AU2007203208B2 - BE9301A tall fescue with endophytes - Google Patents

Abstract

Herein diAlosed is a Neorypkodism endophle (E34) used o inoculate lhe dislosed BE9301A gas plates and seeds In, the en.ph.ye is present in . i. c.. l Wure or cLSdophyLe deposited wiUh Cont .. lbureau vee Schimrmeloultwes, P..Box 5167, 350R AD)Utrechl, The Nethrlnds (13eposit No. CBS L19998) on Jun. 27,2006underiheRudapesitTreaty Thisendophyle willb FIrrvcably and withosi resttion released CI the publhi upon Ohe issuance ofa parn. Therefore, this enadephyre is known, and readily avasileb Is o the puic . Ina onet example, thet disclosure provided end ophlyte depoejied a Cenra Ibtreau voor Selhitnmelcultsmee Deposit No: CBS OI998. This esndophyte an he used to form symbislec hest relationlp, l wth grat plsms. Alto disclosed herein install fescue variety known as breeds code BE9301A that contains the dophy l(E34) an, sdiffrcni frm all knownatictics of tall rescue plans. in Onteexniple. mature8E93OIA planis have aIaematuring dale (suchlaas earlyMay, for cxnmple May2), mash heightfrom aboul]'28 o b out|d 37cmtall, have a high mst tolerance, have a high bacteriall l tolerSee, anLor tfe a high forage yie and persistence. BE9301A tall fescue plani leaves are softer han other tall fesc eBvinge ndophyes. i somec xam Ic, BE93IAplans havc increased periscaedcceplanscebycrattle comnparrd ioather tall freorus, tshe sterall fescue having endephytes.

