 April O9 Root 45ER1 E45 Root Fusarium ngamai (X94174) 221 April.09 Root 45ER2 E45 Root Macrophomina phaseolina (EF545133) 222 April O9 Root 45ER3 E45 Root Periconia macrospinosa (FJ536207) 223 April O9 Root 46ER1 E46 Root Periconia macrospinosa (FJ536207) 224 April O9 Root 46ER2 E46 Root Microdochium sp. (FJ536210) 225 April.09 Root 47ER1 E47 Root Uncultured Nectriaceae (EFO68175) 226 April O9 Root 47ER2 E47 Root Ascomycete sp. (AJ279488) 227 April O9 Root 4ER1 E4 Root Fusarium ngamai (X94174) 228 April O9 Root 4ER2 R Root Fusarium ngamai (X94174) 229 April.09 Root 4ER3 E4 Root Periconia macrospinosa (FJ536207) 230 April O9 Root 4ER4 E4 Root Microdochium sp. (FJ536210) 231 April O9 Root 4ERS E4 Root Fusarium ngamai (X94174) US 8,975,489 B2 17 18 TABLE 2-continued

dentification of fungal en ophytes by rBNA sequence. SEQ Plan ID NO Isolate ID Location* Part Initial ID (Accession No.) 232 April O9 Root SER1 Roo Uncultured Leptosphaeriaceae (AY744286) 233 April.09 Root SER2 Roo Periconia macrospinosa (FJ536207) 234 April O9 Root SER3 Roo Codinaeopsis sp. (EF488392) 235 April O9 Root SWR1 Roo Fusarium ngamai (X94174) 236 April O9 Root SWR2 Roo Fusarium ngamai (X94174) 237 April.09 Root SWR3 Roo Fusarium ngamai (X94174) 238 April O9 Root 6ER1 Roo Ascomycete sp. (DQ657853) 239 April O9 Root 6ER2 Roo Codinaeopsis sp. (EF488392) 240 April O9 Root 7ER1 Roo Fusarium ngamai (X94174) 241 April.09 Root 7ER2 Roo Uncultured fungus (FJ776560) 242 April.09 Root 7ER-A Roo Uncultured Helotiales (FJ475783) 243 April O9 Root 7WR1 Roo Fungal endophyte (DQ979674) 244 April O9 Root 7WR2 Roo Fungal endophyte (FN394.695) 245 April.09 Root 8ER1 Roo Uncultured Hypocreates (FJ708.608) 246 April O9 Root 8ER2 Roo Periconia macrospinosa (FJ536207) 247 April O9 Root 8WR1 Roo Hypocrea ixi (EF392760) 248 July09 Root 10ER1 E10 Roo Periconia macrospinosa (FJ536208) 249 uly09 Root 10ER2 E10 Roo Periconia macrospinosa (FJ536208) 250 July09 Root 10ER3 E10 Roo Periconia macrospinosa (FJ536208) 251 uly09 Root 10ER4 E10 Roo Periconia macrospinosa (FJ536208) 252 July09 Root 10ER5 E10 Roo Fungal endophyte (DQ979674) 253 uly09 Root 10WR1 W10 Roo Fusarium ngamai (X94174) 254 July09 Root 10WR2 W10 Roo Uncultured root-associated fungus (EU144817) 255 July09 Root 12WR1 W12 Roo Gaeumannomyces incrustans (U17214) 256 July09 Root 12WR10 W12 Roo Uncultured root-associated fungus (EU144817) 257 July09 Root 12WR3 Roo Uncultured Hypocreates (FJ708.608) 258 uly09 Root 13ER1 Roo Fusarium ngamai (X94174) 259 July09 Root 13ER2 Roo Fusarium moniiformae (EU364856) 260 July09 Root 13ER4 Roo Sordariomycete sp. (EU680539) 261 uly09 Root 13WR1 Roo Magnaporthe rhizophila (DQ528791) 262 uly09 Root 15WR1 Roo Fusarium oxysporum (FJ478.116) 263 July09 Root 17 ER1 Roo Mycorrhizal fungal sp. (FJ236025) 264 July09 Root 17WR2 Roo Sordariomycete sp. (EU680539) 265 July09 Root 17WR3 Roo Uncultured Hypocreates (FJ708.608) 266 uly09 Root 18ER1 Roo Fusarium ngamai (X94174) 267 July09 Root 18ER2 Roo Fusarium ngamai (X94174) 268 July09 Root 18ER3 Roo Fusarium ngamai (X94174) 269 July09 Root 18ER4 Roo Fusarium ngamai (X94174) 270 uly09 Root 18WR1 Roo Fusarium proliferatum (AF291061) 271 uly09 Root 18WR2 Roo Magnaporthe rhizophila (DQ528791) 272 July09 Root 18WR2 Roo Magnaporthe rhizophila (DQ528791) 273 July09 Root 18WR3 Roo Fusarium moniiformae (EU364856) 274 uly09 Root 18WR4 Roo Fusarium ngamai (X94174) 275 July09 Root 19ER1 Roo Uncultured root-associated fungus (EU144806) 276 July09 Root 19WR2 Roo Fusarium sp. (EU750682) 211 uly09 Root 19WR3 W Roo Fusarium oxysportin f. ciceris (EU442590) 278 July09 Root 1 ER1 Roo Microdochium sp. (FJ536210) 279 July09 Root 1 ER2 Roo Fusarium acuminatum (GQ505462) 280 July09 Root 1 WR1 Roo Fusarium proliferatum (AF291061) 281 uly09 Root 1 WR2 Roo Fusarium moniiformae (EU364865) 282 July09 Root 1 WR3 Roo Uncultured root-associated fungus (EU144806) 283 July09 Root 1 WR4 Roo Uncultured root-associated fungus (FJ362205) 284 July09 Root 1 WR5 Roo Fusarium ngamai (X94174) 285 July09 Root 20ER1 Roo Fusarium ngamai (U34568) 286 July09 Root 20ER10 Roo Magnaporthe rhizophila (DQ528791) 287 July09 Root 20WR2 Roo Uncultured Hypocreates (FJ708.608) 288 July09 Root 21 ER1 Roo Magnaporthe rhizophila (DQ528791) 289 July09 Root 21 ER3 Roo Uncultured soil fungus (DQ420771) 290 July09 Root 21 ER4 Roo Gaeumannomyces incrustans (U17214) 291 July09 Root 21WR1 Roo Uncultured root-associated fungus (EU144817) 292 uly09 Root 21WR2 Roo Dothideomycete sp. (EU680535) 293 July09 Root 21 WR3 Roo Uncultured root-associated fungus (EU144817) 294 July09 Root 22WR2 W22 Roo Didymella fabae (GQ305306) 295 uly09 Root 22WR3 W22 Roo Uncultured root-associated fungus (EU144817) 296 July09 Root 23WR1 Roo Macrophomina phaseolina (EF545133) US 8,975,489 B2 19 20 TABLE 2-continued Identification of fungal endophytes by rDNA sequence. SEQ Plan ID NO Isolate ID Location* Part Initial ID (Accession No.) 297 July09 Root 23WR3 W23 Root Rhizopycnis sp. (DQ682600) 298 July09 Root 23WR4 W23 Root Uncultured root-associated fungus (FJ361993) 299 July09 Root 24WR10 W24 Root Uncultured root-associated fungus (EU144817) 300 July09 Root 24WR4 W24 Root Uncultured root-associated fungus (EU144817) 301 July09 Root 24WR5 W24 Root Phoma medicaginis (EU167575) 302 July09 Root 25 ER1 E25 Root Fusarium proliferatum (AF291061) 303 July09 Root 25WR4 W25 Root Fusarium proliferatum (AF291061) 304 July09 Root 26ER1 E26 Root Fusarium ngamai (X94174) 305 July09 Root 26ER2 E26 Root Magnaporthe rhizophila (DQ528791) 306 July09 Root 26ER3 E26 Root Gaeumannomyces incrustans (U17214) 307 July09 Root 26WR1 W26 Root Gaeumannomyces incrustans (U17214) 308 July09 Root 26WR2 W26 Root Fusarium proliferatum (AF291061) 309 July09 Root 26WR3 W26 Root Uncultured Ascomycete sp. (EU490.093) 310 July09 Root 27WR2 W27 Root Gaeumannomyces incrustans (U17214) 311 July09 Root 27WR3 W27 Root Ascomycete sp. (EF672299) 312 July09 Root 27WR5 W27 Root Fungal endophyte (FN392.299) 313 July09 Root 28ER2 E28 Root Fusarium moniiformae (EU364865) 314 July09 Root 28WR1 W28 Root Uncultured Leptosphaeriaceae (AY744286) 315 July09 Root 29ER1 E29 Root Magnaporthe rhizophila (DQ528791) 316 July09 Root 29WR2 W29 Root Fusarium ngamai (X94174) 317 July09 Root 29WR3 W29 Root Gaeumannomyces incrustans (U17214) 318 July09 Root 2ER2 E2 Root Uncultured fungus (FN397215) 319 July09 Root 2ER3 E2 Root Fusarium ngamai (U34568) 320 July09 Root 2ER5 E2 Root Fusarium oxysporum (AY928417) 321 July09 Root 2WR3 W2 Root Fusarium solani (FJ478.128) 322 July09 Root 31 ER1 E31 Root Fusarium proliferatum (AF291061) 323 July09 Root 31 ER4 E31 Root Fusarium proliferatum (AF291061) 324 July09 Root 31WR1 W31 Root Myrothecium cinctum (DQ135998) 325 July09 Root 31WR2 W31 Root Gaeumannomyces incrustans (U17214) 326 July09 Root 32WR1 W32 Root Fusarium ngamai (U34568) 327 July09 Root 32WR2 W32 Root Fusarium ngamai (U34568) 328 July09 Root 33WR2 W33 Root Gaeumannomyces incrustans (U17214) 329 July09 Root 33WR3 W33 Root Gaeumannomyces incrustans (U17214) 330 July09 Root 35ER2 E35 Root Magnaporthe rhizophila (DQ528791) 331 July09 Root 35ER4 E35 Root Magnaporthe rhizophila (DQ528791) 332 July09 Root 35WR2 W35 Root Uncultured Ascomycete sp. (EF154351) 333 July09 Root 35WR3 W35 Root Fusarium proliferatum (AF291061) 334 July09 Root 35WR4 W35 Root Magnaporthe rhizophila (DQ528791) 335 July09 Root 36ER1 E36 Root Uncultured soil fungus (EU480242) 336 July09 Root 36ER2 E36 Root Uncultured fungus (FN397215) 337 July09 Root 36ER3 E36 Root Periconia macrospinosa (FJ536208) 338 July09 Root 36ER4 E36 Root Periconia macrospinosa (FJ536208) 339 July09 Root 36WR1 W36 Root Fusarium ngamai (X94174) 340 July09 Root 36WR2 W36 Root Eladia saccula (FJ914702) 341 uly09 Root 36WR2-2 W36 Root Gaeumannomyces incrustans (U17214) 342 July09 Root 36WR5 W36 Root Fusarium moniiformae (EU364865) 343 July09 Root 37 ER1 E37 Root Gaeumannomyces incrustans (U17214) 344 July09 Root 37 ER2 E37 Root Uncultured root-associated fungus (EU144817) 345 July09 Root 37 ER3 E37 Root Fusarium ngamai (X94174) 346 July09 Root 39ER1 E39 Root Fusarium sp. (EU750677) 347 July09 Root 39ER3 E39 Root Fusarium ngamai (X94174) 348 July09 Root 39ER5 E39 Root Uncultured endophytic fungus (FJ524302) 349 July09 Root 3ER1-2 E3 Root Fusarium proliferatum (AF291061) 350 July09 Root 3ER2 E3 Root Fusarium oxysporum (FJ154076) 351 July09 Root 3ER3 E3 Root Fusarium ngamai (X94174) 352 July09 Root 3 WR1 W3 Root Uncultured endophytic fungus (EF505610) 353 July09 Root 3 WR2 W3 Root Ascomycete sp. (EF672299) 354 July09 Root 3 WR3 W3 Root Uncultured Ascomycete sp. (EF154351) 355 July09 Root 40ER1 E40 Root Gaeumannomyces incrustans (U17214) 356 July09 Root 40ER2 E40 Root Uncultured root-associated fungus (EU144817) 357 uly09 Root 40ER3 E40 Root Uncultured root-associated fungus (EU144817) 358 July09 Root 41 ER1 E41 Root Fusarium proliferatum (AF291061) 359 July09 Root 42ER1 E42 Root Fusarium proliferatum (AF291061) 360 July09 Root 42ER2 E42 Root Fusarium proliferatum (AF291061) 361 July09 Root 43ER1 E43 Root Uncultured Ascomycete sp. (EU490.093) 362 July09 Root 43ER2 E43 Root Uncultured Hypocreates (FJ708.608) US 8,975,489 B2 21 TABLE 2-continued Identification of fungal endophytes by rDNA sequence. SEQ Plan ID NO Isolate ID Location* Part Initial ID (Accession No.) 363 July09 Root 43ER3 E43 Root Fusarium ngamai (X94174) 364 July09 Root 43ER4 E43 Root Fungal endophyte (FN392.299) 365 July09 Root 44ER1 E44 Root Periconia macrospinosa (FJ536208) 366 July09 Root 45 ER1 E45 Root Magnaporthe rhizophila (DQ528791) 367 July09 Root 45 ER4 E45 Root Fusarium ngamai (X94174) 368 July09 Root 47ER1 E47 Root Fusarium proliferatum (AF291061) 369 July09 Root 47ER2 E47 Root Fusarium proliferatum (AF291061) 370 July09 Root 47ER3 E47 Root Uncultured root-associated fungus (EU144817) 371 July09 Root 47ER4 E47 Root Fusarium ngamai (X94174) 372 July09 Root 48ER1 E48 Root Fusarium ngamai (X94174) 373 July09 Root 48ER2 E48 Root Gaeumannomyces incrustans (U17214) 374 July09 Root 48ER3 E48 Root Magnaporthe rhizophila (DQ528791) 375 July09 Root 4ER1 E4 Root Gaeumannomyces incrustans (U17214) 376 uly09 Root 4ER2 E4 Root Uncultured root-associated fungus (EU144817) 377 July09 Root 4ER3 E4 Root Fusarium sp. (GU257897) 378 July09 Root 4ER4 E4 Root Fusarium sp. (AF158312) 379 July09 Root 4WR1 W4 Root Fusarium proliferatum (AF291061) 380 July09 Root 4WR3 W4 Root Magnaporthe rhizophila (DQ528791) 381 July09 Root 5ER2 E Root Fusarium proliferatum (AF291061) 382 July09 Root 5ER3-1 E Root Periconia macrospinosa (FJ536208) 383 July09 Root 5ER3-2 E Root Codinaeopsis sp. (EF488392), 384 July09 Root 5ER5 E Root Uncultured root-associated fungus (EU144817) 385 July09 Root 5 WR1 W5 Root Fusarium proliferatum (AF291061) 386 July09 Root 5 WR10 W5 Root Gaeumannomyces incrustans (U17214) 387 July09 Root 5 WR2 W5 Root Fusarium ngamai (X94174) 388 July09 Root 5 WR20 W5 Root Magnaporthe rhizophila (DQ528791) 389 July09 Root 5 WR3 W5 Root Fusarium proliferatum (AF291061) 390 July09 Root 6ER E6 Root Fusarium proliferatum (AF291061) 391 July09 Root 6ER2 E6 Root Fusarium proliferatum (AF291061) 392 July09 Root 6WR1 W6 Root Fusarium proliferatum (AF291061) 393 July09 Root 7ER E7 Root Gaeumannomyces incrustans (U17214) 394 July09 Root 7ER2 E7 Root Diaporthe phaseolorum (AF001016) 395 July09 Root 7WR1 W7 Root Fusarium moniiformae (EU364865) 396 July09 Root 7WR10 W7 Root Uncultured fungus (AM260913) 397 July09 Root 8ER Root Trichoderma koningiopsis (EU280108) 398 July09 Root 8ER2 Root Uncultured Helotiales (FJ475783) 399 July09 Root 9ER Root Periconia macrospinosa (FJ536208) 400 Oct09 Root 10W R1 Root Ascomycete sp. (EU520609) 401 Oct09 Root 10W Root Fusarium ngamai (X94174) 402 Oct09 Root 10W Root Fusarium ngamai (X94174) 403 Oct09 Root 11W R1 Root Gaeumannomyces incrustans (U17214) 404 Oct09 Root 11WR2 Root Gaeumannomyces incrustans (U17214) 405 Oct09 Root 11W Root Fusarium ngamai (X94174) 406 Oct09 Root 12W Root Fusarium ngamai (X94174) 407 Oct09 Root 12W Root Sordariomycete sp. (EU680539) 408 Oct09 Root 12W Root Grass root mycorrhizal sp. (AY599235) 409 Oct09 Root 13ER1 Root Periconia macrospinosa (FJ536208) 410 Oct09 Root 13ER2 Root Periconia macrospinosa (FJ536207) 411 Oct09 Root 13ER3 Root Fusarium ngamai (X94174) 412 Oct09 Root 13WR1 Root Fusarium ngamai (X94174) 413 