AU2017309821B2 - Methods of characterising endophytes - Google Patents

Abstract

The present invention relates to fungi, plants infected with fungi, products produced by fungi, and related methods. More particularly, the present invention relates to a method for selecting and/or characterising a fungus, which includes subjecting a fungus to genetic analysis, wherein genetic analysis includes: sequencing one or more non-coding nrDNA regions; sequencing one or more regions of a protein-coding gene; assessing the genetic variation of the sequenced regions; and optionally selecting a fungus having a desired genetic variation.

Description

METHODS OF CHARACTERISING ENDOPHYTES

Field of the invention

The present invention relates to fungi, plants infected with fungi, products produced by fungi, and related methods.

Background of the invention

Microbes represent an invaluable source of novel genes and compounds that have the potential to be utilised in a range of industrial sectors. Scientific literature gives numerous accounts of microbes being the primary source of antibiotics, immunosuppressants, anticancer agents and cholesterol-lowering drugs, in addition to their use in environmental decontamination and in the production of food and cosmetics. A relatively unexplored group of microbes known as endophytes, which reside in the tissues of living plants, offer a particularly diverse source of novel compounds and genes that may provide important benefits to society, and in particular, agriculture.

Endophytes often form mutualistic relationships with their hosts, with the endophyte conferring increased fitness to the host, often through the production of defence compounds. At the same time, the host plant offers the benefits of a protected environment and nutriment to the endophyte.

Members of the Brachiaria-Urochloa species complex belong to the Poaceae family of grasses. Some species of Brachiaria-Urochloa are economically significant tropical forage grasses that have been released as commercial cultivars and include B. brizantha, B. decumbens, B. dictyoneura, B. humidicola, and B. ruziziensis, as well as corresponding interspecific and intraspecific hybrids.

Genetic diversity analysis based on internal transcribed spacer (ITS) nuclear ribosomal DNA sequence data indicates a strong affinity between Urochloa and Brachiaria, supporting morphological and anatomical studies that show a continuous gradation between these grass genera.

Seed-transmitted endophytic fungi have been observed in B. brizantha. These endophytes may play a role in protecting Brachiaria-Urochloa from fungal pathogens, such as Drechslera spp., which cause leaf spots.

There is a general lack of information and knowledge of the fungal endophytes of the Brachiaria-Urochloa species complex as well as of methods for the identification and characterization of endophytes and their deployment in Brachiaria-Urochloa plant improvement programs.

Identification and characterization of endophytes is generally based on morphological characterisation and molecular taxonomy analyses. Morphological characterisation includes analyses of macroscopic and microscopic structures of an endophyte grown on culture media. Molecular taxonomy analysis is mainly based on gene sequence analysis of spacer regions in nuclear ribosomal DNA (nrDNA), particularly in phylogenomics. However, traditional methods of phylogenomics based on nrDNA sequences may not reflect the divergence of closely related species.

It is an objection of the present application to overcome, or at least alleviate, one or more of the difficulties or deficiencies associates with the prior art.

Summary of the Invention

This invention describes methods for the identification, isolation, characterisation and inoculation of novel endophytes from and in Brachiaria-Urochloa, respectively, that may be used to establish novel endophyte-Brachiaria/Urochloa associations for improved pasture production for livestock industries.

The discovery, characterization, and inoculation of novel fungal endophytes in associations with Brachiaria-Urochloa pasture grasses may assist in the varietal development process of these pasture grasses for livestock production in warmer climates around the world.

Many of the commercially developed Brachiaria-Urochloa pasture grasses are aposporous apomicts. These grasses reproduce asexually through seed without a requirement for gamete union, hence propagating the maternal genotype.

Apomictic reproduction has a number of key advantages for research on, and use of, fungal endophyte-grass host associations. The practical implication of seed transmission of endophytes in Brachiaria-Urochloa is significant, once associated with the plant, the fungus can perpetuate itself through seed, provided that seed storage conditions do not reduce the survival of the fungus.

In a first aspect, the present invention provides a substantially purified or isolated fungus purified or isolated from a plant of the Brachiaria-Urochloa species complex and wherein, when said fungus is inoculated into a plant, said plant has improved resistance to diseases and/or pests relative to an uninoculated control plant.

Preferably, the fungus is selected from the group consisting of strain (i.e. isolate) 1IA, strain 3.3.A, strain 3.3.B, strain 3.3.C, strain 4.9.A, strain 4.9.B, strain 5.1.A, strain 5.1.B, strain 5.1.D, strain 5.1.E, strain 7.1.A, strain 8.1.A, strain 8.1.B, strain 8.1.C, strain 9.2.A, strain 9.2.B, strain 9.2.C, strain 10.1A, strain 11.1A, strain 12.1.A, strain 12.1.B, strain 12.1.C, strain 12.1.D strain 12.1.E, strain 14.1.B, strain 14.1.C, strain 15.2.C, strain 15.2.D, strain 15.2.E, as described herein.

The fungus may be an Acremonium species strain, a Phialemonium species strain, a Simplicillium species strain, an Emericellopsis species strain, or a strain of a related or closely related species. Without wishing to be limited by theory, it is believed that strain 1.1A is an Acremonium sclerotigenum species strain, strain 3.3A is a Phialemonium atrogriseum species strain, strain 5.1B is a Phialemonium atrogriseum species strain, strain 9.2A is a Simplicillium species strain, strain 12.1A is an Emericellopsis species strain, and strain 12.1B is an Acremonium sclerotigenum species strain. Thus, in preferred embodiments, the fungus may be an Acremonium sclerotigenum species strain, a Phialemonium atrogriseum species strain (previously Acremonium atrogriseum), a Simplicillium species strain, an Emericellopsis species strain, or a strain of a closely related species.

Representative samples, namely strains 1.1.A (1.1A 3.3.A (33A), 5.1.B (51), 9.2.A (9.2A) and 12.1A (12.1A) were deposited at The National Measurement Institute of 1/153 Bertie Street, Port Melbourne, VIC 3207, Australia on 7 June 2011 with accession numbers V11/011370, V11/011371, V11/011372 V11/011373, and V11/011374, respectively.

By 'substantially purified'is meant that the fungus is free of other organisms. The term therefore includes, for example, a fungus in axenic culture. Preferably, the fungus is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure, even more preferably at least approximately 99% pure.

The term 'isolated' means that the fungus is removed from its original environment (e.g. the natural environment if it is naturally occurring). For example, a naturally occurring fungus present in a living plant is not isolated, but the same fungus separated from some or all of the coexisting materials in the natural system, is isolated.

In its natural environment, the fungus may be an endophyte, i.e. live mutualistically within a plant. Alternatively, the fungus may be an epiphyte, i.e. grow attached to or upon a plant. Preferably, the fungus is a fungal endophyte.

The fungus of the present invention may, in its natural environment, be associated with a plant of the Brachiaria-Urochloa species complex More particularly,the plant of the Brachiaria-Urochloa species complex is selected from the group consisting of Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis, Brachiaria marlothi. Brachiaria nigropedata, Urochloa dictyoneura, Urochloa oligotricha, Urochloa panicoides, Brachiaria obtusiflora, Brachiaria serrifolia, Urochloa advena, Urochloa arrecta, Urchloa brachyura, Urochloa eminii, Urochloa mollis, Urochloa xantholeuca Urochioa oligotricha, Urochloa panicoides, Urochoa pantaginea, Urochoa platynota and Urochloa xantholeuca, as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex.

Rectified Sheet Rule 91 (ISA/AU)

In a particularly preferred embodiment, the plant of the Brachiaria-Urochloa complex is selected from the group consisting of Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola and Urochloa mosambicensis.

By 'associated with' in this context is meant that the fungus lives on, in or in close proximity to the plant. For example, it may be endophytic, for example living within the internal tissues of the plant, or epiphytic, for example growing externally on the plant.

The fungus may be a heterotroph that uses organic carbon for growth, more particularly a saprotroph that obtains nutrients by consuming detritus.

In a further aspect, the present invention provides a plant inoculated with a fungus as hereinbefore described, said plant comprising a fungus-free host plant stably infected with said fungus. Preferably, the plant with which the fungus is associated has improved resistance to pests and/or diseases relative to an uninoculated control plant. In a preferred embodiment, the improved resistance to pests and/or diseases includes insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

In a preferred embodiment, the host plant may be inoculated with more than one fungal strain according to the present invention.

Preferably, the plant is an agricultural plant such as a grass species, preferably forage, turf or bioenergy grasses, such as those belonging to the Brachiaria-Urochloa species complex (panic grasses) including Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, B. dictyoneura, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex, and those belonging to the genera Lolium and Festuca, including L. perenne (perennial ryegrass) and L. arundinaceum (tall fescue) and L. multiflorum (Italian ryegrass).

Preferably, the plant is infected with the fungus by a method selected from the group consisting of inoculation, breeding, crossing, hybridization and combinations thereof.

The fungus-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.

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 a fungus of the present invention. Preferably, the plant, plant seed or other plant part with which the fungus is associated has improved resistance to pests and/or diseases relative to an uninoculated control plant, plant seed or other plant part. In a preferred embodiment, the improved resistance to pests and/or diseases includes insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

Preferably, the plant cell, plant, plant seed or other plant part is from a grass, more preferably a forage, turf or bioenergy grass, such as those belonging to the Brachiaria Urochloa species complex (panic grasses), including Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, B. dictyoneura, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex such as interspecific hybrids between Brachiaria ruziziensis x Brachiaria brizantha, Brachiaria ruziziensis x Brachiaria decumbens, [Brachiaria ruziziensis x Brachiaria decumbens] x Brachiaria brizantha, [Brachiaria ruziziensis x Brachiaria brizantha] x Brachiaria decumbens and those belonging to the genera Lolium and Festuca, including L. perenne (perennial ryegrass) and L. arundinaceum (tall fescue) and L. multiflorum (Italian ryegrass).

By 'plant cell' is meant any self-propagating cell bounded by a semi-permeable membrane and containing plastid. Such a cell also required a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, seeds suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.

In a further aspect, the present invention provides use of a fungus as hereinbefore described to produce a plant stably infected with said fungus. Preferably, the plant with which the fungus is associated has improved resistance to pests and/or diseases relative to an uninoculated control plant. In a preferred embodiment, the improved resistance to pests and/or diseases includes insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

In a further aspect of the present invention, there is provided a method of increasing resistance to pests and/or diseases in a plant, said method including inoculating said plant with a fungus as hereinbefore described. Preferably, the plant with which the fungus is associated has improved resistance to pests and/or diseases relative to an uninoculated control plant. In a preferred embodiment, the improved resistance to pests and/or diseases includes insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

In a further aspect of the present invention, the fungus may be selected and/or characterised by a method including: providing a plurality of samples of fungi; subjecting said fungi to genetic analysis; subjecting said fungi to metabolic analysis; and selecting fungi having a desired genetic and metabolic profile.

