Modified endophyte, methods of modification and uses thereof

Abstract

Described herein is a modified endophyte, methods of modification and uses thereof. The modified endophyte may be modified to enhance traits / characteristics for properties such as fungicide resistance / tolerance. Modification may be via isolation, in vitro culturing, selection and reintroduction to a plant host. The modified endophytes and methods may be used to produce endophytes and related plant:endophyte symbiotic relationships with enhanced properties such as enhanced fungicide resistance.

Description

[0001] MODIFIED ENDOPHYTE, METHODS OF MODIFICATION AND USES THEREOF

[0002] RELATED APPLICATIONS

[0003] This application derives priority from New Zealand patent application number 817804 filed on 10 January 2025 with WIPO DAS code 7209 incorporated herein by reference.

[0004] TECHNICAL FIELD

[0005] Described herein is a modified endophyte, methods of modification and uses thereof. The modified endophyte may be modified to confer fungicide resistance properties (traits).

[0006] Modification may be via isolation, in vitro culturing, selection and reintroduction to a plant host. The modified endophytes and methods may be used to produce endophytes and related plant:endophyte symbiotic relationships with enhanced properties such as enhanced fungicide resistance.

[0007] BACKGROUND

[0008] Producing plants with enhanced characteristics has been a common practice as long as farming exists.

[0009] In more recent decades, it was discovered that some plants are host plants to other organisms termed herein as obligate symbionts. The combination of host plant and obligate symbiont has been a natural evolutionary process. Its discovery led to better understanding of symbiosis and how to use this symbiosis to achieve desired outcomes.

[0010] A known symbiosis exists between fungal endophytes and host grass plants. The fungal endophytes live in the host grass and produce alkaloid compounds that can be found in the host plant such as in the leaves and pseudostem. The alkaloid compounds can improve plant resistance and / or tolerance to pests or drought. Some alkaloids, or some level of some alkaloids, can however be unwanted in agricultural settings because of the negative effect they may have on animals foraging on the host plants and consuming the alkaloid compounds as part of their diet. A great effort has been put into endophyte discovery to find endophytes with preferential alkaloid profiles.

[0011] A review by Ayesha et al. 2021 showed that most endophytes, regardless of the endophyte -host plant combination, are detrimentally affected by fungicide application. In the pastoralscenario, Adusei-Fosu et. al. 2025 indicates for farms that report pasture diseases, 95% are dealing with rust or other fungal infections. Of those farms, 36% use fungicides as pest control strategies. Fungicide application is thus common practice, and likely to become an even more common practice within farms, due to climate change raising temperature and humidity levels. Thus, endophytes are indirectly targeted and will likely be increasingly indirectly targeted in the future. This is particularly important in certain grass seed production scenarios were maintaining a high (>70%) level of desired endophyte viability is crucial to the integrity of the final seed product. This creates a need of either specifically targeted fungicides that do not affect endophytes or fungicide resistant endophytes.

[0012] The genetics of fungicide resistance is well understood. For example, Angelini et. al. 2015 describes how acquired resistance to fungicides in fungal plant pathogens is a challenge in modern crop protection. Fungi are adaptable to changing environmental conditions, such as the introduction of a new fungicide in the agricultural practice. According to Angelini et. al. 2015, several genetic mechanisms may underlie fungicide resistance and influence the chance and time of its appearance and spreading in fungal populations. Resistance may be caused by mutations in major genes (monogenic or oligogenic resistance) or in minor genes (polygenic resistance) which may occur in nuclear genes as well as in cytoplasmic genes. Angelini et. al 2015 provides a table of the genes and genetic bases of resistance to most major fungicide groups in fungal plant pathogens but has no teaching or suggestion around obligate symbionts. Angelini et. al 2015 further describes that resistant mutants can be obtained from samples under laboratory conditions through selection of spontaneous mutations or following chemical or physical mutagenesis and that molecular tools, such as gene cloning, sequencing, site-directed mutagenesis and gene replacement, make genetic studies on fungicide resistance amenable. In general, the development of fungicide resistance or tolerance is seen as a challenge in agriculture (as the crop protecting fungicides are no longer effective), it has not been seen as an opportunity.

[0013] The use of the above knowledge in endophytes is limited. State of the art in endophyte and fungicide resistance / tolerance is currently limited to finding what fungicides can be safely used on endophyte and not trying to make the endophytes more tolerant or resistant to fungicides that may impact the endophyte. For example, Rolston 2002 looked at what fungicide treatments were safe to use with ryegrass endophytes but did not look to enhance the endophyte against non-safe fungicides.

[0014] Hume et. al. 2025 demonstrated that the technology has not changed as they used a similar method to determine what fungicides could be safely used on Epichloe endophytes of Triticumspp. They did not try to modify the endophyte for enhanced fungicide resistance despite realising that fungicides were detrimental to endophytes but needed for crop protection. "A complicating factor is that fungicides may still be reguiredfor crop management, which in turn could be detrimental to the Epichloe endophyte. While this issue has been studied for pasture and amenity grasses, no similar studies have been performed with endophyte-infected cereals". The author is not aware of research to develop fungicide resistant endophyte, rather the current art seems to be avoidance of fungicides that detrimentally affect endophytes.

[0015] Endophytes have been modified to date via genetic modification by knocking out specific genes to adjust the alkaloids produced and hence, avoid deleterious effects on animals yet still have enhanced pest and drought resistance. No commercial products exist from this research to date that the inventors are aware of. Endophyte isolation from a host plant is known as well as in vitro growth on Agar plates (Potato Dextrose Agar (PDA), or similar) to observe endophyte growth and enable the differentiation of endophyte types.

[0016] Published research exists on identifying Epichloe fungi that inhibit the fungal growth in culture of other fungi, but the authors are not aware of research into creating fungicide resistance in the endophyte itself. The research is largely around inhibiting fungi that is pathogenic or disease causing to the host plant species.

[0017] In more recent times, significant effort has gone into use of genetic modification and gene editing or mutagenesis generally to produce modified endophyte variants. A drawback of mutagenesis (especially in asexual material, like Epichloe endophyte) is that the mutagenized populations suffer, due to Muller's Rachet, an accumulation of deleterious mutations over a specific advantageous mutation. Genetic modification and gene editing is associated with high costs of regulation and release may be difficult or impossible in a commercial meaningful manner.

[0018] Another drawback of genetic modification and gene editing or mutagenesis generally is that the methods may be complex and subject to tight proprietary ownership and licensing (if a licence is available) hence, not able to be widely deployed commercially.

[0019] Those skilled in the art have been able to isolate endophytes and replace endophytes in a different host grass using in vitro techniques. However, in the inventor's experience, this process has not fundamentally changed the host genetics or the endophyte genetics (unless biotechnology (genetic engineering or gene editing) has been applied). Such novel host / endophyte combinations may provide altered alkaloid profiles and altered growth profiles of both endophyte and host, due to the new unique relationship created (GxG interactions of newplant host and new endophyte), but this does not involve deliberate (non-biotechnological) alteration of the host or endophyte DNA. The process described herein uses an in vitro endophyte culture stage as a unique selective step to create desired DNA changes in the endophyte that can confer new useful traits / characteristics to the endophyte and or the host endophyte relationship.

[0020] In the inventor's knowledge, random mutagenesis and the biotechnology techniques of genetic modification and gene editing have been used to alter the characteristic(s) of the endophyte and so the endophyte / host plant interaction, but in vitro recurrent selection to alter characteristics of use in the application of the endophyte / host plant has not been used previously. In addition, it is the inventors experience that the art does not describe growing an endophyte in culture to achieve an enhanced characteristic, prior to returning said endophyte to its host or another host and conferring that characteristic (aside from the use of biotechnological techniques) to the symbiotic relationship e.g., developing an endophyte with enhanced fungicide resistance / tolerance, and then returning that endophyte to a host plant and maintaining the capability of fungicide resistance / tolerance within the symbiotic relationship.