Description

AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant: BARENBRUG USA, INC. Invention Title: BE9301A TALL FESCUE WITH ENDOPHYTES The following statement is a full description of this invention, including the best method for performing it known to us: BE9301A TALL FESCUE WITH ENDOPHYTES FIELD This disclosure relates to a new tall fescue grass with endophytes that is more palatable 5 and late maturing compared to other known tall fescues, as well as the known endophytes used to inoculate the grass. One example of this grass is known as breeder's code BE930 I A and the endophyte is known as E34. BACKGROUND 10 The majority of the grass forage pastures in the United States are located in regions that are subjected to extreme climatic conditions with hot, dry summers and very cold winters with no snow cover. The predominant cool season perennial grass grown in the Southeast United States and transition zone is tall fescue (Festuca arundinacea). Summer survival of tall fescue is related to presence of living fungal endophyte 15 (Neotyphodium coenophialum) in the plants. For example, the presence of endophyte in tall fescue as well as perennial ryegrass can enhance drought tolerance, summer survival, insect tolerance and disease tolerance. However, cattle grazing forage from most endophyte-infected tall fescue cultivars suffer from fescue toxicosis. Fescue toxicosis results from ingesting ergot alkaloids. Ergovaline is considered to be the main ergot alkaloid responsible for most animal M0 problems. Therefore, new endophytes and forage grasses containing endophytes are needed, such as those that have a less toxic effect on grazing animals. It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general 25 knowledge in the art, in Australia or any other country. -2 304S555_ (GHNatters) P72452.AU 9-Jan-12 SUMMARY A first aspect provides an isolated endophyte deposited as CBS 119998. A second aspect provides an axenic culture of an endophyte deposited as CBS 119998. A third aspect provides a seed comprising the endophyte of the first or second aspect. 5 A fourth aspect provides a synthetic combination of the endophyte of the first or second aspect with a grass cultivar. A fifth aspect provides a seed of the synthetic combination of the fourth aspect. A sixth aspect provides a grass seed deposited as ATCC No: PTA-7709. A seventh aspect provides grass plant comprising the endophyte of the first or second 10 aspect or germinated from the seed of any one of the third, fifth or sixth aspect. An eighth aspect provides a method of producing grass seed, comprising: germinating the grass seed of any one of the third, fifth or sixth aspect; growing a grass plant from the germinated seed; setting of progeny seed; and 15 harvesting the progeny seed. A ninth aspect provides a method of producing a grass plant, the method comprising: crossing the grass plant of the seventh aspect with at least one other grass plant to produce at least one seed; harvesting the seed; and !0 germinating the seed to produce at least one progeny grass plant. A tenth aspect provides a grass seed produced by the method of the eighth aspect. An eleventh aspect provides a grass plant produced by the method of the ninth aspect. A twelfth aspect provides a progeny grass plant from a self-cross of the grass plant of the seventh or the eleventh aspect. 25 A thirteenth aspect provides a first generation grass plant from an out-cross of the grass plant of the seventh or eleventh aspect. A fourteenth aspect provides hay, comprising the grass plant of the seventh or eleventh aspect. A fifteenth aspect provides a pasture comprising the grass plant of the seventh or eleventh 30 aspect. -3 3045855_1 (GKMfte,)) P72452 AU 94e n12 A sixteenth aspect provides a vegetative sprig or clone of the grass plant of the seventh or eleventh aspect. A seventeenth aspect provides sward, comprising the grass plant of the seventh or eleventh aspect. 5 An eighteenth aspect provides sward, comprising a grass plant germinated from the seed of any one of the third, fifth, sixth or tenth aspect. A nineteenth aspect provides a seed mixture comprising the seed of any one of the third, fifth, sixth or tenth aspect. Herein disclosed is a Neotyphodium endophyte (E34) used to inoculate the disclosed 10 BE9301 A grass plants and seeds. In a particular example, the endophtye is present in an axenic culture of an endophyte deposited with Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD Utrecht, The Netherlands (Deposit No. CBS 119998) on June 27, 2006 under the Budapest Treaty. This endophyte will be irrevocably and without restriction released to the public upon the issuance of a patent. Therefore, this endophyte is known and readily available to 15 the public. In one example, the disclosure provides endophyte deposited as Centraalbureau voor Schimmelcultures Deposit No: CBS 119998. This endophyte can be used to form symbiotic host relationships with grass plants. Also disclosed herein is a tall fescue variety known as breeder's code BE9301A that contains the endophyte (E34) and is different from all known varieties of tall fescue plants. In 20 one example, mature BE930 lA plants have a late maturing date (such as early May, for example May 2), reach a height from about 128 cm to about 137 cm tall, have a high rust tolerance, have a high bacterial wilt tolerance, and/or have a high forage yields and persistence. BE930 A tall fescue plant leaves are softer than other tall fescues having endophytes. In some examples, BE930 IA plants have increased persistence and acceptance by cattle compared to other tall 25 fescues, such as other tall fescues having endophytes. At least 2500 seeds of BE930IA were deposited with The American Type Culture Collection (ATCC, Manassas, VA; ATCC Deposit No. PTA-7709) on July 11, 2006 under the Budapest Treaty. These seeds will be irrevocably and without restriction released to the public upon the issuance of a patent. Therefore, these seeds are known and readily available to the 30 public. In one example, the disclosure provides grass seed deposited as ATCC No: PTA-7709. - 3a 3045855_1 (GHMaer) P72452 AU 9-Jan-12 BRIEF DESCRIPTION OF THE FIGURES Figs. 1A-1C show the % of tillers producing alkaloids from three fescue varieties on May 3, 2006 (Fig. IA), January 12, 2007 (Fig. IB), and February 28, 2007 (Fig. 1C). 5 Fig. 2 is a table showing the allele sizes produced using the markers identified in the table. DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS In one example, the disclosure provides tall fescue plants including the morphological and physiological characteristics of BE9301A. In another example, the 10 disclosure provides tall fescue plants having all of the morphological and physiological characteristics of BE9301A. Also disclosed are seeds of such plants, progeny of such plants, and vegetative sprigs or clones of such plants. In another example, the disclosure provides grass plants having the genotype of BE9301A. The disclosure also encompasses tall fescue plants that are produced by crossing 15 BE9301A with other grass varieties (out-crossing). Seeds and plants generated from those seeds that are the result of a first out-crossing are referred to as an F1 generation (first generation). Any grass variety capable of crossing with BE9301 can be used to create and out-cross, including fescue varieties or rye grass varieties. In some examples the BE9301A is crossed with one of the varieties described in the examples section. 20 Accordingly, the disclosure also provides seeds resulting from such out-cross, grass plants grown from such seeds, and vegetative sprigs or clones from such a cross. In one example, the seeds resulting from the cross are part of a seed mixture. The disclosure also encompasses tall fescue plants that are produced by self crossing BE9301A. Self-crossing as used herein describes the process of letting the 25 same BE9301A plant pollinate itself, or letting two BE9301A plants pollinate each other. Such self-crossing produces more BE9301A seeds and plants. Accordingly, the disclosure provides seeds resulting from such self-cross, grass plants grown from such seeds, and vegetative sprigs or clones from such a cross. In one example, the seeds resulting from the cross are part of a seed mixture. -4- One of ordinary skill in the art readily understands that seed mixtures are desired in many circumstances. Seed mixtures are used to increase the amount of grass that grows in a certain location, such as in a pasture. Generally, the mixture is designed to contain more that one variety of grass seed so that if the weather conditions vary at least 5 one of the included varieties performs well. Seed mixtures include not only BE9301A seeds, seeds from self-crossing BE930 IA, or Fl seeds from out-crossing BE9301A in combination of any other known grass seed, such as other known tall fescue seeds. For example, BE930 I A seeds can be combined with one or more of the varieties described herein. 10 In another example, the disclosure provides a method of producing grass seed, including planting seed from BE9301A under conditions that result in the germination of the seed, growth of grass plants and setting of progeny seed, and then harvesting the progeny seed. Also disclosed is grass seed produced by this method, as well as grass seed mixtures including such grass seed. 15 Also disclosed is a method of producing a grass plant which includes crossing a grass plant produced from BE930IA with at least one other grass plant to produce at least one seed, harvesting the seed, and germinating the seed to produce at least one progeny grass plant. Included in the disclosure are grass plants produced using this method, as well as a vegetative sprig or clone of the grass plant. 20 The grass plants disclosed herein can be planted in a variety of areas, for example in areas where grazing of farm animals is desired. Examples include, but are not limited to pastures, such as areas for animal grazing. The disclosure also includes sward or animal feed, such as hay, which includes the grass plants (such as BE9301A plants) disclosed herein. The sward can be planted 25 in any area where grass plants are desired, such as a pasture. In one example, the grass plants disclosed herein include one or more transgenes. In some examples, the disclosed endophyte, (Neotyphodium endophyte E34), can form a stable synthetic combination with a grass host, such as a tall fescue (Festuca -5arundinacea) or a perennial ryegrass (Lolium perenne). In a particular example, the grass is a temperate tall fescue cultivar. Exemplary tall fescue cultivars include Dovey, Drover, Prosper, Barcarella, BAR FA 6FRD, Barcel, Barolex, BarElite, and Bariane, and exemplary perennial ryegrasses include Barsprinter and Remington. 5 A "synthetic endophyte/grass combination" is the combination of an endophyte and a grass cultivar (such as a tall fescue or perennial ryegrass or other forage variety) each of which has been isolated from nature, but the combination of which does not exist in nature. For example, the grass cultivar can be a culturally improved grass variety, such as having one or more desirable characteristics, such as increased 10 persistence, drought tolerance, resistance to one or more diseases, and the like. Methods of making such a synthetic combination are known in the art, and can include inoculating the desired grass cultivar (such as a tall fescue cultivar) with an axenic culture of endophyte E34, or crossing a synthetic endophyte/grass cultivar combination with an endophyte-free grass cultivar (such as a tall fescue cultivar or a perennial 15 ryegrass cultivar) to form a new cultivar infected with the endophyte. In one example, the endophyte-free grass cultivar includes one or more desirable phenotypes, such as disease resistance, drought resistance, persistence, low toxicity to grazing animals, and so forth. In particular examples, the disclosed combinations provide a grass plant having 20 reduced amounts of one or more alkaloids compared to naturally occurring endophyte/grass combinations, reduced toxicity to livestock compared to naturally occurring endophyte/grass combinations, improved palatability and digestibility compared to naturally occurring endophyte/grass combinations, or combinations thereof. In a specific example, the axenic culture of endophyte E34 in synthetic 25 combination with a tall fescue cultivar, such as the synthetic combination of BE9301 A, produces levels of ergovaline of less than 100 ppb (for example less than 50 ppb, such as 100-10 ppb or 50-20 ppb), such as about 38 ppb compared to 524 ppb produced by Kentucky 31 (containing another natural toxic endophyte). In another specific example, the axenic culture of endophyte E34 in synthetic combination with a tall fescue cultivar, -6such as the synthetic combination of BE9301A, produces levels of ergot alkaloid of less than 500 ppb (for example less than 250 ppb, such as 500-100 ppb or 250-70 ppb), such as about 122 ppb of ergot alkaloid compared to 1369 ppb (numbers are the average of those shown in Table 7) produced by Kentucky 3 1 (containing another natural toxic 5 endophyte). Also provided by the present disclosure are seeds of the disclosed synthetic combinations. For example, such seeds can be harvested from grass plants (such as tall fescue or perennial ryegrass plants inoculated with the disclosed endophyte. In another example, the seeds are harvested from plants of the disclosed synthetic endophyte/grass 10 combination grown from seeds harvested from grass plants inoculated with the endophyte. The following examples are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. 