In a preferred embodiment, this aspect of the invention may include the further step of assessing geographic origin of the fungi and selecting fungi having a desired genetic and metabolic profile and a desired geographic origin.

In a preferred embodiment, the plurality of samples of fungi may be provided by a method including: providing a plurality of plant samples that may contain fungi; and isolating fungi from said plant samples.

In a preferred embodiment, the genetic analysis includes detecting the presence or absence of polymorphic markers such as simple sequence repeats.

Applicant has found that specific detection of fungi in planta with markers such as SSR markers has provided the tools for efficient assessment of fungus genetic diversity in diverse grass populations and the potential discovery of novel fungal strains.

By a 'plurality' of samples of endophytes or plant samples is meant a number sufficient to enable a comparison of genetic and metabolic profiles of individual fungal endophytes. Preferably, between approximately 10 and 1,000,000 samples of endophytes or plant samples are provided, more preferably between approximately 100 and 1,000 samples of endophytes or plant samples.

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 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 co-dominantly inherited. Their detection is based on the polymerase chain reaction (PCR), requiring only small amounts of DNA and suitable for automation. They are ubiquitous in eukaryotic genomes and have been found to occur in fungal genomes and in plant genomes. Consequently, SSRs are ideal markers for a broad range of applications such as genetic diversity analysis, genome mapping, trait mapping and marker-assisted selection.

Alternatively, or in addition, the genetic analysis may involve sequencing genomic and/or mitochondrial DNA and performing sequence comparisons to assess genetic variation between fungi. In a preferred embodiment, the internal transcribed spacer (ITS) sequence may be used for genetic analysis.

By 'metabolic analysis' is meant analysing metabolites, in particular toxins, produced by the fungi. Preferably, this is done by preparation of inoculated plants for each of the fungi and measurement of toxin levels in planta. More preferably, this is done by preparation of isogenically inoculated plants for each of the fungi and measurement of toxin levels in planta.

By a 'desired genetic and metabolic profile' is meant that the fungus includes genetic and metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, the fungus.

Such beneficial properties include improved tolerance to water and/or nutrient stress and improved resistance to pests and/or diseases in the plant with which the fungus is associated. In a preferred embodiment, the beneficial properties include insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

For example, resistance to pests and/or diseases in the plant 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 an uninoculated plant that does not contain the fungal endophyte. Preferably, resistance to pests and/or diseases in the plant may be increased by between approximately 5% and approximately 50%, more preferably between approximately 10% and approximately 25%, relative to an uninoculated plant that does not contain the fungal endophyte.

Alternatively, or in addition, a fungus may be selected and/or characterised based primarily on genetic analysis.

Thus, in a further aspect, the present invention provides a method for selecting and/or characterising a fungus, said method including subjecting a fungus to genetic analysis, wherein genetic analysis includes: sequencing one or more non-coding nrDNA regions; sequencing one or more regions of a protein-coding gene; assessing the genetic variation of the sequenced regions; and optionally selecting a fungus having a desired genetic variation.

In a preferred embodiment of this aspect of the invention, there is provided a method for characterising and selecting a fungus for inoculation into a host plant, said method including subjecting a fungus to genetic analysis, wherein genetic analysis includes: sequencing one or more non-coding nuclear ribosomal DNA (nrDNA) regions; sequencing one or more regions of a protein-coding gene, wherein the protein coding gene is selected from one or more of a tub2 gene, a tefA gene and an act1 gene, and fragments thereof; and assessing the genetic variation of the sequenced regions; subjecting the fungus to a metabolic analysis; and selecting a fungus having a desired genetic variation and/or a desired metabolic profile that will confer a beneficial phenotype to the host plant. ?0 By nrDNA is meant nuclear ribosomal DNA. Eukaryotic nrDNA includes genes encoding small subunit (SSU), large subunit (LSU), and 5.8S nrDNAs. The SSU and LSU nrDNAs are separated by two external transcribed spacer (ETS), and a non-transcribed spacer (NTS), non-coding regions. The 5.8S rDNA is embedded in two internal transcribed spacer (ITS1 and ITS2) non-coding regions.

Thus, the non-coding nrDNA may be an ETS, NTS or ITS spacer. In preferred embodiments, the non-coding nrDNA is an ITS spacer.

The protein-coding gene is not particularly limited, but is preferably one which is not highly conserved. This may aid the differentiation of closely related species which may be characterisable by substantial or total identity in conserved protein-coding gene regions. One or more, for example two or three or more, protein-coding genes may be sequenced for this method.

The region of the non-coding nrDNA and/or the protein-coding gene may be the entire non-coding nrDNA or protein-coding gene, or may be a fragment thereof. For example, for an ITS spacer, the region may be the entire spacer, or may be a fragment thereof. Similarly, for the tub2 gene, the region may be the entire gene, or may be a fragment thereof. A suitable fragment size is not particularly limited, and may depend on the non coding nrDNA or the protein-coding gene, along with a number of factors, i.e. selection of primers for PCR etc. Preferably, a fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, and more preferably at least 200 nucleotides. In preferred embodiments, the region of the non coding nrDNA and the protein-coding gene is substantially the entire non-coding nrDNA and protein-coding gene, respectively.

Assessing the genetic variation of the sequenced regions will usually involve a comparison of the sequenced regions against sequences of the same regions or part thereof of another fungus. The other fungus may be the same or a different fungus, for example a closely-related fungus, or a fungus of the same or similar origin. The sequences of the same regions or part thereof of another fungus may be obtained the same way, i.e. by sequencing the regions. Alternatively, or in addition, the sequences of the same regions or part thereof of another fungus may be obtained from existing resources, for example a database of sequence data. The comparison may be by way of, for example, sequence alignments.

The extent of variation may be complete or none, or any amount there between. For example, when compared with sequences of the same regions or part thereof of another fungus, the variation may be little or none, such that the compared regions are substantially identical or identical, respectively. Conversely, the variation may be complete, such that there is no identity between the compared regions. The extent of variation of the non-coding nrDNA and the region of a protein-coding gene may be independently different.

The extent of variation may be used to characterise a fungus, for example assign a fungus to a clade or to designate it to a taxonomic group. The characterisation may also include assigning a new species.

A fungus having a desired genetic variation may also be selected, for example for further analysis or application. A desired genetic variation may be, for example, complete variation, little or no variation. For example, little or no variation may be desired to indicate a close relationship to another fungus, for example to a fungus that includes genetic and metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, the fungus.

Thus, the fungus may be an endophyte of a plant, for example a grass. Preferably the plant is a forage, turf or bioenergy grass, more preferably a plant of the Brachiaria Urochloa complex as hereinbefore described.

In a preferred embodiment, the method of the present invention may include the further step of subjecting the fungus to a morphological analysis. By morphological analysis is meant studying the form or shape of the fungus or part thereof. In a preferred embodiment, the morphological analysis may be a comparative morphological analysis.

In a preferred embodiment, the method of the present invention may include the further step of subjecting the fungus to a metabolic analysis. By 'metabolic analysis' is meant analysing metabolites, in particular toxins, produced by the fungi. Preferably, this is done by preparation of inoculated plants for each of the fungi and measurement of toxin levels in planta. More preferably, this is done by preparation of inoculated plants for each of the fungi and measurement of toxin levels in planta.

In a preferred embodiment, the method of the present invention may include the further step of assessing geographic origin of the fungus. In a preferred embodiment, this may include the further step of assessing geographic origin of the fungi and selecting fungi having a desired genetic and metabolic profile and a desired geographic origin.

In a further aspect, the present invention provides a method of culturing a fungus as hereinbefore described, said method including growing said fungus on a medium including a source of carbohydrates, for example a starch/sugar-based agar or broth such as potato dextrose agar or potato dextrose broth, or a cereal-based agar or broth such as oatmeal agar or oatmeal broth.

The fungus may be cultured under aerobic or anaerobic conditions.

In a particularly preferred embodiment, the fungus may be cultured in a culture medium including potato dextrose or oatmeal, for example potato dextrose agar, oatmeal agar, potato dextrose broth or oatmeal broth.

The fungus may be cultured for a period of approximately 1 to approximately 100 days, more preferably from approximately 10 to approximately 50 days more preferably from approximately 10 to approximately 30 days.

In a preferred embodiment, the fungus may be cultured in a bioreactor. By a 'bioreactor' is meant a device or system that supports a biologically active environment, such as a vessel in which is carried out a chemical process involving fungi of the present invention and/or products thereof. The chemical process may be aerobic or anaerobic. The bioreactor may have a volume ranging in size from millilitres to cubic metres, for example from approximately 50 ml to approximately 50,000 litres. The bioreactor may be operated via batch culture, batch feed culture, perfusion culture or continuous culture, for example continuous culture in a stirred-tank bioreactor. Fungi cultured in the bioreactor may be suspended or immobilized.

In a preferred embodiment, the method may include the further step of recovering an organic compound produced by the fungus from within fungal cells, including intracellular tissues, from the culture medium (e.g. secreted liquids) or from the air space (e.g. secreted vapours) associated with the culture medium or fungus.

Vapours may arise directly from the fungus or from the secreted liquids which transition between vapour and liquid phases.

The step of recovering the organic compound is preferably done by separating cells from the culture medium or capturing vapours associated with the culture medium or fungus.

Preferably the organic compound is then isolated or purified by a method selected from the group consisting of gas chromatography, liquid chromatography, fractional distillation and absorption chromatography, such as pressure swing adsorption.

By an 'organic compound' is meant a chemical compound whose molecules contain carbon.

In a preferred embodiment, the organic compound may have insecticidal or insect repellent activity. In a particularly preferred embodiment, the organic compound may be peramine or an analogue, derivative or salt thereof.

By a 'derivative' is meant an organic compound obtained from, or regarded as derived from, a compound of the present invention. Examples of derivatives include compounds where the degree of saturation of one or more bonds has been changed (e.g., a single bond has been changed to a double or triple bond) or wherein one or more atoms are replaced with a different atom or functional group. Examples of different atoms and functional groups may include, but are not limited to hydrogen, halogen, oxygen, nitrogen, sulphur, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, amine, amide, ketone and aldehyde.

Preferably, said organic compound is produced by a method as hereinbefore described.

In a preferred embodiment, the organic compound may be obtained from a fungus of the present invention.

In a still further aspect of the present invention, there is provided use of an organic compound according to the present invention as an insecticide or insect repellent.

In a still further aspect of the present invention, there is provided use of an organic compound according to the present invention as an antifungal compound.

In a further aspect of the present invention, there is provided a method of producing an organic compound, said method including culturing a fungus as hereinbefore described under conditions suitable to produce said organic compound. Preferably the conditions are as hereinbefore described.

Preferably the organic compound is peramine or an analogue, derivative or salt thereof.

In a preferred embodiment, the method may include the further step of recovering an organic compound produced by the fungus as hereinbefore described.

