Molluscicidal composition

Abstract

The present invention relates to molluscicidal compositions. In particular, the invention relates to molluscicidal compositions comprising loline species (e.g., loline alkaloids) and endophyte- infected plant biomass and methods and uses thereof.

Description

[0001] MOLLUSCICIDAL COMPOSITION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to molluscicidal bait compositions. In particular, the invention relates to molluscicidal bait compositions comprising: 1) endophyte-infected plant biomass comprising at least one loline species (e.g., loline alkaloids), and 2) one or more phagostimulants. The plant biomass is derived from an endophyte-infected plant and the at least one loline species is not extracted from the plant biomass and is not synthetic. Related methods and uses of the composition are also provided.

[0004] BACKGROUND

[0005] Mollusc pests such as slugs and snails cause over £100 million in annual crop losses in the UK. These pests are particularly harmful to crops such as potatoes, cereals, and oilseed rape.

[0006] Globally, food systems account for over one-third of all greenhouse gas (GHG) emissions, which includes agriculture and pesticide use. Many of the world’s biggest oil companies produce pesticides or their chemical ingredients. Pesticides exacerbate the climate emergency throughout their lifecycle via manufacturing, packaging, transportation, application, and even through environmental degradation and disposal. For example, 99% of synthetic pesticides are derived from petroleum. In 2021 , 3.53 million metric tons of pesticides were used globally. The estimated global carbon equivalent emissions for producing those pesticides used in 2021 was 18 million metric tons.

[0007] In addition to the climate risk, using synthetic pesticides in pest control has caused significant environmental and health issues. The problems associated with synthetic pesticides include toxicity to non-target organisms, contamination of ground water and surface water, persistence that limits crop rotation, and bioaccumulation in the food chain. These issues have a cumulative impact on human health, leading to consequences for life expectancy, healthcare outcomes, and associated costs (Pimentel et al., 1992, BioScience, 42(5), 354-362). Synthetic pesticides may become unsustainable in developed and developing countries in the coming decades. There is a growing demand for growers to reduce chemical pesticides in crop production and cultivate fruits and vegetables with reduced detectable residues.

[0008] Molluscicides in particular are harmful to other animals. Metaldehyde, for example, is a common active ingredient in slug pellets but is harmful to birds and mammals that eat slug pellets, contaminated slugs or other creatures that have consumed pellets or slugs. Further, the UK has recently banned many synthetic molluscicides, including metaldehyde, meaning that the options for effectively controlling molluscs are further limited to pesticides such as ferric phosphate or biological control methods such as slug resistant crop varieties.

[0009] It is, therefore, an object of the present invention to overcome one or more of the above issues and to provide a more accessible alternative to chemical pesticides that is flexible for multiple applications and is user-friendly to aid adoption.

[0010] SUMMARY OF THE INVENTION

[0011] The present inventors have developed novel molluscicidal compositions that both enhance soil health and reduce dependence on synthetic pesticides. The compositions of the present invention are designed to be compatible with standard agricultural practices and therefore can seamlessly integrate into existing farming systems.

[0012] The compositions of the invention can be used as a means of sustainable pest control, offering a scalable, non-toxic alternative to known pesticides whilst also addressing critical environmental, agricultural, and regulatory challenges.

[0013] In particular, the present inventors have demonstrated for the first time that loline species are effective molluscicidal agents. Whilst purified lolines have been described in the art as insecticides, prior to the present invention, the effectiveness of lolines has not previously been considered, let alone demonstrated.

[0014] Further, the present inventors have generated molluscicidal active compositions that do not require expensive or complex extraction of lolines, reducing the cost and increasing the availability of effective, environmentally safe molluscicides. Further, the compositions provided by the inventors provide further advantages, as they are capable of carbon sequestration.

[0015] Accordingly, the present invention provides a biological molluscicidal bait composition comprising: 1) endophyte-infected plant biomass comprising at least one loline species (e.g., loline alkaloids), and 2) one or more phagostimulants. The plant biomass is derived from an endophyte-infected plant and the at least one loline species is not extracted from the plant biomass and is not synthetic.

[0016] Also provided is a method for producing a composition for repelling and / or controlling a pest of the phylum Mollusca, preferably the class gastropoda (slugs and snails), the method comprising: a) harvesting or providing an endophyte-infected plant, wherein the plant comprises at least one loline species and wherein the at least one loline species has molluscicidal activity; b) cutting the harvested plant to produce plant clippings; c) combining the plant clippings with one or more additive, wherein the additive is at least one phagostimulant.

[0017] The method preferably further comprises the steps of extruding and pelletising the mixture. The method may also further comprise a drying step, as needed.

[0018] Also provided is a composition obtainable by the method of the invention.

[0019] Also provided is the use of the composition of the invention for repelling and / or controlling the population size of a pest of the phylum Mollusca, preferably a pest of the class gastropoda (gastropods, e.g., slugs and snails).

[0020] Also provided is a method for controlling mollusc damage to plants in horticulture or agriculture, the method comprising contacting a plant, a plant growth medium or a mollusc with the composition of the invention.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1A shows a grass paddock sown with (left-hand side) and without (right-hand side) Epichloe infected seed (meadow fescue + endo) and the other half was sown with non-infected Epichloe seed (meadow fescue without endo). Presence of the endophytes in the inoculated grass was confirmed by microscopy (B, arrows). Of the two sides, the fescue comprising endophyte-infected grass is healthier. In particular, the grass is greener, longer and more uniform in the presence of endophytes. There are also signs that the endophyte-infected grass is more tolerant to stress, (e.g., drought) and has far less predation by Grass grub (Costelytra giveni).

[0023] FIG. 2 depicts an exemplary method for producing a composition of the invention. Briefly, endophyte-infected grass comprising loline species are mowed (1), further cut to produce grass clippings (2), mixed with additives such as surfactants, binders, fillers and feeding stimulants (phagostimulants) (3), mixed (e.g., using a rotary mixer) (4), extruded (5) and formed into pellets (6).

[0024] FIG. 3 shows how pellets comprising a composition of the invention effectively protect crops (in this case lettuce) from mollusc damage. Four plants were treated with pellets produced according to the method shown in Fig. 2. As a control, four plants were untreated. Four weeks after the initial treatment, the plants were assessed for slug damage. No live slugs were found on the treated plants. As shown in the photograph, the treated plants were healthier (i.e. , had larger and more numerous leaves with no signs of slug damage) compared to the untreated controls.

[0025] DETAILED DESCRIPTION

[0026] Definitions

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 20 Ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedence over any dictionary or extrinsic definition. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary.

[0028] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0029] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure considering the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0030] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

[0031] As used herein, the term "capable of' when used with a verb, encompasses or means the action of the corresponding verb. For example, "capable of controlling" also means controlling, "capable of promoting" also means promoting, and "capable of targeting..." also means targets.

[0032] Numeric ranges are inclusive of the numbers defining the range. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0033] As used herein, the articles "a" and “an” may refer to one or to more than one (e.g. to at least one) of the grammatical object of the article. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting.

[0034] “About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Preferably, the term “about” shall be understood herein as plus or minus (±) 5%, preferably ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, ± 0.1 %, of the numerical value of the number with which it is being used. The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the invention.

[0035] As used herein the term "consisting essentially of" refers to those elements required for a given invention. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that invention (i.e. inactive or non- immunogenic ingredients).

[0036] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and / or “consisting essentially of” such features.

[0037] In the context of this document, "and / or" indicates that each of the two specified features or components, either individually or in combination, is explicitly disclosed. For instance, "A and / or B" should be understood as specifically disclosing (i) A, (ii) B, and (iii) A and B, as if each possibility were presented separately within the document.

[0038] Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0039] The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. The terms "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, "reduction" or "inhibition" encompasses a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100% inhibition (i.e. abrogation) as compared to a reference level.

[0040] The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statistically significant amount. The terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 25%, at least 50% as compared to a reference level, for example an increase of at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, at least about 95%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 250% or more compared with a reference level, or at least about a 1 .5-fold, or at least about a 2-fold, or at least about a 2.5-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 1.5-fold and 10-fold or greater as compared to a reference level. In the context of a yield or titre, an "increase" is an observable or statistically significant increase in such level.

[0041] The term "plants" refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage, and fruits.

[0042] The term "locus" as used herein means any area (e.g. fields) in or on which plants are growing, or where seeds of cultivated plants are sown, or where seed will be placed. It includes soil, seeds, and seedlings, as well as established vegetation.

[0043] The term "plant propagation material" denotes all generative parts of a plant, for example seeds or vegetative parts of plants such as cuttings and tubers. It includes seeds in the strict sense, as well as roots, fruits, tubers, bulbs, rhizomes, and parts of plants.

[0044] The term "plant growth media" is used to describe any media in which a plant (typically the plant to be protected from pests of the phylum Mollusca) is grown. Preferably, the media consists of or comprises soil. Alternative, soil-free media are also encompassed by the present invention.

[0045] The term “bait”, as used herein, refers to any suitable agent (such as a phagostimulant) for attracting a mollusc (e.g. a gastropod such as a slug or snail).

[0046] As used herein, the term “molluscicidally effective amount” refers to an amount which induces mortality, disrupts or impedes growth, interferes functions, effects sterilization, and / or interferes with reproduction of one or more target molluscs.

[0047] The term “population of molluscs” as used herein may refer to a mixed-species, geographically discreet population of molluscs, or single-species.

[0048] As used herein, the terms "controlling a mollusc population" or "controls a mollusc" refers to any effect on a mollusc that results in limiting the damage that the mollusc causes. Controlling a mollusc includes, but is not limited to, killing the mollusc, inhibiting development of the mollusc, altering fertility or growth of the mollusc in such a manner that the mollusc provides less damage to the plant, decreasing the number of offspring produced, producing less fit molluscs, producing molluscs more susceptible to predator attack or deterring the molluscs from eating the plant. “Controlling a population of molluscs” typically means that the number of molluscs within a population of molluscs is reduced, principally through mortality, at a level that is significantly greater than a population to which the method of the present invention is not performed, or a composition of the invention is not present.

[0049] The term “agronomically acceptable” as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Environmental Protection Agency Register, European Environment Agency Register or other generally recognized register.

[0050] The term “biological stability” as used herein refers to the maintenance of viability of the composition over time and / or a range of temperatures. By way of non-limiting example, a preparation with increased biological stability may comprise lolines that remain active for a longer period of time compared to a relevant control (e.g. the corresponding lolines in extracted form or a composition comprising such extracted loline(s)). Alternatively, or in addition, a preparation with increased biological stability may comprise endophytes that remain viable for a longer period of time compared to a relevant control. Further alternatively or in addition, a preparation with increased biological stability may comprise lolines that remain active at higher and / or lower temperatures compared to a relevant control (e.g. the corresponding lolines in extracted form or a composition comprising such extracted loline(s)). Further alternatively or in addition, a preparation with increased biological stability may comprise endophytes that remain viable at higher and / or lower temperatures compared to a relevant control. Maintaining viability may be defined in terms of the activity of the one or more loline species. Activity may be determined using any appropriate assay, such as those exemplified herein. By way of non-limiting example, a composition of the invention may be considered viable if the activity of the at least one loline species is at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%, at least 99% against a suitable control (e.g. the corresponding lolines in extracted form or a composition comprising such extracted loline(s)). Maintaining viability may be defined in terms of the survival of the endophytes. By way of non-limiting example, a composition of the invention may be considered viable if at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%, at least 99%, or more of the endophytes survive. By way of non-limiting example, a composition of the invention may be considered viable if the germination rate of the endophytes is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more. Preferably, a composition of the invention may be considered viable if the germination rate of its endophyte spores is at least 90%, more preferably at least 95%, even more preferably at least 99%. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0051] Composition

[0052] The present invention provides a biological molluscicidal bait composition, comprising: 1) endophyte-infected plant biomass comprising at least one loline species (e.g., loline alkaloids), and 2) one or more phagostimulants. The plant biomass is derived from an endophyte-infected plant (with the endophytes producing the lolines) and the at least one loline species is not extracted from the plant biomass and is not synthetic.