[0021] Previous publications have presented fungicide resistance as a differential characteristic in endophytes and have shown this to be of great interest in the industry, for example, CM 142 (NZ Patent, 747602) and AR584 (US Patent 8,465,963). These examples have a level of natural fungicide resistance / tolerance over and above previously existing commercial endophytes, but they have not been enhanced for additional fungicide resistance.

[0022] It may be useful to be able to develop enhanced versions of endophyte with fungicide resistance; or with additional fungicide resistance; or with fungicide tolerance; or with additional fungicide tolerance. It may further be useful to provide a modified endophyte, methods of modification, or uses to produce or take advantage of enhanced selected for characteristics or at least provide the public with a choice.

[0023] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.Further aspects and advantages of the modified endophyte, methods of modification and uses thereof will become apparent from the ensuing description that is given by way of example only.

[0024] SUMMARY

[0025] Described herein is a modified endophyte, methods of modification and uses thereof. The modified endophyte may be modified to confer fungicide resistance properties (traits).

[0026] Modification may be via isolation, in vitro culturing, selection and reintroduction to a plant host. Selection may be recurrent phenotype selection and / or clonal selection. The modified endophytes and methods may be used to produce endophytes and related plant:endophyte symbiotic relationships with enhanced properties such as enhanced fungicide resistance. In a first aspect, there is provided a method of modifying an obligate symbiont comprising:

[0027] isolating an obligate symbiont from its plant host;

[0028] culturing the isolated obligate symbiont in vitro;

[0029] modifying the obligate symbiont through selective propagation to enhance a selected for characteristic of the obligate symbiont selected from one or more of: fungicide resistance, additional fungicide resistance, fungicide tolerance, additional fungicide tolerance; modification occurring through selection, using no genetic modification, genetic engineering, or gene editing technology;

[0030] placing the modified obligate symbiont into a plant host; and

[0031] growing the plant host and modified obligate symbiont together.

[0032] In a second aspect, there is provided a modified obligate symbiont produced by the method substantially as described above.

[0033] In a third aspect, there is provided the use of the modified obligate symbiont produced by the method substantially as described above to produce a host grass and obligate symbiont combination with an enhanced characteristic provided by the modified obligate symbiont. The above-described modified obligate symbiont, method of modification and uses thereof may provide advantages over existing endophytes and modification methods. Examples of advantages found by the inventor may comprise one or more of the following:Provision of and methods of producing, endophytes with fungicide resistance; additional fungicide resistance; fungicide tolerance; and / or additional fungicide tolerance.

[0034] An advantage over mutation breeding in asexual species (such as Epichloe endophyte), as this method avoids the accumulation of random deleterious mutations (Mullers Ratchet) whilst in the pursuit of a specific advantageous mutation. The method of modification only selects for desirable mutations in the characteristic required and does not create large numbers of random mutations as is observed in mutation breeding.

[0035] The method above also provides an advantage over new breeding technologies (e.g., genetic modification or gene editing) as it avoids complexity associated with genetic modification, genetic engineering and gene editing (the direct manipulation of an organism's DNA using biotechnology to add, delete, or alter specific genes). Further, the method is not bound by the regulatory hurdles that genetic modification, genetic engineering and gene editing are bound by and hence, can be deployed more quickly, in more environments and with fewer restrictions.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Further aspects of the modified endophyte, methods of modification and uses thereof will become apparent from the following description that is given by way of example only and with reference to the accompanying drawings in which:

[0038] Figure 1 is a photograph of three agar plates used to culture similar sized pieces of NEA58 (Control or non-fungicide resistant / tolerant) and NEA58 (Selected or resistant / tolerant) endophyte on 6x carbendazim fungicide containing agar plates, taken on day 0 of a trial;

[0039] Figure 2 is a photograph of three agar plates used to culture similar sized pieces of NEA57 (Control) and NEA57 (Selected) endophyte on 6x carbendazim fungicide containing agar plates, taken on day 0 of a trial;

[0040] Figure 3 is a photograph of two agar plates used to culture similar sized pieces of NEA58 (Control) and NEA58 (Selected) endophyte on plain agar, taken on day 0 of a trial;

[0041] Figure 4 is a photograph of two agar plates used to culture similar sized pieces of NEA57 (Control) and NEA57 (Selected) endophyte on plain agar, taken on day 0 of a trial;Figure 5 is a photograph of four agar plates used to culture selected similar sized pieces of NEA58 (Control) and NEA58 (Selected) endophyte on plain agar, taken after 4 weeks growth;

[0042] Figure 6 is a photograph of four agar plates used to culture selected similar sized pieces of NEA57 (Control) and NEA57 (Selected) endophyte on plain agar, taken after 4 weeks growth;

[0043] Figure 7 is a photograph of four agar plates used to culture selected similar sized pieces of NEA64 (Control) and NEA64 (Selected) endophyte on plain agar, taken after 4 weeks growth;

[0044] Figure 8 is a photograph of four agar plates used to culture selected similar sized pieces of NEA68 (Control) and NEA68 (Selected) endophyte on plain agar, taken after 4 weeks growth;

[0045] Figure 9 is a photograph of four agar plates used to culture selected similar sized pieces of NEA67 (Control) and NEA67 (Selected) endophyte on plain agar, taken after 4 weeks growth;

[0046] Figure 10 is a photograph of four agar plates used to culture selected similar sized pieces of NEA58 (Control) and NEA58 (Selected) endophyte on plain agar, taken after 4 weeks growth;

[0047] Figure 11 depicts a region of the beta-tubulin gene from NEA58 (control) and NEA58 (selected) to show a 3bp deletion that results in a single amino acid loss post modification;

[0048] Figure 12 is the full amino acid sequence of the beta-tubulin gene of NEA58 (Selection) compared to NEA58 (Control) compared also against sequences for the endophytes NEA12, SE and ARI; and

[0049] Figure 13 is a graph showing the average transmission rate of NEA58 (Control) against NEA58 (Selected) after a seed production regime that included carbendazim application.

[0050] DETAILED DESCRIPTION

[0051] As noted above, described herein is a modified endophyte, methods of modification and uses thereof. The modified endophyte may be modified to enhance selected for properties such as fungicide resistance. Modification may be via isolation, in vitro culturing, selection and reintroduction to a plant host. The modified endophytes and methods may be used to produce endophytes and related plant:endophyte symbiotic relationships with enhanced properties such as enhanced fungicide resistance.

[0052] For the purposes of this specification, the term 'about', or 'approximately', or 'substantially' and grammatical variations thereof mean a quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15,10, 9, 8, 7 , 6, 5, 4, 3, 2, or 1% to a reference quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0053] The term 'comprise' and grammatical variations thereof shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements.

[0054] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7).

[0055] Unless otherwise stated, the singular forms "a," "an," and "the" include the plural reference. As used herein the term "and / or" means "and" or "or", or both.

[0056] As used herein "(s)" following a noun means the plural and / or singular forms of the noun. The terms 'genetic modification', 'genetic engineering', or 'gene editing technology' or grammatic variations thereof as used herein refers to the direct manipulation of the DNA or an organism using biotechnology to add, delete, or alter specific genes. These terms exclude natural mutations and human-driven selective breeding.

