Abstract:
The present invention relates to a method for identifying and/or characterising an endophyte strain, said method including providing a plurality of samples of endophytes, subjecting said endophytes to genetic analysis, subjecting said endophytes to metabolic analysis and selecting endophytes having a desired genetic and metabolic profile. 
     The present invention also relates to novel endophytes having a desired toxin profile wherein the endophyte produces significantly less toxic alkaloids compared with a control endophyte such as standard toxic (ST) endophyte; and/or significantly more alkaloids conferring beneficial properties compared with a control endophyte such as ST endophyte. 
     The present invention also relates to endophyte variants having a desired genetic and metabolic profile, wherein said endophyte variants possess genetic and/or metabolic characteristics that result in a beneficial phenotype in a plant harbouring or otherwise associated with the endophyte variant. Preferably said endophyte variants are generated by polyploidisation or induced chromosome doubling.

Description:
STATEMENT OF RELATED CASES 
       [0001]    This application is a continuation in part of PCT/AU2011/000020, filed Jan. 7, 2011, which claims priority from Australian Patent Application filed Jan. 7, 2010, and Australian Patent Application 2010902821, filed Jun. 25, 2010, and also claims priority from Australian Patent Application Nos. 2012902275 and 2012902276, filed Jun. 1, 2012. All of these applications are incorporated herein by reference in their entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to endophytic fungi (endophytes), including modified variants thereof, and to nucleic acids thereof. The present invention also relates to plants infected with endophytes and to related methods, including methods of selecting, breeding, characterising and/or modifying endophytes. 
       BACKGROUND OF THE INVENTION 
       [0003]    Important forage grasses perennial ryegrass and tall fescue are commonly found in association with fungal endophytes. 
         [0004]    Both beneficial and detrimental agronomic properties result from the association, including improved tolerance to water and nutrient stress and resistance to insect pests. 
         [0005]    Insect resistance is provided by specific metabolites produced by the endophyte, in particular loline alkaloids and peramine. Other metabolites produced by the endophyte, lolitrems and ergot alkaloids, are toxic to grazing animals and reduce herbivore feeding. 
         [0006]    Considerable variation is known to exist in the metabolite profile of endophytes. Endophyte strains that lack either or both of the animal toxins have been introduced into commercial cultivars. 
         [0007]    Molecular genetic markers such as simple sequence repeat (SSR) markers have been developed as diagnostic tests to distinguish between endophyte taxa and detect genetic variation within taxa. The markers may be used to discriminate endophyte strains with different toxin profiles. 
         [0008]    However, there remains a need for methods of identifying, isolating, characterising and/or modifying endophytes and a need for new endophyte strains having desired properties. 
         [0009]    In the fungal kingdom, there is no differentiation of individuals into sexes generating different gametes, but instead mating-type identity is determined by inheritance of alleles at specific mating-type loci. 
         [0010]    The mating-type (MAT) genes constitute master regulators of sexual reproduction in filamentous fungi. Although mating-type loci consist of one to a few linked genes, and are thus limited to a small genomic region, alternate sequences at MAT, denoted idiomorphs, lack significant sequence similarity and encode different transcriptional regulators. 
         [0011]    Fusion events are required during sexual reproduction in filamentous ascomycete species. Although cell fusion processes associated with vegetative growth as opposed to sexual development serve different developmental functions, both require extracellular communication and chemotropic interactions, followed by cell wall breakdown, membrane-merger and pore formation. 
         [0012]    A number of genes have been characterised that are required for both sexual reproduction and vegetative hyphal fusion, including components of the MAPK pathway which is activated in response to pheromone perception during mating. The expression of pheromone precursors and pheromone receptor genes is directly controlled by transcription factors encoded by the mating-type genes. 
         [0013]    Hyphal fusion occurs readily within an individual colony during vegetative growth, maintaining the physiological continuity of the organism. Hyphal fusion between different endophyte strains of opposite mating-type may be promoted by treating the mycelia with a combination of cell wall-degrading enzymes and fusion agents such as PEG4000. 
         [0014]    However, there remains a need for methods of molecular breeding of endophytes and for new endophyte strains having desired properties. 
         [0015]      Neotyphodium  endophytes are not only of interest in agriculture, as they are a potential source for bioactive molecules such as insecticides, fungicides, other biocides and bioprotectants, allelochemicals, medicines and nutraceuticals. 
         [0016]    Difficulties in artificially breeding of these endophytes limit their usefulness. For example, many of the novel endophytes known to be beneficial to pasture-based agriculture exhibit low inoculation frequencies and are less stable in elite germplasm. Thus, there remains a need for methods of generating novel, highly compatible endophytes. 
         [0017]    There also remains a need for more endophyte strains with desirable properties and for more detailed characterisation of their toxin and metabolic profiles, antifungal activity, stable host associations and their genomes. 
         [0018]    It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art. 
         [0019]    In a first aspect, the present invention provides a method for selecting and/or characterising an endophyte strain, said method including:
       providing a plurality of samples of endophytes;   subjecting said endophytes to genetic analysis;   subjecting said endophytes to metabolic analysis; and   selecting endophytes having a desired genetic and metabolic profile.       
 
         [0024]    In a preferred embodiment, this aspect of the invention may include the further step of assessing geographic origin of the endophytes and selecting endophytes having a desired genetic and metabolic profile and a desired geographic origin. 
         [0025]    In a preferred embodiment, the plurality of samples of endophytes may be provided by a method including:
       providing a plurality of plant samples; and   isolating endophytes from said plant samples.       
 
         [0028]    In a preferred embodiment, the method may be performed using an electronic device, such as a computer. 
         [0029]    Applicant has surprisingly found that specific detection of endophytes in planta with markers such as SSR markers has provided the tools for efficient assessment of endophyte genetic diversity in diverse grass populations and the potential discovery of novel endophyte strains. 
         [0030]    A large scale endophyte discovery program was undertaken to establish a ‘library’ of novel endophyte strains. A collection of perennial ryegrass and tall fescue accessions was established. 
         [0031]    Genetic analysis of endophytes in these accessions has lead to the identification of a number of novel endophyte strains. These novel endophyte strains are genetically distinct from known endophyte strains. 
         [0032]    Metabolic profiling was undertaken to determine the toxin profile of these strains grown in vitro and/or following inoculation in planta. 
         [0033]    Specific detection of endophytes in planta with SSR markers may be used to confirm the presence and identity of endophyte strains artificially inoculated into, for example, grass plants, varieties and cultivars. 
         [0034]    The endophytes have been genetically characterised to demonstrate genetic distinction from known endophyte strains and to confirm the identity of endophyte strains artificially inoculated into, for example, grass plants, varieties and cultivars. 
         [0035]    By a ‘plurality’ of samples of endophytes or plant samples is meant a number sufficient to enable a comparison of genetic and metabolic profiles of individual endophytes. Preferably, between approximately 10 and 1,000,000 endophytes are provided, more preferably between approximately 100 and 1,000 endophytes. 
         [0036]    Phenotypic screens were established to select for novel ‘designer’ grass-endophyte associations. These screens were for desirable characteristics such as enhanced biotic stress tolerance, enhance drought tolerance and enhanced water use efficiency, and enhanced plant vigour. 
         [0037]    Novel ‘designer’ endophytes were generated by targeted methods including polyploidisation and X-ray mutagenesis. 
         [0038]    These endophytes may be characterised, for example using antifungal bioassays, in vitro growth rate assays and/or genome survey sequencing (GSS). 
         [0039]    Metabolic profiling may also be undertaken to determine the toxin profile of these strains grown in vitro and/or following inoculation in planta. 
         [0040]    These endophytes may be delivered into plant germplasm to breed ‘designer’ grass endophyte associations. 
         [0041]    Specific detection of endophytes in planta with SSR markers may be used to confirm the presence and identity of endophyte strains artificially inoculated into, for example, grass plants, varieties and cultivars. 
         [0042]    The endophytes may be subject to genetic analysis (genetically characterized) to demonstrate genetic distinction from known endophyte strains and to confirm the identity of endophyte strains artificially inoculated into, for example, grass plants, varieties and cultivars. 
         [0043]    By ‘genetic analysis’ is meant analysing the nuclear and/or mitochondrial DNA of the endophyte. 
         [0044]    This analysis may involve detecting the presence or absence of polymorphic markers, such as simple sequence repeats (SSRs) or mating-type markers. SSRs, also called microsatellites, are based on a 1-7 nucleotide core element, more typically a 1-4 nucleotide core element, that is tandemly repeated. The SSR array is embedded in complex flanking DNA sequences. Microsatellites are thought to arise due to the property of replication slippage, in which the DNA polymerase enzyme pauses and briefly slips in terms of its template, so that short adjacent sequences are repeated. Some sequence motifs are more slip-prone than others, giving rise to variations in the relative numbers of SSR loci based on different motif types. Once duplicated, the SSR array may further expand (or contract) due to further slippage and/or unequal sister chromatid exchange. The total number of SSR sites is high, such that in principle such loci are capable of providing tags for any linked gene. 
         [0045]    SSRs are highly polymorphic due to variation in repeat number and are co-dominantly inherited. Their detection is based on the polymerase chain reaction (PCR), requiring only small amounts of DNA and suitable for automation. They are ubiquitous in eukaryotic genomes, including fungal and plant genomes, and have been found to occur every 21 to 65 kb in plant genomes. Consequently, SSRs are ideal markers for a broad range of applications such as genetic diversity analysis, genotypic identification, genome mapping, trait mapping and marker-assisted selection. 
         [0046]    Known SSR markers which may be used to investigate endophyte diversity in perennial ryegrass are described in van 41 de Jong et al (2003). 
         [0047]    Alternatively, or in addition, the genetic analysis may involve sequencing genomic and/or mitochondrial DNA and performing sequence comparisons to assess genetic variation between endophytes. 
         [0048]    The endophytes may be subject to metabolic analysis to identify the presence of desired metabolic traits. 
         [0049]    By ‘metabolic analysis’ is meant analysing metabolites, in particular toxins, produced by the endophytes. Preferably, this is done by generation of inoculated plants for each of the endophytes and measurement of toxin levels in planta. More preferably, this is done by generation of isogenically inoculated plants for each of the endophytes and measurement of toxin levels in planta. 
         [0050]    By a ‘desired genetic and metabolic profile’ is meant that the endophyte includes genetic and metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, the endophyte. 
         [0051]    Such beneficial properties include improved tolerance to water and/or nutrient stress, improved resistance to pests and/or diseases, enhanced biotic stress tolerance, enhanced drought tolerance, enhanced water use efficiency, reduced toxicity and enhanced vigour in the plant with which the endophyte is associated, relative to a control endophyte such as standard toxic (ST) endophyte or to a no endophyte control plant. 
         [0052]    For example, tolerance to water and/or nutrient stress may be increased by at least approximately 5%, more preferably at least approximately 10%, more preferably at least approximately 25%, more preferably at least approximately 50%, more preferably at least approximately 100%, relative to a control endophyte such as standard toxic (ST) endophyte or to no endophyte control plant. Preferably, tolerance to water and/or nutrient stress may be increased by between approximately 5% and approximately 50%, more preferably between approximately 10% and approximately 25%, relative to a control endophyte such as ST or to a no endophyte control plant. 
         [0053]    Such beneficial properties also include reduced toxicity of the associated plant to grazing animals. 
         [0054]    For example, toxicity may be reduced by at least approximately 5%, more preferably at least approximately 10%, more preferably at least approximately 25%, more preferably at least approximately 50%, more preferably at least approximately 100%, relative to a control endophyte such as ST endophyte. Preferably, toxicity may be reduced by between approximately 5% and approximately 100%, more preferably between approximately 50% and approximately 100% relative to a control endophyte such as ST endophyte. 
         [0055]    In a preferred embodiment toxicity may be reduced to a negligible amount or substantially zero toxicity. 
         [0056]    For example, water use efficiency and/or plant vigour may be increased by at least approximately 5%, more preferably at least approximately 10%, more preferably at least approximately 25%, more preferably at least approximately 50%, more preferably at least approximately 100%, relative to a control endophyte such as ST or to a no endophyte control plant. Preferably, tolerance to water and/or nutrient stress may be increased by between approximately 5% and approximately 50%, more preferably between approximately 10% and approximately 25%, relative to a control endophyte such as ST or to a no endophyte control plant. 
         [0057]    The methods of the present invention may be applied to a variety of plants. In a preferred embodiment, the methods may be applied to grasses, preferably forage, turf or bioenergy grasses such as those of the genera  Lolium  and  Festuca , including  L. perenne  (perennial ryegrass) and  L. arundinaceum  (tall fescue). 
         [0058]    The methods of the present invention may be applied to a variety of endophytes. In a preferred embodiment, the methods may be applied to fungi of the genus  Neotyphodium , including  N. lolii  and  N. coenophialum . In another preferred embodiment, the methods may be applied to fungi of the genus  Epichloë , including  E. festucae  and  E. typhina . However, the methods may also be used to identify endophytes of previously undescribed taxa. 
         [0059]    Applicants have surprisingly found that endophyte E1 is a genetically novel, non- Neotyphodium lolii , endophyte. E1 is representative of an as yet un-named taxon. This finding is supported by mitochondrial and nuclear genome sequence analysis. 
         [0060]    While applicants do not wish to be restricted by theory, on the basis of DNA specific content, the predicted alkaloid profile of E1 indicates that lolitrem B toxins deleterious to animal health are not produced by this endophyte. Endophyte E1 has the mating-type MAT1-1, the opposite mating-type to that carried by the  N. lolii  endophytes previously characterized. Endophyte E1 also has a high inoculation success rate in perennial ryegrass as compared to other endophytes. 
         [0061]    Accordingly, in a second aspect, the present invention provides a substantially purified or isolated endophyte selected from the group consisting of E1, NEA10, NEA11 and NEA12, which were deposited at The National Measurement Institute on 5 Jan. 2010 with accession numbers V10/000,001, V10/000,002, V10/000,003 and V10/000,004, respectively. 
         [0062]    The present invention also provides a substantially purified or isolated endophyte selected from the group consisting of NEA13 and NEA14, which were deposited at the National Measurement Institute on 23 Dec. 2010 with accession numbers V10/030,285 and V10/030,284, respectively. 
         [0063]    In a further aspect the present invention provides a substantially purified or isolated endophyte having a desired toxin profile. Preferably the endophyte is isolated from a fescue species, preferably tall fescue. Preferably, the endophyte is of the genus  Neotyphodium , more preferably it is from a species selected from the group consisting of  N. uncinatum, N. coenophialum  and  N. lolii , most preferably  N. coenophialum . The endophyte may also be from the genus  Epichloe , including  E. typhina, E. baconii  and  E. festucae . The endophyte may also be of the non- Epichloe  out-group. The endophyte may also be from a species selected from the group consisting of FaTG-3 and FaTG-3 like, and FaTG-2 and FaTG-2 like. 
         [0064]    By a ‘desired toxin profile’ is meant that the endophyte produces significantly less toxic alkaloids, such as ergovaline, compared with a plant inoculated with a control endophyte such as standard toxic (ST) endophyte; and/or significantly more alkaloids conferring beneficial properties such as improved tolerance to water and/or nutrient stress and improved resistance to pests and/or diseases in the plant with which the endophyte is associated, such as peramine, N-formylloline, N-acetylloline and norloline, again when compared with a plant inoculated with a control endophyte such as ST or with a no endophyte control plant. 
         [0065]    For example, toxic alkaloids may be present in an amount less than approximately 1 μg/g dry weight, for example between approximately 1 and 0.001 μg/g dry weight, preferably less than approximately 0.5 μg/g dry weight, for example between approximately 0.5 and 0.001 μg/g dry weight, more preferably less than approximately 0.2 μg/g dry weight, for example between approximately 0.2 and 0.001 μg/g dry weight. 
         [0066]    For example, said alkaloids conferring beneficial properties may be present in an amount of between approximately 5 and 100 μg/g dry weight, preferably between approximately 10 and 50 μg/g dry weight, more preferably between approximately 15 and 30 μg/g dry weight. 
         [0067]    In a particularly preferred embodiment, the present invention provides a substantially purified or isolated endophyte selected from the group consisting of NEA16, NEA17, NEA18, NEA19, NEA20, NEA21 and NEA23, which were deposited at The National Measurement Institute on 3 Apr. 2012 with accession numbers V12/001413, V12/001414, V12/001415, V12/001416, V12/001417, V12/001418 and V12/001419, respectively. Such endophytes may have a desired toxin profile as hereinbefore described. 
         [0068]    In a further aspect the present invention provides an endophyte variant having a desired genetic and metabolic profile. Preferably the endophyte variant is generated by polyploidisation or induced chromosome doubling, for example by treating the endophyte with colchicine or a similar compound. Alternatively, the endophyte variant may be generated by X-ray mutagenesis or exposing the endophyte to ionising radiation, for example from a caesium source. 
         [0069]    Preferably the endophyte which is treated to generate the endophyte variant is isolated from a  Lolium  species, preferably  Lolium perenne . Preferably, the endophyte is of the genus  Neotyphodium , more preferably it is from a species selected from the group consisting of  N. uncinatum, N. coenophialum  and  N. lolii , most preferably  N. lolii . The endophyte may also be from the genus  Epichloe , including  E. typhina, E. baconii  and  E. festucae . The endophyte may also be of the non- Epichloe  out-group. The endophyte may also be from a species selected from the group consisting of FaTG-3 and FaTG-3 like, and FaTG-2 and FaTG-2 like. 
         [0070]    In a preferred embodiment, the endophyte variant may have a desired toxin profile. By a ‘desired toxin profile’ is meant that the endophyte produces significantly less toxic alkaloids, such as ergovaline, compared with a plant inoculated with a control endophyte such as standard toxic (ST) endophyte; and/or significantly more alkaloids conferring beneficial properties such as improved resistance to pests and/or diseases in the plant with which the endophyte is associated, such as peramine, N-formylloline, N-acetylloline and norloline, again when compared with a plant inoculated with a control endophyte such as ST or with a no endophyte control plant. 
         [0071]    For example, toxic alkaloids may be present in an amount less than approximately 1 μg/g dry weight, for example between approximately 1 and 0.001 μg/g dry weight, preferably less than approximately 0.5 μg/g dry weight, for example between approximately 0.5 and 0.001 μg/g dry weight, more preferably less than approximately 0.2 μg/g dry weight, for example between approximately 0.2 and 0.001 μg/g dry weight. 
         [0072]    For example, said alkaloids conferring beneficial properties may be present in an amount of between approximately 5 and 100 μg/g dry weight, preferably between approximately 10 and 50 μg/g dry weight, more preferably between approximately 15 and 30 μg/g dry weight. 
         [0073]    In a particularly preferred embodiment, the present invention provides an endophyte variant selected from the group consisting of NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14, and NEA12dh17, which were deposited at The National Measurement Institute on 3 Apr. 2012 with accession numbers V12/001408, V12/001409, V12/001410, V12/001411 and V12/001412, respectively. Such endophytes may have a desired genetic and metabolic profile as hereinbefore described. 
         [0074]    In a preferred embodiment, the endlphyte may be substantially purified. 
         [0075]    By ‘substantially purified’ is meant that the endophyte is free of other organisms. The term therefore includes, for example, an endophyte in axenic culture. Preferably, the endophyte is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure. 
         [0076]    The term ‘isolated’ means that the endophyte is removed from its original environment (eg. the natural environment if it is naturally occurring). For example, a naturally occurring endophyte present in a living plant is not isolated, but the same endophyte separated from some or all of the coexisting materials in the natural system, is isolated. 
         [0077]    On the basis of the deposits referred to above, the entire genome of an endophyte selected from the group consisting of E1, NEA10, NEA11, NEA12, NEA13, NEA14, NEA21, NEA23, NEA18, NEA19, NEA16, NEA20, NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14 and NEA12dh17, is incorporated herein by reference. 
         [0078]    Thus, in a further aspect, the present invention includes identifying and/or cloning nucleic acids including genes encoding polypeptides or transcription factors, for example transcription factors that are involved in sexual reproduction or vegetative hyphal fusion, in an endophyte. For example, the nucleic acids may encode mating-type genes, such as MAT1-1. 
         [0079]    Methods for identifying and/or cloning nucleic acids encoding such genes are known to those skilled in the art and include creating nucleic acid libraries, such as cDNA or genomic libraries, and screening such libraries, for example using probes for genes of the desired type, for example mating-type genes; or mutating the genome of the endophyte of the present invention, for example using chemical or transposon mutagenesis, identifying changes in the production of polypeptides or transcription factors of interest, for example those that are involved in sexual reproduction or vegetative hyphal fusion, and thus identifying genes encoding such polypeptides or transcription factors. 
         [0080]    Thus, in a further aspect of the present invention, there is provided a substantially purified or isolated nucleic acid encoding a polypeptide or transcription factor from the genome of an endophyte of the present invention. Preferably, the nucleic acid may encode a polypeptide or transcription factor that is involved in sexual reproduction or vegetative hyphal fusion in an endophyte. 
         [0081]    In a preferred embodiment, the nucleic acid may include a mating-type gene, such as MAT1-1, or a functionally active fragment or variant thereof. 
         [0082]    In a particularly preferred embodiment, the nucleic acid may include a nucleotide sequence selected from the group consisting of sequences shown in  FIG. 1  hereto, and functionally active fragments and variants thereof. 
         [0083]    By ‘nucleic acid’ is meant a chain of nucleotides capable of carrying genetic information. The term generally refers to genes or functionally active fragments or variants thereof and or other sequences in the genome of the organism that influence its phenotype. The term ‘nucleic acid’ includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA or microRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, synthetic nucleic acids and combinations thereof. 
         [0084]    By a ‘nucleic acid encoding a polypeptide or transcription factor’ is meant a nucleic acid encoding an enzyme or transcription factor normally present in an endophyte of the present invention. 
         [0085]    By a ‘nucleic acid encoding a polypeptide or transcription factor involved sexual reproduction or vegetative hyphal fusion’ is meant a nucleic acid encoding an enzyme or transcription factor normally present in an endophyte of the present invention, which catalyses or regulates a step involved in sexual reproduction or vegetative hyphal fusion in the endophyte, or otherwise regulates sexual reproduction or vegetative hyphal fusion in the endophyte. 
         [0086]    The present invention encompasses functionally active fragments and variants of the nucleic acids of the present invention. By ‘functionally active’ in relation to the nucleic acid is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of manipulating the function of the encoded polypeptide, for example by being translated into an enzyme or transcription factor that is able to catalyse or regulate a step involved in the relevant pathway, or otherwise regulate the pathway in the endophyte. For example, the fragment or variant may be capable of manipulating sexual reproduction or vegetative hyphal fusion in an endophyte, for example by being translated into an enzyme or transcription factor that is able to catalyse or regulate a step involved in sexual reproduction or vegetative hyphal fusion in the endophyte, or otherwise regulate sexual reproduction or vegetative hyphal fusion in the endophyte. 
         [0087]    Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes. Examples of suitable nucleic acid changes are also shown in  FIG. 1  hereto. 
         [0088]    Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides. 
         [0089]    By ‘conservative nucleic acid changes’ is meant nucleic acid substitutions that result in conservation of the amino acid in the encoded protein, due to the degeneracy of the genetic code. Such functionally active variants and fragments also include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. 
         [0090]    By ‘conservative amino acid substitutions’ is meant the substitution of an amino acid by another one of the same class, the classes being as follows:
       Nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp   Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln   Acidic: Asp, Glu   Basic: Lys, Arg, His       
 
         [0095]    Other conservative amino acid substitutions may also be made as follows:
       Aromatic: Phe, Tyr, His   Proton Donor: Asn, Gln, Lys, Arg, His, Trp   Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln       
 
         [0099]    In a further aspect of the present invention, there is provided a genetic construct including a nucleic acid according to the present invention. 
         [0100]    By ‘genetic construct’ is meant a recombinant nucleic acid molecule. 
         [0101]    In a preferred embodiment, the genetic construct according to the present invention may be a vector. 
         [0102]    By a ‘vector’ is meant a genetic construct used to transfer genetic material to a target cell. 
         [0103]    The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from  Agrobacterium tumefaciens ; derivatives of the Ri plasmid from  Agrobacterium rhizogenes ; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable or integrative or viable in the target cell. 
         [0104]    In a preferred embodiment of this aspect of the invention, the genetic construct may further include a promoter and a terminator; said promoter, gene and terminator being operatively linked. 
         [0105]    By a ‘promoter’ is meant a nucleic acid sequence sufficient to direct transcription of an operatively linked nucleic acid sequence. 
         [0106]    By ‘operatively linked’ is meant that the nucleic acid(s) and a regulatory sequence, such as a promoter, are linked in such a way as to permit expression of said nucleic acid under appropriate conditions, for example when appropriate molecules such as transcriptional activator proteins are bound to the regulatory sequence. Preferably an operatively linked promoter is upstream of the associated nucleic acid. 
         [0107]    By ‘upstream’ is meant in the 3′-&gt;5′ direction along the nucleic acid. 
         [0108]    The promoter and terminator may be of any suitable type and may be endogenous to the target cell or may be exogenous, provided that they are functional in the target cell. 
         [0109]    A variety of terminators which may be employed in the genetic constructs of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the (CaMV)35S polyA and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes. 
         [0110]    The genetic construct, in addition to the promoter, the gene and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (nptII) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes [such as beta-glucuronidase (GUS) gene (gusA) and the green fluorescent protein (GFP) gene (gfp)]. The genetic construct may also contain a ribosome binding site for translation initiation. The genetic construct may also include appropriate sequences for amplifying expression. 
         [0111]    Those skilled in the art will appreciate that the various components of the genetic construct are operably linked, so as to result in expression of said nucleic acid. Techniques for operably linking the components of the genetic construct of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites. 
         [0112]    Preferably, the genetic construct is substantially purified or isolated. 
         [0113]    By ‘substantially purified’ is meant that the genetic construct is free of the genes, which, in the naturally-occurring genome of the organism from which the nucleic acid or promoter of the invention is derived, flank the nucleic acid or promoter. The term therefore includes, for example, a genetic construct which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (eg. a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a genetic construct which is part of a hybrid gene encoding additional polypeptide sequence. 
         [0114]    Preferably, the substantially purified genetic construct is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure. 
         [0115]    The term “isolated” means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment. 
         [0116]    As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the genetic construct in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical assays (e.g. GUS assays), thin layer chromatography (TLC), northern and western blot hybridisation analyses. 
         [0117]    The genetic constructs of the present invention may be introduced into plants or fungi by any suitable technique. Techniques for incorporating the genetic constructs of the present invention into plant cells or fungal cells (for example by transduction, transfection, transformation or gene targeting) are well known to those skilled in the art. Such techniques include  Agrobacterium -mediated introduction,  Rhizobium -mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos, biolistic transformation, Whiskers transformation, and combinations thereof. The choice of technique will depend largely on the type of plant or fungus to be transformed, and may be readily determined by an appropriately skilled person. For transformation of protoplasts, PEG-mediated transformation is particularly preferred. For transformation of fungi PEG-mediated transformation and electroporation of protoplasts and  Agrobacterium -mediated transformation of hyphal explants are particularly preferred. 
         [0118]    Cells incorporating the genetic constructs of the present invention may be selected, as described below, and then cultured in an appropriate medium to regenerate transformed plants or fungi, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants or fungi may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants or fungi. 
         [0119]    In a further aspect, the present invention provides a plant inoculated with an endophyte or endophyte variant as hereinbefore described, said plant comprising an endophyte-free host plant stably infected with said endophyte or endophyte variant. 
         [0120]    Preferably, the plant is infected with the endophyte or endophyte variant by a method selected from the group consisting of inoculation, breeding, crossing, hybridization and combinations thereof. 
         [0121]    In a preferred embodiment, the plant may be infected by isogenic inoculation. This has the advantage that phenotypic effects of endophytes may be assessed in the absence of host-specific genetic effects. More particularly, multiple inoculations of endophytes may be made in plant germplasm, and plantlets regenerated in culture before transfer to soil. 
         [0122]    The identification of an endophyte of the opposite mating-type that is highly compatible and stable in planta provides a means for molecular breeding of endophytes for perennial ryegrass. Preferably the plant may be infected by hyper-inoculation. 
         [0123]    Hyphal fusion between endophyte strains of the opposite mating-type provides a means for delivery of favourable traits into the host plant, preferably via hyper-inoculation. Such strains are preferably selected from the group including an endophyte strain that exhibits the favourable characteristics of high inoculation frequency and high compatibility with a wide range of germplasm, preferably elite perennial ryegrass and/or tall fescue host germplasm and an endophyte that exhibits a low inoculation frequency and low compatibility, but has a highly favourable alkaloid toxin profile. 
         [0124]    It has generally been assumed that interactions between endophyte taxa and host grasses will be species specific. Applicants have surprisingly found that endophyte from tall fescue may be used to deliver favourable traits to ryegrasses, such as perennial ryegrass. 
         [0125]    In a further aspect of the present invention there is provided a method of analysing metabolites in a plurality of endophytes, said method including:
       providing:
           a plurality of endophytes; and   a plurality of isogenic plants;   
           inoculating each isogenic plant with an endophyte;   culturing the endophyte-infected plants; and   analysing the metabolites produced by the endophyte-infected plants.       
 
