# Patent

## Title
Brachiaria-urochloa endophytes

## Abstract

The present invention relates to fungi of Acremonium spp, wherein said fungi are purified or isolated from plants of the Brachiaria-Urochloa complex and wherein, when said fungi are inoculated into a plant, said plant has improved resistance to diseases and/or pests relative to an uninocualated control plant. The present invention also relates to plants inoculated with such fungi, products produced by the fungi and related genes, proteins and methods.

## Background
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 novel endophytes and their deployment in Brachiaria-Urochloa plant improvement programs.
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 of Acremonium spp, wherein said fungus is 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 uninocualated control plant.
Preferably, the fungus is selected from the group consisting of Acremonium 1.1.A, Acremonium 3.3.A, Acremonium 3.3.B, Acremonium 3.3.C, Acremonium 4.9.A, Acremonium 4.9.B, Acremonium 5.1.A, Acremonium 5.1.B, Acremonium 5.1.D, Acremonium 5.1.E, Acremonium 7.1.A, Acremonium 8.1.A, Acremonium 8.1.B, Acremonium 8.1.C, Acremonium 9.2.A, Acremonium 9.2.B, Acremonium 9.2.C, Acremonium 10.1.A, Acremonium 11.1.A, Acremonium 12.1.A, Acremonium 12.1.B, Acremonium 12.1.C, Acremonium 12.1.D Acremonium 12.1.E, Acremonium 14.1.B, Acremonium 14.1.C, Acremonium 15.2.C, Acremonium 15.2.D, Acremonium 15.2.E, as described herein.
Representative samples, namely Acremonium 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) were deposited at The National Measurement Institute. 1/153 Bertie Street, Port Melbourne, Victoria, Australia, 3207, on 7 Jun. 2011 with accession numbers V11/011370, V11/011371, V11/011372, V11/011373, and V11/011374, respectively. Replacement deposits were made on Apr. 15, 2016 in response to a notification of non-viability, and were assigned the same accession numbers.
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 (eg. 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 marlothii, Brachiaria nigropedata, Urochloa dictyoneura, Urochloa oligotricha, Urochloa panicoides, Brachiaria obtusiflora, Brachiaria serrifolia, Urochloa advena, Urochloa arrecta, Urochloa brachyura, Urochloa eminii, Urochloa mollis, Urochloa xantholeuca, Urochloa oligotricha, Urochloa panicoides, Urochloa plantaginea, Urochloa platynota and Urochloa xantholeuca, as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex.
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×Brachiaria brizantha, Brachiaria ruziziensis×Brachiaria decumbens, [Brachiaria ruziziensis×Brachiaria decumbens]×Brachiaria brizantha, [Brachiaria ruziziensis×Brachiaria brizantha]×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.
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 milliliters to cubic meters, for example from approximately 50 ml to approximately 50,000 liters. 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 repellant.
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 of Acremonium spp., selected from the group consisting Acremonium 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, Gln
        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, Gln, Lys, Arg, His, Trp
        Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln
    
    


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, e.g. 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 (nptII) 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 (eg. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.
As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the genetic construct in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical assays (e.g. GUS assays), 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
FIG. 1. Principal components analysis (PCA) analysis of genetic diversity between Brachiaria-Urochloa grass species using dominantly scored SSR markers.
FIG. 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 25° C. 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.
FIG. 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.
FIG. 4. Morphology of representative fungal endophytes isolated from Brachiaria-Urochloa grass species. Endophyte isolates are grouped based on ITS sequence analysis.
FIG. 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 26° C. 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.
FIG. 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.
FIG. 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.
FIG. 8. LC-MS analysis of Urocholoa 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.
FIG. 9. An example of inhibition reactions in the antifungal assay. Acremonium endophyte isolate 9.2.A was tested for antifungal activity against 8 species of pathogenic fungi.
FIG. 10. DNA sequence alignment of the GAPDH gene from Neurospora crassa with homologues of 5 fungal Acremonium endophyte isolates. The 3 different nuclear rDNA ITS groups to which the 5 Acremonium isolates belong are as follows: 2nd line—Group 2; 3rd line—Group 3; Lines 4, 5 and 6—Group 1.
FIG. 11. Relevant section of a Neighbour-Joining tree derived from alignment of the GAPDH protein displaying the novel identity of 3 Acremonium isolates. Acremonium endophytes are highlighted to indicate ITS groups 1, 2 and 3. Note: only 1 Acremonium 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.
FIG. 12. Alignment of the Epichloe festucae perA gene (1_0) with homologous genes from Acremonium isolates from ITS group 1 (3.3.A, 5.1.B and 12.1.E).



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.)



