WO2004039820A1 - Material - Google Patents

Abstract

The present invention provides an antimicrobial material comprising (i) an antimicrobial compound in a stabilised form (a 'stabilised compound'), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a 'primary precursor') to the antimicrobial compound; and (b) (I) a primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a 'secondary precursor') to the primary precursor; and a secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof, such as compounds having Formula (I), wherein R1 and R2 are independently selected from H and C(=O)R3 wherein R3 is a hydrocarbyl group, and R4 is from H and OH.

Description

MATERIAL

FIELD OF THE INVENTION

The present invention relates an antimicrobial material. In particular the present invention relates to a stabilised antimicrobial compound and to the in situ production of an antimicrobial compound. It further relates to processes for preventing and/or inhibiting the growth of, and/or killing a micro-organism utilising the antimicrobial material.

TECHNICAL BACKGROUND AND PRIOR ART

The compound microthecin has been known for many years. The structure of microthecin is shown below.

It is known to have mild anti-microbial activity against some specific micro-organisms. For examples JP54122796 teaches microthecin may be isolated from strains Melanospora ornata SANL 23077, Microthecium compressum SANL 46472 and Microthecium zobelii SANK 14776 and has anti-biotic action against fungi strains Proteus vulgaris SANK 73266 and Proteus rettgeri SANK 73775.

These findings have been contradicted in some further studies. For example Baute et al. Phytochemistry, Vol 33, No. 1 , pp.44-45, 1993 relates to use of echinosphorin against bacteria. However reference is made to a Baute paper of 1988 which proposes microthecin might play a role as an antimicrobial defence metabolite in fungi.

However, the general teaching in the art has been towards the anti-microbial activity of microthecin. For example, WO 95/10616 teaches microthecin is an antibiotic which can be prepared from glucan lyase. US 6013504 teaches that microthecin is antibiotic

Vole et al., Arch Microbiol (1991) 156:297-301 although primarily directed to the production of cortalcerone makes mention of microthecin. In particular the production of microthecin from pyranosone dehydratase is discussed as is the use of microthecin in an antibiotic

WO 03/037918 relates to production of microthecin from pyranosone dehydratase and in particular the conversion of anhydrofructose to microthecin using pyranosone dehydratase. It is also taught that microthecin may be used as an antimicrobial, such as on a foodstuff, and teaches use against plant fungal pathogens and as plant and seed protectants. Use of microthecin as an anti-fungal against specific pathogens is taught.

WO 03/038107 by reference to JP 54-122796 teaches that microthecin may be anti- fungal. It further teaches production processes for the preparation of Ascopyrone P derivatives such as microthecin.

Broberg et al., 1998, Carbohydrate Research 306, 171-185, indicates that 'after incubation of microthecin in a citrate buffer of pH 6.6 at 30C for 18hr, the relative composition of the mixture was 45% of microthecin and 55% of product.

Microthecin is typically produced by enzymatic means. For example, it has been established that starch can be converted to 1 ,5-anhydro-D-fructose (AF) [S. Yu and J. Marcussen, Recent Advances in Carbohydrate Bioengineering; Gilbert, H. J.; Davies, G. J; Henrissat B.; Svensson, B., Eds.; Royal Society of Chemistry (RS.C) Press, 1999. 242-250]. It has further been shown that several fungal and red algal extracts can convert AF to microthecin possibly enzymatically [Baute, M-A.; Deffieux, G.; Baute, R. (1986), Phytochemistry (Oxf) vol. 25:1472-1473; Broberg, A., Kenne, L, and Pedersen, M. (1996), Phytochemistry (oxf). 41: 151-154].

Derivatives of microthecin, such as cortalcerone and ascopyrone P, are also taught in the art. These derivatives are also typically produced by enzymatic means. It is known that glucose can be oxidised by pyranose 2-oxidase (EC 1.1.3.10, P2O) to form glucosone (D-arabino-hexos-2-ulose), which in turn can be converted to cortalcerone (structure below) by pyranosone dehydratase (PD) [Koths, K.; Halenbeck, R.; Moreland, M. (1992), Carbohydr Res. Vol. 232 No. 1 , PP. 59-75; Gabriel, J.; Vole, J.; Sedmera, P.; Daniel, G.; Kubatova, E. (1993), Arch. Microbi., 160:27-34]. Both P2O and PD have been purified in fungi and P2O has been cloned. PD has been purified from Polyporus obtusus by Koths et al (1992), and from Phanerochaete chrysosporium by Gabriel et al (1993). It has also been documented that ascopyrone P (APP) can be produced from AF [Baute, M-A.; Deffieux, G.; Vercauteren, J.; Baute, R.; Badoc, A. (1993), Phytochemistry (oxf) vol. 33 no. 1 , 41-45].

Cortalcerone

Unlike many alternative anti-microbial compounds, microthecin and its derivatives are cheap to produce in large quantities, and as such is suitable for a wide range of commercial applications. Consequently, one would envisage that it would find may applications as an anti-microbial. However, microthecin has only reported to have had weak antimicrobial activity, or activity against a narrow range of microbes.

SUMMARY OF THE INVENTION

Microthecin has only reported to have had weak antimicrobial activity, or activity against a narrow range of microbes. Surprisingly, we have discovered that this poor performance is due to microthecin rapidly degrading, particularly when formulated as an aqueous solution, and that this instability results in poor anti-microbial activity. Although the prior art describes microthecin as having weak anti-fungal activity, there is no indication that this relatively poor activity is due to the inherent instability of microthecin.

The invention relates to methods of enhancing the stability of the compound microthecin and its derivatives such cortalcerone. It has been found that microthecin is highly unstable in aqueous solution. However microthecin can be stabilised by selecting a suitable formulation that enhances its stability.

We have found that by employing stabilised formulations of microthecin, or by continual production or administration of microthecin, a significant enhancement of the antimicrobial effect is obtained, thereby making microthecin suitable as an anti-microbial active ingredient in numerous formulations, such as personal hygiene products and cosmetics, and agrochemical formulations. Aspects of the present invention are presented in the claims and in the following commentary.

For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.

DETAILED DISCLOSURE OF INVENTION

In a first aspect, the invention provides an antimicrobial material comprising (i) an antimicrobial compound in a stabilised form (a "stabilised compound"), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to the antimicrobial compound; and (b) (I) a primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and a secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof.

In a second aspect, the invention provides a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, comprising the steps of contacting the material with an antimicrobial material comprising (i) an antimicrobial compound in a stabilised form (a "stabilised compound"), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to the antimicrobial compound; and (b) (I) a primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and a secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof.

In a third aspect, the invention provides use of an antimicrobial material for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the antimicrobial material comprises (i) an antimicrobial compound in a stabilised form (a "stabilised compound"), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to the antimicrobial compound; and (b) (I) a primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and a secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof

In a fourth aspect, the invention provides a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, comprising the steps of contacting the material either sequentially or simultaneously with (i) an antimicrobial compound in a stabilised form (a "stabilised compound"), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to the antimicrobial compound; and (b) (I) a primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and a secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof.

In a fifth aspect, the invention provides a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, comprising the steps of contacting the material with microthecin or a derivative thereof, wherein the micro-organism is selected from Alternaha spp. , Albugo spp., Amyloporia spp., Ascochyta spp., Aspergillus spp, Basidiophora spp, Bipolaris spp, Botrytis spp, Bremia spp, Cladospohum spp, Claviceps spp, Coniophora spp., Colletothchum spp, Diplodia spp, Dipiocarpon spp., Donkiopoha spp., Drechslera spp, Erysiphe spp, Eutypa spp, Fibroporia spp., Fusahum spp, Gaeumanomyces spp, Geotrichum spp, Guignardia spp, Gymnosporangium spp, Helmintosporium spp, Hemileia spp, Kabatiella spp, Leptosphaeria spp, Macrophomina spp, Marssonina spp., Monilinea spp, Merulus spp, Mycosphaerella spp, Nectria spp, Paracercospora spp, Penicillium spp, Peronophythora spp, Peronospora spp, Phelle as spp., Phoma spp, Phomopsis spp, Phymatotrichum spp, Phytophthora spp, Plasmophora spp, Podosphaera spp, Porai spp., Pseudocercosporella spp, Pseudoperonospora spp, Puccinia spp, Pyrenophora spp, Pyricularia spp, Rhizopus spp, Sclerophthora spp, Sclerotinia spp, Sclerotium spp, Septoria spp, Serpula spp., Sphaerotheca spp, Stagonospora spp, Taph na spp, Thielaviopsis spp, Tilletia spp, Trichoderma spp, Uncinula spp, Ustilago spp, Ventu a spp, and Verticillium spp

In a sixth aspect, the invention provides use of an antimicrobial compound material for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the antimicrobial compound is microthecin or a derivative thereof, and wherein the micro-organism is selected from Alternaria spp. , Albugo spp., Amyloporia spp., Ascochyta spp., Aspergillus spp, Basidiophora spp, Bipolaris spp, Botrytis spp, Bremia spp, Cladosporium spp, Claviceps spp, Coniophora spp., Colletothchum spp, Diplodia spp, Dipiocarpon spp., Donkiopoha spp., Drechslera spp, Erysiphe spp, Eutypa spp, Fibropo a spp., Fusarium spp, Gaeumanomyces spp, Geotrichum spp, Guignardia spp, Gymnosporangium spp, Helmintosporium spp, Hemileia spp, Kabatiella spp, Leptosphaeria spp, Macrophomina spp, Marssonina spp., Monilinea spp, Merulus spp, Mycosphaerella spp, Nectria spp, Paracercospora spp, Penicillium spp, Peronophythora spp, Peronospora spp, Phellehas spp., Phoma spp, Phomopsis spp, Phymatothchum spp, Phytophthora spp, Plasmophora spp, Podosphaera spp, Porai spp., Pseudocercosporella spp, Pseudoperonospora spp, Puccinia spp, Pyrenophora spp, Pyricularia spp, Rhizopus spp, Sclerophthora spp, Sclerotinia spp, Sclerotium spp, Septo a spp, Serpula spp., Sphaerotheca spp, Stagonospora spp, Taph na spp, Thielaviopsis spp, Tilletia spp, T choderma spp, Uncinula spp, Ustilago spp, Venturia spp, and Verticillium spp

In a seventh aspect the present invention provides an anti-microbial composition comprising microthecin or a derivative thereof in a stabilised form.

In an eighth aspect the present invention provides microthecin or a derivative thereof in a substantially dry form.

In a ninth aspect, the invention provides a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, comprising the steps of contacting the material with microthecin or a derivative thereof, wherein the micro-organism is as described herein.

In a tenth aspect, the invention provides use of an antimicrobial compound material for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the antimicrobial compound is microthecin or a derivative thereof, and wherein the micro-organism is as described herein.

By the term "substantially dry form" it is meant having water present in an amount of less than 5wt%, preferably less than 2wt%, preferably less than 1wt%, preferably less than 0.5wt%, preferably less than 0.25wt%, more preferably less than 0.1wt%. In a eight aspect the present invention provides a kit for preparing an antimicrobial material as defined herein, the kit comprising (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to and antimicrobial compound, wherein the antimicrobial compound is selected from microthecin and derivatives thereof; and (b) (I) a primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and a secondary precursor; in separate packages or containers; optionally with instructions for admixture and/or contacting and/or use.

ADVANTAGES

The present invention provides an antimicrobial material in which an anti-microbial compound, previously reported to have low antimicrobial activity, is delivered in a stabilised form or is delivered by in situ synthesis by a precursor and a conversion agent. Consequently antimicrobial compounds may be provided having potent antimicrobial activity. In addition these compounds may be readily and cheaply synthesised, for example enzymatically either for stabilised delivery or in situ. For example the present invention provides microthecin or cortalcerone in a stabilised form and/or facilitates the in situ production of microthecin or cortalcerone.

The in situ production or stabilised delivery of microthecin according to the present invention surprisingly overcomes the problems of low activity resulting from the instability of the microthecin, particularly when applied to plants.

The in situ production of microthecin, particularly in plants or parts thereof, provides effective pathogen , particularly fungal pathogen, control. The control afforded by the in situ production of microthecin is preferably broad spectrum, i.e. target more than one pathogen.

The present invention may be used to control fungal pathogens on a plant during growth (cultivation) thereof. Suitably, however, the present invention may be used to control fungal pathogens on the plant or part thereof after harvest, for example during storage. Suitably, the plant or part thereof according to the present invention will be protected from attack by pathogens e.g. fungal pathogens, both during cultivation and post- harvest, such as during storage. As will be readily appreciated by the skilled person, the present invention may, therefore, be applicable to preserve stored seeds and/or other plant parts, as well as stored silage for example, or to preserve food of feed for animals, foodstuffs, food products, prepared from plant or other edible materials, including preservation of fruits, seed, grain and vegetables post harvest.

The present invention may be used to control or prevent microbial growth, for example fungal infection of plant seeds after sowing, particularly prior to growth.

The terms "fungus", "fungal" or "fungi" used herein includes micro-organisms from any taxonomic group that is classified within the following taxons: Oomycetes, Ascomycetes, Fungi imperfecta, Deutormycetes, Basidiomycetes, Zygomycetes, or Mastigomycetes. Preferably the terms "fungus", "fungal" or "fungi" used herein relates to micro-organisms selected from one or more of the following taxonomic groups: Oomycetes, Ascomycetes, Fungi imperfecta, Deutormycetes, Basidiomycetes, Zygomycetes, or Mastigomycetes

PREFERRED ASPECTS

In one aspect the antimicrobial material comprises the stabilised compound. Thus in one aspect the present invention provides

• an antimicrobial material comprising an antimicrobial compound in a stabilised form (a "stabilised compound")

• a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, comprising the steps of contacting the material with an antimicrobial material comprising an antimicrobial compound in a stabilised form (a "stabilised compound")

• use of an antimicrobial material for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the antimicrobial material comprises an antimicrobial compound in a stabilised form (a "stabilised compound")

In one aspect the antimicrobial material provides the means to form the antimicrobial compound. Thus in one aspect the present invention provides an antimicrobial material comprising (a) the first conversion agent; and (b) (I) the primary precursor, or (II) the second conversion agent and the secondary precursor. In some applications the precursors, either the primary precursor or the secondary precursor may already be present in the material to be treated. An example of such an application is the treatment of plants in which for example starch may be present. In such applications, the provision of the primary precursor and/or the secondary precursor is optional. Thus in a further aspect the present invention provides

• an antimicrobial material comprising (i) an antimicrobial compound in a stabilised form (a "stabilised compound"), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to the antimicrobial compound; and (b) (I) an optional primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and an optional secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof.

• a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, comprising the steps of contacting the material with an antimicrobial material comprising (i) an antimicrobial compound in a stabilised form (a "stabilised compound"), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to the antimicrobial compound; and (b) (I) an optional primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and an optional secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof.

• use of an antimicrobial material for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the antimicrobial material comprises (i) an antimicrobial compound in a stabilised form (a "stabilised compound"), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to the antimicrobial compound; and (b) (I) an optional primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and an optional secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof

As will be appreciated by one skilled in the art, when the antimicrobial compound is formed in situ it may formed directly from constituents of the antimicrobial material or it may formed indirectly from constituents of the antimicrobial material. Thus the antimicrobial material may provide the first conversion agent; and the primary precursor. In this aspect the first conversion agent converts the primary precursor into the antimicrobial compound. The antimicrobial compound may formed indirectly from constituents of the antimicrobial material when the present invention provides an antimicrobial material comprising (a) the first conversion agent; and (b) the second conversion agent and the secondary precursor.

As will be appreciated by one skilled in the art, the present invention requires only that constituents be supplied which will convert an initial substrate through one or more steps into a final antimicrobial compound. This may involve one, two, three, four, five or more steps. Consequently the present invention is not limited to a first conversion agent and/ optionally a second conversion agent. The invention allows for a third conversion agent, a fourth conversion agent, a fifth conversion agent and so on, so that a chain of conversion may occur to form a final antimicrobial compound.

ANTIMICROBIAL COMPOUND

In one preferred embodiment, the derivative of microthecin is 2-furyl-hydroxymethyl-ketone or 4-deoxy-g/ycero-hexo-2,3-diluose.

In another preferred embodiment, the derivative of microthecin is 2-furylglyoxal.

In one preferred aspect the antimicrobial compound is a compound having Formula I, Formula I

wherein Ri and R₂ are independently selected from H and C(=O)R₃ wherein R₃ is a hydrocarbyl group, and R₄ is from H and OH

The term "hydrocarbyl group" as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo, alkoxy, nitro, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. A non- limiting example of a hydrocarbyl group is an acyl group.

A typical hydrocarbyl group is a hydrocarbon group. Here the term "hydrocarbon" means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.

In some aspects of the present invention, the hydrocarbyl group is selected from optionally substituted alkyl group, optionally substituted haloalkyl group, aryl group, alkylaryl group, alkylarylakyl group, and an alkene group.

In some aspects of the present invention, the hydrocarbyl group is an optionally substituted alkyl group.

In some aspects of the present invention, the hydrocarbyl group is selected from C₁-C-₁0 alkyl group, such as d-C₆ alkyl group, and C^Cs alkyl group. Typical alkyl groups include d alkyl, C₂ alkyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, C₇ alkyl, and C₈ alkyl.

In some aspects of the present invention, the hydrocarbyl group is selected from C₁-C₁0 haloalkyl group, C C₆ haloalkyl group, C C₃ haloalkyl group, CrC^ bromoalkyl group, d-Ce bromoalkyl group, and C-ι-C₃ bromoalkyl group. Typical haloalkyl groups include C-, haloalkyl, C₂ haloalkyl, C₃ haloalkyl, C₄ haloalkyl, C₅ haloalkyl, C₇ haloalkyl, C₈ haloalkyl, d bromoalkyl, C₂ bromoalkyl, C₃ bromoalkyl, C bromoalkyl, C₅ bromoalkyl, C₇ bromoalkyl, and C₈ bromoalkyl.

In some aspects of the present invention, the hydrocarbyl group is selected from aryl groups, alkylaryl groups, alkylarylakyl groups, -(CH₂)ι-ιo-aryl, -(CH₂)ι.₁₀-Ph, (CH₂)₁.₁₀-Ph- Cι.,o alkyl, -(CH^-Ph, (CH₂)₁.₅-Ph-C₁.₅ alkyl, -(CH^-Ph, (CH^-Ph-C^ alkyl, -CH₂- Ph, and -CH₂-Ph-C(CH₃)₃.

When the hydrocarbyl group is or contains an aryl group, the aryl group or one or more of the aryl groups may contain a hetero atom. Thus the aryl group or one or more of the aryl groups may be carbocyclic or more may heterocyclic. Typical hetero atoms include O, N and S, in particular N.

In some aspects of the present invention, the hydrocarbyl group is selected from -(CH₂)ι- ₁₀-cycloalkyl, -(CH₂)ι-ιo-C₃..ιocycloalkyl, -(CH₂)ι-₇-C₃.₇cycloalkyl, -(CH₂)ι^-C₃.5cycloalkyl, - (CH₂)ι-₃-C₃.₅cycloalkyl, and -CH₂- C₃cycloalkyl.

In some aspects of the present invention, the hydrocarbyl group is an alkene group. Typical alkene groups include C^C-io alkene group, C C₆ alkene group, C C₃ alkene group, such as Cι, C₂, C₃, C₄, C₅, C₆, or C₇ alkene group. In a preferred aspect the alkene group contains 1 , 2 or 3 C=C bonds. In a preferred aspect the alkene group contains 1 C=C bond. In some preferred aspect at least one C=C bond or the only C=C bond is to the terminal C of the alkene chain, that is the bond is at the distal end of the chain to the ring system.

