AU2016259448B2

AU2016259448B2 - Novel endophytes (2)

Info

Publication number: AU2016259448B2
Application number: AU2016259448A
Authority: AU
Inventors: Piyumi Ekanayake; John White Forster; Kathryn Michaela Guthridge; Jatinder Kaur; Emma Jane Isobel Ludlow; Maia Andrea Rabinovich; Simone Jane Rochfort; Timothy Ivor Sawbridge; German Carlos Spangenberg
Original assignee: Agriculture Victoria Services Pty Ltd
Current assignee: Agriculture Victoria Services Pty Ltd
Priority date: 2011-01-07
Filing date: 2016-11-18
Publication date: 2018-11-08
Anticipated expiration: 2033-04-10
Also published as: AU2021225232B2; AU2019200890A1; AU2013203581A1; NZ611224A; AU2021225232A1; AU2019200890B2; AU2016259448A1

Abstract

The present invention relates to endophytic fungi (endophytes) and nucleic acids thereof, to plants infected with endophytes and to related methods, including methods of selecting, breeding and/or characterising endophytes. More 5 particularly, the present invention provides novel endophytes having a desired toxin profile, wherein the endophyte produces significantly less toxic alkaloids compared with a control endophyte; and/or significantly more alkaloids conferring beneficial properties compared with a control endophyte.

Description

The present invention relates to endophytic fungi (endophytes) and nucleic acids thereof, to plants infected with endophytes and to related methods, including methods of selecting, breeding and/or characterising endophytes. More particularly, the present invention provides novel endophytes having a desired toxin profile, wherein the endophyte produces significantly less toxic alkaloids compared with a control endophyte; and/or significantly more alkaloids conferring beneficial properties compared with a control endophyte.

P/00/001 Regulation 3.2

2016259448 18 Nov 2016

AUSTRALIA

Patents Act 1990

COMPLETE SPECIFICATION

STANDARD PATENT

Invention title: NOVEL ENDOPHYTES (2)

The following statement is a full description of this invention, including the best method of performing it known to us:

2016259448 18 Nov 2016

-2NOVEL ENDOPHYTES (2)

This application is a divisional of Australian patent application no. 2013203581 which is an application for a patent of addition to Australian Patent Application No. 2011204749, the entire disclosures of which are incorporated herein by reference.

Field of the Invention

The present invention relates to endophytic fungi (endophytes) and nucleic acids thereof, to plants infected with endophytes and to related methods, including methods of selecting, breeding and/or characterising endophytes.

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 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.

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 detect genetic variation within taxa. The markers may be used to discriminate endophyte strains with different toxin profiles.

-32016259448 18 Nov 2016

However, there remains a need for methods of identifying, isolating and/or characterising endophytes and a need for new endophyte strains having desired properties.

Neotyphodium endophytes are not only of interest in agriculture, as they are a 5 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 agriculture exhibit low inoculation frequencies and are less stable in elite germplasm. Thus, there remains a need for methods of molecular breeding of novel, highly compatible endophytes.

International patent application PCT/AU2011/000020 describes a method for identifying or characterising endophyte strains which involves subjecting multiple 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 E1, NEA10, NEA11, NEA12, NEA13and NEA14.

However, there 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.

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’ of novel endophyte strains. A collection of perennial ryegrass and tall fescue accessions was established.

Genetic analysis of endophytes in these accessions has led to the identification of a

-42016259448 18 Nov 2016 number of novel endophyte strains. These novel endophyte strains are genetically distinct from known endophyte strains.

Metabolic profiling may be undertaken to determine the toxin profile of these strains grown in vitro and/or following inoculation in planta.

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.

The endophytes may be subject to genetic analysis (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.

By ‘genetic analysis’ is meant analysing the nuclear and/or mitochondrial DNA of the endophyte.

This analysis may involve detecting the presence or absence of polymorphic 15 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.

SSRs are highly polymorphic due to variation in repeat number and are codominantly inherited. Their detection is based on the polymerase chain reaction

-52016259448 18 Nov 2016 (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.

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 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.

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.

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 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

-62016259448 18 Nov 2016 endophyte control plant.

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.

Such beneficial properties also include reduced toxicity of the associated plant to 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.

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 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

2016259448 18 Nov 2016

-7 control endophyte such as ST or to a no endophyte control plant.