15 The singular forms "a," "an," and "the" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprising a grass plant" includes single or plural plants and is considered equivalent to the phrase "comprising at least one grass plant." The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates 20 otherwise. As used herein, "comprises" means "includes." Thus, "comprising A or B," means "including A, B, or A and B," without excluding additional elements. Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. 25 The foregoing and other objects, and features of the disclosure will become more apparent from the following detailed description of several embodiments. -7- EXAMPLE I Endophyte Isolation and Inoculation of Barcel This example describes methods used to isolate the E34 endophyte and to inoculate the Barcel variety with the E34 endophyte. 5 The Barcel variety of tall fescue (US Plant Variety Protection 8100085 issued July 15, 1982) was developed by Barenbrug Research in Wolfheze, The Netherlands. The endophyte E34 was isolated from its natural host, a tall fescue ecotype. Seeds of various different grasses were analyzed for ergovaline alkaloid. Endophyte was isolated from seeds producing significantly low ergovaline content compared to 10 Kentucky 31 tall fescue seeds. The E34 endophyte was isolated from seed as follows. Seeds (3g) were sterilized in 5% NaHCl with 2 drops 'Tween 20' for 2 hours, and then rinsed with water. This was repeated, with a final rinsing in a laminar flow hood with sterile water. Approximately 50 seeds were placed into an empty Petri dish under sterile conditions. 15 The outer coats of the seeds were removed, then the embryos were removed and suspended in sterile water (to keep moist). Each embryo was cut in half longitudinally to prevent germination, and placed into a bottle containing Mirashige & Skoog media containing 2,4:D media. The bottles were placed on an orbital shaker at 100rpm in the dark at 20'C for 4 weeks, until the first fungus was visible. Balls of fungus were 20 removed from the liquid media, and plated on PDA media. If fungus balls were not visible, the contents of the bottle were poured onto a petri dish, and then the liquid was decanted off (allowing any fungus present to remain on the plate). The E34 endophyte strain does not sporulate and is not fast growing. The color of the colony was white/brown. The aerial mycelium was felted and the texture was 25 dry. Isolated endophytes were further inoculated in developed cultivars such as Barcel and other forage grasses. The methods used for inoculation were as follows. Seeds (3g) were sterilized in 5% NaHCI with 2 drops 'Tween 20' for 2 hours. The seeds were then rinsed with water overnight. The following day, excess water was removed -8and the sterilization was repeated for 2 hours, subsequently the seed were rinsed thoroughly in laminar flow with sterile water. The sterile seeds (10) were placed in a circle in a Petri dish containing 1.5% water agar, and incubated at 20'C or 25*C, and allowed to grow until the shoot began to emerge from coleoptile. Seedlings in the Petri 5 dish were inoculated with fresh endophyte (transferred to fresh media up to fourteen days before inoculation) under sterile conditions. A small piece of endophyte mycelium (about 0.5 mm) was removed with needle or forceps. The base of the shoot was pierced with a needle to generate a hole, and the endophyte mycelium piece placed into the hole. Ideally, the endophyte was positioned in the center of the shoot. 10 The inoculated seedlings were incubated at 20'C for 7 days (12 hrs light), then transferred to test tubes or soil. Test tube transfer was as follows. Inoculated seedlings were removed from the Petri dish and placed into test tubes containing solid M&S media under sterile conditions. Tubes were incubated at 20'C (16 hrs light) for 8 weeks. Subsequently, presence of the endophyte was screened, and those having 15 endophyte transplanted into soil. Inoculated seedlings were removed from the Petri dish or the test tube and placed into a seed tray with potting mix. Root and seed were covered, leaving most of the shoot exposed. The shoot was cut to about 10 cm, and the seedlings were watered and covered to prevent evaporation. Seedlings were incubated at 20'C for 2 weeks (from test tube or Petri dish), then placed in a glasshouse. 20 Seedlings were trimmed to encourage tiller multiplication. After 8 weeks (when more than one tiller is present per plant) plants were screened for the presence endophyte. Out of 44 seedlings of Barcel that were inoculated, 7 seedlings that tested positive for endophyte were planted in a field at Barenbrug Tourneur Recherches, Mas Grenier, France. Seeds from these plants were harvested. The harvested seed was 25 tested for endophyte for verification that inoculated endophyte was stable. Five plants with desirable forage attributes (such as soft leafiness, late maturity, and freedom from diseases) and presence of endophyte were chosen. The seeds from these five was harvested separately. The seed was tested for the presence of endophyte. 100% of the seeds were positive for endophyte. These seeds were coded as BE9399. -9- BE 9399 seed was used to grow 800 plants in a greenhouse. Four rows of 200 plants each were transplanted between alternating rows of two other improved tall fescue varieties (BAR 3FA BTR9 and BAR FA IBTR10). These two other tall fescue varieties are also developed for late maturity, and high palatability and digestibility and 5 used as pollen source for crossing with BE9399 plants. In the field growing plants of BE9399 endophyte presence were rechecked at two times. After pollination, the BAR 3FABTR9 and BAR FA IBTR10 were removed and seeds from only the four rows of BE9399 plants was harvested. This harvested seed was coded as BE9301. The harvested seed tested 80% positive for endophyte. In the 10 fall, BE9301 seed was grown in greenhouse and 2110 live seedlings were analyzed for endophyte and 1650 seedlings tested positive for endophyte and were transplanted in a multiplication block in field. In spring, the multiplication block was checked for uniformity. In summer, the seed was harvested from this block was coded as BE9301A and had 100% endophyte. 15 BE9301A seed has been used for forage trials and further multiplication in U.S and France. The BE9301A variety retains endophyte viability and transfer through at least three generations. Detection of Endophyte 20 Endophyte was detected in grass seed as follows. Seeds were incubated in a 5% NaOH solution overnight then rinsed with water. Seeds were placed on a glass slide and a drop of analine blue (0.325 g aniline blue, 100 ml water, 50 ml lactic acid 85%) was added. The seed was crushed with steady pressure applied to a cover slip on top of the seed. Hyphae are stained blue and located mostly in the area of the darkly stained 25 aleurone cells, indicating the presence of endophyte. Endophyte was detected in grass plants as follows. A tiller from the plant was removed, and all dead leaves removed. The root was trimmed, leaving 0-5 cm above the ground, close to the meristem, were the mycelium density is the highest. A longitudinal section from the adaxial surface of the leaf sheath was removed. The - 10epidermal strip with the inner surface facing upwards was placed onto a glass slide containing a drop of aniline blue. The sample was examined for the presence of characteristic intercellular mycelium as described above. 5 EXAMPLE 2 Seed Deposits Seeds of the tall fescue variety BE9301A inoculated with endophyte described in Example 1 were deposited with the ATCC (Manassas, VA) under the Budapest Treaty on July 11, 2006 under accession number PTA-7709. The identification 10 reference by the depositor is BAR FA BE9301A. The variety is also maintained at, and available from, Barenbrug U.S.A., Inc., P.O. Box 239, Tangent, Oregon 97389. EXAMPLE 3 Endophyte Deposit 15 The endophyte described in Example 1 was deposited with the Centraalbureau voor Schimmelcultures depository in The Netherlands under the Budapest Treaty on June 27, 2006 under Deposit No. CBS 119998. The endophyte is also maintained at, and available from, Barenbrug Holland, Stationstraat 40, Post bus 4, 6678 ZG Oosterhout, The Netherlands. 20 EXAMPLE 4 Analysis of Endophyte by Polymorphic DNA Microsatellite Analysis This example describes methods used to genetically analyze E34 and the 25 commercial isolates, such as ArkPlus. The results from the genetic analysis indicate that E34 is genetically diverse from ArkPlus. E-34 was also identified as a member of the taxon N. coenophialum. Genetic differences (either in allele size or number of alleles) were detected between isolate E34 and an isolate of ArkPlus for more than 18 of the 39 SSR loci (see, - 11- Fig. 2 which provides allele size information). This includes two of the five SSR loci described in US patent number 6,111,170 herein incorporated by reference. Materials and methods To obtain mycelia for genomic DNA extraction, the isolates were grown in 2.4% 5 w/v potato dextrose broth (Difco, Sparks, Maryland, USA) at 22"C in the dark with shaking at 150 rpm for 2-3 weeks. Total genomic DNA was extracted from lyophilised mycelia from each isolate following the method of M6ller et al. (M61ler et al., Nucleic Acids Research. 20: 6115-6116, 1997). Total genomic DNA was extracted from either single seeds or seed bulks (5 or 10 10 seeds) using the Plant DNeasy kit (Qiagen, Hilden, Germany). The E34 isolate as well as two N. lolii isolates (Lp19 and ARl) were genetically characterised with a total of 39 SSR markers (Table 1). Polymerase chain reaction (PCR) amplifications were performed using the markers and methods described in U.S. patent number 6,111,170 (B4, B6, B9, B10 and Bl 1), however, the error in primer 15 B 11.4 was corrected using the sequence described in Moon et al., Applied and Environmental Microbiology 65: 1268-1279, 1999. For the remaining markers, PCR amplifications were performed in 20 sl reaction volumes containing 1 x PCR buffer (Bioline, London, UK), 0.2 mM of each dNTP (GE Healthcare, Chalfont St Giles, UK), 0.5 U Immolase (Bioline), 0.25 pM each primer (Sigma Aldrich, St. Louis, Missouri, 20 USA; Applied Biosystems, Foster City, Calfornia, USA) and 10 ng genomic DNA. The forward primer was 5'-end labelled with a fluorescent phosphoramidite dye (6-FAM, HEX or NED). Amplification was performed in a thermocycler (GeneAmp, PE Applied Biosystems, Foster City, California, USA) using an appropriate touchdown profile depending on the Tm value of the primer pairs: (1) 10 minutes at 95"C, 10 cycles 25 of 30 seconds at 94"C, 30 seconds at 65"C, 1 minute at 72*C with a reduction of annealing temperature of 1*C every cycle, followed by 20 cycles of 30 seconds at 94"C, 30 seconds at 55"C, 1 minute at 72"C; (2) a similar profile as (1) with an initial annealing temperature of 60*C and final annealing temperature of 50"C; (3) a similar profile as (1) with an initial annealing temperature of 55"C and final annealing - 12temperature of 45*C; and (4) a similar profile as (1) with an initial annealing temperature of 50*C and final annealing temperature of 40"C. For each isolate, reactions with different sized-products and fluorochrome labels were combined and purified by isopropanol precipitation. Purified products (2 pl) were mixed with MegaBACE loading 5 solution containing internal size standard ET400-R (GE Healthcare) and denatured at 94*C for 5 minutes before analysis on the MegaBACE 4000 capillary electrophoresis platform (GE Healthcare). Table 1. Endophyte SSR markers. 10 Marker Motif Touchdown program NCESTAIAA09 (GAC) 7 2 NCESTAIAGII (CTCAG) 5 NCESTAIAG12 (GCAC) 6 3 NCESTA I BDO9 (GA) 6 I NCESTAICC10 (GT) 4

