mycoherbicide

Abstract

The invention relates to methods of producing a biocidal composition comprising viable mycelia from a pathogenic fungus. The invention also relates to methods and uses of such biocidal compositions in the control of pests, and in particular weeds.

Description

[0001] MYCOHERBICIDE

[0002] FIELD OF THE INVENTION

[0003] The invention relates to methods of producing a biocidal composition comprising viable mycelia from a pathogenic fungus. The invention also relates to methods and uses of such biocidal compositions in the control of pests, and in particular weeds.

[0004] BACKGROUND

[0005] The most commonly cited explanation for the failure of fungal pathogens used for biological control agents to cause severe disease is the requirement of an extended dew period for adequate infection.

[0006] Previous research studying the use of a strain of Colletotrichum gloeosporioides (the Collego® pathogen) to control Northern Jointvetch (Aeschynomene virginica), found that infection was not limited by the absence of a dew period but that fastest onset of disease was achieved if the inoculated plants were exposed to 80% relative humidity overnight.

[0007] All phytopathogens require the presence of moisture to allow infection of the host. For example, Botrytis spp has an exacting requirement to infect its host which is exactly 93% relative humidity (rH) for 6 hours. Without this niche requirement being achieved, Botrytis is unable to become infectious. This is how greenhouse growers ensure no Botrytis infection, as with the introduction of heating pipes in greenhouses the humidity is deliberately aimed at ensuring this niche requirement is never achieved. It does not matter how many spores of Botrytis are in the greenhouse they will never have the opportunity to infect.

[0008] Phytopathogens attempting to be commercialised as a mycoherbicide are exactly the same in that they require a niche environment of exactly 75%rH to 85%rH for 10h to 12h after spraying. In greenhouses this is achievable with considerable effort but outdoors this requirement is almost impossible.

[0009] Numerous mycoherbicide studies indicate the importance of secondary infection and subsequent dispersal for effective control, both requiring time. It has previously been reported that anthracnose disease caused by Colletotrichum gloeosporioides f.sp. jussiaea, which has an incubation period of 3-5 days, required 28 days to progress from 29% (primary infection) to 94% infection on the winged water primrose in a rice field. However, in practice, pesticides requiring prolonged incubation times between application / primary infection to biocidal effect are not commercially viable.

[0010] In addition, to-date pesticidal products comprising pathogenic or entomopathogenic fungi typically comprise the fungi in the form of spores, as these are robust and amenable to handling. In contrast, fungal mycelia are more fragile, and often lose viability when harvested,and then further processed and formulated into usable compositions. This is particularly the case for the mycelium of Sclerotinia sclerotiorum. However, it is in the mycelial form that pathogenic and entomopathogenic fungi cause disease. Therefore, being able to formulate viable fungal mycelia into pesticidal products is desirable, as this has the potential to reduce the time between application / primary infection to biocidal effect.

[0011] Therefore, there remain unmet needs in the field of biocidal compositions comprising pathogenic or entomopathogenic fungi. These include the ability to formulate viable fungal mycelia into biocidal compositions and the ability to achieve the necessary water content to allow host infection. It is an object of the present invention to address one or more of these problems.

[0012] SUMMARY OF THE INVENTION

[0013] The present inventors have provided for the first time a method of formulating viable mycelial fragments into a biocidal composition. In particular, the present inventors have developed methods for formulating mycelia as small granules that can be suspended in a viscous aqueous medium for application as large drops to the plant surface. This is advantageous, as these drops can adhere to a plant and provide a moist, nutrient rich protective environment conducive to rapid growth of mycelium from the granule and subsequent infection of host plants. The present inventors have demonstrated proof of concept for their methods and compositions using S. sclerotiorum mycelium, demonstrating that S. sclerotiorum mycelium formulated according to the invention remain 100% viable for at least 56 days at 22°C.

[0014] Accordingly, the present invention provides a method of producing a biocidal composition comprising mycelia of one or more pathogenic and / or entomopathogenic fungus, said method comprising the steps of (a) inoculating a carrier material with mycelia of one or more pathogenic and / or entomopathogenic fungus; (b) incubating the inoculated carrier material in the absence of light to grow the mycelia; (c) drying the material from step (b) to reduce the water content to 10% w / w or less; and (d) granulating the dried material.

[0015] The carrier material may (i) be organic or inorganic, preferably organic, and optionally wherein the organic carrier material may be selected from a cereal, a cereal-derived material, or a plant-derived material; and / or (ii) have a % w / w water content of between about 30 % w / w to about 50 % w / w, wherein optionally the carrier material may be dried prior to step (a) to arrive at this % w / w water content.

[0016] The cereal, cereal-derived or plant-derived carrier material may comprise or consist of material derived from rice, wheat, maize, rye, bran or oats. The inorganic carrier material may comprise or consist of diatomaceous earth, perlite, calcium silicate and / or magnesium silicate,bentonite, attapulgite, sepiolite, vermiculite, cellulosic fibre, preferably the carrier material may comprise diatomaceous earth.

[0017] Step (b) of the method of the invention may be for at least 4 days, at least 5 days, at least 6 days, at least 7 days or at least 8 days, preferably for at least 6 days. Step (b) may be performed at a temperature of (i) between about 20°C to about 40°C, optionally between about 20°C to about 30°C or between about 20°C to about 25°C; and / or (ii) about 20°C, at about 21 °C, at about 22°C, at about 23°C, at about 24°C, at about 25°C, preferably about 22°C.

[0018] In step (c) of the method of the invention (i) the water content of material from step (b) may be reduced to 7% w / w or less, 5% w / w or less, 3% w / w or less, 1 % w / w or less, preferably the water content may be reduced to less than 1% w / w; and / or (ii) may be carried out at a temperature of about 30°C or less, about 20°C or less, about 10°C or less, about 5°C or less, about 4°C or less, about 3°C or less, about 2°C or less, or about 1°C or less, preferably about 20°C or less, more preferably about 18°C or less, even more preferably about 4°C or less.

[0019] In step (d), (i) granulating may be carried out by rotary granulation; (ii) granulating may be carried out using two or more granulators in sequence with decreasing aperture size; and / or (iii) granules of average diameter of between about 100 pm to about 1500 pm may be produced, optionally between about 500 pm to about 1500 pm, about 700 pm to about 1500 pm, or between about 700 pm to about 1300 pm.

[0020] The carrier material in step (a) of the method of the invention may be sterilised before being inoculated, optionally wherein the carrier material may be sterilised by autoclaving or microwaving.

[0021] The biocidal composition may be mixed with one or more additional component selected from the list consisting of: (i) a filler, optionally a cereal-based or plant-based product; (ii) nutrients, optionally organic and / or inorganic nutrients; (iii) viscosity increasing agents, optionally a synthetic or natural gum and / or soluble starch; (iv) buffers, optionally to maintain the pH in the range of between about 3.0 to about 6.0, preferably between about 3.5 to about 4.5; (v) humectants, optionally inverted sugars, polyethylene glycols, vegetable mineral and / or synthetic oils; (vi) UV light absorbing material, optionally soya bean oil and / or titanium dioxide; (vii) wetting agents optionally methylcelluloses and / or hydroxypropylmethyl celluloses; (viii) protective agents, optionally PEG, polyvinylpyrrolidone, polyvinyl alcohol, casein salts, natural or synthetic gums, oils and / or activated carbon; (ix) bacteriostatic agents, optionally oxalic acid; (x) scarifying agents, optionally perlite; (xi) enzymes or other agents which destroy plant tissue, optionally C8-C10 fatty acids; and / or (xii) plant defence mechanism inhibitors, optionally CAPE 1 peptide, 2,1,3 Benzothiadiazole (BTH), abscisic acid (ABA), p Aminobutyric acid (BABA), Methyldihydrojasmonate (dihydro - JA) and / or a 1,6 cyclizedpi ,2glucohexadecaose (CBpG16a); wherein optionally the composition may be formulated as a wettable powder.

[0022] The method of the invention may further comprise the steps (e) encapsulating the granulated material from step (d); and (f) suspending the encapsulated material from step (e) in an homogeneous oil phase to form a suspension.

[0023] In step (e) of the method of the invention, the granulated material from step (d) may be suspended in an aqueous solution of gelling agent and then dispensed as droplets into an aqueous solution of calcium chloride. The gelling agent may be selected from the group consisting of sodium alginate, hydroxypropyl methyl cellulose, methyl cellulose, gum Arabic, guar gum, pectine and gellan gum, preferably the gelling agent may be sodium alginate or gellan gum.

[0024] In step (f) of the method of the invention, the homogeneous oil phase may comprise soybean oil and optionally Tween 80. One or more additional agent may be added to the suspension, wherein optionally said one or more additional agent may be selected from the group consisting of glycerol, propylene glycol, xanthan gum, and / or carboxymethyl cellulose, preferably wherein glycerol, propylene glycol, xanthan gum, and carboxymethyl cellulose may be added to the suspension.

[0025] The one or more pathogenic and / or entomopathogenic fungus may be a mycopathogenic fungus, which is optionally selected from the group consisting of S. sclerotiorum, Verticillium dahliae, Colletotrichum gloeosporioides, Sclerotinia minor, Agroathelia rolfsii, Armillaria mellea, and Rhizoctonia sp , wherein preferably the mycopathogenic fungus is S. sclerotiorum. The one or more pathogenic and / or entomopathogenic fungus may remain viable and may be capable of reanimation, penetration, infection and causing disease for a period of no less than 12 months.

[0026] The present invention also provides a biocidal composition obtainable by any method of the invention.

[0027] The present invention further provides a biocidal composition comprising granules containing a carrier material and mycelia of one or more pathogenic and / or entomopathogenic fungus, wherein the water content of the granules is at least 75% w / w for at least 8 hours postapplication. The granules may be encapsulated, the composition may be an aqueous suspension; and / or the composition may have a shelf life of 12 - 14 months.

[0028] The biocidal composition of the invention may further comprise one or more plant defence mechanism inhibitor, wherein optionally the one or more plant defence mechanism inhibitor may be selected from the group consisting of CAPE 1 peptide, 2,1,3 Benzothiadiazole, abscisic acid, Aminobutyric acid, Methyldihydrojasmonate and a 1,6 cyclized pi,2glucohexadecaose.The one or more pathogenic and / or entomopathogenic fungus in the biocidal composition of the invention may be a mycopathogenic fungus, which may be optionally selected from the group consisting of S. sclerotiorum, Verticillium dahliae, Colletotrichum gloeosporioides, Sclerotinia minor, Agroathelia rolfsii, Armillaria mellea, and Rhizoctonia sp, preferably the one or more mycopathogenic fungus may be S. sclerotiorum.

[0029] The water content of the granules in the biocidal composition of the invention may be (i) at least 80% w / w or at least 85% w / w for at least 12 hours post-application, preferably the water content of the granules may be at least 85% w / w for at least 12 hours post-application; (ii) between about 70% w / w to about 90% w / w for at least about 10 hours post-application, optionally at least about 12 hours post-application; and / or (iii) between about 75% w / w to about 85% w / w for at least about 10 hours post-application, optionally at least about 12 hours postapplication.

[0030] The invention further provides the use of a biocidal composition of the invention for preventing, ameliorating, inhibiting, eliminating or delaying the onset of a pest infection or infestation, wherein preferably the pest is a weed, optionally selected from Cirsium avense, Senecio jacobaea, Hieracium pilosella, Eichhornia crassipes or Ranunculus acris.

[0031] The invention further provides a method for treating, preventing, ameliorating, inhibiting, eliminating or delaying the onset of an infestation, comprising the step of applying onto a plant, to a plant material or in the vicinity of said plant or plant material a biocidal composition of the invention; wherein preferably the infestation is a weed infestation, optionally an infestation of Cirsium avense, Senecio jacobaea, Hieracium pilosella, Eichhornia crassipes or Ranunculus acris.