In a first 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. The endophyte may be of the genus

Neotyphodium, for example from a species selected from the group consisting of N. uncinatum, N. coenophialum and 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 FaTG10 2 and FaTG-2 like.

In a preferred embodiment, the present invention provides a substantially purified or isolated endophyte having a desired toxin profile, wherein the endophyte produces significantly less toxic alkaloids compared with a control endophyte; and/or significantly more alkaloids conferring beneficial properties compared with a control endophyte, wherein said control endophyte is standard toxic endophyte; and wherein said endophyte is selected from the group consisting of NEA16, NEA17, NEA18, NEA19, NEA20, NEA21 and NEA23, as deposited at the National Measurement Institute with accession numbers V12/001413, V12/001414, V12/001415, V12/001416, V12/001417, V12/001418 and V12/001419, respectively.

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 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, Nacetylloline and norloline, again when compared with a plant inoculated with a control endophyte such as ST or with a no endophyte control plant.

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,

2016259448 08 Oct 2018

-8preferably 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 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 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 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 April 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.

In another particularly preferred embodiment, the present invention provides a substantially purified or isolated FaTG-3 species endophyte having a desired toxin profile, wherein the endophyte produces significantly less toxic alkaloids compared with a control endophyte; and/or significantly more alkaloids conferring beneficial properties compared with a control endophyte, wherein said control endophyte is standard toxic endophyte; and wherein said endophyte is selected from the group consisting of NEA21 and NEA23, as deposited at the National Measurement

Institute with accession numbers V12/001418 and V12/001419, respectively.

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

2016259448 08 Oct 2018

-8aleast approximately 95% pure, even more preferably at least approximately 98% pure.

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.

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On the basis of the deposits referred to above, the entire genome of an endophyte selected from the group consisting of NEA21, NEA23, NEA18, NEA19, NEA16 and NEA20, is incorporated herein by reference.

Thus, in a further aspect, the present invention includes identifying and/or cloning 5 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 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.

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.

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 doublestranded, 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 of the present invention.

-102016259448 18 Nov 2016

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. 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.

Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides.

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 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 wherein the fragment encodes X, and that retains conserved regions within consensus sequences from species Z, and variants of the wild type sequence or

-11 2016259448 18 Nov 2016 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 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 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.

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

Acidic: Asp, Glu Basic: Lys, Arg, His

Other conservative amino acid substitutions may also be made as follows:

Aromatic: Phe, Tyr, His

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.

-12 2016259448 18 Nov 2016

By ‘genetic construct’ is meant a recombinant nucleic acid molecule.

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 5 target cell.

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, nonchromosomal 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.

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.

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 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.

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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.

A variety of terminators which may be employed in the genetic constructs of the 5 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.

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, antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (nptll) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes [such as betaglucuronidase (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.

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.

Preferably, the genetic construct is substantially purified or isolated.

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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.

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.

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.

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.

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

-152016259448 18 Nov 2016 art. Such techniques include Agrobacterium-medi\aied introduction, Rhizobiummediated 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-med\ated transformation of hyphal explants are particularly preferred.

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.

In a further aspect, the present invention provides a plant inoculated with an endophyte as hereinbefore described, said plant comprising an endophyte-free host plant stably infected with said endophyte.

Preferably, the plant is infected with the endophyte 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 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.

-162016259448 18 Nov 2016

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 hyperinoculation.

Hyphal fusion between endophyte strains of the opposite mating-type provides a means for delivery of favourable traits into the host plant, preferably via hyperinoculation. 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.

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.

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.

-17 2016259448 18 Nov 2016

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 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 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.

In a particularly preferred embodiment, the endophytes may be selected from the group consisting of NEA21, NEA23, NEA18, NEA19, NEA16 and NEA20.

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.

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 of the present invention.

-182016259448 18 Nov 2016

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 5 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 as hereinbefore described to produce a plant stably infected with said endophyte.

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.

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 such as an endophyte gene, for example the peramine biosynthesis perA gene.

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.

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.

Reference to any prior art in the specification is not, and should not be taken as, an

-192016259448 18 Nov 2016 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

In the figures:

Figure 1 shows genotypic analysis of endophyte content in accessions from a targeted fescue germplasm collection.

Figure 2 shows genetic diversity analysis of tall fescue endophytes.

Figure 3 shows diversity analysis of host and endophyte.

Figure 4 shows selection of fescue-endophyte combinations for metabolic profiling, endophyte isolation and isogenic inoculation.