Oct09 Root 13WR2 Root Fusarium ngamai (X94174) 414 Oct09 Root 13WR3 Root Gaeumannomyces incrustans (U17214) 415 Oct09 Root 14WR2 Root Magnaporthe rhizophila (DQ528791) 416 Oct09 Root 15WR1 Root Gaeumannomyces incrustans (U17214) 417 Oct09 Root 15WR2 Root Magnaporthe rhizophila (DQ528791) 418 Oct09 Root 16WR2 Root Fusarium proliferatum (AF291061) 419 Oct09 Root 17ER1 Root Fusarium proliferatum (AF291061) 420 Oct09 Root 17WR1 Root Magnaporthe rhizophila (DQ528791) 421 Oct09 Root 17WR2 Root Magnaporthe rhizophila (DQ528791) 422 Oct09 Root 17WR5 Root Gaeumannomyces incrustans (U17214) 423 Oct09 Root 18ER1 Root Fusarium ngamai (X94174) 424 Oct09 Root 18ER2 Root Fusarium ngamai (X94174) 425 Oct09 Root 18ER3 Root Uncultured Lachnum (FJ440910) 426 Oct09 Root 18WR2 Root Trichoderma aureoviride (AF194010) 427 Oct09 Root 18WR3 Root Magnaporthe rhizophila (DQ528791) 428 Oct09 Root 18WR4 Root Fusarium ngamai (X94174) 429 Oct09 Root 19ER1 Root Fusarium ngamai (X94174) 430 Oct09 Root 19ER2 Root Sordariomycete sp. (EU680539) 431 Oct09 Root 19ER3 Root Gaeumannomyces incrustans (U17214) 432 Oct09 Root 19ER4 Root Fusarium moniiformae (EU364856) 433 Oct09 Root 19WR1 1 Root Fusarium ngamai (U34568) 434 Oct09 Root 1ER1 Root Gaeumannomyces incrustans (U17214) US 8,975,489 B2 23 24 TABLE 2-continued Identification of fungal endophytes by rDNA sequence. SEQ Plan ID NO ISO ate I Location* Part Initial ID (Accession No.) 435 Oc 09 Root Roo Gaeumannomyces incrustans (U17214) 436 Oc 09 Root Roo Gaeumannomyces incrustans (U17214) 437 Oc 09 Root Roo Fusarium sp. 14018 (EU750682) 438 Oc 09 Root Roo Ascomycete sp. (EF672299) 439 Oc 09 Root Roo Ascomycete sp. (EF672299) 440 Oc 09 Root Roo Fusarium ngamai (X94174) 441 Oc 09 Root Roo Fusarium ngamai (X94174) 442 Oc 09 Root Roo Fusarium ngamai (X94174) 443 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) 444 Oc 09 Roo Roo Alternaria alternata (DQ023279) 445 Oc 09 Roo Roo Sordariomycete sp. (EU680539) 446 Oc 09 Roo Roo Sordariomycete sp. (EU680539) 447 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 448 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 449 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 450 Oc 09 Roo Roo Marasmius nigrobrunneus (EU935578) 451 Oc 09 Roo Roo Hypocrea ixi (AF194011) 452 Oc 09 Roo Roo Codinaeopsis sp. (EF488392) 453 Oc 09 Roo Roo Fusarium ngamai (X94174) 454 Oc 09 Roo Roo Fusarium ngamai (X94174) 455 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 456 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 457 Oc 09 Roo Roo Sordariomycete sp. (EU680539) 458 Oc 09 Roo Roo Uncultured root-associated fungus (EU144817) 459 Oc 09 Roo E25 Roo Gaeumannomyces incrustans (U17214) 460 Oc 09 Roo E25 Roo Gaeumannomyces incrustans (U17214) 461 Oc 09 Roo E25 Roo Gaeumannomyces incrustans (U17214) 462 Oc 09 Roo E25 Roo Waitea Circinata var. zeae (GQ221863) 463 Oc 09 Roo W25 Roo Gaeumannomyces incrustans (U17214) 464 Oc 09 Roo W25 Roo Gaeumannomyces incrustans (U17214) 465 Oc 09 Roo W25 Roo Magnaporthe rhizophila (DQ528791) 466 Oc 09 Roo W25 Roo Gaeumannomyces incrustans (U17214) 467 Oc 09 Roo W25 Roo Penicillium sp. (FJ571475) 468 Oc 09 Roo E26 Roo Fusarium ngamai (X94174) 469 Oc 09 Roo E26 Roo Magnaporthe rhizophila (DQ528791) 470 Oc 09 Roo E26 Roo Uncultured root-associated fungus (EU144817) 471 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 472 Oc 09 Roo Roo Sordariomycete sp. (EU680539) 473 Oc 09 Roo Roo Sordariomycete sp. (EU680539) 474 Oc 09 Roo Roo Grass root mycorrhizal sp. (AY599235) 475 Oc 09 Roo Roo Fusarium ngamai (X94174) 476 Oc 09 Roo Roo Fusarium ngamai (X94174) 477 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 478 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 479 Oc 09 Roo Roo Codinaeopsis sp. (EF488392) 480 Oc 09 Roo Roo Myrothecium melanosporum (FJ235086) 481 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) 482 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 483 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 484 Oc 09 Roo Roo Sordariomycete sp. (EU680539) 485 Oc 09 Roo Roo Uncultured root-associated fungus (EU144817) 