[0032] The use or the method for treating, preventing, ameliorating, inhibiting, eliminating or delaying the onset of an infestation of the invention wherein (i) the biocidal composition may have a biocidal activity that may be at least 30% greater than a composition without mycelia of the one or more pathogenic and / or entomopathogenic fungus; (ii) the amount of the biocidal composition required to control the infestation may be 2 times less than the amount of a composition without mycelia of the one or more pathogenic and / or entomopathogenic fungus that is required to treat, prevent, ameliorate, inhibit, eliminate or delay the onset of the same infestation; (iii) the method or use may comprise the controlled release of the mycelia of the one or more pathogenic and / or entomopathogenic fungus, wherein optionally controlled release may comprise contacting the biocidal composition with sodium citrate; and / or (iv) the application rate of the biocidal composition may be between about 10 to about 20 kg of the biocidal composition / 100 L water I hectare, preferably about 15kg of the biocidal composition / 100 L water / hectare.Brief description of the Drawings

[0033] Figure 1 - Average Plant Counts After Pre-Emergence Application of Mycoherbicide Formulation. The figure shows that the mycoherbicide formulation of the invention allows for herbicidal control at 7 days and 20 days of Thistle Seedlings at different application rates of 7 kg / hectare, 10 kg / hectare and 15 kg / hectare compared with control and unformulated Sclerotinia when applied before emergence of the Thistle Seedlings.

[0034] Figure 2 - Average Plant Counts After Post-Emergence Application of Mycoherbicide Formulation. The figure shows that the mycoherbicide formulation of the invention allows for herbicidal control at application time and 13 days of Thistle Seedlings at different application rates of 7 kg / hectare, 10 kg / hectare and 15 kg / hectare compared with control and unformulated Sclerotinia when applied after emergence of the Thistle Seedlings.

[0035] Figure 3 - Viability of mycelia after granulation in function of moisture content. Viability of mycelia after granulation at different water content levels is plotted. Viability of the mycelial fragments were correlated to lower water content especially below 7% after storage for 56 days at 22°C.

[0036] DETAILED DESCRIPTION

[0037] Definitions

[0038] 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.

[0039] 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.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 in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0040] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

[0041] 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.

[0042] 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.

[0043] 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. Inthis 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.

[0044] “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.

[0045] 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.

[0046] 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).

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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" or “elimination” is a 100% inhibition (i.e. abrogation) as compared to a reference level.

[0051] The terms "increased", "increase", "enhance", “ameliorate”, or "activate" are all used herein to mean an increase by a statically significant amount. The terms "increased", "increase", "enhance", “ameliorate”, 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.

[0052] The term "plants" refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage, and fruits.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] As used herein, the term “biocidal composition” is used to refer to a composition comprising at least one mycelium from at one or more pathogenic and / or entomopathogenic fungus and which has a biocidal effect. A biocidal composition may comprise an additional agent(s) with biological effects, such as a microbial biocidal agent and / or a plant defence mechanism inhibitor.

[0057] As used herein, the term “biocidal effect” refers to inducing mortality, disrupting or impeding growth, interfering with functions, effecting sterilization, and / or interfering with reproduction of one or more target pests, or any other effect on a pest that results in limiting the damage that the pest causes. Thus, “biocidal effects” can be useful in controlling a pest population.A microbial biocidal agent (also referred to as a biological control agent) has a biocidal effect. Such microbial biocidal agent include bacteria, fungi, protozoans, viruses, viroids, peptides and mycoplasmas. Preferably microbial biocidal agent refers to fungi.

[0058] As used herein, the term “mycelium”, refers to the vegetative part of a fungus. It is distinct from fungal spores and conidia and should be understood as such by the skilled person. It is known in the art that mycelium is more susceptible to physical damage.

[0059] As used herein, the term “pathogenic fungus”, refers to fungi which can kill or impact the health of one or more pests. Typically, the one or more pathogenic fungus may be mycopathogenic (i.e. herbicidal), insecticidal, fungicidal or molluscicidal.

[0060] The terms “entomopathogenic” and “insect pathogenic” are used herein to refer to microbial biocidal agents, preferably fungi, which can control one or more species of insect, typically wherein said one or more species of insect is an agricultural and / or horticultural pest. Such insect pathogenic activity includes, but is not limited to, killing the insect, inhibiting development of the insect, altering fertility or growth of the insect in such a manner that the insect provides less damage to the plant, decreasing the number of offspring produced, producing less fit insects, producing insects more susceptible to predator attack or deterring the insects from eating the plant.

[0061] The term “population of pests” as used herein may refer to a mixed-species, geographically discreet population of pests, or single-species, geographically discreet population of pests. Typically where a population is mixed species, the pests are all of the same type, e.g. different populations of pest plants or different populations of pest insects.

[0062] As used herein, the terms "controlling a pest population" or "controls a pest " refers to any effect on a pest that results in limiting the damage that the pest causes. This may also be referred to as the “biocidal activity” of a composition. Controlling a pest includes, but is not limited to, killing the pest, inhibiting development of the pest, altering fertility or growth of the pest in such a manner that the pest provides less damage to the plant, decreasing the number of offspring produced, producing less fit pests, producing pests more susceptible to predator attack or deterring the pests from eating the plant. “Controlling a population of pests” typically means that the number of pests within a population of pest 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.

[0063] Control of a pest (i.e. biocidal activity) can be measured against a suitable control and typically the biocidal activity of the composition of the invention may decrease the impact of the pest on the crop or plant of interest by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or atleast 99%. The biocidal activity of the composition of the invention may eliminate the impact of the pest on the crop or plant of interest.

[0064] The term “pest” as used herein refers to a plant, parasite, fungus, mollusc or insect that impacts the health of a crop and / or plant of interest. Any reference herein to “pests” encompasses all categories of pests unless stated to the contrary. Plant pests are particularly preferred.

[0065] As used herein, the term “parasite”, refers to an animal or plant that lives on or in another plant of a different type and feeds from it.

[0066] A pest plant may also be referred to as a weed. As used herein, the term “weed”, refers to any plant that is growing where it is not wanted and in competition with a crop and / or plant of interest.

[0067] As used herein, the term “crop and / or plant of interest”, refers to any cultivated plants that one would want to protect by preventing, ameliorating, inhibiting, eliminating or delaying the onset of a pest infection or infestation.

[0068] As used herein, the term “carrier material” or “carrier” refers to material that can serve as a nutrient source for the mycelium and enable its growth. A carrier material of the invention may be organic or inorganic. Preferably the carrier material is organic such as cereal, a cereal-derived material, or a plant-derived material. By way of non-limiting example, the cereal, cereal-derived or plant-derived carrier material may comprise or consist of material derived from rice, wheat, maize, rye, bran or oats. By way of non-limiting example, an inorganic carrier material may comprise or consist of diatomaceous earth, perlite, calcium silicate, magnesium silicate, Bentonite, Attapulgite, Sepiolite, Vermiculite or Cellulosic fibre, or combination thereof. Preferably the inorganic carrier material may comprise or consist of diatomaceous earth.

[0069] As used herein, the term “water content” relates to the amount of water or moisture present in a product. As described herein, the water content of a biocidal composition of the invention is typically important for the viability and growth of the mycelia of one or more pathogenic and / or entomopathogenic fungus following application. Alternatively or in addition, the invention also relates to the water content of one or more component of the biocidal composition during manufacturing. Water content may be quantified in terms of %w / w or relative humidity (%rH). Water content can be measured by methods known in the art, such as measuring the %w / w lost upon drying.

[0070] As used herein, the term “sterilised” relates to make components of the composition of the invention or equipment free from bacteria or other living microorganisms including potentially competing fungus. Unless stated to the contrary, any suitable technique may be used for sterilisation, such as autoclaving, microwaving or pasteurisation. Suitable techniquesand protocols are known in the art and within the routine practice of one of ordinary skill in the art.

[0071] As used herein, the term “filler” relates to an inert material which can serve as a nutrient source for the mycelium upon hydration and / or application. By way of non-limiting example, the filler may comprise or consist of a cereal-based or a plant-based product.

[0072] As used herein, the term “nutrient” relates to a range of soluble nutrients that may be incorporated in the compositions of the invention. By way of non-limiting example, the nutrient may comprise or consist of organic or inorganic nutrients, or combination thereof.

[0073] As used herein, the term “viscosity increasing agent” relates to agents providing reasonable viscosity to the composition of the invention which may help adherence to the pest, or to the crop plant to facilitate uptake by the pest. By way of non-limiting example, the viscosity increasing agent may comprise or consist of a synthetic or natural gum or soluble starch, or combination thereof.

[0074] As used herein, the term “buffer” relates to materials that maintain the pH of the composition of the invention within a range suitable for the growth of the one or more pathogenic and / or entomopathogenic fungus. Such buffer materials are known in the art and the selection thereof for use in the present invention is within the routine practice of one of ordinary skill in the art. Typically, the pH of the composition of the invention may be acidic. The pH of the composition of the invention may be less than about 7.0, less than about 6.0, pH less than about 5.0 or pH less than about 4.0. Preferably the pH of the composition of the invention may be between about 3.0 to about 6.0, more preferably the pH of the composition of the invention may be between about 3.5 to about 4.5.

[0075] As used herein, the term “humectant” relates to material which can prevent desiccation of the composition of the invention under drying conditions in use. By way of non-limiting example, the humectant may comprise or consist of inverted sugars, polyethylene glycols, vegetable mineral or synthetic oils, or combination thereof.

[0076] As used herein, the term “UV light absorbing material” relates to materials which can protect the growth of the mycelium from strong UV light, especially UVB. Byway of non-limiting example, the UV light absorbing material may comprise or consist of soya bean oil and / or titanium dioxide.

[0077] As used herein, the term “wetting agent” relates to materials which can facilitate wetting, dispersion and hydration of a powder in water or aqueous solution. By way of nonlimiting example, the wetting agent may comprise or consist of methylcelluloses and / or hydroxypropylmethyl celluloses.

[0078] As used herein, the term “protective agent” relates to materials which can enhance the viability of the fungal mycelium. By way of non-limiting example, the protective agent maycomprise or consist of PEG, polyvinylpyrrolidone, polyvinyl alcohol, casein salts, natural or synthetic gums, oils or activated carbon, or combination thereof.

[0079] As used herein, the term “bacteriostatic agent” relates to materials whose mechanism of action can stall bacterial cellular activity without directly causing bacterial death. A bacteriostatic agent may comprise or consist of oxalic acid.

[0080] As used herein, the term “scarifying agent” relates to materials which may facilitate mycelium infection of pest tissue at the point of application. The scarifying agent may comprise or consist of perlite. By way of non-limiting example, further agents that destroy pest tissue may comprise or consist of enzymes or C8-C10 fatty acids, or combination thereof.

[0081] As used herein, the term “plant defence mechanism inhibitor” relates to compounds that substantially suppress essential plant defence mechanisms. By way of non-limiting example, the plant defence mechanism inhibitor may comprise or consist of CAPE 1 peptide, 2,1,3 Benzothiadiazole (BTH), abscisic acid (ABA), p Aminobutyric acid (BABA), Methyldihydrojasmonate (dihydro - JA) and / or a 1,6 cyclized pi,2glucohexadecaose (CB G16a), or combination thereof.

[0082] As used herein, the term “aqueous” in the context of solutions or suspensions of the invention means that the solutions or fluid portion of the suspensions of the invention substantially consist of water as a main solvent. Several other components may be dissolved is such solution or fluid portion of the suspension. Solution or fluid portion of the suspension may comprise further co-solvents in a proportion of less than about 50% v / v, less than about 40% v / v, less than about 30% v / v, less than about 20% v / v, less than about 10% v / v, less than about 5% v / v of the total volume of the solution or fluid portion of the suspension.

[0083] As used herein, the term “homogeneous” relates to process or solutions involving substances in the same phase. Typically an homogeneous solution consists of a single liquid phase.

[0084] As used herein, the term “suspension” relates to a mixture in which granules or encapsulated granules of the invention are dispersed throughout the bulk of a fluid portion. Suspensions may be stable meaning that the granules or encapsulated granules may stay substantially dispersed throughout the bulk of a fluid portion or the suspension may be unstable meaning that the granules or encapsulated granules may slowly settle by gravity.