On the basis of the deposits referred to above, the entire genome of a fungus selected from the group consisting strain 1.1.A (1.1A), 3.3.A (3.3A), 5.1.B (5.1B), 9.2.A (9.2A) and 12.1.A (12.1A) is incorporated herein by reference.

Thus, in a further aspect, the present invention includes identifying and/or cloning nucleic acids including genes encoding polypeptides that are involved in the production of organic compounds of the present invention, for example genes encoding enzymes from one or more biochemical pathways which result in the synthesis of said organic compounds.

By a 'biochemical pathway' is meant a plurality of chemical reactions occurring within a cell which are catalysed by more than one enzyme or enzyme subunit and result in the conversion of a substrate into a product. This includes, for example, a situation in which two or more enzyme subunits (each being a discrete protein coded by a separate gene) combine to form a processing unit that converts a substrate into a product. A 'biochemical pathway' is not constrained by temporal or spatial sequentially.

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 encoding enzymes from synthetic pathways for said organic compounds, or mutating the genome of the fungus of the present invention, for example using chemical or transposon mutagenesis, identifying changes in the production of an organic compound of the present invention, and thus identifying genes encoding enzymes from synthetic pathways for said organic compound.

Thus, in a further aspect of the present invention, there is provided a substantially purified or isolated nucleic acid encoding a polypeptide involved in the production of an organic compound of the present invention.

In a preferred embodiment, the nucleic acid may encode a polypeptide involved in the production of peramine or an analogue, derivative or salt thereof.

In a preferred embodiment, the nucleic acid may include a gene encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), or a functionally active fragment or variant thereof. In a particularly preferred embodiment, the nucleic acid may include a nucleotide sequence selected from the group consisting of sequences shown in Sequence ID Nos. 2, 3, 4, 5 and 6 hereto and functionally active fragments and variants thereof.

In a preferred embodiment, the nucleic acid may include a perA gene, or a functionally active fragment or variant thereof. In a particularly preferred embodiment, the nucleic acid may include a nucleotide sequence selected from the group consisting of sequences shown in Sequence ID Nos. 8, 9 and 10 hereto and functionally active fragments and variants thereof.

By 'nucleic acid' is meant a chain of nucleotides capable of carrying genetic information. The term generally refers to genes or functionally active fragments or variants thereof and/or other sequences in the genome of the organism that influence its phenotype. The term 'nucleic acid' includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA or microRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, synthetic nucleic acids and combinations thereof.

By a 'nucleic acid encoding a polypeptide involved in the production of an organic compound of the present invention' is meant a nucleic acid encoding an enzyme normally present in a fungus of the present invention, which catalyses a step in the pathway that results in synthesis of the organic compound 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 synthesis of an organic compound of the present invention, for example by being translated into an enzyme that is able to participate in the pathway that results in synthesis of the organic compound. 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, more preferably at least 200 nucleotides, more preferably at least 500 nucleotides.

By 'conservative nucleic acid changes' is meant nucleic acid substitutions that result in conservation of the amino acid in the encoded protein, due to the degeneracy of the genetic code. Such functionally active variants and fragments also include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.

By 'conservative amino acid substitutions' is meant the substitution of an amino acid by another one of the same class, the classes being as follows: Nonpolar: Ala, Val, Leu, Ile, Pro, Met Phe, Trp Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, 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.

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 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, non-chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable or integrative or viable in the target cell.

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.

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 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.

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], and reporter genes (such as beta glucuronidase (GUS) gene (gusA)]. 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. 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 (e.g. 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, even more preferably at least approximately 99% pure.

The term "isolated" means that the material is removed from its original environment (e.g. 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), 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 art. Such techniques include Agrobacterium-mediated introduction, Rhizobium-mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos, biolistic transformation, Whiskers transformation, and combinations thereof. The choice of technique will depend largely on the type of plant or fungus to be transformed, and may be readily determined by an appropriately skilled person. For transformation of plant protoplasts, PEG-mediated transformation is particularly preferred. For transformation of fungal protoplasts, electroporation and PEG-mediated transformation are particularly preferred. For transformation of fungal hyphae, Agrobacterium-mediated transformation is 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 may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants or fungi.

The present invention also provides a substantially purified or isolated polypeptide involved in the production of an organic compound of the present invention.

In a preferred embodiment, the polypeptide may be involved in the production of peramine or an analogue, derivative or salt thereof.

In a preferred embodiment, the polypeptide may be encoded by a nucleic acid according to the present invention.

The present invention encompasses functionally active fragments and variants of the polypeptides of the present invention. By 'functionally active' in this context is meant that the fragment or variant has one or more of the biological properties of the corresponding protein from which the fragment or variant is derived. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the 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, 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 amino acid substitutions of one or more residues in the corresponding amino acid sequence.

Preferably the fragment has a size of at least 10 amino acids, more preferably at least 20 amino acids, more preferably at least 50 amino acids, more preferably at least 100 amino acids, more preferably at least 200 amino acids. 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 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

The present invention will now be more fully described with reference to the accompanying examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

Description of the Figures

Figure 1. Principal components analysis (PCA) analysis of genetic diversity between Brachiaria-Urochloa grass species using dominantly scored SSR markers.

Figure 2. Isolation of fungal endophytes from Brachiaria-Urochloa grass species. A. Surface-sterilised inner tiller explants from Brachiaria-Urochloa grass species are placed on potato dextrose agar (PDA) medium and cultured at 250C in the dark for fungal endophyte out-growth, B. After 4 weeks, fungal endophytes grow out of the tiller explants and are subcultured onto fresh PDA medium.

Figure 3. Neighbour-joining tree obtained from sequence analysis of the nuclear rDNA ITS region for 29 fungal endophytes isolated from Brachiaria-Urochloa grass species. After alignment of all ITS sequences, the total contig length was 619 bp and contained 120 parsimony informative sites. The robustness of nodes in the tree was tested by 1000 bootstrap re-samplings. Numbers at branches are bootstrap percentages.

Figure 4. Morphology of representative fungal endophytes isolated from Brachiaria Urochloa grass species. Endophyte isolates are grouped based on ITS sequence analysis.

Figure 5. Seed-derived young seedling inoculation of Brachiaria-Urochloa grasses with fungal endophyte mycelium. A. Seeds are scarified (inset) and sterilised; B. Seed germination following 9 days at 260C in the dark; C. Young seedlings are inoculated with endophyte mycelium; D. After 4 weeks on MS medium, plantlets are transferred to soil; E. Plantlets growing after 7 days in soil; F. Plants established in soil under glasshouse conditions are tested for endophyte presence and identity using a DNA marker-based assay.

Figure 6. Inoculation of in vitro regenerating calli from Brachiaria-Urochloa grasses with isolated subcultured fungal endophytes. A. Generation of meristem-derived proliferating embryogenic calli of Brachiaria-Urochloa grasses; B. Explants from in vitro cultured embryogenic calli of Brachiaria-Urochloa grasses; C. Shoot (and root) regeneration followed by endophyte inoculation; D. Plantlet regeneration; E. After 4 weeks on MS medium, plantlets are transferred to soil; F. Mature plants are tested for endophyte presence and identity using a DNA marker-based assay.

Figure 7. Principal components analysis (PCA) plot of all metabolite compounds following LC-MS (ITMS + p ESI Full ms [80.00-2000.00]) analysis of pseudostem tissue samples of B. brizantha, B. decumbens, B. humidicola and U. mosambicensis associated with corresponding fungal endophytes. Technical replicates are shown clustered together. Components 1, 2 and 3 explain up to 19.2% 11.3% and 5.6% of the variability, respectively.

Figure 8. LC-MS analysis of Urochloa mosambicensis grass-fungal endophyte associations displaying extracted ion chromatogram. A. Positive ion extraction; B. Peramine extracted ion chromatogram m/z 248; C. Mass spectrometry at retention time 3.00 min.

Figure 9. An example of inhibition reactions in the antifungal assay. Endophyte isolate 9.2.A was tested for antifungal activity against 8 species of pathogenic fungi.

Figure 10. DNA sequence alignment of the GAPDH gene from Neurospora crassa with homologues of 5 fungal endophyte isolates. The 3 different nuclear rDNA ITS groups to which the 5 isolates belong are as follows: 2nd line - Group 2; 3 rd line - Group 3; Lines 4, 5 and 6 - Group 1.

Figure 11. Relevant section of a Neighbour-Joining tree derived from alignment of the GAPDH protein displaying the novel identity of 3 isolates. Endophytes are highlighted in boxes, corresponding to the ITS groups 1,2 and 3. Note: only 1 isolate (12.1.E) from ITS group 1 is displayed in the tree due to amino acid identity of GAPDH protein among members within this ITS group.

Figure 12. Alignment of the Epichloe festucae perA gene (1_0) with homologous genes from isolates from ITS group 1 (3.3.A, 5.1.B and 12.1.E).

Figure 13. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial sequence of ITS region in 3.3A and selected reference fungal isolates. Branches with bootstrap values of greater than 60% from 500 bootstrap replication are marked next to each branch. GenBank Accession number and CBS number (where available) for each reference is given next to taxon name and T refers to a TYPE strain.

Figure 14. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial sequence of 28S region in 3.3A and selected reference fungal isolates. Branches with bootstrap values of greater than 60% from 500 bootstrap replication are marked next to next to each branch. GenBank Accession number and CBS number for each reference is given next to taxon name and T refers to a TYPE strain.

Figure 15. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial tub2 gene sequence of 3.3A and selected reference fungal isolates. Branches with bootstrap values of greater than 60% from 500 bootstrap replication are marked next to each branch. GenBank Accession number and CBS number (where available) for each reference is given next to taxon name

Figure 16. Morphological characterisation of Group 1 type specimen isolate 3.3A. Group 1 fits very closely with the morphological descriptions and pictures of Phialemonium atrogriseum described in Perdomo et al. (2013). Phialemoniopsis, a new genus of Sordariomycetes; Mycologia 105 (2) 398-421.

Figure 17. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial sequence of ITS region in 1.1A and selected reference fungal isolates. GenBank Accession number and CBS number (where available) for each reference is given next to taxon name and T refers to a TYPE strain.

Figure 18. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial sequence of 18S region in 1.1A and selected reference fungal isolates. Branches with bootstrap values of greater than 60% from 500 bootstrap replication are marked next to next to each branch. GenBank Accession number and CBS number (where available) for each reference is given next to taxon name and T refers to a TYPE strain.

Figure 19. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial sequence of 28S region in 1.1A and selected reference fungal isolates. Branches with bootstrap values of greater than 60% from 500 bootstrap replication are marked next to next to each branch. GenBank Accession number and CBS number (where available) for each reference is given next to taxon name and T refers to a TYPE strain.