[0057] The term 'fungicide resistance' and grammatical variations thereof as used herein refers to a stable, heritable genetic change that allows a fungus to survive and grow at fungicide concentrations that would normally be lethal to the original population

[0058] The term 'fungicide tolerance' and grammatical variations thereof as used herein refers to the ability of a fungal population to survive or grow slowly in the presence of the fungicide The term 'additional' or grammatical variations thereof when used in respect of fungicide resistance or fungicide tolerance refers to the modified obligate symbiont having a greater resistance or tolerance or both resistance and tolerance relative to an unmodified obligate symbiont.

[0059] Method of Obligate Symbiont Modification

[0060] In a first aspect, there is provided a method of modifying an obligate symbiont comprising: isolating an obligate symbiont from its plant host;

[0061] culturing the isolated obligate symbiont in vitro;modifying the obligate symbiont through selective propagation to enhance a selected for characteristic of the obligate symbiont selected from one or more of: fungicide resistance, additional fungicide resistance, fungicide tolerance, additional fungicide tolerance; modification occurring through selection, the modification using no genetic modification, genetic engineering, or gene editing technology;

[0062] placing the modified obligate symbiont into a plant host; and

[0063] growing the plant host and modified obligate symbiont together.

[0064] Obligate Symbiont

[0065] The obligate symbiont may be an organism living in symbiosis with another organism (the host plant).

[0066] The obligate symbiont may be a fungal endophyte. The obligate symbiont may alternatively be other types of fungi or bacteria. For ease of description, further examples and description will generally refer to fungal endophytes although this may not be limiting. Various crop and pasture species have obligate symbionts (fungal and bacterial). There is no reason in the inventor's experience, why these alternative obligate symbionts cannot be subjected to similar in vitro asexual growth and selection processes to confer similar traits and effect similar DNA changes as identified herein. By way of illustration, fungal endophytes in Brassica (mustard / cabbage family) are microbes living inside plant tissues, offering benefits including plant growth promotion, nutrient uptake (especially phosphorus) and stress tolerance The fungal endophyte maybe an endophyte that lives within a plant host. The plant host is described further below.

[0067] The fungal endophyte may be asexual i.e. the endophyte is clonally propagated from the parent endophyte., No mixing of male and female gametes may occur during reproduction. The endophyte may be an Epichloe endophyte. Epichloe are a genus of ascomycete fungi that form endophytic symbiosis with grasses.

[0068] Plant Host

[0069] The plant host may be a plant that a symbiotic organism lives on, or in.

[0070] A donor plant host may be a plant host from which the obligate symbiont is taken from.A receiver plant host may be a plant host which the modified obligate symbiont is placed into. The donor plant host donor may be the same genus, or species, or variety, as the receiver plant host. Alternatively, the donor plant host and receiver plant host may be of differing genus, or species, or variety.

[0071] The same plant hosts may be used for both the obligate symbiont and modified obligate symbiont. It is considered more likely that different donor or receiver plant hosts (same species but different genotypes) may be used. For clarity, it is noted that the plant host may be different genetically but, still may be the same plant host. Alternatively, the donor host plant maybe different to the receiver plant host or vice versa. For example, a donor ryegrass endophyte and a receiver fescue plant host.

[0072] The plant host to which the modified obligate symbiont may be placed may have no obligate symbiont already present.

[0073] The plant host maybe from a crop or pasture family. For example, the family of Poaceae (e.g., ryegrass species, cereals, fescue species., Alternatively, the plant host may be selected from the Brassicaceae family (e.g., mustards, crucifers, or cabbage genera).

[0074] The plant host may be a grass. The grass may be from the family Poaceae (sub-family pooideae), formally called Gramineae.

[0075] The plant host may be selected from: field; fodder; grass; hay; paddock; silage; sward, turf; ryegrass (Lolium sp.); Festuca sp.; Cocksfoot (Dactylis glomerata), and combinations thereof. The plant host may be a ryegrass cultivar or group of ryegrass cultivars.

[0076] The ryegrass cultivar may be selected from: Perennial ryegrass (Lolium perenne L.); Italian ryegrass (Lolium multiflorum Lam. Spp. Italicum (A. Br.) Vokart, Lolium multiflorum Lam. Ssp. non Alternativum); Westerwolds ryegrass (Lolium multiflorum Lam. Var. westerwoldicum Wittmt, Lolium multiflorum Lam. Ssp. Alternativum); Hybrid ryegrass (Lolium boucheanum Kunth, Lolium x hybridum Hausskn.; Stiff darnel, Wimmera ryegrass (Lolium rigidum Gaudin). The ryegrass cultivar may be naturally occurring; wild type; hybrid; genetically modified; gene edited; populations created by TILLING (Targeting Induced Local Lesions IN Genomes); mutated populations; tetrapioid; polyploid.Isolation

[0077] Isolation refers to the process of removing the obligate symbiont from the original plant host (donor). This is commonly completed by taking a section of plant, placing this section on agar, growing out the obligate symbiont onto the agar, and then subculturing the obligate symbiont onto a fresh agar plate to obtain the obligate symbiont free from its host. For example, a bottom 1cm of tiller from plants containing endophyte may be surface- sterilised using ethanol and sodium hypochlorite, then rinsed in sterile water and placed on PDA plates. The plates may be left at 20°C for 3-6 weeks, until the endophyte grows out of the tiller tissue, onto the agar. The endophyte cultures may then be periodically sub-cultured and moved to fresh PDA plates. Other methods and considerations may be required for different endophytes, such as described methods referenced in the review of Tironi et. al. 2024.

[0078] Culturing

[0079] Culturing refers to the step of maintaining the obligate symbiont cells or other matter in conditions suitable for growth.

[0080] In vitro refers in this context to obligate symbiont growth and culturing occurring outside of the plant host in which the obligate symbiont normally grows.

[0081] Selective propagation

[0082] Selective propagation refers to the process of selectively propagating obligate symbionts for desired characteristics and eliminating or culling those with less desirable characteristics. Selection may be recurrent phenotypic selection and / or clonal selection.

[0083] Selection may enhance a selected for characteristic of the obligate symbiont, via natural mutations and human-driven selective breeding.

[0084] As may be appreciated, these methods of selection of novel obligate symbiont breeds or lines, or cultivars and the modification described does not use genetic modification or gene editing technology. This may be important to the success of the re-introduced obligate symbiont and plant host. No, or minimal, changes are made to the obligate symbiont basic genetic blueprint other than those required to confer the selective advantage. This is unlike a mutagenized obligate symbiont which would contain many different genetic mutations, including changesunrelated to the desirable characteristic. Such changes in the mutagenized obligate symbiont may be detrimental to the endophyte and the symbiosis.

[0085] Selection on media per se is known, for example, non-symbiotic fungi (e.g. penicillin, fusarium) have been grown and selected on media before. Also, endophytes have been modified before. In this method however, selective propagation for endophytes especially in traits producing a useful host / endophyte product differs to the prior art. In this method, it is possible to use the fungicide (that the endophyte has been selected for to confer resistance / tolerance to) to protect the plant from fungal diseases and having a reduced impact on the fitness of the endophytes due to its novel resistant / tolerant phenotype. This is beneficial for survival and transmission of the endophyte.

[0086] Selective propagation may be a means to select obligate symbionts with an upregulated gene corresponding to the desired selected characteristic.

[0087] Selective propagation may be a means to select obligate symbionts with extra gene copies corresponding to the desired selected characteristic.

[0088] Selective propagation may be a means to select obligate symbionts with a change in promotor corresponding to the desired selected characteristic.

[0089] Selective propagation may be a means to select obligate symbionts with a change in DNA sequence corresponding to the desired selected characteristic.