[0085] As used herein, the term “encapsulating” or “encapsulated” refers to a preparation or composition comprising, or consisting of capsules, also referred to herein as particles. Such particles are small spherical structures comprising an enclosed material. The enclosed material in the particle may be referred to as the core, internal phase, or fill. In the case of the present invention, the enclosed material is the granules comprising the one or more pathogenic fungus of the invention.As used herein, the term “viable” in the context of the one or more pathogenic and / or entomopathogenic fungus used in the invention means that the mycelium of the one or more pathogenic and / or entomopathogenic fungus of the invention retains its ability to grow and infect the pest. Maintaining viability may be defined in terms of the survival of the pathogenic and / or entomopathogenic fungi. By way of non-limiting example, a pathogenic and / or entomopathogenic fungus 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 are still able to grow and infect the pest. In particular, “viability” may be defined as capable of reanimation of the fungal mycelia, penetration of the fungal mycelia into a pest, infection of the pest and / or having a biocidal effect (causing disease).

[0086] 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 mycelia that remain active and / or viable for a longer period of time compared to a relevant control (e.g. a composition comprising mycelia made by a method in which the mycelia are not dried before formulating). Alternatively or in addition, a preparation with increased biological stability may comprise mycelia that remain active and / or viable at higher and / or lower temperatures compared to a relevant control. Maintaining viability may be defined in terms of the activity of the mycelia. Activity may be determined using any appropriate assay, such as those exemplified herein.

[0087] As used herein, the term “shelf life” relates to the maintenance of viability of the pathogenic fungi over time and / or a range of temperatures. By way of non-limiting example, a composition with increased shelf-life may comprise pathogenic fungi that remain viable for a longer period of time compared to a relevant control (such as a corresponding composition made by a method other than that described herein; a corresponding composition without pathogenic and / or entomopathogenic fungi; and / or a corresponding composition wherein the mycelia of the pathogenic and / or entomopathogenic fungi are not encapsulated). Alternatively or in addition, a composition with increased biological stability may comprise pathogenic fungi that remain viable at higher and / or lower temperatures compared to a relevant control (such as a corresponding composition made by a method other than that described herein; a corresponding composition without pathogenic and / or entomopathogenic fungi; and / or a corresponding composition wherein the mycelia of the pathogenic and / or entomopathogenic fungi are not encapsulated).

[0088] As used herein, the term “controlled release” relates to the release of the one or more pathogenic fungus for infestation of the pest upon a specific stimulus. Typically, the controlled release of the pathogenic fungi may comprise contacting the biocidal composition with sodiumcitrate. This contacting can be simultaneous or sequential with the providing the biocidal composition of the invention to the pest.

[0089] As used herein, the term “application rate” relates to the amount of the composition of the invention that is applied in use. Such application rate is measured in kg of composition per hectare or kg of composition per litter per hectare in the case of a suspension.

[0090] 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.

[0091] Method of producing a biocidal composition

[0092] The present invention provides a method of producing a biocidal composition.

[0093] In particular, the present invention provides a method of producing a biocidal composition comprising mycelia of one or more pathogenic and / or entomopathogenic fungus. Said method may comprise or consist of the following steps:

[0094] (a) inoculating a carrier material with mycelia of one or more pathogenic and / or entomopathogenic fungus;

[0095] (b) incubating the inoculated carrier material in the absence of light to grow the mycelia; and

[0096] (c) drying the material from step (b).

[0097] In step (c), the drying of the material from step (b) typically reduces the water content to 10% w / w or less, as described further below.

[0098] The dried material produced in step (c) may be granulated. Thus, the invention provides a method of producing a biocidal composition comprising mycelia of one or more pathogenic and / or entomopathogenic fungus. Said method may comprise or consist of the following steps:

[0099] (a) inoculating a carrier material with mycelia of one or more pathogenic and / or entomopathogenic fungus;

[0100] (b) incubating the inoculated carrier material in the absence of light to grow the mycelia; and

[0101] (c) drying the material from step (b); and

[0102] (d) granulating the dried material.

[0103] Pathogenic and entomopathogenic fungi

[0104] As described herein, the one or more pathogenic and / or entomopathogenic fungus present in the biocidal composition of the invention may be selected independently. Acomposition of the invention may comprise one or more pathogenic fungus. A composition of the invention may comprise one or more entomopathogenic fungus. A composition of the invention may comprise one or more pathogenic and one or more entomopathogenic fungus.

[0105] The one or more pathogenic and / or entomopathogenic fungus present in a composition of the invention may be selected based on numerous factors, such as the pest to be controlled and / or the crop or plant of interest. The one or more pathogenic and / or entomopathogenic fungus may be have biocidal activity against any pest of interest, as described herein. Thus, the one or more pathogenic and / or entomopathogenic fungus may be described as pesticidal. For example, the one or more pathogenic and / or entomopathogenic fungus may be mycopathogenic (i.e. herbicidal), insecticidal, or molluscicidal. In some preferred embodiments, the one or more pathogenic and / or entomopathogenic fungus is mycopathogenic.

[0106] Biocidal compositions of the invention may comprise or consist of a combination of one or more pathogenic and / or entomopathogenic fungus. By way of non-limiting example, the one or more pathogenic and / or entomopathogenic fungi present in a composition of the invention may comprise or consist of one, two, three, four or five pathogenic and / or entomopathogenic fungi. The one or more pathogenic and / or entomopathogenic fungus present in a composition of the invention may comprise or consist of no more than three or no more than two pathogenic and / or entomopathogenic fungi. The one or more pathogenic and / or entomopathogenic fungus present in a composition of the invention may comprise or consist of no more than two and / or entomopathogenic fungi. The one or more pathogenic and / or entomopathogenic fungus present in a composition of the invention may comprise or consist of a single pathogenic and / or entomopathogenic fungi.

[0107] Preferably the one or more pathogenic and / or entomopathogenic fungus is substantially biologically pure, which will be appreciated by the skilled reader as meaning that the strain is comprised mostly of the one or more pathogenic fungus of interest substantially without any biological contaminants, within a degree of error as is appreciably feasible using standard manufacturing practices and processes.

[0108] The one or more pathogenic fungus may be a plant pathogen, i.e. may be mycopathogenic (also referred to as herbicidal). By way of non-limiting example, the one or more pathogenic fungus may be a mycopathogenic fungus independently selected from Sclerotinia sclerotiorum, Verticillium dahliae, Colletotrichum gloeosporioides, Sclerotinia minor, Agroathelia rolfsii, Armillaria mellea, Fusarium sp and Rhizoctonia sp. Preferably a biocidal composition of the invention comprises Sclerotinia sclerotiorum (S. sclerotiorum).

[0109] The one or more pathogenic fungus may be an insect pathogen, i.e. may be insecticidal. By way of non-limiting example, the one or more pathogenic fungus may be ainsecticidal fungus independently selected from Metarhizium, Beauveria, Lecanicillium, Isaria, Nomuraea and Hirstiulla species.

[0110] The one or more pathogenic and / or entomopathogenic fungus is comprises as mycelia within a biocidal composition of the invention, as described herein. As discussed in more detail herein, the methods and biocidal compositions of the invention are advantageous as the mycelia of the one or more pathogenic and / or entomopathogenic fungus comprised therein are typically viable and / or have improved viability compared with a relevant control (examples of which are described herein).

[0111] As described herein, one or more pathogenic and / or entomopathogenic fungus may be present in a biocidal composition at a concentration of between about 1% w / w to about 30% w / w. Where two or more pathogenic and / or entomopathogenic fungi are present in a biocidal composition, this may be the concentration of each pathogenic and / or entomopathogenic fungus, or the total concentration of all pathogenic and / or entomopathogenic fungi present in the composition. The concentration of the one or more pathogenic and / or entomopathogenic fungus in a biocidal composition of the invention may depend on factors such as the formulation / route of application. By way of non-limiting example, wherein a biocidal composition of the invention is a solid composition which is placed directly on or in a locus for plant growth and / or plant growth medium, then the composition may comprise granules of the one or more pathogenic and / or entomopathogenic fungus at a concentration of at least 50% w / w, such as at least 60% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, at least 90% w / w, at least 95% w / w, at least 96% w / w, at least 97% w / w, at least 98% w / w, at least 99% w / w or more, up to 100% w / w of the composition.

[0112] By way of non-limiting example, wherein a biocidal composition of the invention is a powder intended for water / solvent dispersion, then the composition may comprise granules at a concentration of at least 5% w / w, such as at least 10% w / w, at least 15% w / w, at least 20% w / w, at least 25% w / w, at least 30% w / w, or more.

[0113] Carrier material

[0114] The carrier material used in a method of producing biocidal compositions of the invention typically provides nutrients to promote growth of the one or more pathogenic and / or entomopathogenic fungus.

[0115] A carrier material of the invention may be organic or inorganic. Preferably, the carrier material is organic such as cereal, a cereal-derived material, or a plant-derived material. A cereal, cereal-derived or plant-derived carrier material may comprise or consist of material derived from rice, wheat, maize, rye, bran and / or oats, or a combination thereof. By way ofnon-limiting example, cereal, cereal-derived or plant-derived carrier material may comprise or consist of Cerebind® (an enzyme deactivated gluten denatured powder from wheat, comprising wheat flour and wheat bran); Avon Gold® (a high protein powder rich in xanthophylls derived from maize); rye flour; bran flake (derived from the external layer of wheat, comprising approximately 11 % protein and 70% carbohydrates); or milk oaties (derived from thinly flaked small cut roasted groats, comprising approximately 12.5% protein and 75% carbohydrates).

[0116] When a carrier material of the invention is inorganic, it is typically combined with an aqueous solution of nutrients to promote growth of the one or more pathogenic and / or entomopathogenic fungus. An inorganic carrier material may comprise or consist of diatomaceous earth, perlite, calcium silicate, magnesium silicate, bentonite, attapulgite, sepiolite, vermiculite or combination thereof. Preferably the inorganic carrier material may comprise or consist of diatomaceous earth. By way of non-limiting example, a non-organic carrier may comprise: 48.1% w / w diatomaceous earth (e.g. Celite 500H®); 2.33% w / w potato or dextrose broth; 0.97% yeast extract; and 48.6% w / w / water.

[0117] The water content of the carrier material may be selected to support growth of the one or more pathogenic and / or entomopathogenic fungus. The carrier material may be prepared to have a water content of less than about 70% w / w, less than about 60% w / w, less than about 50% w / w, less than about 40% w / w, less than about 30% w / w, less than about 20% w / w or less than about 10% w / w. Preferably the carrier material may be prepared to have a water content of less than about 50% w / w. More preferably the carrier material may be prepared to have a water content of between about 30% w / w and about 50% w / w. The preparation of the carrier material may involve combining the carrier material with water or drying the carrier material to obtain the desired water content.

[0118] The carrier material may be prepared prior to its inoculation with mycelia of one or more pathogenic and / or entomopathogenic fungus. This preparation may comprise blending the carrier material to achieve the desired consistency and / or to add nutrients (e.g. when inorganic carrier material is used). This may be achieved using conventional apparatus and / or techniques, such as blending in a pin mixer or ribbon blender. The blended carrier material may be further processed to remove lumps, such as by passing through a hammermill.

[0119] Before inoculation, the carrier material may be sterilised to avoid any competitive growth with the pathogenic fungus mycelia. Sterilisation is a well-known process in the art and the skilled person would be able to select an appropriate protocol. By way of non-limiting example, sterilisation may be carried out by pasteurising, autoclaving or microwaving. Any appropriate autoclaving protocol may be used (e.g. 121°C for 15 minutes).The carrier material may be prepared in such a way to provide optimal inoculation conditions with the mycelia of the one or more pathogenic and / or entomopathogenic fungus. The carrier material may be inoculated with mycelia of the one or more pathogenic and / or entomopathogenic fungus and further incubated to allow mycelial growth.