486 Oc 09 Root Roo Uncultured root-associated fungus (EU144817) 487 Oc 09 Roo Roo Uncultured root-associated fungus (EU144817) 488 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) 489 Oc 09 Roo Roo Fusarium ngamai (X94174) 490 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) 491 Oc 09 Roo Roo Uncultured soil fungus (EU490.117) 492 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 493 Oc 09 Roo Roo Sordariomycete sp. (EU680539) 494 Oc 09 Roo Roo Fusarium moniiforme (EU364856) 495 Oc 09 Roo Roo Codinaeopsis sp. (EF488392) 496 Oc 09 Roo Roo Fusarium ngamai (X94174) 497 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 498 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) 499 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) SOO Oc 09 Roo Roo Gibberella sp. 1893 (FJOO8984) SO1 Oc 09 Roo Roo Uncultured root-associated fungus (EU144817) SO2 Oc 09 Roo 35ER3 Roo Uncultured root-associated fungus (EU144817) US 8,975,489 B2 25 26 TABLE 2-continued Identification of fungal endophytes by rDNA sequence. SEQ Plan ID NO ISO ate I Location Part Initial ID (Accession No.) SO3 Oc 09 Roo 35 ER4 E35 Roo Uncultured root-associated fungus (EU144817) SO4 Oc 09 Roo ER1 Roo Gaeumannomyces incrustans (U17214) SOS Oc 09 Roo Roo Fusarium ngamai (X94174) SO6 Oc 09 Roo Roo Fusarium sp. (EU750677) SO7 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) SO8 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) SO9 Oc 09 Roo Roo Rhizoctonia praticola (DQ223780) S10 Oc 09 Roo Roo Fusarium ngamai (X94174) S11 Oc 09 Roo Roo Ascomycete sp. (EF672299) S12 Oc 09 Roo 40 ER4 Roo Sordariomycete sp. (EU680539) S13 Oc 09 Roo 42 ER1 Roo Gaettmannomyces graminis var. tritici (AJ246153) 514 Oc 09 Root Roo Gaettmannomyces graminis var. tritici (AJ246153) S15 Oc 09 Root Roo Gaettmannomyces graminis var. tritici (AJ246153) S16 Oc 09 Root ER1 Roo Uncultured root-associated fungus (EU144817) S17 Oc 09 Roo Roo Gaeumannomyces incrustans (U17214) S18 Oc 09 Roo Roo Fusarium ngamai (U34568) S19 Oc 09 Roo Roo Uncultured root associated fungus (EU144855) 520 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) S21 Oc 09 Roo ER4 Roo Gaeumannomyces incrustans (U17214) S22 Oc 09 Roo ER1 Roo Uncultured root-associated fungus (EU144817) S23 Oc 09 Root ER1 Roo Uncultured root-associated fungus (EU144817) 524 Oc 09 Root Roo Uncultured root-associated fungus (EU144817) S25 Oc 09 Roo 4 Roo Sordariomycete sp. (EU680539) 526 Oc 09 Roo Roo Sordariomycete sp. (EU680539) S27 Oc 09 Roo Roo Fusarium ngamai (X94174) 528 Oc 09 Roo Roo Ascomycete sp. (EF672299) 529 Oc 09 Roo WR Roo Fusarium sp. (EU750677, EU750686) 530 Oc 09 Roo E R 1 Roo Sordariomycete sp. (EU680539) S31 Oc 09 Roo E R 2 Roo Gaeumannomyces incrustans (U17214) S32 Oc 09 Roo E R 3 Roo Gaeumannomyces incrustans (U17214) S33 Oc 09 Roo Roo Fusarium ngamai (X94174) S34 Oc 09 Roo Roo Fusarium ngamai (X94174) S35 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) S36 Oc 09 Roo E R 2 Roo Magnaporthe rhizophila (DQ528791) 537 Oc 09 Roo E R 3 Roo Diaporthe phaseolorum (AF001016) S38 Oc 09 Roo E R 4 Roo Diaporthe phaseolorum (AF001016) 539 Oc 09 Roo E R 5 Roo Magnaporthe rhizophila (DQ528791) S4O Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) 541 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) 542 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) 543 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) 544 Oc 09 Roo Roo Uncultured root-associated fungus (EU144817) 545 Oc 09 Roo 8 E R 2 Roo Uncultured root-associated fungus (EU144817) 546 Oc 09 Roo E R 4 Roo Fusarium ngamai (X94174) 547 Oc 09 Roo Roo Fusarium sp. (EF152423) 548 Oc 09 Roo Roo Magnaporthe rhizophila (DQ528791) 549 Oc 09 Roo Roo Microdiplodia sp. (EF43.2267) SSO Oc 09 Roo Roo Rhizopycnis sp. (DQ682600) SS1 Oc 09 Roo Roo Penicilium camemberti (DQ681326) SS2 Oc 09 Roo Roo Fusarium moniiformae (EU364856) SS3 Oc 09 Root Roo Rhizopycnis sp. (DQ682600) SS4 Oc 09 Roo y R Roo Codinaeopsis sp. (EF488392) 555 Oc 09 Roo Roo Uncultured root-associated fungus (EU144817) *GPS coordinates for each location are provided in Table 1.