         [0132]    By ‘metabolites’ is meant chemical compounds, in particular toxins, produced by the endophyte-infected plant, including, but not limited to, lolines, peramine, ergovaline, lolitrem, and janthitrems, such as janthitrem I, janthitrem G and janthitem F. 
         [0133]    By ‘isogenic plants’ is meant that the plants are genetically identical. 
         [0134]    The endophyte-infected plants may be cultured by known techniques. The person skilled in the art can readily determine appropriate culture conditions depending on the plant to be cultured. 
         [0135]    The metabolites may be analysed by known techniques such as chromatographic techniques or mass spectrometry, for example LCMS or HPLC. In a particularly preferred embodiment, endophyte-infected plants may be analysed by reverse phase liquid chromatography mass spectrometry (LCMS). This reverse phase method may allow analysis of specific metabolites (including lolines, peramine, ergovaline, lolitrem, and janthitrems, such as janthitrem I, janthitrem G and janthitem F) in one LCMS chromatographic run from a single endophyte-infected plant extract. 
         [0136]    In another particularly preferred embodiment, LCMS including EIC (extracted ion chromatogram) analysis may allow detection of the alkaloid metabolites from small quantities of endophyte-infected plant material. Metabolite identity may be confirmed by comparison of retention time with that of pure toxins or extracts of endophyte-infected plants with a known toxin profile analysed under substantially the same conditions and/or by comparison of mass fragmentation patterns, for example generated by MS2 analysis in a linear ion trap mass spectrometer. 
         [0137]    In a particularly preferred embodiment, the endophytes may be selected from the group consisting of E1, NEA10, NEA11, NEA12, NEA13, NEA14, NEA21, NEA23, NEA18, NEA19, NEA16 and NEA20. 
         [0138]    In a particularly preferred embodiment, the endophyte variant may be selected from the group consisting of NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14, and NEA12dh17. 
         [0139]    In a further aspect, the present invention provides a plant, plant seed or other plant part derived from a plant of the present invention and stably infected with an endophyte or endophyte variant of the present invention. 
         [0140]    Preferably, the plant cell, plant, plant seed or other plant part is a grass, more preferably a forage, turf or bioenergy grass, such as those of the genera  Lolium  and  Festuca , including  L. perenne  and  L. arundinaceum.    
         [0141]    By ‘plant cell’ is meant any self-propagating cell bounded by a semi-permeable membrane and containing plastid. Such a cell also required a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, seeds suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. 
         [0142]    In a further aspect, the present invention provides use of an endophyte or endophyte variant as hereinbefore described to produce a plant stably infected with said endophyte or endophyte variant. 
         [0143]    In a still further aspect, the present invention provides a method of quantifying endophyte content of a plant, said method including measuring copies of a target sequence by quantitative PCR. 
         [0144]    In a preferred embodiment, the method may be performed using an electronic device, such as a computer. 
         [0145]    Preferably, quantitative PCR may be used to measure endophyte colonisation in planta, for example using a nucleic acid dye, such as SYBR Green chemistry, and qPCR-specific primer sets. The primer sets may be directed to a target sequence such as an endophyte gene, for example the peramine biosynthesis perA gene. 
         [0146]    The development of a high-throughput PCR-based assay to measure endophyte biomass in planta may enable efficient screening of large numbers of plants to study endophyte-host plant biomass associations. 
         [0147]    As used herein, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps. 
         [0148]    Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art. 
     
    
     
       DETAILED DESCRIPTION OF THE EMBODIMENTS 
         [0149]    In the figures: 
           [0150]      FIG. 1  shows sequence alignment analysis of mating-type loci of endophyte strains  E. festucae  strain E2368, E1, NEA12 and ST. 
           [0151]      FIG. 2A  shows a UPGMA phenogram of genetic relationships among endophytes in ryegrass accessions of diverse origins and reference  Neotyphodium  and  Epichloë  species. Genetic identity was measured across 18 SSR loci using the Dice coefficient. Detailed annotations for sections A-D are shown in  FIGS. 2B to 2E , respectively. Specifically, accessions analysed in this study are shaded in grey, the number of genotypes host to that endophyte strain from the total number of genotypes analysed are indicated in the round brackets and a representative host genotype is given in the square brackets. Endophyte isolates from the reference collection are specified in the square brackets following the species name.  N. lolii  Group 1 comprises of isolates Aries 1, Banks 5847, Ellett 5837, Fitzroy 2, Fitzroy 3, KT1-2, North African 6, Vedette 6645 and Victorian 2. 
           [0152]      FIG. 3  shows isogenic inoculation methodology for endophyte inoculation. A. Meristem callus induction (4 weeks); B. Embryogenic callus proliferation (4 weeks); C. Shoot (and root) regeneration (5 days, 16 hours light); D. Endophyte inoculation; E. Plantlet growth (4 weeks, 16 hours light); F. Growth in soil (3 months); G. SSR-based analysis. 
           [0153]      FIG. 4  shows the number of hits showing a given percent identity for 250 bp fragments of the NEA12 genome against the  E. festucae  and  N. lolii  genomes. The X-axis shows the percent identity, the Y-axis shows the number of hits. Black:  N. lolii  strain ST; White:  E. festucae  strain E2368. 
           [0154]      FIG. 5  shows the number of hits showing a given percent identity for 250 bp segments of the E1 genome against the genomes of NEA12 , E. festucae  and  N. lolii . The X-axis shows the percent identity, the Y-axis shows the number of hits. Black (1st bar in each group):  E. festucae  strain E2368; Grey (2nd bar in each group): Non- N. lolii  strain NEA12; White (3rd bar in each group):  N. lolii  strain ST. 
           [0155]      FIG. 6  shows the number of hits showing a given percent identity for 250 bp fragments of E1 against NEA12 , E. festucae  and  N. lolii . The X-axis shows the percent identity, the Y-axis shows the number of hits expressed as a fraction of the total matches seen per comparison. Grey (1st bar in each group):  E. festucae  strain E2368; Black (2nd bar in each group): Non- N. lolii  strain NEA12; White (3rd bar in each group):  N. lolii  strain ST. 
           [0156]      FIG. 7  shows a schematic diagram of the mating-type loci in  Neotyphodium/Epichloë.    
           [0157]      FIG. 8  shows ClustalW analysis trees of the sequence flanking the mating-type loci (left), and the NoxR gene (cloned from  E. festucae  strain FL1 gi117413991; right). 
           [0158]      FIG. 9  shows an alignment between mitochondrial genome of  N. lolii  strain Lp19 and a representative of the Clavicipitaceae,  Metarhizium anisopliae  (Genbank reference number NC — 008068.1). While the two mitochondrial genomes vary in size, the genes are present in the same order and strand sense, with differences being due to variable insertions in the  N. lolii  mitochondrial genome. 
           [0159]      FIG. 10  shows a depiction of part of the block structure of the mitochondrial genomes for each of the fungal endophytes sequenced in this study, as well as  E. festucae  strain E2368 and  Metarhizium anisopliae  for comparison. A shared block (e.g. b84) is present in all 12 mitochondria whereas block 85 is present only in the mitochondria of  E. festucae  strain E2368, and Non- N. lolii  strains E1 and NEA12. 
           [0160]      FIG. 11  shows a mitochondrial genome comparison. Parsimony tree of the relationships between the mitochondrial genomes of the 10 perennial ryegrass endophyte strains sequenced,  E. festucae  strain E2368 and  Metarhizium anisopliae.    
           [0161]      FIG. 12  shows a mitochondrial genome comparison. Neighbour joining tree analysis using ClustalW from a DNA alignment of the 40 blocks of sequence (˜40 kb) that are shared across the 10 perennial ryegrass endophyte strains sequenced,  E. festucae  strain E2368 and  Metarhizium anisopliae.    
           [0162]      FIG. 13  shows a standard curve for quantitative assessment of endophyte colonisation (copy number relative to total plant gDNA). (a) Tight clustering of amplification curves (4 technical replicates) ranging from 2×10 2  to 2×10 6  copies of the 73 bp perA amplicon. (b) Dissociation curve analysis of the amplification curves shown in (a), with the presence of a single peak indicating primer pair specificity. (c) Assay performance is determined in terms of efficiency, precision and sensitivity. For a typical reaction, a slope of −3.1 to −3.6 and R 2  value≧0.985 is acceptable. This assay recorded a slope of −3.2 and R 2  value of 0.999. 
           [0163]      FIG. 14  shows a quantitative assessment of endophyte colonisation in diverse ryegrass host panel. (a) Standard curve of perA target sequence (2×10 2  to 2×10 6 ) and amplification curves of the unknown samples. (b) Dissociation curve analysis of the amplification curves shown in (a). (c) Standard curve for perA target (▪) and unknown samples (▴). 
           [0164]      FIG. 15  shows a colchicine kill curve of endophyte strain ST mycelia grown in potato dextrose broth at 22° C., 150 rpm for 21 days. 
           [0165]      FIG. 16  shows phenotype of colchicine treated colonies (0.1 and 0.2%) of endophyte strain ST compared to the untreated ST control. Mycelia were grown on potato dextrose agar at 22° C. in dark. 
           [0166]      FIG. 17  shows an assessment for changes in ploidy level by flow cytometry. a) Dot plots and histogram overlay of control samples, ST, BE9301 and NEA11. b) Dot plots and histogram overlay of two individual ST colonies (13 and 14), showing a shift in peak location relative to the controls. 
           [0167]      FIG. 18  shows high throughput PCR screening method for detection of lolitrem B gene deletion mutants. The lolitrem genes targeted include: ItmM (480 bp), ItmJ (734 bp) and ItmC (583 bp). M: EasyLadder1 (100-2000 bp); 1-13: Individual putative lolitrem B gene deletion mutants; ST: ST DNA (positive control for ItmM, ItmJ and ItmC); AR1: AR1 DNA (positive control for ItmM and ItmC, negative control for ItmJ); H 2 O PCR control. 
           [0168]      FIG. 19  shows geographical origins represented in the tall fescue endophyte incidence assessment. This graph shows the 40 different geographic origins represented in the incidence assessment. The X axis gives geographic origins in the alphabetical order and the Y axis shows the number of accessions. The number of negative accessions is shown with black and the number of positive accessions is shown in grey. 
           [0169]      FIG. 20  shows UPGMA phenogram of genetic relationships among endophytes in tall fescue accessions of diverse origins and reference  Neotyphodium, Epichloë , FaTG-2 and FaTG-3 species. 
           [0170]      FIG. 21  shows production of the insecticidal alkaloids loline, loline formate and peramine by tall fescue endophytes in their endogenous host. 
           [0171]      FIG. 22  shows production of the anti-mammalian alkaloids ergovaline and lolitrem B by tall fescue endophytes in their endogenous host. 
           [0172]      FIG. 23  shows an example of antifungal bioassay of inhibition reactions. Testing for antifungal activity of endophyte NEA12, ST and AR1 against 8 species of pathogenic fungi. 
           [0173]      FIG. 24  shows endophytes selected for metabolic profiling in in vitro culture. Shown in the top left hand corner is the inhibition score. 
           [0174]      FIG. 25  shows a method for sampling material for LCMS analysis. 
           [0175]      FIG. 26  shows a validation assay.  Rhizoctonia cerealis  was grown in the presence of methanol extracts of endophyte mycelia. Shown is an example using the endophyte strain ST. A. Methanol extract of ST grown in the absence of  R. cerealis ; B. Methanol extract ST grown in presence of  R. cerealis ; C. Water only control; D. Methanol only control. 
           [0176]      FIG. 27  shows structures of endophyte metabolites 
           [0177]    1 peramine (MW 247.3); 
           [0178]    2 ergovaline (MW 533.6); 
           [0179]    3 lolitrem B (MW 685.9); 
           [0180]    4 janthitrem I (MW 645.8); 
           [0181]    5 janthitrem G (MW 629.8); 
           [0182]    6 janthitrem F (MW 645.8). 
           [0183]      FIG. 28  shows LCMS analysis of standard materials displaying extracted ion chromatogram for the toxins: 
           [0184]    A. peramine 
           [0185]    NL: 7.47E4 
           [0186]    Base Peak m/z=47.50-248.50 F: ITMS+c ESI Full ms 
           [0187]    [150.00-2000.00] MS 
           [0188]    B. ergovaline 
           [0189]    NL: 1.64E6 
           [0190]    Base Peak m/z=533.40-534.40 F: ITMS+c ESI Full ms 
           [0191]    [150.00-2000.00] MS 
           [0192]    C. lolitrem B 
           [0193]    NL: 2.25E3 
           [0194]    Base Peak m/z=685.50-687.00 F: ITMS+c ESI Full ms 
           [0195]    [150.00-2000.00] MS 
           [0196]      FIG. 29  shows an LCMS comparison of AR37 inoculated perennial ryegrass with NEA12 inoculated perennial ryegrass (IMP04 NEA12 20). 
           [0197]    A. AR37 no peramine 
           [0198]    NL: 3.14E3 
           [0199]    Base Peak m/z=247.50-248.50 F: ITMS+c ESI 
           [0200]    Full ms [150.00-2000.00] MS 
           [0201]    B. AR37 no ergovaline 
           [0202]    NL: 7.39E4 
           [0203]    Base Peak m/z=533.40-534.40 F: ITMS+c ESI 
           [0204]    Full ms [150.00-2000.00] MS 
           [0205]    C. AR37 no lolitrem B 
           [0206]    NL: 1.32E4 
           [0207]    Base Peak m/z=685.50-687.00 F: ITMS+c ESI 
           [0208]    Full ms [150.00-2000.00] MS 
           [0209]    D. AR37 janthitrem 
           [0210]    NL: 8.68E4 
           [0211]    Base Peak m/z=645.50-646.50 F: ITMS+c ESI 
           [0212]    Full ms [150.00-2000.00] MS 
           [0213]    E. NEA12 no peramine 
           [0214]    NL: 6.18E3 
           [0215]    Base Peak m/z=247.50-248.50 F: ITMS+c ESI 
           [0216]    Full ms [150.00-2000.00] MS 
           [0217]    F. NEA12 no ergovaline 
           [0218]    NL: 4.10E3 
           [0219]    Base Peak m/z=533.40-534.40 F: ITMS+c ESI 
           [0220]    Full ms [150.00-2000.00] MS 
           [0221]    G. NEA12 no lolitrem B 
           [0222]    NL: 1.32E4 
           [0223]    Base Peak rn/z=685.50-687.00 F: ITMS+c ESI 
           [0224]    Full ms [150.00-2000.00] MS 
           [0225]    H. NEA12 janthitrem 
           [0226]    NL: 1.04E4 
           [0227]    Base Peak m/z=645.50-646.50 F: ITMS+c ESI 
           [0228]    Full ms [150.00-2000.00] MS 
           [0229]      FIG. 30  shows an MSMS analysis of NEA12 insulated perennial ryegrass metabolite 4. Inset is Table 2 from International patent application WO2004/106487 describing the fragmentations of the janthitrems found. Data for NEA12 metabolite 4 is in good agreement with that of component I in the table. (endol5June09-010 #3184 RT: 49.01 AV: 1 NL: 5.02E2, T: ITMS+cESId Full ms2 646.51@cid35.00 [165.00-660.00]) 
           [0230]      FIG. 31  shows Reverse phase liquid chromatography mass spectrometry (LCMS) analysis of A. TOL03 NEA12 and B. TOL03 ST. Profiles show the presence and absence of specific metabolites including peramine, ergovaline, lolitrem, and janthitrems. 
           [0231]      FIG. 32  shows genotypic analysis of endophyte content in accessions from a targeted fescue germplasm collection. 
           [0232]      FIG. 33  shows genetic diversity analysis of tall fescue endophytes. 
           [0233]      FIG. 34  shows diversity analysis of host and endophyte. 
           [0234]      FIG. 35  shows selection of fescue-endophyte combinations for metabolic profiling, endophyte isolation and isogenic inoculation. 
           [0235]      FIG. 36  shows selection of fescue-endophyte combinations for metabolic profiling, endophyte isolation and isogenic inoculation. 
           [0236]      FIG. 37  shows a desired toxin profile of tall fescue endophytes. 
           [0237]      FIG. 38  shows a metabolic profile analysis. 
           [0238]      FIG. 39  shows endophytes selected for semi-quantitative analysis of metabolites. 
           [0239]      FIGS. 40 and 41  show metabolomics analyses of fescue endophytes. 
           [0240]      FIG. 42  shows a semi-quantitative analysis of metabolic profile under temperature/water stress. 
           [0241]      FIG. 43  shows endophytes selected for isogenic inoculation. 
           [0242]      FIG. 44  shows SSR-based genotyping of isolated endophytes cultures prior to isogenic inoculation. 
           [0243]      FIG. 45  shows endophyte vegetative stability in tall fescue and perennial ryegrass host genotypes (stability at 12 months post inoculation). 
           [0244]      FIG. 46  shows endophytes selected for isogenic inoculation. 
           [0245]      FIGS. 47-50  show metabolic profiling of isogenic tall fescue-endophyte associations. 
           [0246]      FIG. 51  shows anti-fungal bioassays of fescue endophytes. Column 1  Colletotrichum graminicola , Column 2  Drechslera brizae , Column 3  Rhizoctonia cerealis.    
           [0247]      FIG. 52  shows sequencing of selected novel fescue endophytes. 
           [0248]      FIG. 53  shows peramine biosynthetic pathway. 
           [0249]      FIGS. 54  A-C show presence of perA gene within non- Epichloe  out-group endophytes ( FIG. 54A  NEA17;  FIG. 54B  NEA18;  FIG. 54C  NEA19). 
           [0250]      FIG. 55  shows ergovaline biosynthetic pathway. 
           [0251]      FIG. 56  shows genes in the eas gene cluster. 
           [0252]      FIGS. 57  A-D show presence of dmaW gene for ergovaline biosynthesis in endophyte strains ( FIG. 57A  NEA17;  FIG. 57B  NEA16;  FIG. 57C  AR542;  FIG. 57D  NEA20). 
           [0253]      FIGS. 58  A-D show presence of eas gene cluster for ergovaline biosynthesis.  FIG. 58A  FaTG-2 NEA17 (287819);  FIG. 58B  non- Epichloe  out-group NEA18 (FEtc6-75);  FIG. 58C  FATG-3 NEA21 (231557);  FIG. 58D   N. coenophialum  NEA16 (FEtc7-342). 
           [0254]      FIG. 59  shows the Lolitrem B biosynthetic pathway. 
           [0255]      FIG. 60  shows genes in the Lolitrem B biosynthetic gene cluster. 
           [0256]      FIGS. 61A-D  show presence of Lolitrem B biosynthetic gene cluster 1 (ItmG, ItmM and ItmK) in endophyte strains.  FIG. 61A  FaTG-2 NEA17 (287819);  FIG. 61B  non- Epichloe  out-group NEA18 (FEtc6-75);  FIG. 61C  FATG-3 NEA21 (231557);  FIG. 61D   N. coenophialum  NEA16 (FEtc7-342). 
           [0257]      FIGS. 62  A-D show presence of Lolitrem B biosynthetic gene cluster 2 (ItmB, ItmQ, ItmP, ItmF and ItmC) in endophyte strains.  FIG. 62A  FaTG-2 NEA17 (287819);  FIG. 62B  non- Epichloe  out-group NEA18 (FEtc6-75);  FIG. 62C  FATG-3 NEA21 (231557);  FIG. 62D   N. coenophialum  NEA16 (FEtc7-342). 
           [0258]      FIGS. 63  A-D show presence of Lolitrem B biosynthetic gene cluster 3 (ItmE and ItmJ) in endophyte strains.  FIG. 63A  FaTG-2 NEA17 (287819);  FIG. 63B  non- Epichloe  out-group NEA18 (FEtc6-75);  FIG. 63C  FATG-3 NEA21 (231557);  FIG. 63D   N. coenophialum  NEA16 (FEtc7-342). 
           [0259]      FIG. 64  shows the loline biosynthetic pathway. 
           [0260]      FIG. 65  shows the loline biosynthetic gene cluster. 
           [0261]      FIGS. 66  A-D show presence of Loline biosynthetic gene cluster in endophyte strains.  FIG. 66A  FaTG-2 NEA17 (287819);  FIG. 66B  non- Epichloe  out-group NEA18 (FEtc6-75);  FIG. 66C  FATG-3 NEA21 (231557);  FIG. 66D   N. coenophialum  NEA16 (FEtc7-342). 
           [0262]      FIGS. 67  A-F show alkaloid biosynthetic gene analysis for endophyte strain NEA23 (269850).  FIG. 67A  Presence of loline gene cluster;  FIG. 67B  Presence of peramine gene;  FIG. 67C  Analysis of Lolitrem gene cluster 01;  FIG. 67D  Analysis of Lolitrem gene clusters 02 and 03;  FIG. 67E  Analysis of dmaW gene for ergovaline production;  FIG. 67F  Analysis of eas gene cluster for ergovaline production. 
           [0263]      FIG. 68  shows genotypic analysis of NEA23 and NEA21. 
           [0264]      FIG. 69  shows genotypic analysis of NEA16 and NEA20. 
           [0265]      FIG. 70  shows the structures of Lolitrem B, Erogvaline and Peramine, with desirable toxin profiles indicated. 
           [0266]      FIG. 71  shows in vitro bioassays to assess antifungal activity of  Neotyphodium  endophytes. 
           [0267]      FIG. 72  shows a detached leaf assay to assess resistance to crown rust ( Puccinia coronata  f. sp.  Lolii ) of perennial ryegrass plants with and without  Neotyphodium  endophytes. 
           [0268]      FIG. 73  shows glasshouse and field trial screens for drought tolerance and water use efficiency of perennial ryegrass plants with and without  Neotyphodium  endophytes. 
           [0269]      FIG. 74  shows the steps involved in cell division. 
           [0270]      FIG. 75  shows experimental work flow for chromosome doubling of endophyte cells. 
           [0271]      FIG. 76  shows flow cytometry calibrations for DNA content assessment in  Neotyphodium  endophyte strains. Peaks indicate relative nuclear DNA content. 
           [0272]      FIG. 77  shows flow cytometry analysis of NEA12 dh    Neotyphodium  endophyte strains. 
           [0273]      FIG. 78  shows analysis of growth rate in culture after 8 weeks of NEA12 dh    Neotyphodium  endophyte strains compared to control endophyte strains. 
           [0274]      FIG. 79  shows analysis of growth rate in culture over 5 weeks of NEA12 dh    Neotyphodium  endophyte strains compared to control endophyte strains. 
           [0275]      FIG. 80  shows antifungal bioassays of NEA12 dh    Neotyphodium  endophyte strains. 
           [0276]      FIG. 81  shows antifungal bioassays of NEA12 dh    Neotyphodium  endophyte strains. 
           [0277]      FIG. 82  shows analysis of genome survey sequencing read depth of colchicine-treated  Neotyphodium  endophyte strains. 
           [0278]      FIG. 83  shows analysis of genome survey sequencing reads mapping to NEA12 genome survey sequence assembly. 
           [0279]      FIG. 84  shows experimental work flow for X-ray mutagenesis. 
           [0280]      FIG. 85  shows the indole-diterpene biosynthetic pathway of  Neotyphodium  endophytes. 
           [0281]      FIG. 86  shows in vitro growth of X-ray irradiated  Neotyphodium  endophyte strains. 
           [0282]      FIG. 87  shows Itm gene clusters of  Neotyphodium  endophytes. 
           [0283]      FIG. 88  shows determination of genome sequence variation in X-ray irradiated  Neotyphodium  endophyte strains. 
           [0284]      FIG. 89  shows single nucleotide polymorphisms (SNPs) in genome sequences of X-ray irradiated  Neotyphodium  endophyte strains. 
           [0285]      FIG. 90  shows small insertions/deletions (INDELs) in genome sequences of X-ray irradiated  Neotyphodium  endophyte strains. 
           [0286]      FIG. 91  shows deletions in genome sequences of X-ray irradiated  Neotyphodium  endophyte strains. 
           [0287]      FIG. 92  shows numbers of SNPs in genic regions of genome sequences of X-ray irradiated  Neotyphodium  endophyte strains. 
           [0288]      FIG. 93  shows numbers of INDELs in genic regions of genome sequences of X-ray irradiated  Neotyphodium  endophyte strains. 
           [0289]      FIG. 94  shows the spectrum of genome sequence changes (deletions) in genome sequences of X-ray irradiated  Neotyphodium  endophyte strains. 
           [0290]      FIG. 95  shows mutagenesis index of X-ray irradiated strains based on number of genome sequence changes observed in genome sequences of X-ray irradiated  Neotyphodium  endophyte strains. 
           [0291]      FIG. 96  shows metabolic profiling of NEA12 dh    Neotyphodium  endophyte strains. 
           [0292]      FIG. 97  shows metabolic profiling of X-ray irradiated  Neotyphodium  endophyte strains. 
       
    
    
     EXAMPLE 1 
     Identification of Novel Endophytes 
       [0293]    A collection of 244 perennial grass accessions was assembled for the discovery of novel endophyte strains. The collection targeted accessions from the Northern Mediterranean and Eastern Europe for endophytes that lack lolitrems, as well as accessions from the Middle East, the proposed centre of origin of perennial ryegrass and  N. lolii.    
         [0294]    Genotypic analysis of endophyte content was performed across a total of 189 accessions. From each accession 1-5 plant genotypes were analysed for endophyte. Endophyte incidence was low, with endophyte detected in 51% of accessions. Endophyte was consistently detected (with ≧10 SSR markers) in 77 of the accessions. 
         [0295]    Endophytes representing five different taxa were detected across the 77 accessions with 18 SSR markers used to investigate endophyte diversity in perennial ryegrass ( FIG. 2 ).  N. lolii  was predominant, occurring in 63 accessions. Also detected, although less common, were LpTG-2 and putatively new taxa. 
         [0296]    Genetic variation in  N. lolii  appeared to be low. A total of 22 unique genotypes were detected across the 63 accessions host to  N. lolii.    
         [0297]    The likely toxin profiles of 14 of the 22 genotypes were established from comparisons with genetic and phenotypic data from previous studies. Most of these genotypes (12/14) showed genetic similarity to endophytes known to produce lolitrems. 
         [0298]    There were two genotypes that showed genetic similarity to genotypes known to lack lolitrems but produce ergovaline. One of these genotypes was identical to the genotype detected in the endophyte NEA6. The likely toxin profiles of the remaining eight genotypes were not known. These genotypes did not show high levels of genetic similarity to the endophytes AR1, Endosafe, NEA3 or NEA5. 
         [0299]    Plants carrying candidate endophytes were subjected to primary metabolic profiling in the endogenous genetic background, through clonal propagation and measurement of toxin levels. A total of 42 genotypes representing four of the five taxa were selected for toxin profiling, including the eight novel genotypes with unknown toxin profiles. The perennial ryegrass genotype North African 6 (NA 6 ), which contains standard toxic (ST) endophyte, was used as a control. 
         [0300]    For metabolic profiling, a complete randomised block design was used, with four replicate clones for each plant and using four hydroponics tubs as blocks. Following three months in hydroponics, whole shoot (leaf plus basal region) was harvested from each plant. The fresh and dry weights of each plant were measured and powdered sample material from 80 (20 genotypes×4 replicates) samples (three tillers per sample) analysed for alkaloid content (lolitrem, ergovaline and peramine). 
       EXAMPLE 2 
     Candidate Endophytes 
       [0301]    Candidate endophytes for further study were chosen on the basis of their genetic identity and metabolic profile. Host-endophyte combinations producing significant amounts of lolitrem B were eliminated, as the ryegrass staggers syndrome produced by this alkaloid is the most important limitation for livestock production. 
         [0302]    The candidate endophyte NEA10 (originating from Spain) was identified as a novel genotype in this analysis with an unknown toxin profile. Its genetic identity is a unique  N. lolii  strain. Following in planta metabolic profiling analysis, candidate endophyte NEA10 was found to produce ergovaline and peramine, and not lolitrem B. 
         [0303]    The candidate endophyte NEA11 (originating from France) was identified as a novel genotype in this analysis with an unknown toxin profile. Its genetic identity is a unique LpTG-2 strain. Following in planta metabolic profiling analysis, candidate endophyte NEA11 was found to produce ergovaline and peramine, and not lolitrem B. 
         [0304]    The candidate endophyte NEA12 (originating from France) was identified as a novel genotype in this analysis with an unknown toxin profile. NEA12 is a genetically novel, non- Neotyphodium lolii , endophyte representative of an as yet un-named taxon. Following in planta metabolic profiling analysis, candidate endophyte NEA12 was found to not produce the three alkaloids assessed (lolitrem B, ergovaline and peramine). 
         [0305]    The candidate endophyte E1 was identified as a novel genotype in this analysis with an unknown toxin profile. E1 is a genetically novel, non- Neotyphodium lolii , endophyte representative of an as yet un-named taxon. Following in planta metabolic profiling analysis, candidate endophyte E1 was found to not produce the three alkaloids assessed (lolitrem B, ergovaline and peramine). 
       EXAMPLE 3 
     Methodologies for Endophyte Characterisation 
     Endophyte Isolation 
       [0306]    Novel candidate endophytes were isolated from their host plant to establish an in vitro culture. Following isolation, the genotype of each endophyte was confirmed by SSR analysis to ensure a high level of quality control prior to inception of isogenic inoculations. 
       Establishment of Meristem Cultures for a Diverse Perennial Ryeqrass Host Panel 
       [0307]    A set of cultivars representing elite germplasm were obtained, including forage and turf types. Meristem cultures from different cultivars were established to evaluate and compare the phenotypic properties of novel endophyte strains in diverse isogenic host backgrounds. Embryogenic genotypes were identified for each of the cultivars through callus induction and proliferation. Subsequent regeneration of embryogenic genotypes identified primary tissue culture responsive (pTCR) genotypes for each of the cultivars. The number of pTCR genotypes with regeneration frequencies ranging from 80-100% varied from 1-4 per cultivar. pTCR genotypes were then prepared for meristem-derived callus induction to identify highly regenerable genotypes for isogeneic endophyte inoculation. Table 1 shows a selection of cultivars developed, and the tissue culture responsive (TCR) genotype, used for isogenic inoculation. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Summary information for perennial cultivars 
               
               
                 selected for isogenic inoculation. 
               
