AU2013203584A1 - Designer endophytes - Google Patents

Abstract

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 5 selecting, breeding, characterising and/or modifying endophytes. More particularly, the present invention provides 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.

Description

P/00/001 Regulation 3.2 AUSTRALIA Patents Act 1990 CO P 0MVL E TE SP ECI F IC(;A T IO)N STANDARD PATENT Invention title: DESIGNER ENDOPHYTES The following statement is a full description of this invention, including the best method of performing it known to us: -2 DESIGNER ENDOPHYTES This application is an application for a patent of addition to Australian Patent Application No. 2011204749, the entire disclosure of which is incorporated herein by reference. 5 Field of the Invention 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. 10 Background of the Invention Important forage grasses perennial ryegrass and tall fescue are commonly found in association with fungal endophytes. Both beneficial and detrimental agronomic properties result from the association, including improved tolerance to water and nutrient stress and resistance to insect 15 pests. 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. 20 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. Molecular genetic markers such as simple sequence repeat (SSR) markers have been developed as diagnostic tests to distinguish between endophyte taxa and 25 detect genetic variation within taxa. The markers may be used to discriminate -.3 endophyte strains with different toxin profiles. 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. 5 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. Difficulties in artificially breeding of these endophytes limit their usefulness. For example, many of the novel endophytes known to be beneficial to pasture-based 10 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. International patent application PCT/AU2011/000020 describes a method for identifying or characterising endophyte strains which involves subjecting multiple 15 samples of endophytes to genetic and metabolic analyses, and optionally also assessing geographic origin. The application also identifies a number of endophytes which were isolated by this method, including El, NEA10, NEA11, NEA12, NEA13 and NEA1 4. However, there remains a need for more endophyte strains with desirable 20 properties and for more detailed characterisation of their toxin and metabolic profiles, antifungal activity, stable host associations and their genomes. 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. A large scale endophyte discovery program was undertaken to establish a 'library' 25 of novel endophyte strains. A collection of perennial ryegrass and tall fescue accessions was established.

-4 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 and are described in a co pending Australian provisional patent application filed 1 June 2012 entitled 'Novel 5 Endophytes. 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. 10 Novel 'designer' endophytes were generated by targeted methods including polyploidisation and X-ray mutagenesis. These endophytes may be characterised, for example using antifungal bioassays, in vitro growth rate assays and/or genome survey sequencing (GSS). Metabolic profiling may also be undertaken to determine the toxin profile of these 15 strains grown in vitro and/or following inoculation in pianta. These endophytes may be delivered into plant germplasm to breed 'designer' grass endophyte associations. 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 20 example, grass plants, varieties and cultivars. 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. 25 By 'genetic analysis' is meant analysing the nuclear and/or mitochondrial DNA of the endophyte.

-5 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 5 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 10 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. SSRs are highly polymorphic due to variation in repeat number and are co dominantly inherited. Their detection is based on the polymerase chain reaction 15 (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 20 selection. Known SSR markers which may be used to investigate endophyte diversity in perennial ryegrass are described in van Zijll de Jong et al (2003) Genome 46 (2): 277-290. Alternatively, or in addition, the genetic analysis may involve sequencing genomic 25 and/or mitochondrial DNA and performing sequence comparisons to assess genetic variation between endophytes. The endophytes may be subject to metabolic analysis to identify the presence of desired metabolic traits.

-6 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 5 the endophytes and measurement of toxin levels in planta. By a 'desired genetic and metabolic profile' is meant that the endophyte possesses genetic and/or metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, the endophyte. Such beneficial properties include improved tolerance to water and/or nutrient 10 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. 15 For example, tolerance to water, drought, nutrient and/or biotic 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 20 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 standard toxic (ST) endophyte or to a no endophyte control plant. Such beneficial properties also include reduced toxicity of the associated plant to 25 grazing animals. 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. 5 In a preferred embodiment toxicity may be reduced to a negligible amount or substantially zero toxicity. 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 10 preferably at least approximately 100%, relative to a control endophyte such as ST endophyte 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 endophyte or to a no endophyte control plant. 15 In a first 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 20 ionising radiation, for example from a caesium source. 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 Neotyphodiun, more preferably it is from a species selected from the group consisting of N uncinatum, N coenophialum and N lolii, 25 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.