Figure 20. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial sequence of tub2 gene in 1.1A and selected reference fungal isolates. Branches with bootstrap values of greater than 60% from 500 bootstrap replication are marked next to next to each branch. GenBank Accession number for each reference is given next to taxon name.

Figure 21. Morphological characterisation of Group 2 type specimen isolate 1.1A. Morphology of 1.1A fits quite closely with the type description of A. sclerotigenum.

Figure 22. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial sequence of ITS region in 9.2A and selected reference fungal isolates. Branches with bootstrap values of greater than 60% from 500 bootstrap replication are marked next to next to each branch. GenBank Accession number for each reference is given next to taxon name and T refers to a TYPE strain.

Figure 23. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial sequence of tub2 gene in 9.2A and selected reference fungal isolates. GenBank Accession number for each reference is given next to taxon name.

Figure 24. Morphological characterisation of Group 3 type specimen isolate 9.2A. This isolate morphologically fits within Simplicillium.

Figure 25. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial sequence of ITS region in 12.1A and selected reference fungal isolates. Branches with bootstrap values of greater than 60% from 500 bootstrap replication are marked next to next to each branch. GenBank Accession number and CBS number (where available) for each reference is given next to taxon name and T refers to a TYPE strain.

Figure 26. Bootstrap consensus tree generated through Maximum Likelihood analysis of partial sequence of tub2 gene in 12.1A and selected reference fungal isolates. Branches with bootstrap values of greater than 60% from 500 bootstrap replication are marked next to next to each branch. GenBank Accession number and CBS number (where available) for each reference is given next to taxon name and T refers to a TYPE strain.

Figure 27. Morphological characterisation of Group 4 type specimen isolate 12.1A. The best match morphologically is Emericellopsis glabra, at 97% to ITS region.

Figure 28. Brachiaria organs/tissues sampled to determine the in planta distribution of selected endophytes following inoculation into novel hosts species.

Figure 29. Confirmation of the presence of endophyte strain 12.1.B in B. humidicola root and pseudostem. Mycelia was stained with aniline blue and examined at 40x magnification under bright field microscope.

Example 1 - Molecular Characterisation of Brachiaria-Urochloa Grasses

Brachiaria-Urochloa grass species seed batches were sourced from within Australia (Table 1). This resource provided the basis for endophyte discovery and characterisation from the grass species complex Brachiaria-Urochloa.

Table 1. Brachiaria-Urochloa species used for endophyte discovery.

Seed Batch Brachiaria name Urochloa name 5 Brachiaria brizantha Urochloa brizantha (Hochst. ex A. Rich.) (Hochst. ex A. Rich.) R.D. Webster Stapf. 1,6,7,10,13,14 Brachiaria decumbens Urochloa decumbens Stapf. (Stapf) R.D. Webster 2,4,8,9,15 Brachiaria humidicola Urochloa humidicola (Rendle) Schweick (Rendle) Morrone &Zuloaga 3,11,12 Brachiaria stolonifera Urochloa mosambicensis Gooss (Hack.) Dandy

To characterise the diversity of the grass species and confirm their assignment to the Brachiaria-Urochloa complex, genetic diversity analysis was conducted using simple sequence repeat (SSR) markers derived from Brachiaria-Urochloa. The primer pairs BbUNICAMPOO1, BbUNICAMPOO2, BbUNICAMPOO3, BbUNICAMPOO4, BbUNICAMPOO5, BbUNICAMP006 and BbUNICAMP007 were selected (Jungmann et al. 1999) and used to amplify across species of Brachiaria-Urochloa.As the ploidy levels between different Brachiaria-Urochloa species varies, alleles for each SSR locus were scored dominantly (presence/absence) and principal components analysis (PCA) was performed (Figure 1).

Each of the Brachiaria-Urochloa species was effectively discriminated using these markers. No variation within populations was observed, as expected for apomictic species. B. brizantha and B. decumbens are more similar to each other than they are to B. humidicola and U. mosambicensis. There are two B. humidicola populations, with Humidicolal being distinct from Humidicola2 and U. mosambicensis. The genetically distinct nature of the Humidicolal and Humidicola2 plants suggests that there are two different (sub)-species present in the B. humidicola seed batches analysed.

Example 2 - Isolation of Fungal Endophytes from Brachiaria-Urochloa Grasses

Fungal endophytes from Brachiaria-Urochloa grasses were isolated from surface sterilised young tiller explants (Figure 2). A total of 31 fungal endophytes were isolated and subcultured. Twenty nine fungal endophyte isolates were identified by morphological examination in in vitro culture. Two fungal endophyte isolates (14.1.A and 14.1.D) were excluded from further analysis. Table 2 shows a summary of the fungal endophytes isolated from Brachiaria-Urochloa grasses.

Table 2. Summary of purified and subcultured fungal endophytes isolated from Brachiaria-Urochloa grasses. Isolate names are coded such that the first number represents the seed batch and the second number the plant number from 20 seed germinated from each seed batch.

Endophyte Host Plant Identification based on morphological isolate ID characteristics 1.1.A B. decumbens Acremonium sp. 3.3.A U. mosambicensis Acremonium sp. 3.3.B U. mosambicensis Acremonium sp. 3.3.C U. mosambicensis Acremonium sp. 4.9.A B. humidicola (2) Acremonium sp. 4.9.B B. humidicola (2) Acremonium sp. 5.1.A B. brizantha Acremonium sp. 5.1.B B. brizantha Acremonium sp. 5.1.D B. brizantha Acremonium sp. 5.1.E B. brizantha Acremonium sp. 7.1.A B. decumbens Acremonium sp. 8.1.A B. humidicola (1) Acremonium sp. 8.1.B B. humidicola (1) Acremonium sp. 8.1.C B. humidicola (1) Acremonium sp. 9.2.A B. humidicola (1) Acremonium sp. 9.2.B B. humidicola (1) Acremonium sp. 9.2.C B. humidicola (1) Acremonium sp. 10.1.A B. decumbens Acremonium sp. 11.1.A U. mosambicensis Acremonium sp. 12.1.A U. mosambicensis Acremonium sp. 12.1.B U. mosambicensis Acremonium sp. 12.1.C U. mosambicensis Acremonium sp. 12.1.D U. mosambicensis Acremonium sp. 12.1.E U. mosambicensis Acremonium sp. 14.1.A B. decumbens Unknown (Sterile) 14.1.C B. decumbens Acremonium sp. 14.1.D B. decumbens Possibly Paecilomyces 14.1.B B. decumbens Acremonium sp. 15.2.C B. humidicola (1) Acremonium sp. 15.2.E B. humidicola (1) Acremonium sp. 15.2.D B. humidicola (1) Acremonium sp.

Example 3- Genetic Characterization of Fungal Endophytes from Brachiaria Urochloa Grasses

The internal transcribed spacer ITS sequence was used for further characterisation. The entire region of nuclear ribosomal DNA which comprises both internal transcribed spacers ITS1 and ITS2 and the 5.8S subunit was PCR-amplified using primers ITS5 and ITS4 (White et al. 1990). Purified PCR amplification products were sequenced using Sanger sequencing technology. Isolated subcultured endophytes were then grouped based on ITS sequence identity. Sequence data was used in BLASTn analysis to identify matches in the NCBI database (Table 3).

Phylogenetic analysis of 29 fungal endophytes isolated from Brachiaria-Urochloa grasses identified 4 distinct clades based on nuclear rDNA ITS sequence (Figure 3). Morphological differences in the endophytes exist both between and within these ITS groups (Figure 4). Endophyte isolates within each clade matched (599% identity) to a wide range of different Ascomycetes (Table 3). None of the endophyte isolates isolated from the Brachiaria-Urochloa grasses displayed 100% identity to the nuclear rDNA ITS sequence from other fungi within the public database, indicating unique fungal endophytes have been isolated.

Molecular analysis of the 29 endophyte isolates with nuclear rDNA ITS data identified presence of multiple endophyte strains within the same plant for plants 9.2 and 12.1 (Table 4). The presence of multiple endophyte strains within the one host plant is not usually observed in other grass species such as perennial ryegrass and tall fescue, suggesting a novel discovery in Brachiaria.

Table 3. Summary of fungal endophytes isolated from Brachiaria-Urochloa grasses characterised using ITS sequence-based analysis. Fungal endophytes are grouped by ITS sequence identity and the closest BLAST match for each ITS clade is shown.

Group Accession # Species - best BLASTn match 1 AB540569 Acremonium atrogriseum DQ317343 Ascomycete sp. 21 Brachiaria endophytes FJ235936 Fungalsp. AB190399 Phialophora intermedia FM177651 Uncultured compost fungus Group Accession # Species - best BLASTn match 2 U57674 Acremonium alternatum FN706550 Acremonium egyptiacum 1.1.A, 9.2.B, 10.1.A, HQ649793 Acremonium sp. 12.2.B, 12.1.C EU520092 Acremonium strictum EU427036 Cladosterigma sp. EU520121 Cytospora chrysosperma AM176743 Hypocreales sp. EU754963 Uncultured fungus Group Accession # Species - best BLASTn match 3 EF577237 Acremonium sp. AJ292395 Cephalosporium lanoso-niveum 9.2.A HQ270477 Simplicillium lanosoniveum FJ861375 Simplicillium lanosoniveum HQ191403 Uncultured Dikarya EF685278 Uncultured fungus DQ443734 Verticillium fungicola Group Accession # Species - best BLASTn match 4 AB540572 Acremonium dichromosporum AY882946 Acremonium exuviarum 12.1.A, 12.1.D HQ914927 Acremonium sp. AY632658 Emericellopsis donezkii AY632657 Emericellopsis glabra AY632659 Emericellopsis humicola AB425984 Emericellopsis microspora AY632660 Emericellopsis minima AY632667 Emericellopsis pallida AY632666 Emericellopsis salmosynnemata HQ914819 Emericellopsis sp. AY632665 Emericellopsis synnematicola AB425993 Emericellopsis terricola AY632671 Stanjemonium grisellum AY632672 Stanjemonium ochroroseum 1 FJ939394 Stilbella fimetaria

Table 4. Summary of the number of endophytes isolated from each Brachiaria or Urochloa plant and the corresponding number of nuclear rDNA ITS groups identified.

Plant number Species host # Endophytes isolated # ITS groups 1.1 B. decumbens 1 1 3.3 U. mosambicensis 3 1 4.9 B. humidicola 2 2 1 5.1 B. brizantha 4 1 7.1 B. decumbens 1 1 8.1 B. humidicola 1 3 1 9.2 B. humidicola 1 3 3 10.1 B. decumbens 1 1 11.1 U. mosambicensis 1 1 12.1 U. mosambicensis 5 3 14.1 B. decumbens 2 1 1 5.2 B. humidicola 1 3 1

Example 4 - Metabolic Profiling of Brachiaria-Urochloa Grass-Endophyte Associations

Mature plants of Brachiaria-Urochloa grass-endophyte associations that had been maintained in a controlled environment were subjected to metabolic profiling analysis. Three individual plants (biological replicates) from each seed batch were analysed using liquid chromatography-mass spectrometry (LC-MS), with two technical replicates per plant. Additional plants representing the Humidicolal and Humidicola2 sub-groups identified in the SSR-based genetic diversity analysis were selected from seed batches 2, 4 and 8. Freeze-dried pseudostem samples were prepared for LC-MS analysis using an 80% methanol extraction procedure.