             
          
           
               
                   
                   
                 TCR genotype used 
               
               
                 Cultivar 
                 Characteristics 
                 for inoculation 
               
               
                   
               
               
                 Bealey 
                 Tetraploid forage type 
                 Bea 02 
               
               
                 Bronsyn 
                 Standard forage type with robust 
                 Bro 08 
               
               
                   
                 endophyte performance 
               
               
                 Impact 
                 Late flowering, dense tillering 
                 Imp 04 
               
               
                   
                 forage type 
               
               
                 Barsandra 
                 Turf type 
                 San 02 
               
               
                 Tolosa 
                 Distinct forage type 
                 Tol 03 
               
               
                   
               
             
          
         
       
     
       Isogenic Inoculation of Novel Perennial Ryegrass Endophytes 
       [0308]    In order to accurately determine the phenotypic effects of different candidate endophytes in the absence of host-specific genetic effects, a system for isogenic inoculation was developed ( FIG. 3 ). The regenerating callus method of inoculation was chosen, as it results in a relatively high rate of inoculation compared to other tested techniques, and the achieved isogenic inoculation rate was similar to the standard inoculation procedure for non-isogenic seedlings. Novel candidate endophytes NEA10, NEA11, NEA12, E1 and control endophyte ST were individually inoculated into elite germplasm. The logistical approach was to inoculate two cultivars at any given time, with one TCR genotype for each variety chosen for inoculation in this initial study. For each cultivar-endophyte combination, 30 replicate inoculations were performed, 25 of these replicates being transferred to soil. Following inoculation and plantlet regeneration in culture, plants were transferred to soil for three months to allow establishment of endophyte and host-plant associations. After this period, three tillers from each plant were sampled and tested for endophyte presence using SSR-based analysis. 
         [0309]    A quantitative score was used to assess endophyte inoculation frequency (Table 2). Three diagnostic SSR markers were used to determine endophyte presence and identity and samples were scored on a scale of 0-3. 
         [0310]    Of the 570 inoculations tested, 195 (34.2%) could be positively scored with a high degree of confidence (Table 3). Successful inoculations are listed on Table 3. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 SSR screening for endophyte presence in planta. 
               
             
          
           
               
                 Quantitative 
                 Alleles present and of correct 
               
               
                 score 
                 size for given SSR loci 
               
               
                   
               
               
                 3 
                 Endophyte present 
               
               
                 2 
                 Endophyte present 
               
               
                 1 
                 Endophyte absent 
               
               
                 0 
                 Endophyte absent 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Summary statistics for isogenic inoculation of selected candidate  
               
               
                 endophytes into a targeted perennial ryegrass panel of 5 hosts. 
               
               
                   
               
             
             
               
                 A. Number of positive inoculants 
               
             
          
           
               
                   
                 NEA10 
                 NEA11 
                 NEA12 
                 E1 
                 ST 
                 Total 
               
               
                   
               
               
                 Bea02 
                 0 
                 12 
                 3 
                 4 
                 8 
                 27 
               
               
                 Bro08 
                 0 
                 14 
                 1 
                 13 
                 13 
                 41 
               
               
                 Imp04 
                 3 
                 40 
                 4 
                 10 
                 16 
                 73 
               
               
                 San02 
                 0 
                 17 
                 6 
                 6 
                 11 
                 40 
               
               
                 Tol03 
                 0 
                  3 
                 2 
                 6 
                 3 
                 14 
               
               
                 Total 
                 3 
                 86 
                 16 
                 39 
                 51 
                 195  
               
               
                   
               
             
          
           
               
                 B. Total number of inoculations tested 
               
             
          
           
               
                   
                 NEA10 
                 NEA11 
                 NEA12 
                 E1 
                 ST 
                 Total 
               
               
                   
               
               
                 Bea02 
                 24 
                 18 
                 20 
                 19 
                 25 
                 106 
               
               
                 Bro08 
                 19 
                 15 
                 20 
                 18 
                 25 
                 97 
               
               
                 Imp04 
                 31 
                 49 
                 21 
                 12 
                 35 
                 148 
               
               
                 San02 
                 47 
                 39 
                 24 
                 7 
                 32 
                 149 
               
               
                 Tol03 
                 17 
                 7 
                 18 
                 17 
                 11 
                 70 
               
               
                 Total 
                 138  
                 128 
                 103 
                 73 
                 128  
                 570 
               
               
                   
               
             
          
           
               
                 C. Percent of positive inoculants 
               
             
          
           
               
                   
                 NEA10 
                 NEA11 
                 NEA12 
                 E1 
                 ST 
                 Average 
               
               
                   
               
               
                 Bea02 
                 0.0 
                 66.7 
                 15.0 
                 21.1 
                 32.0  
                 25.5 
               
               
                 Bro08 
                 0.0 
                 93.3 
                 5.0 
                 72.2 
                 52.0  
                 42.3 
               
               
                 Imp04 
                 9.7 
                 81.6 
                 19.0  
                 83.3  
                 45.7 
                 49.3 
               
               
                 San02 
                 0.0 
                 43.6 
                 25.0  
                 85.7 
                 34.4  
                 26.8 
               
               
                 Tol03 
                 0.0 
                 42.9 
                 11.1  
                 35.3 
                 27.3  
                 20.0 
               
               
                 Average 
                 2.2 
                 67.2 
                 15.5  
                 53.4  
                 39.8 
                 34.2 
               
               
                   
               
             
          
         
       
     
         [0311]    Variation in inoculation success according to candidate endophyte identity was observed (Table 3). Endophyte NEA10 (2.2%), for example, exhibited relatively lower success rates as compared to NEA11 (67.2%), or the commercial endophyte ST (39.8%; Table 4) and only formed stable associations with one of the five hosts in the panel (Impact). Endophyte E1 is a highly compatible endophyte, which obtained a high rate of success of inoculation into perennial ryegrass (Table 3) compared to other endophytes examined, including the strain ST. 
         [0312]    Variation was also observed between host plant genotypes for successful inoculations (Table 3). Tolosa (20.0%) appears to be more recalcitrant to inoculation compared to host plants such as Bronsyn (42.3%) and Impact (49.3%). 
       Vegetative Stability of Isogenic Perennial Ryegrass-Fungal Endophyte Associations 
       [0313]    Fully confirmed endophyte positive plants from the targeted host-endophyte panel (host plants Bealey, Bronsyn, Barsandra, Tolosa and Impact; endophytes ST, NEA10, NEA11, NEA12) were retested 6-12 months after inoculation and 18-24 months after inoculation, to confirm the presence of endophyte and to assess vegetative stability. In this experiment, 3 replicates of 3 tillers each (total of 9 tillers) were collected for SSR-based analysis. 
         [0314]    Most of the previously confirmed endophyte positive plants were again confirmed in this study at 6-12 months post inoculation, indicating that each of the host—endophyte combinations were stable (Table 4). Endophyte NEA12 appears to be less stable in planta, as 7 of the 13 previously confirmed samples could not be fully confirmed in this experiment (Table 4). ST also showed lower levels of stability compared to NEA11, with 7/21 samples not re-confirmed in this study (Table 4). Following this analysis, up to three independent inoculation events from each host plant—endophyte combination were retained for further study. 
         [0315]    At 18-24 months post inoculation, plants were further assessed for long term vegetative stability (Table 4). ST, NEA10 and NEA11 each exhibit stable associations, with most plants retaining endophyte. NEA12 appears to be less stable in some associations, however does form stable long term associations with Tolosa. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Endophyte frequency in priority ryegrass host panel genotypes in re- 
               
               
                 sampled plants that were previously fully confirmed. Plants were re- 
               
               
                 sampled 6-12 months (shown in bold text) post inoculation and again 
               
               
                 after 18-24 months post inoculation (shown in normal text). 
               
             
          
           
               
                   
                 Plant 
                 Endophyte genotype 
                   
               
             
          
           
               
                   
                 genotype 
                 ST 
                 NEA10 
                 NEA11 
                 NEA12 
               
               
                   
                   
               
               
                   
                 Impact 
                   9/10   
                 
                   2/3 
                 
                 
                   12/12 
                 
                 
                   1/4 
                 
               
               
                   
                 (Imp04) 
                 3/3 
                 2/2 
                 3/3 
                 1/1 
               
               
                   
                 Barsandra 
                 
                   4/6 
                 
                   
                 
                   7/7 
                 
                 
                   2/4 
                 
               
               
                   
                 (San02) 
                 2/3 
                 NA 
                 3/3 
                 1/2 
               
               
                   
                 Tolosa 
                 
                   1/2 
                 
                   
                 
                   3/3 
                 
                 
                   2/2 
                 
               
               
                   
                 (Tol03) 
                 1/1 
                 NA 
                 2/3 
                 2/2 
               
               
                   
                 Bealey 
                 
                   3/3 
                 
                   
                 
                   9/9 
                 
                 
                   0/2 
                 
               
               
                   
                 (Bea02) 
                 2/3 
                 NA 
                 3/3 
                 0/1 
               
               
                   
                 Bronsyn 
                 
                   3/6 
                 
                   
                 
                   9/9 
                 
                 
                   1/1 
                 
               
               
                   
                 (Bro08) 
                 2/2 
                 NA 
                 4/4 
                 0/1 
               
               
                   
                   
               
               
                   
                 NA = not applicable, as no fully confirmed plants were previously identified. 
               
             
          
         
       
     
       Metabolic Profiling of Isogenic Perennial Ryegrass-Fungal Endophyte Associations 
       [0316]    Metabolic profiling was conducted to determine the stability of the predicted endophyte phenotype in a range of different host genotype backgrounds. Four replicates of three tillers each were grown under optimal conditions in hydroponics for six weeks prior to measuring lolitrem B, ergovaline and peramine levels. Each replicate plant was also tested for the presence/identity of endophyte using SSR-based genotyping in order to correlate toxin profile with endophyte presence, in particular for those instances were toxin profiles were negative for the alkaloids measured. 
         [0317]    Table 5 summarises the outcomes of metabolic profiling in hydroponics for both the endophyte discovery phase and the isogenic inoculation phase. Toxin profiles were as predicted from the cluster assignment of the endophyte in the diversity analysis and the toxin profiles measured in the endogenous host plant. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Metabolic profile of candidate endophytes. 
               
             
          
           
               
                 Endophyte 
                 Endogenous 
                 Isogenic 
                   
                   
               
               
                 strain 
                 toxin profile 
                 toxin profile b   
                 Origin 
                 Species 
               
               
                   
               
               
                 NEA10 
                 —/E/n.d a /— 
                 —/E/P/— 
                 Spain 
                 
                   N. lolii 
                 
               
               
                 NEA11 
                 —/E/n.d/— 
                 —/E/P/— 
                 France 
                 LpTG-2 
               
               
                 NEA12 
                 —/—/—/n.d 
                 —/—/—/J 
                 France 
                 non- N. lolii   
               
               
                 E1 
                 n.d 
                 —/—/—/— 
                   
                 non- N. lolii   
               
               
                 ST 
                 L/E/P/— 
                 L/E/P/— 
                   
                 
                   N. lolii 
                 
               
               
                   
               
               
                 Toxins are listed in order: L = Lolitrem B; E = Ergovaline; P = Peramine; J = Janthitrems 
               
               
                   a Peramine not measured in NEA10 and NEA11 samples; Janthitrems not measured in NEA12 samples 
               
               
                   b Toxin profile in isogenic associations 
               
             
          
         
       
     
       Genome Survey Sequencing of Candidate Fungal Endophytes 
     Nuclear Genome Assembly 
       [0318]    Genome Survey Sequencing was performed for non- N. lolii  strains NEA12 and E1, LpTG-2 strain NEA11 and  Neotyphodium lolii  strains including Standard Toxic (ST) and NEA10 using GSFLX Titanium (TI-GSFLX) pyrosequencing technology (Roche; as per manufacturers instructions). A further five  N. lolii  strains were sequenced using either GSFLX Standard or GS20 pyrosequencing technology. Genome assembly for each of the strains was conducted with GSFLX De Novo Assembler (Table 6). 
         [0319]    A new genome assembly was performed for  N. lolii  strain ST (GSFLX De Novo Assembler), combining sequence reads from both GSFLX and TI-GSFLX runs. Table 7 compares the assembly of single and multiple strains. This combined assembly of the ST genome achieves c.12× coverage of the c.32 Mbp haploid genome. The genome is assembled into 7,875 large contigs (0.5 to 47 kb) of which the net length is 31,750,111 bp. 
         [0320]    Analysis using Augustus gene prediction software trained for  Fusarium graminearum  shows that there are 11,517 predicted protein coding genes in the  N. lolii  genome. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Summary statistics for GS-FLX based whole genome sequencing of candidate endophytes. 
               
             
          
           
               
                   
                 
                   N. 
                   lolii 
                 
                 
                   N. 
                   lolii 
                 
                 
                   N. 
                   lolii 
                 
                 
                   N. 
                   lolii 
                 
                 
                   N. 
                   lolii 
                 
                 
                   N. 
                   lolii 
                 
                 
                   N. 
                   lolii 
                 
                 
                   N. 
                   lolii 
                 
                 non- N.   lolii   
                 non- N.   lolii   
                 LpTG-2 
               
               
                   
                 Lp19 
                 ST 
                 NEA3 
                 AR1 
                 E9 
                 G4 
                 NEA10 
                 ST 
                 E1 
                 NEA12 
                 NEA11 
               
               
                   
               
               
                 Genome size (Mb) 
                 ~29 
                 ~29 
                 ~29 
                 ~29 
                 ~29 
                 ~29 
                 ~29 
                 ~28 
                 TBD 
                 TBD 
                 ~55 
               
               
                 Toxin profile a   
                 L + E + P 
                 L + E + P 
                 E + P 
                 P 
                 L + P 
                 L + P 
                 E + P 
                 L + E + P 
                 TBD 
                 J 
                 E + P 
               
               
                 454 Sequencer 
                 GS20 
                 GSFLX 
                 GSFLX 
                 GSFLX 
                 GSFLX 
                 GSFLX 
                 GSFLX 
                 GSFLX 
                 GSFLX 
                 GSFLX 
                 GSFLX 
               
               
                   
                   
                 Standard 
                 Standard 
                 Standard 
                 Standard 
                 Standard 
                 Titanium 
                 Titanium 
                 Titanium 
                 Titanium 
                 Titanium 
               
               
                 Number of sequencing runs 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1½ 
                 ½ 
                 1 
                 ½ 
                 ½ 
                 ½ 
               
               
                 Number of high quality reads  
                 449,408 
                 288,527 
                 361,154 
                 437,465 
                 344,074 
                 631,248 
                 580,060 
                 1,220,036 
                 539,019 
                 399,868 
                 456,111 
               
               
                 Number of bases in high quality reads  
                 47,820,858 
                 71,810,513 
                 84,032,924 
                 97,510,674 
                 85,419,382 
                 146,574,403 
                 221,859,987 
                 451,459,919 
                 202,854,865 
                 165,826,144 
                 177,307,015 
               
               
                 Average read length (bases)  
                 106 
                 249 
                 232 
                 223 
                 249 
                 215 
                 383 
                 370 
                 377 
                 415 
                 389 
               
               
                 Origin of reads assembled b   
                 nuclear + mt 
                 nuclear + mt 
                 nuclear + mt 
                 nuclear + mt 
                 nuclear + mt 
                 nuclear + mt 
                 nuclear + mt 
                 nuclear + mt 
                 nuclear + mt 
                 nuclear + mt 
                 nuclear + mt 
               
               
                 Large contigs (&gt;500 bases) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Number of contigs 
                 6 
                 2,524 
                 5,251 
                 6,070 
                 6,612 
                 12,663 
                 7,272 
                 4,198 
                 9,139 
                 12,399 
                 14,791 
               
               
                 number of bases 
                 99,508 
                 1,834,624 
                 3,911,733 
                 4,650,113 
                 5,208,116 
                 12,393,467 
                 26,931,240 
                 24,382,151 
                 27,150,736 
                 17,300,350 
                 16,306,033 
               
               
                 average contig size  
                 16,584 
                 726 
                 744 
                 766 
                 787 
                 978 
                 3703 
                 5,808 
                 2970 
                 1,395 
                 1,102 
               
               
                 N50 contig size 
                 88,709 
                 680 
                 723 
                 751 
                 774 
                 1039 
                 7668 
                 11,026 
                 5845 
                 1,703 
                 1,214 
               
               
                 largest contig size (bases) 
                 88,709 
                 65,108 
                 15,473 
                 19,024 
                 81,839 
                 29,071 
                 50,291 
                 90,675 
                 40,456 
                 16,319 
                 59,986 
               
               
                 All contigs 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 number of contigs 
                 29,013 
                 28,137 
                 33,262 
                 33,777 
                 33,136 
                 32,796 
                 11.809 
                 6,962 
                 15,589 
                 20,640 
                 39,791 
               
               
                 number of bases 
                 3,532,954 
                 7,999,326 
                 10,842,510 
                 11,755,707 
                 12,022,601 
                 17,790,671 
                 28,155,780 
                 25,104,969 
                 28,916,589 
                 19,862,340 
                 23,307,237 
               
               
                   
               
               
                   a L = Lolitrem B, 
               
               
                 E = Ergovaline, 
               
               
                 P = Peramine, 
               
               
                 J = Janthitrems 
               
               
                   b Newbler Assembler 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Assembly comparison of single and multiple strains of  N.   lolli  endophyte 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 ST + NEA3 +  
               
               
                   
                 Lp19 
                 ST 
                 NEA3 
                 AR1 
                 E9 
                 G4 
                 ST 
                 combined ST 
                 AR1 + E9 + G4 
               
               
                   
               
               
                 454  
                 GS 20 
                 GS FLX 
                 GS FLX 
                 GS FLX 
                 GS FLX 
                 GS FLX 
                 GS FLX 
                 GS FLX 
                 GS FLX 
               
               
                 Sequencer 
                   
                 (Standard) 
                 (Standard) 
                 (Standard) 
                 (Standard) 
                 (Standard) 
                 (Titanium) 
                 (Standard + 
                 (Standard) 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Titanium) 
                   
               
               
                 Number 
                 1 
                 1 a   
                 1 
                 1 
                 1 a   
                 1 b   
                 1 
                 2 
                 5 
               
               
                 of sequencing  
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 runs 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Number of  
                 282,604 
                 191,848 
                 257,381 
                 311,444 
                 267,445 
                 446,017 
                 913,566 
                 1,105,414 
                 1,474,135 
               
               
                 reads 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Number of  
                 28,628,965 
                 46,613,713 
                 58,666,512 
                 68,947,121 
                 65,192,155 
                 101,770,051 
                 334,946,727 
                 381,560,440 
                 341,189,552 
               
               
                 bases in reads 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Large contigs 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (≧500 bases) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 number of contigs 
                 109 
                 1,419 
                 3,210 
                 3,519 
                 4,560 
                 11,895 
                 8,825 
                 7,875 
                 11,905 
               
               
                 number of bases 
                 124,393 
                 909,187 
                 2,111,227 
                 2,317,893 
                 3,041,084 
                 9,249,140 
                 31,669,111 
                 31,750,111 
                 26,515,831 
               
               
                 average contig size 
                 1,141 
                 640 
                 657 
                 658 
                 666 
                 777 
                 3,588 
                 4,031 
                 2,227 
               
               
                 N50 contig size c   
                 1,193 
                 606 
                 632 
                 636 
                 644 
                 774 
                 6,142 
                 7,231 
                 3,436 
               
               
                 largest contig size 
                 7,867 
                 8,639 
                 7,382 
                 8,816 
                 8,016 
                 8,226 
                 46,664 
                 46,668 
                 24,527 
               
               
                 (bases) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Q40 plus bases (%) d   
                 90.62 
                 93.97 
                 93.43 
                 93.90 
                 93.72 
                 94.64 
                 98.03 
                 98.52 
                 98.32 
               
               
                 All contigs  
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 (≧100 bases) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 number of contigs 
                 2,183 
                 8,769 
                 12,434 
                 15,324 
                 15,126 
                 28,456 
                 11,324 
                 10,555 
                 21,836 
               
               
                 number of bases  
                 500,187 
                 2,911,985 
                 4,778,407 
                 5,584,622 
                 6,123,678 
                 14,307,808 
                 32,350,805 
                 32,482,543 
                 29,165,397 
               
               
                   
               
             
          
         
       
     
       Alkaloid Biosynthetic Gene Content 
       [0321]    The content of genes known to be involved in alkaloid production in each of the sequenced endophyte genomes was investigated. Sequence reads for each of the strains were subjected to a BLAST(N) search against each of the known toxin gene sequences (downloaded from NCBI) to determine the degree of gene coverage by sequence reads. Table 8 below shows the correlation between secondary metabolite production and toxin-related gene content in endophyte genomes. 
         [0322]    Based on this analysis, endophyte strain E1 is predicted to produce the alkaloids peramine and ergovaline, but not loline or lolitrem B. In planta analysis of alkaloid content has shown that E1 does indeed not produce loline or lolitrem B. 
         [0323]    NEA10 and NEA11 produce ergovaline and peramine, but not lolitrem B. The NEA11 sequence provides evidence for 2 peramine biosynthesis genes, as might be expected in a heteroploid genome. 
         [0324]    NEA12, known to lack production of ergot alkaloids and lolitrem B, also lacks corresponding biosynthetic genes. 
         [0000]    
       
         
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 Correlation between secondary metabolite production and toxin-related gene content in fungal endophyte genomes. 
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
             
          
         
       
     
       Nuclear Genome Comparison 
       [0325]    Comparison of the NEA12 Nuclear Genome to  E. festucae  E2368 and  N. lolii  ST 
         [0326]    To compare the nuclear genome of NEA12 to  E. festucae  and  N. lolii , the contigs derived from NEA12 were split into 250 bp segments and these segments were used as BLAST(N) queries against  E. festucae  strain E2368 (University of Kentucky, http://www.genome.ou.edu.fungi.html) and  N. lolii  ST contigs. One hit was scored for each 250 bp contig if it was greater than 50 bp long and greater than 80% identity. Summary statistics were taken for NEA12 250 bp fragments against  E. festucae  and  N. lolii  ( FIG. 4 ). 
         [0327]    The number of hits showing a given percent identity shows there are more 250 bp segments that give 100 percent identity matches against an  E. festucae  genome than a  N. lolii  genome. 
         [0328]    The above statistic is independent of the length of the overlap. An identical 250 bp region would give a 250 bp overlap with a percent identity of 100. The number and proportion of these identical reads is given for the two searches below (Table 9). 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 The number and proportion of identical reads between NEA12 
               
               
                 and an  E. festucae  genome and a  N. lolii  genome. 
               