Example 3 Comparisons were made between species from the east and west parts of the tallgrass prairie, as well as between sampling Endophyte Population Analysis 65 dates. In addition, fungal species from shoot and root tissue Two comprehensive endophyte species lists, one for shoot were grouped according to their higher taxonomic level (ordi and one for root, were generated for each collection date. nal level) to allow ordinal frequencies to be evaluated. US 8,975,489 B2 27 28 Species diversity in each of these fungal “communities” TABLE 3 was estimated using the Shannon diversity index (Bowman et al. 1971) as implemented in the PROCIML program, SAS Temporal distributions of endophytic fungal species in software version 9.1.3 (SAS 2004). Differences in Shannon Switchgrass shoot tissues from the tallgrass prairie. diversity indices of any two fungal communities were com Month & Species Distribution pared using Student's t-test at a 95% confidence level. The mean organic matter, macronutrients, sodium, and pH con Species April July October Total Percent tent in Soils from the east and west parts of the tallgrass prairie Acremonium sp. 2 5 O 7 4.90 were compared using PROCTTEST in SAS software version Acremonium Stricium 6 1 8 5.59 9.1.3 (SAS 2004). 10 Alternaria alternata O 2 O 2 140 Alternaria arborescens O 1 O O.70 A total of 736 culturable fungal taxa were obtained from Alternaria mai 2 O 3 2.10 three collection trips. Alternaria sp. O 2 O 2 140 Out of these isolates, 69% originated from roots, while Ascomycete sp. 2 O O 2 140 31% came from shoot tissues. Quality ITS sequences were Biomectria rossmaniae O O O.70 15 Bipolaris heveae O O O.70 obtained for 555 isolates, representing 75.4% of the total Bipolaris Oryzae 1 O 2 140 isolates obtained in this study. Of those sequenced, 74% were Biergeneratia spariinae O O O.70 from root tissue and 26% were from shoot tissues. The results Cladosporium colombiae O O O.70 presented below are based on the sequence data from these Codinaeopsis sp. O 1 O O.70 555 isolates. Colletotrichum graminicola O 2 O 2 140 Dothideomycete sp. O O O.70 Endophyte Community Structure in Shoot Tissues Emericellopsis minima O O O.70 The shoot fungal endophyte community was composed of Emericellopsis terricola 6 O O 6 4.2O 143 fungal taxa representing at least 51 different species Etit-pa scoparia O O O.70 Exserohium rostratum O 1 O O.70 (Table 3). The number of fungal taxa isolated from shoot Fusaritin actiminatin O O 1 O.70 tissues varied between study months. The numbers of taxa Fusarium moniiformae O 8 O 8 5.59 isolated in April, July, and October were 83, 46, and 14, 25 Fusarium nganai 9 5 15 10.49 respectively. Foxysportin f.sp. vaSinfectin 2 O O 2 140 Fusarium proliferatum 2 10 1 13 9.09 Endophyte Community Structure in Root Tissues Fusarium pseudograminearlin O 1 O O.70 The root fungal endophyte community was composed of Fusarium subgiitiinans O 1 O O.70 412 taxa representing at least 58 different species (Table 4). Gibbereia acuminata 1 O O O.70 The number of fungal taxa isolated from root tissue also 30 Gibberella sp. 2 8 O 10 6.99 Hypocrea ixii 3 O O 3 2.10 varied between study months. The numbers of taxa isolated in Leaf litter ascomycetes O O 1 O.70 April, July, and October were 104,156, and 152, respectively. Leptosphaeria avenaria f.sp. 1 O O O.70 Species Diversity in Endophyte Communities in Different triticea Sampling Months Leptosphaeria bicolor O 1 O O.70 35 Monographelia sp. O 1 O O.70 The species diversity in endophyte communities varied Myrothecium melanosporum 3 O O 3 2.10 between sampling months (Table 5). The shoot community Myrothecium verrucaria O 2 O 2 140 had the highest species diversity in April (HS=3.241), and the Nigrospora oryzae O 1 O O.70 lowest in October (HS=1.730). The shoot endophyte commu Parasarcopodium ceratocarvi O 1 O O.70 Penicilium citreonigrum O O O.70 nity was significantly more diverse than the root community Periconia macrospinosa O 2 O 2 140 in April, whereas the root community was significantly more 40 Phaeosphaeria sp. O O O.70 diverse than the shoot community in the October sampling Phoma glomerata O O O.70 date (P<0.001: Table 5). Pleosporaceae sp. O 1 O O.70 Species Diversity in Fungal Communities from East and West Pseudozy mafiocchiosa O 1 O O.70 Parts of the Tallgrass Prairie Sordariomycete sp. 1 2 4 2.80 Sporisorium everhartii O 10 O 10 6.99 Fungal communities from the east and west parts were 45 Stachybotry's bishyi O O O.70 similar in species diversity (Table 6). However, a combined Stachybotry's elegans 2 O 3 5 3.SO analysis of species from three collections revealed that the Uncultured Ascomycete sp. O O O.70 root endophyte community from the west had significantly Uncultured endophytic fungus 2 1 O 3 2.10 higher species diversity than that of the east (HS=2.957 vs. Uncultured root-associated O 1 O O.70 fungus 2.787; P<0.001; Table 6). 