-8 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 or Lolitrem B, compared with a plant inoculated with a control endophyte such as standard toxic (ST) endophyte; and/or significantly 5 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. 10 For example, toxic alkaloids may be present in an amount less than approximately 1 pg/g dry weight, for example between approximately 1 and 0.001 pg/g dry weight, preferably less than approximately 0.5 pg/g dry weight, for example between approximately 0.5 and 0.001 pg/g dry weight, more preferably less than approximately 0.2 pg/g dry weight, for example between approximately 0.2 and 15 0.001 pg/g dry weight. In a particularly preferred embodiment the endophyte may not produce Lolitrem B toxins. For example, said alkaloids conferring beneficial properties may be present in an amount of between approximately 5 and 100 pg/g dry weight, preferably between 20 approximately 10 and 50 pg/g dry weight, more preferably between approximately 15 and 30 pg/g dry weight. 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 25 Measurement Institute on 3 April 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.

-9 In a preferred embodiment, the endophyte may be substantially purified. 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 5 approximately 95% pure, even more preferably at least approximately 98% pure. The term 'isolated' means that the endophyte is removed from its original environment (e.g. 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 10 natural system, is isolated. On the basis of the deposits referred to above, the entire genome of an endophyte selected from the group consisting of NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14, and NEA12dh17, is incorporated herein by reference. Thus, in a further aspect, the present invention includes identifying and/or cloning 15 nucleic acids including genes encoding polypeptides or transcription factors from said genome. 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 20 for genes of the desired type; 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, and thus identifying genes encoding such polypeptides or transcription factors. 25 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.

-10 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 5 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. 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 10 of the present invention. 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 15 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. 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 20 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 25 at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes. Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides.

-11 Preferably, said fragments are able to produce the same activity as the original gene when expressed. Preferably, said fragments maintain conserved regions within consensus sequences of the original gene. Preferably said variants are variants of the original sequences that provide either 5 conserved substitution, or limited modifications in consensus sequences to a level, for example, of no more than approximately 5%, more preferably no more than 1 %, relative to the original gene. For example, fragments and variants of a sequence encoding X may include a wild type sequence from species Z that encodes X, a fragment of a wild type sequence 10 wherein the fragment encodes X, and that retains conserved regions within consensus sequences from species Z, and variants of the wild type sequence or fragments which encode X activity and have only conservative substitutions, a variant X' that encodes X activity and in which sequence differs only by substitutions found in one or more contributing sequences used in formulating the 15 consensus sequence, or a variant X" that encodes X activity in which the variant has not more than approximately 95% amino acid variation, more preferably not more than approximately 99% amino acid variation from the wild type sequence or fragment. By 'conservative nucleic acid changes' or 'conserved substitution' is meant nucleic 20 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. 25 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, lie, Pro, Met, Phe, Trp Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gin -12 Acidic: Asp, Glu Basic: Lys, Arg, His Other conservative amino acid substitutions may also be made as follows: Aromatic: Phe, Tyr, His 5 Proton Donor: Asn, GIn, Lys, Arg, His, Trp Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gin In a further aspect of the present invention, there is provided a genetic construct including a nucleic acid according to the present invention. By 'genetic construct' is meant a recombinant nucleic acid molecule. 10 In a preferred embodiment, the genetic construct according to the present invention may be a vector. By a 'vector' is meant a genetic construct used to transfer genetic material to a target cell. The vector may be of any suitable type and may be viral or non-viral. The vector 15 may be an expression vector. Such vectors include chromosomal, non chromosomal and synthetic nucleic acid sequences, e.g. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacteriun tunefaciens; derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary 20 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. 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 25 being operatively linked.

-13 By a 'promoter' is meant a nucleic acid sequence sufficient to direct transcription of an operatively linked nucleic acid sequence. 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 5 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. By 'upstream' is meant in the 3'->5' direction along the nucleic acid. The promoter and terminator may be of any suitable type and may be endogenous 10 to the target cell or may be exogenous, provided that they are functional in the target cell. 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 15 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. 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 20 combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns, antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt/l) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes [such as beta 25 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.