[0120] Incubation

[0121] The incubation of the mycelia of the one or more pathogenic and / or entomopathogenic fungus in the carrier material (step (b) of a method of the invention) may typically be carried out in the absence of light or in low-light conditions. Such conditions are known to the skilled person in the art and exclude direct sunlight or substitutes thereof, but may encompasses specifically filtered light or substantially reduced lighting to allow the user to manipulate the carrier material and follow growth of the mycelia.

[0122] The length of incubation of the one or more pathogenic and / or entomopathogenic fungus (i.e. the incubation period) may be selected for optimal mycelial growth of the one or more pathogenic and / or entomopathogenic fungus. The length of incubation may therefore be dependent on the nature or type of the one or more pathogenic and / or entomopathogenic fungus. The length of the incubation period is not particularly limited, provided that at the end of the incubation period, sufficient mycelial growth has occurred such that sufficient quantities of mycelia can be harvested to produce a biocidal composition of the invention. The product of this incubation step may be referred to as a mycelial cake.

[0123] Incubation of the one or more pathogenic and / or entomopathogenic fungus in the carrier material may be for at least 4 days, at least 5 days, at least 6 days, at least 7 days or at least 8 days. Preferably the length of incubation is at least 6 days. Incubation of the one or more pathogenic and / or entomopathogenic fungus in the carrier material may be for between about 1 day to about 10 days; such as between about 1 day to about 8 days; between about 1 day to about 7 days; between about 1 day to about 6 days; between about 2 days to about 10 days; between about 2 days to about 8 days; between about 2 days to about 7 days; between about 2 days to about 6 days; between about 3 days to about 10 days; between about 3 days to about 8 days; between about 3 days to about 7 days; between about 3 days to about 6 days; between about 4 days to about 10 days; between about 4 days to about 8 days; between about 4 days to about 7 days; between about 4 days to about 6 days; between about 4 days to about 10 days; between about 4 days to about 8 days; between about 4 days to about 7 days; or between about 4 days to about 6 days. Incubation of the one or more pathogenic and / or entomopathogenic fungus in the carrier material may be for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days or more, preferably about 6 days.The temperature set during the incubation period may be maintained to a certain level, allowing for minor variations, as anticipated by the person skilled in the art. The temperature during incubation of the one or more pathogenic and / or entomopathogenic fungus may be selected for optimal mycelial growth of the one or more pathogenic and / or entomopathogenic fungus. The length of incubation may therefore be dependent on the nature or type of the one or more pathogenic and / or entomopathogenic fungus. The temperature may be kept constant throughout the incubation period, or may be altered during the incubation. Preferably, the temperature during the incubation period is kept constant.

[0124] The temperature for the incubation period may typically be set between below about 40°C. Preferably the temperature for the incubation period may be set between about 20°C and about 40°C, between about 20°C and about 30°C or between about 20°C and about 25°C. The temperature for the incubation period may be set at about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C or about 25°C. Preferably, the temperature for the incubation period may be set at about 22°C.

[0125] Drying of the grown mycelia

[0126] Following the incubation period (step (b)), the carrier material containing the grown mycelia (also referred to as the mycelial cake) is dried to allow for an easier granulation. This drying step (step (c)) is advantageous, as is prevents the need to extrude the mycelial cake. The temperatures, pressures and / or crushing and shearing forces of extrusion can damage the mycelia, reducing their viability. Without being bound by theory, it is believed that, in contrast, drying the mycelial cake allows the dried cake to be granulated with minimal loss of viability of the mycelia of the one or more pathogenic and / or entomopathogenic fungus. The mycelial cake is typically dried to reduce the water content such that the cake becomes frangible. This is believed to prevent crushing and / or distortion of the cake during granulation.

[0127] The drying process is typically carried out at a temperature which minimises any negative effect on the viability of the mycelia of the one or more pathogenic and / or entomopathogenic fungus. Thus, drying may be carried out at a temperature of about 35°C or less, at about 30°C or less, at about 25°C or less, at about 20°C or less, at about 15°C or less, at about 10°C or less, at about 5°C or less, at about 4°C or less, at about 3°C or less, at about 2°C or less or at about 1°C or less. Preferably, the drying process may be carried out at about 20°C or less. More preferably the drying process may be carried out at about 18°C or less. Even more preferably the drying process may be carried out at about 4°C.

[0128] Any appropriate drying apparatus or methodology may be used. Suitable examples of such apparatus are known in the art, such as static bed dryers, drying cabinets and vacuum / refrigeration drying systems.The drying process may be carried out at atmospheric pressure or under reduced pressure conditions. The skilled person may appreciate that reducing pressure allows for the drying process to happen at lower temperature and at a faster rate. Therefore, preferably the drying process is carried out at reduced pressure.

[0129] In conditions known by the skilled person, the drying process may be conducted in temperature of 0°C or less using reduced pressure to allow for sublimation of the residual water content of the material.

[0130] The carrier material containing the grown mycelia (i.e. the mycelial cake) may be dried such that its water content is reduced to 10% w / w or less. Preferably, the dried carrier material containing the grown mycelia has a water content reduced to 7% w / w or less, 6% w / w or less, 5% w / w or less, 4% w / w or less, 3% w / w or less, 2% w / w or less or 1% w / w or less. More preferably, the water content may be reduced to less than 1% w / w.

[0131] Granulation

[0132] The dried carrier material containing the grown mycelia (produced in step (c) of the method of the invention) may be granulated. This may be referred to as step (d) in a method of producing a biocidal composition according to the invention. Granulation may allow for easier distribution of the pathogenic fungus in a biocidal composition, and subsequent dispersion onto a crop or plant of interest. Without being bound by theory, it is believe that granules form of mycelium provide a 'critical mass' of mycelia which are associated with extended viability.

[0133] The granulation process may be carried out by any appropriate methods, examples of which are known in the art and within the routine practice of one of ordinary skill. By way of non-limiting example, granulation may be carried out by rotary granulation or using two or more granulators in sequence with decreasing aperture size (such as using a 10mm aperture mesh, followed by a 2.5mm aperture mesh, followed by a 1.3mm aperture mesh).

[0134] The resulting granules are typically spherical-like particles, although their shape is not particularly limited.

[0135] Viability of granules increases with average (e.g. mean) granule diameter. Thus, granules with a smaller average (e.g. mean) granule diameter lose their viability more rapidly than granules with a larger average (e.g. mean) granule diameter. Conversely, reducing the average (e.g. mean) granule diameter has the potential to provide a given number of propagules per m2in a reduced weight of biocidal composition, with potential cost saving implications. In addition, granules with a smaller average (e.g. mean) granule diameter are more amenable to spraying, as they do not block spray nozzles to the same extent as granules with larger average (e.g. mean) granule diameters. Therefore, the average (e.g. mean)granule diameter may be selected based on factors including the nature or type of the one or more pathogenic and / or entomopathogenic fungus and / or the means by which the biocidal composition is to be applied in use (e.g. spraying or in a solid composition such as a “weed stick”).

[0136] The granules, e.g. when dispensed using a “weed stick” applicator, may have an average (e.g. mean) diameter of about 1500 pm or less, such as about 1300 pm or less, about 1000 pm or less, about 750 pm or less, about 500 pm or less or about 100 pm or less. The average (e.g. mean) diameter of the granules may be between about 100 pm and about 1500 pm, such as between about 100 pm and about 1300 pm, between about 100 pm and about 1000 pm, between about 100 pm and about 750 pm, between about 200 pm and about 1500 pm, between about 200 pm and about 1300 pm, between about 200 pm and about 1000 pm, between about 200 pm and about 750 pm, between about 300 pm and about 1500 pm, between about 300 pm and about 1300 pm, between about 300 pm and about 1000 pm, between about 300 pm and about 750 pm, or between about 100 pm and about 500 pm. The average (e.g. mean) diameter of the granules may be about 1300 pm or less. The average (e.g. mean) diameter of the granules may be about 1000 pm or less. The average (e.g. mean) diameter of the granules may be about 750 pm or less.

[0137] Without being bound by theory, it is believed that particles having an average (e.g. mean) diameter of about 100 pm or less provides increased sprayability, as the particles will not block the spraying nozzle when dispensed using a spraying nozzle.

[0138] The diameter of the granules may be measured by methods known in the art such as Dynamic Light Scattering or simply different mesh sieves. The person skilled in the art would be able to select the appropriate method to measure the diameter of the granules of the invention.

[0139] Mixed formulation

[0140] The granules of the invention may be mixed with one or more additional components, optionally selected from fillers, nutrients, viscosity enhancing agents, buffers, humectants, UV light absorbing material, wetting agents, protective agents, bacteriostatic agents, scarifying agents, enzymes and / or other agents which destroy plant tissue or plant defence mechanism inhibitors or any combination thereof.

[0141] Non-limiting categories of additional components are described herein (see above), and the amounts of each additional components in a biocidal composition of the invention may depend on the use of said biocidal composition. Depending on the pest to be controlled, the one or more pathogenic and / or entomopathogenic fungus used, the locus of application and / orthe crop or plant of interest, the user may vary the amount(s) of any additional components to allow for optimal efficacy.

[0142] In particular, a biocidal composition of the invention may comprise one or more plant defence mechanism inhibitor. As described herein, decreasing the time from primary infection / application of a biocidal composition to the appearance of disease in a pest and hence control of the pest is commercially desirable. When the pest is a pest plant, including one or more plant defence mechanism inhibitor in a biocidal composition may increase the susceptibility of the pest plant to the one or more pathogenic fungus within a biocidal composition of the invention. Therefore, biocidal compositions comprising one or more plant defence mechanism inhibitor in addition to mycelia from one or more pathogenic fungus are particularly preferred. Plant defence mechanism inhibitors are known in the art, such as described in Clemente et al. (Int J Mol Sci. (2019) 20(6):1345. doi: 10.3390 / ijms20061345). Accordingly, it is within the routine practice of one of ordinary skill to select an appropriate plant defence mechanism inhibitor to include in a biocidal composition of the invention. Nonlimiting examples of plant defence mechanism inhibitors include CAPE 1 peptide, 2,1,3 Benzothiadiazole (BTH), abscisic acid (ABA), p Aminobutyric acid (BABA), Methyldihydrojasmonate (dihydro - JA) and / or a 1,6 cyclized pi,2glucohexadecaose (CB G16a).

[0143] The composition of the invention may be formulated as dry granules or wettable powder, depending on its use on the field.

[0144] Encapsulation

[0145] The granules comprised in a biocidal composition of the invention may be encapsulated. This may be step (e) in a method of producing a biocidal composition according to the invention. Accordingly, the methods of the invention may further comprise a step of encapsulating the granules produced (e.g. in step (d) of the claimed method). Further, the invention also provides a method for producing encapsulated preparations of the invention.

[0146] Emulsification Method

[0147] A carrier solution may be prepared by mixing a gelling agent with water and optionally a surfactant. The viscosity of the carrier solution can be adjusted as necessary to achieve a viscosity of between about 200 mPas to about 250 mPas at room temperature (e.g. 22.1 °C). By way of non-limiting example, a carrier solution for use in a method of the invention may have a viscosity of about 225 mPas to about 235 mPas at room temperature (e.g. 22.1 °C).The gelling agent used may be selected from the group consisting of sodium alginate, hydroxypropyl methyl cellulose, methyl cellulose, gum Arabic, guar gum, pectine and gellan gum. Preferably the gelling agent may be sodium alginate or gellan gum.

[0148] Contacting the granulated material comprising mycelia of the one or more pathogenic and / or entomopathogenic fungus with a solution of the carrier to produce a suspension of the granulated material (i.e. granules) in the carrier solution typically involves mixing of the carrier solution and the granulated material. The suspension may be allowed settle for at least 15 minutes prior to emulsification.