[0053] The term “biological molluscicidal composition” refers to a composition comprising an agent derived from natural materials such as microbes, plants, animals and minerals, wherein the agent acts as a pesticide against organisms of the phylum Mollusca. Specifically, the present invention relates to molluscicidal compositions derived from endophyte-infected plants (e.g. grass).

[0054] A composition of the invention may comprise at least one loline species (e.g. any 1 , 2, 3, 4, 5, 6 or more loline species). Any loline species (or combination thereof) may be used in the present invention, provided said loline species (or combination thereof) is molluscicidal. Loline species are known in the art and can be readily identified as such by a skilled person without undue burden. Non-limiting examples of loline species and combinations thereof are described herein.

[0055] According to the invention, the at least one loline species is comprised within the plant biomass. In other words, the loline is present in (and inherent to) the plant from which the plant biomass is obtained and which is used to produce the composition, rather than being added exogenously.

[0056] The invention provides the at least one loline species is produced by an endophyte in the endophyte-infected plant, which loline species is then in turn present within the plant biomass. In other words, the at least one loline species and the plant biomass are derived from the same endophyte-infected plant. Thus, the invention provides a biological molluscicidal composition comprising plant biomass which is derived from an endophyte-infected plant, wherein the plant biomass comprises at least one loline species. Advantageously, according to the invention, the at least one loline species is not extracted from the plant biomass before use in a composition of the invention. Thus, the plant biomass containing the endophyte (and the at least one loline species produced by said endophyte) are harvested together and processed to produce a composition of the invention. Exemplary methods for the production of such compositions are described herein.

[0057] Any endophyte species (or combination thereof) may be used in the present invention, provided said endophyte species (or combination thereof) produces at least one molluscicidal loline species. Endophytes are known in the art and can be readily identified as such by a skilled person without undue burden. Non-limiting examples of endophytes are described herein.

[0058] A composition of the invention may function as a systemic molluscicidal composition, meaning that the active molluscicidal agent(s) (i.e., the at least one loline species) is soluble enough in water that it can be absorbed by a plant and distributed around in its tissues.

[0059] A composition of the invention may act as a passive molluscicide and / or active molluscicide. According to the invention, the composition actively attracts a mollusc through the use of a phagostimulant. Consumption of the plant biomass comprising the loline species may kill the mollusc, inhibit development of the mollusc, alter fertility or growth of the mollusc in such a manner that the mollusc provides less damage to the plant, decrease the number of offspring produced, produce less fit molluscs (i.e. the molluscs have lower levels of fitness), produce molluscs more susceptible to predator attack or deter the molluscs from eating the plant. Preferably, consumption of the plant biomass comprising the loline species will kill the mollusc. As used herein, the term passive means that the composition does not actively attract molluscs. Instead, when the composition is applied to a locus, for example by drilling or placing molluscicidal pellets of the invention into soil (e.g., furrows), the loline species will leach from the composition and be absorbed by the plants (e.g. crops) in the locus. Systemic distribution of the loline species in the plant means that consumption of the plant (e.g. crop) comprising the loline species will be lethal to the mollusc.

[0060] Preferably, a composition of the invention acts as both an active and passive molluscicide. This dual-action system tackles mollusc populations directly and protects crops in the long term, offering significant benefits for (UK) farmers, gardeners, and growers.

[0061] A composition of the invention may be formulated in various ways using formulation adjuvants, such as carriers, solvents and surface-active substances. The formulations can be in various physical forms, e.g. in the form of dusting powders, gels, wettable powders, water-dispersible granules, water-dispersible tablets, effervescent pellets, emulsifiable concentrates, micro- emulsifiable concentrates, oil-in-water emulsions, oil-flowables, aqueous dispersions, oily dispersions, suspo-emulsions, capsule suspensions, emulsifiable granules, soluble liquids, water-soluble concentrates (with water or a water-miscible organic solvent as carrier), impregnated polymer films or in other forms known e.g. from the Manual on Development and Use of FAQ and WHO Specifications for Pesticides, United Nations, First Edition, Second Revision (2010). Such formulations can either be used directly or diluted prior to use. Regardless of the form, the composition does not comprise an extracted loline species. Rather, the loline is comprised in situ within the plant biomass (having been produced by the endophyte-infected plant from which the biomass was obtained), even if the loline is subsequently solubilised and absorbed by a plant to be treated. Typically the composition is a solid formulation, as described further herein, preferably a pellet or granule.

[0062] Advantageously, the compositions of the invention are not toxic to mammals (e.g. humans) and / or bees. Compositions of the invention are therefore safe for widespread use in agricultural and horticultural settings.

[0063] Further advantageously, the composition does not comprise a synthetic loline species. Instead the composition comprises one or more lolines produced by an endophyte in an endophyte- infected plant used to provide the plant biomass.

[0064] A composition of the invention may be produced according to the methods described herein. Advantageously, the methods of the invention do not involve steps to extract the loline species from the plant biomass.

[0065] A composition of the invention may comprise one or more additive. Typically said one or more additive may be selected from a phagostimulant, an active ingredient, an adjuvant, a carrier, a diluent, an emulsifying agent and an inert ingredient. As described herein, the “phagostimulant” means a component that induces sustained feeding and / or a synthetic compound that is added to bait in order to attract molluscs. The term “active ingredient” is intended to mean any substance that is capable of repelling, or otherwise controlling a target pest (e.g., a mollusc). The term “adjuvant” means a chemical that is combined with the composition to improve mixing, application or to enhance molluscicidal activity. The term “carrier” means a substance that aids the effective dispersal of the composition (e.g., talc in a dust formulation). The term “diluent” means anything used to dilute a pesticide. The term “emulsifying agent” is intended to mean any agent which helps to prevent an emulsion from separating. The term “inert ingredient” means any ingredient other than the active ingredient. Inert ingredients may be added to dilute the pesticide or to make it safer, more effective, and easier to measure, mix, apply, and handle.

[0066] Non-limiting examples of such additives are described herein. A composition of the invention comprises at least one phagostimulant. Again, phagostimulants are described in more detail herein, including non-limiting examples thereof. Thus, a composition of the invention may further comprise or consist of one or more phagostimulant, active ingredient, adjuvant, carrier, diluent, emulsifying agent and / or inert ingredient. In particular, a composition of the invention comprises one or more phagostimulant, such as those described herein.

[0067] Additives

[0068] The one or more additive may comprise an active agent. Preferred active agents include one or more phagostimulant that function as bait to attract a mollusc (e.g. a gastropod such as a slug or snail) to the composition. Suitable phagostimulants are well known to the skilled person, and include cereal grains or flour (e.g., wheat, corn or bran grains, wheat flour, cornflour, grain flour or bakers flour), rice, fruit extracts, molasses, sugars (e.g., sucrose, glucose, lactose and / or fructose), yeast extracts, beer, fermented foods, and / or foods with a strong odour. For example, the composition may comprise one or more phagostimulant selected from cereal grains or flour (e.g., wheat, corn or bran grains, wheat flour, corn flour, grain flour or bakers flour), rice, fruit extracts, molasses, sugars (e.g., sucrose, glucose, lactose and / or fructose), yeast extracts, beer, fermented foods, and / or foods with a strong odour. In preferred embodiments, the composition comprises one or more phagostimulant selected from cereal grains or flour (e.g., wheat, corn or bran grains, wheat flour, cornflour, grain flour or bakers flour).

[0069] Additionally, or alternatively, the composition may comprise a further active agent, wherein the active agent is a further pesticide. Suitable known pesticides include those disclosed in the Pesticide Manual (The Pesticide Manual - A World Compendium; Seventeenth edition; Editor: C. D. S. Tomlin; The British Crop Protection Council). For example, the composition may comprise a further active agent selected from an acaricide, bactericide, fungicide, herbicide, insecticide, miticide, molluscicide, or a nematicide. When present, the further pesticide may be a second molluscicide.

[0070] Preferably, the one or more loline species in the composition is the only molluscicidal agent present. Further preferably, a composition of the invention does not comprise an additional pesticide.

[0071] A composition of the invention may further comprise or consist of at least one crop enhancing additive. According to the present invention, ‘crop enhancement’ or ‘enhancing crops’ means an improvement in plant vigour (e.g. early vigour), an improvement in plant quality, improved tolerance to stress factors and / or improved input use efficiency.

[0072] According to the present invention, an ‘improvement in plant vigour’ means that certain traits are improved qualitatively or quantitatively when compared with the same trait in a control plant which has been grown under the same conditions in the absence of a composition of the invention. Such traits include, but are not limited to, early and / or improved germination, improved emergence, the ability to use less seeds, increased root growth, a more developed root system, increased root nodulation, increased shoot growth, increased tillering, stronger tillers, more productive tillers, increased or improved plant stand, less plant verse (lodging), an increase and / or improvement in plant height, an increase in plant weight (fresh or dry), rapid expansion of leaf area in early stages of growth, bigger leaf blades, greener leaf colour, increased pigment content, increased photosynthetic activity, earlier flowering, longer panicles, early grain maturity, increased seed, fruit or pod size, increased pod or ear number, increased seed number per pod or ear, increased seed mass, enhanced seed filling, less dead basal leaves, delay of senescence, improved vitality of the plant, increased levels of amino acids in storage tissues and / or less inputs needed (e.g. less fertiliser, water and / or labour needed). A plant with improved vigour (e.g. early vigour) may have an increase in any of the aforementioned traits or any combination or two or more of the aforementioned traits.

[0073] According to the present invention, an ‘improvement in plant quality’ means that certain traits are improved qualitatively or quantitatively when compared with the same trait in a control plant which has been grown under the same conditions in the absence of a composition of the invention. Such traits include, but are not limited to, improved visual appearance of the plant, reduced ethylene (reduced production and / or inhibition of reception), improved quality of harvested material, e.g. seeds, fruits, leaves, vegetables (such improved quality may manifest as improved visual appearance of the harvested material), improved carbohydrate content (e.g. increased quantities of sugar and / or starch, improved sugar acid ratio, reduction of reducing sugars, increased rate of development of sugar), improved protein content, improved oil content and composition, improved nutritional value, reduction in anti-nutritional compounds, improved organoleptic properties (e.g. improved taste) and / or improved consumer health benefits (e.g. increased levels of vitamins and anti-oxidants)), improved postharvest characteristics (e.g. enhanced shelf-life and / or storage stability, easier processability, easier extraction of compounds), more homogenous crop development (e.g. synchronised germination, flowering and / or fruiting of plants), and / or improved seed quality (e.g. for use in following seasons). A plant with improved quality may have an increase in any of the aforementioned traits or any combination or two or more of the aforementioned traits.