Principal Components Analysis (PCA) based on the full LC-MS dataset reveals differences in metabolic profiles of each Brachiaria-Urochloa grass-endophyte association analysed (Figure 7). Each of the associations forms a distinct cluster, indicating that there is limited variation within a species/population. As for the SSR based genetic analysis, there are two separate B. humidicola populations, with Humidicolal samples forming a separate cluster to the remaining populations. The 3D

PCA plot indicates that B. decumbens and B. brizantha associations share similar metabolic profiles as do Humidicola2 and U. mosambicensis associations.

The fungal endophyte-derived compound peramine, known to have insecticidal activity, was produced in planta and was thus identified in the metabolic profiles of the Urochloa mosambicensis grass-fungal endophyte associations (Figure 3). The presence of peramine was confirmed through MS (ions extracted at the mass-to-charge ratio [m/z] of 248). All samples of the Urochloa mosambicensis grass-fungal endophyte associations tested produced the endophyte-derived insecticidal compound peramine (Table 5 and Figure 8).

Table 5. Determination of presence of the fungal endophyte-derived insecticidal compound peramine in Brachiaria-Urochloa grass-fungal endophyte associations. Samples of Brachiaria-Urochloa grass-fungal endophyte associations were selected for metabolic profiling analysis. Three plants (biological replicates) from each group were analysed. Samples of the Urochloa mosambicensis grass-fungal endophyte associations tested produced the endophyte-derived insecticidal compound peramine.

Seed Batch Species Peramine(+-) i Brachiaria decumbens 2 Brachiaria humidicola1 2 Brachiaria humidicola2 3 Urocholoa mosambicensis +

4 Brachiaria humidicolaI 4 Brachiaria humidicola2 5 Brachiaria brizantha 6 Brachiaria decumbens 7 Brachiaria decumbens 8 Brachiaria humidicolaI 8 Brachiaria humidicola2 9 Brachiaria humidicola 10 Brachiaria decumbens 11Urocholoa mosambicensis +

12 Urocholoa mosambicensis +

13 Brachiaria decumbens 14 Brachiaria decumbens 15 Brachiaria humidicola

Example 5 - Antifungal Activity of endophytes isolated from Brachiaria/Urochloa species complex

A previous publication reported antifungal activity in the Acremonium implicatum endophytic fungus isolated from Brachiaria brizantha (Kelemu et al. 2001). To investigate antifungal activity in the endophytes isolated here, all 29 endophytic fungi were tested against 8 model test fungi: Alternaria alternata, Colletotrichum graminicola, Rhizoctonia cerealis, Trichoderma harzianum, Phoma sorghina, Botrytis cinerea, Bipolaris portulaceae and Drechslera brizae. Petri dishes containing potato dextrose agar were inoculated with a central colony of each endophyte isolate, and incubated 10 days at 240C. Two inoculum of a model test fungus were then placed on opposite sides of each dish. Cultures were incubated at room temperature in the dark during 5 days and the size of the zone of inhibition was visually assessed on a scale of 0-5 (0 - no inhibition; 1 - very weak inhibition; 2 - weak inhibition; 3 - moderate inhibition; 4 - strong inhibition; 5 - very strong inhibition. For each endophyte-fungal pathogen combination five replicates were scored and the scores averaged. Endophyte isolate 9.2.A displayed strong, broad spectrum antifungal activity, inhibiting growth of all but Botrytis cinerea and Trichoderma harzianum (Table 6, Figure 9). There were distinct differences in the level of antifungal activity across ITS groups - with group 3 (isolate 9.2.A) displaying the strongest, followed by group 2 (isolates 12.1.A and 12.1.D) and group 2 (5 isolates). Endophyte isolates within the ITS group 1 showed minimal inhibition of growth of pathogenic fungi (Table 6).

Table 6. Antifungal activity exhibited by endophytes from Brachiaria against plant pathogenic fungi. The size of the zone of inhibition was visually assessed on a scale of 0-5 (0 - no inhibition; 1 - very weak inhibition; 2 - weak inhibition; 3 - moderate inhibition; 4 - strong inhibition; 5 - very strong inhibition).

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Example 6 - Whole Genome Sequencing of Fungal Endophytes Isolated from Brachiaria-Urochloa Grasses

Methodologies for whole genome sequencing of fungal endophytes based on massive parallelisation of sequencing reactions have been established using sequencing platforms such as the Illumina HiSeq2000. High quality genomic DNA is prepared from mycelia samples from fungal endophytes isolated from Brachiaria-Urochloa grasses, sub-cultured in liquid media. DNA from each fungal endophyte strain is prepared for sequencing using established methodologies. Samples may be sequenced in multiplex using an indexing approach. The Illumina HiSeq2000 platform is based upon sequencing by synthesis approach, where millions of DNA fragments are bound to the surface of a glass flow cell and then amplified in situ to produce a discrete cluster of DNA strands. Sequencing is achieved by the addition of polymerase and 4 nucleotides differentially fluorescently labelled with an inactive 3'-OH group that ensures only a single nucleotide is incorporated with each cycle. Each base-incorporation is followed by image capture and then chemical cleavage to remove the fluorescent dye to enable base extension. The sequence is compiled by image overlay after sequence cycling is completed. Compiled sequences are checked for quality prior to genome assembly and analysis.

Five endophyte isolates (1.1.A, 3.3.A, 5.1.B, 9.2.A and 12.1.E) were sequenced using the Illumina HiSeq2000 platform. Paired end reads from each isolate were used as input for de novo genome sequence assembly. Analysis of assembled sequenced revealed isolates from within the same ITS group showed similar sequence assembly characteristics (Table 7).

Table 7.

ITS-Group Isolate Assembled Size# Contigs >100bp Largest Contig N50 #reads input # reads used 1 3.3.A 33,194,262 6,173 282,024 23,771 23,286,068 19,779,082 1 5.1.B 33,453,571 5,937 331,319 34,056 35,030,948 29,733,043 1 12.1.E 33,707,236 6,168 250,614 25,466 17,237,708 15,676,548 2 1.1.A 33,542,777 2,529 1,912,494 302,046 19,152,972 17,145,454 4 9.2.A 29,635,075 1,705 1,830,966 584,893 25,280,459 26,552,756

To investigate the level of diversity among the 5 endophyte strains (1.1.A, 3.3.A, 5.1.B, 9.2.A and 12.1.E), the GAPDH gene was identified by using the Neurospora crassa GAPDH cDNA sequence as a query in a BLASTn search of a sequence database comprising contigs from the 5 endophyte isolates. The GAPDH gene sequences were polymorphic between ITS groups, but highly similar within groups (Figure 10), suggesting isolates 3.3.A, 5.1.B and 12.1.E may be the same strain. Phylogenetic analysis of the GAPDH protein confirmed the 3 ITS groups to which isolates 1.1.A, 9.2.A and 12.1.E belong to are divergent from one another and all other fungi within an in-house fungal database (Figure 11).

To further interrogate the level of diversity among the 3 isolates belonging to ITS group 1, the Epichloe festucae peramine A gene (perA) (GenBank Accession # BAE06845) and homologous genes within ITS group 1 endophytes 3.3.A, 5.1.B and 12.1.E were aligned. Sequence polymorphism was observed within ITS group 1 isolates possibly suggesting different strains (Figure 12).

Example 7 - Molecular Taxonomy and Morphological Analysis

Methods for Molecular Taxonomy

Genomic DNA was extracted from lyophilized fungal mycelia by the cetyltrimethylammonium bromide (CTAB) extraction method, and the quality and quantity of the DNA was assessed by both agarose gel electrophoresis and specific absorbance measurements using the NanoDrop 2000 Spectrophotometer (Thermo Scientific). The entire region of nuclear ribosomal DNA which comprises both internal transcribed spacers ITS1 and ITS2 and the 5.8S subunit was PCR-amplified using primers ITS5 and ITS4. Purified PCR amplification products were sequenced using Sanger sequencing technology. Sequence data was used in BLASTN analysis to identify matches in the NCBI database.

For each fungal DNA sample, paired-end libraries with inserts c. 400 bp in size were prepared using the standard protocol (TruSeq DNA Sample Prep V2 Low Throughput: Illumina Inc., San Diego, USA) with paired-end adaptors. Library quantification was performed using the KAPA library quantification kit (KAPA Biosystems, Boston, USA). Paired-end libraries were pooled according to the nature of the attached adaptors and sequence analysed using the HiSeq2000 platform (Illumina Inc.), following the standard manufacturer's protocol. All generated sequence reads were assured by filtering and trimming of reads based on quality using a custom Python script, which calculates quality statistics and stores trimmed reads in several fastq files.

In order to assemble each of the interested gene sequence such as 18S, 28S, tub2, and tefA, matching reads for each gene were identified from the database of all trimmed reads for each fungal genome, through BLASTN using gene sequence from a closely related species as the reference, and defining the E value threshold as 0.1. From this BLAST output, all corresponding paired reads (both forward and reverse) were extracted from the database, and gene assembly was performed using the Linux based de novo assemblers Velvet ver.1.2.10. Different hash lengths (K-mer sizes) ranging from 21 to 61 were tested as appropriate for different sequence read sets, and the minimum contig length was always defined as 100 bp. Values for estimated coverage and coverage cut-off were set to auto.

Multiple alignments of the complete gene sequences for each gene were performed individually using ClustalW with default parameters. To reconstruct tree topology, maximum likelihood method was used as implemented in MEGA 5.2 with default parameters and 500 bootstrap replicates. All positions containing gaps and missing data were eliminated during the analysis.

Methods for Morpholoqical Analysis

For morphological characterisation, the isolates were examined on oatmeal agar (OA: 30 g oat flakes, 20 g agar, 1 L distilled water), potato dextrose agar (PDA, Difco Laboratories) and malt extract agar (MEA). Cultures were incubated in the dark at 250C. Colony diameters were measured at 9 days and again at 24 days (three replicates per isolate/medium combination). Colony colours were determined at 24 days growth. In descriptions (following), colour colony codes in parenthesis refer to Kornerup and Wanscher (1978): Methuen Handbook of Colour, 3 rd Edition, Methuen London. The microscopic features were examined and measured in lactic acid under a light microscope (Olympus BX-50) using oil immersion (x1000).