[0149] The suspension is then emulsified in a continuous oil phase. Emulsification may be carried out at a temperature of between about 15°C to about 30°C, such as between about 20°C to about 30°C, typically at about 25°C. Any appropriate oil may be used for the continuous oil phase. The concentration of the oil used for the continuous oil phase may be between about 0.5 g / mL to about 5 mg / mL, such as between about 0.5 g / mL to about 2.5 g / mL, between about 0.75 g / mL to about 1.5 g / mL, or between about 0.75g / mL to about 1.0 g / mL, preferably of about 0.9 g / mL. Emulsification may take place by agitating at a speed of between about 200 rpm to about 1000 rpm, such as between about 200 rpm to about 750 rpm, between about 200 rpm to about 500 rpm, between about 250 rpm to about 500 rpm. The duration of emulsification step is not limited, provided that a stable emulsion is achieved. By way of non-limiting example, the emulsification step may be between about 10 minutes to about 2 hours, such as between about 15 minutes to about 90 minutes, between about 15 minutes to about 60 minutes, between about 15 minutes to about 45 minutes.

[0150] CaCI2may be added to facilitate particle formation. The concentration of CaCI2may be between about 40 mg / mL to about 200 mg / mL, such as between about 40 mg / mL to about 150 mg / mL, between about 40 mg / mL to about 100 mg / mL, between about 40 mg / mL to about 75 mg / mL. Preferably, the concentration of CaCI2may be about 50 mg / mL.

[0151] Following encapsulation, agitation may be stopped, and the resulting encapsulated granules extracted by vacuum filtration, optionally using a filter with a pore size of 0.2 pm.

[0152] The extracted granules may be washed with a surfactant solution (such as 0.03% Tween-80 solution) and either dried at room temperature or preserved as a concentrated oil suspension, preferably in soybean oil. The concentrated oil suspension may further be comprising Tween 80.

[0153] The concentrated oil suspension may comprise one or more additional components to modify specific properties of the suspension. The one or more additional components may be selected from the group consisting of glycerol, propylene glycol, xanthan gum, and / or carboxymethyl cellulose. Preferably, glycerol, propylene glycol, xanthan gum, and carboxymethyl cellulose are added to the suspension.Drip Casting Method

[0154] A method of producing an encapsulated preparation of granulated material comprising mycelia from one or more pathogenic and / or entomopathogenic fungus typically comprises the steps of preparing an aqueous solution comprising a gelling agent and the granulated material (i.e. granules) and drip casting said aqueous solution into a solidification solution comprising a divalent metal salt, whereby said drip casting forms encapsulated granules.

[0155] The aqueous solution may be prepared by mixing a gelling agent solution and the granulated material (i.e. granules) prior to the drip casting step.

[0156] The gelling agent solution may be prepared by mixing the gelling agent with water and optionally a suitable surfactant. Suitable surfactants are known in the art, and it is within the routine practice of one of ordinary skill to select a suitable surfactant. Preferably the surfactant comprises or consists of a non-ionic surfactant. By way of non-limiting example, as exemplified herein, polysorbate 80 (Tween-80) may be used. The gelling agent may be present at a concentration of about 0.5% w / w to about 5% w / w. The viscosity of the gelling agent solution can be adjusted as necessary to achieve a viscosity of between about 200 mPas to about 250 mPas at room temperature (e.g. 22.1°C). By way of non-limiting example, a carrier solution for use in a method of the invention may have a viscosity of about 225 mPas to about 235 mPas at room temperature (e.g. 22.1°C).

[0157] Contacting the granulated material (i.e. granules) with the gelling agent solution to produce the aqueous suspension typically involves mixing of the gelling agent solution and the granulated material (i.e. granules). Typically the aqueous suspension is allowed to settle until the foam generated by the surfactant, if used has settled.

[0158] Typically, the gelling agent used may be selected from the group consisting of sodium alginate, hydroxypropyl methyl cellulose, methyl cellulose, gum Arabic, guar gum, pectine and gellan gum. Preferably the gelling agent may be sodium alginate or gellan gum.

[0159] The aqueous suspension then undergoes drip casting to produce encapsulated granules. Drip casting may be carried out by forcing the aqueous suspension through a nozzle and into the solidification solution.

[0160] Drip casting is a well-known term in the art, and refers to the extrusion of a fluid or solution into another fluid or solution or onto a surface. The shape and size of the encapsulated granules produced through drip casting will depend on the pressure applied to the fluid or solution, the diameter of extrusion die and the pulse parameters (amplitude and frequency). Drip casting may be carried out using different well known in the art apparatus. By way of nonlimiting example, as exemplified herein, drip casting may be caried out using a nozzle of a preferred diameter.The diameter of the nozzle used for drip casting may be between about 50 pm to about 200 pm, such as between about 75 pm to about 150 pm, or between about 75 pm to about 125 pm, preferably about 100 pm.

[0161] Drip casting may be carried out at a frequency of between about 5,000 Hz to about 10,000 Hz, such as between about 7,000 Hz to about 10,000 Hz, between about 7,000 Hz to about 9,000 Hz, preferably about 8,000 Hz.

[0162] Drip casting may be carried out at a pressure of between about 200 mBar to about 1000 mBar, such as between about 200 mBar to about 750 mBar, between about 400 mBar to about 750 mBar, or between about 400 mBar to about 600 mBar, preferably at about 500 mBar.

[0163] Drip casting may be carried out at an amplitude of between about 1,000 mV to about 10,000 mV, such as between about 2,000 mV to about 8,000 mV, between about 3,000 mV to about 8,000 mV, between about 4,000 mV to about 6,000 mV, preferably about 5,000 mV.

[0164] The solidification solution is typically a solution comprising or consisting of a divalent metal salt, preferably a calcium salt and more preferably CaCI2in water. The solidification solution optionally further comprising a biocompatible polymer, preferably polyethylene glycol 1500 (PEG 1500). Typically, when CaCI2is the divalent metal salt, the concentration of CaCI2may be between about 40 mg / mL to about 200 mg / mL, such as between about 40 mg / mL to about 150 mg / mL, between about 40 mg / mL to about 100 mg / mL, between about 40 mg / mL to about 75 mg / mL. Preferably, the concentration of CaCI2may be about 40 mg / mL or about 50 mg / mL.

[0165] The method of production of an encapsulated composition may comprise one or more additional step. Said one or more additional step may be include steps such as separating the resulting encapsulated granules from the solidification solution (e.g. by filtration, centrifugation or sedimentation); washing the encapsulated granules, typically with water; and / or packing the encapsulated granules, typically in an oil concentrated suspension in homogeneous oil phase. This oil concentrated suspension in homogeneous oil phase may be a suspension in soybean oil, optionally a surfactant may be added, typically Tween 80. Wherein a method of producing a biocidal composition of the invention comprises a suspension step, this may be referred to as step (f) of the method.

[0166] The concentrated oil suspension may comprise one or more additional components to modify specific properties of the suspension. The one or more additional components may be selected from the group consisting of glycerol, propylene glycol, xanthan gum, and / or carboxymethyl cellulose. Preferably, glycerol, propylene glycol, xanthan gum, and carboxymethyl cellulose are added to the suspension.More detailed drip-casting encapsulation methods are described in UK Patent Application No. 2409303.1, which is herein incorporated by reference. It is within the ability of a skilled person to adapt the methods described therein for use with the biocidal compositions of the present invention.

[0167] Extended shelf-life

[0168] The invention provides methods of production of biocidal compositions with extended shelf life when compared to other compositions comprising mycelia of one or more pathogenic and / or entomopathogenic fungus. Thus, the biocidal compositions of the invention typically have an extended shelf life when compared to other compositions comprising mycelia of one or more pathogenic and / or entomopathogenic fungus.

[0169] The methods described herein may allow the one or more pathogenic fungus mycelia to remain viable for an extended period of time. This extended period of time may be with reference to an appropriate control composition, such as described herein.

[0170] The mycelia of the one or more pathogenic and / or entomopathogenic fungus may remain viable for at least 6 months, at least 12 months, at least 14 months, at least 18 months or at least 24 months or more. Preferably mycelia of the one or more pathogenic and / or entomopathogenic fungus may remain viable for at least 12 months or at least 14 months. Mycelia of the one or more pathogenic and / or entomopathogenic fungus may remain viable for at least between about 12 months to about 14 months. This period of viability may maintained under any appropriate conditions, typically standard storage conditions. Thus, this period of viability may be maintained at a temperature of <35°C, <30°C, <25°C, <23°C, <22°C, <21°C, <20°C, <15°C, <10°C, or<5°C. This period of viability may be interchangeably referred to as the shelf-life of the biocidal composition.

[0171] The mycelia of the one or more pathogenic and / or entomopathogenic fungus may be capable of reanimation, penetration, infection and causing disease for at least 6 months, at least 12 months, at least 14 months, at least 18 months or at least 24 months. Preferably the mycelia of the one or more pathogenic and / or entomopathogenic fungus may be capable of reanimation, penetration, infection and causing disease for at least 12 months or at least about 14 months, the mycelia of the one or more pathogenic and / or entomopathogenic fungus may be capable of reanimation, penetration, infection and causing disease for at least between about 12 months to about 14 months. This ability may maintained under any appropriate conditions, typically standard storage conditions, such as at a temperature of <35°C, <30°C, <25°C, <23°C, <22°C, <21°C, <20°C, <15°C, <10°C, or <5°C.Biocidal compositions

[0172] The invention provides biocidal compositions obtainable or obtained by any of the methods of production described herein.

[0173] Thus, the invention provides a biocidal composition which comprises or consists of granules containing a carrier material and mycelia of one or more pathogenic and / or entomopathogenic fungus, as described herein.

[0174] In particular, the invention provides a biocidal composition comprising granules containing a carrier material and mycelia of one or more pathogenic and / or entomopathogenic fungus, wherein the water content of the granules is at least 75% w / w for at least 8 hours postapplication.

[0175] Preferably the one or more pathogenic and / or entomopathogenic fungus is substantially biologically pure, which will be appreciated by the skilled reader as meaning that the strain is comprised mostly of the one or more pathogenic fungus of interest substantially without any biological contaminants, within a degree of error as is appreciably feasible using standard manufacturing practices and processes.

[0176] A biocidal composition of the invention may comprise or consist of components described herein in the context of the methods of the invention. By way of non-limiting example, a biocidal composition of the invention may comprise mycelia of one or more pathogenic and / or entomopathogenic fungus, such as the exemplary fungi as described herein.

[0177] By way of a further non-limiting example, a biocidal composition of the invention may further comprise a filler, nutrients, viscosity enhancing agents, buffers, humectants, UV light absorbing material, wetting agents, protective agents, bacteriostatic agents, scarifying agents, enzymes or other agents which destroy plant tissue or plant defence mechanism inhibitors or combination thereof, as described herein. Preferably a biocidal composition of the invention may comprise one or more plant defence mechanism inhibitors, a biocidal composition of the invention may additionally comprise one or more plant defence mechanism inhibitor as described herein.

[0178] The one or more pathogenic fungus may be a plant pathogen, i.e. may be mycopathogenic (also referred to as herbicidal). By way of non-limiting example, the one or more pathogenic fungus may be a mycopathogenic fungus independently selected from Sclerotinia sclerotiorum, Verticillium dahliae, Colletotrichum gloeosporioides, Sclerotinia minor, Agroathelia rolfsii, Armillaria mellea, Fusarium spp and Rhizoctonia sp. Preferably a biocidal composition of the invention comprises Sclerotinia sclerotiorum (S. sclerotiorum).

[0179] The one or more pathogenic fungus may be an insect pathogen, i.e. may be insecticidal. By way of non-limiting example, the one or more pathogenic fungus may be ainsecticidal fungus independently selected from Metarhizium, Beauveria, Lecanicillium, Isaria, Nomuraea and Hirstiulla species.

[0180] By way of non-limiting example, the plant defence mechanism inhibitor may be selected from the group consisting of CAPE 1 peptide, 2,1,3 Benzothiadiazole, abscisic acid, P Aminobutyric acid, Methyldihydrojasmonate and a 1,6 cyclized pi,2glucohexadecaose.

[0181] A biocidal composition of the invention may have an extended shelf life (i.e. the mycelia of the one or more pathogenic and / or entomopathogenic fungus remain viable for an extended period of time), as described herein in the context of the methods of the invention. By way of example, a biocidal composition of the invention may have a shelf life of 2 months or more, such as 6 months or more, 12 months or more, 18 months or more or 24 months or more. Typically, a biocidal composition of the invention may have a shelf life of 12 months or more. Preferably a biocidal composition of the invention may have a shelf life of at least between 12 months and 14 months.