[0074] According to the present invention, an ‘improved tolerance to stress factors’ means that certain traits are improved qualitatively or quantitatively when compared with the same trait in a control plant which has been grown under the same conditions in the absence of a composition of the invention. Such traits include, but are not limited to, an increased tolerance and / or resistance to abiotic stress factors which cause sub-optimal growing conditions such as drought (e.g. any stress which leads to a lack of water content in plants, a lack of water uptake potential or a reduction in the water supply to plants), cold exposure, heat exposure, osmotic stress, UV stress, flooding, increased salinity (e.g. in the soil), increased mineral exposure, ozone exposure, high light exposure and / or limited availability of nutrients (e.g. nitrogen and / or phosphorus nutrients). A plant with improved tolerance to stress factors may have an increase in any of the aforementioned traits or any combination or two or more of the aforementioned traits. In the case of drought and nutrient stress, such improved tolerances may be due to, for example, more efficient uptake, use or retention of water and nutrients.

[0075] According to the present invention, an ‘improved input use efficiency’ means that the plants are able to grow more effectively using given levels of inputs compared to the growth of control plants which are grown under the same conditions in the absence of a composition of the invention. In particular, the inputs include, but are not limited to fertiliser (such as nitrogen, phosphorous, potassium, micronutrients), light and water. A plant with improved input use efficiency may have an improved use of any of the aforementioned inputs or any combination of two or more of the aforementioned inputs.

[0076] Other crop enhancements of the present invention include a decrease in plant height, or reduction in tillering, which are beneficial features in crops or conditions where it is desirable to have less biomass and fewer tillers.

[0077] Any of or all the above crop enhancements may lead to an improved yield by improving, for example, plant physiology, plant growth and development and / or plant architecture.

[0078] In the context of the present invention ‘yield’ includes, but is not limited to, (i) an increase in biomass production, grain yield, starch content, oil content and / or protein content, which may result from (a) an increase in the amount produced by the plant per se or (b) an improved ability to harvest plant matter, (ii) an improvement in the composition of the harvested material (e.g. improved sugar acid ratios, improved oil composition, increased nutritional value, reduction of anti-nutritional compounds, increased consumer health benefits) and / or (iii) an increased / facilitated ability to harvest the crop, improved processability of the crop and / or better storage stability / shelf life. Increased yield of an agricultural plant means that, where it is possible to take a quantitative measurement, the yield of a product of the respective plant is increased by a measurable amount over the yield of the same product of the plant produced under the same conditions, but without application of the present invention. According to the present invention, it is preferred that the yield be increased by at least 0.5%, preferably 1 %, 2%, 3% or more. Even more preferred is an increase in yield of at least about 5%, 10%, 15% or 20% or more.

[0079] Any, or all, of the above crop enhancements may also lead to an improved utilisation of land, that is land which was previously unavailable or sub-optimal for cultivation may become available. For example, plants which show an increased ability to survive in drought conditions, may be able to be cultivated in areas of sub-optimal rainfall, for example on the fringe of a desert or even the desert itself.

[0080] By way of non-limiting example, a composition may comprise one or more additive that is a plant growth regulator such as 1 -methylcyclopropene, 1-naphthol, 2,3,5-tri-iodobenzoic acid, 2,3-dihydro-5,6-diphenyl-1 ,4-oxath(ll)ne, 2,4,5-T, 2,4-D, 2,4-DB, 2,4-DEP, 24-epi- brassinolide, 28-homobrassinolide, 2-cyano-3-(2,4-dichlorophenyl)acrylic acid, 2- hydrazinoethanol, 2iP, 4-CPA, 4-hydroxyphenethyl alcohol, abscisic acid, AC 94377, ACC, ancymidol, auxins, aviglycine, bachmedesh, benzofluor, benzyladenine, 24-epi-brassinolide, 28-homo-brassinolide, brassinolide, brassinolide-ethyl, brassinosteroids, BTS 44584, buminafos, butralin, calcium cyanamide, carbaryl, carvone, chlorfluren, chlorflurenol, chlormequat-chloride, chlorphonium, chlorpropham, choline chloride, ciobutide, clofencet, clofibric acid, cloprop, cloxyfonac, cyanamide, cyclanilide, cycloheximide, cyprosulfamide, cytokinins, daminozide, DCPTA, deoxystrigol, dicamba-methyl, dichlorflurenol, dichlorflurenol- methyl, dichlorprop, dikegulac, dimexano, endothal, epocholeone, etacelasil, ethephon, ethychlozate, ethylene, fenoprop, fenridazon, flumetralin, fluoridamid, flurenol, flurprimidol, forchlorfenuron, fosamine, fuphenthiourea, furalane, gibberellic acid, gibberellins, glyoxime, glyphosine, GR-24, heptopargil, hexafluoroacetone trihydrate, holosulf, hymexazol, IAA, IBA, Inabenfide, INCYDE, isoprothiolane, isopyrimol, jasmonates, jasmonic acid, karetazan, karrikins, kinetin, lead arsenate, maleic hydrazide, MCPB, mefluidide, mepiquat, merphos, methasulfocarb, metoxuron, N-(2-ethyl-2H-pyrazol-3-yl)-N'-phenylurea, N-m-tolylphthalamic acid, Npyrrolidinosuccinamic acid, naphthaleneacetamide, naphthoxyacetic acids, n-decanol, nonanoic acid, N-phenylphthalamic acid, orobanchol, paclobutrazol, pentachlorophenol, piproctanyl, potassium naphthenate, polyamines, prohexadione-calcium, prohydrojasmon, propham, propyl 3-tert -butylphenoxyacetate, prosuler, pydanon, pyripropanol, salicylic acid, sintofen, sodium (Z)-3-chloroacrylate, sodium naphthenate, sorgolactone, strigol, strigolactones, tecnazene, tetcyclacis, thidiazuron, tiaojiean, triacontanol, triapenthenol, tribufos, trinexapac, trinexapac-ethyl, uniconazole, zeatin, and a-naphthaleneacetic acid.

[0081] Alternatively or in addition, by way of a further non-limiting example, a composition may comprise one or more additive that enhances plant tolerance to abiotic stress. Exemplary additives may include biostimulant products that contain one or more micronutrients, macronutrients, plant hormones, or amino acids. Examples of biostimulant products include seaweed extracts, Quantis ™, Isa bi on™, Vitazyme ™, Megafol™, Releaseed™, Biozyme™, TerraSorb™, Aminocore™, Radical™, Proplex™, Bioforge ™, Terrabiogen™, Folicist™, Cytozyme™, Cytoplant™, and Greenstim™. Further alternatively or in addition, a composition may also include, or be applied in conjunction with, one or more beneficial organisms.

[0082] A composition of the invention may also comprise one or more carrier. Suitable carriers may be either solid or liquid carriers.

[0083] Suitable solid carriers may be selected from: talc, titanium dioxide, pyrophyllite clay, silica, attapulgite clay, kieselguhr, limestone, calcium carbonate, bentonite, calcium montmorillonite, cottonseed husks, wheat flour, bakers flour, cornflour soybean flour, pumice, wood flour, ground walnut shells, and lignin.

[0084] Liquid carriers may be selected from mineral oils, oils of vegetable or animal origin, modified oils of vegetable or animal origin, organic solvents, water, surface-active substances or combinations thereof, for example: water, toluene, xylene, petroleum ether, vegetable oils, acetone, methyl ethyl ketone, cyclohexanone, acid anhydrides, acetonitrile, acetophenone, amyl acetate, 2-butanone, butylene carbonate, chlorobenzene, cyclohexane, cyclohexanol, alkyl esters of acetic acid, diacetone alcohol, 1 ,2-dichloropropane, diethanolamine, p- diethylbenzene, diethylene glycol, diethylene glycol abietate, diethylene glycol butyl ether, diethylene glycol ethyl ether, diethylene glycol methyl ether, N,N-dimethylformamide, dimethyl sulfoxide, 1 ,4-dioxane, dipropylene glycol, dipropylene glycol methyl ether, dipropylene glycol dibenzoate, diproxitol, alkylpyrrolidone, ethyl acetate, 2-ethylhexanol, ethylene carbonate, 1 ,1 ,1 -trichloroethane, 2- heptanone, alpha-pinene, d-limonene, ethyl lactate, ethylene glycol, ethylene glycol butyl ether, ethylene glycol methyl ether, gamma-butyrolactone, glycerol, glycerol acetate, glycerol diacetate, glycerol triacetate, hexadecane, hexylene glycol, isoamyl acetate, isobornyl acetate, isooctane, isophorone, isopropylbenzene, isopropyl myristate, lactic acid, laurylamine, mesityl oxide, methoxypropanol, methyl isoamyl ketone, methyl isobutyl ketone, methyl laurate, methyl octanoate, methyl oleate, methylene chloride, m- xylene, n-hexane, n- octylamine, octadecanoic acid, octylamine acetate, oleic acid, oleylamine, o-xylene, phenol, polyethylene glycol, propionic acid, propyl lactate, propylene carbonate, propylene glycol, propylene glycol methyl ether, p-xylene, toluene, triethyl phosphate, triethylene glycol, xylenesulfonic acid, paraffin, mineral oil, trichloroethylene, perchloroethylene, ethyl acetate, amyl acetate, butyl acetate, propylene glycol methyl ether, diethylene glycol methyl ether, methanol, ethanol, isopropanol, and alcohols of higher molecular weight, such as amyl alcohol, tetrahydro-furfuryl alcohol, hexanol, octanol, ethylene glycol, propylene glycol, glycerol, N-methyl-2-pyrrolidone and the like.

[0085] Compositions of the invention may include one or more additive selected from the list comprising: flour (e.g., bakers flour and cornflour), maize starch, perlite, potassium sorbate, sepiolite, sorbic acid, sucrose, talc and tylose. Solid formulations

[0086] Preferably, the composition is formulated as a solid composition. Solid compositions include pellets, tablets, granules, powders or gels. The composition may be formulated as a wettable formulation (e.g. a wettable pellet, tablet, granule, powder or gel), a water-dispersible formulation (e.g. a water-dispersible pellet, tablet, granule, powder or gel), an effervescent formulation (e.g. a wettable pellet, tablet, granule, powder or gel).

[0087] Particularly preferred formulations of the compositions of the invention are pellets, coated core prill and / or granules, most preferably pellets. Preferably, the pellets are extruded pellets.

[0088] The size of pellets is not particularly limited, but may be any appropriate size as determined by factors such as the method of application (e.g., for use in furrow), plant to be treated, mollusc to be controlled, ease of application, etc. By way of non-limiting example, average (e.g. mean) diameter of the pellets may be in the region of from about 1mm to about 10mm, particularly from about 1 mm to about 5mm. For example, and according to a preferred feature of the present invention, the average (e.g. mean) diameter of the pellets may be in the region of from about 1 ,4mm to about 3.5mm.

[0089] The mass of the pellets is not particularly limited, but may be any appropriate mass as determined by factors such as the method of application (e.g., for use in furrow), plant to be treated, mollusc to be controlled, ease of application, etc. By way of non-limiting example, average (e.g. mean) mass of the pellets may be in the region of from about 1 mg to about 10mg, particularly from about 5mg to about 10mg. Preferably, average (e.g. mean) mass of the pellets may be about 8mg.