Figure 5 shows selection of fescue-endophyte combinations for metabolic profiling, endophyte isolation and isogenic inoculation.

Figure 6 shows a desired toxin profile of tall fescue endophytes.

Figure 7 shows a metabolic profile analysis.

Figure 8 shows endophytes selected for semi-quantitative analysis of metabolites.

Figures 9 and 10 show metabolomics analyses of fescue endophytes.

Figure 11 shows a semi-quantitative analysis of metabolic profile under 20 temperature/water stress.

Figure 12 shows endophytes selected for isogenic inoculation.

-202016259448 18 Nov 2016

Figure 13 shows SSR-based genotyping of isolated endophytes cultures prior to isogenic inoculation.

Figure 14 shows endophyte vegetative stability in tall fescue and perennial ryegrass host genotypes (stability at 12 months post inoculation).

Figure 15 shows endophytes selected for isogenic inoculation.

Figures 16-19 show metabolic profiling of isogenic tall fescue-endophyte associations.

Figure 20 shows anti-fungal bioassays of fescue endophytes. Column 1 Colletotrichum graminicola, Column 2 Drechslera brizae, Column 3 Rhizoctonia cerealis.

Figure 21 shows sequencing of selected novel fescue endophytes.

Figure 22 shows peramine biosynthetic pathway.

Figures 23 A-C show presence of perA gene within non-Epichloe out-group endophytes (Fig 23A NEA17; Fig 23B NEA18; Fig 23C NEA19).

Figure 24 shows ergovaline biosynthetic pathway.

Figure 25 shows genes in the eas gene cluster.

Figures 26 A-D show presence of dmaW gene for ergovaline biosynthesis in endophyte strains (Fig 26A NEA17; Fig 26B NEA16; Fig 26C AR542; Fig 26D

NEA20).

Figures 27 A-D show presence of eas gene cluster for ergovaline biosynthesis. Fig 27A FaTG-2 NEA17 (287819); Fig 27B non-Epichloe out-group NEA18 (FEtc6-75); Fig 27C FATG-3 NEA21 (231557); Fig 27D N. coenophialum NEA16 (FEtc7-342).

-21 2016259448 18 Nov 2016

Figure 28 shows the Lolitrem B biosynthetic pathway.

Figure 29 shows genes in the Lolitrem B biosynthetic gene cluster.

Figures 30 A-D show presence of Lolitrem B biosynthetic gene cluster 1 (ItmG, ItmM and ItmK) in endophyte strains. Fig 30A FaTG-2 NEA17 (287819); Fig 30B non-Epichloe out-group NEA18 (FEtc6-75); Fig 30C FATG-3 NEA21 (231557); Fig 30D N. coenophialum NEA16 (FEtc7-342).

Figures 31 A-D show presence of Lolitrem B biosynthetic gene cluster 2 (ItmB, ItmQ, ItmP, ItmF and ItmC) in endophyte strains. Fig 31A FaTG-2 NEA17 (287819); Fig 31B non-Epichloe out-group NEA18 (FEtc6-75); Fig 31C FATG-3 NEA21 (231557); Fig 31D N. coenophialum NEA16 (FEtc7-342).

Figures 32 A-D show presence of Lolitrem B biosynthetic gene cluster 3 (ItmE and ItmJ) in endophyte strains. Fig 32A FaTG-2 NEA17 (287819); Fig 32B nonEpichloe out-group NEA18 (FEtc6-75); Fig 32C FATG-3 NEA21 (231557); Fig 32D N. coenophialum NEA16 (FEtc7-342).

Figure 33 shows the loline biosynthetic pathway.

Figure 34 shows the loline biosynthetic gene cluster.

Figures 35 A-D show presence of Loline biosynthetic gene cluster in endophyte strains. Fig 35A FaTG-2 NEA17 (287819); Fig 35B non-Epichloe out-group NEA18 (FEtc6-75); Fig 35C FATG-3 NEA21 (231557); Fig 35D N. coenophialum NEA16 (FEtc7-342).

Figures 36 A-F show alkaloid biosynthetic gene analysis for endophyte strain NEA23 (269850). Fig 36A Presence of loline gene cluster; Fig 36B Presence of peramine gene; Fig 36C Analysis of Lolitrem gene cluster 01; Fig 36D Analysis of Lolitrem gene clusters 02 and 03; Fig 36E Analysis of dmaW gene for ergovaline production; Fig 36F Analysis of eas gene cluster for ergovaline production.