Stapf.
R. D. Webster


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 BbUNICAMP001, BbUNICAMP002, BbUNICAMP003, BbUNICAMP004, BbUNICAMP005, 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 (FIG. 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 Humidicola1 being distinct from Humidicola2 and U. mosambicensis. The genetically distinct nature of the Humidicola1 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 (FIG. 2). A total of 31 fungal endophytes were isolated and subcultured. Twenty nine fungal endophyte isolates were identified as Acremonium species by morphological examination in in vitro culture. Two fungal endophyte isolates (14.1.A and 14.1.D) were not of the Acremonium morpho-type and 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

Identification based on



isolate ID
Host Plant
morphological 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
As Acremonium is an anamorphic genus, 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 (FIG. 3). Morphological differences in the endophytes exist both between and within these ITS groups (FIG. 4). Endophyte isolates within each clade matched (≦99% 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


21 Brachiaria endophytes
DQ317343
Ascomycete sp.



FJ235936
Fungal sp.



AB190399
Phialophora intermedia



FM177651
Uncultured compost fungus


2
U57674
Acremonium alternatum


1.1.A, 9.2.B, 10.1.A,
FN706550
Acremonium egyptiacum


12.2.B, 12.1.C
HQ649793
Acremonium sp.



EU520092
Acremonium strictum



EU427036
Cladosterigma sp.



EU520121
Cytospora chrysosperma



AM176743
Hypocreales sp.



EU754963
Uncultured fungus


3
EF577237
Acremonium sp.


9.2.A
AJ292395
Cephalosporium lanoso-




niveum



HQ270477
Simplicillium lanosoniveum



FJ861375
Simplicillium lanosoniveum



HQ191403
Uncultured Dikarya



EF685278
Uncultured fungus



DQ443734
Verticillium fungicola


4
AB540572
Acremonium dichromosporum


12.1.A, 12.1.D
AY882946
Acremonium exuviarum



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



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


15.2
B. humidicola 1
3
1









EXAMPLE 4—INOCULATION OF FUNGAL ENDOPHYTES INTO BRACHIARIA-UROCHLOA HOST PLANTS
Methodologies for inoculating isolated and subcultured fungal endophytes into seedlings (FIG. 5) and regenerating calli (FIG. 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).
EXAMPLE 5—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 Humidicola1 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 (FIG. 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 Humidicola1 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 Urocholoa mosambicensis grass-fungal endophyte associations (FIG. 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 Urocholoa mosambicensis grass-fungal endophyte associations tested produced the endophyte-derived insecticidal compound peramine (Table 5 and FIG. 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 Urocholoa mosambicensis grass-fungal endophyte


associations tested produced the endophyte-derived insecticidal


compound peramine.









Seed Batch
Species
Peramine (+/−)












1
Brachiaria decumbens
−


2
Brachiaria humidicola1
−


2
Brachiaria humidicola2


3
Urocholoa mosambicensis
+


4
Brachiaria humidicola1
−


4
Brachiaria humidicola2


5
Brachiaria brizantha
−


6
Brachiaria decumbens
−


7
Brachiaria decumbens
−


8
Brachiaria humidicola1
−


8
Brachiaria humidicola2


9
Brachiaria humidicola
−


10
Brachiaria decumbens
−


11
Urocholoa mosambicensis
+


12
Urocholoa mosambicensis
+


13
Brachiaria decumbens
−


14
Brachiaria decumbens
−


15
Brachiaria humidicola
−









EXAMPLE 6—ANTIFUNGAL ACTIVITY OF ACREMONIUM 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 Acremonium 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 24° C. 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, FIG. 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 4 (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).

















Host 
Endophyte
Group 
Bipolaris
Colletotrichum
Rhizoctonia
Altemaria
Drechsiera
Phoma
Botrytis
Trichoderma


Id
Strain
(ITS)
portulaceae
graminicota
cerealis
altemata
brizae
sorghina
cinerea
harzianum