-14 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 5 the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites. Preferably, the genetic construct is substantially purified or isolated. 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 10 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 15 endonuclease digestion) independent of other sequences. It also includes a genetic construct which is part of a hybrid gene encoding additional polypeptide sequence. 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. 20 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 25 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. 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 -15 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. 5 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 AgrobacteriIm-mediated introduction, Rhizobium 10 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 15 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. 20 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 25 asexually, using methods well known in the art, to produce successive generations of transformed plants or fungi. In a further aspect, the present invention provides a plant inoculated with an endophyte variant as hereinbefore described, said plant comprising an endophyte free host plant stably infected with said endophyte variant.

-16 Preferably, the plant is infected with the endophyte variant by a method selected from the group consisting of inoculation, breeding, crossing, hybridization and combinations thereof. In a preferred embodiment, the plant may be infected by isogenic inoculation. This 5 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. The identification of an endophyte of the opposite mating-type that is highly 10 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. 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 15 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 20 favourable alkaloid toxin profile. 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. 25 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 -17 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. 5 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. By 'isogenic plants' is meant that the plants are genetically identical. The endophyte-infected plants may be cultured by known techniques. The person 10 skilled in the art can readily determine appropriate culture conditions depending on the plant to be cultured. 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 15 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. 20 In a particularly preferred embodiment, the endophyte variant may be selected from the group consisting of NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14, and NEA12dh17. In another particularly preferred embodiment, LCMS including EIC (extracted ion chromatogram) analysis may allow detection of the alkaloid metabolites from small 25 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 -18 the same conditions and/or by comparison of mass fragmentation patterns, for example generated by MS2 analysis in a linear ion trap mass spectrometer. 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 5 endophyte variant of the present invention. 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. By 'plant cell' is meant any self-propagating cell bounded by a semi-permeable 10 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. In a further aspect, the present invention provides use of an endophyte variant as 15 hereinbefore described to produce a plant stably infected with said endophyte variant. 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. 20 In a preferred embodiment, the method may be performed using an electronic device, such as a computer. 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 25 such as an endophyte gene, for example the peramine biosynthesis perA gene. The development of a high-throughput PCR-based assay to measure endophyte -19 biomass in planta may enable efficient screening of large numbers of plants to study endophyte-host plant biomass associations. As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are 5 not intended to exclude further additives, components, integers or steps. 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 10 relevant by a person skilled in the art. Detailed Description of the Embodiments In the figures: Figure 1 shows the structures of Lolitrem B, Erogvaline and Peramine, with desirable toxin profiles indicated. 15 Figure 2 shows in vitro bioassays to assess antifungal activity of Neotyphodium endophytes. Figure 3 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. 20 Figure 4 shows glasshouse and field trial screens for drought tolerance and water use efficiency of perennial ryegrass plants with and without Neotyphodium endophytes. Figure 5 shows the steps involved in cell division. Figure 6 shows experimental work flow for chromosome doubling of endophyte -20 cells. Figure 7 shows flow cytometry calibrations for DNA content assessment in Neotyphodium endophyte strains. Peaks indicate relative nuclear DNA content. Figure 8 shows flow cytometry analysis of NEA12dI Neotyphodium endophyte 5 strains. Figure 9 shows analysis of growth rate in culture after 8 weeks of NEA12dh Neotyphodiun endophyte strains compared to control endophyte strains. Figure 10 shows analysis of growth rate in culture over 5 weeks of NEA12dh Neotyphodium endophyte strains compared to control endophyte strains. 10 Figure 11 shows antifungal bioassays of NEA12h Neotyphodium endophyte strains. Figure 12 shows antifungal bioassays of NEA12h Neotyphodium endophyte strains. Figure 13 shows analysis of genome survey sequencing read depth of colchicine 15 treated Neotyphodium endophyte strains. Figure 14 shows analysis of genome survey sequencing reads mapping to NEA12 genome survey sequence assembly. Figure 15 shows experimental work flow for X-ray mutagenesis. Figure 16 shows the indole-diterpene biosynthetic pathway of Neotyphodium 20 endophytes. Figure 17 shows in vitro growth of X-ray irradiated Neotyphodium endophyte strains.