             
          
           
               
                   
                 ST 
                 
                   E. festucae 
                 
                 Total 
               
               
                   
                   
               
             
          
           
               
                 Number of identical reads 
                 16914 
                 28866 
                 89416 
               
               
                 (100% identity between 
               
               
                 250 bp segment) 
               
               
                 Percent of identical reads 
                 18.92 
                 32.28 
               
               
                 (100% identity between 
               
               
                 250 bp segment) 
               
               
                   
               
             
          
         
       
     
         [0329]    There are also segments that have no match to either  N. lolii  (6051) or  E. festucae  (5670). These data suggest that NEA12 is a new endophyte taxon that is genetically closer to  E. festucae  than  N. lolii . This data supports the earlier observation, using SSR-based genetic diversity analysis, that NEA12 is genetically distinct from  N. lolii  and  E. festucae.    
         [0000]    Comparison of E1 Nuclear Genome to NEA12 , E. festucae  E2368 and  N. lolii  ST 
         [0330]    For comparison at the whole genome level, the contigs from endophyte strain E1 were split into 123,258 250 bp segments. Each 250 bp segment was used as a BLAST(N) query against the assembled whole genome DNA sequences from NEA12 , E. festucae  E2368 and  N. lolii  ST ( FIG. 5 ). A BLAST(N) hit was recorded if there was an overlap of greater than 49 bp. The number of overlaps at a given percent identity was counted for each search. The plot of this data reveals that the genome of endophyte strain E1 is more similar to that of  E. festucae  strain E2368 than to either  N. lolii  strain ST or NEA12. 
         [0331]    The assembled contigs from NEA12 sum to c.17.3 Mb, so the level of sequence similarity to that endophyte is probably underestimated due to limited scope for comparison. If the similarity is expressed as a fraction of the total matches observed per comparison, strain E1 is seen to be more similar to strain NEA12 than to  N. lolii  strain ST ( FIG. 6 ). The property of enhance similarity between E1 and  E. festucae  as compared to  N. lolii  is similar to the pattern seen with mitochondrial genome analysis. 
       LpTG-2 Endophyte NEA11 
       [0332]    The LpTG-2 endophyte strain NEA11 is reported to be a hybrid of  N. lolii  and  E. typhina.    
         [0333]    Mitochondrial sequence analysis supports the hybridisation of  E. typhina  with a  N. lolii  with only the  N. lolii  mitochondria being retained. 
         [0334]    Evidence for the hybrid nuclear genome is seen when nuclear genes are used as a query against contigs from the NEA11 genome assembly ( FIGS. 7 and 8 ). 
         [0335]    The panels below show a region of the ‘UDP-N-acetylglucosaminyltransferase’ gene from  E. festucae  being used as a BLAST(N) query against:  E. festucae  (E2368) genome contigs;  N. lolii  (ST) genome contigs; and LpTG-2 (NEA11) genome contigs. This result clearly shows a second variant of this gene in the NEA11 genome that has far more SNPs than the first NEA11 contig hit. This presumably represents the  E. typhina  copy of this gene that has been retained in the NEA11 genome. It is unlikely that this is a localised duplication in NEA11 as neither  E. festucae , nor  N. lolii  has such a duplication. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
             
               
               
               
               
             
               
               
             
               
               
               
               
             
           
               
                   
               
             
             
               
                 1:  E .  festucae  (E2368) genome contigs 
               
             
          
           
               
                 1_0 
                  1141 
                 accagacgatacaatctcgatagtaaccgcctgcctcatagcgggattgattacacccag 
                  1200 
               
               
                 contig01260 
                 42150 
                 ............................................................ 
                 42091 
               
               
                 1_0 
                  1201 
                 gcagctcatgacagtggtatcaatctccaatataagactctacgaacggactctgatata 
                  1260 
               
               
                 contig01260 
                 42090 
                 ............................................................ 
                 42031 
               
               
                 1_0 
                  1261 
                 acgccatcgtccccgactcatgatgcccatgtgaaacctttaccagttgccaacgccgtg 
                  1320 
               
               
                 contig01260 
                 42030 
                 ............................................................ 
                 41971 
               
               
                 1_0 
                  1321 
                 tcctcgttagaggtcctgaacaatctgtgtgaacagagtagttggaaatgggtggaaggt 
                  1380 
               
               
                 contig01260 
                 41970 
                 ............................................................ 
                 41911 
               
               
                 1_0 
                  1381 
                 atgttaattggaggctgtcttcaatacggcctagagcgatacgatgatgcgttcaagtcc 
                  1440 
               
               
                 contig01260 
                 41910 
                 ............................................................ 
                 41851 
               
               
                 1_0 
                  1441 
                 ttctcaaggattgtcgcagttgattccaggtaagttgctcgccacaataccctcactcct 
                  1500 
               
               
                 contig01260 
                 41850 
                 ............................................................ 
                 41791 
               
               
                 1_0 
                  1501 
                 ctgcttgacctcacaatcaccggcttcccagccatgttgaagctatcagtcatatgggcg 
                  1560 
               
               
                 contig01260 
                 41790 
                 ............................................................ 
                 41731 
               
               
                 1_0 
                  1561 
                 cagccttgtattgcctcggacgtcaagatgaagcagagaaaaattggctccgggtgataa 
                  1620 
               
               
                 contig01260 
                 41730 
                 ............................................................ 
                 41671 
               
               
                 1_0 
                  1621 
                 agctacgaccaaattatctcgatgtcatggaacacttggtgggtcatctttataaaaatc 
                  1680 
               
               
                 contig01260 
                 41670 
                 ............................................................ 
                 41611 
               
               
                   
               
             
          
           
               
                 2:  N .  lolii  (ST) genome contigs 
                   
               
             
          
           
               
                 1_0 
                  1141 
                 accagacgatacaatctcgatagtaaccgcctgccccatagcgggattgattacacccag 
                  1200 
               
               
                 contig01260 
                 42150 
                 ............................................................ 
                 42091 
               
               
                 1_0 
                  1201 
                 gcagctcatgacagtggtatcaatctccaatataagaccctacgaacggactctgatata 
                  1260 
               
               
                 contig01260 
                 42090 
                 ............................................................ 
                 42031 
               
               
                 1_0 
                  1261 
                 acgccatcgtccccgactcatgatgcccatgtgaaacctttaccagttgccaacgccgtg 
                  1320 
               
               
                 contig01260 
                 42030 
                 ............................................................ 
                 41971 
               
               
                 1_0 
                  1321 
                 tcctcgttagaggtcctgaacaatctgtgtgaacagagtagttggaaatgggtggaaggt 
                  1380 
               
               
                 contig01260 
                 41970 
                 ............................................................ 
                 41911 
               
               
                 1_0 
                  1381 
                 atgttaattggaggctgtcttcaatacggcctagagcgatacgatgatgcgttcaagtcc 
                  1440 
               
               
                 contig01260 
                 41910 
                 ............................................................ 
                 41851 
               
               
                 1_0 
                  1441 
                 ttctcaaggattgtcgcagttgattccaggtaagttgctcgccacaataccctcactcct 
                  1500 
               
               
                 contig01260 
                 41850 
                 ............................................................ 
                 41791 
               
               
                 1_0 
                  1501 
                 ctgcttgatctcacaatcaccggcttcccagccatgttgaagctatcagtcatatgggcg 
                  1560 
               
               
                 contig01260 
                 41790 
                 ............................................................ 
                 41731 
               
               
                 1_0 
                  1561 
                 cagccttgtattgcctcggacgtcaagatgaagcagagaaaaattggctccgggtgataa 
                  1620 
               
               
                 contig01260 
                 41730 
                 ............................................................ 
                 41671 
               
               
                 1_0 
                  1621 
                 agctacgaccaaattatctcgatgccacggaacacttggtgggccatctttataaaaatc 
                  1680 
               
               
                 contig01260 
                 41670 
                 ............................................................ 
                 41611 
               
               
                   
               
             
          
           
               
                 3: LpTG-2 (NEA11) genome contigs 
                   
               
             
          
           
               
                 1_0 
                 1141 
                 accagacgatacaatctcgatagtaaccgcctgccccatagcgggattgattacacccag 
                 1200 
               
               
                 contig04703 
                 1281 
                 ............................................................ 
                 1340 
               
               
                 contig18455 
                  473 
                                                      .......a..........g.... 
                  451 
               
               
                 1_0 
                 1201 
                 gcagctcatgacagtggtatcaatctccaatataagaccctacgaacggactctgatata 
                 1260 
               
               
                 contig04703 
                 1341 
                 ............................................................ 
                 1400 
               
               
                 contig18455 
                  450 
                 ....t....t.............................ac................g.. 
                  391 
               
               
                 1_0 
                 1261 
                 acgccatcgtccccgactcatgatgcccatgtgaaacctttaccagttgccaacgccgtg 
                 1320 
               
               
                 contig04703 
                 1401 
                 ............................................................ 
                 1460 
               
               
                 contig18455 
                  390 
                 ...................................g........c..c............ 
                  331 
               
               
                 1_0 
                 1321 
                 tcctcgctagaggtcctgaacaatctgtgtgaacagagtagttggaaatgggtggaaggt 
                 1380 
               
               
                 contig04703 
                 1461 
                 ............................................................ 
                 1520 
               
               
                 contig18455 
                  330 
                 ..t...c................c...............g.................... 
                  271 
               
               
                 1_0 
                 1381 
                 atgttaattggaggctgtcttcaatacggcctagagcgatacgatgatgcgttcaagtcc 
                 1440 
               
               
                 contig04703 
                 1521 
                 ............................................................ 
                 1580 
               
               
                 contig18455 
                  270 
                 ......g................g.....t..............a............... 
                  211 
               
               
                 1_0 
                 1441 
                 ttctcaaggattgtcgcagttgattccaggtaagttgctcgccacaataccctcactcct 
                 1500 
               
               
                 contig04703 
                 1581 
                 ............................................................ 
                 1640 
               
               
                 contig18455 
                  210 
                 ..........................a.......c...c...........t.c..t...g 
                  151 
               
               
                 1_0 
                 1501 
                 ctgcttgatctcacaatcaccggcttcccagccatgttgaagctatcagtcatatgggcg 
                 1560 
               
               
                 contig04703 
                 1641 
                 ............................................................ 
                 1700 
               
               
                 contig18455 
                  150 
                 t.............g...c.t.......t............................... 
                   91 
               
               
                 1_0 
                 1581 
                 cagccttgtattgcctcggacgtcaagatgaagcagag-aaaaattggctccgggtgata 
                 1619 
               
               
                 contig04703 
                 1701 
                 ......................................a..................... 
                 1760 
               
               
                 contig18455 
                   90 
                 ......................c.....c.........-c.................g.. 
                   32 
               
               
                 1_0 
                 1620 
                 aagctacgaccaaattatctcgatgccacggaacacttggtgggccatctttataaaaat 
                 1679 
               
               
                 contig04703 
                 1761 
                 ............................................................ 
                 1820 
               
               
                 contig18455 
                   31 
                 ..............c................ 
               
               
                   
               
             
          
         
       
     
         [0336]    The panel below shows the  N. lolii  peramine gene from GenBank used as a query against NEA11 genome assembly contigs. BLAST(N) alignment of LpTG-2 endophyte strain NEA11 reads against the peramine gene (perA) sequence (GenBank accession number: AB205145). The presence of SNP in one set of contigs indicates the presence of two copies of the peramine gene sequence in endophyte strain NEA11. 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
               
             
             
               
                 PerA_A3205145.1 
                 1596 
                 gcgcgtcacgatttcccatttaacaccctcagtcacgcggctgatagacccagattcaca 
                 1655 
               
               
                 FYGH81301D3US2 
                   24 
                 ............................................................ 
                   83 
               
               
                 FYGH813013FIA9 
                   24 
                 ............................................................ 
                   83 
               
               
                 FYGH81301AO93L 
                  450 
                 ...... 
                  453 
               
               
                 FYGH81301CXOSV 
                  247 
                 .............g.........................a.................... 
                  156 
               
               
                 FYGH81301BMMOF 
                  247 
                 .............g.........................a.................... 
                  306 
               
               
                 FYGH81301D5HI6 
                  247 
                 .............g.........................a.................... 
                  306 
               
               
                 FYGH81301CM2KG 
                   52 
                 .............g.........................a............ 
                    1 
               
               
                 FYGH81301AWXAQ 
                  251 
                 .............g.........................a.................... 
                  310 
               
               
                   
               
               
                 PerA_A3205145.1 
                 1656 
                 accttttctaaagacgatggtgtttaccggcgagcctctgtctgtggacgatgctacccg 
                 1715 
               
               
                 FYGH81301D3US2 
                   34 
                 ............................................................ 
                  143 
               
               
                 FYGH81301BFIA9 
                   34 
                 ............................................................ 
                  143 
               
               
                 FYGH81301CXOSV 
                  307 
                 ...cg.c..c.................................................. 
                  366 
               
               
                 FYGH81301BMMOF 
                  307 
                 ...cg.c..c.................................................. 
                  366 
               
               
                 FYGH81301DBHI6 
                  307 
                 ...cg.c..c.................................................. 
                  366 
               
               
                 FYGH81301AWXAQ 
                  311 
                 ...cg.c..c............... 
                  335 
               
               
                   
               
               
                 PerA_A3205145.1 
                 1716 
                 atggtggggaaaggtcgacgtcgtcaacgaatatgggcctgcagagtgcaccatcaacac 
                 1775 
               
               
                 FYGH81301D3U82 
                  144 
                 ............................................................ 
                  203 
               
               
                 FYGH813013FIA9 
                  144 
                 ............................................................ 
                  203 
               
               
                 FYGH81301CXCSV 
                  367 
                 ............................................................ 
                  426 
               
               
                 FYGH81301BMMOF 
                  367 
                 ............................................................ 
                  426 
               
               
                 FYGH81301D3HI6 
                  367 
                 ............................................................ 
                  426 
               
               
                   
               
               
                 PerA_A3205145.1 
                 1776 
                 tgtcaacagccgacctatcagtcctgaagctgctacgaacatagggctgccggttggagt 
                 1835 
               
               
                 FYGH81301D3U82 
                  204 
                 ............................................................ 
                  263 
               
               
                 FYGH813013FIA9 
                  204 
                 ............................................................ 
                  263 
               
               
                 FYGH81301CXOSV 
                  427 
                 ..............................c...g......................... 
                  486 
               
               
                 FYGH81301BMMOF 
                  427 
                 ..............................c...g......................... 
                  486 
               
               
                 FYGH81301D3HI6 
                  427 
                 ..............................c...g......................... 
                  486 
               
               
                   
               
               
                 PerA_AB208145.1 
                 1836 
                 ggccgcttggattaccgacccggaaaaccatcaagtactcgttccgatcggctgtgttgg 
                 1895 
               
               
                 FYGH81301D3U82 
                  264 
                 ............................................................ 
                  323 
               
               
                 FYGH81301BFIA9 
                  264 
                 ............................................................ 
                  323 
               
               
                 FYGH81301CXOSV 
                  487 
                 ...............a........... 
                  513 
               
               
                 FYGH81301BMMOF 
                  487 
                 ...............a........... 
                  513 
               
               
                 FYGH81301DBHI6 
                  487 
                 ............... 
                  501 
               
               
                 FYGH81301EQ6ID 
                    4 
                                 ............tg................a.............. 
                   47 
               
               
                   
               
             
          
         
       
     
       Mating-Type Analysis 
       [0337]    In heterothallic fungi, such as  Epichloë  spp, strains must be of opposite mating-type for sexual reproduction to proceed. In  Epichloë  spp, sexual development is regulated by alternative MAT1-1 (comprising MAT1-1-1, MAT1-1-2 and MAT1-1-3) and MAT1-2 (comprising MAT1-2-1) genes at the MAT locus. Although the flanking regions of MAT1-1 and MAT1-2 are homologous, the nucleotide sequences of MAT1-1 and MAT1-2 idiomorphs are highly dissimilar ( FIG. 7 ). 
         [0338]    The mating-type locus of  E. festucae  E2368 was contained in contig 5 of the original assembly (University of Kentucky, http://www.genome.ou.edu.fungi.html). This contig was aligned with contigs derived from  N. lolii  endophyte strain ST. The MAT1-1 mating-type locus genes found in  E. festucae  (MAT1-1-1, MAT1-1-2, MAT1-1-3) were demonstrated to be absent in the  N. lolii  consensus sequence ( FIG. 7 ). In the corresponding location a single gene for the opposite mating type (MAT1-2) was identified. This opposite mating type gene (MAT1-2-1) was found in all the  N. lolii  strains sequenced as well as NEA12 (Table 10). 
         [0000]    
       
         
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                 GS-FLX based sequence analysis of mating-type loci. Endophyte strain  
               
               
                 E1 is of the same mating-type as  E. festucae  strain E2368. 
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
             
          
         
       
     
         [0339]    To assess the mating type of endophyte strain E1, the two possible mating type contigs were compared to E1 contigs. This activity proved that E1 contained the same three (MAT1-1-1, MAT1-1-2, MAT1-1-3) mating-type genes as  E. festucae  E2368 and is thus of the MAT1-1 mating-type. This is in contrast to the mating type gene of non- N. lolii  strain NEA12, which is of the MAT1-2 , N. lolii -like, mating-type. 
         [0340]    Cluster analysis based on sequence nucleotide diversity shows that endophyte strains E1 and NEA12 cluster with  E. festucae  strain E2368, with their position in the tree switching between analysis based on the mating-type loci flanking sequence and the NoxR gene respectively, and suggesting that recombination has occurred in these lineages ( FIG. 8 ). 
         [0341]    The identification of an endophyte strain of the opposite mating-type to previously characterised perennial ryegrass endophyte strains provides a means for molecular breeding of endophytes to deliver favourable traits into the plant endophyte symbiotum through the use of the novel E1 strain endophyte. 
       Mitochondrial Genome Analysis 
       [0342]    The mitochondrial genome of  N. lolii  endophyte strain Lp19 was present as a single c.88.7 kb contig. This sequence was used to identify contigs containing mitochondrial DNA sequences in the other  N. lolii  strains sequenced through BLAST(N)-based sequence similarity. Homology searches identified mitochondrial contigs in the  E. festucae  strain E2368 assembly the two non- N. lolii  genomes and the LpTG-2 genome that were sequenced. 
         [0343]    The mitochondrial genome sizes for each of the fungal endophytes sequenced in this study as well as the  E. festucae  strain E2368 are shown on Table 11. A representative of the Clavicipitaceae,  Metarhizium anisopliae  (Genbank reference number NC — 008068.1), is shown for comparison. The  N. lolii  mitochondrial genomes are similar in size, ranging from 88,377 bp for G4 to 88,740 bp for AR1. LpTG-2 representative, NEA11 has a mitochondrion genome similar in size to  N. lolii . The two non- N. lolii  genomes, E1 (63,218 bp) and NEA12 (57,818 bp), have relatively smaller mitochondrial genomes more similar in size to that of  E. festucae  strain E2368 (69,614 bp) than that of  N. lolii . 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Mitochondrial genome size of the 10 fungal endophyte strains sequenced  
               
               
                 in this study,  E.   festucae  strain E2368 and  Metarhizium   anisopliae . 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                   
                 non- 
                 non- 
                   
                   Epichlo {umlaut over ( e )} 
                   
               
               
                   
                   N .  lolli   
                   N .  lolli   
                   N .  lolli   
                   N .  lolli   
                   N .  lolli   
                   N .  lolli   
                   N .  lolli   
                   N .  lolli   
                   N .  lolli   
                 LpTG-2 
                 
                   festucae 
                 
                 
                   Metarhizium 
                 
               
               
                   
                 Lp19 
                 ST 
                 NEA3 
                 AR1 
                 E9 
                 G4 
                 NEA10 
                 E1 
                 NEA12 
                 NEA11 
                 2368 
                 
                   anisopliae 
                 
               
               
                   
               
               
                 Approximate Mitochondrial 
                 88709 
                 88711 
                 87526 
                 88740 
                 88738 
                 88377 
                 88734 
                 63219 
                 57818 
                 88692 
                 69614 
                 24674 
               
               
                 Genome Lengths (bp) 
               
               
                   
               
             
          
         
       
     
         [0344]    The multiple mitochondrial DNA sequences were used to generate a mitochondrial genome alignment along with the mitochondrial genome sequence of the Clavicipitaceae fungus  Metarhizium anisopliae . The alignment demonstrated that while the different mitochondrial genomes vary in size, the genes are present in the same order and strand sense in all genomes, with differences being due to variable insertions in each strain ( FIGS. 9 and 10 ). 
         [0345]    Scoring block presence as 1 and absence as 0, a matrix was created to generate a parsimony tree of the relationships between the mitochondrial genomes ( FIG. 11 ). This tree places the E1 and NEA12 mitochondria on a branch with the  E. festucae  strain E2368 mitochondrial genome, these three genomes showing greater variation than that of the  N. lolii  mitochondria. The mitochondrial tree shows that endophyte strains NEA12 and E1 are neither  E. festucae  nor  N. lolii , but are more similar to  E. festucae  than N. Endophyte LpTG-2 NEA11 has a mitochondrial genome that is genetically a  N. lolii  type, being in a Glade with NEA3 and AR1, within the  N. lolii  cluster. 
         [0346]    A similar pattern is observed if a neighbour joining tree is constructed using ClustalW from a DNA alignment of only the 40 blocks of sequence that are shared across all endophyte species (c. 40 kb;  FIG. 12 ). There are still gaps present in the Metarhizium anisopliae sequence in this alignment. 
       A Quantitative PCR Method for Assaying Endophyte Biomass in Planta 
       [0347]    A quantitative PCR (qPCR) method for assaying endophyte biomass in planta has been developed and successfully implemented. The development of a high-throughput PCR-based assay to measure endophyte biomass in planta enables efficient screening of large numbers of plants to study endophyte-ryegrass biomass associations. qPCR-specific primer sets have been designed for the peramine biosynthesis gene (perA). To quantitatively assess in planta endophyte biomass, a standard curve, ranging from 2×10 2  to 2×10 6  copies of the target sequence, has been generated from endophyte DNA template ( FIG. 16 ). The standard curve is used to quantitatively determine in planta endophyte biomass of unknown samples ( FIG. 17 ). 
         [0348]    A proof-of-concept study was conducted using a subset of plants which had been previously analysed using established SSR methodology. The analysis clearly shows a correlation between the quantitative SSR allele scoring and the presence of endophyte in planta (Table 12). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Association between SSR-based analysis of endophyte presence and 
               
               
                 endophyte colonisation as determined by qPCR-based analysis. Each 
               
               
                 host genotype-endophyte combination represented three independent 
               
               
                 biological replicates. An SSR-based quality score was used to assess 
               
               
                 endophyte presence, a score of 3 indicated 3 out of 3 SSR markers 
               
               
                 were efficiently amplified and of the correct size. 
               
             
          
           
               
                 Host plant- 
                   
                   
               
               
                 endophyte 
                   
                 qPCR results 
               
               
                 combination 
                 SSR-based assay 
                 (copies/ng gDNA) 
               
               
                   
               
             
          
           
               
                 1 
                 1 
                 Negative 
               
               
                 2 
                 3 
                 16.638 
               
               
                 3 
                 3 
                 68.98 
               
               
                 4 
                 1 
                 Negative/very low 
               
               
                 5 
                 3 
                 24.3 
               
               
                 6 
                 3 
                 1.48 
               
               
                 7 
                 3 
                 14.386 
               
               
                 8 
                 2 
                 0.7646 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 4 
     Molecular Breeding—E1 as a Vehicle for Trait Delivery into Perennial Ryegrass by Hyper-Inoculation 
       [0000]    
       
         
           
             Endophyte E1 is a genetically novel, non- Neotyphodium lolii  endophyte. E1 is representative of an as yet un-named taxon 
             This supposition is supported by mitochondrial and nuclear genome sequence analysis 
             On the basis of DNA specific content, the predicted alkaloid profile of E1 indicates that the lolitrem B toxins deleterious to animal health are not produced by this endophyte. 
             The E1 endophyte does not produce lolitrem B, ergovaline, peramine, lolines or janthitrems in planta. 
             Endophyte E1 has the mating-type MAT1-1, the opposite mating-type to that carried by all  N. lolii  endophytes previously characterised 
             Endophyte E1 has a high inoculation success rate in perennial ryegrass as compared to other endophytes 
             The identification of an endophyte of the opposite mating-type that is highly compatible and stable in planta provides a means for molecular breeding of endophytes for perennial ryegrass through hyper-inoculation 
           
         
       
     
         [0356]    Hyphal fusion between endophyte strains of the opposite mating-type provides a means for delivery of favourable traits into the host plant via hyper-inoculation. Such strains would include: 1) an endophyte strain that exhibits the favourable characteristics of high inoculation frequency and high compatibility with a wide range of elite perennial ryegrass host germplasm and; 2) an endophyte that exhibits a low inoculation frequency and low compatibility, but has a highly favourable alkaloid toxin profile. 
         [0357]    The E1 endophyte strain is genetically novel and is compatible with a wide range of elite germplasm as it can be inoculated with a high degree of success. E1 also is of the opposite mating-type to all of the previously characterised perennial ryegrass endophytes. Molecular breeding may therefore be applied by combining the highly compatible E1 endophyte traits with the favourable toxin profile traits of endophytes such as NEA12. 
         [0358]    The process of molecular breeding through vegetative (hyphal) fusion may occur in planta by co-inoculation of two endophyte into the same plant. However, molecular breeding may be more efficiently achieved through vegetative fusion in in vitro culture of endophytes of the opposite mating-type, followed by hyper-inoculation of the resultant endophyte. 
         [0359]    The following experimental design is applied for molecular breeding of fungal endophytes 
         [0000]    1. Determine vegetative compatibility of known endophytes using established co-culturing methodologies.
 
2. Generation of auxotrophic mutants (e.g. by gene silencing techniques such as RNAi) for two strains of endophyte, such as E1 and NEA12, exhibiting opposite mating-types.
 
3. Development of vegetative (hyphal) fusion protocol using a combination of cell well degrading enzymes and PEG-4000.
 
4. Screen for regenerated endophytes based on survival (indicating complementarity of auxotrophic mutations).
 
5. Genetic screen using SSR and/or mating-type markers to confirm presence of the hybrid genome in a single nuclear compartment.
 
6. Inoculation and compatibility/stability assessment of endophytes using established methodologies.
 
7. Phenotypic assessment of endophyte-host associations using established methodologies.
 
       EXAMPLE 5 
     Generation of Artificial Polyploids of Fungal Endophytes 
       [0360]    Colchicine has been widely used for induction of polyploidy in plant species such as perennial ryegrass, as compared to the application to fungi, which has been limited to a few species. 
         [0361]    The mitotic spindle inhibitor colchicine is capable of inducing autopolyploidisation, and may be applicable to the production of artificial polyploid endophytes. 
         [0362]    Artificial polyploids were generated by colchicine induced chromosome doubling of the endophyte strains ST and NEA12. 
         [0363]    NEA12, a janthitrem only producing endophyte, with superior bioprotective properties forms stable associations with a limited range of perennial ryegrass hosts. An artificial polyploid of NEA12 that is non-toxic to mammals, with enhanced bioprotective properties, that is broadly compatible and highly stable is highly desirable to industry. 
       Generation of Artificial Polyploids 
       [0364]    Experiments were conducted to determine the range of colchicine concentrations in which the mycelia of the fungal endophyte  N. lolii  (strain ST) would grow successfully. Mycelia were grown in colchicine concentrations ranging from 0% to 1% for 21 days and monitored for growth ( FIG. 15 ). At greater than or equal to 0.2% colchicine mycelium growth halted whereas at 0.1% or less colchicine mycelium growth was prolific. 
         [0365]    Artificial polyploids were generated for endophyte strains ST and NEA12. Endophyte strains ST and NEA12 (n) were grown in 0, 0.1 and 0.2% colchicine and potato dextrose broth for 21 days followed by a 7-10 day recovery period in potato dextrose broth only. Protoplasts were generated from all colchicine concentrations and single colonies isolated ( FIG. 16 ). 
         [0366]      N. coenophialum  strain BE9301 and LpTG-2 strain NEA11 which are natural heteroploids (3n and 2×n fused respectively) have been utilised as control material for assessment of ploidy changes using flow cytometry. An optimised protocol was established allowing analysis of fungal protoplasts via flow cytometry. A number of colonies have been identified with changes in nuclear DNA content relative to the control samples ( FIGS. 20 and 21 ). 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 Summary of individual endophyte colonies, ST and NEA12, treated 
               
               
                 with colchicine and subjected to flow cytometry analysis. 
               
             
          
           
               
                   
                 Colchicine 
                 Number of 
                 Number colonies 
               
               
                 Endophyte 
                 treatment (%) 
                 colonies 
                 analysed 
               
               
                   
               
             
          
           
               
                   N. lolii  ST 
                 0.2 
                 12 
                 12 
               
               
                   N. lolii  NEA12 
                 0.1 
                 60 
                 2 
               
               
                   N. lolii  NEA12 
                 0.2 
                 60 
                 18 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 6 
     Generation of Novel Endophyte Variation Using Ionising Radiation 
     Summary 
       [0000]    
       
         
           
             Lolitrem B is the major alkaloid leading to ryegrass staggers in grazing animals. 
             A method has been developed to eliminate the production of the detrimental alkaloid lolitrem B, using X-ray mutagenesis induced deletion of genes in the lolitrem B biosynthetic gene cluster, in the ST endophyte. 
             Such an endophyte would be advantageous over existing commercial endophytes, as ST is highly stable and broadly compatible. 
           
         
       
     
       Introduction 
       [0370]    Ionising radiation is capable of introducing a broad range of mutagenic lesions and has been found to be very effective in many species. Published methods are available to readily detect deletion mutants in targeted plant genes (Li et al, 2002). Experiments have been performed to determine if  N. lolii  mycelia are amenable to production of mutagenic lesions by ionising radiation, in particular deletion mutations. 
       Generation of Novel Endophyte Variation Using Ionising Radiation 
       [0371]      N. lolii  strain ST was grown in potato dextrose broth for different periods of time ranging from 2-14 days before exposure to ionising radiation. Radiation from a caesium source was applied to the liquid cultures in doses ranging from 10-30 Gy. Following a recovery period (10-14 days) the radiation dose was repeated. Protoplasts were generated and recovery of individual colonies monitored over a 4-6 week period. 
         [0372]    Lolitrem B is the major alkaloid leading to ryegrass staggers in grazing animals. Three genes within the lolitrem B gene cluster, which contains 10 genes all required for synthesis of lolitrem B, were targeted to identify individual  N. lolii  colonies with deletions (Young et al, 2005). A high throughput PCR screening method was developed to detect for the presence and absence of the three lolitrem B genes ( FIG. 18 ). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Analysis of ionising radiation experiments. Protoplast regeneration, concentration 
               
               
                 of recovered protoplasts and number of PCR analysed colonies. 
               