In one aspect R₃ contains from 1 to 10 carbon atoms.

In one aspect R₃ contains from 1 to 5 carbon atoms.

In one aspect R₃ is a C-|.₅ alkyl group.

In one aspect F is H. Thus, in this aspect the antimicrobial compound is a compound having Formula II

Formula II

wherein R-i and R₂ are independently selected from H and C(=O)R₃ wherein R₃ is a hydrocarbyl group. In one aspect of the present invention when R₄ is OH, R¹ is H and R² is H.

In one aspect preferably at least one of and R is H

Preferably R¹ is H.

Preferably R² is H.

In one aspect preferably R¹ is H and R² is H.

In one highly preferred aspect R¹ is H, R² is H and R⁴ is H. Thus in this aspect the antimicrobial compound has the Formula

This compound is commonly referred to as microthecin

In one highly preferred aspect R¹ is H, R² is H and R⁴ is OH. Thus in this aspect the antimicrobial compound has the Formula

This compound is commonly referred to as cortalcerone.

In one aspect the microthecin is prepared in accordance with a process described in WO03/037918. Thus in one aspect the microthecin is prepared by reacting pyranosone dehydratase with 1 ,5-anhydro-D-fructose.

It will be recognised by the skilled person that the antimicrobial compound of the invention, when produced in situ in an organism (for example in a transgenic organism such as a plant, a fungus, a filamentous fungus, a yeast, or a bacteria), can be further modified by endogenous or heterologously introduced enzymes within the organism. As such the invention also relates to derivatives of the antimicrobial compounds of the invention generated from said antimicrobial compounds in situ in the organism, where the derivatives of the microbial compounds also exhibit anti-microbial activity.

FIRST CONVERSION AGENT

In one aspect the first conversion agent is an enzyme.

In one preferred aspect the first conversion agent is a dehydratase enzyme.

In one highly preferred aspect the first conversion agent is a pyranosone dehydratase [EC4.2.1.X (x can be any no.)]. A pyranosone dehydratase is defined an enzyme that will remove water from a five carbon sugar, preferably AHF or derivative thereof.

In one aspect the pyranosone dehydratase is a pyranosone dehydratase as described in WO03/037918.

In one aspect the pyranosone dehydratase may be obtainable, or obtained, from one or more of Phanerochaete chrysosporium, Polyporus obtusus or Corticium caeruleum.

In one aspect the pyranosone dehydratase may be obtainable, or obtained, from an organism in the order of Pezizales, Auriculahales, Aphyllophorales, Agaricales or Gracilariales.

In one aspect the pyranosone dehydratase may be obtainable, or obtained, from one or more of Aleuria aurantia, Peziza badia, P. succosa, Sarcophaera eximia, Morchella conica, M. costata, M. elata, M. esculenta, M. esculenta var. rotunda, M. hortensis, Gyromitra infula, Auricularia mesentehca, Pulcherricium caeruleum, Peniophora quercina, Phanerochaete sordida, Vuilleminia comedens, Stereum gausapatum, S. sanguinolentum, Lopharia spadicea, Sparassis laminosa, Boletopsis subsquamosa, Bjerkandera adusta, Trichaptum biformis, Cerrena unicolor, Pycnoporus cinnabarinus, P. sanguineus, Junghunia nitida. Ramaria flava, Clavulinopsis helvola, C. helvola var. geoglossoides, V. pulchra, Clitocybe cyathiformis, C. dicolor, C. gibba, C odora, Lepista caespitosa, L inversa, L. luscina, L nebularis, Mycena seynii, Pleurocybella porrigens, Marasmius oreales, Inocybe pyhodora,Gracilaha varrucosa, Gracilaria tenuistipitata, Gracilariopsis sp, Gracilariopsis lemaneiformis. Melanosopora spp, Melanospora ornata, Microthecium spp., Microthecium compressum, and Microthecium zobelii.

SECOND CONVERSION AGENT

In one aspect the second conversion agent is an enzyme.

In one preferred aspect the second conversion agent is a lyase or oxidase enzyme.

In one preferred aspect the second conversion agent is a lyase.

In one preferred aspect the second conversion agent is an oxidase enzyme.

In one highly preferred aspect the second conversion agent is a glucan lyase or a pyranose-2-oxidase [EC 1.1.3.10}

In one aspect the glucan lyase is a glucan lyase as described in our copending PCT application of attorney reference P015628WO.

In one aspect the pyranose-2-oxidase is a pyranose-2-oxidase as described in our copending PCT application of attorney reference P015628WO.

In one aspect the pyranose-2-oxidase is a pyranose-2-oxidase as described in US 6146865 or US 5,712,139.

In one highly preferred aspect the second conversion agent is a glucan lyase.

In one highly preferred aspect the second conversion agent is a pyranose-2-oxidase.

PRIMARY PRECURSOR

In one preferred aspect the primary precursor is glucosone or 1 ,5-D-anhydrofructose. In one preferred aspect the primary precursor is glucosone.

In one preferred aspect the primary precursor is 1 ,5-D-anhydrofructose.

SECONDARY PRECURSOR

In one preferred aspect the secondary precursor is a starch dextrin or glucose.

In one preferred aspect the secondary precursor is glucose.

In one preferred aspect the secondary precursor is a starch.

CONVERSION AGENTS/PRECURSORS

As will be appreciated by one skilled in the art the combination of conversion agent(s) and primary or secondary precursor will be chosen so that they interact to provide the desired antimicrobial compound. For example, a primary precursor will be chosen which is a substrate of a first conversion agent so as form the required antimicrobial compound. Alternatively a secondary precursor and second conversion agent will be chosen such that the second conversion agent acts on the secondary precursor to provide a primary precursor. The first conversion agent will be chosen in tandem with this combination so that the formed primary precursor is then converted finally to antimicrobial compound.

Preferred combinations of conversion agents/precursors include

• first conversion agent: pyranosone dehydratase; primary precursor: glucosone or 1,5- D-anhydrofructose.

• first conversion agent: pyranosone dehydratase; primary precursor: glucosone.

• first conversion agent: pyranosone dehydratase; primary precursor: 1 ,5-D- anhydrofructose.

• first conversion agent: pyranosone dehydratase; second conversion agent: a glucan lyase; secondary precursor: starch

• first conversion agent: pyranosone dehydratase; second conversion agent: pyranose- 2-oxidase; secondary precursor: glucose. STABILISED COMPOUND

Stabilising Formulations

There are a number of mechanisms by which microthecin or derivatives thereof may be stabilised.

Dry Formulation

In a preferred embodiment microthecin or derivative thereof is prepared/used/administered in a solid formulation, for example as a powder or as a tablet, lozenge or pessary.

In a preferred aspect microthecin or derivative thereof is kept in a dry state during storage and transportation. The dry state may be achieved by freeze-drying or spray- dry. In one aspect the antimicrobial material is obtained or is obtainable by freeze-drying or spray-drying. In spray drying is chosen the microthecin or derivative thereof may be spray dried with a carrier. Preferred carriers are dextrins.

Spray drying may be performed as described generally in the "Spray Drying Handbook", δ.sup.th ed., K. Masters, John Wiley & Sons, Inc., NY, N.Y. (1991), and in Platz, R., et al., Patent Publication No. WO 97/41833 (1997). Granulation may be performed as described US6499984, US6170269 and US6428724. Congealing may be performed as described US638622. Flash drying may be performed as described in US4043049 and US6085440. Fluid bed drying may be performed as described in US6148541. Swirl fluidising or evaporation may be performed as described in US6120795. Crystallisation may be performed as described in US5898067 or US6416585. Agglomeration, or freeze-drying may be performed as described in US6361813. Pulse drying may be performed as described in US4695248. Ultra sonic atomisation may be performed as described in US6127429. Air drying or oven drying may be performed as described in US6058625.

In a preferred aspect the invention relates to a composition comprising microthecin or a derivative thereof in a solid or powder form. In one aspect the microthecin or derivative thereof is formulated such that in a dry form it is suitable for application to the material to be treated. This is in contrast to materials which may be in a form suitable for application which requires drying after application to provide a dry and stable compound.

If the microthecin or derivative thereof is provided in a dry form it may be dissolved or dispersed in a liquid medium for application. Dry or solid microthecin or derivatives thereof may be dissolved or dispersed in deionised water. Suitably prepared solutions are preferably be kept in the dark and chill temperature prior to use.

Stabilising Agents

The present composition may contain a stabilising agent which stabilises the microthecin or derivative thereof.

In order to determine whether an specific agent can be used in the composition as a stabilising agent, the stability of microthecin or derivative thereof in the presence of a suitable range of concentration of the agent can assess and compared to a control without the agent. A suitable control is a 0.1 M buffer pH (6.5) buffer or citrate buffer. Agents that increase the stability to an extent greater than 10%, preferably greater than 20%, preferably greater than 30, preferably greater than 50 , preferably greater than 70%, preferably greater than 80%, preferably greater than 90%, preferably greater than 100%, preferably greater than 200%, are considered suitable for the purpose of this invention. The stability of microthecin can be determined over a 10 hour, preferably 50 hour, preferably 100 hour, preferably 1000 hour time period using the methodology in example 8 or 9.. Numerous agents and or formulation supports for inclusion in pharmaceutical, veterinary, personal hygiene, domestic and industrial detergents and anti-fungal agents formulations are known, and include the following.

Auxiliary Components - in a preferred embodiment microthecin or derivative thereof is formulated with one or more auxiliary components which improves the stability of the microthecin or derivative thereof. The one or more auxiliary components may be selected from anti-oxidants, free radical quenchers, (metal) chelators, emulsifiers and UV protectants. Suitable UV filter substances which are used in combination with the formulations to be used according to the invention are any UV-A and UV-B filter substances. Examples which may be mentioned are: 4-Aminobenzoic acid, 3-(4'- Trimethylammonium)benzylidenebornan-2-one methylsulfate, 3,3,5-Trimethylcyclohexyl salicylate (homosalate), 2-Hydroxy-4-methoxy-benzophenone (oxybenzone), 2- Phenylbenzimidazole-5-sulfonic acid and its 27503-81-7, potassium, sodium and triethanolamine salts, 3,3'-(1 ,4-Phenylenedimethine)-bis(7,7-dimethyl- 2- oxobicyclo[2.2.1]heptane-1-methanesulfonic acid) and its salts, Polyethoxyethyl 4- bis(polyethoxy)aminobenzoate, 2-Ethylhexyl 4-dimethylaminobenzoate, 2-Ethylhexyl salicylate, 2-lsoamyl 4-methoxycinnamate, 2-Ethylhexyl 4-methoxycinnamate, 2- Hydroxy-4-methoxy-benzophenone-5-sulfonic acid (sulisobenzone) and the sodium salt, 3-(4'-Sulfobenzylidene)bornan-2-one and salts, 3-Benzylidenebornan-2-one, 1-(4'- lsopropylphenyl)-3-phenylpropane-1 ,3- dione, 4-lsopropylbenzyl salicylate, 2,4,6- Trianiline(o-carbo-2'-ethylhexyl-1'-oxy)- 1,3,5-triazine, 3-lmidazol-4-ylacrylic acid and its ethyl ester, Ethyl 2-cyano-3,3-diphenylacrylate, 2'-Ethylhexyl 2-cyano-3,3- diphenylacrylate, Menthyl o-aminobenzoate, 5-methyl-2-(1-methylethyl)-2- aminobenzoate, Glyceryl p-aminobenzoate or: 1-glyceryl 4-aminobenzoate, 2,2'- Dihydroxy-4-methoxybenzophenone (dioxybenzone), 2-Hydroxy-4-methoxy-4- methylbenzophenone (mexenone), Triethanolamine salicylate, Dimethoxyphenylglyoxalic acid, sodium 3,4-dimethoxyphenylglyoxalate, 3-(4'Sulfobenzylidene)bornan-2-one and its salt, 4-tert-Butyl-4'-methoxydibenzoylmethane, 2,2',4,4'-Tetrahydroxybenzophenone, 2,2'-Methylenebis[6(2H-benzotriazol-2-yl)-4-(1 , 1 ,3,3-tetramethylbutyl)phenol], 2,2'-(1 ,4- Phenylene)-bis-1 H-benzimidazole-4,6- disulfonic acid, Na salt, 2,4-bis[4-(2- Ethylhexyloxy)-2-hydroxy]phenyl-6- (4-methoxyphenyl)-(1 ,3,5)-triazine, 3-(4-

Methylbenzylidene)camphor, Polyethoxyethyl, 4-bis(polyethoxy)paraaminobenzoate, 2,4- Dihydroxybenzophenone, 2,2'-Dihydroxy-4,4'-dimethoxybenzophenone-5,5'- disodium sulphonate, cinnamic derivatives; dibenzoylmethane derivatives; salicylic derivatives, camphor derivatives; triazine derivatives (such as those described in US. 4,367,390, EP- 863,145, EP-517,104, EP-570,838, EP-796,851, EP-775,698, EP-878,469 and EP- 933,376); benzophenone derivatives; .beta.,.beta.'-diphenylacrylate derivatives, benzimidazole derivatives; bisbenzoazolyl derivatives (such as those described in EP-A- 0,669,323 and US. 2,463,264); bishydroxyphenolbenzotriazol derivatives such as those described in US. 5,237,071 and 5,166,355, GB-A-2,303,549, DE-197,26, 184 and EP-A- 893,119); p-aminobenzoic acid derivatives; screening hydrocarbon polymers and screening silicones (such as those described in WO-93/04665), p-aminobenzoic acid, oxyethylenated p-aminobenzoate (25 mol), 2-ethylhexyl p-dimethylaminobenzoate, N- oxypropylenated ethyl p-aminobenzoate, glyceryl p-aminobenzoate, homomenthyl salicylate, 2-ethylhexyl salicylate, triethanolamine salicylate, 4-isopropylbenzyl salicylate, menthyl anthranilate, 2-ethylhexyl-2-cyano-3,3'-diphenylacrylate, ethyl 2-cyano-3,3'- diphenylacrylate, 2-phenylbenzimidazole-5-sulfonic acid and salts thereof, 3-(4'- trimethylammonium)benzylidenebornan-2-one methyl sulfate, 2-hydroxy-4- methoxybenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfonate, 2,4- dihydroxybenzophenone, 2,2',4,4'-tetrahydroxybenzophenone, 2,2'-dihydroxy-4,4'- dimethoxybenzophenone, 2-hydroxy-4-n-octoxybenzophenone, 2-hydroxy-4-methoxy-4'- methylbenzophenone, .alpha.-(2-oxoborn-3-ylidene)-tolyl-4-sulfonic acid and salts thereof, 3-(4'-sulfo)benzylideneboman-2-one and salts thereof, 3-(4'-methylbenzylidene)- d, 1 -camphor, 3-benzylidene-d, 1 -camphor, 1 ,4-benzene[di(3-methylidene-10- camphorsulfonic)] acid and salts thereof, urocanic acid, 2,4-bis{[4-2-ethylhexyloxy)]-2- hydroxy]phenyl}-6-(4-methoxyphenyl)-1 ,3,5-tr iazine, polymers of N-(2 and 4)-[(2- oxoborn-3-ylidene)methyl]benzyl]acrylamide, 1 ,4-bisbenzimidazolylphenylene-3,3',5,5'- tetrasulfonic acid and salts thereof, polyorganosiloxanes containing a benzalmalonate functional group, polyorganosiloxanes containing a benzotriazole functional group, such as Drometrizole Trisiloxane, 2,2'-methylenebis[6-(2H-benzotriazol-2-yl)-4-(1, 1 ,3,3- tetramethylbutyl)phen ol] in a soluble form marketed under the trademark MIXXIM BB/100 by FAIRMOUNT CHEMICAL, and in micronized insoluble form under the trademark TINOSORB M, 2,2'-methylenebis[6-(2H-benzotriazol-2-yl)-4-(methyl)phenol] marketed under the trademark MIXXIM BB/200 by FAIRMOUNT CHEMICAL.

Suitable Free radical quenchers are Lipophilic free-radical quenchers, such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.

The antioxidants are advantageously similar to those disclosed in US 6419938 and are selected from the group consisting of Ascopyrone, such as Ascopyrone P Ascorbic acid, anhydrofructose, amino acids (for example glycine, histidine, tyrosine, tryptophan) and derivatives thereof, imidazoles (e.g. urocanic acid) and derivatives thereof, peptides such as D,L-carnosine, D-carnosine, L-carnosine and derivatives thereof (e.g. anserine), carotenoids, carotenes (e.g. .alpha.-carotene, .beta.-carotene, .psi.-lycopene) and derivatives thereof, chlorogenic acid and derivatives thereof, lipoic acid and derivatives thereof (e.g. dihydrolipoic acid), aurothioglucose, propylthiouracil and other thiols (e.g. thioredoxin, glutathione, cysteine, cystine, cystamine and the glycosyl, N-acetyl, methyl, ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl, .gamma.-linoleyl, cholesteryl and glyceryl esters thereof) and salts, thereof, dilauryl thiodipropionate, distearyl thiodipropionate, thiodipropionic acid and derivatives thereof (esters, ethers, peptides, lipids, nucleotides, nucleosides and salts) and sulphoximine compounds (e.g. buthionine sulphoximines, homocysteine sulphoximine, buthionine sulphones, penta-, hexa-, heptathionine sulphoximine) in very small tolerated doses (e.g. pmol to .mu.mol/kg), also (metal) chelating agents (e.g. .alpha.-hydroxy fatty acids, palmitic acid, phytic acid, lactoferrin), .alpha.-hydroxy acids (e.g. citric acid, lactic acid, malic acid), humic acid, bile acid, bile extracts, bilirubin, biliverdin, EDTA, EGTA and derivatives thereof, unsaturated fatty acids and derivatives thereof (e.g. .gamma.-linolenic acid, linoleic acid, oleic acid), folic acid and derivatives thereof, furfurylidenesorbitol and derivatives thereof, ubiquinone and ubiquinol and derivatives thereof, vitamin C and derivatives (e.g. ascorbyl palmitate, Mg ascorbyl phosphate, ascorbyl acetate), tocopherols and derivatives (e.g. vitamin E acetate), vitamin A and derivatives (vitamin A palmitate) and coniferyl benzoate of benzoin, rutinic acid and derivatives thereof, .alpha.-glycosylrutin, fenulic acid, furfurylideneglucitol, carnosine, butyl hydroxytoluene, butyl hydroxyanisole, nordihydroguaiac resin acid, nordihydroguaiaretic acid, trihydroxybutyrophenone, uric acid and derivatives thereof, mannose and derivatives thereof, zinc and derivatives thereof (e.g. ZnO, ZnSO.sub.4), selenium and derivatives thereof (e.g. selenium methionine), stilbenes and derivatives thereof (e.g. stilbene oxide, trans-stilbene oxide) and the derivatives (salts, esters, ethers, sugars, nucleotides, nucleosides, peptides and lipids) of said active ingredients which are suitable according to the invention.

In a preferred aspect the invention relates to a composition comprising microthecin or a derivative thereof formulated with an anti-oxidant.

In a preferred aspect the invention relates to a composition comprising microthecin or a derivative thereof formulated with a UV protectant.