-21 Figure 18 shows Itm gene clusters of Neotyphodium endophytes. Figure 19 shows determination of genome sequence variation in X-ray irradiated Neotyphodium endophyte strains. Figure 20 shows single nucleotide polymorphisms (SNPs) in genome sequences of 5 X-ray irradiated Neotyphodium endophyte strains. Figure 21 shows small insertions/deletions (INDELs) in genome sequences of X-ray irradiated Neotyphodium endophyte strains. Figure 22 shows deletions in genome sequences of X-ray irradiated Neotyphodium endophyte strains. 10 Figure 23 shows numbers of SNPs in genic regions of genome sequences of X-ray irradiated Neotyphodium endophyte strains. Figure 24 shows numbers of INDELs in genic regions of genome sequences of X ray irradiated Neotyphodium endophyte strains. Figure 25 shows the spectrum of genome sequence changes (deletions) in genome 15 sequences of X-ray irradiated Neotyphodium endophyte strains. Figure 26 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. Figure 27 shows metabolic profiling of NEA12dh Neotyphodium endophyte strains. 20 Figure 28 shows metabolic profiling of X-ray irradiated Neotyphodiun endophyte strains. The invention will now be described with reference to the following non-limiting examples.

- 22 Example 1 - Overview of generation of novel designer Neotyphodium endophyte variant strains through mutagenesis The objective of this work was to create novel variants of the perennial ryegrass endophyte, Neotyphodium lolii, through induced polyploidisation and mutagenesis, 5 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. 10 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 The experimental activities thus included: 15 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 20 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) 25 germplasm development process.

-23 Example 2 - Establishment of phenotypic screens for novel 'designer' grass endophyte associations Assessment of enhanced biotic stress tolerance using NEA12 is shown in Figures 2 and 3. Figure 2 shows in vitro bioassays to assess antifungal activity of 5 Neotyphodium endophytes. Figure 3 shows a detached leaf assay to assess resistance to crown rust (Puccinia coronata f.sp. 1olii). Assessment of enhanced drought tolerance and enhanced water use efficiency is shown in Figure 4. This involved glasshouse and field trial screens for drought tolerance, survival and recovery, regrowth after drought, metabolic profiling and 10 detailed phenotypic characterisation including multiple trait dissection (based on assessments and measurements associated with plant morphology, plant physiology, plant biochemistry). Example 3 - Generation of designer N. lol genotypes by polyploidisation This involved creation of novel variation in Neotyphodium endophytes without the 15 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 Figure 5). This enables the generation of novel endophytes through induced chromosome doubling and may be applicable to the production of artificial polyploid 20 endophytes. The experimental work flow for chromosome doubling is shown in Figure 6. Flow cytometry calibrations to assess DNA content in Neotyphodium endophytes are shown in Figure 7. Peaks indicate relative nuclear DNA content. Flow cytometry analysis of NEA12oh strains is shown in Figure 8 and Table 1. 25 1. ST endophyte strain is highly stable, broadly compatible and produces lolitrems, peramine and ergovaline. 2. NEA12 endophyte strain produces janthitrem only. 3.