(GC)I(GT)

4 NCESTAIDHO4 (GTC) 8 2 NCESTA I FC04 (GCAAA) 4 3 NCESTAIFHO3 (GCC) 7 4 NCESTAIGA07 (CAT) 7 4 NCESTAIHA02 (CAG)I NCESTAlIC04 (GCA) 4

(AG)

1

(AAC)

4 2 NCESTA I G08 (GCA) 8 2 NLESTAIBB08 (TC), 2 NLESTAICCO2 (AT) 4

(GT),(AT)

4

(A),(AT)

4 NLESTA I CCO5 (TGT) 1 7 NLESTAIEE05 (GCATC) 6 (GTATC),(GCATC),(GTATC), 2 NLESTAIGA07 (CT) 5

(A),(CT)

4 3 NLESTA I GF09 (GAG) 4 NLESTA I G04 (CAC) 6 NLESTAI LB02 (ACA) 12 2 NLESTA I LB04 (CGG) 6 NLESTA I LF05 (ACA) 4 (GCA), NLESTAINB03 (CCA) 3 (TCA),(CCA)I NLESTAINF04 (TGC) 8

(TGA)

3 (TGG),(TGA) NLESTAINGO3 (GTC) 6 I NLESTAIPF06 (TA) 7 2 NLESTAIQA09 (GA) 2 0

(G)

1

(GA)

3 3 NLESTAIQE05 (GCA), 3 NLESTAIRDO4 (GGA) 4 NLESTAITAIO (TC) 3 1 2 NLESTAIXBOI (GTG) 4 NLESTAIXCO6 (GAC) 8 2 NCSSRA I AHO2 (CT) 1

(C):(CT)

6 2 EFSSRA1AH07 (GTT)o(GCT),(GTT), 2 B4 (CA) 3 (GCG) (CA) 3

(ACG),(CA)

3

(A),(CA)

4 - 13 - B6 (CAT) 2

(CAC),(CAT)

3 B9 (G) 5

(AG)

9 and (GAGAG) 2

(C)

1

(GAGGA)

2 B10 (AGC) 3

(CG),(CAT)

3

(CAA)