-22 2016259448 18 Nov 2016

Figure 37 shows genotypic analysis of NEA23 and NEA21.

Figure 38 shows genotypic analysis of NEA16 and NEA20.

The invention will now be described with reference to the following non-limiting examples.

Example 1 - Tall fescue endophyte discovery

The objectives of this work on discovery and characterization of endophytes in tall fescue (Lolium arundinaceum) were:

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.

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.

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

-232016259448 18 Nov 2016

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.

New accessions were included in the analysis from the under-represented 5 geographic origins; with a total of 568 accessions from 40 countries tested for endophyte incidence.

	Number of geographic origins	Percentage positive accessions
	FEtc collection	GRIN collection	FEtc collection	GRIN collection
Incidence assessment 01	7	23	96%	30%
Incidence assessment 02	-	10	-	45%

Table 1: Genotypic analysis of endophyte content in accessions from a targeted fescue germplasm collection

Genotypic analysis of endophyte content in accessions from a targeted fescue germplasm collection is shown in Table 1. 233 endophyte positive accessions (41%) were detected. The geographical origins are represented in the endophyte incidence assessment.

A genetic diversity analysis of tall fescue endophytes is shown in Figure 2. A

-242016259448 18 Nov 2016 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 Λ/. coenophialum. Twenty were FaTG-2. Six were putative FaTG-3. Thirteen were FaTG-3 like.

Diversity of host and endophyte is shown in Figure 3.

Selection of fescue-endophyte combinations for metabolic profiling, endophyte isolation and isogenic inoculation is shown in Figure 4. 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.

Selection of fescue-endophyte combinations for metabolic profiling, endophyte isolation and isogenic inoculation is shown in Figure 5. Initial selections are shown in red. Additional selections are shown in blue.

The desired toxin profile of tall fescue endophytes is shown in Figure 6.

Example 2 - Metabolic profiling

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.

-252016259448 18 Nov 2016

Experimental design for semi-quantitative analysis of metabolites

Trim and re-pot	16h L: 21 C	Trim 5 cm
the plants	8h D: 16°C, 2 Weeks	above ground

zr σ

σ>

; Notes:

! Light intensity: 620mmol m-1s-1 ; Water supply: 50 ml/day ! Maintain 4 copies per accession in ;_aeneral oottina mix σ>

<

Ο ro φ

φ ττ w

ro <

O

	Harvest 10 tillers	16h L: 21 C	Trim 5 cm
10	(2-4 weeks age)	8h D: 16°C,^{2 Weeks}	above ground

A metabolic profile analysis for detection of ergovaline and peramine is shown in Figure 7.

Endophytes selected for semi-quantitative analysis of metabolites are shown in 15 Figure 8.

Metabolic profile analysis for the detection of alkaloid production of different fescue endophytes

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 Figure 9. The alkaloid profile

-262016259448 18 Nov 2016 (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 2.

Tall fescue accession details	Alkaloid profile
Tall fescue accession	Endophyte strain	Endophyte 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	+	+	+	-

-272016259448 08 Oct 2018

FEtc6- 129	NEA31	N. coenophialum	+	+	+	-
287819	NEA17	FaTG-2	-	+	M +	-
231557	NEA21	FaTG-3	+	+	-	-
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	+	+	+	-

Table 2 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 (*

Published data; nd = not determined).

Further metabolic analysis of the fescue endophytes is shown in Figure 10.

Example 3 - Semi-quantitative Analysis of Metabolic Profile under Temperature/Water Stress

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 (Figures 7 - 10), a semiquantitative 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 16h Light and 30°C; 18h Dark and 20°C, and then sampled for alkaloid profile analysis as described below:

Harvest (control) —> freeze dry —> 50 mg pseudostem material —> 80%

-282016259448 18 Nov 2016 methanol extraction —> LCMS analysis • Recovery and water stress • Second harvest (stress) —> freeze dry —> SSR confirm all of the plant material again.

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.

Figure 11 shows a semi-quantitative analysis of metabolic profile of tall fescue10 endophyte associations grown under high temperature and water stress conditions.