-24 AR1 produces peramine only. Niolli ST 0.2 12 12 N.iolii NEA12 0.1 60 2 M/ofii NEA 12 j0,2 60 18_______ NJo/ii AR1 0.1 60 0 NoIii AR1 0.2 60 0 Table 1: Colchicine treated endophyte strains (ST, NEA12 and AR1 endophyte strains) subjected to colchicine treatments (at different colchicine 5 concentrations in %) leading to the recovery of endophyte colonies (# of colonies) used for flow cytometry analysis Example 4 - Analysis of in vitro growth of NEA12& Neotyphodium variant endophyte strains Analysis of growth rate of NEA12dh Neotyphodium variant endophyte strains in in 10 vitro culture after 8 weeks is shown in Figure 9. In an initial screen, analysis of variance identified two NEA12dh Neotyphodium variant endophyte strains (NEA12dhl and NEA12 dh 4 ) showing significantly different in vitro growth rate to the control NEA12 endophyte: NEA1 2 "h17 grows significantly faster (p<0.01**) 15 NEA12dh4 grows significantly slower (p<0.05*) Analysis of growth rate of NEA12dh Neotyphodium variant endophyte strains in in vitro culture over 5 weeks is shown in Figure 10. In a validation screen, Student's t tests identified two NEA12dh Neotyphodium variant endophyte strains (NEA12dhl7 and NEA12")hl5) showing significantly different in vitro growth rate to the control 20 NEA12 endophyte: NEA1 2d11 7 grows significantly faster (p<0.01**) NEA12dhI 5 grows significantly slower (p<0.01**) -25 Example 5 - Antifungal bioassays of NEA12 & Neotyphodium variant endophyte strains 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 5 NEA12 d Neotyphodium variant endophyte strains to assess their spectrum of antifungal activities is shown in Table 2. Fungus Disease Hosts Alternaria alternata leaf spot, rot, blight Numerous (dead plant materials) Bipolaris portulacae Damping-off Asteraceae (daisies), Portulacaceae (purslane) Botrytis cinerea Stem rot, mould, seedling Many dicots, few monocots wi It Colletotrichum Leaf spot, stalk rot Poaceae (especially Zea graminicola mays) Drechslera brizae Leaf blight Poaceae (Briza spp.) Phoma sorghina Spot (leaf, glume, seed), Poaceae (grasses) Root rot, Dying-off Rhizoctonia cerealis Spot (wheat) Poaceae (grasses) Yellow patch (turfgrass) Trichoderma Green mould, Many dicots, few monocots, harzianum Parasite of other fugni Fungi Table 2: Fungal pathogens (causing a range of fungal diseases and infecting a range of different plant hosts) included in antifungal bioassays to analyse NEA1 2 dh Neotyphodium variant endophyte strains to assess their spectrum of 10 antifungal activities Antifungal bioassays of NEA12 d Neotyphodium variant endophyte strains are shown in Figures 11 and 12. Twenty NEA1 2 dh strains were screened for changes in antifungal activity. Four NEA1201 strains (i.e. dh5, dh6, dh13 and dh14) were - 26 identified as having greater antifungal activity compared to NEA12. Example 6 - Genome survey sequencing and sequence analysis of NEA12dh Neotyphodium variant endophyte strains NEA1 2 dh Neotyphodium variant endophyte strains with enhanced antifungal activity, 5 showing faster in vitro growth rate and higher DNA content were subjected to genorne survey sequencing (GSS). Sequence data was generated for 10 NEA12 dh strains and control NEA12 strain (highlighted in blue on Table 3). Endophyte Antifungal Growth NEA12 Std Std NEA12dhi 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 NEA1 2dh11I 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 Table 3: List of NEA12 dh Neotyphodiurn variant endophyte strains showing 10 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 Genomne survey sequencing (GSS) data obtained for NEA12 d Neotyphodium variant endophyte strains derived from colchicine treated NEA12 control strain -27 (highlighted in blue on Table 3) 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 NEA12dh Neotyphodium variant endophyte strains 5 - Map the GSS data sequence reads from the NEA12 d 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 10 Analysis of GSS read depth of NEA12)h Neotyphodium variant endophyte strains is shown in Figure 13. 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 15 NEA12d" Neotyphodium variant endophyte strains and the control NEA12 strain. Analysis of GSS sequence assemblies for the NEA12h Neotyphodium variant endophyte strains and the control NEA12 strain is shown in Table 4. 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 Table 4: Analysis of GSS sequence assemblies for the NEA1 2 dh Neotyphodium variant endophyte strains and the control NEA12 strain 20 Independent de novo sequence assemblies were performed using parameters -28 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 NEA12dh Neotyphodium variant endophyte strains and used in the sequence assemblies 5 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. Analysis of sequence reads mapping to the NEA12 genome sequence assembly is shown in Figure 14. While we do not wish to be restricted by theory, if the genomes 10 were the same no difference in the number of sequence reads mapping to the reference genome sequence would be expected. NEA12 d" Neotyphodium variant endophyte strains range from 35-70% sequence reads mapping to NEA12 sequence contigs > 5kb in size. There are differences between the genome sequences of the NEA12d& Neotyphodium variant endophyte strains and the control 15 NEA12 strain. Summary of results on generation and characterisation of novel designer Neotyphodium variant endophyte strains through colchicine treatment based mutagenesis Sequence read depth changes were analysed in NEA12 d" Neotyphodium variant 20 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 NEA12h Neotyphodium variant endophyte strains cannot be excluded based on the GSS sequence analysis. De novo sequence assemblies were independently performed on GSS data 25 obtained from the NEA12dh Neotyphodium variant endophyte strains. Differences in sequence assembly statistics indicate that genomic changes were caused by the colchicine-treatment in the NEA12d& Neotyphodium variant endophyte strains. The number of sequence reads from NEA12dh Neotyphodium variant endophyte strains mapping to the NEA12 reference genome sequence varies between strains. All -29 GSS data analyses performed on the NEA12 dh Neotyphodium variant endophyte strains indicate genomic differences. In summary, the following novel designer endophytes were generated by colchicine treatment of NEA12 endophytes: 5 - Four NEA1 2 dh Neotyphodium variant endophyte strains (dh5, dh6, dh13 and dh14) with enhanced bioprotective properties (i.e. antifungal bioactivities); - One NEA12 h Neotyphodium variant endophyte strain (dh17) with higher in vitro growth rate than control NEA12 strain (i.e. potentially with enhanced stability/host colonization ability); 10 - Ten NEA12dh Neotyphodium variant endophyte strains (including dh5, dh6, dhl3, dhl4 and dhl7) and control NEA12 strain subjected to genome survey sequencing; and - Five NEA12dh Neotyphodium variant endophyte strains (including dh5, dh13 and dhl 7) selected and subjected to isogenic inoculation in planta. 15 Example 7 - In planta isogenic inoculation in perennial ryegrass with NEA12 d Neotyphodium variant endophyte strains The following NEA1 2 dh Neotyphodium variant endophyte strains and control NEA12 strain were used for in planta isogenic inoculation in perennial ryegrass: 20 - 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 25 - NEA12dh17 showing faster in vitro growth rate than control NEA1 2 - 30 Plant Genotype NEA12 NEA12 NEA12 NEA12 NEA12 NEA12 dh4 dh5 dh13 dh15 dh17 IMPO4 30 30 30 30 32 30 TOLO3 25 30 30 20 30 20 Table 5: Isogenic inoculation of perennial ryegrass genotypes (IMPO4 and TOLO3) 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 5 strains. Example 8 - Generation of designer N lolli genotypes by X-ray mutagenesis 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 10 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. Such an novel designer endophyte would be advantageous over existing commercial endophytes, such as AR1 and AR37, as it would be highly stable and 15 broadly compatible and with optimal toxin profile. Figure 15 shows an experimental work flow for X-ray mutagenesis of endophyte strains. Figure 16 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 20 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.