[0090] Lolines

[0091] Loline species (also referred to interchangeably as loline alkaloids) are produced through the symbiosis of fungal endophytes such as Epichloe or Neotyphodium with host plants, particularly grasses such as meadow fescue.

[0092] Loline species have the following general chemical formula: A composition of the invention may comprises at least one loline species falling within this general formula.

[0093] Typically, a composition of the invention may comprise one or more loline species having the following chemical formula (I): wherein:

[0094] R = H or CH3; and

[0095] R' = H, CH3, CHO, COCH3.

[0096] For example, a composition of the invention may comprise one or more loline species having the formula (I), wherein:

[0097] R = CH3and R’ = H (i.e. , the loline species is loline);

[0098] R = H and R’ = H (i.e., the loline species is norloline);

[0099] R = CH3and R’ = CH3(i.e., the loline species is N-methylloline);

[0100] R = CH3and R’ = CHO (i.e., the loline species is N-formylloline);

[0101] R = H and R’ = CHO (i.e., the loline species is N-formylnorloline);

[0102] R = CH3and R’ = COCH3(i.e., the loline species is N-acetylloline); and / or

[0103] R = H and R’ = COCH3(i.e., the loline species is N-acetylnorloline).

[0104] At least one of the loline species may be a saturated pyrrolizidine alkaloid. The composition of the invention may comprise any loline species of the disclosure or a mixture (combination) thereof.

[0105] The composition may comprise one or more loline species selected from the list comprising: N-acetylloline, N-formylloline, N-acetylnorloline, loline, norloline, N-formylnorloline, and N- methylloline or a variant thereof. In some aspects, the composition comprises two or more loline species selected from the list comprising: N-acetylloline, N-formylloline, N-acetylnorloline, loline, norloline, N- formylnorloline, and N-methylloline or a variant thereof.

[0106] In some aspects, the composition comprises three or more loline species selected from the list comprising: N-acetylloline, N-formylloline, N-acetylnorloline, loline, norloline, N- formylnorloline, and N-methylloline or a variant thereof.

[0107] In some aspects, the composition comprises four or more loline species selected from the list comprising: N-acetylloline, N-formylloline, N-acetylnorloline, loline, norloline, N- formylnorloline, and N-methylloline or a variant thereof.

[0108] In some aspects, the composition comprises five or more loline species selected from the list comprising: N-acetylloline, N-formylloline, N-acetylnorloline, loline, norloline, N- formylnorloline, and N-methylloline or a variant thereof.

[0109] In some aspects, the composition comprises six or more loline species selected from the list comprising: N-acetylloline, N-formylloline, N-acetylnorloline, loline, norloline, N- formylnorloline, and N-methylloline or a variant thereof.

[0110] In some aspects, the composition comprises N-acetylloline, N-formylloline, N-acetylnorloline, loline, norloline, N-formylnorloline, and N-methylloline or a variant thereof.

[0111] The at least one loline species in the composition may comprises or consist of one of the combinations shown below:

[0112] I. N-acetylloline

[0113] II. N-formylloline

[0114] III. N-acetylnorloline

[0115] IV. loline

[0116] V. norloline

[0117] VI. N-formylnorloline

[0118] VII. N-methylloline

[0119] VIII. N-acetylloline, and N-formylloline

[0120] IX. N-acetylloline, and N-acetylnorloline

[0121] X. N-acetylloline, and loline

[0122] XI. N-acetylloline, and norloline

[0123] XII. N-acetylloline, and N-formylnorloline

[0124] XIII. N-acetylloline, and N-methylloline

[0125] XIV. N-formylloline, and N-acetylnorloline

[0126] XV. N-formylloline, and loline XVI. N-formylloline, and norloline

[0127] XVII. N-formylloline, and N-formylnorloline

[0128] XVIII. N-formylloline, and N-methylloline

[0129] XIX. N-acetylnorloline, and loline

[0130] XX. N-acetylnorloline, and norloline

[0131] XXI. N-acetylnorloline, and N-formylnorloline

[0132] XXII. N-acetylnorloline, and N-methylloline

[0133] XXIII. loline, and norloline

[0134] XXIV. loline, and N-formylnorloline

[0135] XXV. loline, and N-methylloline

[0136] XXVI. norloline, and N-formylnorloline

[0137] XXVII. norloline, and N-methylloline

[0138] XXVIII. N-formylnorloline, and N-methylloline

[0139] XXIX. N-acetylloline, N-formylloline, and N-acetylnorloline

[0140] XXX. N-acetylloline, N-formylloline, and loline

[0141] XXXI. N-acetylloline, N-formylloline, and norloline

[0142] XXXII. N-acetylloline, N-formylloline, and N-formylnorloline

[0143] XXXIII. N-acetylloline, N-formylloline, and N-methylloline

[0144] XXXIV. N-acetylloline, N-acetylnorloline, and loline

[0145] XXXV. N-acetylloline, N-acetylnorloline, and norloline

[0146] XXXVI. N-acetylloline, N-acetylnorloline, and N-formylnorloline

[0147] XXXVII. N-acetylloline, N-acetylnorloline, and N-methylloline

[0148] XXXVIII. N-acetylloline, loline, and norloline

[0149] XXXIX. N-acetylloline, loline, and N-formylnorloline

[0150] XL. N-acetylloline, loline, and N-methylloline

[0151] XLI. N-acetylloline, norloline, and N-formylnorloline

[0152] XLII. N-acetylloline, norloline, and N-methylloline

[0153] XLI II. N-acetylloline, N-formylnorloline, and N-methylloline

[0154] XLIV. N-formylloline, N-acetylnorloline, and loline

[0155] XLV. N-formylloline, N-acetylnorloline, and norloline

[0156] XLVI. N-formylloline, N-acetylnorloline, and N-formylnorloline

[0157] XLVII. N-formylloline, N-acetylnorloline, and N-methylloline

[0158] XLVIII. N-formylloline, loline, and norloline

[0159] XLIX. N-formylloline, loline, and N-formylnorloline

[0160] L. N-formylloline, loline, and N-methylloline

[0161] LI. N-formylloline, norloline, and N-formylnorloline

[0162] Lil. N-formylloline, norloline, and N-methylloline LIII. N-formylloline, N-formylnorloline, and N-methylloline

[0163] LIV. N-acetylnorloline, loline, and norloline

[0164] LV. N-acetylnorloline, loline, and N-formylnorloline

[0165] LVI. N-acetylnorloline, loline, and N-methylloline

[0166] LVII. N-acetylnorloline, norloline, and N-formylnorloline

[0167] LVI II. N-acetylnorloline, norloline, and N-methylloline

[0168] LIX. N-acetylnorloline, N-formylnorloline, and N-methylloline

[0169] LX. loline, norloline, and N-formylnorloline

[0170] LXI. loline, norloline, and N-methylloline

[0171] LXI I. loline, N-formylnorloline, and N-methylloline

[0172] LXI II. norloline, N-formylnorloline, and N-methylloline

[0173] LXIV. N-acetylloline, N-formylloline, N-acetylnorloline, and loline

[0174] LXV. N-acetylloline, N-formylloline, N-acetylnorloline, and norloline

[0175] LXVI. N-acetylloline, N-formylloline, N-acetylnorloline, and N-formylnorloline

[0176] LXVII. N-acetylloline, N-formylloline, N-acetylnorloline, and N-methylloline

[0177] LXVIII. N-acetylloline, N-formylloline, loline, and norloline

[0178] LXIX. N-acetylloline, N-formylloline, loline, and N-formylnorloline

[0179] LXX. N-acetylloline, N-formylloline, loline, and N-methylloline

[0180] LXXI. N-acetylloline, N-formylloline, norloline, and N-formylnorloline

[0181] LXXII. N-acetylloline, N-formylloline, norloline, and N-methylloline

[0182] LXXIII. N-acetylloline, N-formylloline, N-formylnorloline, and N-methylloline

[0183] LXXIV. N-acetylloline, N-acetylnorloline, loline, and norloline

[0184] LXXV. N-acetylloline, N-acetylnorloline, loline, and N-formylnorloline

[0185] LXXVI. N-acetylloline, N-acetylnorloline, loline, and N-methylloline

[0186] LXXVII. N-acetylloline, N-acetylnorloline, norloline, and N-formylnorloline

[0187] LXXVIII. N-acetylloline, N-acetylnorloline, norloline, and N-methylloline

[0188] LXXIX. N-acetylloline, N-acetylnorloline, N-formylnorloline, and N-methylloline LXXX. N-acetylloline, loline, norloline, and N-formylnorloline

[0189] LXXXI. N-acetylloline, loline, norloline, and N-methylloline

[0190] LXXXII. N-acetylloline, loline, N-formylnorloline, and N-methylloline

[0191] LXXXIII. N-acetylloline, norloline, N-formylnorloline, and N-methylloline

[0192] LXXXIV. N-formylloline, N-acetylnorloline, loline, and norloline

[0193] LXXXV. N-formylloline, N-acetylnorloline, loline, N-formylnorloline

[0194] LXXXVI. N-formylloline, N-acetylnorloline, loline, and N-methylloline

[0195] LXXXVII. N-formylloline, N-acetylnorloline, norloline, and N-formylnorloline LXXXVIII. N-formylloline, N-acetylnorloline, norloline, and N-methylloline

[0196] LXXXIX. N-formylloline, N-acetylnorloline, N-formylnorloline, and N-methylloline XC. N-formylloline, loline, norloline, and N-formylnorloline

[0197] XCI. N-formylloline, loline, norloline, and N-methylloline

[0198] XCII. N-formylloline, loline, N-formylnorloline, and N-methylloline

[0199] XCI II. N-formylloline, norloline, N-formylnorloline, and N-methylloline

[0200] XCIV. N-acetylnorloline, loline, norloline, and N-formylnorloline

[0201] XCV. N-acetylnorloline, loline, norloline, and N-methylloline

[0202] XCVI. N-acetylnorloline, loline, N-formylnorloline, and N-methylloline

[0203] XCVII. N-acetylnorloline, norloline, N-formylnorloline, and N-methylloline XCVI II. loline, norloline, N-formylnorloline, and N-methylloline

[0204] XCIX. N-acetylloline, N-formylloline, N-acetylnorloline, loline, and norloline

[0205] C. N-acetylloline, N-formylloline, N-acetylnorloline, loline, and N-formylnorloline

[0206] Cl. N-acetylloline, N-formylloline, N-acetylnorloline, loline, and N-methylloline

[0207] CH. N-acetylloline, N-formylloline, N-acetylnorloline, norloline, and N- formylnorloline

[0208] CHI. N-acetylloline, N-formylloline, N-acetylnorloline, norloline, and N-methylloline CIV. N-acetylloline, N-formylloline, N-acetylnorloline, N-formylnorloline, and N- methylloline

[0209] CV. N-acetylloline, N-formylloline, loline, norloline, and N-formylnorloline

[0210] CVI. N-acetylloline, N-formylloline, loline, norloline, and N-methylloline

[0211] CVH. N-acetylloline, N-formylloline, loline, N-formylnorloline, and N-methylloline CVIH. N-acetylloline, N-formylloline, norloline, N-formylnorloline, and N-methylloline CIX. N-acetylloline, N-acetylnorloline, loline, norloline, and N-formylnorloline

[0212] CX. N-acetylloline, N-acetylnorloline, loline, norloline, and N-methylloline

[0213] CXI. N-acetylloline, N-acetylnorloline, loline, N-formylnorloline, and N-methylloline