50 Soils from East and West Parts of the Tallgrass Prairie Uncultured soil fungus O O O.70 Soils from the east and west parts of tallgrass prairie were significantly different in organic matter, phosphorus, cal cium, and sodium contents (P<0.049; Table 7). The organic TABLE 4 matter and calcium content was high in Soils from the east 55 part, whereas phosphorus and sodium content was high in the Temporal distributions of endophytic fungal species in soils from the west part. Switchgrass root tissues from the tallgrass prairie. Diversity in Endophyte Communities at the Ordinal Level Month & Species Distribution Endophytic fungal isolates from the 2009 collections belonged to at least 18 orders (Table 8). Isolates from shoot 60 Species April July October Total Percent tissues were assigned to nine orders and root isolates to 15 Alternaria alternata O O 1 1 O.24 orders. Three orders were unique to shoot tissues, whereas Alternaria longissina 1 O O 1 O.24 nine orders were unique to root tissues. At least seven orders Alternaria mai 2 O O 2 O49 were common between shoot and root tissues. Members of Amyloathelia crassiusculia 1 O O 1 O.24 Anthostomella braibei 1 O O 1 O.24 the order Hypocreales were the most commonly isolated 65 Ascomycete sp. 2 2 5 9 2.18 fungi, constituting approximately 64% and 39% of the fungal Codinaeopsis sp. 3 1 4 8 1.94 communities in shoot and root tissue, respectively. US 8,975,489 B2

TABLE 4-continued TABLE 4-continued Temporal distributions of endophytic fungal species in Temporal distributions of endophytic fungal species in Switchgrass root tissues from the tallgrass prairie. Switchgrass root tissues from the tallgrass prairie. Month & Species Distribution 5 Month & Species Distribution Species April July October Total Percent Species April July October Total Percent CoprintiSatiricomits 2 O O 2 O.49 Microdochium sp. 4 1 O 5 1.21 Diaporihephaseolorum O 2 3 0.73 Mycorrhizal fungal sp. O 1 O O.24 Didymella fabae O O 0.24 10 Myrothecium cinctum O 1 O O.24 Dothideomycete sp. O O O.24 Myrothecium melanosporum O O 1 O.24 Eladia sacchia O O O.24 Penicium camemberii O O 1 O.24 Eui-pella sp. 1 O O O.24 Penicilium sp. O O 1 O.24 Fungal endophyte 3 3 O 6 1.46 Penicium verrucatiosum 1 O O O.24 Fusaritin actiminatin 2 O 3 0.73 Periconia macrospinosa 2O 9 2 31 7.52 Fusarium moniiformae 2 6 3 1 2.67 15 Phoma medicaginis O 1 O O.24 Fusarium nganai 31 23 29 83 20.15 Rhizoctonia praticola O O 1 O.24 Fusarium oxysportin O 3 O 3 0.73 Rhizopycnis sp. O 1 2 3 0.73 Fusarium oxysportin f Cicer is O O O.24 Sordariomycete sp. O 2 13 15 3.64 Fusarium proliferatum O 2 2 23 5.58 Trichoderma aureoviride O O 1 1 O.24 Fusarium Soiani O O O.24 Trichoderma koningiopsis O 1 O 1 O.24 Fusarium sp. 4 4 4 12 2.91 Uncultured Ascomycete sp. 4 4 O 8 1.94 Gaettmannomyces graminis var. O O 3 3 0.73 20 Uncultured endophytic fungus O 2 O 2 O49 tritici Uncultured Fungus 1 3 O 4 0.97 Gaettmannomyces incrisians 7 16 34 57 13.83 Uncultured Helotiales 1 1 O 2 O49 Gibberella sp. 1 O 1 2 O.49 Uncultured Hypocreates 1 3 O 4 0.97 Grass root mycorrhizal sp. O O 2 2 O.49 Uncultured Lachnum O O 1 1 O.24 Halorosellinia sp. 2 O O 2 O.49 Uncultured Leptosphaeriaceae 1 2 O 3 0.73 Hypocrea ixii 1 O 1 2 0.49 25 Uncultured Nectriaceae 1 O O 1 O.24 Kabaieia microsticia 1 O O 1 O.24 Uncultured root-associated 1 17 16 34 8.25 Macrophomina phaseolina 1 1 O 2 O.49 fungus Magnaporthe rhizophila O 14 22 36 8.74 Uncultured soil fungus 1 2 1 4 0.97 Marasnii is nigrobrunnetts O O 1 O.24 Waitea circinata war. zeae O O 1 1 O.24 Microdiplodia sp. O O 1 O.24

TABLE 5

Fungal taxa, species, and species diversity in Switchgrass endophytic fungal communities from the tallgrass prairie.