- 31 Example 9 - Screening of X-ray irradiated N. lol strains In a preliminary primary screen >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. lo/ii endophyte 5 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 10 lolitrem B genes (Itm J). Table 6: Putative X-ray irradiation-induced /tm gene deletion mutants of N. ll derived from irradiation with 30 Gy dose. The colony number represents the unique identifier of the putative X-ray irradiation-induced itm gene deletion mutant 15 (i.e. 139-6 and 145-15). Black represents FOR-negative result for respective itm gene analysis, grey represents FOR-positive result for respective /tm gene analysis. Example 10 - Antifungal bioassays of designer X-ray irradiated N. llivariant strains There were eight X-ray irradiated N. loli variant strains (i.e. X-ray mutagenesis 20 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. loli strain (i.e. ST endophyte strain). 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. loli variant strains, as follows: 25 -Bipolaris portulacae - 32 - Colletotrichum graminicola - Drechslera brizae - Phoma sorghina - Rhizoctonia cerealis 5 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 11 - In vitro growth of designer X-ray irradiated N. lolii variant strains 10 Results from the analysis of in vitro growth rate of designer X-ray irradiated N. lolii variant strains are shown in Figure 17, 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: p< 0.05* (for X-irradiated N. lolii variant strain 139-6) 15 p<0.01** (for all other mutants) Example 12 - Genome survey sequencing of designer X-ray irradiated N. lolli variant strains 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 20 genome sequence coverage for the different strains as shown in Table 7.