5 B1i (GACA) 1 8 Products or alleles detected across each of the isolates were characterized by size (i.e. number of base pairs) using MegaBACE Fragment Profiler version 1.2 software (GE Healthcare). Isolates were scored for the presence (1) and absence (0) of 5 each allele for each SSR locus. For seed samples, due to inconsistent detection of multiple alleles, the data score for the various SSR loci was taken from different single and bulk seed samples to ensure all multiple alleles were accounted for. Data matrices were generated and analysed with NTSYS version 2.02i software (Exeter Software, Setaukat, New York, USA). Genetic identity between isolates was measured using the 10 simple matching coefficient. Phenograms were constructed from the genetic distances by the unweighted pair group method of arithmetic averages (UPGMA). The goodness of fit of the clustering method was tested by Mantel test statistics comparing the genetic identity matrix with the cophentic matrix. 15 EXAMPLE 5 Morphological Characteristics of BE9301A This example describes field trials that were conducted to compare morphological characteristics of the BE9301A variety to other tall fescue grass varieties. 20 Seeds from fourteen different tall fescue varieties were sown in greenhouse potting medium in August 2003. After germination, the seedlings were transplanted into plug trays in September 2003. Seedlings were grown in the greenhouse until the plugs were filled with roots. During December 2003, the plug seedlings were transplanted in a field nursery near Albany, Oregon. The seedlings were transplanted 25 two feet apart. Sixty seedlings from each variety were grown in this field nursery. The field nursery was maintained with crop management practices used for grass seed production systems in the region. - 14 - In mid July 2004 and 2005, the following morphological characteristics were measured: (1) heading Day (the day on which a plant has three emerged panicles); (2) flag leaf height (the flag leaf is the highest leaf on a grass stem); (3) flag leaf blade length; (4) flag leaf blade width; (5) panicle length; (6) tiller leaf width; (7) tiller leaf 5 length; (8) first internode length; and (10) plant height. The trial consisted of commercially available cultivars and the BE9301A variety. Tables 2-3 show comparisons of several morphological characteristics of the BE9301A variety with other tall fescue varieties. Table 2: Morphological characteristics in 10 2004 Data Collection year 2004 Heading Flag Leaf Flag Leaf Flag Leaf Panicle Tiller Leaf Tiller Leaf First Intemode Plant Dav Height Length Width Length Width Length Length Height Julian cm cm mm cm mm CM cm cm Variety Name BAR 3FA BTR9 162.1 a 54.2 e 19.8 a 7.3 28.2 ab 9.2 27.4 abc 17.4 ef 106.1 d Barianne 160.6 a 60.7 a-d 18.6 a 7.7 30.9 a 10.7 27.8 a 20.1 cde 112 cd BAR FA 1BTRi0 157.8 ab 53.1 e 18.4 a 8.6 28.3 ab 10.6 26 a-e 18.2 def 111.8 cd Barcel 157.8 ab 55.8 de 19.7 a 8.4 28.3 ab 10.3 27.2 a-d 19.1 def 115.6 a-d BE9301 A 157.3 abc 55.8 de 19.3 a 8.4 28.5 ab 11.1 28.3 a 2D.7 bcd 122.1 ab Barolex 156.9 abc 52.2 e 19.3 a 7.4 27.6 b 9.3 27.7 ab 17 f 113.1 bcd Kentucky 31+ 154 4 bcd 65 a 13.5 Cd 7.9 28.6 ab 11.7 22.9 fg 23.9 a 121.7 abc Barcarella 153.6 b-e 64.7 ab 15.3 bc 7.6 27.4 bc 10.1 23.4 ef 24.2 a 119.4 abc Kentucky 31- 151.4 cde 59.8 bcd 12.7 d 7.5 27.8 b 10.6 20.3 g 22.1 abc 128.9 abc BTR 6 150.5 def 64.7 ab 16 b 7.7 28.7 ab 10.9 24.5 c-f 23.4 ab 125.5 a Dovey 148.3 efg 57.1 cde 15.2 bc 7.3 26.3 bcd 10.5 24.2 def 23.8 a 117.5 abc Forager 147.6 efg 60.3 a-d 15.8 bc 7.1 24.6 cd 10.2 27.9 a 24.8 a 112.8 bcd Bar Fa 6FRD 145.3 fg 57 cde 15.3 bc 7.4 26.5 bcd 10 24.7 b-f 23 ab 120.6 abc Fawn 142.5 g 62 abc 13.8 bcd 6.4 23.7 d 9.1 24.8 b-f 22.9 abc 113.2 bcd [SD (P=.05) 612 4 94 2.44 NS 2.83 NS 3.03 2.79 10.02 CV 1 2.38 5.01 8.761 9.61 6.12 10.26 7.07 7.75 5.12 Means followed by same letter do not significantly differ (P=.05. LSD) NS=Non Significant - 15- Table 3: Morphological characteristics in 2005 Morphological measurements in various forage tall fescue cultivars Trial ID: 03-Fa-Morph Location: Albany Data Collection year: 2005 Heading Flag Leaf Flag Leaf Flag Leaf Panicle Tiller Leaf Tiller Leaf First Internode Plant Day Height Length Width Length Width Length Length Height Julian cm cm mm cm mm cm cm cm Variety Name Barianne 129.8 a 82.8 a-d 27.3 a 7.5 35.4 a 8.5 b-e 38.6 ab 54.7 133.2 BTR 10 126 ab 76.9 cde 25 ab 7.2 29.4 bc 9.2 bcd 35.4 bcd 57.6 128.5 BTR 9 124.8 bc 76.6 de 24.5 bc 7.1 28.8 b-e 8.4 b-e 43.2 a 51.4 124.3 Barolex 123.5 bcd 78.2 cde 22.2 cd 6.5 29.9 bc 7.6 def 35 bcd 55.7 129.2 Barcel 122.8 bcd 74.4 de 24.1 bc 6.2 30.2 b 7.5 def 35.2 bcd 51.9 124.4 BE9301 A 122 cd 80.1 bcd 24.1 bc 6.7 28.8 b-e 8.1 b-f 36.5 abc 55.3 131.2 Barcarella 121.5 cd 90.2 abc 19.1 efg 6.5 28.8 b-e 9.8 abc 29.9 c-f 49 126.9 BTR 6 120.7 d 85.2 a-d 22.7 bcd 8.5 30 bc 8.4 b-e 35.3 bcd 52.6 135.8 K31+ 116.7 e 95.7 a 20.5 def 8.2 31.5 b 9.9 ab 30.7 c-f 57 145.2 Bar Fa 6FRD 114 e 80.8 bcd 22.7 bcd 7.3 30.1 bc 11.2 a 33.7 b-e 54.5 125.2 K31- 113.8 e 91.5 ab 21.2 de 6.9 29.2 bcd 8 c-f 28.1 d-g 57.4 140.2 Forager 106.3 f 65.9 e 15.6 h 5.3 25.5 e 6.9 ef 22.2 g 56.3 120.9 Fawn 103.3 f 75.7 de 17.8 gh 6.9 26.7 cde 6.4 f 26.4 efg 56.1 128.1 covey 97.6 g 76.1 de 17.9 fgh 6.3 25.8 de 8.1 b-f 23.2 fg 58.3 147.7 LSD(P=.05) 3.95 13.31 2.65 NS 3.49 1.88 7.74 NS NS CV 2.01 9.82 7.24 14.53 7.09 13.3 14.24 6.61 9.68 Means followed by same letter do not significantly differ (P=.05. LSD) NS=Non Significant Table 4 provides a summary of the analysis of persistence for tall fescue, 5 perennial ryegrass and Kentucky bluegrass varieties. Grasses were sown in September 2002 in Princeton, Kentucky, and persistence analyzed on the dates shown in the Table. Table 4. Percent stand of tall fescue, perennial ryegrass (PRG) and Kentucky bluegrass (BG) varieties sown September 25, 2002 in a cattle grazing tolerance study at Princeton, Kentucky. Percent Stand 2003 2004 2005 Variety Apr 3 Dec 2 Apr Dec 21 Apr 15 Nov 3 Commercial Varieties-Available for Farm Use Select 90 78 77 78 68 83* KY31+' 90 78 78 75 72 81* Festival 90 76 75 80 72 78* Calibra (PRG) 90 85 79 67 72 37 - 16- Certified Kenblue (BG) 90 23 25 25 33 33 Experimental Varieties FA 2845 90 80 84 80 78 87* KYFA9301 90 79 78 80 77 83* KYFA9304 90 79 78 80 78 83* AGRFA 104 90 78 80 77 77 82* AGRFA 106 90 77 78 75 78 82* AGRFA 114 90 78 78 85 73 82* KY3I J 90 81 79 78 72 81* FA 2458 90 75 77 77 73 78* FA 2651 90 77 76 75 72 78* FA2720 90 78 80 80 73 77* BE9301A 90 78 78 75 72 77* HMll 90 75 70 80 77 72 AGRFAI1 90 70 73 67 68 62 EC411(PRG) 90 83 84 77 70 53 KYPP 9901 (BG) 86 12 26 28 33 40 HB96(BG) 6 13 5 10 18 22 HB95(BG) 5 10 13 15 18 17 Mean 83 68 67 67 65 68 CV,% 2 11 12 12 15 15 LSD,0.05 2 9 9 9 11 11 * Not significantly different from the highest value in the column, based on the 0.05 LSD. "+" indicates variety is endophyte infected; "-" indicates variety is endophyte free. EXAMPLE 6 Resistance to Rust and Bacterial Wilt The susceptibility of BE9301A tall fescue to rust (Stem rust, Puccinia grammis) 5 and bacterial wilt (Xanthomonas spp) was measured. Seeds were grown as described in Example 5. Symptoms of a rust disease and bacterial wilt (which occurred naturally) were visible. Initial symptoms of rust were visible damage of leaves by rust spores, and there were also brown stripes visible on the leaves. - 17- Differences in rust and bacterial wilt susceptibility were observed for BE9301A, which was superior to other known cultivars. EXAMPLE 7 Alkaloids Characterization 5 This example describes methods used to characterize the amount of alkaloids (such as ergovaline) present in BE9301A grass. Endophytic funguses can produce alkaloids. Of the