Example 4 - In planta isogenic inoculation in tall fescue with novel endophytes

Summary:

A total of 36 fescue endophytes have been isolated from a range of fescue accessions from different geographic origin as described in Table 3, and found to belong to different taxa as follows: 19 of them being N. coenophialurrr, 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

-292016259448 08 Oct 2018

Fescue Accession	Endophyte Strain	Origin	Cluster	Taxon	Fescue Accession	Endophyte Strain	Origin	Cluster	Taxon
1 8PC	8PC		C01.1	N. coenophialum	23231557	NEA21	Morocco	C09	FaTG-3
2 BE9301	E34		C01.1	N. coenophialum	24287819	NEA17	Spain	C09	FaTG-2
3E77	E77		C01.2	N. coenophialum	25598834		Morocco	C09	FaTG-2
4 FEtc6-62		Catalunya (Spain) 4	C01.2	N. coenophialum	26231559		Morocco	C09	FaTG-2
5 FEtc6-68	NEA26	Catalunya (Spain) 14	C01.2	N. coenophialum	27598852		Morocco	C09	FaTG-2
6FEtc7-127	NEA29	Aragon (Spain)14	C01.2	N. coenophialum	28598934		Italy	C10	Outgroup
7 FEtc7-289	NEA25	Aragon (Spain)14	C01.2	N. coenophialum	29231553	NEA19	Algeria	C10	Outgroup
8 FEtc7-58	NEA15	Aragon (Spain) 1	C01.2	N. coenophialum	30FEtc6-75	NEA18	Sardegna (NW Italy) 5	C10	Outgroup
9 234746	NEA22	Spain	C01.2	N. coenophialum	31269850	NEA23	Tunisia	C12	FaTG-3
10 632582		Italy	C02.1	N. coenophialum	32610918		Tunisia	C12	FaTG-3
11 Kentucky 31	KY31		C02.1	N. coenophialum	33610919		Tunisia	C12	FaTG-3
12FEtc6-128	NEA30	Pyrenees13	C02.2	N. coenophialum	34598829		Morocco	C13	FaTG-3 like
13FEtc6-129	NEA31	Pyrenees17	C02.2	N. coenophialum	35598863		Morocco	C13	FaTG-3 like
14FEtc7-180	NEA14	PaySardegna (Basque (FreCfiS.)B	N. coenophialum	36598870		Morocco	C13	FaTG-3 like
15 440364		Kazakhstan	C03	N. coenophialum	37 M311046		Russion Federation	C14	N. uncinatum
16619005		China	C03	N. coenophialum	38M595026		United Kingdom	C14	N. uncinatum
17FEtc6-83	NEA24	Corsica (France)7	C04	N. coenophialum	39 M611046		Russion Federation	C14	N. uncinatum
18FEtc6-85	NEA27	Corsica (France) 15	C04	N. coenophialum
19FEtc6-87	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

Table 3 - Isolation of fungal endophyte cultures from endophyte-containing fescue accessions

Establishment of Meristem Cultures for Diverse Host Panel for In Planta 5 Inoculation of Fescue Endophytes

Table 4 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 >80%.

-302016259448 18 Nov 2016

Cultivar	Genotype 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

Table 4 - 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

Isolated fungal endophytes from endophyte-containing fescue accessions selected for in planta isogenic inoculation into the diverse host panel are shown in Figure 12. Figure 13 shows SSR-based genotyping of isolated endophyte cultures prior to in planta isogenic inoculation to confirm their identity.

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 5.

Endophyte Strain ID	Tall Fescue Accession ID	NCESTA1DH04 (FAM)	NLESTA1TA10 (FAM)	NCESTA1HA02 (HEX)	NCESTA1CC10 (HEX)
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

Table 5 - Presence of alleles in endophyte strains

-31 2016259448 18 Nov 2016

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 6.	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.
A. Number of inoculations tested
	E77	E34	NEA13	NEA15	NEA14	AR542	NEA16	NEA17	NEA18	NEA19
	E77	BE9301	8PC	Fetc7-58	FEtc7-180	AR542	FEtc7-342	287819	FEtC&75	231553	Total
BARI 27	23	25	30	34	38	38	24	32	40	21	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
	E77	E34	NEA13	NEA15	NEA14	AR542	NEA16	NEA17	NEA18	NEA19
	E77	BE9301	8PC	Fetc7-58	FEtc7-180	AR542	FEtc7-342	287819	FEtC&75	231553	Total
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
	E77	E34	NEA13	NEA15	NEA14	AR542	NEA16	NEA17	NEA18	NEA19
	E77	BE9301	8PC	Fetc7-58	FEtc7-180	AR542	FEtc7-342	287819	FEtC&75	231553	Total
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

Table 6 - Inoculation Ability of Tall Fescue Endophytes in Tall Fescue and Perennial Ryegrass Hosts

Example 5 - Endophyte Vegetative Stability in Tall Fescue and Perennial Ryegrass Host Genotypes

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:

-32 2016259448 18 Nov 2016 • 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.