- 33 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 Table 7: Genome sequence coverage obtained in genome survey sequencing for for 8 X-ray irradiated N. loli ST variant strains and corresponding control ST strain 5 Example 13 - Detecting genome sequence variation in designer X-ray irradiated N. ilolivariant strains Results from the analysis to detect genome sequence variation in X-ray irradiated N. lolii variant strains are shown in Figure 19. Corresponding results on the detection of single nucleotide polymorphisms (SNPs) are shown in Figure 20 and 10 results on the detection of small insertions/deletions (INDELs) are shown in Figure 21. Differences in sequence read depth and pair insert size in X-ray irradiated N. lo/ii variant deletion mutant strains are shown in Figure 22. Results on sequence analysis for Ltm gene clusters are shown in Figure 18. No deletions, large or small, were found in the coding or regulatory sequences of /tm 15 gene clusters. No SNPs, insertions or translocations were found in the coding or regulatory sequences of tm gene clusters.

- 34 Example 14 - Spectrum of genome sequence changes detected in the X-ray irradiated N. loii variant strains Figure 23 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 5 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. lo/ii 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 10 per Mb. Figure 24 shows numbers of INDELs in genic regions of X-ray irradiated N. loli 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. lo/ii variant deletion 15 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 1OGy*2 treatment, consistent with the results obtained in the SNP detection analysis. Figure 25 shows the spectrum of genome sequence changes in the form of 20 deletions detected in X-ray irradiated N. lolli variant deletion mutant strains. Table 8 shows examples of some of these genome sequence deletions detected in X-ray irradiated N. lolii variant deletion mutant strains.

-35 Radiation Strain Treatment Deletion 123_20 30Gy*2 Contig00915 (268bp) 124_6 30Gy*2 Partial duplication 139_6 30Gy Partial duplication 144_16 30Gy 145_15 30Gy Partial duplication 1_35 1OGy Contig00831 (3.6kb) 47 1OGy 7_22 10Gy*2 ContigO1131 (0.6kb), contigO1082 (4.2kb), contig02985 7_47 1OGy*2 (1kb), contig02725 (83bp), contigOl 095 (130bp) Table 8: Deletions detected in genome sequences of X-ray irradiated N. loll variant deletion mutant strains. Bold indicates deletions confirmed by changes in sequence read coverage. The remainder are potential transposon deletions. 5 The X-ray irradiated N. loii variant deletion mutant strain # 7_47, which was generated following two X-irradiation treatments at 10 Gy dose (lOGy*2) of N. lolii ST endophyte, had the greatest number of large deletions. Example 15 - Annotation of deleted sequences in the genomes of X-ray irradiated N. ll variant deletion mutant strains 10 X-Ray Irradiated N. lo/ii Variant Mutant Strain 1_35: 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: 15 ST454contig00831_AUGUSTUS_gene_3318:6018 (847 letters) 1) ref XP_386347.1| hypothetical protein FG06171.1 [Gibberella 660x0.0 gblEAW12630.1| DUF500 domain protein [Aspergillus NRRL 1]; 253 x 9e-66, and - 36 ST454contig00831_AUGUSTUS_gene_3958:4728 (183 letters); and 2) gblEAW13545.11 2,3-cyclic-nucleotide 2-phosphodiesterase [Aspergillus 32 x 2.4 X-Ray Irradiated N. lolii Variant Mutant Strain 7_47: 5 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: Query= ST454contig01082 length=9120 numreads=287 gblAAA21442.1| putative pol polyprotein [Magnaporthe grisea] 145 1e-32 10 Query= ST454contigO2985 length=2414 numreads=99 gblAAA21442.11 putative pol polyprotein [Magnaporthe grisea] 92 2e-1 7 Example 16 - Mutagenesis index of X-ray irradiated N. ilol variant deletion mutant strains Figure 26 shows SNPs and Indels per Mb in genic regions of X-ray irradiated N. lolii 15 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 lenght). Strain 124_6 has a partial duplication. Strains 139_6 and 145_15 have partial duplications. Given that ST endophyte has approximately 443.5 genes per Mb, using 10Gy*2 20 treatment, the expected rate of SNP/INDEL occurrence is 0.33 per gene in the genome. Summary 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 25 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. loili 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 - 37 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 - 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. loli variant endophyte mutant strains were identified; - 9 X-ray irradiated N. lo/ii variant endophyte mutant strains were subjected to 10 antifungal bioassays; - 9 X-ray X-ray irradiated N. lo/ii 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; 15 - 2 X-ray irradiated N. lolii variant endophyte mutant strains with gene deletions (135 and 7_47) were identified; and - 3 X-ray irradiated N. lolii variant endophyte mutant strains with gene duplications (1246, 139_6 and 14515) were identified. Example 17 - In planta isogenic inoculation in perennial ryegrass with X-ray 20 irradiated N. 1olii variant endophyte mutant strains Plant Genotype ST-IRM ST-IRM ST-IRM ST-IRM ST-IRM ST 139-6 145-15 144-16 1-35 7-47 IMPO4 30 25 30 30 30 25 TOLO3 25 0 25 30 30 20 Table 9: Isogenic inoculation of perennial ryegrass genotypes (IMPO4 and -38 TOLO3) with X-ray irradiated N. oilli 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 5 IRM 1-35 and ST-IRM 7-47) and control ST endophyte strain. Example 18 - Metabolic profiling of colchicine treatment-derived NEA12dh and X-ray irradiation-derived Neotyphodium variant endophyte strains Results from metabolic profiling of colchicine treatment derived NEA12dh endophyte variant strains is shown in Figure 27. 10 Results from metabolic profiling of X-ray irradiation treatment derived N. lolii ST endophyte variant strains is shown in Figure 28. 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 15 - 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. loli ST endophyte variant strain 145-15 and subjected to metabolic profiling using LCMS on corresponding 1. Liquid filtrate 20 2. Mycelial extract The X-ray irradiation treatment derived N. lo/ii ST endophyte variant strains could be readily distinguished from control N. lo/ii ST strain using mycelia extracts or filtrates alone.