[0090] Selective propagation may be a means to select obligate symbionts for improved gene performance.

[0091] Selective propagation may be a means to select obligate symbionts with a deletion in a gene corresponding to the desired selected characteristics.

[0092] Selective propagation may be by via any of the methods above. Selective propagation may be via other DNA change or changes that has / have occurred via natural mutation and human-driven selective breeding and which correspond to the desired selected characteristics e.g. fungicide resistance, additional fungicide resistance, fungicide tolerance, additional fungicide tolerance.

[0093] Selective propagation may take multiple rounds of recurrent selection to achieve a desired change in a selected characteristic. A first round may be the isolation of obligate symbiont and growth in a culture on fungicide. The most resistant results may then be selected and the culturing process repeated. 2 to 10 repeats (or more) may occur to arrive at a superior selected obligate symbiont.A first or earlier rounds may utilise a relatively low fungicide rate in the media and this may increase to a relatively higher fungicide rate in the media in subsequent or later rounds. This may be useful to allow for recurrent selection progresses to arrive at a superior obligate symbiont.

[0094] In a further variation, selective propagation may further comprise enhancing the chances of genetic change by use of chemical or physical mutagens, e.g. exposure to electromagnetic radiation such as ultra-violet (UV) light and / or chemical modification techniques such as exposure to ethyl methane sulfonate (EMS). Although this moves into the realms of random mutagenesis, the concept here is to keep the random mutation to a minimum by using low levels of mutagen and still providing selective propagation pressure to enable identification of the desired changes quickly.

[0095] Genetic Changes

[0096] As noted above, the method of modifying the obligate symbiont may create a variety of genetic changes e.g. an upregulated gene, extra gene copies, a change in promotor, a change in DNA sequence, a deletion in a gene corresponding to the desired selected characteristics.

[0097] Research completed by the applicant has identified distinct and measurable differences in the genome of the modified obligate symbiont. These may vary depending on the fungicide, plant host or obligate symbiont.

[0098] In one case, the selective propagation may result in deletion in a genome of the modified obligate symbiont corresponding to a site or region to which a fungicide would bind to. The selected endophytes and methods described create a deletion in a gene and, as a result, the fungicide does not have the same fungicidal impact on the selected for endophyte.

[0099] The deletion may be a 3 base pair deletion.

[0100] The deletion may span two codons. Typically, the deletion of two codons would also alter the amino acid sequence. In this case, the neighbouring nucleotides re-form to encode the same amino acid, leaving the deletion of just a single first Phe (F) amino acid, which prior to the deletion was two Phe (F) amino acids.

[0101] To summarise:NEA58 (Control) :

[0102] Codon : 263 264 265 266 267 268

[0103] Nucleotides : CTG CAC TTC TTC ATG GTC

[0104] Amino Acids : L H F F M V

[0105] NEA58 (Selected) :

[0106] Codon : 263 264 265 266 267 268

[0107] Nucleotides : CTG CA- - -C TTC ATG GTC

[0108] Amino Acids : L H [del] F M V

[0109] The deletion may remove a third base of a codon and first and second bases of a subsequent codon.

[0110] The deletion may occur about codon 265. The carbendazim binding site is primarily in the regions 135-142 and 175-185 of beta-tubulin gene. Although previous resistance hotspots for resistance mutations have been in codons 198-200, it is understood that these mutations alter the overall protein structure reducing carbendazim affinity. It is understood that the above deletion sits at codon 265.

[0111] The selective propagation may result in an in-frame mutation, and wherein the in-frame mutation gene still produces a functional protein, but with at least one fewer amino acid. Specifically, a first Phenylalanine (F) amino acid may be omitted in the in-frame mutation. The neighbouring nucleotides may re-form to remain in frame, with the deletion of a first Phe (F).

[0112] The above genome differences were established based on experiments comparing the genomes of NEA58 (control) endophyte against that of NEA58 (selected) endophyte. NEA58 (control) is an existing known endophyte that has negligible / no fungicide resistance (control sample) and NEA58 (selected) is an endophyte selected according to the above methods to have an enhanced resistance to fungicide.Selected Characteristics

[0113] The selected characteristics referred to may be a desired fungicide resistance. Other important factors for commercial success that may influence selection may be alkaloid profile produced by the obligate symbiont, distribution in a plant host, transmission improvement, stability in host plant seed improvement, and combinations thereof.

[0114] A fungicide resistant / tolerant endophyte for example means that a farmer can apply fungicide to a crop and know they will be targeting the fungal disease but not affecting (or reducing the effect) on the endophyte (obligate symbiont) and host grass combination. Essentially, the farmer produces a crop 'free' (or at least reduced) of fungal disease but still containing high levels of endophyte. This is particularly useful for pasture seed producers who often need to produce pasture seed containing a high level of desired fungal endophyte (the NZ industry standard is for >70% viable endophyte infection in seed).

[0115] Fixed Selected Characteristic

[0116] The selected characteristics may be a fixed characteristic in the selected obligate symbiont. This results because a genetic change has been made, via natural mutation and human-driven selective breeding, that impacts that ability of the selective agent to work e.g., the over expression of the enzyme so it can cope with the toxin (fungicide), or as in the example above, the removal of a fungicide binding site, so that the fungicide has no / reduced activity on its protein target. Several rounds of selection on increasing levels of fungicide may be required. One or several DNA changes may be required in one or several genes, or genomic regions to build up a comprehensive resistance / tolerance.

[0117] Growing of the modified obligate symbiont and plant host

[0118] As noted above, re-inoculation of the modified endophyte occurs into the plant host / receiver plant host. In one example, this may occur by:

[0119] Selecting receiver plant host seeds,

[0120] Sterilising the seeds,

[0121] Growing the sterilised seeds on agar,

[0122] After a period of time, cutting a hole in the shoot and inserting a small piece of endophyte from culture into the opening,

[0123] Completing further growth.Using ryegrass as a more specific example, ryegrass seeds may be surface sterilised in sodium hypochlorite, rinsed in sterile water, and placed on bacteriological agar for 5 days at 20°C. Under a stereomicroscope, a hole may be made in the meristematic tissue of the shoot, and a small piece of endophyte (from culture) may be stuffed into the hole (cut and stuff technique). Seedlings may be returned to 20°C for 7 days, then transferred to potting mix in glasshouse for 6-8 weeks. Plant tillers may then be checked for endophyte infection, by microscopy or other published methods. Other inoculation methods, such as those described by Latch and Christensen 1985, or Becker et. al. 2018 may also be used.

[0124] Growing of the modified obligate symbiont and plant host may be completed using normal growing conditions for the plant host.

[0125] Adding back the modified obligate symbiont to a plant host does not necessarily change the general plant:host interaction. The modified characteristics / trait however remains in the modified obligate symbiont as the host plant receiver and obligate symbiont grow thus conferring the selected for characteristic / trait to the new plant host / obligate symbiont combination.

[0126] Growing the plant host and modified obligate symbiont together noted above may be a first step used to show stability and maintenance of other plant host and obligate symbiont characteristics.

[0127] Re-Isolation

[0128] The modified obligate symbiont grown with the plant host may be re-isolated and placed into a further plant host(s). Re-isolation and re-inoculation may occur via the same techniques as noted when discussing isolation above.

[0129] Fungicide

[0130] The fungicide to which the modified obligate symbiont may have resistance and / or tolerance to may be varied. The fungicide may be systemic, protectant or a type that disrupts fungal cell walls or disrupts their energy processes. The fungicide may have one or more of these properties.