             
          
           
               
                 Endophyte 
                 Age of 
                 Dose 
                 Irradiation 
                 Protoplast 
                   
                 Colonies 
                 PCR screened 
               
               
                 strain 
                 Culture 
                 (Gy) 
                 events 
                 regeneration 
                 Concentration 
                 plated 
                 colonies 
               
               
                   
               
             
          
           
               
                 ST 
                 2 
                 wks 
                  0 
                 1 
                 ✓ 
                 1.8 × 10 8  pp/ml 
                 — 
                 — 
               
               
                 ST 
                 2 
                 wks 
                 10 
                 1 
                 ✓ 
                 5.8 × 10 5  pp/ml 
                 700 
                 450 
               
               
                 ST 
                 2 
                 wks 
                 15 
                 1 
                 ✓ 
                 7.5 × 10 5  pp/ml 
                 200 
                 200 
               
               
                 ST 
                 2 
                 wks 
                 20 
                 1 
                 ✓ 
                 2.2 × 10 6  pp/ml 
                 2950  
                 400 
               
               
                 ST 
                 2 
                 wks 
                  30* 
                 1 
                 — 
                 — 
                 — 
                 — 
               
               
                 ST 
                 2 
                 wks 
                 30 
                 1 
                 ✓ 
                 1.94 × 10 7  pp/ml  
                 400 
                 350 
               
               
                 ST 
                 2 
                 wks 
                  0 
                 2 
                 ✓ 
                 1.1 × 10 8  pp/ml 
                 — 
                 — 
               
               
                 ST 
                 2 
                 wks 
                 10 
                 2 
                 ✓ 
                 2.6 × 10 5  pp/ml 
                 150 
                 150 
               
               
                 ST 
                 2 
                 wks 
                 15 
                 2 
                 slow/reduced 
                 2.2 × 10 7  pp/ml 
                 200 
                 200 
               
               
                   
                   
                   
                   
                   
                 numbers 
               
               
                 ST 
                 2 
                 wks 
                 20 
                 2 
                 ✓ 
                 1.38 × 10 7  pp/ml  
                 — 
                 — 
               
               
                 ST 
                 2 
                 wks 
                 30/25 
                 2 
                 ✓ 
                 6.38 × 10 5  pp/ml  
                 1000  
                 750 
               
               
                 ST 
                 2 
                 wks 
                 30 
                 2 
                 ✓ 
                 1.3 × 10 8  pp/ml 
                 900 
                 300 
               
               
                 ST 
                 4 
                 Days 
                  0 
                 1 
                 ✓ 
                 2.5 × 10 8  pp/ml 
                 — 
                 — 
               
               
                 ST 
                 4 
                 Days 
                 10 
                 1 
                 slow/reduced 
                 3.75 × 10 8  pp/ml  
                 200 
                 200 
               
               
                   
                   
                   
                   
                   
                 numbers 
               
               
                 ST 
                 4 
                 Days 
                 15 
                 1 
                 slow/reduced 
                 1.38 × 10 8  pp/ml  
                 200 
                 200 
               
               
                   
                   
                   
                   
                   
                 numbers 
               
               
                 ST 
                 4 
                 Days 
                 20 
                 1 
                 slow/reduced 
                 2.7 × 10 5  pp/ml 
                 — 
                 — 
               
               
                   
                   
                   
                   
                   
                 numbers 
               
               
                 ST 
                 4 
                 Days 
                 25 
                 1 
                 slow/reduced 
                 1.38 × 10 5  pp/ml  
                  50 
                  50 
               
               
                   
                   
                   
                   
                   
                 numbers 
               
               
                 ST 
                 4 
                 Days 
                 30 
                 1 
                 slow/reduced 
                 1.38 × 10 7  pp/ml  
                 — 
                 — 
               
               
                   
                   
                   
                   
                   
                 numbers 
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 Total 
                 6950  
                 3250 
               
               
                   
               
               
                 *30 Gy dose for first irradiation 
               
             
          
         
       
     
       EXAMPLE 7 
     Tall Fescue Endophyte Discovery and Characterisation 
     Summary 
     Tall Fescue Endophyte Discovery 
       [0373]    The strategies implemented for perennial ryegrass endophyte discovery were extended to the resident endophytes of tall fescue (including the FaTG-2 and FaTG-3 taxonomic groups). 
         [0374]    A targeted collection of tall fescue germplasm was made from throughout the range of natural growth and domesticated cultivation. 
         [0375]    A total of 568 tall fescue accessions obtained from 40 different countries were tested for endophyte incidence using endophyte-specific simple sequence repeat (SSR) genetic markers. Twelve to twenty seeds from each accession were tested for endophyte presence. Total genomic DNA was extracted from two independent seed bulks of 6-10 seeds from each accession and endophytes were detected by PCR amplification with six endophyte-specific SSR markers. 
         [0376]    Endophyte was detected in 40% (228/568) of the tall fescue accessions tested. Furthermore, accessions from 23 out of the 40 countries screened were endophyte positive ( FIG. 19 ) showing the highest incidence in Morocco and Pyrenees, where the majority of accessions tested (80%-100%) were endophyte positive. Accessions originating from Italy, Spain, and United States exhibited a higher endophyte incidence among the tall fescue accessions tested. 
         [0377]    A subset of selected endophyte positive samples, were selected for further analysis using 32 endophyte-specific SSR markers. The selected genotypes represent a broad range of known geographical origins, hence representing an effective survey of tall fescue endophyte genotypic variation. A set of 52 reference isolates representing several endophyte species, including the resident endophyte of tall fescue and meadow fescue were also included to the diversity analysis. 
         [0378]    The UPGMA phenogram, constructed using average taxonomic distance based on SSR polymorphism across 203 endophyte positive accessions, represented six different known taxa, and two out-grouped clusters ( FIG. 20 ). The phenogram was supported by Mantel test statistics showing a high correlation coefficient (r=0.95) which indicated a high goodness-of-fit for the data. Endophytes representing six different taxa were detected in the 203 accessions ( FIG. 20 ). The majority of endophytes (60%; 122/203) appeared to belong to the taxon  Neotyphodium coenophialum , clustering in the phenogram with  N. coenophialum  isolates from the reference endophyte collection ( FIG. 20 ). This species occurred in 72% (122/170) of tall fescue collection accessions. 
         [0379]    As defined by the  N. coenophialum  reference isolates, the  N. coenophialum  cluster comprised five main sub-clusters, of which the fifth sub-cluster is rather out grouped from the other four ( FIG. 20 ). 
         [0380]    The genetic variation observed within  N. coenophialum  was high when comparing it with other taxonomic groups. In the phenogram  N. coenophialum  strains clustered for the most part according to their geographical origin ( FIG. 20 ). The first sub-cluster of  N. coenophialum  comprised mainly tall fescue accessions from Spain (28) and few accessions from Pyrenees (3) and France (4) ( FIG. 20 ). Italian (7) and French (14) accessions were clustered in the second sub-cluster ( FIG. 20 ). The third sub-cluster clearly shows the genetic similarity among accessions collected from geographic area surrounding Russian Federation [Slovenia (3), Russian Federation (6), Kazakhstan (7), Former Soviet Union (4) and China (3)] ( FIG. 20 ). Furthermore within the third sub-cluster a set of accessions from France (11) and Pyrenees (1) have formed a separate cluster from Russian Federation and its surrounding geographic origins. The fourth sub-cluster comprises only five endophytes of which two are Moroccan accessions and two are AR endophytes (AR542 and AR584) which were initially isolated from tall fescue originated in Morocco (Latch et al, 2000). The accessions collected from Portugal (4) have formed a distinct sub-cluster which is separated from all the other four sub-clusters ( FIG. 20 ). 
         [0381]    FaTG-2 accessions formed a cluster close, but distinct from isolates of  N. lolii  ( FIG. 20 ). There were 20 FaTG-2 endophyte genotypes tall fescue collection which clustered with the FaTG-2 reference genotype. Among them, a set of six accessions formed sub-clusters having lesser genetic similarity to the FaTG-2 reference genotype. Therefore, the endophytes of those sub-clusters were named “FaTG-2 like” endophyte genotypes. 
         [0382]    A set of six endophyte genotypes formed a distinct cluster with putative FaTG-3 reference isolates as defined by the previously-analysed AR endophytes. Furthermore, 13 accessions primarily originating from Morocco (9/13) formed a sub-cluster with putative FaTG-3 isolates and those unidentified accessions, forming a cluster distinct to putative FaTG-3 were named “FaTG-3 like” endophytes ( FIG. 20 ). 
         [0383]    The identities of selected putative FaTG-2 and FaTG-3 accessions are largely consistent with geographical provenance, as these taxa are known to be characteristic of populations from southern Europe and North Africa. 
         [0384]    Two out grouped clusters were also identified and they were named as “out-group I” and “out-group II” ( FIG. 20 ). Accessions of Mediterranean origin primarily clustered in “out-group I”, whereas one accession from Former Soviet Union formed the second out-group. Moreover, within “out-group I” Italian accessions clearly group separately from Moroccan and Algerian accessions. 
         [0385]    A number of candidate novel endophytes have been identified. 
       Metabolic Profiling of Tall Fescue—Endophyte Associations 
       [0386]    Representative tall fescue—endophyte associations were selected for metabolic profiling analysis in order to determine the endophyte derived alkaloid profile, in particular, lolitrem B, ergot alkaloids, peramine and lolines. 
         [0387]    Analysis of metabolite production was assessed under controlled conditions using a growth chamber. Tall fescue—endophyte associations were each replicated four times by clonal splitting and arranged in a randomised block design in the growth chamber. Plants were maintained in soil for six weeks, with trimming every two weeks to encourage growth. Following 6 weeks growth, pseudostem tissue was harvested and freeze dried prior to performing a metabolite extraction and LCMS analysis. The perennial ryegrass— N. lolii  designer association Bronsyn-ST was used as a control as ST is known to produce lolitrem B, ergovaline and peramine. For each of the accessions, the presence and identity of the resident endophyte was confirmed through SSR analysis of the plant material harvested for metabolic profile analysis and endophyte negative samples were removed from further analysis. 
         [0388]    The results of the qualitative assessment alkaloid of production for 20 novel tall fescue endophytes are summarised in Table 15. Relative quantitation data for Batch three, comprising 13 endophytes assessed in their endogenous hosts, are shown in  FIG. 21  and  FIG. 22 . A number of novel endophytes with favourable toxin profiles (low/no ergovaline production combined with loline and peramine production) have been identified. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 Summary of alkaloid profiles for selected tall fescue endophytes in their endogenous host. 
               
             
          
           
               
                 Tall fescue accession 
                   
               
             
          
           
               
                 details 
                 Batch # 
                   
                   
               
             
          
           
               
                 Tall 
                   
                 for 
                   
               
             
          
           
               
                 fescue 
                 Endophyte 
                 alkaloid 
                 Alkaloid profile 
                 Confirmed 
               
             
          
           
               
                 accession 
                 species 
                 profiling 
                 Lolines 
                 Peramine 
                 Ergovaline* 
                 Lolitrem B 
                 profile 
               
               
                   
               
             
          
           
               
                 1 
                 
                   N. coenophialum 
                 
                 1 &amp; 3 
                 + 
                 + 
                 + M   
                 − 
                 Y 
               
               
                 NEA13 
                 
                   N. coenophialum 
                 
                 2 
                 n.d 
                 + 
                 + 
                 n.d 
                 n.a 
               
               
                 3 
                 
                   N. coenophialum 
                 
                 3 
                 + 
                 + 
                 + L   
                 − 
                 n.a 
               
               
                 4 
                 
                   N. coenophialum 
                 
                 1 
                 n.d 
                 + 
                 + 
                 n.d 
                 n.a 
               
               
                 5 
                 
                   N. coenophialum 
                 
                 2 
                 n.d 
                 + 
                 + 
                 n.d 
                 n.a 
               
               
                 6 
                 
                   N. coenophialum 
                 
                 2 
                 n.d 
                 − 
                 + 
                 n.d 
                 n.a 
               
               
                 7 
                 
                   N. coenophialum 
                 
                 2 &amp; 3 
                 + 
                 + 
                 + H   
                 − 
                 Y 
               
               
                 8 
                 
                   N. coenophialum 
                 
                 2 
                 n.d 
                 + 
                 + 
                 n.d 
                 n.a 
               
               
                 9 
                 
                   N. coenophialum 
                 
                 2 
                 n.d 
                 + 
                 + 
                 n.d 
                 n.a 
               
               
                 10 
                 
                   N. coenophialum 
                 
                 2 
                 n.d 
                 − 
                 + 
                 n.d 
                 n.a 
               
               
                 NEA14 
                 
                   N. coenophialum 
                 
                 1 &amp; 3 
                 + 
                 + 
                 + H   
                 − 
                 Y 
               
               
                 12 
                 
                   N. coenophialum 
                 
                 2 &amp; 3 
                 + 
                 − 
                 + H   
                 − 
                 Y 
               
               
                 13 
                 
                   N. coenophialum 
                 
                 1 &amp; 3 
                 + 
                 + 
                 + L   
                 − 
                 Y 
               
               
                 14 
                 
                   N. coenophialum 
                 
                 2 &amp; 3 
                 + 
                 + 
                 + L   
                 − 
                 Y 
               
               
                 15 
                 
                   N. coenophialum 
                 
                 1 &amp; 3 
                 + 
                 + 
                 + M   
                 − 
                 Y 
               
               
                 16 
                 FaTG-2 
                 3 
                 + 
                 + 
                 + M   
                 − 
                 n.a 
               
               
                 17 
                 FaTG-2 
                 2 &amp; 3 
                 − 
                 + 
                 + M   
                 − 
                 N 
               
               
                 18 
                 FaTG-3 
                 3 
                 + 
                 + 
                 − 
                 + 
                 n.a 
               
               
                 19 
                 Out group 1 
                 2 &amp; 3 
                 − 
                 − 
                 + L   
                 − 
                 Y 
               
               
                 20 
                 Out group 1 
                 1 &amp; 3 
                 − 
                 − 
                 + L   
                 − 
                 Y 
               
               
                 ST 
                 
                   N. lolii 
                 
                 3 
                 − 
                 + 
                 + 
                 + 
                 Y 
               
               
                   
               
               
                 *Relative quantitation of ergovaline levels: 
               
               
                   L = Low; 
               
               
                   M = Medium; 
               
               
                   H = High. 
               
             
          
         
       
     
       Establishment of Meristem Cultures for a Diverse Fescue Host Panel 
       [0389]    Tissue culture responsive genotypes from selected germplasm material have been generated (Drover, Dovey, Bariane, Barolex). Table 16 shows the host cultivars, and their tissue culture responsive genotype, selected for further study. Each of the selected genotypes has a regeneration frequency greater than 80% 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 16 
               
             
             
               
                   
               
               
                 Establishment of meristem cultures for 
               
               
                 a diverse tall fescue host panel. 
               
             
          
           
               
                   
                 TCR genotype 
                   
                   
               
               
                   
                 used for 
               
               
                 Cultivar 
                 inoculation 
                 Species 
                 Characteristics 
               
               
                   
               
               
                 Bariane 
                 BARI 27 
                 
                   L. arundinaceum 
                 
                 Soft leaved, later 
               
               
                   
                   
                   
                 maturing, highly 
               
               
                   
                   
                   
                 palatable 
               
               
                 Dovey 
                 DOV 24 
                 
                   L. arundinaceum 
                 
                 High yielding, fast 
               
               
                   
                   
                   
                 establishing 
               
               
                 Quantum 
                 QUAN 17 
                 
                   L. arundinaceum 
                 
                 Soft leaved with 
               
               
                   
                   
                   
                 improved rust 
               
               
                   
                   
                   
                 resistance 
               
               
                 Jesup 
                 JESS 01 
                 
                   L. arundinaceum 
                 
                 Cool season 
               
               
                   
                   
                   
                 perennial forage 
               
               
                 Bronsyn 
                 BRO 08 
                 
                   L. perenne 
                 
                 Standard perennial 
               
               
                   
                   
                   
                 ryegrass forage type 
               
               
                   
               
             
          
         
       
     
       Tall Fescue Endophyte Isolation 
       [0390]    Selected novel endophytes were isolated from tall fescue accessions (Table 17). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 17 
               
             
             
               
                   
               
               
                 Summary of endophytes isolated from tall fescue accessions 
               
             
          
           
               
                 Endophyte Accession 
                 Origin 
                 Taxon 
               
               
                   
               
             
          
           
               
                 1 
                 Spain 
                 
                   N. coenophialum 
                 
               
               
                 NEA13 
                   
                 
                   N. coenophialum 
                 
               
               
                 4 
                 Pyrenees 
                 
                   N. coenophialum 
                 
               
               
                 5 
                 Pyrenees 
                 
                   N. coenophialum 
                 
               
               
                 6 
                 Catalunya (Spain) 
                 
                   N. coenophialum 
                 
               
               
                 7 
                 Corsica (France) 
                 
                   N. coenophialum 
                 
               
               
                 8 
                 Corsica (France) 
                 
                   N. coenophialum 
                 
               
               
                 9 
                 Corsica (France) 
                 
                   N. coenophialum 
                 
               
               
                 10 
                 Aragon (Spain) 
                 
                   N. coenophialum 
                 
               
               
                 NEA14 
                 PaySardegna (France) 
                 
                   N. coenophialum 
                 
               
               
                 12 
                 Aragon (Spain) 
                 
                   N. coenophialum 
                 
               
               
                 13 
                 Gaurda (Portugal) 
                 
                   N. coenophialum 
                 
               
               
                 14 
                 Gaurda (Portugal) 
                 
                   N. coenophialum 
                 
               
               
                 15 
                 Aragon (Spain) 
                 
                   N. coenophialum 
                 
               
               
                 17 
                 Spain 
                 FaTG-2 
               
               
                 18 
                 Tunisia 
                 FaTG-3 
               
               
                 19 
                 Algeria 
                 outgroup1 
               
               
                 20 
                 Sardegna (NW Italy) 
                 outgroup1 
               
               
                 21 
                 Catalunya (Spain) 
                 
                   N. coenophialum 
                 
               
               
                   
               
             
          
         
       
     
       Isogenic Inoculation of Novel Tall Fescue Endophytes 
       [0391]    A set of ten novel tall fescue endophytes were selected for inoculation based on genetic novelty using SSR-based diversity analysis and the toxin profile based on qualitative metabolic profiling (Table 18). Included in the set was the endophyte AR542 a commercial endophyte in use globally. AR542 was discovered and isolated by AgResearch NZ and is marketed as MaxP™ and MaxQ™. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 18 
               
             
             
               
                   
               
               
                 Endophytes selected for isogenic inoculation based on 
               
               
                 analysis of genetic diversity and metabolic profile 
               
             
          
           
               
                 Tall fescue accession details 
                   
               
             
          
           
               
                 Tall fescue 
                 Endophyte 
                 Alkaloid profile 
               
             
          
           
               
                 accession 
                 species 
                 Lolines 
                 Peramine 
                 Ergovaline 
                 Lolitrem B 
               
               
                   
               
               
                 NEA13 
                 
                   N. coenophialum 
                 
                 n.d 
                 + 
                 + 
                 n.d 
               
               
                 3 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + L   
                 − 
               
               
                 22 
                 
                   N. coenophialum 
                 
                 n.d 
                 n.d 
                 n.d 
                 n.d 
               
               
                 NEA14 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + H   
                 − 
               
               
                 13 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + L   
                 − 
               
               
                 15 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + M   
                 − 
               
               
                 17 
                 FaTG-2 
                 − 
                 + 
                 + M   
                 − 
               
               
                 19 
                 Out group 1 
                 − 
                 − 
                 + L   
                 − 
               
               
                 20 
                 Out group 1 
                 − 
                 − 
                 + L   
                 − 
               
               
                 AR542* 
                 
                   N. coenophialum 
                 
                 n.d 
                 n.d 
                 − 
                 n.d 
               
               
                   
               
               
                 *toxin profile from Bouton et al, 2002. 
               
             
          
         
       
     
         [0392]    In order to accurately determine the phenotypic effects of different candidate endophytes in the absence of host-specific genetic effects, a system for isogenic inoculation was used. Novel candidate endophytes were individually inoculated into elite tall fescue germplasm as well as the perennial ryegrass host genotype Bronsyn (Bro08). Following inoculation and plantlet regeneration in culture, plants were transferred to soil for three months to allow establishment of endophyte and host-plant associations. After this period, three tillers from each plant were sampled and tested for endophyte presence using SSR-based analysis. 
         [0393]    Of the 498 isogenic inoculations tested, 109 (21.9%) could be positively scored with a high degree of confidence. Successful inoculations are listed on Table 19. 
         [0394]    Variation in inoculation success according to candidate endophyte identity was observed. Endophyte strain 3 (4.3%), for example, exhibited relatively lower success rates as compared to strain 20 (51.1%), or the commercial endophyte AR542 (44.4%; Table 19) and only formed stable associations with one of the five hosts (Bariane). No successful inoculations were identified for endophyte strain 15. FaTG-2 endophyte, strain 17, is a highly compatible endophyte which obtains a high rate of success of inoculation into tall fescue (Table 19) compared to other endophytes examined, and is comparable to AR542. Out-group 1 endophyte strain 20 exhibits the highest level of compatibility as measured by its ability to be inoculated. 
         [0395]    Both tall fescue endophytes inoculated into perennial ryegrass host Bro08, strain NEA13 and strain NEA14, were taken up successfully, establishing that endophyte inoculation across a range of host species is possible. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 19 
               
             
             
               
                   
               
               
                 Summary statistics for isogenic inoculations of selected candidate endophytes 
               
               
                 in a targeted isogenic tall fescue and perennial ryegrass panel of 5 hosts. 
               
               
                 C. Percent of successful inoculations 
               
             
          
           
               
                   
                 Endophyte strain 
               
             
          
           
               
                 Host plant genotype 
                 22 
                 3 
                 NEA13 
                 15 
                 NEA14 
                 AR542 
                 13 
                 17 
                 20 
                 19 
                 Total 
               
               
                   
               
               
                 BARI 24 
                 13.0 
                 12.5  
                 22.2 
                 0.0 
                  0.0 
                 42.3 
                 16.7 
                 56.5 
                 54.5 
                  8.3 
                 24.3 
               
               
                 BRO 08 
                 TBD 
                 TBD 
                 18.2 
                 TBD 
                 11.8 
                 TBD 
                 TBD 
                 TBD 
                 TBD 
                 TBD 
                 14.3 
               
               
                 DOV 24 
                 30.0 
                 0.0 
                 TBD 
                 TBD 
                 TBD 
                 TBD 
                 TBD 
                 TBD 
                 TBD 
                 TBD 
                 12.5 
               
               
                 JESS 01 
                 30.4 
                 0.0 
                 17.9 
                 0.0 
                 35.0 
                 47.4 
                 20.0 
                 10.0 
                 41.7 
                 20.0 
                 22.2 
               
               
                 QUAN 17 
                 37.5 
                 0.0 
                 10.0 
                 0.0 
                 TBD 
                 TBD 
                 TBD 
                 TBD 
                 TBD 
                 TBD 
                 17.5 
               
               
                 Total 
                 25.0 
                 4.3 
                 17.9 
                 0.0 
                 13.4 
                 44.4 
                 18.2 
                 41.5 
                 51.1 
                 13.6 
                 21.9 
               
             
          
           
               
                 Species 
                 
                   N. coenophialum 
                 
                 Fa TG-2 
                 Outgroup 1 
               
               
                   
               
               
                 TBD Be Determined 
               
             
          
         
       
     
       EXAMPLE 8 
     Antifungal Activity of  Neotyphodium/Epichloé  Endophytes 
     Introduction 
       [0396]      Neotyphodium  endophytes at present are largely unexplored in terms of their production of novel antimicrobials. 
         [0397]    While some  Epichloé/Neotyphodium  endophytes have been shown to inhibit the growth of plant-pathogenic fungi in vitro, the inhibitory substances produced have not been identified. 
         [0398]    Endophytes with anti-fungal properties may benefit host plants by preventing pathogenic organisms from colonising them and causing disease. This is of particular interest to the turf grass industry. 
       A Bioassay to Assess Antifungal Activity of  Neotyphodium  Endophytes 
       [0399]    To determine if endophytes of the species  Neotyphodium  produce anti-fungal substances in vitro representative species/strains from  Neotyphodium  were tested for the presence of anti-fungal activity against eight species of fungal plant pathogens. 
         [0400]    Three types of inhibition reactions were observed. In the first reaction, pathogenic fungal growth was unaffected. In the second, growth of the pathogenic fungi was initially unaffected, but growth ceased when the colony margin approached a “critical” distance from the central endophyte colony. In the third stronger reaction type, the overall growth of the colony of the pathogenic fungi was reduced. Examples of inhibition reactions are shown in  FIG. 23 . 
         [0401]    Variation was observed within and between endophyte taxa. Non- N. lolii  strain NEA12 exhibits the strongest and most broad spectrum antifungal activity. Variation was also observed among genetically distinct strains of  N. lolii . Within  N. lolii , strains with strongest to weakest effects were ST&gt;AR1&gt;NEA3&gt;NEA10. ST exhibited the broadest spectrum of antifungal activity, inhibiting the growth of 7/8 fungi strains tested. The bioassay results showed that endophytes in vitro exhibit variation in anti-fungal activity that does not correlate with known toxin production (specifically, lolitrem B, ergovaline and peramine). For example NEA12 does not produce lolitrem B, ergovaline and peramine and has strong antifungal activity and ST does produce lolitrem B, ergovaline and peramine and also has strong antifungal activity. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 20 
               
             
             
               
                   
               
               
                 Antifungal activity exhibited by representative strains of  N. lolii   
               
               
                 and related endophyte taxa. Assays were scored visually from 0-5. 
               
             
          
           
               
                   
                 Fungal species 
               
             
          
           
               
                 Endophyte 
                 Endophyte 
                 
                   Alternaria 
                 
                 
                   Colletrichum 
                 
                 
                   Rhizoctonia 
                 
                 
                   Trichoderma 
                 
                 
                   Phoma 
                 
                 
                   Botrytis 
                 
                 
                   Bipolaris 
                 
                 
                   Drechslera 
                 
               
               
                 strain 
                 species 
                 
                   alternata 
                 
                 
                   graminicola 
                 
                 
                   cerealis 
                 
                 
                   harzianum 
                 
                 
                   sorghina 
                 
                 
                   cinerea 
                 
                 
                   portulaceae 
                 
                 
                   brizae 
                 
               
               
                   
               
               
                 AR510 
                 FaTG-3 
                 0 
                 0 
                 5 
                 1 
                 NT 
                 NT 
                 NT 
                 NT 
               
               
                 NEA11 
                 LpTG-2 
                 0 
                 1 
                 2 
                 0 
                 NT 
                 NT 
                 NT 
                 NT 
               
               
                 AR1 
                 
                   N. lolii 
                 
                 0 
                 0 
                 3 
                 0 
                 2 
                 0 
                 1 
                 1 
               
               
                 NEA10 
                 
                   N. lolii 
                 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
               
               
                 NEA3 
                 
                   N. lolii 
                 
                 0 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
               
               
                 ST 
                 
                   N. lolii 
                 
                 0 
                 1 
                 3 
                 2 
                 2 
                 2 
                 4 
                 3 
               
               
                 NEA12 
                 Non- N. lolii   
                 3 
                 4 
                 4 
                 3 
                 3 
                 3 
                 3 
                 2 
               
               
                   
               
               
                 NT—not tested. 
               
               
                 Samples are scored visually from 0-5. 0 is no antifungal activity, 1 is low antifungal activity, 5 is strong antifungal activity. 
               
               
                 NT—not tested 
               
             
          
         
       
     
       Mass Spectrometry for Identification of Antifungal Metabolites 
       [0402]    Mass spectrometry was used to determine the relationship between antifungal activity and metabolite expression. 
         [0403]    Endophyte strains representing the full spectrum of antifungal activity were selected for analysis in order to identify those alkaloids that may be associated with antifungal activity ( FIG. 24 ). 
         [0404]    Endophyte strains were grown both in the presence and absence of the pathogenic fungi  Rhizoctonia cerealis  ( FIG. 25 ). Freeze dried endophyte mycelia was then extracted for metabolic profiling analysis. 
         [0405]    Following extraction, a validation assay was done to ensure that the alkaloids associated with antifungal activity had been appropriately extracted ( FIG. 26 ). The antifungal activity of the extract used for LCMS analysis was confirmed. The expression of antifungal alkaloids is constitutive as extracts taken from endophyte in the absence of  Rhizoctonia cerealis  also exhibit antifungal activity ( FIG. 26 ). 
       EXAMPLE 9 
     Metabolic Profiling 
     Summary 
       [0406]    Perennial ryegrass cultivars inoculated with the NEA12 endophyte were analysed using LCMS. The toxins peramine, ergovaline and lolitrem B were not detected in the extract. The AR37 metabolite 11,12-epoxy janthitrem G was detected and its structure assigned based on retention time and MS analysis of an extract of the AR37 inoculated perennial ryegrass. 
       Metabolic Profiling of Endophyte Nea12 in Perennial Ryegrass. 
       [0407]    Perennial ryegrass cultivars inoculated with different endophytes were analysed for peramine (1), ergovaline (2), lolitrem B (3) and the AR37 isolated metabolites janthitrem I (4) (11,12-epoxy janthitrem G (janthitrem G (5)) by LCMS. Janthitrem G is an isomer of the previously described janthitrem F (6) and its structure was determined by NMR in the original patent describing AR37 (Latch et al, 2000; structures shown in  FIG. 27 ). 
         [0408]    Standards were analysed to provide reference for the perennial ryegrass analyses. The lolitrem B standard had deteriorated significantly but a peak matching the expected m/z and approximate retention time could be found ( FIG. 28 ). 
         [0409]    Data for AR37 inoculated endophyte and NEA12-inoculated ryegrass gave comparable results. Neither contained detectable levels of peramine, ergovaline or lolitrem B. Both contained 11,12-epoxy-janthitrem G (4) ( FIG. 29 ). MSMS analysis of the ion m/z 646 (4) is shown in  FIG. 30 . The data is a good match for that described in the original patent application. 
         [0410]    Analysis of NEA12 was carried out in a number of perennial ryegrass cultivars. It was present to a greater or lesser extent in the majority of those examined (Table 21). No attempt was made to quantitate the amount found. A standard toxic (ST) endophyte was analysed in the same perennial ryegrass cultivars. The ST endophyte produced peramine and ergovaline but not janthitrems (Table 21). The toxin profiles for ST and NEA12 are shown in  FIG. 31 . 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 21 
               
             
             
               
                   
               
               
                 Analysis of endophytes in different perennial ryegrass cultivars. 
               