In a preferred aspect the invention relates to a composition comprising microthecin or a derivative thereof formulated and a metal chelator or a mixture of metal chelators, such as EDTA, and EGTA. The emulsifier may selected from distilled monoglycerides, acetic acid esters of mono and diglycerides, citric acid esters of mono and diglycerides, lactic acid esters of mono and diglycerides, mono and diglycerides, polyglycerol esters of fatty acids, polyglycerol polyricinoleate, propylene glycol esters of fatty acids, sorbitan monostearates, sorbitan tristearates, sodium stearoyl lactylates, calcium stearoyl lactylates, diacetyl tartaric acid esters of mono- and diglycerides, Tween 20, 40, 60 and 80, Triton X-100, SDS, and octy- glucoside.

Without being bound by theory it is believed that the presence of an emulsifier will stabilize microthecin or a derivative thereof in solution. The model based on microthecin is as below:

The hydrophobic part of microthecin molecule interacts with lipid tails while its hydrophilic part interacts with the lipid head.

This may have a further advantage in particular applications. For example if the composition is applied to hydrophobic materials such as plant materials and in particular leaves. In these aspects the emulsifier will reduce the surface tension so the microthecin solution will not form drops on the material e.g. leaves, instead forming an evenly distributed thin layer of solution the material surface.

In one aspect the auxiliary component may be an agent that increases the hydrophobicity of the composition. Suitable agents include isopropoanol, 1-butanol, and ethanol. Polar Solvent - in a preferred embodiment microthecin or derivative thereof is formulated with one or more polar solvents, preferably one or more polar organic solvents. Typically polar solvents include alcohols and in particular ethanol, methanol, and glycerol.

Polar solvents include acetic acid and alcohols. The alcohols may contain one or more OH group and may be selected, in particular, from the group including methanol, ethanol, 1-propanol, 2- propanol, 1-butanol, 2-methyl-1-propanol, 2-butanol, 2-methyl-2-propanol, ethylene glycol, 1,3 propandiol, 1 ,2-propandiol, glycerin, 1,2-butandiol,.

The polar aprotic solvent preferably consist of dimethyl sulfoxide (DMSO), dimethylformamide(DMF), dimethylacetamide (DMA), aceto nitril, N-methyl pyrrilidone, diethylene glycol, dimethyl ether, tetra ethylene glycol dimethyl ether, sulfolane, 1,3- dimethyl-2-imidazolidinon, 1,3-dimethyl tetra hydro-2(1H-) pyrimidonon, 2-methoxy-1- methyl ethyl acetate or propylene carbonate.

In a preferred aspect the invention relates to a composition comprising microthecin or a derivative thereof formulated with a polar solvent, preferably an alcohol.

pH Alteration -in a preferred embodiment microthecin or derivative thereof is formulated in a composition which has a pH of at least 7.

Methods of stabilising pharmaceutical formulations are well known in the art and include

In order to produce soluble products, it may be possible prepare prodrugs by attaching the compound to solubilizing moieties such as succinate and amino acids, or to solubilize the compound by microencapsulation or by conjugation to a water soluble polymer such as a polyglutamic acid or a polyaspartic acid, for example, or to a water soluble metal chelator. In those embodiments in which the compound is conjugated to a water soluble metal chelator, the composition may further comprise a chelated metal ion. The chelated metal ion may be an ionic form of any one of aluminium, boron, calcium, chromium, cobalt, copper, dysprosium, erbium, europium, gadolinium, gallium, germanium, holmium, indium, iridium, iron, magnesium, manganese, nickel, platinum, rhenium, rubidium, ruthenium, samarium, sodium, technetium, thallium, tin, yttrium or zinc. Preferred water soluble chelators to be used may include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1 ,4,7, 10-tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA), tetraazacyclotetra- decane-N,N^,,N",N"'-tetraacetic acid (TETA), hydroxyethylidene diphosphonate (HEDP), dimercaptosuccinic acid (DMSA), diethylenetriaminetetramethylenephosphonic acid (DTTP) and 1-(p-aminobenzyl)-DTPA, 1,6-diamino hexane-N,N,N',N'-tetraacetic acid, DPDP, and ethylenebis (oxyethylenenitrilo)-tetraacetic acid.

In certain embodiments of the present invention, the compound may be conjugated to a water soluble polymer. A degradable linkage, such as an ester, may be used to ensure that the active material is released from the polymeric carrier. Preferred polymers include, but are not limited to polyethylene glycol, poly(1-glutamic acid), poly(d-glutamic acid), poly(d1-glutamic acid), poly(1-aspartic acid), poly(d-aspartic acid), poly(d1-aspartic acid), polyethylene glycol, copolymers of the above listed polyamino acids with polyethylene glycol, polycaprolactone, polyglycolic acid and polylactic acid, as well as polyacrylic acid, poly(2-hydroxyethyl1-glutamine), carboxymethyl dextran, hyaluronic acid, human serum albumin and alginic acid, with polyethylene glycol, polyaspartic acids and polyglutamic acids being particularly preferred.

In a preferred aspect the invention relates to microthecin in a micro-encapsulated form.

In a preferred aspect the invention relates to a method of prolonging the stability/shelf life of microthecin or a derivative thereof by formulating by any of the methods above, preferably as a solid, preferably in the absence of light, preferably in the absence of UV light

PROCESS

In one aspect, the present invention provides a process for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, comprising the steps of contacting the material either sequentially or simultaneously with (i) an antimicrobial compound in a stabilised form (a "stabilised compound"), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to the antimicrobial compound; and (b) (I) a primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and a secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof.

In one aspect the constituents to be contacted with the material are contacted sequentially with the material.

In one aspect the constituents to be contacted with the material are contacted simultaneously with the material. For example the constituents to be contacted may be formulated in a composition prior to contact.

When more than two constituents are to be contacted with the material, some may be contacted simultaneously and some sequentially.

FURTHER COMPONENTS

The antimicrobial of the present invention may comprise additional components which may be required for the application to which the antimicrobial is to utilised.

For example, in one preferred aspect the antimicrobial material further comprises an agriculturally acceptable carrier, excipient or diluent.

For example, in one preferred aspect the antimicrobial material further comprises an edible carrier, excipient or diluent.

TREATED MATERIAL

The compounds of the invention are particularly advantageous both food and such non-food applications due to their biodegradability and instability. The antimicrobial is unstable in the environment so unlike many prior antimicrobials it will be readily biodegradable. This is an important advantage in many industrial and agricultural settings where large quantities of material may intentionally or accidentally be release in the environment. Moreover, since many of the compounds of formula I may be derived from starch, they are ideally suited for cosmetic, medical and surface cleaning purposes in view of their low toxicity.

Stabilised formulations of microthecin can be used in the following applications

• anti-fungal - pharmaceutical/therapeutic applications, for example for the treatment/prevention of fungal pathogen infection in humans and animals, e.g. including Candida infections, (e.g. thrush), and Aspergillus fumigatus infections. • anti-bacterial - pharmaceutical/therapeutic applications, for example for the treatment/prevention of bacterial pathogen infection in humans and animals, including e.g. E.coli & Staphylococcus aureus.

• anti-parasitic -pharmaceutical/therapeutic applications, for example for the treatment/prevention of parasitic infections in humans/animals, for example Malaria, Eimeria, Cryptosporidium

• anti-microbial - plant protectant

• anti-fungal - plant protectant

• domestic/industrial detergent/fungicide

Agrochemical Applications

In one preferred aspect the material is a seed, a plant or a part thereof. In one aspect the plant or part thereof is a foodstuff.

In one preferred aspect the material is a seed.

The present invention is particularly advantageous as it allows use of direct application of microthecin or derivatives thereof in agricultural or agrochemical application.

An additional advantage of the present invention is that the compositions of the invention have been found to be less phytotoxic to plant material that some previously used antimicrobials. Thus in a preferred aspect the present invention relates to compositions for preventing and/or inhibiting the growth of, and/or killing phytopathogenic fungi of crop plants and to a method of combating phytopathogenic diseases on crop plants.

Thus in a preferred aspect the present invention relates to compositions for preventing and/or inhibiting the growth of, and/or killing phytopathogenic bacteria of crop plants, especially phytopathogenic bacteria, and to a method of combating phytopathogenic diseases on crop plants.

In one aspect microthecin or a derivative thereof may be used in specific direct applications, for example to seed coat or foliar application. In one preferred aspect microthecin or a derivative thereof is used as other than a systemic fungicide.

Preferably the microthecin is applied to the plant or part of a plant, in a stabilising formulation, such formulations include application as a dry powder, in a microcapsulated form, or in a polar solvent. Typical formulations are discussed herein.

The active ingredient mixtures according to the invention have very advantageous properties in the protection of plants against the outbreak of disease.

With the present active ingredient mixtures, the micro-organisms appearing on plants or plant parts (fruits, flowers, foliage, stems, tubers, roots) of different useful plants can be stopped or destroyed, whereby plant parts growing later also remain free from such micro-organisms. They may also be used as dressings for the treatment of plant propagation material, especially seeds (fruits, tubers, seed grain) and plant cuttings (e.g. rice) to provide protection from fungal and bacterial infections, and against phytopathogenic fungi and bacteria appearing in the soil. The active ingredient mixtures according to the invention are notable for their especially good plant tolerance and their environmental acceptability.

Target crops for the indicated fields disclosed herein are, within the context of this invention, e.g. the following species of plants: cereals (wheat, barley, rye, oats, rice, sorghum and the like); beets: (sugar beet and fodder beet); pip, stone and berry fruit: (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries); leguminous plants: (beans, lentils, peas, soybeans); oil plants: (rape, mustard, poppy, olives, sunflowers, coconut, castor oil plant, cacao, peanut); cucumber plants (gourds, cucumbers, melons); fibre plants: (cotton, flax, hemp, jute); citrus fruits: (oranges, lemons, grapefruit, mandarins); vegetable varieties (spinach, lettuce, asparagus, cabbage varieties, carrots, onions, tomatoes, potatoes, peppers); Lauraceae: (avocado, cinnamon, camphor) or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, grapevines, hops, banana and natural rubber plants, as well as ornamental plants (flowers, shrubs, deciduous trees and coniferous trees such as conifers). This is not intended to be a limiting list of plants.

The active ingredient are normally employed in the form of compositions. The active ingredients may be applied to the area or plants to be treated either simultaneously or in succession on the same day, together with further optional fillers, surfactants or other application-enhancing additives which are customary in formulation techniques.

Appropriate fillers and additives may be solid or liquid, and correspond to the substances which are efficient in formulation techniques, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, adhesion agents, thickeners, binding agents or fertilisers.

A preferred method of applying an active ingredient is application to the parts of the plant above ground, especially the leaf system (leaf application). The number of applications and application rate depend on the biological and climatic living conditions of the instigator. However, the active ingredient may also reach the plant through the root system (systemic action) via the soil or the water, whereby the plant site is drenched with a liquid formulation (e.g. in rice cultivation) or the substances are incorporated into the soil in solid form, e.g. in the form of granules (soil application). The compounds may also be applied to seed grain in a seed treatment (coating), whereby the tubers or grains are either immersed in succession with a liquid formulation of each active ingredient, or are coated with an already combined, wet or dry formulation. In addition, in particular cases, further types of plant application are possible, e.g. the targeted treatment of buds or syncarpy. Here, the compounds are used in unchanged form or preferably together with excipients which are usual in formulation techniques, and they are processed in known manner e.g. into emulsion concentrates, coatable pastes, directly sprayable or diluable solutions, diluted emulsions, wettable powders, soluble powders, dusts, granules, or by encapsulation into, for example, polymeric substances. The application methods, such as spraying, misting, dusting, dispersing, coating or drenching, are selected according to the targeted aims and the given conditions, in the same way as for selection of the type of agent. Favourable application rates for the active ingredient mixture are in general 50 g to 2 kg active substance per ha, especially 100 g to 700 g active substance per ha, most preferably 75 g to 450 g active substance per ha. For the treatment of seeds, the application rates are 0.5-600 g, preferably 5-80 g active substance per 100 kg seeds.

The formulations are produced in known manner, e.g. by intimately mixing and/or grinding the active ingredients with diluting agents, e.g. solvents, solid fillers, and optionally surface-active compounds (surfactants).

The solvents in question may be: aromatic hydrocarbons, preferably fractions C8 to C12, such as xylene mixtures or substituted naphthalenes, phthalic acid esters such as dibutyl or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols, as well as the ethers and esters thereof, such as ethanol, ethylene glycol, ethylene glycol monomethyl ether or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulphoxide or dimethyl formamide, as well as optionally epoxidated vegetable oils such as epoxidated coconut oil or soybean oil; or water.

The solid fillers, e.g. for dusting agents and dispersible powders, are normally natural mineral powder, such as calcitol, talcum, kaolin, montmorillonite or attapulgite. To improve the physical properties, highly disperse silicic acid or highly disperse, absorbent polymerisates may also be added. The granulated, adsorptive granulate fillers may be porous types such as pumice stone, brick fragments, sepiolite or bentonite, and non- sorptive fillers are e.g. calcitol or sand.

Furthermore, a number of pregranulated materials of inorganic or organic nature may also be used, especially dolomite or pulverised plant residues.

Depending on the type of active ingredients to be formulated, the surface-active compounds may be non-ionic, cationic and/or anionic surfactants with good emulsifying, dispersing and wetting properties. By surfactants are also understood surfactant mixtures. Especially advantageous, application-enhancing admixtures are also natural or synthetic phospholipids from the series cephalins and lecithins, e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, lysollecithin.

The agrochemical compositions normally contain 0.1 to 99%, especially 0.1 to 95% active ingredients, 99.9 to 1%, especially 99.9 to 5% of a solid or liquid additive and 0 to 25%, especially 0.1 to 25% of a surfactant.

While concentrated formulations are preferred as commercial products, the final user normally uses diluted formulations.

The following examples serve to illustrate the invention, whereby "active ingredient" indicates microthecin or a derivative thereof, optionally combined with other active ingredients

Microthecin or derivatives thereof may be mixed with other agrochemical grade fungicides, or other agrochemicals such as herbicides, pesticides, fertilisers etc.

Other formulation technologies suitable for carrying out the invention are:

As discussed in WO02/074080 the present agriculturally acceptable compounds may prepared as a solid formulation. A solid formulation of an agrochemical is prepared by forming a melt containing at least one agrochemical and at least one thermoplastic binder having a melting point or glass temperature of greater than 35°C, briquetting the melt by dividing it into drops in a first step and solidifying these drops by cooling in a second step, characterised in that the melt additionally comprises a liquid non-volatile solvent for the agrochemical.

As discussed in JP2001089304 the present agriculturally acceptable compounds may prepared as a granular agrochemical formulation which can rapidly be collapsed and dispersed or dissolved in water, when charged in the water. This granular agrochemical formulation contains a low substituted hydroxypropylcellulose. The compositions of the present invention may also be used in the area of protecting technical material against attack of fungi. Technical areas include wood, paper, leather, constructions, cooling and heating systems, ventilation and air conditioning systems, and the like. The combinations according the present invention can prevent the disadvantageous effects such as decay, discoloration or mould.

The antimicrobial material may be contacted at time of planting, either by spraying, dipping in the composition or by a powder treatment. Alternatively the material may be applied at any time subsequent to harvest as a preservative.

Food and Feed application:

In one preferred aspect the material is a foodstuff.

In one preferred aspect the material is an animal feed.

By the term "foodstuffs" it is meant food and feed, raw and processed plant material and non plant material.

Modem food animal production depends on use of large amounts of antibiotics for disease control. This provides favourable conditions for the spread and persistence of antimicrobial- resistant zoonotic bacteria such as Campylobacter and E. coli 0157. The occurrence of antimicrobial resistance to antimicrobials used in human therapy is increasing in human pathogenic Campylobacter and E. coli from animals. There is an urgent need to implement strategies for prudent use of antibiotics in food animal production to prevent further increases in the occurrence of antimicrobial resistance in food-borne human pathogenic bacteria such as Campylobacter and E. coli (Aarestrup FM, Wegener HC Microbes Infect 1999 Jul;1(8):639-44). Although microthecin can be employed as a therapeutic to prevent or treat microbial infections, it has not been granted regulatory approval and is not regarded as a therapeutic antibiotic. Due to its comparatively cheap method of production, microthecin is useful for inclusion in the manufacture, processing and/or preparation of food for inhibition or prevention of pathogenic micro-organisms in foodstuffs.

The use of anti-microbials in food and feed is highly regulated, and microbial contamination of food in a non-sterile environment is, over time, almost inevitable. A specific advantage of the present invention is that a naturally occurring compound with low toxicity and low persistence can be used in the preparation of a foodstuff. In a specific embodiment the anti-microbial compound of the invention is used during the preparation of a foodstuff, but is not-present in the foodstuff immediately before consumption. By the term not-present, it should be understood that the functionality /anti-microbial activity is deemed not significant.

The present formulations of microthecin or derivatives thereof can be included in the food stuff itself to prevent or reduce pathogen contamination, and/or as a disinfectant for use in decontaminating premises for food production, and including slaughter houses, milk and dairy production facilities, other food production and processing facilities, commercial and domestic kitchens. Microthecin can also be used as a disinfectant for personal use, particularly for people who prepare food stuffs, thereby preventing or reducing bacterial contamination.

Microthecin in solid, granulated, powdered form or as a stabilised solution can be used to prevent fungal contamination of plant materials and products during storage. It is considered that although the following examples refer to the inclusion of microthecin per se, in some applications it may be preferable to produce microthecin in situ by the use of including enzymes capable of synthesising microthecin, such as the pyranose dehydratase enzyme or the combination of glucan lyase and pyranose-2-oxidase as disclosed in British Patent Application No. 0226159.2, in such situations a starch or dextran substrate can be supplied as part of the formulation.

The plant materials can be seed or grain or other propagative plant tissues (e.g. tubers) being stored for future use as seed (sowing)

Alternatively the plant materials can be seed, grain or other plant materials, or plant derived materials for future use as animal feed.

The formulations containing microthecin according to the invention are suitable, for example, for preserving grass, agricultural crop plants and/or mixed livestock nutrition and the materials used for producing them, such as barley, wheat, rye, oats, corn, rice, oilseed rape, legumes, sunflower seeds, soybeans, sugar beet and sugar cane and residues thereof, hay, straw, peanuts, fishmeal, meat or bonemeal. The formulations containing microthecin according to the invention are used in livestock nutrition, especially as additive to feed for pigs, piglets and poultry, such as chickens and turkeys. They are particularly suitable for preserving animal feed, preferably liquid feed and compound feed, from unwanted microbial decomposition.

Another application of the formulations containing microthecin according to the invention is in the production of silage (ensiling). In silage, the required lactic acid fermentation is frequently accompanied by unwanted microbial decomposition, especially by moulds and putrefactive bacteria. The formulations containing microthecin according to the invention can be added to the animal feed to prevent this unwanted putrefaction.

The formulations containing microthecin according to the invention can be added to the feedstuffs or feedstuff mixtures in a manner known per se. The addition to the feedstuff can take place immediately after harvesting or production in the form of the solutions according to the invention, or in the form of particles or porous carriers to which the solutions have initially been applied. These particles or carriers are in this case mixed with the feedstuff. Examples of suitable particles or carriers are vermiculite, pumice or dried sugar beet pulp. The formulations containing microthecin according to the invention can advantageously be added via a metering device to the feedstuff while the feed is being conveyed by a screw into the silo.