[0131] The modified obligate symbiont is resistant and / or tolerant to multiple fungicides. The modified obligate symbiont is resistant and / or tolerant to fungicides of varying groups,classifications, and / or mechanisms of action. Combining resistances or tolerances through multiple selective passages through a first fungicide and then a second fungicide may also be completed. For example, group 1 fungicide tolerance selected for and, then and group 2 fungicide resistance selected for to produce a combined group 1 and group 2 resistant / tolerant endophyte. The fungicide may be selected from the chemical classes: azoles, strobilurins, dithiocarbamates, carboxamides, chloronitriles, anilinopyrimidines, inorganics such as copper salts and sulphurs, phthalimides and combinations thereof. In one example, the fungicides may be those described in the FRAC Code list 2024.

[0132] The fungicide may be selected from the benzimidazole family of compounds, one example being carbendazim. Carbendazim is classified as a group 1 fungicide.

[0133] The fungicide may be selected from the pyrazole carboxamide chemical family of compounds. One example may be benzovindiflupyr. Benzovindiflupyr is classified as a group 7 fungicide.

[0134] Modified Obligate Symbiont

[0135] In a second aspect, there is provided a modified obligate symbiont produced by the method substantially as described above.

[0136] Use of a Modified Endophyte

[0137] In a third aspect, there is provided the use of the modified obligate symbiont produced by the method substantially as described above to produce a host grass and obligate symbiont combination with an enhanced characteristic provided by the modified obligate symbiont.

[0138] Advantages

[0139] The above-described modified obligate symbiont, method of modification and uses thereof may provide advantages over existing obligate symbionts and modification methods. Examples of advantages found by the inventor may comprise one or more of the following:

[0140] Provision of and methods of producing, endophytes with fungicide resistance; additional fungicide resistance; fungicide tolerance; and / or additional fungicide tolerance.

[0141] An advantage over mutation breeding in asexual species (such as Epichloe endophyte), as this method avoids the accumulation of deleterious mutations (Mullers Ratchet)whilst in the pursuit of a specific advantageous mutation. The method only selects for desirable mutations in the characteristic required and does not create large numbers of random mutations as is observed in mutation breeding.

[0142] The method above also provides an advantage over new breeding technologies (e.g., genetic modification or gene editing) as it avoids complexity associated with genetic modification and gene editing or mutagenesis generally and, it uses techniques not bound by the same strong regulatory hurdles as genetic modification, genetic engineering, or genetic editing hence can be deployed more quickly, in more environments and with fewer restrictions.

[0143] The embodiments described above may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features. Further, where specific integers are mentioned herein which have known equivalents in the art to which the embodiments relate, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0144] EXAMPLES

[0145] The above-described modified endophyte, method of modification and uses thereof are now described by reference to specific examples and the figures and item numbering as summarised below:

[0146] Figure 1:

[0147] 10A Day 0 fungicide resistant NEA58 (Selected) endophyte inoculated on 6x carbendazim fungicide containing agar (potato dextrose agar, PDA).

[0148] 10B Day 0 non-resistant NEA58 (Control) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0149] IOC Day 0 NEA58 (Selected) endophyte maintained in culture (i.e., not passaged through a plant).Figure 2:

[0150] 20A Day 0 fungicide resistant NEA57 (Selected) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0151] 20B Day 0 non-resistant NEA57 (Control) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0152] Figure 3:

[0153] 50A Day 0 NEA58 (Control) endophyte on plain agar (PDA).

[0154] 50B Day 0 NEA58 (Selected) endophyte on plain agar (PDA).

[0155] 50C Day 0 NEA58 (Selected) endophyte maintained in culture (i.e., not passaged through a plant).

[0156] Figure 4:

[0157] 60A Day 0 NEA57 (Control) endophyte on plain agar (PDA).

[0158] 60B Day 0 NEA57 (Selected) endophyte on plain agar (PDA).

[0159] Figure 5:

[0160] 10A Day 28 fungicide resistant NEA58 (Selected) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0161] 10B Day 28 non-resistant NEA58 (Control) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0162] IOC Day 0 NEA58 (Selected) endophyte maintained in culture (i.e., not passaged through a plant).

[0163] 50A Day 28 NEA58 (Control) on plain agar (PDA), and as colonies on fungicide containing agar (PDA).

[0164] 50C Day 0 NEA58 (Selected) endophyte maintained in culture (i.e., not passaged through a plant).

[0165] Figure 6:

[0166] 20A Day 28 fungicide resistant NEA57 (Selected) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0167] 20B Day 28 non-resistant NEA57 (Control) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).60A Day 28 NEA58 (Control) on plain agar (PDA) to show control was alive and capable of growth.

[0168] Figure 7:

[0169] 30A Day 28 fungicide resistant NEA64 (Selected) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0170] 30B Day 28 non-resistant NEA64 (Control) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0171] 70A Day 28 NEA64 (Control) on plain agar (PDA) to show control was alive and capable of growth.

[0172] Figure 8:

[0173] 40A Day 28 fungicide resistant NEA68 (Selected) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0174] 40B Day 28 non-resistant NEA68 (Control) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0175] 80A Day 28 NEA68 (Control) on plain agar (PDA) to show control was alive and capable of growth.

[0176] Figure 9:

[0177] 110A Day 28 fungicide resistant NEA67 (Selected) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0178] HOB Day 28 non-resistant NEA67 (Control) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0179] 120A Day 28 NEA67 (Control) on plain agar (PDA) to show control was alive and capable of growth.

[0180] * Indicates contamination.

[0181] Figure 10:

[0182] 90A Day 28 fungicide resistant NEA58 (Selected) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).

[0183] 90B Day 28 non-resistant NEA58 (Control) endophyte inoculated on 6x carbendazim fungicide containing agar (PDA).100A Day 28 NEA58 (Control) on plain agar (PDA) to show control was alive and capable of growth.

[0184] Figure 11: Figure depicting a region of the beta-tubulin gene from NEA58 (Control) and NEA58 (Selected) to show a 3bp deletion that results in a single amino acid loss post modification. Figure 12: Figure depicting the full amino acid sequence of the beta-tubulin gene from NEA58 (Control) and NEA58 (Selected), along with the endophyte strains NEA12, Standard Endophyte (SE) and ARI to further show the loss of amino acid 265 (F) in the NEA58 (Selected). The amino acid sequence of the beta-tubulin gene from NEA12, SE and ARI were sourced from the NCBI repository, KP834583.1, KP834586.1 and KP834572.1, respectively.

[0185] Figure 13: Shows a graph illustrating an average transmission rate of NEA58 (Control) against that observed for NEA58 (Selected) after a seed production regime that included carbendazim application.

[0186] EXAMPLE 1

[0187] In an experiment, isolation of two Epichloe endophytes of taxonomic group LpTG-3 was completed from host plants. The two endophytes were cultured in vitro and various selections and further culturing made to develop increasing levels of fungicide resistance to carbendazim.

[0188] Aim

[0189] To produce and assess consistent resistance of NEA57 and NEA58 endophytes to carbendazim fungicide, after sub-culturing, inoculation and re-isolation from host plants.