Group 01 - Type specimen isolate 3.3A - Molecular taxonomy

ITS sequence comparison for 3.3A using sequences from TYPE material provided 99% match with 87% coverage to Phialemonium atrogriseum CBS 604.67 TYPE (NR 111521.1). Phylogenetic relationships were reconstructed for 3.3A fungal strain and Phialemonium atrogriseum as well as closely related species of Phialemonium atrogriseum based on partial sequences for ITS (Figure 13), 28S (Figure 14) and tub2 gene (Figure 15) using the Maximum Likelihood method as implemented in MEGA 5.2. Similar tree topologies were observed for each genomic region (Figures 13 to 15) and in each phylogram, fungal strain 3.3A clustered with reference sequences from Phialemonium atrogriseum as indicated by the type specimen with CBS number 604.67.

Group 01 - Type specimen isolate 3.3A - Morpholoqical description

Colonies on OA: 18-23 mm diameter after 9 days and 57-62 mm diameter after 24 days at 25 0C, flat, olive-grey (7E3) with white margins, exudate lacking, margin entire, soluble pigment not produced.

Colonies on PDA: 19-24 mm diameter after 9 days and 52-61 mm diameter after 24 days at 250 C, powdery pale grey (7B1), exudate lacking, reverse pink-brown (7D4), margin entire, soluble pigment not produced.

Colonies on MEA: 22-27 mm diameter after 9 days and 47-65 mm diameter after 24 days at 250C, floccose olive-grey (28D2) with pale margins, exudate lacking, reverse chocolate brown (7F8), margin entire, soluble pigment not produced.

Profuse sporulation on aerial aggregated hyphae growing directly upwards on PDA and MEA. Growth on OA is very different; granular appearance, no aerial mycelium. Dark clusters of phialides scattered over surface of OA media. Hyphae pigmented brown, fused in aggregates.

Phialides single arising laterally off aerial hyphae (PDA, MEA) or in clusters (on OA); flask shaped, occasionally with a markedly inflated base, with a barely visible collarette. (5-)9-

11(-12) x (1-)3-4 pm wide at base, no basal septum. Conidia ovoid, smooth-walled, 1 celled, 3-5(-7) x (2.5-)3-4 pm, subhyaline to pale brown, in slimy heads (Figure 16).

Group 01 - Type specimen isolate 3.3A - Suqqested taxonomy

Based on the above, the suggested taxonomy is Phialemonium atrogriseum.

Group 02 - Type specimen isolate 1.1 A - Molecular taxonomy

ITS sequence comparison for 1.1A using sequences from TYPE material provided 100% match to Acremonium sclerotigenum [CBS 124.42, from dune sand, TYPE (AJ61772.1)] with 85% coverage and 99% match to Acremonium egyptiacum [CBS 114785, clinically relevant specimen, TYPE, (FN706550.1)] with 93% coverage. Phylogenetic relationships were reconstructed for 1.1A fungal strain and Acremonium sclerotigenum as well as closely related Acremonium species based on partial sequences for ITS (Figure 17), 18S (Figure 18), 28S (Figure 19) and tub2 (Figure 20), gene using the Maximum Likelihood method as implemented in MEGA 5.2. Although phylogram derived from ITS sequence shows close affinity of 1.1A to Acremonium sclerotigenum, as well as to Acremonium egyptiacum, and Acremonium alternatum, phylograms of 18S, 28S, and tub2 sequences clearly reveals taxon status of 1.1A as Acremonium sclerotigenum.

Group 02 - Type specimen isolate 1.1 A - Morphological description

Colonies on OA: 32-33 mm diameter after 9 days and to the edge of the plate (85 mm diameter) after 24 days at 25C, floccose pale brown aerial mycelium (5B3), exudate lacking, reverse pale brown (5B3), margin entire, soluble pigment not produced.

Colonies on PDA: 32-41 mm diameter after 9 days and 72-75 mm diameter after 24 days at 25 0C, floccose white aerial mycelium (4A1), exudate lacking, reverse cream (4A3), margin entire, soluble pigment not produced.

Colonies on MEA: 34-41 mm diameter after 9 days and 72-80 mm diameter after 24 days at 25 0C, floccose pale cream to salmon aerial mycelium (4A2-5A2), radially folded, exudate lacking, reverse pale orange-brown (5B6), margin entire, soluble pigment not produced.

Profuse sporulation on aerial hyphae on all media. In older parts of the colony the hyphae are aggregated in thick ropes. Some hyphae with intercalary swollen cells (may be thin walled chlamydospores).

Phialides exclusively solitary, arising from aerial hyphae, slender, gradually tapering towards apex (19-)26-38(-60) x 1-2 pm, with a basal septum. Conidia formed in globose slimy heads. Conidia ovoid to ellipsoid (most shaped like lemon pips), smooth-walled, 1 celled, 3.5-4.5(-5.5) x (1.0-)1.5-2.0(-3.0) pm (Figure 21).

Group 02 - Type specimen isolate 1.1 A - Suqqested taxonomy

Based on the above, the suggested taxonomy is Acremonium sclerotigenum, even though A. sclerotigenum type has more cylindrical-shaped conidia, whereas 1.1A has lemon-pip shaped conidia.

Group 03 - Type specimen isolate 9.2A - Molecular taxonomy

ITS sequence comparison for 9.2A using sequences from TYPE material provided 99% match to Simplicillium aogashimaense [JCN 18167 from soil in Japan, TYPE (NR 111026.1)] with 97% coverage and 96-97% match to 6 other Simplicillium spp. types. Phylogenetic relationships were reconstructed for 9.2A fungal strain and Simplicillium aogashimaense as well as closely related Simplicillium species based on partial sequences for ITS (Figure 22) and tub2 (Figure 23), gene using the Maximum Likelihood method as implemented in MEGA 5.2. Phylogram derived from ITS sequence shows close relationship of 9.2A to Simplicillium aogashimaense.

Group 03 - Type specimen isolate 9.2A - Morpholoqical description

Colonies on OA: 29-32 mm diameter after 9 days and 62-66 mm diameter after 24 days at 250C, velvety, white aerial mycelium, exudate lacking, margin entire, soluble pigment not produced.

Colonies on PDA: 32-43 mm diameter after 9 days and 61-66 mm diameter after 24 days at 250C, velvety, white, exudate lacking, reverse cream-yellow (4A3) with dark brown centre (9F7), margin entire, soluble pigment not produced.

Colonies on MEA: 28-32 mm diameter after 9 days and 67-72 mm diameter after 24 days at 250C, velvety, white, exudate lacking, reverse dark brown (9F7), margin entire, soluble pigment not produced.

Moderate sporulation on aerial mycelium, no hyphal aggregation observed.

Phialides solitary, arising at right angles directly from aerial hyphae, very long and slender, 72-115 x 0.7-1.0 pm, no basal septum. Conidia formed in globose slimy heads. Conidia cylindrical, smooth-walled, 1-celled, 3.5-5.5 x 1.0-1.5(-2.0) pm (Figure 24).

Group 03 - Type specimen isolate 9.2A - Suqqested taxonomy

Based on the above, the suggested taxonomy is Simplicillium sp. The phialides are much longer than other described species; 9.2A may also be a closely related species of Simplicillium.

Group 04 - Type specimen isolate 12.1A - Molecular taxonomy

ITS sequence comparison for 12.1A using sequences from TYPE material provided 99% match to Emericellopsis humicola [CBS 180.56, associated with the seaweed Fucus, TYPE, (AY632659.1)] with 93% coverage, 98% match to Acremonium exuviarum [a lizard associated fungus with affinity to Emericellopsis; TYPE (NR 077167.1)] with 98% coverage and 97% match to Emericellopsis glabra [CBS 119.40, associated with the seaweed Fucus, TYPE (AY632657.1)] with 93% coverage. Phylogenetic relationships were reconstructed for 12.1A fungal strain and Emericellopsis species based on partial sequences for ITS (Figure 25) and tub2 (Figure 26), gene using the Maximum Likelihood method as implemented in MEGA 5.2. Both phylograms revealed a similar topology indicating unique nature of the 12.1A genome.

Group 04 - Type specimen isolate 12.1A - Morpholoqical description Colonies on OA: 33-39 mm diameter after 9 days and to the edge of the plate (85 mm diameter) after 24 days at 250C, powdery, salmon pink (6A4) with tufts of fluffy white aerial mycelium, exudate lacking, margin entire, soluble pigment not produced.

Colonies on PDA: 36-38 mm diameter after 9 days and 52-72 mm diameter after 24 days at 250C, powdery salmon pink (6A4) with darker centre (6A3) and tufts of fluffy white aerial mycelium, exudate lacking, reverse salmon pink (6A7), margin entire, soluble pigment not produced.

Colonies on MEA: 29-32 mm diameter after 9 days and 43-70 mm diameter after 24 days at 250C, powdery, greyish pink (6B3) with cream sectors (5A2), radially folded, exudate forming sparse pale yellow drops, reverse yellow-brown (5C8), margin entire, soluble pigment not produced. On MEA, at the edge of the colony, the conidiophores grow directly upwards with droplets of conidia at the apices.

Profuse sporulation on surface of all media giving slimy appearance, in older parts of the colony the hyphae are aggregated in thick ropes.

Phialides single, slender, gradually tapering towards apex (25-)27-33(-39) x 1.5-2 pm, with a basal septum. Conidia formed in globose slimy heads (possibly in chains). Conidia shape variable: ovoid, ellipsoid or cylindrical, smooth-walled, 1-celled, 4.0-5.5 x 1.5-2.0 pm (Figure 27).

Group 04 - Type specimen isolate 12.1A - Suqqested taxonomy

Based on the above, the suggested taxonomy is Emericellopsis sp., possibly a new species of Emericellopsis.

Example 8 - Inoculation of Fungal Endophytes into Brachiaria-Urochloa Host Plant Panel

Methodologies for inoculating isolated and subcultured fungal endophytes into seedlings (Figure 5) and regenerating calli (Figure 6) from Brachiaria-Urochloa grass species were developed to enable the generation of novel grass host-fungal endophyte associations between Brachiaria-Urochloa grass species and endophytes isolated from a range of pasture grass species (including species within the Brachiaria-Urochloa complex).

Endophyte selection

Five fungal endophytes representing four of the rDNA sequence-defined clades were identified as candidates for inoculation and characterisation into a Brachiaria-Urochloahost panel (Table 8).

Table 8. Endophyte isolates selected for inoculation.

Endophyte Host Plant cla Species Deposit isolate ID ded Identificationa reference number (in vitro)

5.1.B B. brizantha 1 Acremonium V11/011372 Low atrogriseumb 12.1.B U. mosambicensis 2 Acremonium Not Medium sclerotigenum deposited 1.1.A B. decumbens 2 Acremonium V11/011370 Medium sclerotigenum 9.2.A B. humidicola (1) 3 Simplicillium sp. V11/011373 High 12.1.A U. mosambicensis 4 Emericellopsis sp. V11/011374 Medium aMolecular phylogeny was determined based on detailed morphological study combined with sequence

analysis at multiple loci. bAcremonium atrogriseum was renamed Phialemonium atrogriseum in 2013 based on based on a detailed phenetic and genetic study [Mycologia. 105(2):398-421]. cDeposited at The National Measurement Institute on 15 June 2011. drDNA-sequence defined clade (ITS group).