             
          
           
               
                   
                 Perennial ryegrass 
                   
               
               
                 Endophyte 
                 cultivar/inoculation event 
                 alkaloids detected 
               
               
                   
               
               
                 NEA12 
                 IMP04 20 
                 janthitrem 
               
               
                 NEA12 
                 TOL03 18 
                 janthitrem 
               
               
                 NEA12 
                 TOL03 16 
                 janthitrem 
               
               
                 ST 
                 TOL03 01 
                 peramine, ergovaline, lolitrem B 
               
               
                 ST 
                 TOL03 12 
                 peramine, ergovaline, lolitrem B 
               
               
                 ST 
                 IMP04 44 
                 peramine, ergovaline, lolitrem B 
               
               
                 ST 
                 IMP04 04 
                 peramine, ergovaline, lolitrem B 
               
               
                 ST 
                 BRO08 02 
                 peramine, ergovaline, lolitrem B 
               
               
                 ST 
                 BRO08 01 
                 peramine, ergovaline, lolitrem B 
               
               
                   
               
             
          
         
       
     
         [0411]    The NEA12 endophyte appears to have the same alkaloid profile as AR37 and is distinctly different from the ST endophyte. 
       EXAMPLE 10 
     Tall Fescue Endophyte Discovery 
       [0412]    The objectives of this work on discovery and characterization of endophytes in tall fescue ( Lolium arundinaceum ) were: 
         [0000]    1. Identification and characterisation of novel tall fescue endophytes for evaluation in germplasm.
 
2. Development and evaluation of optimised associations between novel endophytes and elite germplasm.
 
         [0413]    The endophyte discovery was based on screening 568 accessions to identify endophyte positive plants followed by genotyping 210 endophytes to identify novel endophytes in tall fescue. 
         [0414]    The characterisation in planta of novel endophytes from tall fescue was based on the following steps:
       Meristem cultures for tall fescue cultivars were established for isogenic host panel   Endogenous metabolic profiles were determined for 48 samples   Isolation of 38 endophytes was undertaken   Inoculation of 15-20 endophytes into isogenic host panel was undertaken   Isogenic host-endophyte associations were characterised
 
Genotypic Analysis of Endophyte Content in Accessions from a Targeted Fescue Germplasm Collection
       
 
         [0420]    Initially, 472 accessions from 30 countries were tested for endophyte incidence; with 2 replicates of 6-10 seeds in each bulk per accession used in the analysis and endophyte incidence assessed with 6 SSRs. 
         [0421]    New accessions were included in the analysis from the under-represented geographic origins; with a total of 568 accessions from 40 countries tested for endophyte incidence. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 22 
               
             
             
               
                   
               
               
                 Genotypic analysis of endophyte content in accessions 
               
               
                 from a targeted fescue germplasm collection 
               
             
          
           
               
                   
                 Number of 
                 Percentage 
               
               
                   
                 geographic origins 
                 positive accessions 
               
             
          
           
               
                   
                 FEtc 
                 GRIN 
                 FEtc 
                 GRIN 
               
               
                   
                 collection 
                 collection 
                 collection 
                 collection 
               
               
                   
                   
               
             
          
           
               
                 Incidence 
                 7 
                 23 
                 96% 
                 30% 
               
               
                 assessment 01 
               
               
                 Incidence 
                 — 
                 10 
                 — 
                 45% 
               
               
                 assessment 02 
               
               
                   
               
             
          
         
       
     
         [0422]    Genotypic analysis of endophyte content in accessions from a targeted fescue germplasm collection is shown in Table 22. 233 endophyte positive accessions (41%) were detected. The geographical origins are represented in the endophyte incidence assessment. 
         [0423]    A genetic diversity analysis of tall fescue endophytes is shown in  FIG. 33 . A selected set of 210 accessions were used to assess genetic diversity of tall fescue endophytes. Genetic diversity was assessed with 38 SSR markers. Six different taxa were detected. The majority were  N. coenophialum . Twenty were FaTG-2. Six were putative FaTG-3. Thirteen were FaTG-3 like. 
         [0424]    Diversity of host and endophyte is shown in  FIG. 34 . 
         [0425]    Selection of fescue-endophyte combinations for metabolic profiling, endophyte isolation and isogenic inoculation is shown in  FIG. 35 . 52 accessions were initially selected for metabolic profiling and endophyte isolation. Endophyte presence was consistently detected in 25 accessions (red). An additional 48 accessions from under-represented clusters were established in the glasshouse and screened for endophyte presence. 20 accessions were endophyte positive (blue) and were selected for further analysis. 
         [0426]    Selection of fescue-endophyte combinations for metabolic profiling, endophyte isolation and isogenic inoculation is shown in  FIG. 36 . Initial selections are shown in red. Additional selections are shown in blue. 
         [0427]    The desired toxin profile of tall fescue endophytes is shown in  FIG. 37 . 
       EXAMPLE 11 
     Metabolic Profiling 
       [0428]    The experimental design used for semi-quantitative metabolic profile analysis of tall fescue-endophyte associations for the detection of alkaloid production in the endogenous host background is described below. 

 
         [0429]    A metabolic profile analysis for detection of ergovaline and peramine is shown in  FIG. 38 . 
         [0430]    Endophytes selected for semi-quantitative analysis of metabolites are shown in  FIG. 39 . 
       Metabolic Profile Analysis for the Detection of Alkaloid Production of Different Fescue Endophytes 
       [0431]    A metabolic analysis of tall fescue-endophyte associations for the detection of alkaloid production including loline, loline formate, peramine, ergovaline and lolitrem B in the endogenous host background is shown in  FIG. 40 . The alkaloid profile (i.e. lolines, peramine, ergovaline and lolitrem B) of tall fescue-endophyte associations in the endogenous host background for a range of endophyte strains belonging to different endophyte species is shown in Table 23. 
         [0000]    
       
         
               
             
               
               
             
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 23 
               
             
             
               
                   
               
               
                 Alkaloid profile (i.e. lolines, peramine, ergovaline and lolitrem B) of 
               
               
                 tall fescue-endophyte associations in the endogenous host background for 
               
               
                 a range of endophyte strains belonging to different endophyte species 
               
             
          
           
               
                 Tall fescue accession details 
                   
               
             
          
           
               
                 Tall 
               
             
          
           
               
                 fescue 
                 Endophyte 
                 Endophyte 
                 Alkaloid profile 
               
             
          
           
               
                 accession 
                 strain 
                 species 
                 Lolines 
                 Peramine 
                 Ergovaline* 
                 Lolitrem B 
               
               
                   
               
               
                 BE9301 
                 E34 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + L   
                 − 
               
               
                 8PC 
                 NEA13 
                 
                   N. coenophialum 
                 
                 n.d 
                 + 
                 + 
                 n.d 
               
               
                 FEtc7-180 
                 NEA14 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + H   
                 − 
               
               
                 FEtc7-58 
                 NEA15 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + M   
                 − 
               
               
                 FEtc7-342 
                 NEA16 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 − 
                 − 
               
               
                 FEtc7-343 
                 NEA20 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 − 
                 − 
               
               
                 234746 
                 NEA22 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + M   
                 − 
               
               
                 FEtc6-83 
                 NEA24 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + H   
                 − 
               
               
                 FEtc7-289 
                 NEA25 
                 
                   N. coenophialum 
                 
                 + 
                 − 
                 + H   
                 − 
               
               
                 FEtc6-68 
                 NEA26 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 FEtc6-85 
                 NEA27 
                 
                   N. coenophialum 
                 
                 n.d 
                 + 
                 + 
                 n.d 
               
               
                 FEtc6-87 
                 NEA28 
                 
                   N. coenophialum 
                 
                 n.d 
                 + 
                 + 
                 n.d 
               
               
                 FEtc7-127 
                 NEA29 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 FEtc6-128 
                 NEA30 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 FEtc6-129 
                 NEA31 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 287819 
                 NEA17 
                 FaTG-2 
                 − 
                 + 
                 + M   
                 − 
               
               
                 231557 
                 NEA21 
                 FaTG-2 
                 + 
                 + 
                 − 
                 − 
               
               
                 269850 
                 NEA23 
                 FaTG-3 
                 + 
                 + 
                 − 
                 − 
               
               
                 231553 
                 NEA19 
                 Out group 1 
                 − 
                 − 
                 − 
                 − 
               
               
                 FEtc6-75 
                 NEA18 
                 Out group 1 
                 − 
                 − 
                 − 
                 − 
               
               
                 ST 
                 ST 
                 
                   N. lolii 
                 
                 − 
                 + 
                 + 
                 + 
               
               
                 AR542* 
                 AR542 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 − 
                 − 
               
               
                 KY31* 
                 KY31 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 E77* 
                 E77 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                   
               
               
                 (*Published data; nd = not determined). 
               
             
          
         
       
     
         [0432]    Further metabolic analysis of the fescue endophytes is shown in  FIG. 41 . 
       EXAMPLE 12 
     Semi-Quantitative Analysis of Metabolic Profile Under Temperature/Water Stress 
       [0433]    In addition to the metabolic analysis of tall fescue-endophyte associations grown under standard conditions, for the detection of alkaloid production conferred by the endopohytes in the endogenous host background ( FIGS. 38-41 ), a semi-quantitative analysis of metabolic profiles of tall fescue-endophyte associations grown under high temperature and water stress conditions was undertaken. Corresponding tall fescue-endophyte associations were grown under 16 h Light and 30° C.; 18 h Dark and 20° C., and then sampled for alkaloid profile analysis as described below:
       Harvest (control)→freeze dry→50 mg pseudostem material→80% methanol extraction→LCMS analysis   Recovery and water stress   Second harvest (stress)→freeze dry→SSR confirm all of the plant material again.       
 
         [0437]    This was performed in a controlled (growth chamber) environment simulating summer conditions, with light watering as required. Nine copies per accession were planted in general potting mix. A Randomized Complete Block with subsampling was used. 
         [0438]      FIG. 42  shows a semi-quantitative analysis of metabolic profile of tall fescue-endophyte associations grown under high temperature and water stress conditions. 
       EXAMPLE 13 
     In Planta Isogenic Inoculation in Tall Fescue with Novel Endophytes 
     Summary 
       [0439]    A total of 36 fescue endophytes have been isolated from a range of fescue accessions from different geographic origin as described in Table 24, and found to belong to different taxa as follows: 19 of them being  N. coenophialum ; 5 of them being FaTG-2; 3 of them being Outgroup; 3 of them being FaTG-3; 3 of them being FaTG-3 like; and 3 of them being  N. uncinatum   
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 24 
               
             
             
               
                   
               
               
                 Isolation of fungal endophyte cultures from endophyte-containing fescue accessions 
               
               
                 Establishment of Meristem Cultures for Diverse Host 
               
               
                 Panel for In Planta Inoculation of Fescue Endophytes 
               
             
          
           
               
                 Fescue 
                 Endophyte 
                   
                   
                   
               
               
                 Accession 
                 Strain 
                 Origin 
                 Cluster 
                 Taxon 
               
               
                   
               
             
          
           
               
                 1 
                 8PC 
                 8PC 
                   
                 C01.1 
                 
                   N. coenophialum 
                 
               
               
                 2 
                 BE9301 
                 E34 
                   
                 C01.1 
                 
                   N. coenophialum 
                 
               
               
                 3 
                 E77 
                 E77 
                   
                 C01.2 
                 
                   N. coenophialum 
                 
               
               
                 4 
                 FEtc6-62 
                   
                 Catalunya (Spain) 4 
                 C01.2 
                 
                   N. coenophialum 
                 
               
               
                 5 
                 FEtc6-68 
                 NEA26 
                 Catalunya (Spain) 14 
                 C01.2 
                 
                   N. coenophialum 
                 
               
               
                 6 
                 FEtc7-127 
                 NEA29 
                 Aragon (Spain)14 
                 C01.2 
                 
                   N. coenophialum 
                 
               
               
                 7 
                 FEtc7-289 
                 NEA25 
                 Aragon (Spain)14 
                 C01.2 
                 
                   N. coenophialum 
                 
               
               
                 8 
                 FEtc7-58 
                 NEA15 
                 Aragon (Spain) 1 
                 C01.2 
                 
                   N. coenophialum 
                 
               
               
                 9 
                 234746 
                 NEA22 
                 Spain 
                 C01.2 
                 
                   N. coenophialum 
                 
               
               
                 10 
                 632582 
                   
                 Italy 
                 C02.1 
                 
                   N. coenophialum 
                 
               
               
                 11 
                 Kentucky 31 
                 KY31 
                   
                 C02.1 
                 
                   N. coenophialum 
                 
               
               
                 12 
                 FEtc6-128 
                 NEA30 
                 Pyrenees13 
                 C02.2 
                 
                   N. coenophialum 
                 
               
               
                 13 
                 FEtc6-129 
                 NEA31 
                 Pyrenees17 
                 C02.2 
                 
                   N. coenophialum 
                 
               
               
                 14 
                 FEtc7-180 
                 NEA14 
                 PaySardegna (Basque (Fran           
                 C02.2 
                 
                   N. coenophialum 
                 
               
               
                 15 
                 440364 
                   
                 Kazakhstan 
                 C03 
                 
                   N. coenophialum 
                 
               
               
                 16 
                 619005 
                   
                 China 
                 C03 
                 
                   N. coenophialum 
                 
               
               
                 17 
                 FEtc6-83 
                 NEA24 
                 Corsica (France)7 
                 C04 
                 
                   N. coenophialum 
                 
               
               
                 18 
                 FEtc6-85 
                 NEA27 
                 Corsica (France) 15 
                 C04 
                 
                   N. coenophialum 
                 
               
               
                 19 
                 FEtc6-87 
                 NEA28 
                 Corsica (France) 17 
                 C04 
                 
                   N. coenophialum 
                 
               
               
                 20 
                 AR542 
                 AR542 
                 Morocco 
                 C05 
                 
                   N. coenophialum 
                 
               
               
                 21 
                 FEtc7-342 
                 NEA16 
                 Gaurda (Portugal) 
                 C06 
                 
                   N. coenophialum 
                 
               
               
                 22 
                 FEtc7-343 
                 NEA20 
                 Gaurda (Portugal) 
                 C06 
                 
                   N. coenophialum 
                 
               
               
                 23 
                 231557 
                 NEA21 
                 Morocco 
                 C09 
                 Fa TG-2 
               
               
                 24 
                 287819 
                 NEA17 
                 Spain 
                 C09 
                 Fa TG-2 
               
               
                 25 
                 598834 
                   
                 Morocco 
                 C09 
                 Fa TG-2 
               
               
                 26 
                 231559 
                   
                 Morocco 
                 C09 
                 Fa TG-2 
               
               
                 27 
                 598852 
                   
                 Morocco 
                 C09 
                 Fa TG-2 
               
               
                 28 
                 598934 
                   
                 Italy 
                 C10 
                 Outgroup 
               
               
                 29 
                 231553 
                 NEA19 
                 Algeria 
                 C10 
                 Outgroup 
               
               
                 30 
                 FEtc6-75 
                 NEA18 
                 Sardegna (NW Italy) 5 
                 C10 
                 Outgroup 
               
               
                 31 
                 269850 
                 NEA23 
                 Tunisia 
                 C12 
                 Fa TG-3 
               
               
                 32 
                 610918 
                   
                 Tunisia 
                 C12 
                 Fa TG-3 
               
               
                 33 
                 610919 
                   
                 Tunisia 
                 C12 
                 Fa TG-3 
               
               
                 34 
                 598829 
                   
                 Morocco 
                 C13 
                 Fa TG-3 like 
               
               
                 35 
                 598863 
                   
                 Morocco 
                 C13 
                 Fa TG-3 like 
               
               
                 36 
                 598870 
                   
                 Morocco 
                 C13 
                 Fa TG-3 like 
               
               
                 37 
                 M311046 
                   
                 Russion Federation 
                 C14 
                 
                   N. uncinatum 
                 
               
               
                 38 
                 M595026 
                   
                 United Kingdom 
                 C14 
                 
                   N. uncinatum 
                 
               
               
                 39 
                 M611046 
                   
                 Russion Federation 
                 C14 
                 
                   N. uncinatum 
                 
               
               
                   
               
               
                             indicates data missing or illegible when filed 
               
             
          
         
       
     
         [0440]    Table 25 shows selected tall fescue and perennial ryegrass cultivars used to identify representative plant genotypes included in the diverse host panel for in planta inoculation of fescue endophytes. All the selected plant genotypes have a high regeneration frequency of &gt;80%. 
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                 TABLE 25 
               
             
             
               
                   
               
               
                 Selected tall fescue and perennial ryegrass cultivars used to 
               
               
                 identify representative plant genotypes included in the diverse 
               
               
                 host panel for in planta inoculation of fescue endophytes 
               
             
          
           
               
                   
                 Genotype 
                   
                   
               
               
                 Cultivar 
                 code 
                 Species 
                 Characteristics 
               
               
                   
               
               
                 Bariane 
                 BARI 27 
                 
                   L. arundinaceum 
                 
                 Soft leaved, later maturing, 
               
               
                   
                   
                   
                 highly palatable 
               
               
                 Dovey 
                 DOV 24 
                 
                   L. arundinaceum 
                 
                 High yielding, fast 
               
               
                   
                   
                   
                 establishing 
               
               
                 Quantum 
                 QUAN 17 
                 
                   L. arundinaceum 
                 
                 Soft leaved with improved 
               
               
                   
                   
                   
                 rust resistance 
               
               
                 Jesup 
                 JES 01 
                 
                   L. arundinaceum 
                 
                 Cool season perennial forage 
               
               
                 Bronsyn 
                 BRO 08 
                 
                   L. perenne 
                 
                 Standard perennial ryegrass 
               
               
                   
                   
                   
                 forage type 
               
               
                   
               
             
          
         
       
     
         [0441]    Isolated fungal endophytes from endophyte-containing fescue accessions selected for in planta isogenic inoculation into the diverse host panel are shown in  FIG. 43 .  FIG. 44  shows SSR-based genotyping of isolated endophyte cultures prior to in planta isogenic inoculation to confirm their identity. 
         [0442]    Results from the SSR genotyping indicating the allele number and sizes for different SSR markers for the different fescue endophyte strains are shown in Table 26. 
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                 TABLE 26 
               
             
             
               
                   
               
               
                 Presence of alleles in endophyte strains 
               
             
          
           
               
                   
                   
                 NCESTA1DH04 
                 NLESTA1TA10 
                 NCESTA1HA02 
                 NCESTA1CC10 
               
               
                 Endophyte 
                 Tall Fescue 
                 (FAM) 
                 (FAM) 
                 (HEX) 
                 (HEX) 
               
             
          
           
               
                 Strain ID 
                 Accession ID 
                 Allele 1 
                 Allele 2 
                 Allele 3 
                 Allele 1 
                 Allele 2 
                 Allele 3 
                 Allele 1 
                 Allele 2 
                 Allele 3 
                 Allele 1 
                 Allele 2 
                 Allele 3 
               
               
                   
               
               
                 AR542 
                 — 
                 212 
                 218 
                 227 
                 165 
                 175 
                   
                 322 
                 327 
                 330 
                 198 
                 201 
                 211 
               
               
                 E34 
                 BE_9301 
                 212 
                 218 
                 224 
                 165 
                 175 
                   
                 322 
                 329 
                 330 
                 198 
                 201 
                 211 
               
               
                 E77 
                 — 
                 212 
                 218 
                 224 
                 165 
                 175 
                   
                 308 
                 322 
                 330 
                 197 
                 201 
                 211 
               
               
                 NEA13 
                 8PC 
                 212 
                 218 
                 224 
                 165 
                 175 
                   
                 322 
                 330 
                   
                 197 
                 200 
                 210 
               
               
                 NEA14 
                 FEtc7-180 
                 215 
                 218 
                 229 
                 165 
                 175 
                   
                 322 
                 329 
                 330 
                 198 
                 201 
               
               
                 NEA15 
                 FEtc7-58 
                 212 
                 218 
                 224 
                 165 
                 175 
                   
                 322 
                 329 
                 330 
                 197 
                 201 
                 211 
               
               
                 NEA16 
                 FEtc7-342 
                 215 
                 227 
                   
                 165 
                 175 
                   
                 309 
                 322 
                 330 
                 198 
                 201 
                 211 
               
               
                 NEA17 
                 287819 
                 215 
                 221 
                 227 
                 171 
                 175 
                   
                 322 
                   
                   
                 201 
                 203 
               
               
                 NEA18 
                 FEtc6-75 
                 218 
                 227 
                   
                 171 
                 175 
                   
                 304 
                 322 
                   
                 201 
               
               
                 NEA19 
                 231553 
                 221 
                 227 
                   
                 171 
                 175 
                   
                 304 
                 325 
                   
                 201 
               
               
                   
               
             
          
         
       
     
         [0443]    Results from the in planta isogenic inoculation into the diverse host panel of selected isolated fungal endophytes from endophyte-containing fescue accessions are shown in Table 27. Data on number of inoculations tested, number of successful inoculations and % of successful inoculations are provided in Table 6 to illustrate the inoculation ability of tall fescue endophytes in tall fescue and perennial ryegrass hosts. 
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                 TABLE 27 
               
             
             
               
                   
               
               
                 Inoculation Ability of Tall Fescue Endophytes in Tall Fescue and Perennial Ryegrass Hosts 
               
             
          
           
               
                   
                 E77 
                 E34 
                 NEA13 
                 NEA15 
                 NEA14 
                 AR542 
                 NEA16 
                 NEA17 
                 NEA18 
                 NEA19 
                   
               
               
                   
                 E77 
                 BE9301 
                 8PC 
                 Fetc7-58 
                 FEtc7-180 
                 AR542 
                 FEtc7-342 
                 287819 
                 FEtc6-75 
                 231553 
                 Total 
               
               
                   
                   
               
             
          
           
               
                 A. Number of inoculations tested 
               
             
          
           
               
                 BARI 27 
                 23 
                 25 
                 30 
                 34 
                 38 
                 38 
                 24 
                 32 
                 40 
                 27 
                 311 
               
               
                 BRO 08 
                 39 
                 31 
                 24 
                 27 
                 35 
                 36 
                 30 
                 33 
                 48 
                 22 
                 325 
               
               
                 DOV 24 
                 10 
                 14 
                 NI 
                 NI 
                 NI 
                 17 
                 8 
                 18 
                 14 
                 16 
                 97 
               
               
                 JESS 01 
                 23 
                 23 
                 39 
                 27 
                 20 
                 36 
                 33 
                 17 
                 28 
                 14 
                 260 
               
               
                 QUAN 17 
                 8 
                 31 
                 20 
                 15 
                 17 
                 21 
                 18 
                 16 
                 15 
                 8 
                 169 
               
               
                 Total 
                 103 
                 124 
                 113 
                 103 
                 110 
                 148 
                 113 
                 116 
                 145 
                 87 
                 1162 
               
             
          
           
               
                 B. Number of successful inoculations 
               
             
          
           
               
                 BARI 27 
                 3 
                 3 
                 4 
                 0 
                 1 
                 11 
                 3 
                 17 
                 18 
                 2 
                 62 
               
               
                 BRO 08 
                 0 
                 0 
                 2 
                 0 
                 2 
                 0 
                 0 
                 4 
                 2 
                 5 
                 15 
               
               
                 DOV 24 
                 3 
                 0 
                 NI 
                 NI 
                 NI 
                 1 
                 0 
                 1 
                 4 
                 0 
                 9 
               
               
                 JESS 01 
                 7 
                 0 
                 5 
                 0 
                 7 
                 10 
                 3 
                 2 
                 1 
                 2 
                 37 
               
               
                 QUAN 17 
                 3 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 6 
                 5 
                 3 
                 18 
               
               
                 Total 
                 16 
                 3 
                 12 
                 0 
                 10 
                 22 
                 6 
                 30 
                 30 
                 12 
                 141 
               
             
          
           
               
                 C. Percent of successful inoculations 
               
             
          
           
               
                 BARI 27 
                 13.0 
                 12.0 
                 13.3 
                 0.0 
                 2.6 
                 28.9 
                 12.5 
                 53.1 
                 45.0 
                 7.4 
                 18.8 
               
               
                 BRO 08 
                 0.0 
                 0.0 
                 8.3 
                 0.0 
                 5.7 
                 0.0 
                 0.0 
                 12.1 
                 4.2 
                 22.7 
                 5.3 
               
               
                 DOV 24 
                 30.0 
                 0.0 
                 NI 
                 NI 
                 NI 
                 5.9 
                 0.0 
                 5.6 
                 28.6 
                 0.0 
                 10.0 
               
               
                 JESS 01 
                 30.4 
                 0.0 
                 12.8 
                 0.0 
                 35.0 
                 27.8 
                 9.1 
                 11.8 
                 3.6 
                 14.3 
                 14.5 
               
               
                 QUAN 17 
                 37.5 
                 0.0 
                 5.0 
                 0.0 
                 0.0 
                 0.0 
                 0.0 
                 37.5 
                 33.3 
                 37.5 
                 15.1 
               
               
                 Total 
                 22.2 
                 2.4 
                 9.9 
                 0.0 
                 10.8 
                 12.5 
                 4.3 
                 24.0 
                 22.9 
                 16.4 
                 12.7 
               
               
                 Cluster 
                 1 
                 1 
                 1 
                 1 
                 2 
                 3 
                 3 
                 7 
                 8 
                 8 
               
             
          
           
               
                 Species 
                 
                   N. coenophialum 
                 
                 Fa TG-2 
                 Outgroup 1 
               
               
                   
               
               
                 NI Not inoculated 
               
             
          
         
       
     
       EXAMPLE 14 
     Endophyte Vegetative Stability in Tall Fescue and Perennial Ryegrass Host Genotypes 
       [0444]    Following in planta isogenic inoculation with a range of selected isolated endophytes from fescue accessions, the endophyte vegetative stability of these endophytes in the different tall fescue and perennial host genotypes (i.e. BRO 08, BARI 27, DOV 24) was assessed, showing that:
       Several tall fescue endophytes (e.g. NEA17, NEA18, NEA19) were stable in perennial ryegrass (BRO08).   BARI27 formed stable associations with all endophytes except for NEA15.   NEA15 failed to form stable associations with any of host genotypes tested.   DOV24 formed few stable associations.       
 
         [0449]    The stability of these associations of novel tall fescue endophytes inoculated in different tall fescue and perennial ryegrass genotypes from the diverse host panel was assessed 12 months post-inoculation. Corresponding results are shown in Table 28. 
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                 TABLE 28 
               
             
             
               
                   
               
               
                 Stability of associations of novel tall fescue endophytes (e.g. NEA13, NEA14, NEA15, NEA16, NEA17, 
               
               
                 etc.) inoculated in different tall fescue and perennial ryegrass genotypes (BARI 27, BRO 08, DOV 
               
               
                 24, JESS 01 and QUAN 17) from the diverse host panel assessed 12 months post-inoculation. 
               