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 7.

Plant Genotyp e	E7 7	E34	NEA1 3	NEA1 5	NEA1 4	AR54 2	NEA1 6	NEA1 7	NEA1 8	NEA1 9
E7 7	BE930 1	8PC	Fetc7- 58	FEtc7- 180	AR54 2	FEtc7- 342	28781 9	FEtc6- 75	23155 3
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

Table 7 - 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.

NA - not applicable, NI - not inoculated, number of stable association/number of associations

-332016259448 18 Nov 2016

Figure 14 shows stability at 12 months post inoculation of selected endophytes in tall fescue and perennial ryegrass host genotypes from the diverse host panel.

The range of novel fescue endophytes selected for in planta isogenic inoculation is shown in Figure 15.

Table 8 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, JESS 01 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

	NEA20	NEA21	NEA22	NEA23	NEA24	NEA27	NEA30
	FEtc7- 343	231557	234746	269850	FEtc6- 83	FEtc6- 85	FEtc6- 128
	Neo	FaTG-3	Neo	FaTG-3	Neo	Neo	Neo
	Lol/-/P/-	L0I/-/P/-	Lol/E/P/-	L0I/-/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

Table 8 - Additional novel tall fescue endophytes (e.g. NEA20, NEA21, NEA22, etc.) selected for in planta isogenic inoculations in tall fescue genotypes (i.e.

-342016259448 18 Nov 2016

BARI 27, JESS 01 and QUAN 17) from the diverse host panel. Neo = N. coenophialum; ? = alkaloid profile not tested; TBI = To Be Inoculated.

Example 6 - 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

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 Figures 16, 18 and 19. 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

Figure 17 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.

Table 9 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.

-352016259448 18 Nov 2016

			Endophyte genotype
Host genotypes neais	NEA17	8PC	AR542 E34	E77 NEA1S	NEA14	NEA16	NEA15
231553	287819		3E3301	FEtcS-75	Fe!c7-130	Fste7-342	Fetc7-53

Bariane (Bari27j

2/5

215

3/3

51/11

3/3

15/1: 5/5

10110

1/4

3112 NA

3/14

515

12/12

3/4

5/12

1/4

15! 13/25

Dovey(OOV24)

NA

2/5

3/3

NA

3/5

S/12

3/5

3/12

NA

Jessup ίJessOI)

2M

·?.··/··

NA

313

12+2 4/4

12/12

NA

213

5/11

NA

214

7/13

2/3

12/12 NA

Quantum (Quant 7)

2/5

317

4/5

12/12

NA

415

12/12

:214

SA2

NA

Bronsyn (BroOSj

3/5

10/11

515

11/:2

1/3

0/3

NA

314

7/7

0/5

NA

Table 9 - 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.

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 9). In total, 29 isogenic host-endophyte associations were subject to LCMS analysis, following the experimental design described below:

Experimental design • Trim and re-pot plants • 16h Light, 30°C; 18h 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 —> 50mg pseudostem material —> 80% methanol extraction —> LCMS analysis.

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

-362016259448 18 Nov 2016 subsampling was used.

Example 7 - Bio-protective properties of fescue endophytes

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. Figure 20 shows results from anti-fungal bioassays of isolated fescue endophytes. Results of antifungal bioassays are also shown in Table 10. A range of endophytes were found to have high (H) and medium (M) antifungal activity (Table 10).