Claims (18)

1. An endophyte variant having a desired genetic and metabolic profile, wherein said endophyte variant possesses genetic and/or metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, 5 the endophyte variant.

2. An endophyte variant according to claim 1, wherein said beneficial phenotype is selected from the group consisting of 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, 10 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.

3. An endophyte variant according to claim 1 or 2, wherein said endophyte variant is generated by polyploidisation or induced chromosome doubling. 15

4. An endophyte variant according to claim 3, wherein said endophyte variant is generated by treating an endophyte with colchicine or a similar compound that induces polyploidisation or induced chromosome doubling.

5. An endophyte variant according to claim 1 or 2, wherein said endophyte variant is generated by subjecting an endophyte to X-ray mutagenesis or exposing 20 an endophyte to ionising radiation.

6. An endophyte variant according to any one of claims 1 to 5, wherein said endophyte variant is generated from an endophyte which is isolated from a Lolium species.

7. An endophyte according to claim 6, wherein said Lolium species is Loliun 25 perenne. -40

8. An endophyte variant according to any one of claims 1 to 6, wherein said endophyte variant is generated from an endophyte of the genus Neotyphodium

9. An endophyte variant according to claim 8, wherein said endophyte variant is generated from an endophyte of a species selected from the group consisting of N 5 uncinatun, N coenophialum and N. lolii.

10. An endophyte according to claim 9, wherein said species is N. lolii.

11. An endophyte variant according to any one of claims 1 to 10, wherein said endophyte variant has a desired toxin profile, wherein the endophyte variant produces significantly less toxic alkaloids and/or significantly more alkaloids 10 conferring beneficial properties, in the plant with which the endophyte is associated, when compared with a control endophyte such as standard toxic (ST) or with a no endophyte control plant.

12. An endophyte variant according to claim 11, wherein said toxic alkaloids are present in an amount less than approximately 1pg/g dry weight. 15

13. An endophyte variant according to claim 11 or 12, wherein said alkaloids conferring beneficial properties are present in an amount of between approximately 5 and 100 pg/g dry weight.

14. An endophyte variant according to any one of claims 1 to 13, selected from the group consisting of NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14, and 20 NEA12dh17.

15. A plant inoculated with an endophyte variant according to any one of claims 1 to 14, said plant comprising an endophyte-free host plant stably infected with said endophyte variant.

16. A plant, plant seed or other plant part derived from a plant according to claim 25 15 and stably infected with an endophyte variant according to any one of claims 1 to 14. -41

17. Use of an endophyte variant according to any one of claims 1 to 14 to produce a plant stably infected with said endophyte variant.

18. An endophyte variant according to claim 1, substantially as hereinbefore described with reference to any one of the figures or examples.