[0214] CXII. N-acetylloline, N-acetylnorloline, norloline, N-formylnorloline, and N- methylloline

[0215] CXIH. N-acetylloline, loline, norloline, N-formylnorloline, and N-methylloline

[0216] CXIV. N-formylloline, N-acetylnorloline, loline, norloline, and N-formylnorloline

[0217] CXV. N-formylloline, N-acetylnorloline, loline, norloline, and N-methylloline

[0218] CXVI. N-formylloline, N-acetylnorloline, loline, N-formylnorloline, and N-methylloline CXVH. N-formylloline, N-acetylnorloline, norloline, N-formylnorloline, and N- methylloline

[0219] CXVIH. N-formylloline, loline, norloline, N-formylnorloline, and N-methylloline CXIX. N-acetylnorloline, loline, norloline, N-formylnorloline, and N-methylloline CXX. N-acetylloline, N-formylloline, N-acetylnorloline, loline, norloline, and N- formylnorloline CXXI. N-acetylloline, N-formylloline, N-acetylnorloline, loline, norloline, and N- methylloline

[0220] CXXII. N-acetylloline, N-formylloline, N-acetylnorloline, loline, N-formylnorloline, and N-methylloline

[0221] CXXIII. N-acetylloline, N-formylloline, N-acetylnorloline, norloline, N-formylnorloline, and N-methylloline

[0222] CXXIV. N-acetylloline, N-formylloline, loline, norloline, N-formylnorloline, and N- methylloline

[0223] CXXV. N-acetylloline, N-acetylnorloline, loline, norloline, N-formylnorloline, and N- methylloline

[0224] CXXVI. N-formylloline, N-acetylnorloline, loline, norloline, N-formylnorloline, and N- methylloline

[0225] CXXVII. N-acetylloline, N-formylloline, N-acetylnorloline, loline, norloline, N- formylnorloline, and N-methylloline

[0226] Typically, the composition comprises at least one loline species selected from the group consisting of N-acetylloline, N-formylloline, and N-acetylnorloline. For example, the composition may comprise at least two loline species selected from the group consisting of N- acetylloline, N-formylloline, and N- acetylnorloline. For example, the composition may comprise N-acetylloline, N-formylloline, and N- acetylnorloline. Optionally, the composition may include further loline alkaloids. Preferably, the loline species in the composition comprises or consists of N-acetylloline and N-formylloline.

[0227] When the composition comprises N-acetylloline [NAL] and N-formylloline [NFL], the weight ratio of [NAL]:[NFL] in the composition may be greater than about 1 :8, about 1 :6, about 1 :4, about 3:11 , about 1 :3, or about 1 :2.

[0228] When the composition comprises N-acetylnorloline [NANL] and N-formylloline [NFL], the weight ratio of [NANL]:[NAL] in the composition may be greater than about 3:4, about 2:3, about 1 :2, about 6:11 , or about 1 :1.

[0229] When the composition comprises N-acetylnorloline [NANL] and N-acetylloline [NAL], the weight ratio of [NANL]:[NAL] in the composition may be greater than about 6:5, about 5:4, about 3:2, about 2:1 , about 3:1 , or about 4:1.

[0230] When the composition comprises N-acetylloline, N-formylloline and N-acetylnorloline, the weight ratio of the three loline compounds is optionally from 1 :3:1 to 1 :6:3. For example, the aforementioned loline compounds (i.e., N- acetylloline, N-formylloline and N-acetylnorloline) may be present in the following weight ratios: about 1 :3:1 , about 1 :3:2, about 1 :3:3, about 1 :4:1 , about 1 :4:2, about 1 :4:3, about 1 :5:1 , about 1 :5:2, about 1 :5:3, about 1 :6:1 , about 1 :6:2, about 1 :6:3.

[0231] Preferably, the weight ratio of N-acetylloline, N-formylloline and N-acetylnorloline is about 1 :4:2.

[0232] Amount of loline species

[0233] As disclosed herein, the composition typically comprises at least one loline species present in an amount effective to act as a molluscicide. For example, the loline species may be present in an amount effective to repel and / or control the population size of a pest of the phylum Mollusca, preferably a pest of the class gastropoda (e.g., slugs and snails), when applied to a given locus. The term “repel” is intended to mean discouraging a mollusc from contacting a plant or plant growth medium treated with the composition of the invention. The skilled person will be aware of suitable methods for quantifying the efficacy of a repellent; for example, by recording changes in mollusc location and feeding behaviour in treated vs control plants.

[0234] Optionally, the composition may comprise at least one loline species in the range of from 100 to 500 ppm. For example, the composition may comprise loline in the range of from 100 to 500 ppm, from 200 to 500 ppm, from 300 to 500 ppm or from 400 to 500 ppm. For example, the composition may comprise Norloline in the range of from 100 to 500 ppm, from 200 to 500 ppm, from 300 to 500 ppm or from 400 to 500 ppm. For example, the composition may comprise N-formylnorloline in the range of from 100 to 500 ppm, from 200 to 500 ppm, from 300 to 500 ppm or from 400 to 500 ppm. For example, the composition may comprise N- acetylloline in the range of from 100 to 500 ppm, from 200 to 500 ppm, from 300 to 500 ppm or from 400 to 500 ppm. For example, the composition may comprise N-formylloline in the range of from 100 to 500 ppm, from 200 to 500 ppm, from 300 to 500 ppm or from 400 to 500 ppm. For example, the composition may comprise N-acetylnorloline in the range of from 100 to 500 ppm, from 200 to 500 ppm, from 300 to 500 ppm or from 400 to 500 ppm. Preferably, the loline species is present in the range of from 200 to 500 ppm.

[0235] Wherein a composition comprises a combination of loline species (e.g., two, three, four, five or six), the concentration of each loline species may be selected independently. For example, the concentration of each loline may be independently selected from a concentration in the range of from 100 to 500 ppm. For example, the composition may comprise any one of the combinations of loline species shown in (VIII) to (CXXVII) above each at a concentration in the range of from 100 to 500 ppm, from 200 to 500 ppm, from 300 to 500 ppm or from 400 to 500 ppm. Preferably, the concentration of each loline species in a composition comprising a combination of loline species is in the range of from 200 to 500 ppm.

[0236] Alternatively, when a composition comprises a combination of loline species (e.g., two, three, four, five or six), the total concentration of all loline species may be in the range of from 100 to 500 ppm. Optionally the concentration of each individual loline is selected independently. For example, the composition may comprise any one of the combinations of loline species shown in (VIII) to (CXXVII) above, wherein the total concentration of loline species within the composition is in the range of from 100 to 500 ppm, from 200 to 500 ppm, from 300 to 500 ppm or from 400 to 500 ppm. Preferably, the total concentration of loline species in a composition is in the range of from 200 to 500 ppm.

[0237] Typically, a composition of the invention may comprise at least 0.45 mg loline species per gram of composition (e.g. dry composition). Preferably, a composition of the invention may comprise about 1 mg, 1.25mg, 1.5mg, 1 ,75mg or 2 mg loline species per gram of composition (e.g., dry composition).

[0238] Plant biomass

[0239] In preferred embodiments, the composition comprises plant biomass derived from an endophyte-infected plant. The term "plant" refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, rhizomes, stems, stalks, foliage, and fruits. In preferred embodiments, the at least one loline species and the plant biomass are derived from the same endophyte-infected plant. Therefore, the composition may comprise a mixture of at least one loline species derived from an endophyte-infected plant and plant biomass derived from the same endophyte-infected plant. For example, the endophyte-infected plant may be a grass, preferably a Festuca species. Therefore, the composition may comprise a mixture of at least one loline species derived from an endophyte-infected grass (e.g., a Festuca species such as Adenocarpus or Argyreia) and plant biomass derived from the same endophyte- infected grass (e.g., a Festuca species).

[0240] In some embodiments, the composition comprises from 50 to 99% by weight plant biomass, for example, the composition may comprise from 60 to 99% by weight plant biomass, from 70 to 99% by weight plant biomass, from 80 to 99% by weight plant biomass, from 90 to 99% by weight plant biomass, or from 95-99% by weight plant biomass. Endophytes

[0241] Endophytes are organisms, often fungi and bacteria, that live between living plant cells. Endophytes act as reservoirs of novel bioactive secondary metabolites, such as alkaloids, phenolic acids, quinones, steroids, saponins, tannins, and terpenoids.

[0242] Typically, the at least one loline species present in a composition of the invention is derived from a plant infected with an alkaloid-producing endophyte.

[0243] Any endophyte species (or combination thereof) may be used in the present invention, provided said endophyte species (or combination thereof) produces at least one loline species with molluscicidal activity. Endophyte species are known in the art and can be readily identified as such by a skilled person without undue burden. Non-limiting examples of endophyte species and combinations thereof are described herein. For example, the endophyte may be selected from the genus Epichloe or Neotyphodium. For example, when the endophyte is selected from the genus Epichloe, the endophyte may be Epichloe uncinate.

[0244] The composition may comprise loline species derived from one or more different species of endophyte.

[0245] The endophyte(s) may be comprised within the plant biomass, such that the at least one loline species produced by said endophyte(s) is also comprised within the plant biomass.

[0246] Preferably, both the loline species and the plant biomass are derived from a plant infected with an alkaloid-producing endophyte. In some embodiments, the endophyte is selected from the genus Epichloe or Neotyphodium. For example, when the endophyte is selected from the genus Epichloe, the endophyte may be Epichloe uncinate.

[0247] The endophyte-infected plant, plant biomass or harvested plant may comprise N-acetylloline [NAL] and N-formylloline [NFL], In particular, the weight ratio of [NAL]:[NFL] in the endophyte- infected plant, plant biomass or harvested plant may be greater than about 1 :8, about 1 :6, about 1 :4, about 3:11 , about 1 :3, or about 1 :2.

[0248] The endophyte-infected plant, plant biomass or harvested plant may comprise N- acetylnorloline [NANL] and N-formylloline [NFL], the weight ratio of [NANL]:[NAL] in the endophyte-infected plant, plant biomass or harvested plant may be greater than about 3:4, about 2:3, about 1 :2, about 6:11 , or about 1 :1.

[0249] The endophyte-infected plant, plant biomass or harvested plant may comprise N- acetylnorloline [NANL] and N-acetylloline [NAL], the weight ratio of [NANL]:[NAL] in the endophyte-infected plant, plant biomass or harvested plant may be greater than about 6:5, about 5:4, about 3:2, about 2:1 , about 3:1 , or about 4:1.

[0250] The endophyte-infected plant may be a plant that comprises at least N-acetylloline [NAL] and N-formylloline [NFL] at a ratio of about 3:11. The endophyte-infected plant may be a plant that comprises at least N-acetylnorloline [NANL] and N-formylloline [NFL] at a ratio of about 6:11. The endophyte-infected plant may be a plant that comprises at least N-acetylnorloline [NANL] and N-acetylloline [NAL] at a ratio of about 2:1. Preferably, the relative amounts indicated above are the relative amounts of loline species in the leaves of the endophyte- infected plant.

[0251] The plant clippings used in a method of the invention may be plant clippings that comprise at least N-acetylloline [NAL] and N-formylloline [NFL] at a ratio of about 3:11. The plant clippings used in a method of the invention may be plant clippings that comprise at least N- acetylnorloline [NANL] and N-formylloline [NFL] at a ratio of about 6:11. The plant clippings used in a method of the invention may be plant clippings that comprise at least N- acetylnorloline [NANL] and N-acetylloline [NAL] at a ratio of about 2:1. Preferably, the relative amounts indicated above are the relative amounts of loline species in the leaves of the endophyte-infected plant.