Tai! Fescue endophytes Antifungal activity against

<	Strain ID 440364	Accession	Taxon N. coenophialum	Cotletotrichum graminicola R	Drechslera brizae H	Rhizoctonia cerealis H
2	AR542	AR542	N. coenophialum	hl	R	R
3	E34	BE9301	N. coenophialum		hl	H
4	NEA13	8PC	N. coenophialum	hl	H	H
5	NEA14	FEte7-180	N. coenophialum	hi	hi	H
6	NEA15	EEtc7-58	N coenophialum	hit	R	R
7	NEA16	FEtc7-342	N. coenophialum	hi	H	H
8	NEA22	234746	N. coenophialum	H	hl	ht
g	NEA27	FEtc6-85	hi. coenophialum	L	hl	L
•0	NEA30	F£tc6-128	N. coenophialum	hl	R	H
11	El		Non-Μ lolii	L	L	ht
12	NEA18	FEtc6-75	Outgroup 1	hl	R	R
13	598852		FaTG-2	hl	R	H
14	610918		FaTG-3	hl	H	H
15	NEA21	231557	FaTG-3	hl	H	iVS
16	598829		FaTG-3 like	hl	L	hi

Antifungal activity: Low, Medium, High

Table 10 - Anti-fungal bioassays of isolated novel fescue endophytes

Example 8 - Genome survey sequencing of novel tall fescue endophytes

A range of novel tall fescue endophtyes were subjected to genome survey 15 sequencing (GSS).

-37 2016259448 18 Nov 2016

Figure 21 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 11.

Table 11 - Alkaloid profiles of sequenced endophytes. Mid grey: alkaloid 5 present, Light grey: Alkaloid absent, White: alkaloid profile not determined * Profiles are taken from published data

-382016259448 18 Nov 2016

Figure 22 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 outgroup endophytes is shown in Figure 23.

Figure 24 shows the ergovaline biosynthetic pathway. Genes in the eas gene cluster which are involved in ergovaline biosynthesis are shown in Figure 25 and Table 12. 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 Figure 26 and presence of the eas gene cluster in novel fescue endophytes is shown in Figure 27.

Gene Cluster	Gene	GenBank Accession No
	dmaW	AY259838
	easA	EF125025
	easE	EF125025
eas gene cluster	easF	EF125025
	easG	EF125025
	easH	EF125025
	IpsA	AF368420
	IpsB	EF125025

Table 12 - Genes in the eas cluster

Figure 28 shows the Lolitrem B biosynthetic pathway. Genes in the gene cluster which are involved in Lolitrem B biosynthesis are shown in Figure 29 and Table 13. Presence of gene cluster 1 (ItmG, ItmM and ItmK) in endophytes is shown in Figure

30, presence of gene cluster 2 (ItmB, ItmQ, ItmP, ItmF and ItmC) is shown in Figure and presence of gene cluster 3 (ItmE and ItmJ) is shown in Figure 32.

-392016259448 18 Nov 2016

Gene Cluster	Gene	GenBank Accession No
	ItmG	AY742903
gene cluster 01	ItmM	AY742903
	ItmK	AY742903
	ItmB	DQ443465
	ItmQ	DQ443465
gene cluster 02	ItmP	DQ443465
	ItmF	DQ443465
	ItmC	DQ443465
gene cluster 03	ItmJ	DQ443465
ItmE	DQ443465

Table 13 - Genes in the gene cluster involved in Lolitrem B biosynthesis

Figure 33 shows the Loline biosynthetic pathway. Genes in the gene cluster which are involved in Loline biosynthesis are shown in Figure 34 and Table 14. Presence of Loline biosynthetic gene cluster in novel fescue endophytes is shown in Figure

35.

Gene Cluster	Gene	GenBank Accession No
	lolF	EF012269
	lolC	EF012269
	lolD	EF012269
	lolO	EF012269
LOL gene cluster	lolA	EF012269
	lolU	EF012269
	lolP	EF012269
	lolT	EF012269
	lolE	EF012269

Table 14 - Genes in the Loline biosynthetic gene cluster

Figure 36 shows an alkaloid biosynthetic gene analysis for endophyte strain NEA23. Tables 15 and 16 show alkaloid biosynthetic gene analyses for various endophyte strains. Table 15 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.

2016259448 18 Nov 2016

-41 2016259448 18 Nov 2016

Table 16 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 fescue5 endophyte associations.