             
          
           
               
                   
                   
                   
                   
                 NEA15 
                 NEA14 
                   
                 NEA16 
                   
                 NEA18 
                   
               
               
                 Plant 
                 E77 
                 E34 
                 NEA13 
                 Fetc7- 
                 FEtc7- 
                 AR542 
                 FEtc7- 
                 NEA17 
                 FEtc6- 
                 NEA19 
               
               
                 Genotype 
                 E77 
                 BE9301 
                 8PC 
                 58 
                 180 
                 AR542 
                 342 
                 287819 
                 75 
                 231553 
               
               
                   
               
               
                 BARI 27 
                 1/2 
                 2/2 
                 1/4 
                 NA 
                 1/1 
                 7/7 
                 1/1 
                 1/2 
                  8/10 
                 1/1 
               
               
                 BRO 08 
                 NA 
                 NA 
                 0/1 
                 NA 
                 0/2 
                 NA 
                 NA 
                 5/5 
                 2/2 
                 3/5 
               
               
                 DOV 24 
                 1/2 
                 NA 
                 NI 
                 NI 
                 NI 
                 0/1 
                 NA 
                 2/2 
                 2/4 
                 NA 
               
               
                 JESS 01 
                 5/5 
                 NA 
                 4/6 
                 NA 
                 5/6 
                  5/10 
                 2/3 
                 0/1 
                 0/1 
                 3/3 
               
               
                 QUAN 17 
                 2/3 
                 NA 
                 0/1 
                 NA 
                 NA 
                 NA 
                 NA 
                 3/6 
                 3/5 
                 1/2 
               
               
                   
               
               
                 NA—not applicable, NI—not inoculated, number of stable association/number of associations 
               
             
          
         
       
     
         [0450]      FIG. 45  shows stability at 12 months post inoculation of selected endophytes in tall fescue and perennial ryegrass host genotypes from the diverse host panel. 
         [0451]    The range of novel fescue endophytes selected for in planta isogenic inoculation is shown in  FIG. 46 . 
         [0452]    Table 29 shows additional novel tall fescue endophytes (e.g. NEA20, NEA21, NEA22, etc.) selected for in planta isogenic inoculations in tall fescue genotypes (i.e. BARI 27, JESS01 and QUAN 17) from the diverse host panel, based on the following selection criteria:
       1. Produce little or no ergovaline   2. Produce no lolitrem B   3. Produce lolines and/or peramine       
 
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                 TABLE 29 
               
             
             
               
                   
               
               
                 Additional novel tall fescue endophytes (e.g. NEA20, NEA21, NEA22, etc.) 
               
               
                 selected for in planta isogenic inoculations in tall fescue genotypes 
               
               
                 (i.e. BARI 27, JESS 01 and QUAN 17) from the diverse host panel. 
               
             
          
           
               
                   
                 NEA20 
                 NEA21 
                 NEA22 
                 NEA23 
                 NEA24 
                 NEA27 
                 NEA30 
               
               
                   
                   
               
             
          
           
               
                   
                 FEtc7- 
                 231557 
                 234746 
                 269850 
                 FEtc6- 
                 FEtc6- 
                 FEtc6- 
               
               
                   
                 343 
                   
                   
                   
                 83 
                 85 
                 128 
               
               
                   
                 Nco 
                 FaTG-3 
                 Nco 
                 FaTG-3 
                 Nco 
                 Nco 
                 Nco 
               
               
                   
                 Lol/—/P/— 
                 Lol/—/P/— 
                 Lol/E/P/— 
                 Lol/—/P/— 
                 Lol/E/P/— 
                 ?/E/P/? 
                 ?/E/P/? 
               
               
                 BARI 27 
                 28 
                 30 
                 30 
                 TBI 
                 30 
                 25 
                 30 
               
               
                 JESS 01 
                 23 
                 20 
                 20 
                 TBI 
                 20 
                 20 
                 30 
               
               
                 QUAN 17 
                 30 
                 30 
                 40 
                 TBI 
                 30 
                 35 
                 25 
               
               
                   
               
               
                 Nco =  N. coenophialum ; ? = alkaloid profile not tested; TBI = To Be Inoculated. 
               
             
          
         
       
     
       EXAMPLE 15 
     Metabolic Profiling of Endophyte-Tall Fescue Associations Established Following in Planta Isogenic Inoculations of Novel Tall Fescue Endophytes in Tall Fescue Genotypes from the Diverse Host Panel 
       [0456]    Metabolic profiling of endophyte-tall fescue associations established following in planta isogenic inoculations of novel tall fescue endophytes in tall fescue genotypes from the diverse host panel is shown in  FIGS. 47 ,  49  and  50 . These figures:
       Compare semi-quantitative alkaloid profiles of selected endophytes across different isogenic hosts   Compare semi-quantitative alkaloid profiles for diverse endophytes in an isogenic host   Compare semi-quantitative alkaloid profiles of tall fescue and perennial ryegrass endophytes in the perennial ryegrass genotype Bro08       
 
         [0460]      FIG. 48  shows the presence of peramine and ergovaline in endophyte-tall fescue associations established following in planta isogenic inoculations of novel tall fescue endophytes in tall fescue genotypes from the diverse host panel. 
         [0461]    Table 30 shows metabolic profiling of endophyte-tall fescue associations established following in planta isogenic inoculations of novel tall fescue endophytes in tall fescue genotypes from the diverse host panel. Confirmed endophyte positive (E+) plants were split to 5 replicates and regularly trimmed to promote tillering. Four months later E+ plants were re-potted in 12 replicates. One month later E+ plants were re-potted if less than 9 positive copies were available at the time. Endophyte status was tested using SSR markers after each re-potting. 
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                 TABLE 30 
               
               
                   
               
               
                 Endophyte-tall fescue associations established following in planta isogenic inoculations of novel tall 
               
               
                 fescue endophytes in tall fescue genotypes from the diverse host panel used for metabolic profiling. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Endophyte genotype 
               
             
          
           
               
                   
                 NEA19 
                 NEA17 
                   
                 E34 
                   
               
             
          
           
               
                 Host genotypes 
                 231553 
                 287819 
                 8PC 
                 AR542 
                 BE9301 
                 E77 
               
               
                   
               
             
          
           
               
                 Bariane (Bari27) 
                 2/5 
                 2/5 
                 3/3 
                 11/11 
                 3/3 
                 10/11 
                 5/5 
                 10/10 
                 1/4 
                 8/12 
                 NA 
                 9/14 
               
               
                 Dovey (DOV 24) 
                 NA 
                   
                 2/5 
                 8/8 
                 NA 
                   
                 NA 
                   
                 NA 
                   
                 3/5 
                 6/12 
               
               
                 Jessup (Jess01) 
                 2/4 
                 4/8 
                 NA 
                   
                 3/3 
                 12/12 
                 4/4 
                 12/12 
                 NA 
                   
                 2/3 
                 8/11 
               
               
                 Quantum (Quan17) 
                 2/5 
                 8/7 
                 4/5 
                 12/12 
                 NA 
                   
                   
                   
                 NA 
                   
                 4/5 
                 12/12  
               
               
                 Bronsyn (Bro08) 
                 9/9 
                 10/11 
                 5/5 
                 11/12 
                 1/9 
                 0/8 
                   
                   
                 NA 
                   
                 NA 
               
               
                   
               
             
          
           
               
                   
                 Endophyte genotype 
               
             
          
           
               
                   
                   
                 NEA18 
                 NEA14 
                 NEA16 
                 NEA15 
               
               
                   
                 Host genotypes 
                 FEtc6-75 
                 Fetc7-180 
                 Fetc7-342 
                 Fetc7-58 
               
               
                   
                   
               
             
          
           
               
                   
                 Bariane (Bari27) 
                 5/5 
                 12/12 
                 3/4 
                 5/12 
                 1/4 
                 1/6 
                   
                 16/25 
               
               
                   
                 Dovey (DOV 24) 
                 3/5 
                  3/12 
                 NA 
                   
                 NA 
                   
                 NA 
               
               
                   
                 Jessup (Jess01) 
                 NA 
                   
                 2/4 
                 7/19 
                 2/3 
                 12/12 
                 NA 
               
               
                   
                 Quantum (Quan17) 
                 2/4 
                  5/12 
                 NA 
                   
                 NA 
                   
                 NA 
               
               
                   
                 Bronsyn (Bro08) 
                 3/4 
                 7/7 
                 0/5 
                   
                 NA 
                   
                 NA 
               
               
                   
                   
               
             
          
         
       
     
         [0462]    A range of endophyte-tall fescue associations established following in planta isogenic inoculations of novel tall fescue endophytes in tall fescue genotypes from the diverse host panel were selected for metabolic profiling (Table 30). In total, 29 isogenic host-endophyte associations were subject to LCMS analysis, following the experimental design described below: 
       Experimental Design 
       [0000]    
       
         
           
             Trim and re-pot plants 
             16 h Light, 30° C.; 18 h Dark, 20° C. 
             Harvest (control)→freeze dry→50 mg pseudostem material→80% methanol extraction→LCMS analysis 
             Recovery and water stress 
             Second harvest (stressed)→freeze dry→50 mg pseudostem material→80% methanol extraction→LCMS analysis. 
           
         
       
     
         [0468]    This was performed in a controlled (growth chamber) environment simulating summer conditions, with light watering as required. Nine copies per accession were planted in general potting mix. A Randomized Complete Block with subsampling was used. 
       EXAMPLE 16 
     Bio-Protective Properties of Fescue Endophytes 
       [0469]    Three fungal pathogens (i.e.  Colletrotrichum graminicola, Drechslera brizae  and  Rhizoctonia cerealis )—causing a range of fungal diseases and infecting a range of different plant hosts—were included in antifungal bioassays used to analyse the potential anti-fungal activities of isolated fescue endophytes.  FIG. 51  shows results from anti-fungal bioassays of isolated fescue endophytes. Results of anti-fungal bioassays are also shown in Table 31. A range of endophytes were found to have high (H) and medium (M) antifungal activity (Table 31). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 31 
               
             
             
               
                   
               
               
                 Anti-fungal bioassays of isolated novel fescue endophytes 
               
             
          
           
               
                   
                 Antifungal activity against 
               
             
          
           
               
                 Tall Fescue endophytes 
                   
                 
                   Colletotrichum 
                 
                 
                   Drechslera 
                 
                 
                   Rhizoctonia 
                 
               
             
          
           
               
                   
                 Strain ID 
                 Accession 
                 Taxon 
                 
                   graminicola 
                 
                 
                   brizae 
                 
                 
                   cerealis 
                 
               
               
                   
                   
               
             
          
           
               
                 1 
                 440364 
                   
                 
                   N. coenophialum 
                 
                 H 
                 H 
                 H 
               
               
                 2 
                 AR542 
                 AR542 
                 
                   N. coenophialum 
                 
                 M 
                 H 
                 H 
               
               
                 3 
                 E34 
                 BE9301 
                 
                   N. coenophialum 
                 
                 M 
                 M 
                 H 
               
               
                 4 
                 NEA13 
                 8PC 
                 
                   N. coenophialum 
                 
                 M 
                 H 
                 H 
               
               
                 5 
                 NEA14 
                 FEtc7-180 
                 
                   N. coenophialum 
                 
                 M 
                 M 
                 H 
               
               
                 6 
                 NEA15 
                 FEtc7-58 
                 
                   N. coenophialum 
                 
                 M 
                 H 
                 H 
               
               
                 7 
                 NEA16 
                 FEtc7-342 
                 
                   N. coenophialum 
                 
                 M 
                 H 
                 H 
               
               
                 8 
                 NEA22 
                 234746 
                 
                   N. coenophialum 
                 
                 H 
                 M 
                 M 
               
               
                 9 
                 NEA27 
                 FEtc6-85 
                 
                   N. coenophialum 
                 
                 L 
                 M 
                 L 
               
               
                 10 
                 NEA30 
                 FEtc6-128 
                 
                   N. coenophialum 
                 
                 M 
                 H 
                 H 
               
               
                 11 
                 E1 
                   
                 Non- N. lolii   
                 L 
                 L 
                 M 
               
               
                 12 
                 NEA18 
                 FEtc6-75 
                 Outgroup 1 
                 M 
                 H 
                 H 
               
               
                 13 
                 598852 
                   
                 FaTG-2 
                 M 
                 H 
                 H 
               
               
                 14 
                 610918 
                   
                 FaTG-3 
                 M 
                 H 
                 H 
               
               
                 15 
                 NEA21 
                 231557 
                 FaTG-3 
                 M 
                 H 
                 M 
               
               
                 16 
                 598829 
                   
                 FaTG-3 like 
                 M 
                 L 
                 M 
               
               
                   
               
               
                 Antifungal activity: Low, Medium, High 
               
             
          
         
       
     
       EXAMPLE 17 
     Genome Survey Sequencing of Novel Tall Fescue Endophytes 
       [0470]    A range of novel tall fescue endophtyes were subjected to genome survey sequencing (GSS). 
         [0471]      FIG. 52  shows a strategy for GSS of selected novel fescue endophytes. The alkaloid profiles of novel fescue endophytes subjected to GSS analysis are shown in Table 32. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 32 
               
             
             
               
                   
               
               
                 Alkaloid profiles of sequenced endophytes. 
               
             
          
           
               
                 Tall fescue accession details 
                   
               
             
          
           
               
                 Endophyte 
                 Accession 
                 Endophyte 
                 Alkaloid profile in Endogenous Host 
               
             
          
           
               
                 strain 
                 No/isolated ID 
                 species 
                 Lolines 
                 Peramine 
                 Ergovaline 
                 Lolitrem B 
               
               
                   
               
               
                 E34 
                 BE9301 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 NEA13 
                 8PC 
                 
                   N. coenophialum 
                 
                 ND 
                 + 
                 + 
                 ND 
               
               
                 NEA14 
                 FEtc7-180 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 NEA15 
                 FEtc7-58 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 NEA16 
                 FEtc7-342 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 − 
                 − 
               
               
                 NEA20 
                 FEtc7-343 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 − 
                 − 
               
               
                 NEA22 
                 234746 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 NEA24 
                 FEtc6-83 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 NEA17 
                 287819 
                 FaTG-2 
                 − 
                 + 
                 + 
                 − 
               
               
                 NEA21 
                 231557 
                 FaTG-3 
                 + 
                 + 
                 − 
                 − 
               
               
                 NEA23 
                 269850 
                 FaTG-3 
                 + 
                 + 
                 − 
                 − 
               
               
                 NEA19 
                 231553 
                 non-Epichloë out-group 
                 − 
                 − 
                 − 
                 − 
               
               
                 NEA18 
                 FEtc6-75 
                 non-Epichloë out-group 
                 − 
                 − 
                 − 
                 − 
               
               
                 AR542* 
                 AR542* 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 − 
                 − 
               
               
                 E77* 
                 E77* 
                 
                   N. coenophialum 
                 
                 + 
                 + 
                 + 
                 − 
               
               
                 598852 
                 598852 
                 FaTG-2 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AR501* 
                 AR501* 
                 FaTG-3 
                 + 
                 + 
                 − 
                 − 
               
               
                 598829 
                 598829 
                 FaTG-3 like 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 E81 
                 E81 
                 
                   N. uncinatum 
                 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 9340 
                 9340 
                 
                   E. typhina 
                 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 9707 
                 9707 
                 
                   E. baconii 
                 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                   
               
               
                 + Alkaloid present, − Alkaloid absent, ND: alkaloid profile not determined 
               
               
                 *Profiles are taken from published data 
               
             
          
         
       
     
         [0472]      FIG. 53  shows the peramine biosynthetic pathway. PerA encodes a single multifunctional enzyme that catalyses all the biosynthetic steps. GenBank accession Number: AB205145. The presence of the perA gene in non- Epichloe  out-group endophytes is shown in  FIG. 54 . 
         [0473]      FIG. 55  shows the ergovaline biosynthetic pathway. Genes in the eas gene cluster which are involved in ergovaline biosynthesis are shown in  FIG. 56  and Table 33. The dmaW gene encodes DMAT synthase enzyme, which catalyzes the first committed step in ergovaline biosynthesis. Presence of the dmaW gene in novel fescue endophytes is shown in  FIG. 57  and presence of the eas gene cluster in novel fescue endophytes is shown in  FIG. 58 . 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 33 
               
             
             
               
                   
               
               
                 Genes in the eas cluster 
               
             
          
           
               
                   
                 Gene Cluster 
                 Gene 
                 GenBank Accession No 
               
               
                   
                   
               
               
                   
                   
                 dmaW 
                 AY259838 
               
               
                   
                 eas gene 
                 easA 
                 EF125025 
               
               
                   
                 cluster 
                 easE 
                 EF125025 
               
               
                   
                   
                 easF 
                 EF125025 
               
               
                   
                   
                 easG 
                 EF125025 
               
               
                   
                   
                 easH 
                 EF125025 
               
               
                   
                   
                 lpsA 
                 AF368420 
               
               
                   
                   
                 lpsB 
                 EF125025 
               
               
                   
                   
               
             
          
         
       
     
         [0474]      FIG. 59  shows the Lolitrem B biosynthetic pathway. Genes in the gene cluster which are involved in Lolitrem B biosynthesis are shown in  FIG. 60  and Table 34. Presence of gene cluster 1 (ItmG, ItmM and ItmK) in endophytes is shown in FIG.  61 , presence of gene cluster 2 (ItmB, ItmQ, ItmP, ItmF and ItmC) is shown in  FIG. 62  and presence of gene cluster 3 (ItmE and ItmJ) is shown in  FIG. 63 . 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 34 
               
             
             
               
                   
               
               
                 Genes in the gene cluster involved in Lolitrem B biosynthesis 
               
             
          
           
               
                   
                 Gene Cluster 
                 Gene 
                 GenBank Accession No 
               
               
                   
                   
               
               
                   
                 gene cluster 01 
                 ltmG 
                 AY742903 
               
               
                   
                   
                 ltmM 
                 AY742903 
               
               
                   
                   
                 ltmK 
                 AY742903 
               
               
                   
                 gene cluster 02 
                 ltmB 
                 DQ443465 
               
               
                   
                   
                 ltmQ 
                 DQ443465 
               
               
                   
                   
                 ltmP 
                 DQ443465 
               
               
                   
                   
                 ltmF 
                 DQ443465 
               
               
                   
                   
                 ltmC 
                 DQ443465 
               
               
                   
                 gene cluster 03 
                 ltmJ 
                 DQ443465 
               
               
                   
                   
                 ltmE 
                 DQ443465 
               
               
                   
                   
               
             
          
         
       
     
         [0475]      FIG. 64  shows the Loline biosynthetic pathway. Genes in the gene cluster which are involved in Loline biosynthesis are shown in  FIG. 65  and Table 35. Presence of Loline biosynthetic gene cluster in novel fescue endophytes is shown in  FIG. 66 . 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 35 
               
             
             
               
                   
               
               
                 Genes in the Loline biosynthetic gene cluster 
               
             
          
           
               
                   
                 Gene Cluster 
                 Gene 
                 GenBank Accession No 
               
               
                   
                   
               
               
                   
                 LOL gene 
                 lolF 
                 EF012269 
               
               
                   
                 cluster 
                 lolC 
                 EF012269 
               
               
                   
                   
                 lolD 
                 EF012269 
               
               
                   
                   
                 lolO 
                 EF012269 
               
               
                   
                   
                 lolA 
                 EF012269 
               
               
                   
                   
                 lolU 
                 EF012269 
               
               
                   
                   
                 lolP 
                 EF012269 
               
               
                   
                   
                 lolT 
                 EF012269 
               
               
                   
                   
                 lolE 
                 EF012269 
               
               
                   
                   
               
             
          
         
       
     
         [0476]      FIG. 67  shows an alkaloid biosynthetic gene analysis for endophyte strain NEA23. Tables 36 and 37 show alkaloid biosynthetic gene analyses for various endophyte strains. Table 36 shows results from the assessment of alkaloid biosynthetic gene presence/absence for different endophytes by mapping genome survey sequence reads corresponding to the different alkaloid biosynthetic genes/gene clusters. 
         [0000]    
       
         
               
             
           
               
                 TABLE 36 
               
               
                   
               
               
                 Assessment of alkaloid biosynthetic gene presence/absence for different endophytes by mapping genome survey sequence reads 
               
               
                 corresponding to the different alkaloid biosynthetic genes/gene clusters. 
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                                           
(-) No alkaloid detected 
               
               
                 (nd) Not determined 
               
             
          
         
       
     
         [0477]    Table 37 shows results from the assessment of alkaloid biosynthetic gene presence/absence for different endophytes by mapping genome survey sequence reads corresponding to the different alkaloid biosynthetic genes/gene clusters as well as corresponding alkaloid profile observed for corresponding tall fescue-endophyte associations. 
         [0000]    
       
         
               
             
           
               
                 TABLE 37 
               
               
                   
               
               
                 Alkaloid biosynthetic gene and alkaloid production analysis. 
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 A+: alkaloid present, 
               
               
                 A−: Alkaloid absent, 
               
               
                 Grey: alkaloid profile not determined, 
               
               
                 *Profiles are taken from published data, 
               
               
                 G+ = gene/gene cluster present, 
               
               
                 G− = gene/gene cluster absent, 
               
               
                 PG+ = gene/gene cluster partially present 
               
             
          
         
       
     
         [0478]    Table 38 shows novel fescue endophytes (NEA16, NEA18, NEA19, NEA20, NEA21 and NEA23) with favourable toxin profiles. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 38 
               
             
             
               
                   
               
               
                 Novel fescue endophytes (NEA16, NEA18, NEA19, NEA20, 
               
               
                 NEA21 and NEA23) with favourable toxin profiles and 
               
               
                 antifungal activities observed in bioassays. 
               
             
          
           
               
                 Tall fescue 
                   
                 Alkaloid profile 
                   
               
               
                 accession 
                 Taxon 
                 (Lol/P/E/L) 
                 Antifungal 
               
               
                   
               
               
                 NEA21 
                 FaTG-3 
                 +/+/−/− 
                 High 
               
               
                 NEA23 
                 FaTG-3 
                 +/+/−/− 
                 Not tested 
               
               
                 AR501* 
                 FaTG-3 
                 +/+/−/− 
                 — 
               
               
                 NEA18 
                 Non- Epichloë   
                 −/−/−/− 
                 High 
               
               
                   
                 Outgroup 
               
               
                 NEA19 
                 Non- Epichloë   
                 −/−/−/− 
                 Not tested 
               
               
                   
                 Outgroup 
               
               
                 NEA16 
                 
                   N. coenophialum 
                 
                 +/+/−/− 
                 High 
               
               
                 NEA20 
                 
                   N. coenophialum 
                 
                 +/+/−/− 
                 Not tested 
               
               
                 AR542* 
                 
                   N. coenophialum 
                 
                 +/+/−/− 
                 — 
               
               
                   
               
               
                 *Control commercial endophyte 
               
             
          
         
       
     
         [0479]    A genotypic analysis of the novel fescue endophytes NEA23 and NEA21 is shown in  FIG. 68 . 
       EXAMPLE 18 
     Overview of Generation of Novel Designer  Neotyphodium  Endophyte Variant Strains Through Mutagenesis 
       [0480]    The objective of this work was to create novel variants of the perennial ryegrass endophyte,  Neotyphodium lolii , through induced polyploidisation and mutagenesis, with desirable properties such as enhanced bioactivities (e.g. antifungal acitivity), and/or altered plant colonization ability and stability of grass host-endophyte variant associations (e.g. altered in vitro growth), and/or altered growth performance (e.g. enhanced plant vigour, enhanced drought tolerance, enhanced water use efficiency) of corresponding grass host—endophyte variant associations. These grass host-endophyte variant associations are referred to as novel ‘designer’ grass-endophyte associations. 
       Experimental Strategies for the Generation and Characterisation of Novel Designer  Neotyphodium  Endophyte Variant Strains Through Mutagenesis 
       [0481]    The experimental activities thus included: 
         [0000]    1. Establishment of phenotypic screens for novel ‘designer’ grass-endophyte associations such as:
       Enhanced biotic stress tolerance   Enhanced drought tolerance and enhanced water use efficiency   Enhanced plant vigour
 
2. Targeted generation (i.e. polyploidisation and X-ray mutagenesis) and characterisation (i.e. antifungal bioassays, in vitro growth rate, genome survey sequencing [GSS]) of novel ‘designer’ endophytes
 
3. Breeding of ‘designer’ grass-endophyte associations
   Delivery of ‘designer’ endophytes into grass (e.g. perennial ryegrass) germplasm development process.       
 
       EXAMPLE 19 
     Establishment of Phenotypic Screens for Novel ‘Designer’ Grass-Endophyte Associations 
       [0486]    Assessment of enhanced biotic stress tolerance using NEA12 is shown in  FIGS. 71 and 72 .  FIG. 71  shows in vitro bioassays to assess antifungal activity of  Neotyphodium  endophytes.  FIG. 72  shows a detached leaf assay to assess resistance to crown rust ( Puccinia coronata  f.sp.  lolii ). 
         [0487]    Assessment of enhanced drought tolerance and enhanced water use efficiency is shown in  FIG. 73 . This involved glasshouse and field trial screens for drought tolerance, survival and recovery, regrowth after drought, metabolic profiling and detailed phenotypic characterisation including multiple trait dissection (based on assessments and measurements associated with plant morphology, plant physiology, plant biochemistry). 
       EXAMPLE 20 
     Generation of Designer  N. lolii  Genotypes by Polyploidisation 
       [0488]    This involved creation of novel variation in  Neotyphodium  endophytes without the use of transgenic technology. Colchicine has been widely and successfully used for chromosome doubling in plants, e.g. perennial ryegrass. It inhibits chromosome segregation during mitosis inducing autopolyploidisation (chromosome doubling; see  FIG. 74 ). This enables the generation of novel endophytes through induced chromosome doubling and may be applicable to the production of artificial polyploid endophytes. 
         [0489]    The experimental work flow for chromosome doubling is shown in  FIG. 75 . 
         [0490]    Flow cytometry calibrations to assess DNA content in  Neotyphodium  endophytes are shown in  FIG. 76 . Peaks indicate relative nuclear DNA content. 
         [0491]    Flow cytometry analysis of NEA12 dh  strains is shown in  FIG. 77  and Table 39. 
         [0492]    1. ST endophyte strain is highly stable, broadly compatible and produces lolitrems, peramine and ergovaline. 2. NEA12 endophyte strain produces janthitrem only. 3. AR1 produces peramine only. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 39 
               
             
             
               
                   
               
               
                 Colchicine treated endophyte strains (ST, NEA12 and AR1 endophyte 
               
               
                 strains) subjected to colchicine treatments (at different colchicine 
               
               
                 concentrations in %) leading to the recovery of endophyte colonies 
               
               
                 (# of colonies) used for flow cytometry analysis 
               
             
          
           
               
                   
                   
                 Colchicine 
                 # of 
                 # colonies 
               
               
                   
                 Endophyte 
                 treatment (%) 
                 colonies 
                 analysed 
               
               
                   
                   
               
             
          
           
               
                   
                   N. lolii  ST 
                 0.2 
                 12 
                 12 
               
               
                   
                   N. lolii  NEA12 
                 0.1 
                 60 
                 2 
               
               
                   
                   N. lolii  NEA12 
                 0.2 
                 60 
                 18 
               
               
                   
                   N. lolii  AR1 
                 0.1 
                 60 
                 0 
               
               
                   
                   N. lolii  AR1 
                 0.2 
                 60 
                 0 
               
               
                   
                   
               
             
          
         
       
     
       EXAMPLE 21 
     Analysis of In Vitro Growth of NEA12 dh    Neotyphodium  Variant Endophyte Strains 
       [0493]    Analysis of growth rate of NEA12 dh    Neotyphodium  variant endophyte strains in in vitro culture after 8 weeks is shown in  FIG. 78 . In an initial screen, analysis of variance identified two NEA12 dh    Neotyphodium  variant endophyte strains (NEA12 dh17  and NEA12 dh4 ) showing significantly different in vitro growth rate to the control NEA12 endophyte: 
         [0000]    NEA12 dh17  grows significantly faster (p&lt;0.01**)
 
NEA12 dh4  grows significantly slower (p&lt;0.05*)
 
         [0494]    Analysis of growth rate of NEA12 dh    Neotyphodium  variant endophyte strains in in vitro culture over 5 weeks is shown in  FIG. 10 . In a validation screen, Student&#39;s t-tests identified two NEA12 dh    Neotyphodium  variant endophyte strains (NEA12 dh17  and NEA12 dh15 ) showing significantly different in vitro growth rate to the control NEA12 endophyte: 
         [0000]    NEA12 dh17  grows significantly faster (p&lt;0.01**)
 
NEA12 dh15  grows significantly slower (p&lt;0.01**)
 
       EXAMPLE 22 
     Antifungal Bioassays of NEA12 dh    Neotyphodium  Variant Endophyte Strains 
       [0495]    A list of fungal pathogens (causing a range of fungal diseases and infecting a range of different plant hosts) that were included in antifungal bioassays used to analyse NEA12 dh    Neotyphodium  variant endophyte strains to assess their spectrum of antifungal activities is shown in Table 40. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 40 
               
             
             
               
                   
               
               
                 Fungal pathogens (causing a range of fungal diseases and infecting 
               
               
                 a range of different plant hosts) included in antifungal bioassays to analyse 
               
               
                 NEA12 dh    Neotyphodium  variant endophyte strains to assess their 
               
               
                 spectrum of antifungal activities 
               
             
          
           
               
                 Fungus 
                 Disease 
                 Hosts 
               
               
                   
               
               
                 
                   Alternaria 
                 
                 leaf spot, rot, 
                 Numerous (dead plant 
               
               
                 
                   alternata 
                 
                 blight 
                 materials) 
               
               
                 
                   Bipolaris 
                 
                 Damping-off 
                 Asteraceae (daisies), 
               
               
                 
                   portulacae 
                 
                   
                 Portulacaceae (purslane) 
               
               
                 
                   Botrytis 
                 
                 Stem rot, mould, 
                 Many dicots, few monocots 
               
               
                 
                   cinerea 
                 
                 seedling wilt 
               
               
                 
                   Colletotrichum 
                 
                 Leaf spot, stalk rot 
                 Poaceae 
               
               
                 
                   graminicola 
                 
                   
                 (especially  Zea mays ) 
               
               
                 
                   Drechslera 
                 
                 Leaf blight 
                 Poaceae 
               
               
                 
                   brizae 
                 
                   
                 ( Briza  spp.) 
               