[0190] Method

[0191] Cultures of two commercial LpTG-3 endophytes were isolated from their host by placing a surface sterilized host plant segment on PDA (potato dextrose agar). From the resulting fungal colony on the media, a sub-sample was transferred to a fresh PDA plate and allowed to grow. The resulting colony was assessed to be the desired endophyte by its phenotypic growth and by use of a Kompetitive Allele Specific Primer (KASP) assay to confirm the presence of a discriminatory genotypic marker. These colonies or subsequent sub-cultures of such colonies were used to create the required resistance and / or tolerance to the fungicide. They were renamed NEA57 and NEA58 for the respective endophyteFour-week-old fungal colony material, ~lcm2of tissue from was excised and ground into 1ml of sterile water. This solution containing small endophyte particles was spread on top of PDA containing a low (lx) concentration of Carbendazim fungicide (CHIEF® FUNGICIDE, ADAMA). PDA plates were made with either 1 x (30ul carbendazim fungicide mixed in 30ml water), 2 x (60ul carbendazim fungicide mixed in 30ml water), 3 x (90ul carbendazim fungicide mixed in 30ml water), 4x (120pl carbendazim fungicide mixed in 30ml water) or 6x (180pl carbendazim fungicide mixed in 30ml water) concentrations of carbendazim fungicide. The term lx, 2x, 3x, 4x and 6x refers to the concentration of fungicide above normal field rates, e.g. 4x is four times the normal rate that would be expected to be sprayed on a grass crop. 1.2ml of these fungicide solutions were added to 200ml PDA solution and used to pour individual agar plates to create the different selective rates.

[0192] After 4 weeks, the largest endophyte colonies from the lx plates were selected, and small pieces placed on 2x fungicide / PDA plates. Colonies of the original endophyte were placed alongside these to act as no / minimal growth controls. Original endophyte cultures were also put on PDA without fungicide to compare growth rates. Plates were assessed after 6 weeks, and comparisons made between resistant and original culture growth.

[0193] Cultures showing healthy growth on 2x fungicide were selected and subsequently grown on increasing concentrations (3x, 4x, 6x) of the Carbendazim fungicide in the same manner as described above.

[0194] Resistant cultures on 6x Carbendazim (NEA57 selected and NEA58 selected) were inoculated into Lolium perenne host plants in 2022 and left growing for several months, before being reisolated from their host plants onto PDA in March 2023.

[0195] June 2023

[0196] Pieces of re-isolated NEA57 selected and NEA58 selected endophyte cultures were transferred to 4x and 6x Carbendazim-PDA plates further termed herein as NEA57 (selected) or NEA58 (Selected).

[0197] Controls of non-resistant starting endophyte cultures (NEA57 control and NEA58 control) were put on the same carbendazim-PDA plates for comparison.

[0198] As a further comparison, pieces of NEA58 (Selected) endophyte maintained in culture (i.e., not passaged through a plant) were also put alongside the NEA58 (Control) side to compare growth between selected NEA58 (Selected - no passage through plant), NEA58 (Selected - passagethrough plant) and NEA58 (Control). This was completed to rule out the possibility of the resistance trait being altered by passage through a plant host.

[0199] Photos showing the findings are described further below in Figures 1-10, taken of plates at culture transfer (day 0), and after 4 weeks growth (trial end).

[0200] Results

[0201] Figure 1 illustrates a photo of the similar sized pieces of NEA58 (Selected) and NEA58 (Control) endophyte on 6x carbendazim containing agar as taken on day 0 of the trial. The plates are split in half with the NEA58 (selected) endophyte samples on the left label 10A and the non-resistant NEA58 (Control) samples on the fungicide agar on the right half 10B. NEA58 (Selected) and not passaged through a plant are on the right hand side of each plate labelled 10C.

[0202] Figure 2 illustrates a photo of the similar sized pieces of NEA57 (Selected) and NEA57 (Control) endophyte on 6x carbendazim containing agar as taken on day 0. The plates are split in half with the NEA57 (Selected) endophyte samples on the left label 20A and the NEA57 (Control) samples on the fungicide agar on the right half 20B.

[0203] Figure 3 illustrates a photo of control pieces of NEA58 endophyte on PDA agar, showing size at transfer on day 0. The left hand side sample 50A is NEA58 (Control) endophyte on plain agar and the right hand side sample 50B is NEA58 (Selected) on plain agar. An NEA58 (Selected) and not passaged through a plant sample is labelled 50C.

[0204] Figure 4 illustrates a photo of control pieces of NEA57 endophyte on PDA agar, showing size at transfer on day 0. The left hand side sample 60A is NEA57 (Control) on plain agar and the right hand side sample 60B is NEA57 (Selected) on plain agar.

[0205] Figure 5 is taken on after 4 weeks of growth on the PDA plates. The photo includes the NEA58 (Control) on plain agar sample 50A. As shown in this photo, NEA58 (Selected) endophyte cultures 10A grew well on the fungicide-infused agar. The NEA58 (Control) endophyte with no fungicide resistance 10B did not grow at all from the Day 0 sample on the fungicide agar. The NEA58 (Control) endophyte 50A grew well as expected on normal PDA agar with no fungicide. The NEA58 (Selected) and not passaged 50C, 10C grew well on all plates confirming no impact from plant passage. These results showed that the steps of selection and re-inoculation completed prior to testing for fungicide resistance was successful in achieving a fungicideresistant NEA58 (Selected) endophyte that could be re-introduced into plant hosts post selection.

[0206] Figure 6 is taken after 4 weeks of growth on the PDA plates. The photo includes the NEA57 (Control) on plain agar sample 60A. As shown in this photo, NEA57 (Selected) endophyte cultures 20A grew well on the fungicide-infused agar. The NEA57 (Control) endophyte cultures with no fungicide resistance 20B did not grow at all since Day 0 on the fungicide control PDA agar. The NEA57 (Control) endophyte 60A grew well as expected on normal PDA agar with no fungicide. These results showed that the steps of selection and re-inoculation completed prior to testing for resistance was successful in achieving a NEA57 (Selected) endophyte that could be re-introduced into plant hosts post selection.

[0207] EXAMPLE 2

[0208] In a further experiment, isolation of further (different to Example 1) Epichloe endophyte from taxonomic group LpTG-3 was completed from a host plant. The endophyte was cultured in vitro and various selections and further culturing made to develop increasing levels of fungicide resistance to a different fungicide to that used in Example 1, benzovindiflupyr (Elatus® Plus). Methodology, based on that used in Example 1 above was used to develop NEA64 (Selected). A sample of this endophyte was subjected to the same selective process as above except that increasing rates of Elatus® Plus fungicide were used instead of Chief®. From this recurrent selection experiment NEA64 (Control) and NEA64 (Selected) endophytes were developed. Figure 7 is taken after 4 weeks of growth on the PDA plates. The photo shows the same protocol being followed using a different fungicide and achieving the same result through selective propagation. The photo includes the non-fungicide resistant NEA64 (Control) on plain agar sample 70A that grew normally as expected without fungicide. NEA64 (Selected) endophyte cultures 30A grew well on the fungicide-infused agar. The NEA64 (Control) endophyte cultures with no fungicide resistance 30B did not grow at all since Day 0 on the fungicide control PDA agar. These results showed the applicability of the method to other selective pressures.

[0209] EXAMPLES

[0210] In a further experiment, isolation of a further (different to Example 1 and 2) Epichloe endophyte from taxonomic group LpTG-3 was completed from a host plant. The endophytewas cultured in vitro and various selections and further culturing made to develop increasing levels of fungicide resistance to the same fungicide as used in Example 1. Methodology was based on Example 1. From this recurrent selection experiment NEA68 (Control) and NEA68 (Selected) endophytes were developed.

[0211] Figure 8 is taken after 4 weeks of growth on the PDA plates. The photo shows the same protocol being followed as in Example 1. The photo includes NEA68 (Control) on plain agar 80A. As shown in this photo, NEA68 (Selected) cultures 40A grew well on the fungicide-infused agar. The NEA68 (Control) endophyte cultures with no fungicide resistance 40B did not grow much since Day 0 on the fungicide control PDA agar. The NEA68 (Control) endophyte 80A grew well as expected on normal PDA agar with no fungicide.