Number of Shannon Degree of

Parameter Comparisons taxa species Index (Hs) t-value freedom P value

Shoot April vs. July 46 wS. 83 3O wS. 28, 3.241 wS. 2.898 2.775 127 <0.001 Shoot April vs. October 46 vs. 14 30 vs. 7 3.241 vs. 1.73O 7.158 58 <0.001 Shoot July vs. October 83 wS. 14 28 wS. 7 2898ws. 1.73O 6167 95 <0.001 Root April vs. July 104 vs. 152 30 wS. 34 2.610 vs. 2.865 4.476 254 <0.001 Root April vs. October 104 vs. 156 30 wS. 28 2.610 vs. 2.512 1631 258 >0.100 Root July vs. October 152 vs. 156 34 wS. 28 2.865 wS. 2.512 6.920 306 <0.001 April Shoot vs. Root 46 wS. 104 30 wS. 30 3.241 wS. 2.61O 5.311 148 <0.001 July Shoot vs. Root 83 vs. 152 28 wS. 34 2.898 wS. 2.865 O497 233 >OSOO October Shoot vs. Root 14 wS. 156 7 wS. 28 1730 wS. 2.512 4.237 168 <0.001 All three months Shoot vs. Root 143 vs. 412 S1 vs. 58, 3.456 vs. 3.OO6 9.857 553 <0.001

TABLE 6 Fungal taxa, species, and species diversity in Switchgrass endophytic fungal communities from the east and west parts of the tallgrass prairie. Number of Shannon Degree of

Month Plant Part Part of Prairie taxa species Index (Hs) t-value freedom P value April Shoot East vs. West 18 wS. 28 14 wS. 19 2.553 vs. 2.818 1,060 44 >0.2OO Root East vs. West 67 vs. 37 20 vs. 17 2.292 vs. 2.358 OSSS 102 >OSOO July Shoot East vs. West 44 wS. 39 21 wS. 15 2.752 wS. 2.486 1965 81 >O.OSO Root East vs. West 82 wS. 70 23 vs. 25 2.647 vs. 2.77O 1477 150 >0.100 October Shoot East vs. West 7 vs. 7 5 wS. 3 1550 vs. 1.004 1491 12 >0.100 Root East vs. West 81 vs. 75 19 wS. 19 2.319 wS. 2.42O 1407 154 >0.100 All three months Shoot East vs. West 69 wS. 74 33 wS. 29 3166 wS. 3.071 O.949 141 >O.300 Root East vs. West 230 wS. 182 38 wS. 42 2.787 wS. 2.957 4.464 410 <0.001 US 8,975,489 B2 31 32 TABLE 7 REFERENCES Organic matter, macronutrients and sodium contents, and pH content The references listed below are incorporated herein by in the soils from the east and west parts of the tallgrass prairie. reference to the extent that they supplement, explain, provide Part of Standard t-value a background for, or teach methodology, techniques, and/or Parameter Prairie Mean eror (at 22 dif) P value compositions employed herein. U.S. Pat. No. 6,111,170 Organic matter (%) East 4.90 O.34 4.460 O.OOO2 International PCT Publication WO99/29 177 West 2.48 O.42 Phosphorus (ppm) East 9 O.99 2.370 O.O267 Abbott L. K. Robson AL (1984) The effect of VA mycorrhizae West 35 11 10 on plant growth. In: VA Mycorrhiza (eds. Powell C L. Potassium (ppm) East 3S4 63 O3SO O.7310 Bagyaraj DJ), pp. 113-130. CRC Press, Boca Raton, Fla. West 395 99 Ahmad A, et al. (2001) Fungal endophyte enhances biomass Calcium (ppm) East 7684 654 3.540 O.OO18 West 4154 751 production and essential oil yield of east Indian lemon Magnesium (ppm) East 412 41 1540 O.1380 grass. Symbiosis 30, 275-285. West 599 114 15 Arnold A E, et al. (2003) Fungal endophytes limit pathogen Sodium (ppm) East 40 8 2.08O O.0491 damage in a tropical tree. Proc. Natl. Acad. Sci. USA 100, West 96 26 pH (1 to 14) East 7.5 O16 O.O90 O.9310 15649-15654. West 7.5 O.24 Bischoff KM, et al. (2009) Extracellular hemicellulolytic enzymes from the maize endophyte Acremonium zeae. Current Microbiology 58,499-503. TABLE 8 Ordinal distributions of Switchgrass fungal endophytes in shoot and root tissues at different Sampling months. Sampling months & number of taxon Total Number of Percentage of April July October Isolates Population

Order Shoot Root Shoot Root Shoot Root Shoot Root Shoot Root

Agaricales O 2 O O O 1 O 3 O.OO 0.73 Boletales O 1 O O O O O 1 O.OO O.24 Botryosphaeriales O 7 O 2 O 1 O 10 O.OO 2.43 Cantharellales O O O O O 1 O 1 O.OO O.24 Capnodiales 1 O O O O O 1 O O.70 O.OO Chaetosphaeriales O 1 1 1 O 4 1 6 O.70 1.46 Corticiales O O O O O 1 O 1 O.OO O.24 Diaporthales O O O 1 O 2 O 3 O.OO 0.73 Dothideales O 1 O O O O O 1 O.OO O.24 Eurotiales 1 1 O 1 O 2 1 4 O.70 0.97 Helotiales O 1 O 1 O 1 O 3 O.OO 0.73 Hypocreales 28 43 52 75 11 44 91 162 63.64 39.32 Incertae sedis 1 O 1 2 2 13 4 15 2.80 3.64 Magnaporthales O 7 O 19 O 39 O 65 O.OO 15.78 Notassigned 2 O 2 16 O 22 4 38 2.80 9.22 Pleosporales 6 24 12 12 O 3 18 39 12.59 9.47 Trichosphaeriales O O 1 O O O 1 O O.70 O.OO Unknown 6 12 2 22 1 22 9 56 6.29 13.59 Ustilaginales O O 11 O O O 11 O 7.69 O.OO Xylariales 1 4 1 O O O 2 4 140 0.97

Total 46 104 83 152 14 156 143 412 1OO 100

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