Preferably, the compositions of the invention or microthecin, cortalcerone, or derivatives or isomers thereof, are used in the treatment of one or more of the following: a crop plant, a monocotyledonous crop plant, a cereal, barley, wheat, maize, Triticale, rice, oats, rye, field beans, fruit crops, vegetables, apple, pear, strawberry, pea, tomato, grape, Brassicas, tobacco, lettuce, sorghum, cotton, sugar cane, legumes, ornamentals, pot plants, turf grasses, sugar beet, celery, Crucifers, plantain, banana, grasses, agricultural crops, livestock nutritional plants, oilseed rape, sunflowers, soybean, peanuts, broccoli, cabbage, carrot, citrus, garlic, onion, pepper (Capsicum), potato, and strawberry, including the seeds thereof.

In one preferred aspect the seed or plant to which the present antimicrobial material may be applied is one of a cereal, barley, wheat, maize, Triticale, rice, oats, rye, field beans, apple, pear, strawberry, pea, tomato, grape, Brassicas, tobacco, lettuce, sorghum, cotton, sugar cane, legumes, ornamentals, pot plants, turf grasses, sugar beet, celery, Crucifers, plantain, banana, grasses, oilseed rape, sunflower, soybean, and peanut. Preferably the seed or plant material is sugar beet seeds or barley.

Seed production

In seeds production, germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds, to control seedborne diseases, and to produce seed with good germination, fairly extensive and well-defined seed production practices have been developed by seed producers, who are experienced in the art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof.

Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD@), methalaxyl (Apron@), and pirimiphos-methyl (Actellic@). If desired, these compounds are formulated together with further carriers, surfactants or application promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.

It is a further aspect of the present invention to provide new agricultural methods, such as the methods exemplified above, which are characterised by the use of the antimicrobial material according to the present invention.

The seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds. The bag, container or vessel may be designed for either short term or long term storage, or both, of the seed. Examples of a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal. Desirably the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type. In one embodiment the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed. In one example, the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering. In another embodiment water absorbent materials are placed between or adjacent to packaging material layers. In yet another embodiment the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed.

Antifungal Compositions

The present invention further provides an antifungal composition, comprising an antifungal effective amount of the antimicrobial material, and an acceptable carrier. The antifungal composition can be used for inhibiting the growth of, or killing, pathogenic fungi. These compositions can be formulated by conventional methods such as those described in, for example, Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Veriag, Munich; van Falkenberg (1972-1973) Pesticide Formulations, Second Edition, Marcel Dekker, N.Y.; and K Martens (1979) Spray Drying Handbook, Third Edition, G. Goodwin, Ltd., London. Necessary formulation aids, such as carriers, inert materials, surfactants, solvents, and other additives are also well known in the art, and are described, for example, in Watkins, Handbook of Insecticide Dust Diluents and Carriers, Second Edition, Darland Books, Caldwell, N.J., and Winnacker- Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Veriag, Munich. Using these formulations, it is also possible to prepare mixtures of the present antimicrobial material with other pesticidally active substances, fertilisers and/or growth regulators, etc., in the form of finished formulations or tank mixes.

Numerous conventional fungal antibiotics and chemical fungicides with which the present antimicrobial material can be combined are known in the art and are described in Worthington and Walker (1983) The Pesticide Manual, Seventh Edition, British Crop Protection Council. These include, for example, polyoxines, nikkomycines, carboxyamides, aromatic carbohydrates, carboxines, morpholines, inhibitors of sterol biosynthesis, and organophosphorus compounds. Other active ingredients which can be formulated in combination with the present antimicrobial material include, for example, insecticides, attractants, sterilising agents, acaricides, nematocides, and herbicides. US5,421,839 contains a comprehensive summary of the many active agents with which substances such as the present antifungal polypeptide can be formulated.

Personal Hygiene

Personal hygiene compositions are applied topically to the skin to treat skin conditions including acne, fine lines and age spots, itching and pain from insect bites, bee stings, fungi (including athletes foot and jock itch), flaking and/or scaly skin (including dandruff, seborrheic dermatitis, psoriasis and heat rash), and burns. Different compositions are presented for use as an acne treatment, a face and body wash, a der atophyte (nail fungus) treatment. Still another is intended for use in makeup, and another in lipstick. Microthecin and derivatives thereof are found to be active in these applications.

As discussed in WO99/59540 micro-organisms, present upon the skin exacerbate many minor human afflictions, such as acne, dandruff and athletes foot. Despite the considerable amount of research conducted upon these subjects, these are still common complaints. Efforts have focused upon developing agents which will kill off the microorganisms, and are suitable for application to the human body. Not only do microorganisms continuously develop resistance but many successful antimicrobials cause burning or irritation to the skin. Thus, there is an enormous need for new antimicrobials.

Many antimicrobials are harsh reagents that cause burning and irritation to human skin, and so are unsuitable for application to the human body. Microthecin and derivatives thereof are mild reagents which do not cause burning and irritation to the skin. Thus they are is suitable for incorporation into formulations designed to be applied to human skin and may be included in formulations designed to treat some of the more common human afflictions which are microbial in origin or exacerbated by subsequent microbial growth.

Microbes are commonly taken to mean any organism too small to be seen unless by microscopy. For the purposes of this invention it is further defined to include bacteria and microscopic fungi. Microscopic fungi is defined to include yeasts. The present invention provides topical antimicrobial compositions containing a cosmetically acceptable diluent or carrier, and an antimicrobially effective amount of microthecin or a derivatives thereof.

Acne is a common affliction of many people in their teenage years and sometimes beyond. As a result of puberty, teenagers often have increased levels of sebum. The initial inflammation of the follicle wall in the development of acne results from the presence of free fatty acids derived from the sebum. The normal bacterial flora in the sebaceous duct produce the enzymes responsible for splitting triglycerides in the sebum and releasing these fatty acids. The main micro-organisms in the sebaceous duct are Propionibacterium acnes and one or two species of Staphylococcus aureus. Therefore in the presence of excess sebum these micro-organisms result in the development of acne.

Most approaches to a cure for acne focus on trying to absorb the excess sebum or to act on the bacteria present.

Microthecin and derivatives thereof inhibit the growth of Staphylococcus aureus and Propionibacterium acnes. By inhibiting their growth, less of the sebum is split into the free fatty acids which act to inflame the follicle wall. It may be used as a bacteriostatic agent. So microthecin and derivatives thereof may be used to treat acne. Microthecin and derivatives thereof may be used in the preparation of a medicament for the treatment of acne.

Athletes foot is the loose term applied to a skin eruption on the foot, usually between the toes. It is a cutaneous fungal infection, most commonly caused by Tinea pedis, Tricophyton rubrum, Tricophyton mentagrophytes or Epidermophyton floccosum. In addition to the effect of the micro-organisms, other factors such as wetness or an increase in temperature can contribute to disease development by providing ideal conditions for the initiation and growth of fungal infections.

The condition is commonly treated by careful foot hygiene, removing the damp conditions helpful to fungal growth and by the use of antifungal agents. Microthecin and derivatives thereof has been found to be an antifungal agent. This invention teaches microthecin and derivatives thereof as a potent antifungal agent and the use of microthecin and derivatives thereof as a treatment for athletes foot. Microthecin and derivatives thereof may be used in the preparation of a medicament for the treatment of athletes foot. Preferably the microthecin is applied externally, typically in a dry powder composition, cream or spray.

Dandruff is a common human condition characterised by the excess scaling of scalp tissue. Pityrosporum ovale is a yeast whose growth is accelerated as a result of the dandruff condition, resulting in secondary infection and a worsening of the condition.

It has been found that microthecin and derivatives thereof is a potent agent against microbial growth particularly at inhibiting the growth of P. ovale and S. aureus. So microthecin and derivatives thereof may be used to prevent microbial growth occurring during dandruff conditions. Microthecin and derivatives thereof may be used in the preparation of a medicament for the treatment of dandruff.

Nappy rash, the skin eruption which tends to occur on the buttocks of infants is due to infrequent changing of soiled nappies. The condition is often worsened by secondary infection with Candida albicans. To prevent this condition, nappies are changed regularly and care is taken to ensure that the baby's bottom is dried properly. The buttocks are commonly treated with an agent to absorb any surplus liquid.

Microthecin and derivatives thereof has been found to be highly efficacious when used in the treatment of nappy rash. It is a potent antifungal, and by stopping the growth of Candida albicans it prevents the associated secondary infection.

Personal Hygiene Formulations

The formulation can be a liquid, a lotion, a cream, a dry powder, or a suspension, and can be used as a spray, wash, rinse, bandage ointment, or lotion, Embodiments of the composition of the invention can be liquids, gels, ointments, salves, lotions, powders, dusting mixtures and bandages or compresses

The compositions can be in a solid form, such as a powder, are dusting compositions comprising microthecin or a derivative thereof, Suitable carriers of the dusting composition can include talc, starch, modified starch, boric acid, zinc oxide, kaolin, light calcined magnesia, potassium alum, or combinations thereof..

In one aspect, the present invention provides methods for treatment of poorly healing wounds and/or for resolution of skin infections. Such methods are taught in

US2001046526. In some embodiments, the methods comprise administering, to an affected area, an effective amount of microthecin. In some embodiments, the methods further comprise administering microthecin and an antibacterial agent to an affected area. In some embodiments, the methods further comprise topically administering to an affected area an effective amount of microthecin or derivative thereof together with an antibacterial agent optionally dispersed in a lipid-based carrier or other pharmaceutically acceptable carrier. The foregoing active ingredients can be administered in any sequence (e.g., sequentially) or substantially at the same time period (e.g., contemporaneously).

In one aspect, the present invention provides a treatment that can be applied as a prophylactic treatment to prevent the onset or spread of a fungal infection.

In one aspect, the present invention provides a treatment for skin wounds, whereby healing is improved. The treatment comprises application of a composition comprising microthecin or derivative thereof to the wound.

In one aspect, the present invention provides a method for skin softening and rejuvenation, comprising applying a composition comprising microthecin or derivative thereof, whereby the skin appears softer.

In one aspect, the present invention provides compositions and methods applicable to human and/or veterinary use.

Specific embodiments of the invention may be directed to one, some, or all of the above- indicated aspects, as well as other aspects ascertainable from this disclosure, and may encompass one, some, or all of the above- and below-indicated embodiments, as well as other embodiments ascertainable from this disclosure. Lipid-Based Carrier-Any carrier, preferably a pharmacologically acceptable carrier, comprising lipid or fatty components, especially those having a hydrophobic moiety and a hydrophilic moiety. The carrier optionally enables the transdermal transport of an active ingredient from the surface of the skin to the regions of the body below the skin. Once below the skin, the active ingredient(s) may remain localised in or around the region(s) to which the combination or composition of interest, which is partially comprised of the lipid- based carrier, has been applied. Alternatively, the active ingredient(s) may become available systemically. In some embodiments, lipids include those that form membranes, bilayers, vesicles, liposomes and the like, particularly biological membranes. Examples of such membrane-forming lipids include but are not limited to phospholipids, glycolipids and cholesterol-type lipids. Specific lipids include phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl inositol and the like.

Biocompatible Organic Solvent-An organic solvent, for example, comprising at least one ester of fatty acids. The fatty acids contain long chain saturated or unsaturated aliphatic groups comprising about 8 to about 50 carbon atoms. In some embodiments the long- chain groups comprise about 12 to about 30 carbon atoms. In other embodiments the long-chain groups comprise about 14 to about 24 carbon atoms. In yet other embodiments the long-chain groups comprise about 16 to about 18 carbon atoms. Examples of fatty acids include lauric, myristic, palmitic, stearic, arachidic, behenic, lignoceric, palmitoleic, oleic, linoleic, linolenic, arachidonic acid and the like. The alcohol portion of the ester group is generally a linear or branched lower alkyl group comprising about 1 to about 8 carbon atoms. Examples of the alcohol portion include methanol, ethanol, propanol, isopropanol, butanol, isobutanol, sec-butanol, tert-butanol, pentanol, isopentanol, hexanol, heptanol, octanol, isooctanol, cyclooctanol and the like. Accordingly, esters of fatty acids suitable for use as, or as part of, biocompatible organic solvents include, but are not limited to, ethyl palmitate, ethyl myristate, ethyl oleate, isopropyl palmitate, isopropyl myristate, isopropyl oleate, and the like. Other biocompatible organic solvents include, but are not limited to, isooctane and cyclooctane.

Hydroxyl Group Containing Aliphatic Organic Solvent-An organic solvent containing a hydroxyl group, including but not limited to those described in the preceding paragraph relating to the "alcohol portion of the ester group." Surfactant-Any compound or surface active agent that reduces surface tension when dissolved in water or water solutions, or which reduces interfacial tension between two liquids, or between a liquid and a solid. Surfactants suitable for use in the present invention include ionic and non-ionic detergents, dispersing agents, wetting agents, emulsifiers and the like. Examples of surfactants include but are not limited to the sodium salt of an N-(alkylsulfonyl)glycine (Emulsifier STH), the salts of linear alkyl sulfonates (LAS), the salts of alkyl benzene sulfonates (ABS), the sodium salt of dodecylsulfate (SDS), a nonionic series of 28 related difunctional block-polymers terminating in primary hydroxyl groups with molecular weights ranging from about 1,000 to over about 15,000, polyoxyalkylene derivatives of propylene glycol (PLURONIC), and the like. The surfactant used in the present invention is preferably pharmaceutically acceptable and biodegradable. In some embodiments, the surfactant is a polyol, a polyoxyalkylene, a fatty ester, a fatty acid or salts thereof. In some embodiments, the polyoxyalkylene comprises a block polymer, for example, of poly(oxyethylene-co- oxypropylene-co-oxyethylene).

An ingredient in compositions intended for topical application can be a material that assists in causing the active ingredients to pass through the dermal layer to permit the subcutaneous attack of the condition sought to be ameliorated. Such dermal layer penetration assistants include lipids and alcohols, including water-soluble alcohols, and other mutual solvents that cause the active ingredients to have increased dermal penetrability. Examples of solvents include dimethyl sulfoxide (DMSO) and ethanol. Ethanol, for example, is readily available, inexpensive and pharmaceutically acceptable.

Another constituent of topically applicable compositions can be an emollient. Isopropyl palmitate is one example of such emollient materials. Other such materials are well known in this art and will be apparent to those of ordinary skill in the art. Surfactants are common ingredients in cremes and ointments for transdermal delivery of medicaments. Non-ionic, cationic and anionic surfactants are all considered suitable for use in the preparation of the topically applied composition of this invention. One example of a suitable surfactant is a commercially available material known by the name PLURONIC.

In an embodiment of the invention, the compositions may be formulated as a gel. Such gels may be formulated in a manner known to those skilled in the art, and may include, but not limited to: a) antibacterials such as dichlorobenzyl alcohol, triclosan, chlorhexidine digluconate and salicylic acid; b) oil absorbers such as silica; c) alcohols such as denatured ethanol, isopropyl alcohol; d) humectants such as panthenol, butylene glycol, glycerin and propylene glycol; e) preservatives such as methyldibromo glutaronitrile, phenoxyethanol, magnesium chloride, magnesium nitrate, methylchloroisothiazolinone, methylisothiazolinone or any paraben, for example such as methylparaben, ethyl paraben and propyl paraben; solubilisers such as polysorbate 20, polyethylene glycol-40 hydrogenated castor oil; g) gelling agents such as xanthan gum, hydroxyethylcellulose, sodium magnesium silicate ; h) emollients such as glycerin, propylene glycol and butylene glycol.

In a further embodiment of the invention, the antimicrobial formulation may be a skin wash, such as a cleanser, moisturiser, face wash, lotion, stick or cream.

The composition may be formulated in a manner known to those skilled in the art.

Such compositions may include, though not limited to: a) alcohols such as propanol, stearyl alcohol, denatured ethanol. b) emulsifiers such as steareth-2, glyceryl stearate, hydrogenated vegetable glycerides, steareth-21, ceteth-20, cetyl alcohol, cetearyl alcohol, stearic acid, paraffin, stearyl alcohol, polawax, tribehenin, ceteareth-7, ceteth-5. c) emollients such as Polypropylene Glycol 5-ceteth-20, methyl gluceth-10, Dicaprytyl maleate, cetearyl isononanoate, silicones, paraffinum liquidum and octyl palmitate petrolatum, dioctyl maleate, isohexadecane, cetearyl octanoate and isopropyl myristate. d) solubilisers such as polysorbate 80, polysorbate 20, polyethylene glycol-40 hydrogenated castor oil, any polysorbate. e) antibacterials such as triclosan, chlorhexidine digluconate, salicylic acid and dichlorobenzyl alcohol, f) thickeners such as hydroxyethylcellulose, xanthan gum, sodium magnesium silicate, magnesium aluminium silicate and cellulose, g) detergents such as sodium laureth sulfate, ammonium lauryl sulfate, magnesium lauryl sulfate, disodium undecylenamido MEA- sulfosuccinate. h) preservatives such as phenoxyethanol, 2-bromo-2-nitropropane-1 ,3- diol, methyldibromo glutaronitrile, imidazolidinyl urea, magnesium chloride, magnesium nitrate, methylchloroisothiazolinone, methylisothiazolinone, or any paraben, for example such as propyl paraben, butyl paraben, ethyl paraben, methyl paraben. i) absorbents such as hydrated silica, clays, talcs, j) antioxidants such as butylated hydroxytoluene or butylated hydroxyacetone. k) moisturisers such as butylene glycol, propylene glycol, sorbitol and glycerin, panthenol, sodium hyaluronate, sodium PCA. In another embodiment of the invention the composition may be formulated as a shampoo. Such a composition may be formulated in a manner known to those skilled in the art, and may include, but not limited to: a) surfactants such as cocamidopropyl betaine and sodium laureth sulphate; b) thickeners such as xanthan gum, hydroxyethylcellulose, laureth 3, sodium chloride, polyethylene glycol-55 propylene glycol oleate and polyethylene glycol-40 hydrogenated castor oil; c) pearlising agents such as formaldehyde, stearic acid, cocamide MEA, glycol distearate glycol stearate and methyldibromo glutaronitrile; d) solubilisers such as laureth 3, polyethylene glycol-40 hydrogenated castor oil, polyethylene glycol-55 propylene glycol oleate and propylene glycol; e) conditioners such as polyquaternium-39, polyquaternium-7, poly quaternium-10 and hydroxypropyl guar hydroxypropylthrimonium chloride;

Oral Health

In one preferred aspect the antimicrobial material may be utilized for preventing and/or inhibiting caries (tooth decay). A suitable toothpaste base formulation into which the present material may be incorporated may be found in WO 03/037906. The formulation is recited below

Ingredients Amount (%)

Glycerine 22.0

Dicalcium phosphate 49.0

Sodium lauryl sulfate 2.0 Sodium saccharin 0.2

Sodium monofluorophosphate 0.75

Tetrasodium pyrophosphate 0.25

Thickening agent 1.0

Colour and flavour to suit Demineralised water to 100.0

ORGANISMS

In one preferred aspect the antimicrobial material may be utilised for preventing and/or inhibiting the growth of, and/or killing a plant pathogen selected from downey mildew, powdery mildew, Mycosphaerella, Paracercospora, Ascomycetes, Leptosphaeria, Phoma, Xanthomonas, Pseudomonas, Fusarium, Rhizoctonia, Pythium, Phytophthora, Thielaviopsis, Aspergillus, Alternaria; Ascochyta; Botrytis; Cercospora; Colletothchum; Diplodia; Erysiphe; Gaeumanomyces; Helminthosporium; Macrophomina; Nectria; Peronospora; Phoma; Phymatotrichum; Plasmopara; Podosphaera; Puccinia; Puthium; Pyrenophora; Pyricularia; Scerotium; Sckerotinia; Septoria; Uncinula; Venturia; Verticillium or Penicillium,

In one preferred aspect the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a plant pathogen selected from the following genera: Mycosphaerella, Paracercospora, Ascomycetes, Leptosphaeria, Phoma, Xanthomonas,

Pseudomonas, Fusarium, Rhizoctonia, Pythium, Phytophthora, Thielaviopsis,

Aspergillus, Alternaria; Ascochyta; Botrytis; Cercospora; Colletothchum; Diplodia;

Erysiphe; Gaeumanomyces; Helminthosporium; Macrophomina; Nectria; Peronospora;

Phoma; Eutypa; Phymatotrichum;; Plasmopara; Podosphaera; Puccinia; Puthium; Pyrenophora; Pyricularia; Scerotium; Sckerotinia; Septoria; Uncinula; Venturia;

Verticillium or Penicillium, including both the anamorph and/or teleomorph stage of any one thereof.