[0212] EXAMPLE 4

[0213] In a further experiment, isolation of an Epichloe endophyte from taxonomic group LpTG-2 (different to all LpTG-3 examples above) was completed from a host plant. The endophyte was cultured in vitro and various selections and further culturing made to develop increasing levels of fungicide resistance to the same fungicide as used in Example 1. Methodology was based on Example 1. From this recurrent selection experiment NEA67 (Control) and NEA67 (Selected) endophytes were developed

[0214] Figure 9 is taken after 4 weeks of growth on the PDA plates. The photo shows the same protocol being followed as in Example 1. The photo includes NEA67 (Control) 120A on plain agar sample 80A. As shown in this photo, NEA67 (Selected) 110A cultures grew well on the fungicide-infused agar. NEA67 (Control) 110B endophyte cultures with no fungicide resistance did not grow much since Day 0 on the fungicide control PDA agar. The NEA67 (Control) 120A endophyte grew well as expected on normal PDA agar with no fungicide. Note that some confounding contamination occurred in these plates labelled with an asterisk *.

[0215] EXAMPLES

[0216] In a further experiment, the generational stability of the modified endophyte from Example 1 was tested. NEA58 (Selected) endophyte from Example 1 was used to demonstrate the stability of the modified endophyte after multiple passages through ryegrass generations. The NEA58 (Selected) was re-inoculated into a new ryegrass host using the 'cut and stuff' methodology described in the above description. Inoculated plants from the method weregrown and inoculated seed was produced from these plants. This seed was germinated and grown, and again, seed produced. This second-generation seed was then germinated and endophyte NEA58 (Selected) isolated from the seedling plant tissue. This equates to a method comprising three passages through plant and two through seed.

[0217] The endophyte was isolated onto agar plates that contained the respective fungicide that NEA58 (Selected) was resistant / tolerant to. NEA58 (Control) was plated onto the agar to act as a control.

[0218] The NEA58 (Selected) was still tolerant after three passages through plant somatic material as shown in Figure 10 below. This demonstrates the stability of the modified endophyte trait over multiple plant generations.

[0219] Figure 10 shows results from inoculated agar plates 4 weeks post inoculation with the NEA58 (Selected) 90A, after multiple passages through the plant host, showing no deleterious effects from fungicide presence whereas, NEA58 (Control) 90B shows minimal if any growth. The NEA58 (Control) samples 100A grew as normal on the control non-fungicide plate as expected.

[0220] Conclusion

[0221] The endophyte taxa LpTG-1, LpTG-2, and LpTG-3 are different genetic groups of Epichloe fungal endophytes that form symbiotic associations with grasses. Epichloe endophyte cultures derived from different LpTG groups can be selected for to modify the endophyte, such that the modified endophyte has a new trait of fungicide resistance, or enhanced fungicide resistance, or fungicide tolerance or enhanced tolerance.

[0222] The different endophytes obtained resistance through recurrent selection on fungicide media. Different families, or groups of fungicide (e.g. group 1 - carbendazim, and group 7 benzovindiflupyr) were used to demonstrate the broad nature of the resistance, or tolerances, that could be obtained to demonstrate the potential wide functionality that can be obtained. Combining resistances or tolerances through multiple selective passages through a first fungicide and then a second fungicide should also be possible (e.g., groupl and group 2 resistance combined). This example of passing the endophyte through multiple host generations and maintaining the fungicide resistance demonstrates the stability and retained the resistance of the modified endophyte. Therefore, the process can be used to introduce diverse fungicide resistance into diverse endophyte types. This fungicide resistance is stable inthe selected for fungicide resistant endophyte even after multiple passages through a host plant.

[0223] EXAMPLE 6

[0224] Further experiments were completed to compare the genomes of the NEA58 (Control) and NEA58 (Selected) from Example 1. NEA58 (Control) is an existing known endophyte that has negligible / no fungicide resistance (control sample) and NEA58 (Selected) is an endophyte selected using NEA58 (Control) as a start point, modified according to the above methods to have an enhanced resistance to fungicide (NEA58 (Selected)).

[0225] NEA58 (Selected) and NEA58 (Control) endophyte samples were sent to Lincoln University between January and November of 2025. DNA samples were extracted and the genomes of both endophytes sequenced using long read sequencing equipment. Sequence information was provided to bioinformaticists, and the sequences were analysed using a suite of bioinformatic software and aligned, along with sequence information from other commercial endophytes. Key differences were identified, and some key regions of difference were identified and the results presented here.

[0226] Figure 11 shows a gene map comparing part of the genomes and DNA of NEA58 (Control) endophyte and NEA58 (Selected) endophyte which illustrates a DNA difference between the endophytes.

[0227] Figure 12 shows a full amino acid sequence of the beta-tubulin gene from NEA58 (Control) and NEA58 (Selected), along with commercial NEA12 endophyte, a wildtype, Standard Endophyte (SE) and commercial ARI endophyte strains. The comparison sequences clearly show the deletion of amino acid 265 (F) in NEA58 (Selected) endophyte.

[0228] As maybe understood from Figure 11 and Figure 12, the applicant has identified a 3 bp deletion between NEA58 (Control) and NEA58 (Selected) in a beta-tubulin gene. The beta-tubulin gene ortholog from Epichloe festucae (strain Fll) maps to this region. The fungicide carbendazim binds to the beta-tubulin gene as part of the carbendazim mode of action. The selected endophytes and methods described modify this gene and as a result, the fungicide does not have the same fungicidal impact on the selected for endophyte. This binding site, and type of genetic change may also be relevant to other types of fungicide and other binding sites hence, reference to carbendazim should not be seen as limiting. Other deletions may also occur toother sites depending on the mode of action of other types of fungicide, yet the methods remain the same as those described.

[0229] Returning to the deletion observed, the deletion appears to be an in-frame mutation. As a result, the gene may still produce a functional protein, but with one fewer amino acid, specifically, the first phenylalanine (F) in that region. The deletion spans two codons, which typically would also alter an amino acid because two codons collapse into one. In this case, the neighbouring nucleotides re-form to leave just the deletion of that first Phe (F).

[0230] To summarise:

[0231] NEA58 (Control) :

[0232] Codon : 263 264 265 266 267 268

[0233] Nucleotides : CTG CAC TTC TTC ATG GTC

[0234] Amino Acids : L H F F M V

[0235] NEA58 (Selected) :

[0236] Codon : 263 264 265 266 267 268

[0237] Nucleotides : CTG CA- - -C TTC ATG GTC

[0238] Amino Acids : L H [del] F M V

[0239] Given that NEA58 (Selected) is fungicide resistant and NEA58 (Control) is not, it is highly unlikely that the mutation is a mere coincidence and not contributing in some way to the resistance.

[0240] EXAMPLE 7

[0241] In this example, endophyte transmission, of selected vs control endophyte, through to the new plant seed was tested in a plant host after fungicide treatment in a practical scenario to demonstrate the advantage of conferring fungicide resistance to an endophyte.

[0242] Two Epichloe endophytes, NEA58 (Control) and NEA58 (Selected) were grown in host ryegrass plants. Single plants from seed of the host ryegrass plants, known to contain the twoendophytes were planted into isolation plots (a NEA58 (Control) plot and a NEA58 (Selected) plot) and given individual plant numbers. A fungicide regime of three sprays of carbendazim (CHIEF® FUNGICIDE, ADAMA) at 2-week intervals was applied at a rate equivalent to 500mL product in 200L water per Ha, after flowering and seed set.