[0182] As described herein, a biocidal composition of the invention typically retains moisture for an extended period of time to allow for the pathogenic fungus mycelia to reanimate, penetrate, infect and cause disease in a pest.

[0183] The biocidal composition of the invention may comprise or consist of encapsulated granules of mycelia of one or more pathogenic and / or entomopathogenic fungus, as described herein. The encapsulation of the granules may be conducted according to any of the encapsulation methods described herein.

[0184] The biocidal composition of the invention may be an aqueous suspension. The aqueous suspension may comprise granules of mycelia of one or more pathogenic and / or entomopathogenic fungus as described herein or encapsulated granules of mycelia of one or more pathogenic and / or entomopathogenic fungus suspended in an aqueous solution. The aqueous solution may comprise one or more further agent. By way of non-limiting example, the aqueous solution may comprise nutrients, viscosity enhancing agents, buffers, protective agents, bacteriostatic agents, enzymes or other agents which destroy plant tissue or plant defence mechanism inhibitors or combination thereof. Such agents are described in more detail elsewhere herein. Thus, a method of the invention may comprise adding the granules of mycelia of one or more pathogenic and / or entomopathogenic fungus to an aqueous solution. A method of the invention may comprise adding the encapsulated granules of mycelia of one or more pathogenic and / or entomopathogenic fungus to an aqueous solution. Such aqueous solutions are described herein.Biocidal composition water content

[0185] A biocidal composition of the invention may retain an elevated water content for an extended period of time. Thus, the granules comprised in the biocidal composition of the invention may retain an elevated water content for an extended period of time.

[0186] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 50% w / w for at least 4h postapplication, for at least 6h post-application, for at least 8h post-application, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of the granules may be at least 50% w / w for at least 8h postapplication. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 50% w / w for at least 10h post-application. Still more preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 50% w / w for at least 12h post-application.

[0187] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 60% w / w for at least 4h postapplication, for at least 6h post-application, for at least 8h post-application, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of the granules may be at least 60% w / w for at least 8h postapplication. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 60% w / w for at least 10h post-application. Still more preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 60% w / w for at least 12h post-application.

[0188] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 70% w / w for at least 4h postapplication, for at least 6h post-application, for at least 8h post-application, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of the granules may be at least 70% w / w for at least 8h postapplication. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 70% w / w for at least 10h post-application. Still more preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 70% w / w for at least 12h post-application.

[0189] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 75% w / w for at least 4h post-application, for at least 6h post-application, for at least 8h post-application, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of the granules may be at least 75% w / w for at least 8h postapplication. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 75% w / w for at least 10h post-application. Still more preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 75% w / w for at least 12h post-application.

[0190] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 80% w / w for at least 4h postapplication, for at least 6h post-application, for at least 8h post-application, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of the granules may be at least 80% w / w for at least 8h postapplication. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 80% w / w for at least 10h post-application. Still more preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 80% w / w for at least 12h post-application.

[0191] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 85% w / w for at least 4h postapplication, for at least 6h post-application, for at least 8h post-application, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of the granules may be at least 85% w / w for at least 8h postapplication. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 85% w / w for at least 10h post-application. Still more preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 85% w / w for at least 12h post-application.

[0192] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 90% w / w for at least 4h postapplication, for at least 6h post-application, for at least 8h post-application, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of the granules may be at least 90% w / w for at least 8h postapplication. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 90% w / w for at least 10h post-application. Still more preferably, the water content of a biocidalcomposition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 90% w / w for at least 12h post-application.

[0193] In some preferred embodiments, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 75% w / w for at least 4h post-application, for at least 6h post-application, for at least 8h post-application, for at least 12h post-application or for at least 24h post-application. In some preferred embodiments, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be at least 75% w / w for at least 8h post-application.

[0194] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 50% w / w to about 90% w / w for at least 4h post-application, for at least 6h post-application, for at least 8h postapplication, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 50% w / w to about 90% w / w for at least 10h post-application. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 50% w / w to about 90% w / w for at least 12h post-application.

[0195] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 60% w / w to about 90% w / w for at least 4h post-application, for at least 6h post-application, for at least 8h postapplication, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 60% w / w to about 90% w / w for at least 10h post-application. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 60% w / w to about 90% w / w for at least 12h post-application.

[0196] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 70% w / w to about 90% w / w for at least 4h post-application, for at least 6h post-application, for at least 8h postapplication, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 70% w / w to about 90% w / w for at least 10h post-application. More preferably, the watercontent of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 70% w / w to about 90% w / w for at least 12h post-application.

[0197] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 75% w / w to about 90% w / w for at least 4h post-application, for at least 6h post-application, for at least 8h postapplication, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 75% w / w to about 90% w / w for at least 10h post-application. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 75% w / w to about 90% w / w for at least 12h post-application.

[0198] The water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 75% w / w to about 85% w / w for at least 4h post-application, for at least 6h post-application, for at least 8h postapplication, for at least 10h post-application, for at least 12h post-application or for at least 24h post-application. Preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 75% w / w to about 85% w / w for at least 10h post-application. More preferably, the water content of a biocidal composition of the invention (or the granules comprised in the biocidal composition of the invention) may be between about 75% w / w to about 85% w / w for at least 12h post-application.

[0199] Water loss from biocidal compositions comprising encapsulated granules (as described herein) may be reduced compared with a composition without the encapsulated granules. Said comparator composition may be a corresponding composition without the encapsulated granules, e.g. the composition may comprise granules of mycelia of the same one or more pathogenic and / or entomopathogenic fungus, but in an unencapsulated form.

[0200] Methods and use of biocidal compositions

[0201] The invention provides uses and methods of use of the biocidal compositions described herein for the preventing, ameliorating, inhibiting, eliminating or delaying the onset of a pest infection or infestation.

[0202] Also provided is a method for treating, preventing, ameliorating, inhibiting, eliminating or delaying the onset of an infestation, comprising the step of applying onto a plant, to a plantmaterial or in the vicinity of said plant or plant material (i.e. a locus or plant growth media (e.g., soil)) a biocidal composition as described above.

[0203] Also provided is a biocidal composition for repelling and / or controlling the population size of a pest, wherein the biocidal composition is as described above.

[0204] Also provided is the use of biocidal composition for repelling and / or controlling the population size of a pest, wherein the biocidal composition is as described above.

[0205] 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.

[0206] Also disclosed is a method for repelling and / or controlling the population size of a pest, the method comprising contacting a plant, plant growth media (e.g., soil) or a pest with the composition as defined herein.

[0207] Also disclosed is a method for controlling damage to plants in horticulture or agriculture by a pest, the method comprising contacting a plant, plant growth media (e.g., soil) or a pest with the composition as defined herein.

[0208] The pest may be any pest as described herein, such as a parasite, insect, fungus, mollusc or pest plant (i.e. a weed) that impacts the health of a crop and / or plant of interest. Preferably, the pest may be a weed. By way of non-limiting example, the pest may be a weed selected from the group consisting of Cirsium arvense, Senecio jacobaea, Hieracium pilosella, Eichhornia crassipes, Solanum mauritianum, Onopordum acanthium, Striga spp, and Ranunculus acris.

[0209] A biocidal composition of the invention typically comprises granules comprising mycelia of one or more pathogenic and / or entomopathogenic fungus as described above. Said granules may be applied directly to crops and plants, such as by using a weed-stick. Alternatively, a biocidal composition of the invention may be a powder, which be mixed with a liquid (e.g. water) and applied directly to a plant, plant growth media (e.g., soil) or a pest as a solution, a dispersion, a suspension and / or a mixture. The composition can preferably be comprised within a spray which can preferably be a foliar spray.

[0210] Biocidal compositions comprising encapsulated granules (as described herein) may provide improved adhesion and / or retention on treated plants compared with a composition without the encapsulated granules. Said comparator composition may be a corresponding composition without the encapsulated granules, e.g. the composition may comprise granules of mycelia of the same one or more pathogenic and / or entomopathogenic fungus, but in an unencapsulated form.

[0211] A biocidal composition of the invention may have a biocidal activity that is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater or at least 50% greater than a composition without mycelia of the one or more pathogenic and / or entomopathogenicfungus. Preferably, a biocidal composition of the invention may have a biocidal activity that is at least 30% greater than a composition without mycelia of the one or more pathogenic and / or entomopathogenic fungus. This may be easily measured with comparison to the appropriate control.

[0212] The amount of the biocidal composition required to control the infestation may be 2 times less, 3 times less, 4 times less, 5 times less or 10 times less than the amount of a composition without mycelia of the one or more pathogenic and / or entomopathogenic fungus that is required to treat, prevent, ameliorate, inhibit, eliminate or delay the onset of the same infestation. Preferably the amount of the biocidal composition required to control the infestation may be 2 times less than the amount of a composition without mycelia of the one or more pathogenic and / or entomopathogenic fungus that is required to treat, prevent, ameliorate, inhibit, eliminate or delay the onset of the same infestation.

[0213] The use or method of use may comprise the controlled release of the mycelia of the one or more pathogenic and / or entomopathogenic fungus. Typically, the controlled release of the pathogenic and / or entomopathogenic fungi may comprise contacting the biocidal composition with sodium citrate. This contacting can be simultaneous or sequential with the providing the biocidal composition of the invention to the plant, plant growth media (e.g., soil) or pest.

[0214] The biocidal 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 cereale), 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 batatus), 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.

[0215] 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.

[0216] Turf grasses include, but are not limited to: annual bluegrass (Poa annua)’, annual ryegrass (Lolium multifloruni)’, 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.)’, Bahia grass (Paspalum notatum)’, carpet grass (Axonopus ajfinis)’, centipede grass (Eremochloa ophiuroides)’, kikuyu grass (Pennisetum clandesinum)’, seashore paspalum (Paspalum vaginatum)’, blue gramma (Bouteloua gracilis)’, buffalo grass (Buchloe dactyloids)’, sideoats gramma (Bouteloua curtipendula).

[0217] Preferably, the plant to be protected 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.

[0218] 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®.

[0219] 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.

[0220] 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).

[0221] A biocidal composition of the invention may be applied at various different times. A biocidal composition of the present invention may be applied pre-emergence or postemergence. For example, it may be applied as a preventative (before pest establishment) or curative (after pest establishment) treatment.

[0222] The present invention envisages application of the biocidal compositions of the invention to plant propagation material prior to, during, or after planting, or any combination of these.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.

[0223] 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, a biocidal composition is applied at seeding as a seed or propagation media treatment.

[0224] 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 pest 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, the application rate of the biocidal composition may be between about 5 to about 25 kg of the biocidal composition / 100 L water I hectare, 10 to about 20 kg of the biocidal composition / 100 L water I hectare. Preferably the application rate of the biocidal composition may be about 15kg of the biocidal composition / 100 L water I hectare.

[0225] The skilled person will appreciate that the amount of viable mycelial fragments in spray solution will vary depending on, for example, the pest population to be controlled, the nature of the soil, the target crop plant and the prevailing climatic conditions. By way of non-limiting example, the amount of viable mycelial fragments in spray solution may be between about 103to about 106viable mycelial fragments I mL of spray solution, about 104to about 105viable mycelial fragments I mL of spray solution. Preferably the amount of viable mycelial fragments in spray solution may be about 104viable mycelial fragments I mL of spray solution. More preferably, the amount of viable mycelial fragments in spray solution may be about 105viable mycelial fragments I mL of spray solution.

[0226] Compositions of the invention may be used in combination with one or more additional agronomic chemical or biological agent.

[0227] EXAMPLES

[0228] 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 be appreciate that suitable equivalents could be used within the scope of the present invention. Thus, theExamples may be considered component parts of the invention, and the individual aspects described therein may be considered as disclosed independently, or in any combination.