In one preferred aspect the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing one or more of the following plant pathogens: Alternaria brassicol; Alternaria solani, Ascochyta pisi, Botrytis cinerea, Cercospora kikuchii, Cercospora zaea-maydis; Colletothchum lindemuthianum; Diplodia maydis; Erysiphe graminis fsp. Graminis; Erysiphe graminis f.sp. hordei; Fusarium nivale; Fusarium oxysporum; Fusarium graminearum; Fusarium culmorum; Fusarium solani; Fusarium moniliforme; Fusarium roseum; Gaeumanomyces graminis f.sp. tritici; Helminthosporium turcicum; Helminthosporium carbonum; Helminthosporium maydis; Macrophomina phaseolina; Maganaporihe grisea; Nectria heamatococca; Peronospora manshurica; Peronospora tabacina; Phoma betae; Phymatotrichum omnivorum; Phytophthora cinnamomi; Phytophthora cactorum; Phytophthora phaseoli; Phytophthora parasitica; Phytophthora citrophthora; Phytophthora megasperma fsp. sojae; Phytophthora infestans; Plasmopara viticola; Podosphaera leucotricha; Puccinia sorghi; Puccinia striiformis; Puccinia graminis fsp. tritici; Puccinia asparagi; Puccinia recondita; Puccinia arachidis; Puthium aphanidenmatum; Pyrenophora t tici-repentens; Pyricularia oryzae; Pythium ultimum; Rhizoctonia solani; Rhizoctonia cerealis; Scerotium rolfsii; Sclerotinia sclerotiorum; Septoria lycopersici; Septoria glycines; Septoria nodorum; Septoria tritici; Thielaviopsis basicola; Uncinula necator; Venturia inaequalis; Verticillium dahliae; Verticillium albo-atrum and Eutypa lata.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a plant pathogen selected from the following: Blumeria graminis, Erysiphe graminis, Botrytis cinerea, Peronospora, Bremia lactucae, Phytophthora, Puccinia, Uromyces, Alternaria, Bipolaris, Drechslerea, Helmintosporium, Exserohilum, Sclerotinia, Fusarium oxysporum, Fusarium, Rhizoctonia, Pythium, Aphanomyces, Cercospora, Septoria (tritici), Stagonospora (nodorum), Phoma (lingam), Mycosphaerella fijiensis, Paracercospora fijiensis, Ascomycetes spp, Leptosphaeria maculans, and Eutypa lata.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a plant pathogen selected from the following: Blumeria graminis, Erysiphe graminis, Botrytis cinerea, Peronospora, Bremia lactucae, Phytophthora, Puccinia, Uromyces, Alternaria, Bipolaris, Drechslerea, Helmintosporium, Exserohilum, Sclerotinia, Fusarium oxysporum, Fusarium, Rhizoctonia, Pythium, Aphanomyces, Cercospora, Septoria (tritici), Stagonospora (nodorum), and Phoma (lingam).

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a plant pathogen selected from the following: Blumeria graminis, Erysiphe graminis, Botrytis cinerea, Peronospora, Bremia lactucae, Phytophthora, Puccinia, Uromyces, Alternaria, Bipolaris, Drechslerea, Helmintosporium, Exserohilum, Sclerotinia, Fusarium oxysporum, Fusarium, Cercospora, Septoria (tritici), Stagonospora (nodorum), Phoma (lingam), and Eutypa lata.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing one or more of the following classes of related phytopathogenic fungi: ascomycetes (e.g. Venturia, Podosphaera, Erysiphe, Monilinia, Mycosphaerella, Uncinula); basidiomycetes (e.g. the genus Hemileia, Rhizoctonia, Puccinia); Fungi imperfecti (e.g. Botrytis, Helminthosporium, Rhynchosporium, Fusarium, Septoria, Cercospora, Alternaria, Pyricularia and in particular Pseudocercosporella herpotrichoides); oomycetes (e.g. Phytophthora, Peronospora, Bremia, Pythium, Plasmopara). In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a plant pathogen selected from the following: Xanthomonas, Pseudomonas, Fusarium, Rhizoctonia, Pythium, Phytophthora, Thielaviopsis, downey mildew, powdery mildew, Aspergillus and Penicillium.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a microorganism assopciated with a storage disease or a disease of fruits and/or vegetables. In many products diseases appear after harvest - during the storage of the product. For example, often more than half of a harvest of carrots is discarded due to damages caused by pathogens blooming during storage. We have shown in the laboratory, that microthecin efficiently control Pythium sulcatum - a fungal pathogen causing cavity spot in carrots.. As microthecin might be a very "mild" antifungal compound, it can be used in washing or spraying of products in storage or fruits/vegetables shortly before harvest. This not only includes carrots, but many other vetables and fruits (citrus-fruits etc.).

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a gram negative bacteria.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a plant pathogen selected from or associated with bacterial leaf blight, bacterial mosaic, bacterial sheath rot, basal glume rot, black chaff (bacterial streak), spike blight (gummosis), leaf spot, blights, cankers, and galls.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from Pseudomonas spp., Clavibacter spp., Xanthomonas spp., Rathayibacter spp., Corynebacterium spp., Erwinia spp. Agrobacterim spp., Xylella spp.

Preferably the antimicrobial compounds of the invention are fungicidal with activity against plant pathogenic fungi selected from one or more of the following taxonomic groups: Oomycetes, Ascomycetes, Fungi imperfecta, Deutormycetes, Basidiomycetes, Zygomycetes, or Mastigomycetes In a preferable emdomiment the anti-microbial compounds of the invention are fungicidal or fungistatic against one or more of fungal plant diseases selected from Downey mildew, powdery mildew, grey mould, canker, black scurf, rots, soft rot, fruit rot, basal rot, crown rot, root rot, rusts, stem rusts, stripe rusts, blight, early blight, late blight, pithium blight, leaf spot, wilt, leaf blotch, glume blotch, black leg, and black sigitoka.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from Alternaria spp. , Albugo spp., Aphanomyces spp. , Amyloporia spp., Ascochyta spp., Aspergillus spp, Basidiophora spp, Bipolaris spp, Botrytis spp, Bremia spp, Cercospora spp, Cladosporium spp, Claviceps spp, Coniophora spp., Colletothchum spp, Diplodia spp, Dipiocarpon spp., Donkiopoha spp., Drechslera spp, Erysiphe spp, Eutypa spp, Fibroporia spp., Fusarium spp, Gaeumanomyces spp, Geotrichum spp, Guignardia spp, Gymnosporangium spp, Helmintosporium spp, Hemileia spp, Kabatiella spp, Leptosphaeria spp, Macrophomina spp, Marssonina spp., Monilinea spp, Merulus spp, Mycosphaerella spp, Nectria spp, Paracercospora spp, Penicillium spp, Peronophythora spp, Peronospora spp, Phellehas spp., Phoma spp, Phomopsis spp, Phymatotrichum spp, Phytophthora spp, Plasmophora spp, Podosphaera spp, Porai spp., Pseudocercosporella spp, Pseudoperonospora spp, Puccinia spp, Pyrenophora spp, Pyricularia spp, Pythium spp, Rhizoctonia spp, Rhizopus spp, Sclerophthora spp, Sclerotinia spp, Sclerotium spp, Septoria spp, Serpula spp., Sphaerotheca spp, Stagonospora spp, Taphrina spp, Thielaviopsis spp, Tilletia spp, Trichoderma spp, Uncinula spp, Ustilago spp, Venturia spp, and Verticillium spp.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from Pythium spp, Aphanomyces spp. Peronospora spp., Phytophthora spp., Albugo spp., Basidiophora spp., Bremia spp., Plasmopara spp., Pseudoperonospora spp., Peronophythora spp.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from P. acanthicum, P. acanthophoron, P. aphanidermatum, P. aquatile, P. aristosporum, P. arrhenomanes, P. bifurcatum, P. buismaniae, P. butleri , P. campanulatum, P. canariense, P. carbonicum, P. carolinianum, P. catenulatum, P. chamaehyphon , P. citrinum, P. coloratum, P. contiguanum, P. cylindrosporum, P. Debaryanum, P. Deliense, P. destruens, P. diclinum, P. dimorphum, P. Dissotocum, P. echinulatum, P. erinaceum,

P. glomeratum, P. graminicola, P. grandisporangium, P. helicoides, P. heterothallicum, P. hydnosporum, P. inflatum, P. insidiosum, P. intermedium, P. irregulare, P. iwagamai, P. jasmonium, P. longandrum, P. macrosporum, P. mamillatum, P. mastophorum, P. megacarpum, P. megalacanthum, P. middletonii, P. monospermum, P. montanum, P. myriotylum, P. nodosum, P. nunn, P. oedochilum,

P. okanoganense, P. oligandrum, P. ornacarpum, P. orthogonon, P. ostracodes, P. pachycaule, P. pachycaule var. ramificatum, P. paddicum, P. paroecandrum, P. pectinolyticum, P. pehilum, P. periplocum, P. perplexum, P. polymastum, P. porphyrae, P. prolatum, P. proliferatum, P. pulchrum, P. ramificatum, P. regulare, P. rhizosaccharum, P. rostratum, P. salpingophorum, P. segnitium, P. spinosum, P. splendens, P. sulcatum, P. sylvaticum, P. terrestris, P. torulosum, P. tracheiphilum, P. ultimum, P. ultimum var. sporangiiferum, P. ultimum var. ultimum, P. uncinulatum, P. undulatum, P. vanterpoolii, P. aff. vanterpoolii, P. vexans, P. violae, P. volutum, P. zingiberum, and P. sp. abappressorium.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from P. acanthicum, P. acanthophoron, P. aphanidermatum, P. aquatile, P. aristosporum, P. arrhenomanes, P. bifurcatum, P. buismaniae, P. butle , P. campanulatum, P. canariense, P. carbonicum, P. carolinianum, P. catenulatum, P. chamaehyphon , P. citrinum, P. coloratum, P. contiguanum, P. cylindrosporum, P. Debaryanum, P. Deliense, P. destruens, P. diclinum, P. dimorphum, P. Dissotocum, P. echinulatum, P. erinaceum,

P. glomeratum, P. graminicola, P. grandisporangium, P. helicoides, P. heterothallicum, P. hydnosporum, P. inflatum, P. insidiosum, P. intermedium, P. irregulare, P. iwagamai, P. jasmonium, P. longandrum, P. macrosporum, P. mamillatum, P. mastophorum, P. megacarpum, P. megalacanthum, P. middletonii, P. monospermum, P. montanum, P. myriotylum, P. nodosum, P. nunn, P. oedochilum,

P. okanoganense, P. oligandrum, P. ornacarpum, P. orthogonon, P. ostracodes, P. pachycaule, P. pachycaule var. ramificatum, P. paddicum, P. paroecandrum, P. pectinolyticum, P. pehilum, P. periplocum, P. perplexum, P. polymastum, P. porphyrae, P. prolatum, P. proliferatum, P. pulchrum, P. ramificatum, P. regulare, P. rhizosaccharum, P. rostratum, P. salpingophorum, P. segnitium, P. spinosum, P. splendens, P. sulcatum, P. sylvaticum, P. terrestris, P. torulosum, P. tracheiphilum, P. uncinulatum, P. undulatum, P. vanterpoolii, P. aff. vanterpoolii, P. vexans, P. violae, P. volutum, P. zingiberum, and P. sp. abappressorium

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from A. astaci, A. cochlioides, A. euteiches, A. helicoides, A. invadans, A. laevis, A. piscicida, A. stellatus, A. sp. 84-1240, A. sp. AR_11, A. sp. TF33, A. sp. TF5. A. euteiches causing Aphanomyces root rot in pea and alfalfa

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from A. astaci, A. euteiches, A. helicoides, A. invadans, A. laevis, A. piscicida, A. stellatus, A. sp. 84-1240, A. sp. AR_11, A. sp. TF33, A. sp. TF5. A. euteiches causing Aphanomyces root rot in pea and alfalfa

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from Rhizoctonia cerealis, orvzae-sativae, R. solani, Rhizina undulata, Rhizoctonia leguminicola, Rhizoctonia zeae, Rhizoctonia repens, Rhizoctonia crocorum, Rhizoctonia muneratii, and Rhizoctonia stahlii.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from Rhizoctonia cerealis, R orvzae-sativae, Rhizina undulata, Rhizoctonia leguminicola, Rhizoctonia zeae, Rhizoctonia repens, Rhizoctonia crocorum, Rhizoctonia muneratii, and Rhizoctonia stahlii.

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from C. apii, C. asparagi, C. beticola, C. canescens, C. caricis, C. coffeicola, C. hayi, C. kalmiae, C kikuchii, C. nicotianae, C. piaropi, C. sojina, C. violae, C. zeae-maydis, C. zebrina, C. sp. PoGDS8-2

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from C. apii, C. asparagi, C. canescens, C caricis, C. coffeicola, C hayi, C. kalmiae, C. kikuchii, C. nicotianae, C. piaropi, C. sojina, C. violae, C. zeae-maydis, C. zebrina, C. sp. PoGDS8-2

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from the following Oomycete plant fungal diseases:

Genera Example of disease

Pythium Root rot and damping off in many important crop plants. P. sulcatum is causing cavity spot in carrots

Aphanomyces A. euteiches causing Aphanomyces root rot in pea and alfalfa. A cochlioides in sugar beet

Peronospora Downy mildew in many important crops

Phytophthora Late Blight in potato, tomato, eggplant Albugo White rust on beet, cabbage, cauliflower, horseradish, kale.

Basidiophora Downy mildew

Bremia Downy mildew in lettuce

Plasmopara Downy mildew in grapes

Pseudoperonospora Hop Downy mildew Peronophythora; P. litchii in Litchi

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from Pythium sp. (widespread: root rot diseases - i.e. Root rot in soybean, Rice Seedling Blight etc.) P. ultimum in beet (causing Damping off and root rot), P. ultimum (other crops), P. aphanidermatum (Pythium blight in turfs), and P. graminicola (Pythium blight in turf).

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from Rhizoctonia sp. (Widespread: Root rot diseases), R. solani in beet (Damping off, root rot and violet disease), R. solani (i.e., Black scurf in potatoes; Sheath Blight of Rice), Aphanomyces sp. (Widespread: Root rot diseases) A. cochlioides in beet (Damping off and root rot), A. euteiches (pea, alfalfa), Fusarium sp. (Widespread: Root rot diseases.Causing fusariose (Fusarium wilt) in many different crops), F. oxysporum (i.e., Fusarium root rot in soybeans, conifers, Spinach Fusarium wilt), F. solani (Sudden Death Syndrome, soybean), Sclerotinia sp., S. sclerotiorium (i.e., white mould (stem rot) in soybean), Sclerotium sp., S. rolfsii (Sclerotium rot, sugar beet), Phytophthora sp., P. megasperma (Stem rot in soybean), Cercospora sp., C. kikuchii (Purple seed stain in soybean), C. beticola (Leaf spot, sugarbeet), Phoma sp., P. betae in beet, P. lingam (oil seed rape), Alternaria sp., A. solani (Early Blight, tomato), A. dauci (Early Blight, tomato, carrot), A. radicina (Early Blight, carrots), A. triticina (Alternaria Leaf Blight, Wheat), Thielaviopsis sp., T. basicola (Black Root Rot in tobacco and many other crops: Vinca), U. tritici (Loose Smut of Wheat), Gaeumannomyces graminis (Take All in Wheat), Albugo Candida (White Rust, radish), Bipolaris sp., and B. sorokiniana (Spot Blotch in Barley; Root Rot in wheat).

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen in a Potato seed tubers, a pathogen associated with Black scurf (stem canker) or the pathogen Rhizoctonia solani. The antimicrobial material may be contacted at time of planting, either by spraying the potatoes, dipping the potatoes in the composition or by a powder treatment.

More preferably, the antimicrobial material of the invention are used in preventing and/or inhibiting the growth of, and/or killing Botrytis, crown and root rot, or mildew.

Preferably, the antimicrobial material of the invention are used in preventing and/or inhibiting the growth of, and/or killing a plant pathogen selected from the following downey mildew or powdery mildew.

Particular powdery mildew organism against which the invention may be effective include Erysiphe spp., Erysiphe cichoracearum (found in for example cucumbers, endive, lettuce, melons, potato, pumpkin, squash), Erysiphe cruciferarum (found in for example broccoli, Brussels sprouts, cauliflower, and other cole crops; radicchio, radishes, turnips) Erysiphe lycopersici & Oidium lycopersicum: (found in for example tomatoes), Erysiphe pis (found in for example peas), Erysiphe heracler. (found in for example carrots, parsley, parsnips), Erysiphe polygoni: (found in for example beets), Leveillula taurica: (found in for example artichoke, eggplant, peppers, tomatillo, tomatoes), Sphaerotheca spp., and Sphaerotheca fuliginea: (found in for example beans, black-eyed peas, cucurbits, okra) Preferably, the antimicrobial material of the invention are used in preventing and/or inhibiting the growth of, and/or killing a plant fungal pathogens.

Preferably, the antimicrobial material of the invention can be used to prevent and/or inhibit the growth of, and/or kill fungi, i.e. it may be used as a fungicide.

Preferably, the antimicrobial material of the invention can be used as an agrochemical, particularly effective as a plant protectant against fungal attack, i.e. as a fungicide.

Preferably, the antimicrobial material of the invention are used in preventing and/or inhibiting the growth of, and/or killing a micro-organisms selected from the orders Rhizoctonia, Pythium, Aphanomyces and Cercospora.

Preferably, the antimicrobial material of the invention are used in preventing and/or inhibiting the growth of, and/or killing a micro-organisms selected from Rhizoctonia solani, Pythium ultimum, Aphanomyces cochlioides and Cercospora beticola.

Preferably, the antimicrobial material of the invention are used in preventing and/or inhibiting the growth of, and/or killing the pathogen Aphanomyces. Preferably, the pathogen is Aphanomyces cochlioides.