[0252] Molluscicidal activity

[0253] Typically, the molluscicidal activity of a composition of the invention is at least 30% greater, such as at least 35% greater, at least 40% greater, at least 50% greater or more than a composition without the at least one loline species. Said comparator composition may be a corresponding composition without the at least one loline species, e.g. the composition may comprise the same plant biomass, but derived from plants not infected by endophytes.

[0254] Alternatively or in addition, a composition of the invention is of such molluscicidal activity that the amount of composition required control the population of molluscs is at least 2 times less, such as at least 2.5 times less, at least 3 times less or lower than the amount of a composition without the at least one loline species. Said comparator composition may be a corresponding composition without the at least one loline species, e.g. the composition may comprise the same plant biomass, but derived from plants not infected by endophytes.

[0255] The skilled person will appreciate that molluscicidal activity may be determined based on observational studies (e.g., molluscs may stop feeding, become immobile or will be repelled in the presence of a molluscicidal composition. Additionally, in the case of forced fed trials, molluscs will get as far away from the treated crop as the boundaries will allow them to. Carbon sequestration

[0256] Production of the compositions of the invention produces significantly less carbon emissions than the production of synthetic pesticides (e.g. molluscicides). On the one hand, the compositions of the invention are not derived from petroleum. In addition, cultivating the endophyte infected plants functions to sequester carbon. For example, research conducted by the inventors indicates that a grass paddock will sequester about 1-3 metric tons of CO2 / Ha / Annum. This figure is even higher if the paddock is mowed; after four mowing passes, the dry matter / Ha / annum was calculated as 12000kg.

[0257] Further, application of a composition of the invention to a plant growth medium can sequester carbon in said plant growth media.

[0258] Therefore, in addition to its pest control capabilities, a composition of the invention can be used to sequester carbon. The plant biomass in the composition may also serve as a useful carbon source for the growing crops, which advantageously improves plant heath, vigour and quality.

[0259] Other beneficial activities

[0260] A composition of the invention may have one or more additional beneficial activity. For example, the composition of the invention may enhance plant yields (e.g., cereal or potato) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%. For example, the composition of the invention may enhance plant yields by at least 15%. For example, the composition of the invention may enhance plant yields by at least 20%. For example, the composition of the invention may enhance plant yields by at least 25%. For example, the composition of the invention may enhance plant yields by at least 30%. For example, the composition of the invention may enhance plant yields by at least 35%. For example, the composition of the invention may enhance plant yields by at least 40%. For example, the composition of the invention may enhance plant yields by at least 45%. For example, the composition of the invention may enhance plant yields by at least 50%. Yield improvements may be of the whole plant or a crop obtained therefrom.

[0261] By way of further example, alternatively or in addition, the composition of the invention may improve plant vigour (or any trait(s) associated with plant vigour as described herein) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%. By way of further example, alternatively or in addition, the composition of the invention may improve plant quality (or any trait(s) associated with plant quality as described herein) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%.

[0262] By way of further example, alternatively or in addition, the composition of the invention may improve plant tolerance to stress factors (or any trait(s) associated with plant tolerance to stress factors as described herein) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%.

[0263] By way of further example, alternatively or in addition, the composition of the invention may improve input use efficiency (or any trait(s) associated with input use efficiency as described herein) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%.

[0264] It has been shown that crop productivity and carbon sequestration can be affected by the balance of microorganisms in the soil. Synthetic pesticides, which are known to disrupt this balance, may negatively affect the quality of soil and thus crop productivity and carbon sequestration. For example, synthetic pesticides have been shown to either decrease soil microbial biomass or reduce the abundance and diversity of soil organisms. Some organisms can be suppressed while others may proliferate in the resulting vacant ecological niches, so organisms that were rare become abundant and vice versa. Research also shows that synthetic pesticides can impact larger fauna that help maintain the structure and fertility of the soil. For example, by impairing the reproductive capacity and survival of earthworms and springtails.

[0265] An advantage of the present composition is that it does not negatively affect soil quality. Instead, alternatively or in addition, the composition of the invention may improve the quality of a plant growth medium such as soil (or any trait(s) associated with the quality of a plant growth media as described herein) by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%. The improvement may be quantified relative to a plant growth medium such as soil that has not been treated with the composition of the invention.

[0266] Hence, the composition of the invention may improve at least one of the following traits associated with soil quality: microbial biomass (for example, increased mycorrhizal fungi), abundance and / or diversity of soil microorganisms, carbon sequestration, nitrogen fixation, soil fertility, nutrient cycling, respiration of soil-dwelling organisms, reproductive capacity and / or survival of fauna such as earthworms and springtails. Alternatively, the composition of the invention may decrease the abundance of pathogenic microorganisms in the soil.

[0267] For example, the composition of the invention may increase the microbial biomass of the soil by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%.

[0268] Methods of production

[0269] Disclosed herein is a method for producing a composition for repelling and / or controlling a pest of the phylum Mollusca. Said methods are particularly preferred for producing compositions for repelling and / or controlling pests of the class gastropoda, such as slugs and snails.

[0270] Such a method of the invention may comprise or consist of the following steps: a) harvesting or providing an endophyte-infected plant, wherein the plant comprises at least one loline species and wherein the at least one loline species has molluscicidal activity; b) cutting the plant to produce plant clippings; and c) combining the plant clippings with one or more additives comprising at least one phagostimulant.

[0271] Exemplary additives are disclosed herein. For example, the one or more additive may comprise a phagostimulant that functions to attract a mollusc (e.g. a gastropod such as a slug or snail) to the composition. Suitable phagostimulants are well known to the skilled person, and include cereal grains or flour (e.g., wheat or bran grains or flour), rice, fruit extracts, molasses, sugars (e.g., sucrose, glucose, lactose and / or fructose), yeast extracts, beer, fermented foods, and / or foods with a strong odour. For example, the composition may comprise one or more phagostimulant selected from cereal grains or flour (e.g., wheat or bran grains or flour), rice, fruit extracts, molasses, sugars (e.g., sucrose, glucose, lactose and / or fructose), yeast extracts, beer, fermented foods, and / or foods with a strong odour. In preferred embodiments, the composition comprises one or more phagostimulant selected from cereal grains or flour (e.g., wheat or bran grains or flour). The additives may be dry additives, liquid additives, or a combination thereof. Where the additives comprise a combination of wet and dry additives, the dry additives may be added simultaneously or separately to the wet additives (preferably separately).

[0272] The plant clippings or the combination of plant clippings and one or more additive may be substantially homogenised (e.g., using a blender). The plant clippings or the combination of plant clippings and one or more additive may be processed (e.g., homogenised) to produce plant powder or granules.

[0273] For example, the method may comprise the following steps: a) harvesting or providing an endophyte-infected plant, wherein the plant comprises at least one loline species and wherein the at least one loline species has molluscicidal activity; b) cutting the plant to produce plant clippings; c) processing the plant clippings to produce a plant powder or granules; and d) combining the powder or granules with one or more additive comprising at least one phagostimulant.

[0274] Alternatively, the method may comprise the following steps: a) harvesting or providing an endophyte-infected plant, wherein the plant comprises at least one loline species and wherein the at least one loline species has molluscicidal activity; b) cutting the plant to produce plant clippings; c) combining the plant clippings with one or more additive comprising at least one phagostimulant; and d) processing the mixture of (c) to produce a powder or granules.

[0275] Any production method of the invention may further comprise extruding the output of any of the above methods. Preferably, the extruded material is formed into pellets. Any production method of the invention may further include a drying step, as needed.

[0276] Thus, an exemplary method comprises the following steps: a) harvesting or providing an endophyte-infected plant, wherein the plant comprises at least one loline species and wherein the at least one loline species has molluscicidal activity; b) cutting the plant to produce plant clippings; c) combining the plant clippings with one or more additive comprising at least one phagostimulant; and d) extruding the output of step (c); optionally in the form of pellets.

[0277] Alternatively, the method may comprise the following steps: a) harvesting or providing an endophyte-infected plant, wherein the plant comprises at least one loline species and wherein the at least one loline species has molluscicidal activity; b) cutting the plant to produce plant clippings; c) processing the plant clippings to produce a plant powder or granules; d) combining the powder or granules with one or more additive comprising at least one phagostimulant; and e) extruding the output of step (d); optionally in the form of pellets.

[0278] Alternatively, the method may comprise the following steps: a) harvesting or providing an endophyte-infected plant, wherein the plant comprises at least one loline species and wherein the at least one loline species has molluscicidal activity; b) cutting the plant to produce plant clippings; c) combining the plant clippings with one or more additive comprising at least one phagostimulant; d) processing the mixture of (c) to produce a powder or granules; and e) extruding the output of step (d); optionally in the form of pellets.

[0279] Any of the above methods may include one or more quality control step, for example, to assess moisture content of the composition. The mixture of plant clippings and additives, for example, may be assessed to determine whether the moisture content is from 20-50%, from 25-45% or from 30-40%. Preferably, the composition may be assessed to determine whether the moisture content of the mixture of plant clippings and additives is from 30-40%. Prior to extrusion, for example, the composition may be assessed to determine whether the moisture content of the composition is from 30-60%, from 35-55% or from 40-50%. Preferably, the composition may be assessed to determine whether the moisture content of the composition is from 40-50%. Following extrusion, for example, the moisture content of the composition may be from 0.5-5%, from 0.5%-3% or from 1-2%. Preferably, the composition may be assessed to determine whether the moisture content of the composition is from 1-2%.

[0280] The skilled person will appreciate that one or more drying step may be used to ensure that the composition has an appropriate moisture content.

[0281] For example, where the method comprises the following steps: a) harvesting or providing an endophyte-infected plant, wherein the plant comprises at least one loline species and wherein the at least one loline species has molluscicidal activity; b) cutting the plant to produce plant clippings; c) combining the plant clippings with one or more additive comprising at least one phagostimulant; and d) extruding the output of step (c); optionally in the form of pellets; the method may comprise a drying step between one or more of: steps (a) and (b), steps (b) and (c), steps (c) and (d), and / or after step (d).

[0282] Drying may be achieved using any number of suitable methods, all of which will be well known to the skilled person. Exemplary methods include the use of a fluid bed dryer, tray dryer or hot air oven. In preferred embodiments, the method comprises the use of a fluid bed dryer.

[0283] Any of the above methods may include the step of combining the powder or granules with one or more liquid ingredients. For example, the homogenised, powder or granules may additionally be combined with water and / or liquid phagostimulants (such as sugar syrup). In preferred embodiments, liquid ingredients are added before extrusion.

[0284] Any production method of the invention may further comprise a step of cultivating the endophyte-infected plant. In such methods, the plants may be naturally infected with endophytes, or the plant may be inoculated with endophytes as part of the cultivating step.

[0285] Importantly, the method of the invention is significantly different to the methods for producing loline species comprising the step of extracting loline alkaloids from endophyte-infected plant material (e.g., as described in WO 2008 / 111861) or methods comprising production of loline alkaloids by fermentation of fungal endophytes in chemically defined growth media (e.g., as described in Blankenship et al., (2001 , Phytochemistry 58, 395-401).