[0229] Example 1: Preparation of viable S. sclerotiorum mycelium concentrate

[0230] Suitable cereal and plant derived materials that have proved effective as a carrier for growth medium of S. sclerotiorum mycelium include the following:

[0231] Cerebind - An enzyme deactivated gluten denatured powder from wheat

[0232] Avon Gold - A high protein powder rich in xanthophylls derived from maize

[0233] Rye Flour - A low gluten product produced by roller milling ryecorn

[0234] Bran Flake - Derived from the external layer of wheat, grains approx. 11% protein, 70% carbohydrate

[0235] Milk Oaties - Derived from thinly flaked small cut roasted groats approx. 12.5% protein, 75% carbohydrate

[0236] A suitable medium based on an inorganic carrier is described in Table 1.

[0237]

[0238] Tab e 1 : Suitable inorganic carrier for growth medium of S. sclerotiorum mycelium

[0239] A nutrient carrier medium is first prepared by blending the ingredients in a pin mixer, ribbon blender or other suitable device and optionally passing the damp blended material through a hammermill with the screen removed to reduce any lumps. Most of the required water is added at the blending stage.

[0240] The nutrient medium (in the form of a damp powder) is then packed into suitable bulk trays or for convenience into high density polyethylene bags for sterilising preferably by autoclaving, but a microwave is also suitable leaving the medium more friable. Each bag is fitted with an aeration tube covered with a gauze / cotton wool filter to restrict entry of contaminant micro-organisms. Bulk trays are covered with deep lids prior to autoclaving and equipped with filtered aeration vents.

[0241] After autoclaving and cooling each pack is inoculated aseptically with an aqueous suspension of S. sclerotiorum mycelium derived from a broth culture. The inoculumsuspension and contents of the pack are crudely mixed by gentle external kneading of the bag by hand or rotating the entire contents of the bag.

[0242] After inoculation the bags are incubated in the absence of light for 4-8 days at 22°C. At the end of this period the mycelium will have grown extensively throughout the medium and transformed same into a rigid mycelium rich mass or 'cake'.

[0243] Example 2: Preparation of viable S. sclerotiorum mycelia granules

[0244] The mycelium in the wet cake is easily damaged by exposure to the crushing and shearing forces encountered during extrusion. However, we have found that if the wet cake is first dried to a low moisture content it can be reduced to granular form by the crumbling action of a reciprocating granulator with minimal loss of viability.

[0245] The drying process stabilizes the mycelium physically within the cake and the crumbling action of the granulator could be compared to breaking of a biscuit or wafer by snapping. It was hypothesised that in dry mycelium, only the mycelium at the fracture point is damaged, and the extent of the damage is directly proportional to the ultimate granule size or fracture area.

[0246] It is important to reduce the moisture content of the mycelium cake to a low level, typically less than 10% w / w and preferably less than 7% w / w. We have demonstrated that substantial destruction of mycelium (loss of viability) occurs during granulation if the mycelium cake is not dried adequately. If the moisture content is high the cake does not develop a brittle or frangible characteristic and can be crushed and distorted during passage through the reciprocating granulator.

[0247] A low moisture content is also important in enhancing the shelf life of the product. Mycelia will die at temperatures in excess of 30°C such that, low temperature drying systems are preferred. Static bed dryers or drying cabinets using warmed de-humidified air can be used, with vacuum / refrigeration drying systems being preferred.

[0248] Domestic food desiccators were used in the present trial. The unit draws air at 2 - 4°C from a large cool store which serves as a dry air reservoir. Mycelium cake from the bags is reduced by hand to small granules around 1-3 cm in diameter prior to drying.

[0249] The dried segments of mycelium cake are reduced to granules by means of a reciprocating granulator such as Manesty Rotogran. Preferably several granulators will be employed in sequence each fitted with a woven wire mesh of decreasing aperture sizes. For example, if it is desired to produce granules less than 1.3mm diameter the dried segments may be granulated in the following sequence:

[0250] A via a 10 mm aperture meshB via a 2.5 mm aperture mesh

[0251] C via a 1.3 mm aperture mesh

[0252] Depending on the type of reciprocating granulator used, and mesh tension adjustment it may be desirable to pass the product over a vibrating screen at each granulation stage to remove the minus 1.3 mm product and return only oversize material for subsequent regranulation.

[0253] This avoids unnecessary working and possible damage to the mycelia and assists in achieving a higher recovery of granules in the upper size range e.g. from 710 -1300 pm.

[0254] For other applications the granulates diameter may be adjusted by way of changing the settings on the granulators or in adapting the mesh size of the vibrating screen.

[0255] Example 3: Preparation of a wettable powder

[0256] In certain situations, it may be desirable to apply the dry mycelial concentrate granules direct to target plants in the field.

[0257] The mycelium granules are formulated with other ingredients as a dry wettable powder. The powder is mixed into clean cold water to form a viscous suspension which is applied as large drops to the target plants. These drops may vary in size from 20mg to 80 mg but larger or smaller quantities of the suspension may be deposited as a discrete unit on the plant surface.

[0258] In developing a water dispersible powder formulation of this type a number of additives may be used to confer specific benefits such as a filler, nutrients, viscosity enhancing agents, buffers, humectants, UV light absorbing material, wetting agents, protective agents, bacteriostatic agents, scarifying agents, enzymes or other agents which destroy plant tissue or plant defence mechanism inhibitors or combination thereof.

[0259] It is important to reduce the moisture content of the mycelium granules and water dispersible powder formulations of same to a low level.

[0260] The moisture content of other ingredients of the powder formulation may be higher than desired and it may be necessary to subject these to a drying step before use.

[0261] On the basis of trial results to date this is beneficial in enhancing the shelf life of the formulated product.

[0262] The carrier used in the exemplified mycelium cake formulation comprised 58.83% w / w 'Cerebind', 41.17% w / w water.

[0263] Experiment M.36 compares Cerebind' only as a carrier and in combination with other fillers. The water content of the mycelium cake of each formulation was 41.17% w / w.The moisture content is not narrowly critical but is calculated to be sufficient for rapid mycelium growth consistent with the desired physical characteristics. If the quantity of water is in excess, air exchange necessary for good mycelium growth will be restricted.

[0264] Depending on the physical characteristics of the carrier medium used, the water content may vary from 30% - 50% w / w but these may be situations where more or less water is required.

[0265] All the examples given in experiments were prepared by the same method which is described as follows:

[0266] 1) Carrier material(s) weighed into a batch pin mixer and blended with 57.14% of the total water required. The water was delivered to the carrier via a pressure vessel and spray nozzle bar assembly.

[0267] 2) 210 g quantities of the damp carrier were weighed into 300 mm x 190 mm x 70 mm open top high density polyethylene bags and a 50 mm diameter stainless steel aeration tube partially inserted. The open neck of the bag was sealed around the tube using an 'elastrator' rubber ring and a cotton wool / gauze filter pad securely fastened over the tube outlet by means of a tight-fitting rubber band.

[0268] 3) The filled packs were stacked into trays and autoclaved for 60 min at 138 kPa.

[0269] 4) After cooling, each pack was inoculated aseptically in a laminar air-flow cabinet with 45 mL of a suspension of S. sclerotiorum mycelium. This suspension comprised 50% w / w broth, 50% w / w sterile water

[0270] 5) The inoculum suspension was crudely mixed with the carrier medium by gentle external kneading of the bag as described previously.

[0271] 6) After inoculation the packs were placed in a darkened incubation room and left undisturbed for 6 days at 22°C. At the end of the incubation period dense mycelium growth was evident throughout the medium. Extending the incubation time will result in the development of sclerotia and is the preferred method for the active ingredient in the granule formulation.

[0272] 7) The contents of each pack were gently fragmented by hand into units ranging from 1 cm - 3cm in diameter. These large units were then dried on a modified domestic food dehydrator as described previously at a temperature not exceeding 28°C.

[0273] 8) The dried mycelium cake was then granulated via a reciprocating granulator in two passes. At the first pass the granulator was fitted with a 2.4 mm aperture stainless steel woven wire mesh and on the second pass a 1.3 mm aperture mesh.

[0274] 9) Water dispersible powders were prepared finally by gently blending the dry mycelium granules with the other ingredients of the formulation.Preparation of an aqueous suspension of the powder is achieved by adding the powder to the water whilst gently stirring. Small quantities can be prepared in a bucket using a flat blade or other suitable stirring device. Larger quantities are prepared in a spray tank equipped with a mechanical stirrer.

[0275] In line filters would be removed and conventional spray nozzles replaced with 'rain drop' nozzles or foam nozzles that will discharge the suspension as large foamy droplets:

[0276] Application rates may vary but a current preferred rate is 15kg of the dispersible powder formulation in 100L water per hectare. This equates to 3 kg / ha of mycelium concentrate granules.

[0277] This is an improved method of producing S. sclerotiorum mycelium in a semi-solid nutrient medium. Because the mycelium is grown, dried and granulated in situ it is not subjected to the level of physical damage that occurs when handling and processing mycelium produced by conventional broth fermentation. The granulation method is also important because it is carried out on dry material and is relatively non-damaging to the mycelium.

[0278] Efficacy of the granules is markedly enhanced by formulating them finally as a water dispersible powder with appropriate additives for application as an aqueous suspension in the form of large drops.

[0279] Example 4: Use of S. sclerotiorum to invasive noxious broadleaves (“Weed stick” formulation)

[0280] Granules to be delivered via a penetrating dispensing “weed stick” which dispenses granules directly into the resetting stage of the target weed.

[0281] Granules Formulation 1 is described in Table 2.

[0282]

[0283] Table 2: Formulation 1 information. Carrier material is 100% Cerabind and S. sclerotiorum isolate S13.

[0284] Details of Water Dispersible Powder Formulation are described in Table 3. This formulation proved to be 100% viable when subjected to the standard test initially and after 56 days at 22°C with promising results.

[0285]

[0286] Table 3: Water Dispersible Powder Formulation information. Using the Granules as described in Formulation 1 (Table 2)

[0287] Example 5: Use of S. sclerotiorum to invasive noxious broadleaves (“Dual-Phase” formulation)

[0288] The formulation consists of a dual-phase delivery system comprising both hydrogel microcapsules and an oil-based suspension.

[0289] Hydrogel Microcapsules (e.g. Gellan Gum or Alginate) contain encapsulated mycelium based on Formulation 1 above. These beads release the mycelium upon exposure to the plant surface, where humidity is higher.

[0290] Oil-based (Soya Bean Oil) suspension surrounds the microcapsules, augmenting adhesion and retention (Xantham Gum) on the plant surface while preventing desiccation through an additional stored moisture barrier (Carboxymethyl Cellulose CMC).

[0291] Preparation Steps

[0292] 1. Microencapsulation

[0293] a. Dissolve Sodium Alginate or Gellan Gum in water and mix thoroughly with formulation 1 (Fungal mycelium fragments).

[0294] b. Drop the mixture into a calcium chloride solution to form hydrogel beads encapsulating the conidia.

[0295] c. Wash and collect the hydrogel beads. (Note beads must be below 1000 pm)

[0296] 2. Oil Suspension:

[0297] a. Mix soybean oil with Tween 80 to form a homogeneous oil phase.

[0298] b. Disperse the hydrogel beads into the oil phase while continuously stirring to ensure homogeneous distribution.

[0299] c. Add glycerol, propylene glycol, xanthan gum, and CMC to the oil phase and mix again to beads and mixture is homogeneous.

[0300] 3. Application:a. Apply the formulated suspension using a sprayer or similar device tailored for agricultural use. (Nozzle filters removed and a white 32 mesh primary pump filter is best) b. The formulation provides an immediate protective oil layer CMC and long-term moisture retention via the hydrogel beads.

[0301] Example 6

[0302] Pre-Emergence Application

[0303] Mycoherbicidal formulation according to the formulation of Example 4 herein was applied at different application rates (7 kg / hectare, 10 kg / hectare and 15 kg / hectare) on thistle seedlings in pastures in Bombay Hills (New Zealand). The mycoherbicidal formulation was applied using a “weed stick” method before emergence of the thistle seedlings then plants were counted 7 days and 20 days after application. Herbicidal activity was compared with inactive control and unformulated Sclerotinia.