Foliar & other diseases

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from Botrytis sp. (Attack many different plants), B. cinerea (i.e. Botrytis Blight, Bunch Rot and Grey Mould of grape, Kiwifruit Grey Mould, Douglas fir grey mould), B. squamosa (Leaf Blight in onion),

Colletrotrichum sp., C. sublineolum (formerly C. graminicola; Anthracnose Leaf Blight, corn, sorghum), Kabatiella zeae (Eyespot, corn), Puccinia sp. (Rust diseases, especially important in cereals), P. sorghi (Common Rust, corn), P. hordei (Brown rust/Leaf Rust,

Barley), P. poae-nemoralis and P. poarum (Rust), P. striiformis (Yellow rust/Stripe

Rust, Wheat), P. horiana (White Rust of chysanthemum), P. graminis f.sp. triticale

(Stem rust, Wheat), Drechslera sp., D. teres (Net Blotch, barley), D. graminea (Leaf

Stripe, Barley), Erysiphe sp. (Powdery Mildew), E. graminis f. sp. hordei (Powdery Mildew, Barley/Wheat) = Blumeria graminis, E. betae (Powdery Mildew, Beet), Ustilago. sp. (Smut fungi - very widespread), U. maydis (Corn Smut), U. nuda (Loose Smut, Barley), U. tritici (Loose Smut of Wheat), U. scita inea (Sugar Cane Smut), Claviceps purpurea (Ergot, cereals, grasses), Tilletia sp., T. caries (Bunt of Wheat), Septoria sp. and Staganospora sp., S. nodorum (Leaf Blotch, wheat), S. tritici (Leaf Blotch, wheat), S. avenae f.sp. triticea (Leaf Blotch, wheat), Phytophthora sp., P. infestans (Late Blight, Very important pathogen of Potato/Tomato/Eggplants)., Pseudocercosporella herpotrichoides (Eyespot, Cereals), Pseudoperonospora sp., P. cubensis (Downy Mildew, cucumber), Sphaerotheca sp., S. fuliginea (Powdery Mildew, cucumber), S. pannosa (Powdery Mildew, Roses), Cercospora sp., C. maydis (Gray leaf spot, Corn), Helmintosporium sp., H. maydis, H. carbonum, H. turcicum (All 3 gives Helmintosporium Leaf Blight in Corn), H. sativum (Spot Blotch, Wheat, Barley), Pyrenophora, P. triticirepensis (Tan spot, Wheat), Fusarium sp., F. oxysporum (Fusarium Wilt in Banana and many other species), Mycosphaerella sp., M. musicola (Banana Leaf spot, Sigatoka disease), M. brassicicola (Black Blight in cabbage, cauliflower, Brussels sprouts, broccoli), Phomopsis sp., P. viticola (Cane and Leaf Spot in grape), Sphaerotheca sp., S. macularis (Powdery Mildew in Strawberry, Raspberry, Blackberry), Verticillium sp. (Verticillium wilt), V. albo-atrum (Wilt in alfalfa, tomato, hop), Gymnosporangium sp. (i.e. apple rust), Hemileia vastatrix (Coffee Rust), Guignardia bidwellii (Black Rot, Grape), Taphrina sp. (Leaf curl diseases), and Pyricularia oryzae (Rice blast).

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from post harvest antimicrobial, preferably fungal, diseases, preferably one or more fungal diseases selected from the following ( e.g. of fruits, vegetables): Cladosporium sp., Rhizopus rot, Penicillium expansum (Blue mould) Alternaria rot, Botrytis cinerea (Grey Mould), Aspergillus niger (Black mold in many fruit and vegetable species, i.e. onion) Monilinia sp. (Brown rot), Fusarium sp. (Pink or Yellow Molds), Geotrichum (sour rots).

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a bacterial pathogen selected from: Pseudomonas syringae is responsible for a number of economically important diseases in a wide variety of fruits and vegetables, and in ornamental plants (Bacterial Blight, Flower Blast, Necrotic Leaf Spots, Spots and blisters on fruits), Erwinia carotovora (Erwinia Soft Rot in many vegetables), Xanthomonas sp. (Bacterial Leaf spots in many vegetables), and X. campestris cause Walnut Blight)

Other plant Pathogens

Banana - A leaf spot disease is the most important of these problems. Black Sigatoka, which is also known as black leaf streak, causes significant reductions in leaf area, yield losses of 50% or more, and premature ripening, a serious defect in exported fruit. It is more damaging and difficult to control than the related yellow Sigatoka disease, and has a wider host range that includes the plantains and dessert and ABB cooking bananas that are usually not affected by yellow Sigatoka.

Black Sigatoka often affects plantain and banana, and is caused by the ascomycete Mycosphaerella fijiensis (Anamorph: Paracercospora fijiensis). Thus the present invention further provides a method for preventing and/or inhibiting the growth of, and/or killing, Mycosphaerella fijiensis (Anamorph: Paracercospora fijiensis), preferably in or on plantain or banana crops.

In export plantations, Black Sigatoka is controlled with frequent applications of fungicides and cultural practices, such as the removal of affected leaves, and adequate spacing of plants and efficient drainage within plantation. In total, these are very expensive practices. For example, fungicide application includes the use of aeroplanes or helicopters, permanent landing strips and facilities for mixing and loading the fungicides, and the high recurring expense of the spray materials themselves. In total, it has been estimated that the costs of control are ultimately responsible for 15-20% of the final retail price of these fruit in the importing countries.

The antimicrobial material of the invention may also be used in the prevention and/or inhibition of leaf spot disease, in particular Black Sigatoka (which is also known as black leaf streak), and/or black leg.

Blackleg, one of the predominant fungal diseases in rape plants, typically results in losses of tens of millions of dollars annually. Sclerotinia, another predominant fungal disease of Cruciferae plants, which includes Brassica plants, as well as 400 other species of plants, including Composite plants such as sunflower and leguminous plants such as pea, also can result in significant economic losses.

Blackleg disease is caused by an Ascomycetes fungus whose perfect or sexual form is Leptosphaeria maculans and whose imperfect or asexual form is Phoma lingam

Turf Grass Diseases

In one preferred aspect, the antimicrobial material may be utilised in preventing and/or inhibiting the growth of, and/or killing a pathogen selected from diseases of turf grass:

Anthracnose And Basal Rot (Colletotrichum graminicola), Blister Smut (Entyloma dactylidis), Brown Patch (Rhizoctonia solani), Damping-off(Pythium, Fusarium,

Helminthosporium And Rhizoctonia spp.), Dollar Spot (Sclerotinia homoeocarpa), Fairy

Ring (Lycoperdon, Psalliota, Clitocybe Species And Others), Fairy Ring (Marasmius oreades), Leaf Spots, Blight, Foot Rots And Melting-out (Drechslera spp.), Powdery

Mildew (Erysiphe graminis), Red Thread (Laetisaria fuciformis), Rust (Puccinia spp.),

Slime Molds (Usually Physarum cinereum), Snow Mold, Cottony Or Ltb/Sltb Snow Molds

(Coprinus psychromorbidus), Snow Mold, Gray Or Speckled (Typhula spp.), Snow Mold,

Pink Or Fusarium And Microdochium Patch (F. nivale And M. nivale), Snow Scald (Myrioscierotinia borealis Or Scierotinia borealis), Stripe Smut (Ustilago striiformis),

Take-all Or Ophiobolus Patch (Gaeumannomyces graminis var. avehae), and Pythium blight (Pithium spp.)

Domestic and Industrial Application of Fungicides

Fungal species can grow and reproduce in most natural, domestic and industrial environments, and can cause significant loss of yield and quality to plant crops, result in considerable damage to industrial and personal property, and cause ill health, disease and allergies in humans and animals.

The control of fungal contamination can be achieved via use of fungicides in addition to other fungal management measures. For example, in the domestic and industrial environments, fungal contaminations can, in many instances be reduced by ensuring good ventilation to avoid the build up of fungal spores and ensure appropriate control of moisture. However, in modern domestic and industrial property, the widespread use of heating, and poor ventilation, results in a environment that is ideal for fungal contamination and growth. Therefore the indoor environment is a creation of the modern era. Previously, buildings were notable for the extent to which they were really open to the outside air, a system that could be referred to as natural ventilation. But, technological advances have permitted us to seal buildings tightly, recirculate the air within them, and fill them with a variety of particle- and chemical-emitting materials and objects. Shelton, B.G. Kirkland, K.H. Flanders, W.D. Morris, G.K. Profiles of airborne fungi in buildings and outdoor environments in the United States. Applied and Environmental Microbiology 68, 1743 - 1753 (2002).

Therefore, there is increasing awareness and concern over the rise in fungal related disorders caused by fungal mycotoxins, allergies to fungal spores, and pathological disease caused by human infection (particularly in immune compromised individuals). In addition fungal contamination is highly unsightly, can damage the structural integrity of surfaces, furnishings, materials, carpets, wooden flows, paints and wall papers, plaster, masonry, etc. and even cause major structural damage, particularly in wood frames properties.

The foloowing are examples of fungal species that contaminate domestic and industrial environments: Wet and dry rots (also known as 'building rot'), Merulius lacrymans (Serpula lacrymans), - dry rot, Coniophporan cerebella and c. puteanaw (borwn rot), Poria incrassata, - Wet rot, Amyloponia xantha, Fibroporia vaillanti, Poria placenta, phellinas contignus, Donkioporia expansa, Asterostrama sp., Paxillus pansuides, white rot:

Mildew

We have particularly found that the present antimicrobial material is effective in preventing and/or inhibiting the growth of, and/or killing mildew.

Fungal species encompassed by the common terms, "mildew" and "mould" or "house fungus" include: Aspergillus sp., Penicillium sp. Aspergillus niger, Rhizopus sp. Stachybotrys chartarum (Black mould), Alternaria, sp., and Cladosporium sp. By the term "mildew" it is meant (1) a visible growth of a mould on a plant surface or other substrate, or (2) a growth on a plant surface by a member of the Erysiphales; a disease so signalized, or a powdery mildew fungus. Mildew may refer to infection by a member of the Peronosporaceae; a disease so signalized, or a downy mildew fungus. Mildew may refer to infection by one of the Meliolaceae.

By the term "mould" it is meant a mycelial microfungus or a visible growth of such a fungus. Blue mould or green mould is caused by Penicillium spp.; grey mould, by Botrytis cinerea; sooty mould is caused by one of the Capnodiaceae but the term has been used incorrectly for growth of Cladosporium spp. and other fungi on foliage.

In many areas of the world, the unwanted growth of mould and mildew is an annoying problem. The rapid growth of mould and mildew on surfaces, such as house walls, bathroom walls, and the like, is particularly troublesome in hot, humid climates.

Typically once mould and mildew appears on a surface it is not only very difficult to remove but its regrowth is almost certain.

A costly problem occurs when mould and mildew is present on a surface that needs to be painted. In order to paint or repaint properly a surface that has become infested with mould or mildew, it is necessary to remove the mould and mildew completely.

The use of fungicides in domestic and industrial environments is therefore deemed in many instances as necessary and/or desirable, both as a preventative and as a treatment of inanimate objects.

Many fungicides are toxic and harmful to both human, animal health and wildlife, and can cause environmental damage, particularly to environmental water such as ground water, lakes, rivers, streams etc. It is therefore highly preferable to use fungicides with low inherent toxicity, and that rapidly degrade once in the environment, particularly in environmental water. Surprisingly microthecin and derivatives and in particular the present antimicrobial material has been found to be highly effective in preventing the growth and infestation of mildew and other fungi/moulds. The anti-microbial composition of the invention is highly preferable to use as a fungicide in view of its low inherent toxicity. In addition, fungicides with low residual toxicity and low environmental persistence are highly advantageous for application of crop plants and for use in domestic gardens and homes, where the risk for poisoning of humans and animals is considered high.

Thus in a further aspect the present invention relates to novel formulations for the prevention of the growth and infestation of mildew and other fungi/moulds.

In a further aspect the present invention relates to the application of microthecin in industrial and domestic environments to inhibit or prevent the unwanted growth of mould and mildew.

In a further aspect the present invention provides a method for removing mould and mildew from an infested surface and inhibiting regrowth thereon.

The invention relates to products, formulations, methods and applications involving the anti-fungal activity of microthecin to reduce, prevent, inhibit, or remove living fungal material from non-living surfaces. In some aspects the invention does not extent directly to the applications on, in or within the immediate vicinity of animals (including humans) or plants or parts thereof (i.e. including seeds, leaves, roots).

It is considered that although the following examples refer to the inclusion of microthecin per se, in some applications it may be preferable to produce microthecin in situ by the use of the precursor(s) and conversion agent(s) of the present invention, such as the pyranose dehydratase enzyme or the combination of glucan lyase and pyranose-2- oxidase, in such situations a starch or dextran substrate can be supplied as part of the formulation. The inclusion of enzymes in detergent formulations is well established.

It will be recognised by a person skilled in the art that the optimal concentration of microthecin and the time period of efficacy with in a formulation will depend on the particular formulation and application of the formulation. The optimal concentration of microthecin for any particular formulation and application may typically be the minimum level at which effective anti-fungal activity as measured by an 80% inhibition of regrowth over a predetermined efficacy time period. One manner in which the optimal concentration may be determined is by preparation of a range of formulations containing microthecin the following levels of microthecin, 1%, 5%, 10%, 20%, 30%, 40% 50%, 75%, 90%, 100% and the applied to a fungally contaminated surface of choice. % inhibition of regrowth, determined by area of fungal contamination is compared to a control surface treated with an equivalent formulation that does not contain microthecin at suitable time points.

The following applications are provided as examples of the manner in which microthecin (or derivatives thereof may be used). The term detergent is used to refer to anti-fungal and/or cleaning products. References to microthecin include derivatives thereof in accordance with the invention and the definitions provided herein.

1. Solid detergent formulation for applying to solid surfaces

Formulations may be in a solid form, preferably a powder form. Pure microthecin powder can be used or microthecin can be formulated with a suitable carrier such as talcum powder. The solid formulation can include other components such as surfactants and bleaching agents.

Treatment of materials such as fabrics, carpets, and curtains - used as a dusting powder - in a solid detergent compositions of cleaning surfaces and destroying and preventing the regrowth of mould and mildew.

Suitable methods for the production of solid or powder detergent formulations are known in the art, for example US 4,059,538 and EP0462723. Alternatively dried crude or pure preparations of microthecin can be used. Microthecin preparations can be prepared as those disclosed in JP54122796 and powder/solid can be obtained by freeze drying the preparations obtained.

2. Liquid detergent formulation for applying to solid surfaces

The microthecin may be either in a solid or powder form, or in a liquid formula formulated to enhance the stability of microthecin, or provided as a stabilised concentrate for dilution prior or during use. In certain applications stabilised microthecin in solid or liquid form can be added directly to a liquid prior or during use. In an embodiment the solid microthecin formulation can be a solid detergent formulation and the liquid a suitable solvent, such as water. In an embodiment the liquid can be a liquid detergent formulation. The liquid detergent formulation composition of the invention can be used to clean and remove fungi and algae from any one of a number of solid surfaces such as exterior painted surfaces, stone, brick, stucco, interior painted surfaces, tile, ceramics, metal, wood and plastic. As used herein the term "solid surface" is intended to mean that the density and coherence in the mass of the respective material is such as to enable the material to maintain a fixed form. Thus, for example a solid surface is intended to include the surface of a material such as painted wood surface or a ceramic tile surface as well as the surface of a material such as fabric utilised in outdoor furniture or tents. The phrase "solid surface" is not typically intended to refer to the skin of humans or animals or the surface of a plant or part of a plant. The compositions are particularly useful in cleaning and removing fungi and algae from surfaces of organic films, such as painted surfaces.

The liquid detergent formulation composition of the invention can be applied to the affected surface using any one of a number of methods. The composition is applied using an applicator such as a garden sprayer, airless paint sprayer, compressor paint sprayer, aerosol can, or hand pump sprayer. The composition can also be applied using a sponge or brush.

The liquid detergent formulation can be water based or alternatively an organic solvent, preferably a polar-organic solvent is used such as alcohol. The liquid detergent formulation can contain a range of other ingredients including, thickening compositions, surfactants, bleaching agents, anti-bacterial agents, other anti-fungal agents.

3. Solid or textile liquid washing formulation, such as domestic washing powder, liquids or tablets.

In a specific embodiment the detergent formulation is a washing powder/liquid/tablets for domestic or industrial cleaning of fabrics, such as clothes, carpets, curtains and/or of solid surfaces. The addition of microthecin and/or enzymes capable of synthesising microthecin in situ, results in reduced build up of fungi/algae in washing environments, such as washing machines and dish washers, and more effective removal of fungal material during the washing process and lower incidence of fungal infections from fungal contamination in clothes, particularly at lower temperature washes. US2002123449 and others relates to the production of washing powder, liquids and tablets (or other suitable formulations).

4. Decorating materials such as paint, tile grout, wall paper pastes.

In a specific embodiment microthecin is added during the preparation of decorating materials to prevent or inhibit the growth of fungi such as mildew and moulds on or within the decorating materials. For example microthecin can be added to paints, tile grouts, wall papers, wall paper pastes, fillers, glues, adhesives, mouldings, fabric protectants.

5. Other cleaning products

In other specific embodiments microthecin may be added to other cleaning products such as furniture polish, tile/grout cleaner, hard surface cleaner, foam surface cleaners, window cleaners, leather cleaner, abrasive surface cleaners, drain cleaners, dishwasher compositions.

SUMMARY

In summation, the present invention relates to an antimicrobial material comprising an antimicrobial compound in a stabilised form (a "stabilised compound"), or a composition comprising precursor(s) and conversion agent(s) capable of providing the antimicrobial compound in situ.

The invention is further illustrated in the following non-limiting examples, and with reference to the following figures wherein:

Figure 1 illustrates the use of 1,5-anhydro-D-fructose and pyranosone dehydratase for the production of microthecin. The reaction mixture consisted of 1 ,5-Anhydro-D-fructose 5μl (3.0%), pyranosone dehydratase preparation 5μl, 65μl sodium phosphate buffer (pH 6.0) and water to a final volume of 0.7 ml. The reaction was monitored by scanning between 350-190 nm. Reaction time at zero min was used as blank. The absorbance peak at around 230 nm indicates the formation of microthecin. The absorbance at 265 nm indicate the first formation of an intermediate from AF before it converts to microthecin. Figure 2 illustrates the production of microthecin and its intermediate. The reaction mixture consisted of 10μl partially purified pyranosone dehydratase (a ammonium sulfate fraction between 25-50% saturation of the cell-free extract from Phanerochaete chrysosporium), 25μl AF (3.0%, w/v), 100μl sodium phosphate buffer (0.1M, pH6.5) and 0.84 ml water. The reaction was started by the addition of the substrate AF. The reaction was performed at 22°C. The formation of microthecin and its intermediate was monitored at 230 nm and 263 nm, respectively. One can see that the intermediate was first formed and levelled off after around 20 min. There was a delay for the formation of microthecin but its formation continued until nearly all the AF in the reaction mixture was consumed.

Figure 3 shows the final emergence of sugar beet seeds treated in accordance with Example 3.

Figure 4 shows the screening effect of microthecin in different concentrations against the sugar beet root rot causing pathogen Aphanomyces cochlioides.

Figures 5 and 6 show the screening effect of microthecin in different concentrations against the sugar beet root rot causing pathogens Pythium ultimum and Rhizoctonia solani respectively.

Figure 7 shows a graph.

Figure 8 shows a graph.

Figure 9 shows a graph

EXAMPLES

The term pyranosone dehydratase may be referred to herein as PD.

Pyranosone Dehydratase purified from the fungus Phanerochaete chrysosporium Phanerochaete chrysosporium (white rot fungus) is a biotechnologically important fungus due to its higher growth optimum temperature (40°C) and its ability to produce a range of extracellular oxidative enzymes. Accordingly, this fungus has been used for treatment of various wastes, including explosive contaminated materials, pesticides, and toxic wastes. Furthermore, Phanerochaete chrysosporium is the first basidiomycete genome to be sequenced (University of California and Department of Energy, USA).

In the search for enzymes that metabolise anhydrofructose (AF), a purified a heat-stable pyranosone dehydratase (PD) was obtained from P. chrysosporium. Studies have shown that this purified PD not only uses AF as substrate, but uses it more efficiently than its natural substrate, glucosone. Furthermore, the product was shown to be microthecin, an antifungal useful in plant protection.