alkaloids produced ergovaline can cause animal health problems at certain concentrations. Toxicosis due to ergovaline is induced in livestock at the following concentrations: horses, 300-500 ppb 10 (0.3-0.5 ppm); cattle, 400-750 ppb (0.4-0.75 ppm); and sheep, 88-1200+ ppb (0.8-1.2 ppm). Therefore, BE9301A grass samples and seed were analyzed for the presence of alkaloids. The presence of ergovaline was determined using chromatogram analysis. Experimentation was performed by the Oregon State University College of Veterinary 15 Medicine. To detect ergovaline alkaloid, fluorometric HPLC assays were conducted. BE9301A and other grasses (BAR FA 1004, Kentucky 31+ and Kentucky 31-) were grown in Lexington, Kentucky. Samples were oven-dried and then analyzed. Fescue straw was analyzed as a control. As shown in Table 5, BE9301A grass plants were found to have about 27-56 ppb of ergovaline. This indicates that the levels of 20 ergovaline in BE9301A hay are safe for animal consumption. - 18- Table 5. Alkaloids in BE9301A* Sample Sample collected May 3, Sample collected July 24, Sample collected Oct. 22, 2002, reported June 19, 2002, reported Aug. 28, 2002, reported Nov. 18, 2002 2002 2002 Ergovaline Ergovaline Ergovaline BAR FA 1004 <10 <10 <10 BE 9301A 38 27 56 *Kentucky 31+ 524 307 792 *Kentucky 31- <10 <10 <10 e amounts of ergovaline in ppb * Samples noted as "+" include endophyte and samples notes as "-" do not include endophyte 5 BE9301A and other tall fescues grown in Mas Grenier, France were analyzed for the presence of ergovaline using HPLC with fluorimetric detection from an adaptation of the method of Hill et al. (Crop Science 33:331-3, 1993). Grass was grown in 2002. As shown in Table 6, BE9301A grass had about 0.05 - 0.12 ppm of 10 ergovaline, which is significantly less than the toxic tall fescue Kentucky 31+. - 19- Table 6. Amount of ergovaline present in tall fescues growing in Mas Grenier. Variety Ergovaline % Endophytes Ergovaline Ergotamine pg/g (ppm) in plot ug/g with ug/g with 100% E+ 100% E+ Cut 21/6/02 BE9301A 0.12 53.30% 0.225 0.169 Kentucky 31+ 0.27 36.60% 0.737 0.205 Cut 26/09/02 BE9301A 0.12 53.30% 0.225 0.182 Kentucky 31+ 0.22 36.60% 0.601 0.273 Cut 13/11/02 BE9301A 0.05 53.30% 0.093 Kentucky 31+ 0.10 36.60% 0.273 Tall fescues including BE9301A were grown in Lexington, Kentucky. Forage clipping samples were obtained and freeze dried, then analyzed for the presence of 5 ergovaline and a complex of all ergot alkaloids that are considered harmful to animals. (ELISA test, Agrinostics Ltd., developed by Dr. Nick Hill, University of Georgia). As shown in Table 6, BE9301A contains an average of about 98-309 ppb alkaloid, less than Kentucky 31 (KY3I+ ), indicating it is safe for animal consumption. - 20 - Table 7. Comparison of alkaloids in tall fescues. Samples collected August 15, 2003, Sample collected Nov. reported on September 14, 2003 18, 2003, ppb alkaloid ppb alkaloid Variety Rep ELISA I ELISA 2 AVE Barolex 1 7 0 3 603 Barolex 2 4 0 2 Kentucky3l + 1 1249 1182 1216 2235 Kentucky3l + 2 1532 1509 1521 BE9301A 1 100 98 99 BE9301A 2 136 152 144 309 This data indicates that BE9301A grass plants and straw are safe for livestock. Therefore, BE9301A straw and grass plants can be used in pastures for grazing, and to 5 make hay to feed to livestock. Additional data was collected in 2006 and 2007, further indicates that BE9301A produces low levels of alkaloids (see, Figs. 1A-IC). EXAMPLE 8 10 Grazing Trial This example describes methods used to demonstrate that BE9301A is not toxic to livestock, is persistent in pastures, and is acceptable to cattle. The data in example 7 indicate that BE9301A is safe for animal consumption. However, endophytes produce many alkaloids besides ergovaline whose effects are not clearly understood. Therefore 15 these grazing and feeding trial methods were used to validate the safety and productivity of an inoculated endophyte grass combination. Animal grazing trials were performed as follows. Varieties Kentucky 31 (KY3 1+), BE9301A, and Barcel (see Example 1) were sown in October 2004 in Hope, Arkansas, with three replications. Each replication was a two acre pasture. In the -21 spring-summer 2006 and 2007, each pasture was stocked with six calves, for two grazing seasons (2 years from mid-January to early June). As shown in Tables 8 and 9, in 2006, the Average Daily Weight gain (ADG) was significantly higher for BE9301A compared to the toxic Kentucky 31. In 2007, and 5 the total gain/calf in BE9301A pasture is significantly higher than Kentucky 31 and Barcel. Table 8. Performance of steers grazing native infected endophyte (Kentucky 31), endophyte free (Barcel), or novel endophyte infected tall fescue (BE930IA) during the 10 2006 production year. Date Kentuky-31 Barcel BE 9301A SE Body Weight, lb January 4 503 503 500 14.74 February 1 568 603 588 17.92 March 1 610 674 637 17.21 March 29 642 734 694 23.8 April 26 690a 822b 780b 22.57 May 24 683a 828 798 23.63 June 21 719a - 824b 23.63 Average Daily Gain, Ib Jan. 4 to Feb. 1 2.30 3.57 3.11 0.35 Feb. 1 to Mar. 1 1.48 2.51 1.76 0.30 Mar. 1 to Mar. 29 1.15 2.17 2.04 0.35 Mar. 29 to Apr. 26 1.71a 3.13c 3.07bc 0.20 Apr.26 to May 24 -0.25 0.21 0.63 0.56 May 24 to Jun. 21 1.34 - 0.93 0.26 Overall ADG 1.29a 2.32 d 1.93bc 0.13 Total Gain/calf, lb 213a 3b 324b 24.8 Grazing-d/acre 27 5 bc 227a 252ab 9.8 Gain/acre, lb 3312 512_'_ l 485" 43.5 abed: Least-square means with uncommon superscripts differ (p 6) SE: Standard Error of mean abcd: Least-square means with uncommon superscripts differ (p !) - 22 - SE: Standard Error of mean OSL: Similar grazing trials were performed in 2007 and the results are shown in Table 9, below. 5 Table 9, Performance of steers grazing native infected endophyte (Kentucky 31), endophyte free (Barcel), ArkPlus, or novel endophyte (BE9301 A) infected tall fescue during the 2007 production year. KY-31 Barcel BE SE 9301 A Body Weight, lb January 9 583 580 572 44.5 February 6 612 631 617 44.1 March 6 654 676 666 33.6 April 3 687 724 723 29.6 May 1 730 748 793 53.2 May 29 - - 826 55.7 Average Daily Gain, lb Jan. 9 to Feb. 6 1.03 1.82 1.62 0.22 Feb. 6 to Mar. 6 1.36 1.33 1.65 0.41 Mar. 6 to Apr. 3 1.18 2.36 2.05 0.54 Apr. 3 to Mayl 1.55 0.86 2.43 0.49 May I to May 29 - - 1.17 0.58 Overall ADG 1.24 1.67 1.80 0.18 Total Gain/calf, lb 131a 94a 236 35.9 Grazing-d/acre' 15_4_ ___ 84a 196c 24.0 Gain/acre, lb 196a 140a 354" 53.9 10 ' Calves were removed from 2 Barcel, 1 KY-3 1, and 1 BAR FA BE 9301A pasture due to flooding, cattle were subsequently returned to I Barcel, the KY-3 1, the BAR FA BE 9301 A, pasture. The trial was ended for 1 Barcel pasture on 6 February, and I Barcel pasture on 6 March, because of low stand counts and forage availability. 