[0286] Also disclosed is a composition obtainable by any one of the aforementioned methods.

[0287] Methods and uses

[0288] Also provided is a composition for repelling and / or controlling the population size of a pest of the phylum Mollusca, preferably a pest of the class gastropoda (slugs and snails), wherein the composition is as described above. Also provided is the use of composition for repelling and / or controlling the population size of a pest of the phylum Mollusca, preferably a pest of the class gastropoda (slugs and snails), wherein the composition is as described above.

[0289] Also disclosed is the use of a composition for controlling mollusc damage to plants in horticulture or agriculture wherein the composition is as described above.

[0290] Also disclosed is a method for repelling and / or controlling the population size of a pest of the phylum Mollusca, preferably a pest of the class gastropod (slugs and snails), the method comprising contacting a plant, plant growth media (e.g., soil) or a mollusc with the composition as defined herein.

[0291] Also disclosed is a method for controlling mollusc damage to plants in horticulture or agriculture, the method comprising contacting a plant, plant growth media (e.g., soil) or a mollusc with the composition as defined herein.

[0292] Preferably, in any of said methods or uses, said composition also functions to sequester carbon within the plant growth media.

[0293] Additionally, or alternatively, contacting a plant or plant growth media according to a method or use of the invention further improves at least one growing characteristic of a plant; optionally wherein the growing characteristic is selected from an improvement in plant vigour, plant quality, tolerance to stress factors and / or input use efficiency. Such characteristics are as described herein.

[0294] The molluscicidal compositions of the invention may be useful in repelling and / or controlling pests from the phylum Mollusca, particularly the class gastropoda (slugs and snails). By way of non-limiting example, the molluscicidal compositions of the invention may be useful in controlling molluscs from the Family Arionidae, such as Arion ater, A. ruus, A. hortensis, and A. subfuscus molluscs from the Family Limacide, such as Deroceras reticulatunr, and molluscs from the Family Helicidae, such as Helix aperta and H. aspersa.

[0295] The molluscicidal compositions of the invention may be useful in the protection of monocots and / or dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica spp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereaie), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), saffiower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas'), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Per sea americana), fig (Ficus casica), guava (Psidium guajava), strawberry (Fragaria x ananassa), raspberry (Rubus spp., e.g. R. idaeus and R. occidentalis), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables ornamentals, and conifers. In some embodiments, protection of strawberry (Fragaria x ananassa) and / or raspberry (Rubus spp., e.g. R. idaeus and R. occidentalis) may be preferred.

[0296] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. meld). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Conifers that may be employed in practicing the embodiments include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponder osa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata)’ Douglas-fir (Pseudotsuga menziesii)’, Western hemlock (Tsuga canadensis)’, Sitka spruce (Picea glauca)’, redwood (Sequoia sempervirens)-, true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea)’, and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Plants of the embodiments include crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

[0297] Turf grasses include, but are not limited to: annual bluegrass (Poa annua)’, annual ryegrass (Lolium multiflorum)’, Canada bluegrass (Poa compressa)’, Chewing's fescue (Festuca rubra)’, colonial bentgrass (Agrostis tenuis)’, creeping bentgrass (Agrostis palustris)’, crested wheatgrass (Agropyron desertorum)’, fairway wheatgrass (Agropyron cristatum)’, hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis)’, orchardgrass (Dactylis glomerata)’, perennial ryegrass (Lolium perenne)’, red fescue (Festuca rubra)’, redtop (Agrostis alba)’, rough bluegrass (Poa trivialis)’, sheep fescue (Festuca ovina)’, smooth bromegrass (Bromus inermis)’, tall fescue (Festuca arundinacea)’, timothy (Phleum pratense)’, velvet bentgrass (Agrostis canina)’, weeping alkaligrass (Puccinellia distans)’, western wheatgrass (Agropyron smithii)’, Bermuda grass (Cynodon spp.)’, St. Augustine grass (Stenotaphrum secundatum)’, zoysia grass (Zoysia spp.y Bahia grass (Paspalum notatum carpet grass (Axonopus ajfinisy centipede grass (Eremochloa ophiuroidesy kikuyu grass (Pennisetum clandesinumy seashore paspalum (Paspalum vaginatumy blue gramma (Bouteloua gracilisy buffalo grass (Buchloe dactyloidsy sideoats gramma (Bouteloua curtipendulay

[0298] Preferably, the plant is a crop plant. For example, the crop may be a vegetable crop (e.g., potatoes, carrots, lettuce, and broccoli), a fruit crop (e.g., strawberries), leafy greens, a cereal / grain crop (e.g., wheat, corn, rice, barley, or millet), a pulse crop (e.g., peas, gram, or lentils) or an ornamental crop. More preferably, the crop is a potato crop or a cereal / grain. In particularly preferred embodiments, the crop is a cereal.

[0299] Crops are to be understood as being those which are naturally occurring, obtained by conventional methods of breeding, or obtained by genetic engineering. They include crops which contain so-called output traits (e.g. improved storage stability, higher nutritional value and improved flavour). Crops are to be understood as also including those crops which have been rendered tolerant to herbicides like bromoxynil or classes of herbicides such as ALS-, EPSPS-, GS-, HPPD- and PPG-inhibitors. An example of a crop that has been rendered tolerant to imidazolinones, e.g. imazamox, by conventional methods of breeding is Clearfield® summer canola. Examples of crops that have been rendered tolerant to herbicides by genetic engineering methods include e.g. glyphosate- and glufosinate-resistant maize varieties commercially available under the trade names RoundupReady®, Herculex I® and Libertylink®.

[0300] Crops are also to be understood as being those which naturally are or have been rendered resistant to harmful insects. This includes plants transformed by the use of recombinant DNA techniques, for example, to be capable of synthesising one or more selectively acting toxins, such as are known, for example, from toxin-producing bacteria. Examples of toxins which can be expressed include d-endotoxins, vegetative insecticidal proteins (Vip), insecticidal proteins of bacteria colonising nematodes, and toxins produced by scorpions, arachnids, wasps and fungi.

[0301] An example of a crop that has been modified to express the Bacillus thuringiensis toxin is the Bt maize KnockOut (Syngenta Seeds). An example of a crop comprising more than one gene that codes for insecticidal resistance and thus expresses more than one toxin is VipCot® (Syngenta Seeds). Crops or seed material thereof can also be resistant to multiple types of pests (so-called stacked transgenic events when created by genetic modification). For example, a plant can have the ability to express an insecticidal protein while at the same time being herbicide tolerant, for example Herculex I® (Dow AgroSciences, Pioneer Hi-Bred International). As described herein, the composition may be applied at various different times. The composition of the present invention may be applied pre-emergence or postemergence. For example, it may be applied as a preventative (before mollusc establishment) or curative (after mollusc establishment) treatment.

[0302] The present invention envisages application of the compositions of the invention to plant propagation material prior to, during, or after planting, or any combination of these.

[0303] Methods for applying or treating active ingredients on to plant propagation material or to the locus of planting are known in the art and include dressing, coating, pelleting, nursery tray application, in furrow application, or incorporation into soil (broad cast or in band). Alternatively or in addition the composition may be applied on a suitable plant growth medium sown together with the plant propagation material.

[0304] The rates of application of compositions of the present invention may vary within wide limits and depend on the nature of the soil, the method of application (e.g. pre- or postemergence etc.), the crop plant, the prevailing climatic conditions, and other factors governed by the method of application, the time of application and the target crop. By way of example, in some preferred embodiments, the composition is applied at seeding as a seed or propagation media treatment.

[0305] Thus, the skilled person will appreciate that the amount of composition to be applied to a given locus will vary depending on, for example, the mollusc population to be controlled, the nature of the soil, the method of application, the target crop plant and the prevailing climatic conditions. By way of non-limiting example, a composition of the invention may be applied at a rate of 250 pellets / m2. By way of further non-limiting example, a composition of the invention may be applied at a rate of 20kg / hectare.

[0306] Compositions of the invention may be used in combination with one or more additional agronomic chemical or biological agent.

[0307] ITEMS

[0308] The present invention will now be described with reference to the following Items.

[0309] 1. A biological molluscicidal composition comprising a mixture of at least one loline species within a plant biomass.

[0310] 2. The composition of Item 1 , wherein the at least one loline compound: a) has the following chemical formula: wherein:

[0311] R = H or CH3;and

[0312] R' = H, CH3, CHO, COCH3; b) is selected from the list comprising: loline, norloline, N-formylnorloline, N-acetylloline, N-formylloline, N-acetylnorloline, and N-methylloline or a variants thereof; further optionally wherein when the composition comprises N-acetylloline, N- formylloline, and N-acetylnorloline, the weight ratio of N-acetylloline, N-formylloline, and N-acetylnorloline is from 1 :3:1 to 1:6:3; c) is not synthetic; and / or d) is derived from an endophyte-infected plant; optionally wherein the endophyte-infected plant is a grass, preferably a Festuca species.

[0313] 3. The composition of any one of the preceding Items, wherein the plant biomass is derived from an endophyte-infected plant; optionally wherein the at least one loline species and the plant biomass are derived from the same endophyte-infected plant.

[0314] 4. The composition of Item 3, the at least one loline species is not extracted from the plant biomass.

[0315] 5. The composition of any one of Items 2-4, wherein the endophyte is selected from the genus Epichloe or Neotyphodiunr, preferably wherein the endophyte is Epichloe uncinata.

[0316] 6. The composition of any one of the preceding Items, wherein the at least one loline compound is present in an amount effective to act as a molluscicide; optionally wherein the loline compound is present in the range of from 100 to 500 ppm; preferably from 400 to 500 ppm. 7. The composition of any one of the preceding Items, wherein the composition has molluscicidal activity against a mollusc of the class gastropoda (slugs and snails).

[0317] 8. The composition of any one of the preceding Items, further comprising one or more additive, optionally wherein the one or more additive is a phagostimulant.

[0318] 9. The composition according to Item 8, wherein the phagostimulant is a compound that induces sustained feeding and / or attracts molluscs.

[0319] 10. The composition according to Item 8 or 9, wherein the one or more phagostimulant is selected from cereal grains or flour (e.g., wheat, corn or bran grains, wheat flour, cornflour, grain flour or bakers flour), rice, fruit extracts, molasses, sugars (e.g., sucrose, glucose, lactose and / or fructose), yeast extracts, beer, fermented foods, and / or foods with a strong odour; and / or wherein the one or more phagostimulant is a liquid phagostimulants (such as sugar syrup).

[0320] 11. The composition of any one of the preceding Items, further comprising at least one additive that improves plant vigour, plant quality, tolerance to stress factors and / or input use efficiency.

[0321] 12. The composition of any one of the preceding Items, wherein the composition is a solid; optionally wherein the solid formulation is a pellet or granule.

[0322] 13. A method for producing a composition for repelling and / or controlling a pest of the phylum Mollusca, preferably the class gastropoda (slugs and snails), the method comprising: a) harvesting or providing an endophyte-infected plant, wherein the plant comprises at least one loline species and wherein the at least one loline species has molluscicidal activity; b) cutting the plant to produce plant clippings; and c) combining the plant clippings with at least one phagostimulant.

[0323] 14. The method of Item 13, further comprising the steps of: d) extruding the mixture; and f) forming pellets.