Brachiaria host panel

A host panel comprising commercially relevant Brachiaria-Urochloa germplasm was established to enable inoculation of genetically novel and highly diverse endophyte isolates into a single host genotype. An optimised method for endophyte inoculation into host plants free of microbial organisms in axenic conditions was developed, facilitating a high frequency of successful inoculation (i.e. as above, Figure 6).

The host panel represents a wide variety of cultivars of commercial relevance, including Brachiaria distachya, Brachiaria decumbens, Hybrid brachiaria, Urochloa mosambicensis, Brachiaria brizantha and Brachiaria humidicola.

Endophyte inoculation into the Brachiaria-Urochloa host panel

Selected endophyte strains were inoculated into the host panel to create a matrix of Brachiaria-Urochloa species-endophyte associations. Approximately 50 inoculations were performed for each endophyte strain (Table 9). Commercial Brachiaria species and endophyte isolates with greatest commercial application (inoculation efficiency) were prioritised in developing this matrix.

Table 9. Number of inoculations performed.

Host plant species Endophyte isolate (rDNA-sequence defined clade) 5.1.B 12.1.B 1.1.A 9.2.A 12.1.A 1 2 2 3 4 Brachiaria brizantha (Mekong Briz) 48 46 46 47 47 Brachiaria brizantha (Marandu) 85 65 54 114 56 Urochloa mosambicensis 59 54 - - Brachiaria humidicola 47 40 - - Brachiaria Hybrid (d,r,b) 55 58 - - Brachiaria decumbens 56 53 - - Brachiaria distachya 65 45 - -

Endophyte inoculation frequency was determined for each candidate endophyte, approximately 6 months post inoculation, using a diagnostic (i.e. specific allele sizes at each SSR loci for each endophyte) set of simple sequence repeat (SSR) markers for each endophyte isolate.

Successful inoculation was achieved for representative endophytes from ribosomal DNA sequence-defined clades 1, 2 and 4 (Table 10). Data for Table 10 is for stable Brachiaria Urochloa endophyte associations identified 3 to 6 months post inoculation. A diagnostic set of SSR markers specific for Brachiaria-Urochloa endophytes are used to test for endophyte presence and identity in planta. Shown is the percentage of endophyte positive plants identified from the total number of plants harvested. The Brachiaria-Urochloa species from which the endophyte was isolated is highlighted in light grey.

Table 10. Summary of inoculation frequencies(%).

Host plant species Endophyte isolate (rDNA-sequence defined clade) 5.1.B 12.1.B 5.1.B 12.1.B 5.1.B 1 2 1 2 1 Brachiaria brizantha (Mekong Briz) 7% 24% 15% 0% 0% Brachiaria brizantha (Marandu) 14% 15% 0% 0% 4% Urochloarmosamnbicensis 0% 3%f - - ||||||||||||||||||||||||||| Brachiaria humidicola 2% 42%- -

Brachiaria Hybrid (d,r,b) 0% 8%- -

Brachiaria decumbens 2% 15% -

Brachiaria distachya 4% 0%- -

Variation between endophyte isolates representing different ITS groups was observed. ITS2 (12.1.B) >ITSI(5.1B)> IT2 (.1A) > ITS3(12.1.A)>ITS4 (9.2.A). Cross species compatibility was also observed. In this study, isolates 5.1.B and 12.1.B exhibit broader compatibility compared to the moderately compatible 1.1.A and 12.1.A. No associations were identified for candidate endophyte 9.2A (IT3), most likely due to the observed detrimental effect of this endophyte onplant survival following inoculation.

Example 9 - Methods for determining the physical nature of Brachiaria endophyte in planta

Distribution of inoculated endophyte in the brachiaria host plant

The distribution of Brachiaria-Urochloa endophytes in planta was determined for selected endophyte strains. To characterise the distribution of these endophytes without the confounding effects of the presence of other fungal endophytes, isolates were inoculated into the Brachiaria-Urochloa host panel (as described above in example 8). The distribution of endophyte strains 5.1.B (ITS1) and 12.1.B (ITS2) was determined through molecular analysis for the presence of endophyte in different organs/tissues of endophyte inoculated mature Brachiaria plants. Tissues analysed represent the entire host plant: fine brown roots (FR), white roots (WR), lower pseudostem (LP), mid pseudostem (MP), upper pseudostem (UP), leaves (L), and, where available, inflorescence (I) (Figure 28).

For each host-endophyte combination 2-5 plants were tested independently. Samples were analysed in replicate and 50-60 mg of (dry weight) plant material was used for each sample. Harvested plant material was freeze-dried, and DNA was extracted using the DNeasy plant 96 kit (Qiagen, Hilden, Germany) following the manufacturer's recommended method. Purified DNA was stored at -20°C.

For each sample, endophyte presence was detected by amplicon generation from total genomic DNA template with endophyte strain specific SSR markers. PCR amplification was performed in a 20 pL reaction containing 5 pL genomic DNA (5-10 ng), 1 X PCR buffer (Bioline, London, UK), 0.2 mM of each dNTP (GE Healthcare, Chalfont St Giles, UK), 0.5 U Immolase (Bioline), and 0.25 pM each primer (Applied Biosystems, Foster City, California, USA; Invitrogen, Van Allen Way, Carlsbad, USA). Cycling conditions for each reaction consisted of 10 min at 95 0C followed by 10 cycles of 30 s at 940 C, 60 s at the initial annealing temperature (65 0C), (reducing the temperature at 1 0C per cycle), and 1 min at 72 0C followed by 20 cycles of 30 s at 94C, 60 s at the final annealing temperature (55C), and 1 min at 720 C followed by a final extension of 10 min at 720 C. Templates from pre determined endophyte-positive and -negative plant genotypes were included as controls.

Amplification products were diluted with sterile ultrapure water (Sartorius AG) at a ratio of 1:10, and 2 pL of the diluted product was mixed with 7.95 pL Hi-Di Formamide (Applied Biosystems) and 0.05 pL 500 LIZ (Applied Biosystems) size standard before analysing on the ABI 3730xl automated capillary electrophoresis platform (Applied Biosystems). Products were detected using the GeneMapper version 3.7 software (Applied Biosystems, 2004), and endophyte-positive samples were identified by the presence of amplification peaks, specific to a minimum of two of the strain specific SSR markers.

The distribution of selected candidate endophytes is summarised in Table 11 (Codes for tissue/organ type are described in Figure 28). Endophyte strain 5.1.B appears to be predominately located in the roots of B. humidicola. Endophyte strain 12.1.B appears to be distributed throughout the host plants tested, B. humidicola and B. decumbens. Further characterisation of the inflorescence is required to determine endophyte presence in this organ.

Table 11. Summary of endophyte incidence in selected host-endophyte combinations.

Host species Endophyte strain 5.1.B 12.1.B Brachiaria decumbens Not tested WR/FR/LP/MP/L Brachiaria humidicola WR/FR WR/LP/MP/UP/L

The presence of endophyte strains was further confirmed by microscopic examination. Pseudostem samples prepared using a modified procedure described by Hignight et al. (1993). Briefly, samples were killed and fixed in Carnoy's solution (6:3:1 ethyl alcohol: chloroform: 85% glacial acetic acid) for 24 hr, stained in aniline blue and cleared using a gradient of methyl salicylate:ethyl alcohol. Root samples were fixed and stained with aniline blue. Endophyte strain 12.1.B was observed in root and pseudostem tissue of B. humidicola by microscopic examination (Figure 29).

Re-isolation of inoculated fungal endophytes

Inoculated fungal endophytes 5.1.B and 12.1.B were directly isolated from pseudostem and root tissue. Five tillers as well as root material were removed from each endophyte inoculated Brachiaria plant and washed in running tap water to remove any visible dirt. The tissue pieces were surface-sterilised with 80% (v/v) ethanol for 2 minutes, followed by 5% (w/v) NaOCI (bleach) for 5 minutes in a laminar flow cabinet. Subsequently, tissue pieces were rinsed three times in sterile water to remove any residual bleach, and allowed to completely dry on sterile paper (Whatman Qualitative 3, 9 cm diameter), with the bottom of the tiller facing to the front of the laminar flow hood. Both the root and the tiller material was chopped into approximately 2 mm pieces and placed downwards on potato dextrose agar (PDA) supplemented with 250 mg/I cefotaxime. Petri dishes were sealed and incubated at 250C for up to 4 weeks, and monitored for contamination on each day. Outgrowing mycelium was sub-cultured onto new PDA medium and incubated at 220C in the dark. Strain identity will be confirmed by ITS sequence analysis and strain specific SSR analysis.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

References

Jungmann, L., A. C. B. Sousa, et al. (2009). Isolation and characterization of microsatellite markers for Brachiaria brizantha (Hochst. ex A. Rich.) Stap. Conservation Genetics 10(6): 1873-1876.

Kelemu, S., White J.F, Jr., et al. (2001). "An endophyte of the tropical forage grass Brachiaria brizantha: Isolating, identifying, and characterizing the fungus, and determining its antimycotic properties." Canadian Journal of Microbiology 47(1): 55-62.

White, T.J., Bruns, T., Lee, S., and Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications pp. 315-322. Academic Press.

Hignight, K.W., Muilenburg, G.A., van Wijk, A.J.P. (1993) A clearing technique for detecting the fungal endophyte Acremonium sp. in grasses. Biotechnic and Histochemistry 68 (2): 87 90.