Assay methods

Measuring of the pyranosone dehydratase (PD) activity

The reaction mixture consisted of 25 μl of anhydrofructose solution (3.0%), 10 μl PD preparation, 93 μl 0.1 M sodium phosphate (pH 6.5), and water to a final volume of 1 ml. The reaction was mixed and scanned between 190 and 320 nm at room temperature (22°C) every 5 min or after 30 min on a Perkin Elmer Lambda 18 UV/vis spectrophotometer. Absorbance values at 265 and 230 nm were recorded. One activity unit of PD is defined as the increase of 0.01 of absorbance unit at 230 nm at 22°C per min.

A protein assay was carried out using the Bio-Rad Method (Bradford method) using the reagent and instructions form Bio-Rad laboratories [Peterson, GL: Determination of total protein, Methods Enzymol. 91 , 95-119 (1983)]

TLC for separation of glucosone, AF and microthecin was performed as described before using a solvent system of ethylacetate, acetic acid, methanol and water (12:3:3:2) [Yu S, Ahmad T, Pedersen M, Kenne L: α-1 ,4-Glucan lyase, a new class of starch/glycogen degrading enzyme. III. Substrate specificity, mode of action, and cleavage mechanism, Biochim Biophys Acta 1244: 1-9 (1995)]. A Merck silica gel 60 (20x20cm) plate with a thickness of 0,15 mm was used. 1 ,5-Anhydro-D-fructose was assayed by the DNS method [Yu S, Olsen CE, Marcussen J: Methods for the assay of 1,5-anhydro-D-fructose and α-1 ,4-glucan lyase, Carbohydr. Res. 305: 73-82 (1998)].

Purification of PD

The purification procedure used was essentially the same as that described by Gabriel et al., (1993), Arch. Microbi., 160:27-34 except the strains used were different. In addition, an extra ammonium sulfate fractionation step was included. The strain used in this application was Phanerochaete chrysosporium from American Type Culture Collection (ATCC 32629) and (ATCC 24725), while the strain used by Gabriel et al (1993) was Phanerochaete chrysosporium k-3 obtained from a Czechish collection centre.

The cell-free extract of Phanerochaete crysosporium was brought up to 55% ammonium sulphate saturation. It was then blended gently for 2 hours and centrifuged for 20 minutes at 4°C at 10000xg. The precipitate that had the PD activity was dissolved in the same volume of extraction buffer, centrifuged again and the supernatant was then used for the purification of PD using the procedure described by Gabriel et al. (1993).

The purification of PD procedure was followed by SDS-PAGE, and native-PAGE using PhastSystem (Pharmacia) using 8-25 % gradient gels according to the manufacturer's instructions. Visualisation of protein bands on the gels was made with Coomassie brilliant blue staining (PhastGel Blue R). From Fig. 1A, PD1 is estimated to have a molecule mass 97 kDa it had a similar migration rate as the protein marker phosphorylase b (97.4 kDa).

Example 1: Use of 1,5-anhydro-D-fructose and PD for the production of microthecin

The reaction mixture consisted of 1,5-Anhydro-D-fructose 5μl (3.0%), PD preparation 5μl, 65μl sodium phosphate buffer (pH 6.0) and water to a final volume of 0.7 ml. The reaction was monitored by scanning between 350-190 nm. Reaction time at zero min was used as blank. The absorbance peak at around 230 nm indicates the formation of microthecin. The absorbance at 265 nm indicate the first formation of an intermediate from AF before it converts to microthecin. The microthecin formed was further confirmed by relative migration rate on TLC and its conversion of 2-furyhydoroxymethylketone that exhibits a typical absorbance peak at 275 nm [Baute M.-A. et al., 1986].

In larger scale production of microthecin, AF used was from 0.4% to 20%. The reaction was followed by AF disappearing from the reaction mixture using the DNS method [Yu. S.; Christensen TMIE, Kragh KM, Bojsen K, Marcussen J, Biochim Biophys Acta 1339: 311-320 (1997)]. The formation of microthecin was monitored at 265nm and its shift to 230nm, and was further monitored by TLC method.

1.5-Anhydro-D-fructose is found to be a much better substrate for the pyranosone dehydratase (PD) than for its natural substrate glucosone. The Vmax is around 4.7 times higher with AF than with glucosone (Table 1).

Table 1

The reaction system consisted of AF or glucosone 1-15μl, 25μl sodium phosphate buffer (6.5. 0.1M), water, 1.4μl PD to a final volume of 200μl. The reaction was performed at 22°C for 5.5 hours. The formation of microthecin from AF and cortalcerone from glucosone were monitored at 226nm.

Example 2: Production of Cortalcerone

Cortalcerone may be produced in one step by incubating a starch-type substrate, such starch, waxy starch, dextrins, with starch hydrolases, such amyloglucosidase and a debranching enzyme or cyclodextrin transferase, pyranose 2-oxidase, and PD. After incubation Cortalcerone can be separated from the reaction mixture by ultrafiltation using membrane cut-off of 300-30,000, preferably 10,000.

Example 3: Use of 1,5-anhydro-D-fructose, PD and ascopyrone P synthase for the production of APP

The reaction mixture consisted of 1 ,5-Anhydro-D-fructose 50μl (3.0%), PD preparation 5μl, ascopyrone P synthase 5 μl, 0.1 ml sodium phosphate buffer (pH 6.0) and water to a final volume of 0.8 ml. The reaction was monitored by the formation of APP at 289 nm spectrophotometrically. The reaction temperature was 22°C and reaction time was 24 hours. At the end of 90% of AF had been converted to APP. The structure of APP was confirmed using NMR as described earlier [WO 00/56838 filed 16/3/00, claiming priority from GB9906457.8, filed 19/3/99].

Microthecin as an Anti-Fungal

Fungal growth in plant causes enormous economical damages. Examples are their damage to sugar beet seedlings and their leaves. As soon as the sugar beet seed is germinated in the soil it is immediately exposed to fungal attack by the species such as Rhizoctonia solani, Pythium ultimum, Aphanomyces cochlioides. In the present invention, it was found microthecin was able to inhibit the growth of these disease- causing fungi. Hence, the seeds of economical crops, sugar as sugar beet seeds are coated with a paste containing microthecin at 50-2000 ppm and dried before use for planting. Alternatively, aqueous solution of microthecin may be directly sprayed on the plant and its leaves.

Experimental

Basic microthecin solution: 24 mg/ml Batch no. Mic20011016 Dilutions used:

All solutions were filtered through a 0.22 μm filter for sterilisation. The solutions were tested against the following fungi:

A circular plug (diameter 10 mm) of fresh mycelium was placed at the centre on a petri- dish (diameter 9 cm) containing PDA medium. (PDA= Potato dextrose agar Difco no. 213400). Wells with a diameter of 5 mm were cut along the periphery of the agar plate. In each well were placed 50 μl of a test solution. Alternatively, 20 μl of each test solution were placed directly on the agar along the periphery of the plate. Also, 50μl of each test solution were placed directly on top of the fungal mycelium plug.

The agar plates were placed at room temperature in daylight, but protected from direct sunlight.

The reaction (inhibition zones) of the fungi to the test substance was judged as follows:

Rhizoctonia solani: after 2-3 days of growth

Pythium ultimum: after 1-2 days of growth Aphanomyces cochlioides: after 3-4 days of growth

Cercospora beticola after 3-4 weeks of growth

Results

Microthecin as a fungal growth regulator was inhibitory against Rhizoctonia solani, Pythium ultimum, Aphanomyces cochlioides and Cercospora beticola. The minimum inhibition concentration (mic) of microthecin against these fungi were 240, 480, 1200 and 2400 ppm, respectively.

Example 4: Effect of Microthecin on pelleted sugar beet seeds The effect of microthecin on the plant pathogenic fungi Pythium ultimum, Rhizoctonia solani and Aphanomyces cochlioides in vitro was investigated by screening for growth inhibition of the pathogens on agar-plates (Figures 4, 5, 6).

Figure 4 shows the screening effect of microthecin in different concentrations against Aphanomyces cochlioides, whereas Figures 5 and 6 show the screening effect against Pythium ultimum and Rhizoctonia solani respectively. In each case, microthecin was dissolved in water and placed in wells in the periphery. An agar block containing the pathogen was placed in the centre. The pathogens were allowed to grow out on the PDA-agar plates for 3-5 days. These investigations showed that microthecin in very low concentrations was able to reduce the growth of Aphanomyces.

Similar tests with other micro-organisms showed that Microthecin has no effect on Cercospora. Pseudomonads (P. fluorescens DS96.578, P. mendocina DS98.124) are slightly affected, whereas it has no effect on the growth of Bacillus (β. . Pumilus DS96.734, B. megaterium DS98.124).

Based on these findings, the efficiency of microthecin was further investigated in a field emergence trial. The trial was sown relatively late giving it a higher chance for presence of the pathogen Aphanomyces in the trial field.

Materials and methods

Pellet TKW

1. Manhattan CAC-7-2306 kb5, 3,0-4,25mm, 19,2 (7) 19,1 (1)

2. Tower MIT-1-0290 kb5, 3,0-4,25mm. 17,3 (8) 17,8 (2)

Seeds were pelleted with standard P1 pelleting mass with (1 ,2) or without (7,8)Thiram.

Standard seed coating:

Inner coating: 0.3 gai/U microthecin as a 0,5% solution in water or 14.7 gai/U Hymexazol.

60 gai/U Imidacloprid. Standard metallic green seed cover film. The following combinations were included in the trial:

R F0 Without fungicides

R FT With Thiram (in pellet)

R FH With Hymexazol

R FM With Microthecin

R P1 STD With Thiram (in pellet) + Hymexazol

R FTM With Thiram (in pellet) + Microthecin

Trial place Bukkehave, DK. (4 reps, 200 seeds/plot)

Trial sown 21.05.2002

1. Count 28.05.2002 (speed)

2. Count 29.05.2002 (speed)

3. Count 24.06.2002 (final)

Results

Lab and field emergence figures can be found in Table 2 (Trial FEHCP034 Aphanomyces). The final emergence is shown in Figure 3.

In the field trial experiments microthecin is less phytotoxic than the commercial available fungicide for treatment of sugar beet seeds (Hymexazol). Surprisingly, preliminary results show that seedlings derived from seeds treated with microthecin emerge a faster than the non-fungicide treated controls and faster than the commercial fungicide treated seeds. Surprisingly, these preliminary results indicate that microthecin can be used to enhance germination rates of crop plant seeds.

In laboratory germination tests, a specific parameter that is found to correlated well with the field germination data - namely the number of germs having a root length longer than 15mm at day 4, shows that Hymexazol (commercial name Tachigaren, active against Aphanomyces) suppress the seed germination, whereas microthecin does not

The results of the lab studies indicate that the inclusion of microthecin decreases the speed of laboratory germination (4d), but this is not reflected in the 4d>15mm figures. This is the opposite effect of Hymexazol that has a low 4d>15mm germination.

With regard to the speed of germination, the field emergence trials indicate that pellets containing Hymexazol - either alone, or in combination with Thiram - germinate relatively slow (as expected from the 4d>15mm lab germination). Pellets containing microthecin show a speed of germination comparable with pellets only containing Thiram.

In contrast to the fast germinating Thiram containing pellets the pellets containing Microthecin show a high final germination (comparable with Hymexazol containing pellets). Although the actual attack by root rot causing pathogens was rather limited, the 4% (approximately) missing plantlets in the FT-plots arise from attack of plantlets by pathogens that can be controlled by Hymexazol (most probably Aphanomyces). The final number of plantlets in the FT-plots are lower than the number of plantlets in the F0 (no fungicides) plots. This can be explained by the action of Thiram, that controls other microbes, but not Aphanomyces, thereby allowing easier access of Aphanomyces to the plantlets.

The microthecin containing pellets are the only pellets that both show a fast germination and a high final germination. It is believed that microthecin might therefore be an alternative to the rather expensive chemical Hymexazol.

Example 5: Test of microthecin and selected compositions against powdery mildew

Method Reference:

Lyngs Jørgensen, H.J., Andresen, H. and Smedegaard-Petersen, V. (1996).

Control of Drechslera teres and Other Barley Pathogens by Preinoculation with Bipolaris maydis and Septoria nodorum. Phytopathology 86, 602-607.)

Barley plants, isoline P01 of the cultivar Pallas, were grown in a growth chamber, at approximately 200 μE m^"2 s (16 h of light at 19°C with 50 to 60 % relative humidity and 8 h of darkness at 16°C with 80 to 90 % relative humidity) in plastic pots (12 by 13.5 cm) containing the soil mix 'Weibulls Enhetsjord' (K jord, Svalόf Weibull AB, Hammenhδg, Sweden).

11 days after sowing the first leaf of each of 10 plants per pot was fixed in a horizontal position, adaxial side upwards, on bent plastic plates using unbleached cotton strings. 2.5 ml of each test solution was sprayed on the fixed leaves of a total of 4 pots. After 1 hour the treatment was repeated with extra 2.5 ml of the same test solution.

The experimental set-up was as shown in the Table 3 below. Table 3

Test solutions Pyranosone dehydratase Stock solution 46.4 units/ml in 25 mM phosphate buffer (pH 6.5).

Used solution: 50μl stock solution in 10 ml 20 mM sodium phosphate buffer (pH 6.5) => 0.23 units/ml.

Anhydrofructose: Stock solution: 49.5 mg/ml of water.

Used solution: 436 μl stock solution was diluted with 20 mM sodium phosphate buffer (pH 6.5) to a total of 10.00 ml => 2.1 mg anhydrofructose/ml.

Anhydrofructose + pyranosone dehydratase: 436 μl stock solution, 50 μl pyranosone dehydratase stock solution and 9514 μl sodium phosphate buffer (pH 6.5) were mixed VA hours before use.

Microthecin:

Stocksolution: Approximately 44mg/ml 20 mM sodium phosphate buffer pH=6.5 Used solution: 460 μl stock solution was diluted with 20 mM sodium phosphate buffer (pH 6.5) to a total of 10.00 ml => 2.0 mg microthecin/ml.

Inoculum of Powdery Mildew, Blumeria graminis f. sp. hordei was produced on barley plants, isoline P01 of the cultivar Pallas. After 7 days of incubation (16 h of light at 18 to 20°C and 8 h of darkness at 15 to 16°C) the powdery mildew fungus sporulated abundantly and was used for inoculation of the treated barley leaves. An inoculum concentration of 3 conidia/mm² leaf area was applied. The pots were randomly placed in the growth chamber (200 μE m^"2 s (16 h of light at 19°C with 50 to 60 % relative humidity and 8 h of darkness at 16°C with 80 to 90 % relative humidity) for 7 days before disease assessment.

Disease assessment

Seven days after treatment the disease development was assessed by judging percentage leaf coverage with mildew colonies on the ten fixed leaves on each pot.

Results

Table 4

As can be seen from Table 4, treatments with microthecin as well as the combined anhydrofructose + pyranosone dehydratase (AF + PD) significantly reduces the mildew colony formation.

Example 6: Testing of microthecin against various plant pathogens

The effect of microthecin against a range of different plant pathogens was investigated, including those listed below in Table 5. Table 5

^* Isolate received from Section for Plantpathology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.

The results are shown below in Table 6, where "+" indicates a positive effect against a particular pathogen.

Table 6

Example 7: Testing microthecin and other products on agar plates

The effect of microthecin and the products for the enzymatic production of microthecin on different plant pathogens were tested on agar plates. The tests were performed on 9-cm agar plates, pH 6,1-pH 6,5. On each plate a 9mm PDA-block of an actively growing pathogen was placed in the centre and allowed to grow out. Different products and enzymes were placed in 5mm diameter cut holes in the periphery of the agar plates, in total about 45-50μl/hole.

The growth and growth inhibition of the pathogenic fungi in the vicinity of the holes were monitored:

+++ pronounced growth inhibition, ++ good inhibition, + some inhibition, no growth inhibition.

Test of Anhydrofructose (AF) + Pyranosone dehydratase (PD) Growth medium: Potato Dextrose Agar (PDA) plates, pH 6,1.

Table 7

1x PD corresponds to 0,02U/mg AF.

Table 7 shows that Aphanomyces and Pythium are sensitive to AF+PD, whereas Rhizoctonia is less sensitive and finally will overgrow the holes. Time-effect study of Anhydrofructose (AF) + Pyranosone dehydratase (PD)

The AF and PD were mixed and at given times filtered through a MW 10.000 sieve to stop the enzymatic reaction. The starting mixture contained AF 44,6mg/ml and PD 40U/mi.

The set-up of the experiment was the same as in Example 1. 15μl probe + 30μl 20mM sodium phosphate buffer, pH 6,5 were added to each hole. Controls were sodium phosphate buffer pH 6,5 and concentrated pyranosone dehydratase ( 46U/ml). The results are shown below in Table 8.

Table 8

As can be seen from Table 8 above, the effect of the mixture is most pronounced on Aphanomyces cochlioides in the time span from 1 hr to 2 days. After 2 days the inhibitory effect decreases. There is no inhibitory effect on Rhizoctonia solani and only weak inhibitory effect on Pythium ultimum.

The time restricted inhibitory effect corresponds to the peak of microthecin in the mixture. Microthecin starts to decompose to other compounds after short time and after 7 days the microthecin in the mixture is halved compared to the maximum amount present in the mixture.

Pyranosone dehydratase in very high concentrations (PD control) is in itself controlling the growth of the tested plant pathogens.

Test of inhibitory effect of dextrin and amylopectin-derived products formed directly in the holes on the agar plates

To rule out if it was possible to produce inhibitory compounds directly from the starch derivatives dextrin and amylopectin using the combined enzyme reaction Glucan Lyase (GL) plus Pyranosone dehydratase (PD), an experiment was set up where the mixtures were added to the holes immediately after blending. The inhibitory effect is shown in Table 9 below.

Dextrin 10 (Fluka 31410, CASno 9050-36-6) 1 ,25% in 20mM sodium phosphate buffer, pH 6,5. Amylopectin (Sigma A8515) 1 ,25% in 20mM sodium phosphate buffer, pH 6,5. Dextrin and amylopectin was boiled for 20min and cooled before adding to the holes (approx. 45μl/hole).

Glucan Lyase (GL) 1 U per hole, Pyranosone dehydratase (PD) 0,25U per hole. Growth medium: Potato Dextrose Agar (PDA) plates, pH 6,1.

Table 9

As can be seen from Table 9 above, the starch derivative plus Glucan lyase has no effect on fungal growth. Addition of Pyranosone dehydratase to the starch derivatives has a slight inhibitory effect, whereas the best inhibitory effect was obtained by adding both Glucan lyase and Pyranosone dehydratase to the starch derivatives. Water Agar

To clarify if the inhibitory effect of the Pyranosone dehydratase was due to assessable substrate present in the potato dextrose agar plates, an experiment was carried out using water agar as the growth medium. The pH in the water agar was adjusted to pH 6,2 with 20mM sodium phosphate buffer.

Also tested was the effect of boiling versus sterile filtration of the dextrin 10 used as the starch derivative (1 ,25%) in this experiment. No difference in inhibitory effect between boiling and sterile filtration was found. The experiment was performed using actively growing Aphanomyces cochlioides and Pythium ultimum on PDA-block placed in the middle of the test plates. Growth inhibition was monitored after 5days (for Pythium also after 2 days). The results are shown below in Table 10.