[0324] 15. The method of Item 14, wherein the mixture is dried, preferably to a 1-2% moisture content.

[0325] 16. The method of Item 14 or 15, wherein the pellets are 1.4-3.5 mm in diameter. 17. The method of any one of Items 13 to 16, wherein the composition is as defined in any one of Items 1-12.

[0326] 18. The method of any one of Items 13 to 17, further comprising the step of cultivating the endophyte-infected plant.

[0327] 19. The method of any one of Items 13-18, wherein the plant clippings are further processed to produce plant powder or granules, optionally wherein the plant clippings are further processed before or after combining with one or more additive, such as the phagostimulant.

[0328] 20. The method of any one of Items 13-19, further comprising at least one drying step; optionally wherein the drying step comprises: a) drying the harvested plant, plant clippings, and / or molluscicidal mixture; b) drying the plant powder or granulates; and / or c) using a fluid bed dryer.

[0329] 21. The method of any one of Items 13-20, further comprising extruding the plant clippings and additives, or the plant powder or granules; optionally wherein the extruded material is formed into pellets.

[0330] 22. A composition obtainable by the method of any one of Items 13 to 21.

[0331] 23. The composition of any one of Items 1 to 12 or 22 for controlling the population size of a pest of the phylum Mollusca, preferably a pest of the class gastropoda (e.g. slugs and snails).

[0332] 24. Use of the composition as defined in any one of Items 1 to 12, 22 or 23 for repelling and / or controlling the population size of a pest of the phylum Mollusca, preferably a pest of the class gastropoda (e.g., slugs and snails).

[0333] 25. The use of Item 24, wherein said composition further sequesters carbon within the plant growth media.

[0334] 26. A method for controlling mollusc damage to plants in horticulture or agriculture, the method comprising contacting a plant, a plant growth medium or a mollusc with the composition as defined in any one of Items 1 to 12, 22 or 23.

[0335] 27. The method of Item 26, wherein contacting a plant growth medium with said composition sequesters carbon within the plant growth medium. 28. The method of Item 26 or 27, wherein the contacting a plant or a plant growth medium further improves at least one growing characteristic of a plant; optionally wherein the growing characteristic is selected from an improvement in plant vigour (such as early vigour), plant quality, tolerance to stress factors and / or input use efficiency.

[0336] 29. The method of any one of Items 26 to 28, wherein the plant is crop; optionally wherein the crop is a potato, strawberry, leafy green, cereal grain, pulse or ornamental crop; preferably wherein the crop is a cereal grain crop.

[0337] 30. The method of any one of Items 26 to 29, wherein the composition is applied at seeding as a seed or propagation media treatment.

[0338] 31 . The method according to any one of Items 26 to 30, wherein the composition is applied to a locus, for example, where molluscs are present, such as by drilling or placing the composition of the invention onto the soil (e.g., furrows), thereby providing systemic crop protection.

[0339] EXAMPLES

[0340] The invention is now described with reference to the Examples below. These are not limiting on the scope of the invention, and a person skilled in the art would appreciate that suitable equivalents could be used within the scope of the present invention. Thus, the Examples may be considered component parts of the invention, and the individual aspects described therein may be considered as disclosed independently, or in any combination.

[0341] Example 1 : Harvesting plant biomass from endophyte-infected pasture

[0342] A grass paddock was divided in two. One half was sown with Epichloe infected seed (meadow fescue + endo) and the other half was sown with non-infected Epichloe seed (meadow fescue without endo). The Meadow fescue seed cultivar was the same for both sides of the paddock. Presence of the endophytes in the inoculated grass was confirmed by microscopy (B, arrows).

[0343] Of the two sides, the fescue comprising endophyte-infected grass is healthier. In particular, the grass is greener, longer and more uniform in the presence of endophytes. There are also signs that the endophyte-infected grass is more tolerant to stress, (e.g., drought) and has far less predation by Grass grub (Costelytra giveni).

[0344] The endophyte-infected grass was harvested to use as plant biomass in the production of exemplary molluscicidal compositions. Example 2: Exemplary method of producing a biological, systemic molluscicide

[0345] As shown in Figure 2, in an exemplary production method, endophyte-infected grass comprising loline species may be mowed (1), further cut to produce grass clippings (2), mixed with additives such as surfactants, binders, fillers and feeding stimulants (phagostimulants) (3), mixed (e.g., using a rotary mixer) (4), extruded (5) and formed into pellets (6).

[0346] By way of particular example, harvested grass chips from Example 1 were mixed with Sepiolite, Bakers Flour, Cornflour, Maize Starch, Sorbic Acid, Potassium Sorbate,

[0347] Talc and Perlite in a ribbon blender and blended until a homogenous mixture was formed.

[0348] A sample of the material was analysed for its water content to ensure that the water content was between about 30.0-40.0%. The mixture was then sieved and mixed further.

[0349] To this mixed material, Water, Tylose, and Sucrose were added, and the mixture further mixed for 10 minutes until the pre agglomerate is formed. This has the appearance of a crumbly extrudable granular substance which does not stick to the side of the bowl.

[0350] A sample of the above agglomerate was analysed for its water content to ensure that the water content was between about 40.0-50.0%.

[0351] The pre agglomerate was then fed into a reciprocating granulator fitted with 2-3 mm stainless steel wire aperture and then placed into the Sherwood fluid bed dryer. The final pellets were analysed for their water content to ensure that the water content was between about 1-2%.

[0352] Example 3: Loline compounds prevent slug damage to crops

[0353] Four lettuce plants were treated with pellets produced according to the method shown in Fig. 3. As a control, four lettuce plants were untreated. Four weeks after the initial treatment, the plants were assessed for slug damage. No live slugs were found on the treated plants. As shown in the photograph, the treated plants were healthier (i.e., had larger and more numerous leaves with no signs of slug damage) compared to the untreated controls.

[0354] As such, pellets comprising a composition of the invention effectively protected crops from mollusc damage.

Claims

CLAIMS1. A biological molluscicidal bait composition comprising: 1) endophyte-infected plant biomass comprising at least one loline species, and 2) one or more phagostimulant, wherein the loline species is derived from the endophyte-infected plant and is not extracted from the plant biomass and is not synthetic.

2. The composition of claim 1, wherein the at least one loline compound: a) has the following chemical formula:wherein:R = H or CH3;andR' = H, CH3, CHO, COCH3; b) is selected from the list comprising: loline, norloline, N-formylnorloline, N-acetylloline, N-formylloline, N-acetylnorloline, and N-methylloline or a variants thereof; further optionally wherein when the composition comprises N-acetylloline, N- formylloline, and N-acetylnorloline, the weight ratio of N-acetylloline, N-formylloline, and N-acetylnorloline is from 1 :3:1 to 1:6:3, preferably about 1 :4:2; and / or c) wherein the endophyte-infected plant is a grass, preferably a Festuca species.

3. The composition of any one of the preceding claims, wherein the at least one loline species and the plant biomass are derived from the same endophyte-infected plant.

4. The composition of claim 3, wherein the at least one loline species is produced by the endophytes in the endophyte-infected plant.

5. The composition of any one of claims 2-4, wherein the endophyte is selected from the genus Epichloe or Neotyphodiunr, preferably wherein the endophyte is Epichloe uncinata.

6. The composition of any one of the preceding claims, wherein the at least one loline compound is present in an amount effective to act as a molluscicide; optionally wherein the loline compound is present in the range of from 100 to 500 ppm; preferably from 400 to 500 ppm.

7. The composition of any one of the preceding claims, wherein the composition has molluscicidal activity against a mollusc of the class gastropoda (slugs and snails).

8. The composition according to any preceding claim, wherein the one or more phagostimulant is a compound that induces sustained feeding and / or attracts molluscs.

9. The composition according to any claim 8, wherein the one or more phagostimulant is selected from cereal grains or flour (e.g., wheat, corn or bran grains, wheat flour, cornflour, grain flour or bakers flour), rice, fruit extracts, molasses, sugars (e.g., sucrose, glucose, lactose and / or fructose), yeast extracts, beer, fermented foods, and / or foods with a strong odour; and / or wherein the one or more phagostimulant is a liquid phagostimulants (such as sugar syrup).

10. The composition of any one of the preceding claims, further comprising one or more additive.

11. The composition of any one of the preceding claims, further comprising at least one additive that improves plant vigour, plant quality, tolerance to stress factors and / or input use efficiency.

12. The composition of any one of the preceding claims, wherein the composition is a solid; optionally wherein the solid formulation is a pellet or granule.

13. A method for producing a composition for repelling and / or controlling a pest of the phylum Mollusca, preferably the class gastropoda (slugs and snails), the method comprising: a) harvesting or providing an endophyte-infected plant, wherein the plant comprises at least one loline species and wherein the at least one loline species has molluscicidal activity; b) cutting the plant to produce plant clippings; andc) combining the plant clippings with one or more additive comprising at least one phagostimulant.

14. The method of claim 13, further comprising the steps of: d) extruding the mixture; and e) forming pellets.

15. The method of claim 13 or 14, wherein the composition is as defined in any one of claims 1-12.

16. The method of claim 15, further comprising the step of cultivating the endophyte- infected plant.

17. The method of any one of claims 13-16, wherein the plant clippings are further processed to produce plant powder or granules, wherein optionally the plant clippings are further processed before or after combining with the one or more additive.

18. The method of any one of claims 13-17, further comprising at least one drying step; optionally wherein the drying step comprises: f) drying the plant, plant clippings, and / or molluscicidal mixture of claim 13; g) drying the plant powder or granulates of claim 17; and / or h) using a fluid bed dryer.

19. The method of any one of claims 13-18, further comprising extruding the plant clippings and additives of claim 13(c) or the plant powder or granules of claims 17 or 18; optionally wherein the extruded material is formed into pellets.

20. A composition obtainable by the method of any one of claims 13-19.

21. The composition of any one of claims 1-12 or 20 for controlling the population size of a pest of the phylum Mollusca, preferably a pest of the class gastropoda (e.g. slugs and snails).

22. Use of the composition as defined in any one of claims 1-12, 20 or 21 for repelling and / or controlling the population size of a pest of the phylum Mollusca, preferably a pest of the class gastropoda (e.g., slugs and snails).

23. The use of claim 22, wherein said composition further sequesters carbon within the plant growth media.

24. A method for controlling mollusc damage to plants in horticulture or agriculture, the method comprising contacting a plant, a plant growth medium or a mollusc with the composition as defined in any one of claims 1-12, 20 or 21.

25. The method of claim 24, wherein contacting a plant growth medium with said composition sequesters carbon within the plant growth medium.

26. The method of claim 24 or 25, wherein the contacting a plant or a plant growth medium further improves at least one growing characteristic of a plant; optionally wherein the growing characteristic is selected from an improvement in plant vigour (such as early vigour), plant quality, tolerance to stress factors and / or input use efficiency.

27. The method of any one of claims 24-26, wherein the plant is crop; optionally wherein the crop is a potato, strawberry, leafy green, cereal grain, pulse or ornamental crop; preferably wherein the crop is a cereal grain crop.

28. The method of any one of claims 24-27, wherein the composition is applied at seeding as a seed or propagation media treatment.

29. The method according to any one of claims 24-28, wherein the composition is applied to a locus where molluscs are present, for example, by drilling or placing the composition as defined in any one of claims 1-12, 20 or 21 onto the soil (e.g., furrows), thereby providing systemic crop protection.

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