15 a- Least-squares means with differing superscripts differ (P < 0.08). SE: Standard Error of mean - 23 - At various times since the grazing trial was established, stand density of the pastures was calculated by conducting stand counts at 20 places in each pasture. The average percent stand density for all pastures of a given variety are shown in Table 10, below. While BE9301A provided better weight gains than Kentucky 31 and Barcel; its 5 persistence was good as the stand density of BE930IA did not decline during the length of the grazing trial. The stand density of endophyte free Barcel was drastically reduced, indicating that BE9301A with endophyte E-34 has better stand persistence. Table 10. Stand Density of indicated varieties. Dec-05 May-06 Jan-07 1-Mar-07 4-May-07 Kentucky3l+ 89% 86% 80% 85% 85% BE9301A 68% 69% 64% 59% 61% 10 Barcel 45% 32% 19% 10% 13% Animal Behavior observations were also conducted in the grazing trial. Cattle were monitored daily from January 2006 to May 2006, and the percent of the day that they spent grazing, cooling in the shade, or sunning was recorded. Cattle that were in 15 the BE9301A pastures spent significantly more time grazing compared to Kentucky 31 during the middle of the day. The cattle on Kentucky 31 pastures were standing in shade. This shows the classical symptom of the Kentucky 31 endophyte toxicity where the cattle is heat-stressed and spends more time cooling off rather than grazing resulting in loss of productivity. Cattle on BE9301A pastures did not show such behaviors of 20 toxicity. Thus, this indicates that the endophyte E-34 in BE9301A does not have toxic symptoms like the endophyte in Kentucky 31. The animal weight gains; stand persistence data; and behavioral data indicate that BE930IA grass is useful, inter alia, as a pasture grass. - 24 - EXAMPLE 9 Production of BE9301A Grasses BE9301A can be grown under normal conditions for growing turf grasses, and bulk seed for large-scale planting can be obtained by methods known in certified seed 5 production. For example, bulk seed may be produced by planting BE9301A seeds and allowing the mature plants to produce seed by cross-pollination with each other and then collecting the seed. Standard precautions should be taken to prevent cross pollination from other grasses, such as growing the variety in an isolated plot of sterilized soil, removing adjacent vegetation, and so forth. The BE9301A seeds 10 deposited with ATCC are breeder seeds; propagation of plants from these seeds can be performed under standard conditions known to those skilled in the art. EXAMPLE 10 Exemplary Uses of BE9301A 15 BE9301A can be used in the same way as other tall fescue varieties. However, the increased palatability and late maturing date of this grass affords BE9301A particular advantages over other varieties. Therefore, this grass and seed can be used in areas where high forage quality grass for animals is desired, for example on a farm or in a pasture. Thus, BE9301A is especially marketable and therefore useful. 20 EXAMPLE 11 Introducing Traits of BE9301A Into Other Grass Varieties The morphological and physiological characteristics of BE9301A, including increased digestibility, palatability, decreased toxicity, later maturing date, or 25 combinations thereof, can be introduced into other grass varieties (such as other forage grasses) by conventional breeding techniques. For example, BE9301A can be grown in pollination proximity to another variety of tall fescue grass, allowing cross-pollination to occur between BE9301A and the other variety, and then harvesting the hybrid seeds. Plants grown from these hybrid seeds can then be tested for the maintenance of the - 25 molecular characteristics described above for BE9301A, and/or the plants can simply be observed to see if they display the same growth characteristics described in the above tables. Ideally, the hybrid plants and seeds retain the E34 endophyte. For example, plants grown from these hybrid seeds can be tested for any of the 5 morphological characteristics described herein, for the presence of the endophyte, for lower amounts of alkaloids (for example as compared to Kentucky 31), or combinations thereof. In this way, these desirable characteristics can be combined with other desirable plant characteristics. Thus, the provision of BE9301A enables the production of progeny plants of BE9301A having the desired characteristics for grazing animals 10 (such as increased palatability). "Progeny plants" of BE9301A are any plants that are the offspring of a cross between BE9301A and any other plant or plants. Progeny plants also include successive generations of the offspring, for example those selected for the desired BE9301A characteristics using the methods described herein. First generation progeny plants may retain the desired characteristics of the BE930IA parent. 15 However, if a first-generation progeny plant does not retain the desired level of digestibility and palatability (or alkaloid levels) observed with BE9301A, subsequent generations of offspring can be recycled for digestibility and palatability (or alkaloid levels) which have at least the same digestibility and palatability and alkaloid characteristics of BE9301A described herein. In one example, subsequent generations 20 of offspring can have alkaloid levels below that of BE9301A or have better digestibility and palatability than BE9301 A. In addition, BE9301A can be used as transformation targets for the production of transgenic grasses. In certain examples, the present disclosure contemplates the transformation of cells derived from BE9301A with at least one transgene. For 25 example, transgenes that can be used, include, but are not limited to, transgenes that confer resistance to herbicides (such as glyphosate), insect, disease (viral, bacterial, fungal, nematode) or drought resistance, standability, prolificacy, salt damage resistance, and quality are useful. Examples of such genes and methods of transforming plants are described in U.S. Patent No. 6,025,545 (Lundquist et al.). - 26 - EXAMPLE 12 Inoculation of Perennial Ryegrass with Endophyte The E34 endophyte can also be used to inoculate other grasses, such as forage 5 perennial ryegrass. Such synthetic combinations of endophyte and grass or endophyte and seed can be used for grazing or for planting in a pasture. For example, the inoculation methods described in Example I can be used to inoculate seeds from any desired grass, such as a forage grass (such as those listed in the summary section). 10 EXAMPLE 13 Forage Yield and Palatability This example provides results indicating that BE9301A has acceptable forage yield and palatability. The trial was conducted in Mas Grenier, France. Palatability measured through sheep preference trials. Briefly, sheep were given the opportunity to 15 graze on the varieties described below and their preferences were recorded using standard methodology. The results indicate that the forage yield and palatability of BE9301A is higher than Kentucky 31+. Table 11. Forage Yield and Palatability of BE930IA. 20 Palatability Forage Yield (/o (1=worst; Variety of average) 9=best) BarElite 94 7.5 Barolex 94 6.2 BE9301A 105 5.8 Kentucky 31+ 92 4.7 BAR FA 9BTR6 106 2.3 cv. 3 4 LSD (.05) 9 0.5 Forage Yield: Average of 8 cuttings in 2006 and 2007 Palatability: Average of 7 observations in 2006 and 2007 -27- Cell wall digestibility measured by NIRS (Near infrared reflectance spectroscopy). The samples were collected from two year old tall fescue trial grown in Crossville, Alabama. The samples used for forage quality analysis are from the first 5 cutting in 2006. The data indicates that BE9301A has better digestibility than Kentucky 31. Table 12. Cell Wall digestibility of BE9301A Variety %CWD BarElite 52.3 BE 9301A 48.9 Barolex 48.9 BAR FA 9BTR 6 47.8 Kentucky 31+ 46.4 Kentucky 31- 44.9 LSD (P=.05) 2.76 CV 3.55 Example 14 10 Genetic Engineering of Endophyte E34 The endophyte provided herein can be used as a host for the introduction or deletion of various nucleic acid sequences (for example through the use of recombinant nucleic acid sequences). For example, the genes responsible for alkaloid biosynthesis can be attenuated through deletion or alteration of their control sequences using the 15 methods described in U.S. patent number 6,335,188, which is herein incorporated by reference. In view of the many possible embodiments to which the principles of the disclosed invention can be applied, it should be recognized that the illustrated embodiments are only examples of the invention and should not be taken as limiting the 20 scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims. - 28 -