               
                 
                   Phoma 
                 
                 Spot (leaf, glume, seed), 
                 Poaceae (grasses) 
               
               
                 
                   sorghina 
                 
                 Root rot, Dying-off 
               
               
                 
                   Rhizoctonia 
                 
                 Spot (wheat) 
                 Poaceae (grasses) 
               
               
                 
                   cerealis 
                 
                 Yellow patch (turfgrass) 
               
               
                 
                   Trichoderma 
                 
                 Green mould, 
                 Many dicots, few 
               
               
                 
                   harzianum 
                 
                 Parasite of other fugni 
                 monocots, Fungi 
               
               
                   
               
             
          
         
       
     
         [0496]    Antifungal bioassays of NEA12 dh    Neotyphodium  variant endophyte strains are shown in  FIGS. 80 and 81 . Twenty NEA12 dh  strains were screened for changes in antifungal activity. Four NEA12 dh  strains (i.e. dh5, dh6, dh13 and dh14) were identified as having greater antifungal activity compared to NEA12. 
       EXAMPLE 23 
     Genome Survey Sequencing and Sequence Analysis of NEA12 dh    Neotyphodium  variant endophyte strains 
       [0497]    NEA12 dh    Neotyphodium  variant endophyte strains with enhanced antifungal activity, showing faster in vitro growth rate and higher DNA content were subjected to genome survey sequencing (GSS). Sequence data was generated for 10 NEA12 dh  strains and control NEA12 strain (highlighted in blue on Table 41). 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 41 
               
             
             
               
                   
               
               
                 List of NEA12 dh    Neotyphodium  variant endophyte strains showing 
               
               
                 different antifungal activity [higher than control or equal 
               
               
                 to control (standard, Std)] and different in vitro growth 
               
               
                 [slower than control, faster than conrol or equal to control 
               
               
                 (standard, Std)] compared to control NEA12 strain 
               
             
          
           
               
                   
                 Endophyte 
                 Antifungal 
                 Growth 
               
               
                   
                   
               
               
                   
                 NEA12 
                 Std 
                 Std 
               
               
                   
                 NEA12dh1 
                 Std 
                 Std 
               
               
                   
                 NEA12dh2 
                 Std 
                 Std 
               
               
                   
                 NEA12dh3 
                 Std 
                 Std 
               
               
                   
                 NEA12dh4 
                 Std 
                 Slower 
               
               
                   
                 NEA12dh5 
                 Higher 
                 Std 
               
               
                   
                 NEA12dh6 
                 Higher 
                 Std 
               
               
                   
                 NEA12dh7 
                 Std 
                 Std 
               
               
                   
                 NEA12dh8 
                 Std 
                 Std 
               
               
                   
                 NEA12dh9 
                 Std 
                 Std 
               
               
                   
                 NEA12dh10 
                 Std 
                 Std 
               
               
                   
                 NEA12dh11 
                 Std 
                 Std 
               
               
                   
                 NEA12dh12 
                 Std 
                 Std 
               
               
                   
                 NEA12dh13 
                 Higher 
                 Std 
               
               
                   
                 NEA12dh14 
                 Higher 
                 Std 
               
               
                   
                 NEA12dh15 
                 Std 
                 Slower 
               
               
                   
                 NEA12dh16 
                 Std 
                 Std 
               
               
                   
                 NEA12dh17 
                 Std 
                 Faster 
               
               
                   
                 NEA12dh18 
                 Std 
                 Std 
               
               
                   
                 NEA12dh19 
                 Std 
                 Std 
               
               
                   
                 NEA12dh20 
                 Std 
                 Std 
               
               
                   
                   
               
             
          
         
       
     
         [0498]    Genome survey sequencing (GSS) data obtained for NEA12 dh    Neotyphodium  variant endophyte strains derived from colchicine treated NEA12 control strain (highlighted in blue on Table 41) were analysed as follows:
       De-novo assembly of the GSS data from NEA12 control strain—to act as a reference genome sequence for the analysis of the NEA12 dh    Neotyphodium  variant endophyte strains   Map the GSS data sequence reads from the NEA12 dh    Neotyphodium  variant endophyte strains to the NEA12 reference genome sequence   Identify potentially duplicated regions, i.e. regions with higher than expected sequence coverage   Identify gene sequences that may have been duplicated       
 
         [0503]    Analysis of GSS read depth of NEA12 dh    Neotyphodium  variant endophyte strains is shown in  FIG. 82 . Analysis of sequence contigs that appeared to have higher than expected read depth indicates that no major duplication event has occurred (excepting whole genome events). The patterns of read depth across these contigs are not identical between strains. This suggests there are differences between the NEA12 dh    Neotyphodium  variant endophyte strains and the control NEA12 strain. 
         [0504]    Analysis of GSS sequence assemblies for the NEA12 dh    Neotyphodium  variant endophyte strains and the control NEA12 strain is shown in Table 42. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 42 
               
             
             
               
                   
               
               
                 Analysis of GSS sequence assemblies for the NEA12 dh    Neotyphodium   
               
               
                 variant endophyte strains and the control NEA12 strain 
               
             
          
           
               
                 Strain 
                 # contigs 
                 N50 
                 Max contig 
                 # bases 
               
               
                   
               
               
                 NEA12 
                 143202 
                 28621 
                 181461 
                 32734984 
               
               
                 NEA12dh5 
                 305031 
                 29444 
                 191191 
                 30994592 
               
               
                 NEA12dh17 
                 274394 
                 37802 
                 209957 
                 30777017 
               
               
                 NEA12dh18 
                 282692 
                 30717 
                 177813 
                 30889903 
               
               
                   
               
             
          
         
       
     
         [0505]    Independent de novo sequence assemblies were performed using parameters identical to those used in assembling the genome sequence for the control NEA12 endophyte strain. Differences in sequence assembly statistics may indicate genomic differences between strains. GSS data obtained for the NEA12 dh    Neotyphodium  variant endophyte strains and used in the sequence assemblies reveal fewer bases incorporated into the sequence assembly and produce more sequence contigs. Increased numbers of smaller sequence contigs may be caused by transposon movement/replication. 
         [0506]    Analysis of sequence reads mapping to the NEA12 genome sequence assembly is shown in  FIG. 83 . While we do not wish to be restricted by theory, if the genomes were the same no difference in the number of sequence reads mapping to the reference genome sequence would be expected. NEA12 dh    Neotyphodium  variant endophyte strains range from 35-70% sequence reads mapping to NEA12 sequence contigs &gt;5 kb in size. There are differences between the genome sequences of the NEA12 dh    Neotyphodium  variant endophyte strains and the control NEA12 strain. 
       Summary of Results on Generation and Characterisation of Novel Designer  Neotyphodium  Variant Endophyte Strains Through Colchicine Treatment Based Mutagenesis 
       [0507]    Sequence read depth changes were analysed in NEA12 dh    Neotyphodium  variant endophyte strains compared with the control NEA12 strain. Whilst no large partial genome sequence duplication events were detected, the occurrence of full genome duplication events in the NEA12 dh    Neotyphodium  variant endophyte strains cannot be excluded based on the GSS sequence analysis. 
         [0508]    De novo sequence assemblies were independently performed on GSS data obtained from the NEA12 dh    Neotyphodium  variant endophyte strains. Differences in sequence assembly statistics indicate that genomic changes were caused by the colchicine-treatment in the NEA12 dh    Neotyphodium  variant endophyte strains. The number of sequence reads from NEA12 dh    Neotyphodium  variant endophyte strains mapping to the NEA12 reference genome sequence varies between strains. All GSS data analyses performed on the NEA12 dh    Neotyphodium  variant endophyte strains indicate genomic differences. 
         [0509]    In summary, the following novel designer endophytes were generated by colchicine treatment of NEA12 endophytes:
       Four NEA12 dh    Neotyphodium  variant endophyte strains (dh5, dh6, dh13 and dh14) with enhanced bioprotective properties (i.e. antifungal bioactivities);   One NEA12 dh    Neotyphodium  variant endophyte strain (dh17) with higher in vitro growth rate than control NEA12 strain (i.e. potentially with enhanced stability/host colonization ability);   Ten NEA12 dh    Neotyphodium  variant endophyte strains (including dh5, dh6, dh13, dh14 and dh17) and control NEA12 strain subjected to genome survey sequencing; and   Five NEA12 dh    Neotyphodium  variant endophyte strains (including dh5, dh13 and dh17) selected and subjected to isogenic inoculation in planta.       
 
       EXAMPLE 24 
     In Planta Isogenic Inoculation in Perennial Ryegrass with NEA12 dh    Neotyphodium  Variant Endophyte Strains 
       [0514]    The following NEA12 dh    Neotyphodium  variant endophyte strains and control NEA12 strain were used for in planta isogenic inoculation in perennial ryegrass:
       NEA12   NEA12dh5 showing higher antifungal activity than control NEA12   NEA12dh13 showing higher antifungal activity than control NEA12   NEA12dh4 showing slower in vitro growth rate than control NEA12   NEA12dh15 showing slower in vitro growth rate than control NEA12   NEA12dh17 showing faster in vitro growth rate than control NEA12       
 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 43 
               
             
             
               
                   
               
               
                 Isogenic inoculation of perennial ryegrass genotypes (IMP04 and TOL03) with 
               
               
                 NEA12dh  Neotyphodium  variant endophyte strains. Numbers indicate number of 
               
               
                 perennial ryegrass plants of the two genotypes subjected to isogenic inoculation 
               
               
                 with the different NEA12dh  Neotyphodium  variant endophyte strains. 
               
             
          
           
               
                 Plant Genotype 
                 NEA12dh4 
                 NEA12dh5 
                 NEA12dh13 
                 NEA12dh15 
                 NEA12dh17 
                 NEA12 
               
               
                   
               
               
                 IMP04 
                 30 
                 30 
                 30 
                 30 
                 32 
                 30 
               
               
                 TOL03 
                 25 
                 30 
                 30 
                 20 
                 30 
                 20 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 25 
     Generation of Designer  N. lolii  Genotypes by X-Ray Mutagenesis 
       [0521]    The generation of designer  Neotyphodium  endophytes genotypes by X-ray mutagenesis offers the opportunity to create novel endophyte variant strains with enhanced properties, such as enhanced stability in grass hosts, broader host compatibility as well as improved toxin profiles e.g. following elimination of the production of the detrimental alkaloid lolitrem B in the highly stable and broadly compatible ST endophyte. 
         [0522]    Such an novel designer endophyte would be advantageous over existing commercial endophytes, such as AR1 and AR37, as it would be highly stable and broadly compatible and with optimal toxin profile. 
         [0523]      FIG. 84  shows an experimental work flow for X-ray mutagenesis of endophyte strains. 
         [0524]      FIG. 85  shows the indole-diterpene biosynthetic pathway. Lolitrem B is the major toxin that causes ryegrass staggers, a disease of grazing animals. Ten genes in 3 gene clusters are required for lolitrem biosynthesis. We focused initial analysis on 3 Ltm genes, one from each gene cluster. Optimised multiplex PCR analysis was designed and implemented. 
       EXAMPLE 26 
     Screening of X-Ray Irradiated  N. lolii  Strains 
       [0525]    In a preliminary primary screen &gt;5,000 colonies of X-ray irradiated  N. lolii —established as an initial resource of novel variation of  N. lolii  endoophytes induced through X-ray mutagenesis and representing a mutagenised  N. lolii  endophyte strain collection—of were screened by multiplex PCR analysis for the presence of targeted Ltm genes leading to a preliminary identification of ˜140 putative lolitrem B gene cluster PCR-negative colonies (˜2.5% of 5,000 colonies screened). In a secondary screen high quality DNA was extracted (140 liquid cultures) and PCR analysis conducted. This identified 2 putative deletion mutants for one of the lolitrem B genes (ltm J). 
         [0000]    
       
         
               
             
           
               
                 TABLE 44 
               
               
                   
               
               
                 Putative X-ray irradiation-induced ltm gene deletion mutants of  
               
               
                   N. lolii  derived from irradiation with 30 Gy dose. 
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 The colony number represents the unique identifier of the putative X-ray irradiation-induced ltm gene deletion mutant (i.e. 139-6 and 145-15). Black represents PCR-negative result for respective ltm gene analysis, white represents PCR-positive result for respective ltm gene analysis. 
               
             
          
         
       
     
       EXAMPLE 27 
     Antifungal Bioassays of Designer X-Ray Irradiated  N. lolii  Variant Strains 
       [0526]    There were eight X-ray irradiated  N. lolii  variant strains (i.e. X-ray mutagenesis derived variant strains 1-35, 4-7, 7-22, 7-47, 123-20, 124-6, 139-6, 144-16 and 145-15) and one control  N. lolii  strain (i.e. ST endophyte strain). 
         [0527]    Five fungal pathogens (causing a range of fungal diseases and infecting a range of different plant hosts) were included in antifungal bioassays used to analyse the X-ray irradiated  N. lolii  variant strains, as follows:
         Bipolaris portulacae        Colletotrichum graminicola        Drechslera brizae        Phoma sorghina        Rhizoctonia cerealis          
 
         [0533]    No significant difference in antifungal activities of X-ray irradiated  N. lolii  variant strains tested was observed compared to the spectrum of antifungal activities observed for the control ST endophyte strain. 
       EXAMPLE 28 
     In Vitro Growth of Designer X-Ray Irradiated  N. lolii  Variant Strains 
       [0534]    Results from the analysis of in vitro growth rate of designer X-ray irradiated  N. lolii  variant strains are shown in  FIG. 86 , with a statistical analysis of in vitro growth undertaken at week 5 for the X-irradiated  N. lolii  variant strains compared to the control ST strain, revealing significant differences in in vitro growth rates as follows: 
         [0000]    p&lt;0.05* (for X-irradiated  N. lolii  variant strain 139-6)
 
p&lt;0.01** (for all other mutants)
 
       EXAMPLE 29 
     Genome Survey Sequencing of Designer X-Ray Irradiated  N. lolii  Variant Strains 
       [0535]    Eight X-ray irradiated  N. lolii  ST variant strains and corresponding control ST strain were subjected to genome survey sequencing (GSS), leading to 46-fold to 79-fold genome sequence coverage for the different strains as shown in Table 45. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 45 
               
             
             
               
                   
               
               
                 Genome sequence coverage obtained in genome survey sequencing 
               
               
                 for for 8 X-ray irradiated  N. lolii  ST variant strains 
               
               
                 and corresponding control ST strain 
               
             
          
           
               
                   
                 Strain 
                 Description 
                 Coverage 
               
               
                   
                   
               
               
                   
                 ST 
                 ST 
                 23x 
               
               
                   
                 139-6 
                 ST irradiated 
                 61x 
               
               
                   
                 145-15 
                 ST irradiated 
                 52x 
               
               
                   
                 144-16 
                 ST irradiated 
                 46x 
               
               
                   
                 1_35 
                 ST irradiated 
                 79x 
               
               
                   
                 4_7 
                 ST irradiated 
                 46x 
               
               
                   
                 7_22 
                 ST irradiated 
                 53x 
               
               
                   
                 7_47 
                 ST irradiated 
                 38x 
               
               
                   
                 123-20 
                 ST irradiated 
                 54x 
               
               
                   
                 124-6 
                 ST irradiated 
                 75x 
               
               
                   
                   
               
             
          
         
       
     
       EXAMPLE 30 
     Detecting Genome Sequence Variation in Designer X-Ray Irradiated  N. lolii  Variant Strains 
       [0536]    Results from the analysis to detect genome sequence variation in X-ray irradiated  N. lolii  variant strains are shown in  FIG. 88 . Corresponding results on the detection of single nucleotide polymorphisms (SNPs) are shown in  FIG. 89  and results on the detection of small insertions/deletions (INDELs) are shown in  FIG. 90 . Differences in sequence read depth and pair insert size in X-ray irradiated  N. lolii  variant deletion mutant strains are shown in  FIG. 91 . 
         [0537]    Results on sequence analysis for Ltm gene clusters are shown in  FIG. 87 . No deletions, large or small, were found in the coding or regulatory sequences of ltm gene clusters. No SNPs, insertions or translocations were found in the coding or regulatory sequences of ltm gene clusters. 
       EXAMPLE 31 
     Spectrum of Genome Sequence Changes Detected in the X-Ray Irradiated  N. lolii  variant strains 
       [0538]      FIG. 92  shows numbers of SNPs detected in genic regions of X-ray irradiated  N. lolii  variant deletion mutant strains. There are large differences in the number of SNPs detected in the X-ray irradiated  N. lolii  variant deletion mutant strains and compared to the control ST strain. All X-ray irradiated  N. lolii  variant deletion mutant strains have over double the number of SNPs per Mb across genic regions compared to the control ST strain. X-ray irradiated  N. lolii  variant deletion mutant strains have on average 6 SNPs per Mb, where the control ST strain has 2 SNPs per Mb. 
         [0539]      FIG. 93  shows numbers of INDELs in genic regions of X-ray irradiated  N. lolii  variant deletion mutant strains. All X-ray irradiated  N. lolii  variant deletion mutant strains contain more indels in genic regions than the control ST strain. The difference in indel numbers between the X-ray irradiated  N. lolii  variant deletion mutant strains and the control ST strain is on average 134 indels per Mb. When grouped by irradiation treatment (i.e. irradiation dose applied and number of repeat irradiations) there appears to be a peak in number of indels at 10Gy*2 treatment, consistent with the results obtained in the SNP detection analysis. 
         [0540]      FIG. 94  shows the spectrum of genome sequence changes in the form of deletions detected in X-ray irradiated  N. lolii  variant deletion mutant strains. 
         [0541]    Table 46 shows examples of some of these genome sequence deletions detected in X-ray irradiated  N. lolii  variant deletion mutant strains. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 46 
               
             
             
               
                   
               
               
                 Deletions detected in genome sequences of X-ray irradiated 
               
               
                   N. lolii  variant deletion mutant strains. Bold indicates 
               
               
                 deletions confirmed by changes in sequence read coverage. 
               
               
                 The remainder are potential transposon deletions. 
               
             
          
           
               
                   
                 Radiation 
                   
               
               
                 Strain 
                 Treatment 
                 Deletion 
               
               
                   
               
               
                 123_20 
                 30Gy*2 
                 Contig00915 (268 bp) 
               
               
                 124_6 
                 30Gy*2 
                 Partial duplication 
               
               
                 139_6 
                 30Gy 
                 Partial duplication 
               
               
                 144_16 
                 30Gy 
               
               
                 145_15 
                 30Gy 
                 Partial duplication 
               
               
                 1_35 
                 10Gy 
                   Contig00831  ( 3.6 kb ) 
               
               
                 4_7 
                 10Gy 
               
               
                 7_22 
                 10Gy*2 
               
               
                 7_47 
                 10Gy*2 
                   Contig01131  ( 0.6 kb ),  contig01082   
               
               
                   
                   
                 ( 4.2 kb ),  contig02985  ( 1 kb ), 
               
               
                   
                   
                 contig02725 (83 bp), contig01095 (130 bp) 
               
               
                   
               
             
          
         
       
     
         [0542]    The X-ray irradiated  N. lolii  variant deletion mutant strain #7 — 47, which was generated following two X-irradiation treatments at 10 Gy dose (10Gy*2) of  N. lolii  ST endophyte, had the greatest number of large deletions. 
       EXAMPLE 32 
     Annotation of Deleted Sequences in the Genomes of X-Ray Irradiated  N. lolii  Variant Deletion Mutant Strains 
     X-Ray Irradiated  N. lolii  Variant Mutant Strain 1 — 35: 
       [0543]    For the X-ray irradiated  N. lolii  variant mutant strain 1 — 35 the following deleted sequences in ST454Contig00831 contig with a ˜4,400-8,000 bp length was detected, with this genome sequence region containing the following two predicted genes: 
         [0544]    ST454contig00831_AUGUSTUS_gene — 3318:6018 (847 letters) 
         [0000]    1) ref |XP — 386347.1| hypothetical protein FG06171.1  [Gibberella  660×0.0 gb|EAW12630.1| DUF500 domain protein [ Aspergillus  NRRL 1]; 253×9e-66, and ST454contig00831_AUGUSTUS_gene — 3958:4728 (183 letters); and
 
2) gb|EAW13545.1| 2,3-cyclic-nucleotide 2-phosphodiesterase [ Aspergillus  32×2.4
 
X-Ray Irradiated  N. lolii  Variant Mutant Strain 747:
 
         [0545]    For the X-ray irradiated  N. lolii  variant mutant strain 7 — 47 the following deleted sequences in ST454Contig01082, ST454Contig01131 and ST454Contig02985, with these genome sequence regions containing no predicted genes: 
         [0000]    Query=ST454contig01082 length=9120 numreads=287
 
gb|AAA21442.1| putative pol polyprotein [ Magnaporthe grisea]  145 1e-32
 
Query=ST454contig02985 length=2414 numreads=99
 
gb|AAA21442.1| putative pol polyprotein [ Magnaporthe grisea]  92 2e-17
 
       EXAMPLE 33 
     Mutagenesis Index of X-Ray Irradiated  N. lolii  Variant Deletion Mutant Strains 
       [0546]      FIG. 95  shows SNPs and Indels per Mb in genic regions of X-ray irradiated  N. lolii  variant deletion mutant strains derived from X-ray irradiation of  N. lolii  at different levels of irradiation. Strain 1 — 35 has a 3.6 kb deletion; Strain 7 — 47 has 3 deletions (4.2 kb, 1 kb, 0.6 kb in length). Strain 124 — 6 has a partial duplication. Strains 139 — 6 and 145 — 15 have partial duplications. 
         [0547]    Given that ST endophyte has approximately 443.5 genes per Mb, using 10Gy*2 treatment, the expected rate of SNP/INDEL occurrence is 0.33 per gene in the genome. 
       Summary 
       [0548]    X-ray irradiated  N. lolii  variant deletion mutant strains were analysed for many types of genome sequence variation i.e. deletions, SNPs, INDELs, inversions and translocations. SNPs, INDELs, deletions and duplications were identified in the genome survey sequences of X-ray irradiated  N. lolii  variant deletion mutant strains. There was an apparent peak in number of SNPs and INDELs in X-ray irradiated  N. lolii  variant deletion mutant strains recovered from administering 10Gy*2 X-ray irradiation treatment to  N. lolii  ST endophyte. The X-ray irradiated  N. lolii  variant deletion mutant strain 7 — 47 had 3 large deletions. It was demonstrated that this mutagenesis method based on X-ray irradiation can be used to create novel designer  Neotyphodium  endophyte strains, and enabled:
       5,000 X-ray irradiated  N. lolii  variant endophyte strains derived from X-ray irradiation of ST  N. lolii  endophyte were screened;   140 putative X-ray irradiated  N. lolii  variant endophyte mutant strains were identified;   9 X-ray irradiated  N. lolii  variant endophyte mutant strains were subjected to antifungal bioassays;   9 X-ray X-ray irradiated  N. lolii  variant endophyte mutant strains were subjected to in vitro growth assays;   9 X-ray irradiated  N. lolii  variant endophyte mutant strains were subjected to genome survey sequencing;   2 X-ray irradiated  N. lolii  variant endophyte mutant strains with gene deletions (1 — 35 and 7 — 47) were identified; and   3 X-ray irradiated  N. lolii  variant endophyte mutant strains with gene duplications (124 — 6, 139 — 6 and 145 — 15) were identified.       
 
       EXAMPLE 34 
     In Planta Isogenic Inoculation in Perennial Ryegrass with X-Ray Irradiated  N. lolii  Variant Endophyte Mutant Strains 
       [0556]      
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 47 
               
             
             
               
                   
               
               
                 Isogenic inoculation of perennial ryegrass genotypes (IMP04 and 
               
               
                 TOL03) with X-ray irradiated  N. lolii  variant endophyte mutant 
               
               
                 strains. Numbers indicate number of perennial ryegrass plants of the 
               
               
                 two genotypes subjected to isogenic inoculation with the different 
               
               
                 X-ray irradiated  N. lolii  variant endophyte mutant strains (i.e. 
               
               
                 ST-IRM 139-6, ST-IRM 145-15, ST-IRM 144-16, ST-IRM 1-35 and ST- 
               
               
                 IRM 7-47) and control ST endophyte strain. 
               
             
          
           
               
                   
                 ST-IRM 
                 ST-IRM 
                 ST-IRM 
                 ST-IRM 
                 ST-IRM 
                   
               
               
                 Plant Genotype 
                 139-6 
                 145-15 
                 144-16 
                 1-35 
                 7-47 
                 ST 
               
               
                   
               
             
          
           
               
                 IMP04 
                 30 
                 25 
                 30 
                 30 
                 30 
                 25 
               
               
                 TOL03 
                 25 
                 0 
                 25 
                 30 
                 30 
                 20 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 35 
     Metabolic Profiling of Colchicine Treatment-Derived NEA12dh and X-Ray Irradiation-Derived  Neotyphodium  Variant Endophyte Strains 
       [0557]    Results from metabolic profiling of colchicine treatment derived NEA12dh endophyte variant strains is shown in  FIG. 96 . 
         [0558]    Results from metabolic profiling of X-ray irradiation treatment derived  N. lolii  ST endophyte variant strains is shown in  FIG. 97 . 
         [0559]    The following endophytes were grown on PDB for 3 weeks:
       Control  N. lolii  ST endophyte strain   X-ray irradiation treatment derived  N. lolii  ST endophyte variant strain 4-7   X-ray irradiation treatment derived  N. lolii  ST endophyte variant strain 139-6   X-ray irradiation treatment derived  N. lolii  ST endophyte variant strain 144-16   X-ray irradiation treatment derived  N. lolii  ST endophyte variant strain 145-15
 
and subjected to metabolic profiling using LCMS on corresponding
   1. Liquid filtrate   2. Mycelial extract       
 
         [0567]    The X-ray irradiation treatment derived  N. lolii  ST endophyte variant strains could be readily distinguished from control  N. lolii  ST strain using mycelia extracts or filtrates alone. 
         [0568]    It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. 
       REFERENCES 
       [0000]    
       
         Bouton, J. H., G. C. M. Latch, N. S. Hill, C. S. Hoveland, M. A. McCann, R. H. Watson, J. A. Parish, L. L. Hawkins and F. N. Thompson (2002) Agronomy Journal 94(3): 567 574. 
         Latch, G. C. M, Christensen, M. J, Tapper, B. A, Easton, H. S, Hume, D. E, Fletcher, L. R. (2000) U.S. Pat. No. 6,111,170 and references therein. 
         Li, X and Zhang, Y., (2002) Comparative and Functional Genomics 3: 158-160. 
         Tapper, B. A, Cooper, B. M, Easton, H. S, Fletcher, L. R, Hume, D. E, Lane, G. A, Latch, G. C. M, Pennell, C. G. L, Popay, A. J, Christensen, M. J. (2004) International Patent Application No. WO 2004/106487 and references therein. 
         Van Zijll de Jong E, Guthridge K M, Spangenberg G C, Forster J W (2003) Genome 46 (2): 277-290 
         Young, C. A., Bryant, M. K., Christensen, M. J., Tapper, B. A., Bryan, G. T., Scott, B. (2005) Molecular Genetics and Genomics, 274: 13-39.