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PCTAU2017050848‐seql‐000001‐EN‐20170814.txt SEQUENCE LISTING

<110> Agriculture Victoria Services Pty Ltd <120> METHODS OF CHARACTERISING ENDOPHYTES

<130> PC‐DPI‐0111

<150> AU2016903175 <151> 2016‐08‐12

<160> 10

<170> PatentIn version 3.5

<210> 1 <211> 360 <212> DNA <213> Neurospora crassa

<400> 1 gacctcgtcg tcaacggcaa gaaggtcaag ttctacactg agcgcgaccc cgctgccatc 60

ccctggtccg agaccggtgc cgactacatt gtcgagtcca ctggtgtctt caccaccacc 120

gagaaggcct ccgcccactt gaagggtggt gccaagaagg tcatcatctc tgccccctct 180

gctgatgccc ccatgtacgt tatgggtgtc aacaacgaga cctacgatgg ctccgccgac 240

gtcatctcca acgcctcttg caccaccaac tgcttggctc ccctcgccaa ggtcatccac 300

gacaacttca ccatcgtcga gggtctcatg accaccgtcc actcctacac cgccacccag 360

<210> 2 <211> 352 <212> DNA <213> Acremonium

<400> 2 cgtcaacggc aagaaggtca agttctacac cgagcgcgac cccgccgcca ttccctggaa 60

ggacaccggc gccgagtaca tcgttgagtc caccggtgtc ttcaccacca ccgacaaggc 120

tgccgctcac ttgaagggcg gtgccaagaa ggtcatcatc tccgcccctt cggccgatgc 180

ccccatgtac gtgatgggtg tcaacgagaa gacctacgac ggcaaggccg atgtcatctc 240

caacgcttct tgcaccacca actgcctggc tcccctcgcc aaggtcatcc acgacaagtt 300

cggcattgtc gagggtctca tgaccaccgt ccactcctac actgccaccc ag 352

Page 1

PCTAU2017050848‐seql‐000001‐EN‐20170814.txt

<210> 3 <211> 360 <212> DNA <213> Acremonium

<400> 3 gaccttgtcg tgaacggcaa gaagatccgt ttctacggtg agcgcgaccc cgccgccatc 60

ccctggaagg agactgccgc cgagtacgtt gtcgagtcca ctggtgtctt caccactacc 120

gacaaggcca aggcccatct tcagggtggt gccaagaagg ttgtcatctc tgctccttct 180

gccgacgccc ccatgtacgt tatgggtgtc aacgagaaga cctacgacgg caaggccgat 240

gtcatctcta acgcttcttg caccaccaac tgcctggctc ccctcgccaa ggtcctccac 300

gacaagttcg gtatcgttga gggtctcatg accaccatcc actcttacac cgccacccag 360

<210> 4 <211> 360 <212> DNA <213> Acremonium

<400> 4 gacctcgtcg tcaatggcaa gaaggtcaag ttctacaccg agagggatcc ggctgccatc 60

ccgtggaagg acaccggcgc cgagtacatc gtcgagtcca ccggtgtctt caccaccact 120

gagaaggccg gtgctcactt gaagggtggt gccaagaagg tcatcatctc ggccccctct 180

gccgatgccc ccatgtacgt catgggcgtc aacgagaagt cgtacgacgg cagcgccaac 240

gtcatctcca acgcgtcgtg caccaccaac tgcctggctc ccctggccaa ggtcatcaac 300

gacaagttca ccattgtcga gggcctgatg accaccatcc acgcctacac cgccacccag 360

<210> 5 <211> 360 <212> DNA <213> Acremonium

<400> 5 gacctcgtcg tcaatggcaa gaaggtcaag ttctacaccg agagggatcc ggctgccatc 60

ccgtggaagg acaccggcgc cgagtacatc gtcgagtcca ccggtgtctt caccaccact 120

gagaaggccg gtgctcactt gaagggtggt gccaagaagg tcatcatctc ggccccctct 180

gccgatgccc ccatgtacgt catgggcgtc aacgagaagt cgtacgacgg cagcgccaac 240 Page 2

PCTAU2017050848‐seql‐000001‐EN‐20170814.txt

gtcatctcca acgcgtcgtg caccaccaac tgcctggctc ccctggccaa ggtcatcaac 300

gacaagttca ccattgtcga gggcctgatg accaccatcc acgcctacac cgccacccag 360

<210> 6 <211> 360 <212> DNA <213> Acremonium

<400> 6 gacctcgtcg tcaatggcaa gaaggtcaag ttctacaccg agagggatcc ggctgccatc 60

ccgtggaagg acaccggcgc cgagtacatc gtcgagtcca ccggtgtctt caccaccact 120

gagaaggccg gtgctcactt gaagggtggt gccaagaagg tcatcatctc ggccccctct 180

gccgatgccc ccatgtacgt catgggcgtc aacgagaagt cgtacgacgg cagcgccaac 240

gtcatctcca acgcgtcgtg caccaccaac tgcctggctc ccctggccaa ggtcatcaac 300

gacaagttca ccattgtcga gggcctgatg accaccatcc acgcctacac cgccacccag 360

<210> 7 <211> 600 <212> DNA <213> Epichloe festucae

<400> 7 tcatcgtcga gggccccgtc ctcgctgcgg gttacctcaa cgatgacgct aagacggcga 60

gggcgtacat cgagaatccc gcctgggtcc gtaaggcgca cttccggccc gctcagcccc 120

gccgccggtt ctaccgcacg ggggatcttg ggcgtcaggc tgtcgacgga tctattacat 180

tcataggccg tgctgatttc caggttaagg ttcgtggcca gcgtatggag ctcggggagg 240

tgcggtcgca tattgtggct tgcctgcctg aggctgttga cattcacgtc gacgtcatct 300

gtcccgaggg ggagaaggtc ctcgcggcct tcctctcgtt cggcaagggt ggcgatgatg 360

gccagcagca gaagcagcag cagcagcagc agcagggcgc tatccgagtc caccagcccg 420

accaggctct ggcggattcg ctccgctcca tggtcgaaaa gctgagacag actctgccac 480

ctgctgcggt tccatcgttc ttcgttccca taaccgggtt tccctacctc gtctcgggga 540

aggtagatcg gcggagcctg ttgagcttcg ccaacgggtc gtcggtggag gagctggcgt 600

Page 3

PCTAU2017050848‐seql‐000001‐EN‐20170814.txt <210> 8 <211> 600 <212> DNA <213> Acremonium

<400> 8 tcatcgtcga gggccccgtc ctcgctgcgg gttacctcaa cgatgacgct aagacggcga 60

gggcgtacat cgagaatccc gcctgggtcc gtaaggcgca cttccggccc gctcagcccc 120

gccgccggtt ctaccgcacg ggggatcttg ggcgtcaggc tgtcgacgga tctattacat 180

tcataggccg tgctgatttc caggttaagg ttcgtggcca gcgtatggag ctcggggagg 240

tgcggtcgca tattgtggct tgcctgcctg aggctgttga cattcacgtc gacgtcatct 300

gtcccgaggg ggagaaggtc ctcgcggcct tcctctcgtt cggcaagggt ggcgatgatg 360

gccagcagca gaagcagcag cagcagcagc agcagggcgc tatccgagtc caccagcccg 420

accaggctct ggcggattcg ctccgctcca tggtcgaaaa gctgagacag actctgccac 480

ctgctgcggt tccatcgttc ttcgttccca taaccgggtt tccctacctc gtctcgggga 540

aggtagatcg gcggagcctg ttgagcttcg ccaacgggtc gtcggtggag gagctggcgt 600

<210> 9 <211> 597 <212> DNA <213> Acremonium

<400> 9 tcatcgtcga gggccccgtc ctcgctgcgg gttacctcaa cgatgacgct aagacggcga 60

gggcgtacat cgagaatccc gcgtgggtcc gcaaggcgca cttccggccc gctcagcccc 120

gccgccggtt ctaccgcacg ggggatcttg ggcgtcaggc tgtcgacgga tctattacct 180

tcatcggccg cgctgatttt caggttaagg ttcgtggcca gcgtatggag ctcggggagg 240

tgcgatcgca tattgtggct tgcctgcctg aggctgttga tatccacgtc gacgtcatct 300

gtcccgaggg ggagaaggtc ctcgcggcct tcctatcgtt cggcgagggt ggcgatgacg 360

gccagcagca gaagcagcag cagcaacagc agggcgctat ccgagtccac cagcccgacc 420

aggctctagc ggattcgctc cgctccatgg tcgagaagct gagacagact ctgccacctg 480

ctgcggttcc atcgttcttc gttcccgtaa ccgggtttcc ctacctggtc tcggggaagg 540

tagatcggcg gagcctgttg agcttcgcca atgggtcgtc ggtggagcag ctggcgt 597 Page 4

PCTAU2017050848‐seql‐000001‐EN‐20170814.txt

<210> 10 <211> 528 <212> DNA <213> Acremonium

<400> 10 tcgagaatcc cgcgtgggtc cgtaaggcgc acttccggcc cgctcagccc cgccgccggt 60

tctaccgcac gggggatctt gggcgtcagg ctgtcgacgg atctatcacc ttcatcggcc 120

gcgctgattt ccaggttaag gttcgtggtc agcgtatgga gctcggggag gtgcggtcgc 180

atattgtggc ttgcctgcct gaggctgttg atatccacgt cgacgtcatc tgtcccgagg 240

gggagaaggt cctcgcggcc ttcctatcgt tcggcgaggg tggcgatgac ggccagcagc 300

agaagcagca gcagcaacag cagggcgcta tccgagtcca ccagcccgac caggctctag 360

cggattcgct ccgctccatg gtcgagaagc tgagacagac tctgccacct gctgcggttc 420

catcgttctt cgttcccgta gccgggtttc cctacctggt ctcggggaag gtagatcggc 480

ggagcctgtt gagcttcgcc aatgggtcgt cggtggagca gctggcgt 528

Page 5

Claims (14)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for characterising and selecting a fungus for inoculation into a host plant, said method including subjecting a fungus to genetic analysis, wherein genetic analysis includes: sequencing one or more non-coding nuclear ribosomal DNA (nrDNA) regions; sequencing one or more regions of a protein-coding gene, wherein the protein-coding gene is selected from one or more of a tub2 gene, a tefA gene and an act1 gene, and fragments thereof; and assessing the genetic variation of the sequenced regions; subjecting the fungus to a metabolic analysis; and selecting a fungus having a desired genetic variation and/or a desired metabolic profile that will confer a beneficial phenotype to the host plant.

2. A method according to claim 1, wherein the nrDNA is selected from one or more of genes encoding small subunit (SSU), large subunit (LSU), and 5.8S nrDNAs, and fragments thereof.

3. A method according to claim 2, wherein the non-coding nrDNA is selected from one ?0 or more of an ETS, NTS or ITS spacer.

4. A method according to claim 3, wherein the non-coding nrDNA is an ITS spacer.

5. A method according to any one of claims 1 to 4, wherein the protein-coding gene is a tub2 gene.

6. A method according to any one of claims 1 to 5, wherein the fungus is an endophyte of a plant.

7. A method according to claim 6, wherein the plant is a grass.

8. A method according to claim 7, wherein the grass is a forage, turf or bioenergy grass.

9. A method according to claim 8, wherein the grass is a plant of the Brachiaria-Urochloa complex.

10. A method according to any one of claims 1 to 9, wherein the beneficial phenotype includes improved tolerance to water and/or nutrient stress and/or improved resistance to pests and/or disease relative to a plant lacking the fungus.

11. A method according to claim 10, wherein the beneficial phenotype includes improved resistance to pests.

12. A method according to claim 10 or 11, wherein the improved resistance to pests includes an insecticidal, insect repellent, or antifungal phenotype.

13. A method according to any one of claims 1 to 12, wherein the method includes the further step of subjecting the fungus to a morphological analysis.

14. A method according to any one of claims 1 to 13, wherein the method includes the further step of assessing geographic origin of the fungus.