Claims (31)

1. An isolated endophyte deposited as CBS 119998. 5

2. An axenic culture of an endophyte deposited as CBS 119998.

3. The endophyte of claim I or claim 2, further comprising a recombinant nucleic acid sequence. 10

4. A seed comprising the endophyte of claim I or claim 2.

5. The seed of claim 4, wherein the seed comprises about 10-100 ppb of ergovaline.

6. A synthetic combination of the endophyte of any one of claims I to 3 with a grass cultivar. 15

7. The synthetic combination of claim 6, wherein the grass cultivar comprises a tall fescue cultivar or a perennial ryegrass cultivar.

8. The synthetic combination of claim 7, wherein the tall fescue cultivar is Bar 3FABTR9 or !0 Barcel.

9. The synthetic combination of claim 7 or claim 8, which has been made by inoculating the grass cultivar with an axenic culture of the endophyte. 25

10. The synthetic combination of claim 7 or claim 8, which has been made by crossing the synthetic combination with an endophyte-free grass cultivar to form a new grass cultivar infected with the endophyte.

I1. A seed of the synthetic combination of any one of claims 6 to 10. 30 - 29 30458551 (GHMatters) P72452.AU 9jn-12

12. The seed of claim 11, wherein the seed has been harvested from a grass plant inoculated with the endophyte.

13. The seed of claim 11, wherein the seed has been harvested from a plant of the synthetic 5 combination grown from seeds harvested from grass plants inoculated with the endophyte or from seeds of subsequent generations of grass plants inoculated with the endophyte.

14. A grass seed deposited as ATCC No: PTA-7709. 10

15. A grass plant comprising the endophyte of any one of claims I to 3 or germinated from the seed of any one of claims 4, 5 or I I to 14.

16. The grass plant of claim 15 further comprising a transgene that confers upon the grass plant resistance to a herbicide. 15

17. A method of producing grass seed, comprising: germinating the grass seed of any one of claims 4, 5 or 11 to 14; growing a grass plant from the germinated seed; setting of progeny seed; and 20 harvesting the progeny seed.

18. A method of producing a grass plant, the method comprising: crossing the grass plant of claim 15 or claim 16 with at least one other grass plant to produce at least one seed; 25 harvesting the seed; and germinating the seed to produce at least one progeny grass plant.

19. The method of claim 18 further comprising transforming the grass plant with a transgene that confers upon the grass plant resistance to a herbicide. 30 - 30 3045855_1 (GHMatters) P72452AU 9Jan-12

20. A grass seed produced by the method of claim 17.

21. A grass plant produced by the method of claim 18 or claim 19. 5

22. A progeny grass plant from a self-cross of the grass plant of any one of claims 15, 16 or 21.

23. A first generation grass plant from an out-cross of the grass plant of any one of claims 15, 16 or 21. 10

24. Hay, comprising the grass plant of any one of claims 15, 16 or 21.

25. A pasture comprising the grass plant of any one of claims 15, 16 or 21.

26. A vegetative sprig or clone of the grass plant of any one of claims 15, 16 or 21. 15

27. Sward, comprising the grass plant of any one of claims 15, 16 or 21.

28. Sward, comprising a grass plant germinated from the seed of any one of claims 4, 5, 11 to 14 or 20. 20

29. A seed mixture comprising the seed of any one of claims 4, 5, 11 to 14 or 20.

30. The endophyte of claim I or claim 2, the seed of any one of claims 4, I1, 14 or 20, the synthetic combination of claim 6, the grass plant of any one of claims 15, 21, 22 or 23, the 25 method of claim 17 or claim 18, the hay of claim 24, the pasture of claim 25, the vegetative sprig or clone of claim 26, the sward of claim 27 or claim 28, or the seed mixture of claim 29, substantially as hereinbefore described with reference to the examples and figures.

-31 3045S85_1 (GHMaer) P72452AU 9-Jan-12