[0243] Seed from individual plants were harvested and tested for endophyte, via growing seedlings and immunoblotting.

[0244] The data from this provided a transmission rate of viable endophyte to the next generation. The hypothesis being that the fungicide selected endophyte (NEA58 (Selected)) would transmit better in the presence of a fungicide treatment than the non- fungicide selected endophyte (NEA58 (Control)).

[0245] Results

[0246] Individual plant transmission of endophyte

[0247]

[0248] Average transmission rate of NEA58 (Control) vs NEA58 (Selected) after a seed production regime that included carbendazim application was greater for selected endophytes as further shown in Figure 13.

[0249] Conclusion

[0250] Endophytes selected to be fungicide resistant can confer endophyte transmission benefits, in plant: endophyte combinations, to the next generation of plant:endophyte seed when the plant:endophyte is grown under a production regime that includes a fungicide treatment.

[0251] EXAMPLES

[0252] In this example, samples of selected and control LpTG-3 endophytes were used to demonstrate the difference in growth rate of the two modified endophytes compared to the unmodified control endophyte when grown on PDA agar plates or PDA agar plates containing fungicide 6x Chief® or 6x Elatus®Plus.

[0253] Small pieces ~4.0mm2of control and selected endophyte were sub-cultured onto the centre of an Agar plate (as per the treatments above). One agar plate was used for each treatment, and this was repeated five times to represent 5 replicates for each treatment. Colony growth was measured after 4 weeks by measuring the diameter of the colony north to south and east to west to provide an average colony diameter. The average diameter was then used to calculate the growth area of each endophyte in each treatment in each rep. The sum of the reps / 5 was then used to provide an average growth area.

[0254] Results

[0255] On plain PDA with no fungicides all endophytes grew approximately the same with a total experimental range between 113.1 and 201.6 mm2but an average range of between 143.0 and 158.6mm2. This is a l.lx difference between the largest and smallest average colony size. On the 6x Chief plates the NEA58 (Selected) grew similar to its rate on plain agar (139.9 vs 143.0mm2). All other endophytes grew much less with the final average area being between 16.7 and 21.6mm2 similar to the starting area (16mm2). The growth of NEA58 (Selected) on this media was ~6.5 to 8.4x the growth of endophytes not selected to grow on the fungicide Chief.On the 6x Elatus plates the NEA64 (Selected) grew less than it did on the plain agar (91.3 vs 149.0mm2). However, this was still much greater than the other endophytes which grew much less on the Elatus with the final average area of endophytes not selected to grow on Elatus being between 18.1 and 24.2mm2slightly greater than the starting area (16mm2). The growth of NEA64 (Selected) on this media was ~3.8 to 5. Ox the growth of endophytes not selected to grow on the fungicide Elatus.

[0256] Plates with PDA

[0257] only

[0258] Averages

[0259] Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Area average Std dev Selected NEA58 113.10 165.13 201.06 113.10 122.72 143.02 38.92493 Selected NEA64 113.10 201.06 122.72 153.94 153.94 148.95 34.41991 Control NEA57 165.13 122.72 143.14 143.14 143.14 143.45 15.0009 Control NEA58 143.14 165.13 153.94 176.71 153.94 158.57 12.77978

[0260] Plates with PDA + 6x Chief

[0261] Averages

[0262] Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Area average Std dev Selected NEA58 143.14 113.10 113.10 165.13 165.13 139.92 26.07852 Selected NEA64 12.57 19.63 19.63 15.90 15.90 16.73 2.982316 Control NEA57 15.90 19.63 28.27 15.90 28.27 21.60 6.281651 Control NEA58 15.90 15.90 15.90 12.57 28.27 17.71 6.079547

[0263] Plates with PDA + 6x Elatus

[0264] Averages

[0265] Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Area average Std dev Selected NEA58 33.18 19.63 23.76 15.90 28.27 24.15 6.8399 Selected NEA64 103.87 122.72 56.75 86.59 86.59 91.30 24.4106 Control NEA57 15.90 19.63 15.90 19.63 19.63 18.14 2.043356 Control NEA58 19.63 19.63 23.76 19.63 19.63 20.46 1.844014

[0266] Aspects of the modified endophyte, method of modification and uses thereof have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the claims herein.

Claims

WHAT IS CLAIMED IS:

1. A method of modifying an obligate symbiont comprising:isolating an obligate symbiont from its plant host;culturing the isolated obligate symbiont in vitro;modifying the obligate symbiont through selective propagation to enhance a selected characteristic of the obligate symbiont selected from one or more of: fungicide resistance, additional fungicide resistance, fungicide tolerance, additional fungicide tolerance; modification occurring through selection, the modification using no genetic modification, genetic engineering, or gene editing technology;placing the modified obligate symbiont into a plant host; andgrowing the plant host and modified obligate symbiont together.

2. The method as claimed in claim 1 wherein the obligate symbiont is a fungal endophyte.

3. The method as claimed in claim 1 or claim 2 wherein the obligate symbiont is an Epichloe endophyte.

4. The method as claimed in any one of the above claims wherein the plant host to which the modified obligate symbiont is placed has no obligate symbiont already present.

5. The method as claimed in any one of the above claims wherein the plant host is a grass from the family Poaceae.

6. The method as claimed in any one of the above claims wherein the modified obligate symbiont is resistant and / or tolerant to multiple fungicides.

7. The method as claimed in any one of the above claims wherein the modified obligate symbiont is resistant and / or tolerant to fungicides of varying groups, classifications, and / or mechanisms of action.

8. The method as claimed in any one of the above claims wherein the selective propagation comprises selecting obligate symbionts with an upregulated gene corresponding to the selected characteristic.

9. The method as claimed in any one of claims 1 to 7 wherein the selective propagation comprises selecting obligate symbionts with extra gene copies corresponding to the selected characteristic.

10. The method as claimed in any one of claims 1 to 7 wherein the selective propagation comprises selecting obligate symbionts with a change in DNA sequence, the change corresponding to the selected characteristic.

11. The method as claimed in any one of claims 1 to 7 wherein the selective propagation results in deletion in a gene of the modified obligate symbiont commensurate to a site or region to which a fungicide would bind to.

12. The method as claimed in claim 11 wherein the deletion is a 3 base pair deletion.

13. The method as claimed in claim 11 or claim 12 wherein the deletion spans two codons.

14. The method as claimed in any one of claims 111 ol3 wherein the deletion removes a third base of a codon and first and second bases of a subsequent codon.

15. The method as claimed in any one of claims 11 to 14 wherein the deletion occurs about approximately codon 265.

16. The method as claimed in any one of the above claims wherein the selective propagation results in an in-frame mutation wherein the in-frame mutation still produces a functional protein, but with at least one fewer amino acid.

17. The method as claimed in claim 16 wherein neighbouring nucleotides re-form to encode a same amino acid, with deletion of a first Phe (F).

18. The method as claimed in any one of the above claims wherein the selective propagation is completed over multiple rounds to achieve a desired change in the selected characteristic.

19. The method as claimed in claim 18 wherein the selected characteristic further comprises at least one of: alkaloid profile produced by the obligate symbiont, distribution in a plant host, transmission improvement, stability in host plant seed improvement, and combinations thereof.

20. The method as claimed in any one of the above claims wherein the modified obligate symbiont grown with the plant host is further re-isolated and placed into a further plant host(s).

21. A modified obligate symbiont produced by the method as claimed in any one of the above claims.

22. Use of a modified obligate symbiont produced by the method as claimed in any one of claims 1 to 20 to produce a host grass and obligate symbiont combination with an enhanced characteristic provided by the modified obligate symbiont.

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