Tall fescue accession details	Alkaloid profile and Gene presence
Endophyte strain	Accession No/isolated ID	Endophyte species	Lolines	Peramine	Ergovaline*	Lolitrem B
E34	BE9301	N. coenophialum				PG+
8PC	8PC	N. coenophialum	G+			PG+
NEA14	FEtc7-180	N. coenophialum				llllllllll
NEA15	FEtc7-58	N. coenophialum				1111011111
NEA16	FEtc7-342	N. coenophialum			G-	iiiiiiii
NEA20	FEtc7-343	N. coenophialum			G-	iiiiiiiii
NEA22	234746	N. coenophialum			11111111	llllllllll
NEA24	FEtc6-83	N. coenophialum
NEA17	287819	FaTG-2	G-
NEA21	231557	FaTG-3	IIIIllM		lllllllll	lllllllll
NEA23	269850	FaTG-3			lllllllll	lllllllll
NEA19	231553	non- Epichloe out-group	lllllllll	llllllllll	lllllllll	lllllllll
NEA18	FEtc6-75	non- Epichloe out-group	lllllllll	llllllllll	lllllllll	lllllllll
AR542*	AR542*	N. coenophialum			111111!!!	PG+
E77*	E77*	N. coenophialum
598852	598852	FaTG-2
AR501*	AR501*	FaTG-3			G-	lllllllll
598829	598829	FaTG-3 like
E81	E81	N. uncinatum
9340	9340	E. typhina
9707	9707	E. baconii	G-	PG+	G-	G-

Table 16 - Alkaloid biosynthetic gene and alkaloid production analysis. Green: alkaloid present, Yellow: Alkaloid absent, Grey: alkaloid profile not

-42 2016259448 18 Nov 2016 determined, ‘Profiles are taken from published data, G+ = gene/gene cluster present, G- = gene/gene cluster absent, PG+ = gene/gene cluster partially present

Table 17 shows novel fescue endophytes (NEA16, NEA18, NEA19, NEA20, NEA21 5 and NEA23) with favourable toxin profiles.

Tall fescue accession	Taxon	Alkaloid profile (Lol/P/E/L)	Antifungal
NEA21	FaTG-3	+/+/-/-	High
NEA23	FaTG-3	+/+/-/-	Not tested
AR501*	FaTG-3	+/+/-/-	-
NEA18	Non-Epichloe Outgroup	-/-/-/-	High
NEA19	Non-Epichloe Outgroup	-/-/-/-	Not tested
NEA16	N. coenophialum	+/+/-/-	High
NEA20	N. coenophialum	+/+/-/-	Not tested
AR542*	N. coenophialum	+/+/-/-	-

Table 17 - Novel fescue endophytes (NEA16, NEA18, NEA19, NEA20, NEA21 and NEA23) with favourable toxin profiles and antifungal activities observed in bioassays. * Control commercial endophyte

A genotypic analysis of the novel fescue endophytes NEA23 and NEA21 is shown 10 in Figure 37.

2016259448 08 Oct 2018

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A substantially purified or isolated FaTG-3 species endophyte having a desired toxin profile, wherein the endophyte produces significantly less toxic alkaloids compared with a control endophyte; and/or significantly more alkaloids

5 conferring beneficial properties compared with a control endophyte, wherein said control endophyte is standard toxic endophyte; and wherein said endophyte is selected from the group consisting of NEA21 and NEA23, as deposited at the National Measurement Institute with accession numbers V12/001418 and V12/001419, respectively.

10 2. An endophyte according to claim 1, wherein said beneficial properties include improved tolerance to water and/or nutrient stress and/or improved resistance to pests and/or diseases in the plant with which the endophyte is associated.

3. An endophyte according to claim 1 or 2, wherein said alkaloids conferring

15 beneficial properties are selected from the group consisting of peramine, Nformylloline, N-acetylloline and norloline.

4. An endophyte according to claim 1, wherein said toxic alkaloids are selected from the group consisting of ergovaline and Lolitrem B.

5. An endophyte according to any one of claims 1 to 4, wherein said endophyte

20 is isolated from a fescue species.

6. An endophyte according to claim 5, wherein said fescue species is tall fescue.

7. An endophyte according to any one of claims 1 to 6, wherein said toxic alkaloids are present in an amount less than approximately 1 pg/g dry weight.

2016259448 08 Oct 2018

-44 8. An endophyte according to any one of claims 1 to 7, wherein said alkaloids conferring beneficial properties are present in an amount of between approximately 5 and 100 pg/g dry weight.

9. A plant inoculated with an endophyte according to any one of claims 1 to 8, 5 said plant comprising an endophyte-free host plant stably infected with said endophyte.

10. A plant, plant seed or other plant part derived from a plant according to claim 9 and stably infected with an endophyte according to any one of claims 1 to 8.

11. Use of an endophyte according to any one of claims 1 to 8 to produce a plant 10 stably infected with said endophyte.

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Fig. 38