[0304] All application rates (7 kg / hectare, 10 kg / hectare and 15 kg / hectare) showed herbicidal activity and especially for the application rate of 15 kg / hectare compared with inactive control. The mycoherbicidal formulation showed at all application rates a superior herbicidal activity compared with unformulated Sclerotinia.

[0305]

[0306] Table 4 - Average Plant Counts per m2After Pre-Emergence Application of Mycoherbicide Formulation. The average plant count is calculated on 3 different replicates. Results shown in Figure 1.

[0307] Post-Emergence Application

[0308] Mycoherbicidal formulation according to the formulation of Example 4 herein was applied at different application rates (7 kg / hectare, 10 kg / hectare and 15 kg / hectare) on thistle seedlings in pastures in Bombay Hills (New Zealand). The mycoherbicidal formulation was applied using a “weed stick” method after emergence of the thistle seedlings then plants were counted on application and 13 days after application. Herbicidal activity was compared with inactive control and unformulated Sclerotinia.

[0309] All application rates (7 kg / hectare, 10 kg / hectare and 15 kg / hectare) showed herbicidal activity and especially for the application rate of 15 kg / hectare compared with inactive control.The mycoherbicidal formulation showed at all application rates a superior herbicidal activity compared with unformulated Sclerotinia.

[0310]

[0311] Table 5 - Average Plant Counts per m2After Post-Emergence Application of Mycoherbicide Formulation. The average plant count is calculated on 3 different replicates. Results shown in Figure 2.

[0312] Example 7: Mycoherbicide viability correlation of dried mycelium fragments vs moisture rate

[0313] After the growth of the Sclerotinia sclerotiorum the growth bags were cut open to begin to dry down. The temperature was turned down to 18°C, the heat pump turned to dehumidify and the bags were left for a period of 4 days to dry down.

[0314] 36 bags were then picked up, granulated and the fragments tipped into a 20 L sterile bucket roughly mixed to make the granules more homogenous and a 10 g sample taken to be tested for which 2 g was tested for moisture rate.

[0315] The remaining 8 g was carefully sieved to remove the larger particles with the finer grains and mycelium fragments collected to test for viability.

[0316] The sieved samples were packed with silica beads and left at 22°C for 56 days. Each sample was divided into two: (i) one tested again for moisture rate; (ii) the other added to sterile water and spread on a microscope slide with a film of 2% SABDEX Agar. This slide was then placed into a petri dish and incubated for 48 hours at 24°C. The sample was then checked under the microscope for presence of growth. A percentage was calculated for viability (Figure 3).

[0317] In this experiment viability of the mycelial fragments were correlated to lower moisture rates, especially below 7% after storage for 56 days at 22°C.

Claims

CLAIMS1. A method of producing a biocidal composition comprising mycelia of one or more pathogenic and / or entomopathogenic fungus, said method comprising the steps of:(a) inoculating a carrier material with mycelia of one or more pathogenic and / or entomopathogenic fungus;(b) incubating the inoculated carrier material in the absence of light to grow the mycelia;(c) drying the material from step (b) to reduce the water content to 10% w / w or less;and(d) granulating the dried material.

2. The method of claim 1 , wherein the carrier material:(i) is organic or inorganic, preferably organic, and optionally wherein the organic carrier material is selected from a cereal, a cereal-derived material, or a plant- derived material; and / or(ii) has a % w / w water content of between about 30 % w / w to about 50 % w / w, wherein optionally the carrier material is dried prior to step (a) to arrive at this % w / w water content.

3. The method of claim 2, wherein:(i) the cereal, cereal-derived or plant-derived carrier material comprises or consists of material derived from rice, wheat, maize, rye, bran or oats; or(ii) the inorganic carrier material comprises or consists of diatomaceous earth, perlite, calcium silicate and / or magnesium silicate, bentonite, attapulgite, sepiolite, vermiculite, cellulosic fibre, preferably the carrier material comprises diatomaceous earth.

4. The method of any one of the preceding claims, wherein step (b) is at least 4 days, at least 5 days, at least 6 days, at least 7 days or at least 8 days, preferably at least 6 days.

5. The method of any one of the preceding claims, wherein step (b) is performed at a temperature of:(i) between about 20°C to about 40°C, optionally between about 20°C to about 30°C or between about 20°C to about 25°C; and / or(ii) about 20°C, at about 21°C, at about 22°C, at about 23°C, at about 24°C, at about 25°C, preferably about 22°C.

6. The method of any one of the preceding claims, wherein in step (c):(i) the water content of material from step (b) is reduced to 7% w / w or less, 5% w / w or less, 3% w / w or less, 1% w / w or less, preferably the water content is reduced to less than 1 % w / w; and / or(ii) is carried out at a temperature of about 30 °C or less, about 20 °C or less, about 10 °C or less, about 5 °C or less, about 4 °C or less, about 3 °C or less, about 2°C or less, or about 1 °C or less, preferably about 20 °C or less, more preferably about 18 °C or less, even more preferably about 4 °C or less.

7. The method of any one of the preceding claims, wherein in step (d):(i) granulating is carried out by rotary granulation;(ii) granulating is carried out using two or more granulators in sequence with decreasing aperture size; and / or(iii) granules of average diameter of between about 100 pm to about 1500 pm are produced, optionally between about 500 pm to about 1500 pm, about 700 pm to about 1500 pm, or between about 700 pm to about 1300 pm.

8. The method of any one of the preceding claims, wherein the carrier material in step (a) is sterilised before being inoculated, optionally wherein the carrier material is sterilised by autoclaving or microwaving.

9. The method of any one of the preceding claims, wherein the biocidal composition is mixed with one or more additional component selected from the list consisting of:(i) a filler, optionally a cereal-based or plant-based product;(ii) nutrients, optionally organic and / or inorganic nutrients;(iii) viscosity increasing agents, optionally a synthetic or natural gum and / or soluble starch;(iv) buffers, optionally to maintain the pH in the range of between about 3.0 to about 6.0, preferably between about 3.5 to about 4.5;(v) humectants, optionally inverted sugars, polyethylene glycols, vegetable mineral and / or synthetic oils;(vi) UV light absorbing material, optionally soya bean oil and / or titanium dioxide; (vii) wetting agents optionally methylcelluloses and / or hydroxypropylmethyl celluloses; (viii) protective agents, optionally PEG, polyvinylpyrrolidone, polyvinyl alcohol, casein salts, natural or synthetic gums, oils and / or activated carbon;(ix) bacteriostatic agents, optionally oxalic acid;(x) scarifying agents, optionally perlite;(xi) enzymes or other agents which destroy plant tissue, optionally C8-C10 fatty acids;and / or(xii) plant defence mechanism inhibitors, optionally CAPE 1 peptide, 2,1,3 Benzothiadiazole (BTH), abscisic acid (ABA), p Aminobutyric acid (BABA), Methyldihydrojasmonate (dihydro - JA) and / or a 1,6 cyclized P1,2glucohexadecaose (CB G16a);wherein optionally the composition is formulated as a wettable powder.

10. The method of any one of the preceding claims, which further comprises the steps:(e) encapsulating the granulated material from step (d); and(f) suspending the encapsulated material from step (e) in an homogeneous oil phase to form a suspension.

11. The method of any one of the preceding claims, wherein in step (e) the granulated material from step (d) is suspended in an aqueous solution of gelling agent and then dispensed as droplets into an aqueous solution of calcium chloride.

12. The method of claim 11 , wherein the gelling agent is selected from the group consisting of sodium alginate, hydroxypropyl methyl cellulose, methyl cellulose, gum Arabic, guar gum, pectine and gellan gum, preferably the gelling agent is sodium alginate or gellan gum.

13. The method of any one of claims 10 to 12, wherein in step (f) the homogeneous oil phase comprises soybean oil and optionally Tween 80.

14. The method of any one of the claims 10 to 13, wherein in step (f) one or more additional agent is added to the suspension, wherein optionally said one or more additional agent is selected from the group consisting of glycerol, propylene glycol, xanthan gum, and / or carboxymethyl cellulose, preferably wherein glycerol, propylene glycol, xanthan gum, and carboxymethyl cellulose are added to the suspension.

15. The method of any one of the preceding claims, wherein the one or more pathogenic and / or entomopathogenic fungus is a mycopathogenic fungus, which is optionally selected from the group consisting of S. sclerotiorum, Verticillium dahliae, Colletotrichum gloeosporioides, Sclerotinia minor, Agroathelia rolfsii, Armillaria mellea, and Rhizoctonia sp , wherein preferably the mycopathogenic fungus is S. sclerotiorum.

16. The method of any one of the preceding claims, wherein the one or more pathogenic and / or entomopathogenic fungus remains viable and is capable of reanimation, penetration, infection and causing disease for a period of no less than 12 months.

17. A biocidal composition obtainable by the method of any of the preceding claims.

18. A biocidal composition comprising granules containing a carrier material and mycelia of one or more pathogenic and / or entomopathogenic fungus, wherein the water content of the granules is at least 75% w / w for at least 8 hours post-application.

19. The biocidal composition of claim 17 or 18, wherein:(i) the granules are encapsulated;(ii) the composition is an aqueous suspension; and / or(iii) the composition has a shelf life of 12 - 14 months.

20. The biocidal composition of any one of claims 17 to 19, wherein the composition further comprises one or more plant defence mechanism inhibitor, wherein optionally the one or more plant defence mechanism inhibitor is selected from the group consisting of CAPE 1 peptide, 2,1,3 Benzothiadiazole, abscisic acid, Aminobutyric acid, Methyldihydrojasmonate and a 1,6 cyclized pi,2glucohexadecaose.

21. The biocidal composition of any one of claims 17 to 20, wherein the one or more pathogenic and / or entomopathogenic fungus is a mycopathogenic fungus, which is optionally selected from the group consisting of S. sclerotiorum, Verticillium dahliae, Colletotrichum gloeosporioides, Sclerotinia minor, Agroathelia rolfsii, Armillaria mellea, and Rhizoctonia sp, preferably the one or more mycopathogenic fungus is S. sclerotiorum.

22. The biocidal composition of any one of claims 17 to 21 , wherein the water content of the granules is:(i) at least 80% w / w or at least 85% w / w for at least 12 hours post-application, preferably the water content of the granules is at least 85% w / w for at least 12 hours post-application;(ii) between about 70% w / w to about 90% w / w for at least about 10 hours postapplication, optionally at least about 12 hours post-application; and / or (iii) between about 75% w / w to about 85% w / w for at least about 10 hours postapplication, optionally at least about 12 hours post-application.

23. Use of a biocidal composition as defined in any of claims 16 to 22 for preventing, ameliorating, inhibiting, eliminating or delaying the onset of a pest infection or infestation, wherein preferably the pest is a weed, optionally selected from Cirsium avense, Senecio jacobaea, Hieracium pilosella, Eichhornia crassipes or Ranunculus acris.

24. A method for treating, preventing, ameliorating, inhibiting, eliminating or delaying the onset of an infestation, comprising the step of applying onto a plant, to a plant material or in the vicinity of said plant or plant material a biocidal composition of any one of claims 16 to 22; wherein preferably the infestation is a weed infestation, optionally an infestation of Cirsium avense, Senecio jacobaea, Hieracium pilosella, Eichhornia crassipes or Ranunculus acris.

25. The use of claim 23 or the method of claim 24, wherein:(i) the biocidal composition has a biocidal activity that is at least 30% greater than a composition without mycelia of the one or more pathogenic and / or entomopathogenic fungus;(ii) the amount of the biocidal composition required to control the infestation is 2 times less than the amount of a composition without mycelia of the one or more pathogenic and / or entomopathogenic fungus that is required to treat, prevent, ameliorate, inhibit, eliminate or delay the onset of the same infestation;(iii) the method or use comprises the controlled release of the mycelia of the one or more pathogenic and / or entomopathogenic fungus, wherein optionally controlled release comprises contacting the biocidal composition with sodium citrate; and / or (iv) the application rate of the biocidal composition is between about 10 to about 20 kg of the biocidal composition / 100 L water / hectare, preferably about 15kg of the biocidal composition / 100 L water / hectare.

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