B.b
5.1.A
1
1-2
1-2
1-2
0
1-2
3-4
1-2
0


B.b
5.1.B
1
1-2
1-2
3-4
0
1-2
3-4
1-2
0


B.b
5.1.D
1
3-4
1-2
1-2
0
3-4
3-4
1-2
0


B.b
5.1.E
1
1-2
1-2
1-2
0
1-2
3-4
1-2
0


B.d
14.1.B
1
1-2
1-2
3-4
1-2
3-4
3-4
3-4
0


B.d
14.1.C
1
1-2
1-2
1-2
0
1-2
1-2
3-4
0


B.d
7.1.A
1
1-2
1-2
1-2
0
1-2
1-2
3-4
0


B.h1
15.2.C
1
1-2
1-2
1-2
1-2
1-2
3-4
3-4
0


B.h1
15.2.D
1
1-2
1-2
1-2
0
1-2
1-2
3-4
0


B.h1
15.2.E
1
1-2
0
1-2
0
1-2
1-2
1-2
0


B.h1
8.1.A
1
1-2
1-2
1-2
0
1-2
1-2
1-2
0


B.h1
8.1.B
1
1-2
1-2
1-2
0
3-4
3-4
3-4
0


B.h1
8.1.C
1
1-2
1-2
1-2
0
3-4
3-4
3-4
0


B.h1
9.2.C
1
1-2
1-2
3-4
0
1-2
1-2
3-4
0


B.h2
4.9.A
1
1-2
1-2
1-2
0
1-2
3-4
1-2
0


B.h2
4.9.B
1
1-2
1-2
1-2
0
1-2
3-4
1-2
0


U.m
11.1.A
1
3-4
1-2
3-4
0
3-4
1-2
3-4
0


U.m
12. 1.E
1
1-2
1-2
3-4
0
1-2
1-2
3-4
0


U.m
3.3.A
1
1-2
1-2
1-2
0
1-2
1-2
1-2
0


U.m
3.3.B
1
1-2
1-2
1-2
0
1-2
1-2
3-4
0


U.m
3.3.C
1
1-2
1-2
1-2
0
1-2
1-2
1-2
0


B.d
1.1.A
2
1-2
3-4
5
1-2
1-2
1-2
1-2
1-2


B.d
10.1.A
2
1-2
3-4
3-4
1-2
3-4
1-2
1-2
1-2


B.h1
9.2.B
2
1-2
5
3-4
0
1-2
3-4
1-2
1-2


U.m
12.1.B
2
1-2
5
5
1-2
3-4
1-2
0
1-2


U.m
12.1.C
2
1-2
3-4
5
3-4
3-4
1-2
0
1-2


B.h1
9.2.A
3
5
5
5
5
5
5
3-4
1-2


U.m
12.1.A
4
3-4
5
5
1-2
3-4
1-2
3-4
1-2


U.m
12.1.D
4
1-2
3-4
5
1-2
3-4
3-4
3-4
3-4









EXAMPLE 7—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 >100 bp
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 (FIG. 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 (FIG. 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 (FIG. 12).
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.

## Claims

1. A method of increasing resistance to pests and/or diseases in a plant, said method comprising the step of inoculating said plant with a fungi of Acremonium spp., wherein, when said fungus is inoculated into the plant, said plant has improved resistance to diseases and/or pests relative to an uninoculated control plant and wherein the internal transcribed spacer (ITS) of said fungi has 99% or greater sequence identity with the internal transcribed spacer of Acremonium 9.2A or Acremonium 3.3A.

2. The method according to claim 1, wherein said increased resistance to pests and/or diseases is selected from the group consisting of insecticidal activity, insect repellent activity and antifungal activity.

3. The method according to claim 1, wherein said fungus is purified or isolated from a plant of the Brachiaria-Urochloa species complex.

4. The method according to claim 3, wherein said plant of the Brachiaria-Urochloa complex is selected from the group consisting of Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola and Urochloa mosambicensis.

5. The method according to claim 3, wherein said fungus is isolated 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.

6. The method according to claim 5, wherein said method further includes the step of assessing geographic origin of the fungi and selecting fungi having a desired genetic and metabolic profile and a desired geographic origin.

7. The method according to claim 5, wherein said genetic analysis includes detecting the presence or absence of polymorphic markers.

8. The method according to claim 3, wherein said plant of the Brachiaria-Urochloa 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 marlothii, Brachiaria nigropedata, Urochloa dictyoneura, Urochloa oligotricha, Urochloa panicoides, Brachiaria obtusiflora, Brachiaria serrifolia, Urochloa advena, Urochloa arrecta, Urochloa brachyura, Urochloa eminii, Urochloa mollis, Urochloa xantholeuca, Urochloa oligotricha, Urochloa panicoides, Urochloa plantaginea, Urochloa platynota and Urochloa xantholeuca.

9. The method according to claim 1, wherein said fungus is selected from the group consisting of Acremonium 9.2A, Acremonium 3.3A and Acremonium 5.1B.

10. The method of claim 1, wherein the inoculated plant is a grass species.

11. The method of claim 10, wherein the inoculated plant is a forage, turf or bioenergy grass.

12. The method of claim 11, wherein the inoculated plant is a member of the Brachiaria-Urochloa species complex.

13. The method of claim 11, wherein the inoculated plant is a member of the genera Lolium or Festuca.

14. A method of increasing resistance to pests and/or diseases in a plant, said method comprising the step of inoculating said plant with a fungi of Acremonium spp., wherein, when said fungus is inoculated into the plant, said plant has improved resistance to diseases and/or pests relative to an uninoculated control plant and wherein the internal transcribed spacer (ITS) of said fungi has 99% or greater sequence identity with the internal transcribed spacer of Acremonium 3.3A.