Table 10

The inhibitory effect on Pythium seems to be transient as the PDA plates are completely overgrown after 5 days. In Aphanomyces, the combined Dextrin + Glucan lyase + Pyranosone dehydratase has a pronounced effect also after 5 days on PDA-plates.

Test of seeds coated with microthecin or anhydrofructose + Pyranosone dehydratase complex

Pelleted sugar beet (Beta vulga s L.) seeds (1 Unit = 100.000 seeds) were coated with

1) 0,3g/Unit Microthecin (FM, FM2)

2) 0,9g/Unit Anhydrofructose (FA)

3) 0,9g/Unit Anhydrofructose + 4 enzymeU/Unit Pyranosone dehydratase (FAD) in the underfilm. The underfilm also contained the insecticide Imidacloprid (60gai/Unit). The coating was finished with a standard green coverfilm. Treatments FT (Thiram) and FH (Hymexazol) are standard fungicide treatments for sugar beet seeds. Treatment FM differs from FM2 in being prepared 6 months before agar plate testing.

After coating, the seeds were tested on PDA-agar plates by placing seeds in the periphery with actively growing pathogenic fungi in the middle of the agar plate. Inhibitory effect was monitored after 2 days. The results are shown below in Table 11.

Table 11

(1) No overgrowth of seed with pathogen.

Treatment of seeds with low levels of Microthecin act at a level comparable with the standard sugar beet fungicides Thiram and Hymexazol against the pathogen Aphanomyces. The level of Microthecin is too low to act against the other two tested pathogens in this experiment.

No phytotoxic effects were observed on seed germination (The pelleted seeds germinated after a few days on the agar-plates).

PDA plates. Action against other plant pathogenic diseases

A number of other plant pathogenic fungi were tested on agar plates. Test system consisted of actively growing fungi placed in the centre of the 9-cm agar plate (pH 6.2) and 9mm holes were cut in the periphery of the agar plate. The 7 holes per plate contained the following:

GL: Glucan lyase

PD: Pyranosone dehydratase

The results are shown below in Table 12. Table 12

^* Septoria tritici was tested after plating of spores on PDA-plates. The spores seem not able to germinate in presence of media containing Pyranosone dehydratase. Substitution of dextrin with amylopectin resulted in the same pattern of fungal growth inhibition. In general treatment 3 (Substrate+GL+PD) seems to control the growth of a variety of fungal species.

Example 8 - Investigation of the stability of Microthecin in water by NMR- spectroscopy.

Microthecin 1 was prepared by enzymatic dehydration of 1 ,5-anhydro-D-fructose (Scheme 1).

1 ,5-Anhydro-D-fructose Microthecin 1 Scheme 1

A sample was delivered to for NMR spectroscopic investigations. After dissolving the compound (ca. 30 mg) in D₂O (1 ml) a ¹³C NMR spectrum was measured immediately. This showed the presence of 1 and another major compound, together with minor impurities. It was thus decided to investigate the stability of microthecin in water by running NMR spectra at intervals, as well as elucidating the structures of the compounds involved.

³C-NMR spectra were measured on day 1 , day 10, day 21 , day 32, day 46 and day 100. One ¹H-NMR experiment was performed day 101. A span of three months is thus covered. In the following description sample # 1 refers to the first experiment, sample # 2 to the second and so forth. All experiments were run at room temperature, on a Varian 300 MHz.

Sample # 1-5 (¹³C-NMR)

Sample # 1 :

The spectrum shows the presence of two major compounds A and B, together with a number of peaks in the ppm-region 60-100, which are currently unassigned. The two compounds A and B each shows 6 peaks : A (194.0, 152.5, 124.6, 96.4, 63.6 and 61.2 ppm) and B (190.0, 150.1 , 149.4, 120.9, 113.4 and 64.7 ppm).

Sample # 2 : Some peaks have disappeared, most prominently the two carbonyl peaks above 200 ppm. Also the amount of A has decreased to about % of the amount of B. The impurities are still present in the same amount.

Sample # 3 : Compound A has now completely disappeared. B is intact and so are the impurities.

Sample # 4 + 5 :

The spectra are identical with spectrum #3. Thus no further changes had taken place. Compound B is present as well as the impurities. The most prominent of these are peaks at 94.0, 76.5, 70.9, 64.3 and 37.5 ppm.

Sample # 5 (¹H-NMR)

This proton spectrum shows peaks at the following chemical shifts : 7.75, 7.74, 7.39 4.73, 4.66 (D₂O) and a number of small peaks in the region 3.4-3.8 ppm The pH value of the sample was now 3. The pH of the original solution has not been measured.

Conclusion :

Surveying the literature of microthecin revealed that a number of compounds closely related to 1 often appear in the same environment, and it was thus among these compounds we looked for candidates, which would fit with the observed NMR-data. A summary of the ¹³C and ¹H chemical shifts for compounds 1-4 are given in table 13 and 15, respectively. Table 14 and 16 show the observed values, ¹³C and ¹H respectively, of the sample under investigation.

Microthecin 1 Ascopyrone P 2 Echinosporin 3 2-furyl-hydroxy- methylketon 4 Scheme 2

Table 13 ¹³C chemical shifts: Literature Values

(d) = D₂O, (c) = CDCI₃ , nd = no data

Table 14 C chemical shifts: Observed Values; Compounds A and B

^X L. Kenne et al. Phytochemistry, 1996, 41, 151-154 ² R. Baute et al. Phytochemistry 1993, 33, 41-45

³ R. Baute et al. Phytochemistry, 1991, 30, 1419-1423

⁴ Matsumoto et al. JOC 1985, 50, 603-606

⁵ Fujimoto et al. Chem. Pharm. Bull. 1996, 44, 1843-1848

Table 15 ¹H chemical shifts: Literature Values

(c) = CDCI₃

Table 16 ¹H chemical shifts: Observed Values

(d)= D₂O

The ¹H NMR spectrum of the final compound shows the presence of 3 vinylic protons. This indicates that a substituted furane might have been formed. Comparison of the observed proton chemical shifts with those from compound 4, indicates that the compounds are identical. The differences in chemical shifts are due to the different solvents used, CDCI₃ and D₂O, respectively. The ¹³C chemical shifts are in good agreement (Tables 1 and 2). Furthermore, the ¹³C δ values of compound B correspond well with the ones from microthecin. Thus, the two compounds which were present in almost equal amounts , are believed to be microthecin 1 (equals A), and 1-(2-furyl)-2- hydroxy-ethanon 4 (equals B). (scheme 3) The NMR data are in good agreement the small difference i ppm-values are likely to be due to different solvents being used.

1-(2-furyl)-2- Microthecin 1 hydroxy-ethanon 4

Scheme 3. Compound A and B

It is known from literature that hot, acidic conditions will convert 1 into 4⁷ . A likely proposed mechanism for this conversion is presented in scheme 4 :

⁶ Fujimoto et al. Chem. Pharm. Bull. 1996, 44, 1843-1848

Scheme 4. A proposed mechanism for the conversion of 1 to 4

The stability of the product is likely to drive the reaction to completion.

In order to produce microthecin by dehydration of AF, it is therefore of extreme importance to avoid acidic conditions during the reaction and work up/purification procedures.

Example 9 - Formulations of microthecin an antimicrobial agent.

Example 9.1. Formation of microthecin and its degradation

The reaction mixture consisted of 0.77ml water, 0.2ml 0.1M pi buffer pH (6.5) and 0.02ml pyranosone dehydratase, 0.01 ml anhydrofructose (containing 130 micromole AF). The reaction was monitored at 226nm and 263nm for every 5min at 22.5°C.

From Figure 7 one can see that at 270 min absorbance reached the highest 0.8659 after that it started to decay. Absorbance at 263 nm was used as a reference wavelength.

Example 9.2. Microthecin is more stable in deionised water than in solution containing various salts.

Microthecin is significant more stable in a solution which is basically free from metal ions, especially transition metals and buffer substances.

⁷ R. Baute et al. Phytochemistry 1986, 25, 1472-1473 When microthecin is dissolved in deionised water (milliQ water for example) and kept at 22°C under room light, no loss of microthecin was observed after 1 day, and a loss of 8% was observed after 2 days, a loss of 63% after 8 days (see Figure 8 and 9). No loss was observed after 95°C for 7 min.

This results shown in Figure 8 are in contrast to what is reported in the literature where 55% of microthecin has been lost after 18 hours in citrate buffer (By Anders Broberg, Lennart Kenne, and Marianne Pedersen, Carbohydrate Research Volume 306, Issues 1- 2 , January 1998, Pages 171-175, Formation of 4-deoxy-glycero-hexo-2,3-diulo-furanose from microthecin)

Example 10 - Formulation Examples

The agrochemical formulation of the invention can be prepared as a wettable powder

Wettable Powder

The active ingredient is mixed well with the additives and ground thoroughly in an appropriate mill. Spray powders are obtained, which can be diluted with water to form suspensions of any desired concentration. Coated granule active ingredient (l:ll = 1:10) 8% polyethylene glycol (molecular weight 200) 3% kaolin 89%

The finely ground active ingredient is applied evenly to the kaolin which is moistened with polyethylene glycol, in a mixer. In this way, dust-free coated granules are obtained. Example 11 - Activity Against Erysiphe graminis on Barley

a) Residual Protective Activity

Barley plants of approximately 8 cm height are sprayed until dripping wet with an aqueous spray mixture prepared from wettable powder of the active ingredient mixture, and 3 to 4 hours later are dusted with conidia of the fungus. The infected plants are placed in a greenhouse at 22°C. 12 days after infection, the fungal attack is evaluated.

b) Systemic Activity

An aqueous spray mixture prepared from wettable powder of the active ingredient mixture is poured onto barley plants of approximately 8 cm height. Care is taken that the spray mixture does not come into contact with the parts of the plants that are above ground. 48 hours later, the plants are dusted with conidia of the fungus. The infected plants are placed in a greenhouse at 22°C. 12 days after infection, the fungal attack is evaluated.

Example 12 - Toxicity:

We have found that microthecin is less phytotoxic than the commercial available fungicide for treatment of sugar beet seeds against Aphanomyces. It was found that seedlings derived from seeds treated with microthecin emerge as fast (in fact a little faster) as the non-fungicide treated controls and faster than the commercial fungicide treated seeds.

In laboratory germination, the number of germs after 4 days and after 7 days were counted. No differences are observed at the 7 day germination, but in the 4 day germination the microthecin treated seems to be a little slower. In contrary, a specific parameter - that usually correlated well with the field germination data - namely the number of germs having a root length longer than 15mm at day 4, shows that Hymexazol (commercial name Tachigaren, active against Aphanomyces) suppress the speed, whereas microthecin did not. See table bvelow

Mention is made of U.S. applications Serial Nos. 10/283,940 (attorney ref. P11937US), 10/283,988 (attorney ref. P12627US), 10/283,987 (attorney ref. P11933US), 10/283,936 (attorney ref. P11934US), and 10/283,963 (attorney ref. P11938US) each filed on 30 October 2002.

Mention is also made of U.S. provisional applications Serial Nos. 60/343,485 (attorney ref. P11937USO), 60/343,313 (attorney ref. P12627USO), 60/343,447 (attorney ref. P11933USO), 60/343,368 (attorney ref. P11934USO), and 60/343,316 (attorney ref. P11938USO) each filed 21 December 2001.

Mention is also made of UK patent application Nos. 0126164.3 (attorney ref. P11937GB), 0126163.5 (attorney ref. P12627GB), 0126165.0 (attorney ref. P11933 GB), 0126186.6 (attorney ref. P11934 GB), and 0126162.7 (attorney ref. P11938 GB), each filed on 31 October 2001.

Mention is also made of UK patent application No. 0306312.0 (attorney ref. P16679GB) filed on 19 March 2003. Mention is also made of UK patent application Nos. 0226159.2 (attorney ref. P15628GB) filed on 8 November 2002, 0310479.1 (attorney ref. P15628GBR) filed on.7 May 2003, 0306315.3 (attorney ref. P16678GB) filed on 19 March 2003, and 0310480.9 (attorney ref. P16679GB) filed on 7 May 2003.

Mention is also made of U.S. provisional applications Serial No. 60/468,954 (attorney ref. P15628USO) filed on 7 May 2003.

Each of these applications, together with any document cited or referenced in each of these applications, is hereby incorporated herein by reference. All documents cited herein and all documents cited or referenced in herein cited documents (including any manufacturer's specifications, instructions, etc. as to products mentioned herein or in documents cited or referenced in herein cited documents) are hereby incorporated herein by reference.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following.

Claims

1. An antimicrobial material comprising

(i) an antimicrobial compound in a stabilised form (a "stabilised compound"), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to the antimicrobial compound; and (b) (I) a primary precursor, or

(II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and a secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof.

2. An antimicrobial material according to claim 1 wherein the antimicrobial compound is a compound having Formula I,

wherein R^ and R₂ are independently selected from H and C(=O)R₃ wherein R₃ is a hydrocarbyl group, and R₄ is from H and OH

3. An antimicrobial material according to claim 2 wherein at least one of R¹ and R² is H.

4. An antimicrobial material according to claim 2 or 3 wherein R₃ is a hydrocarbon group.

5. An antimicrobial material according to claim 2, 3 or 4 wherein R₃ is an alkyl group.

6. An antimicrobial material according to any one of claims 2 to 5 wherein R₃ is a straight chain alkyl group.

7. An antimicrobial material according to any one of claims 2 to 6 wherein R₃ contains from 1 to 5 carbon atoms.

8. An antimicrobial material according to of any one of claims 2 to 7 wherein R₃ is a C_LS alkyl group.

9. An antimicrobial material according to claim 2 wherein R is H, R is H and R is H.

10. An antimicrobial material according to claim 2 wherein R¹ is H, R² is H and R⁴ is OH.

11 An antimicrobial material according to claim 1 wherein the antimicrobial compound is microthecin.

12. An antimicrobial material according to of any one of claims 1 to 11 comprising the stabilised compound.

13. An antimicrobial material according to of any one of claims 1 to 11 comprising (a) the first conversion agent; and

(b) (I) the primary precursor, or

(II) the second conversion agent and the secondary precursor.

14. An antimicrobial material according to of any one of claims 1 to 11 comprising (a) the first conversion agent; and

(b) a primary precursor.

15. An antimicrobial material according to of any one of claims 1 to 11 comprising (a) the first conversion agent; and (b) the second conversion agent and the secondary precursor.

16. An antimicrobial material according to of any one of claims 1 to 15 wherein the first conversion agent is an enzyme.

17. An antimicrobial material according to of any one of claims 1 to 16 wherein the first conversion agent is a dehydratase enzyme.

18. An antimicrobial material according to of any one of claims 1 to 17 wherein the first conversion agent is a pyranosone dehydratase.

19. An antimicrobial material according to of any one of claims 1 to 18 wherein the second conversion agent is an enzyme.

20. An antimicrobial material according to of any one of claims 1 to 19 wherein the second conversion agent is a lyase or oxidase enzyme.

21. An antimicrobial material according to of any one of claims 1 to 20 wherein the second conversion agent is a glucan lyase or a pyranose-2-oxidase.

22. An antimicrobial material according to of any one of claims 1 to 21 wherein the primary precursor is glucosone or 1 ,5-D-anhydrofructose.

23. An antimicrobial material according to of any one of claims 1 to 22 wherein the secondary precursor is a starch dextrin or glucose.

24. An antimicrobial material according to of any one of claims 1 to 23 wherein the first conversion agent is a pyranosone dehydratase and the primary precursor is glucosone or 1 ,5-D-anhydrofructose.

25. An antimicrobial material according to of any one of claims 1 to 25 wherein the first conversion agent is a pyranosone dehydratase, the second conversion agent is a glucan lyase and the secondary precursor is a starch.

26. An antimicrobial material according to of any one of claims 1 to 25 wherein the first conversion agent is a pyranosone dehydratase, the second conversion agent is a pyranose-2-oxidase and the secondary precursor is glucose.

27. An antimicrobial material according to of any one of the preceding claims wherein the stabilised compound is in the solid phase.

28. An antimicrobial material according to claim 27 wherein the solid phase is in the form of a powder, a tablet, lozenge or pessary..

29. An antimicrobial material according to claim 27 or 28 wherein the solid phase stabilised compound is obtained or is obtainable by freeze-drying or spray-dry.

30. An antimicrobial material according to claim 29 wherein the solid phase stabilised compound is obtained or is obtainable by spray drying the antimicrobial compound with a carrier.

31. An antimicrobial material according to claim 30 wherein the carrier is a dextrin.

32. An antimicrobial material according to of any one of the preceding claims wherein the stabilised compound comprises (i) the antimicrobial compound and (ii) a stabilising agent which stabilises the antimicrobial compound.

33. An antimicrobial material according to claim 32 wherein the stabilising agent is selected from anti-oxidants, free radical quenchers, (metal) chelators, emulsifiers and UV protectants.

34. An antimicrobial material according to claim 33 wherein the emulsifier is selected from distilled monoglycerides, acetic acid esters of mono and diglycerides, citric acid esters of mono and diglycerides, lactic acid esters of mono and diglycerides, mono and diglycerides, polyglycerol esters of fatty acids, polyglycerol polyricinoleate, propylene glycol esters of fatty acids, sorbitan monostearates, sorbitan tristearates, sodium stearoyl lactylates, calcium stearoyl lactylates, diacetyl tartaric acid esters of mono- and diglycerides, Tween 20, 40, 60 and 80, Triton X-100, SDS, and octy-glucoside.

35. An antimicrobial material according to claim 32, 33 or 34 wherein the stabilising agent increases the hydrophobicity of the composition.

36. An antimicrobial material according to claim 32, 33, 34 or 35 wherein the stabilising agent is a polar solvent.

37. An antimicrobial material according to claim 36 wherein the polar solvent is selected from ethanol, methanol, glycerol and mixtures thereof.

38. An antimicrobial material according to of any one of the preceding claims wherein the stabilised compound is formulated in a composition which has a pH of at least 7.

39. An antimicrobial material according to of any one of the preceding claims wherein the stabilised compound is micro-encapsulated antimicrobial compound.

40. An antimicrobial material according to of any one of the preceding claims further comprising an agriculturally acceptable carrier, excipient or diluent.

41. An antimicrobial material according to of any one of the preceding claims further comprising an edible carrier, excipient or diluent.

42. A process for preventing and/or inhibiting the growth of, and/or killing a microorganism in a material, comprising the steps of contacting the material with an antimicrobial material as defined in any one of claims 1 to 41.

43. Use of an antimicrobial material for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the antimicrobial material is as defined in any one of claims 1 to 41.

44. A process according to claim 42 wherein the first conversion agent is a pyranosone dehydratase.

45. A process according to claim 42 wherein the second conversion agent is a glucan lyase or a pyranose-2-oxidase.

46. A process according to claim 42 wherein the primary precursor is glucosone or 1 ,5-D-anhydrofructose.

47. A process according to claim 42 wherein the secondary precursor is a starch dextrin or glucose.

48. A process according to claim 42 wherein the antimicrobial material comprises

(a) the first conversion agent, where the first conversion agent is a pyranosone dehydratase; and

(b) the primary precursor, wherein the primary precursor is glucosone or 1,5-D- anhydrofructose.

49. A process according to claim 42 wherein the antimicrobial material comprises

(a) the first conversion agent, wherein the first conversion agent is a pyranosone dehydratase; and (b) the second conversion agent, wherein the second conversion agent is a glucan lyase, and the secondary precursor, wherein the secondary precursor is a starch.

50. A process according to claim 42 wherein the antimicrobial material comprises

(a) the first conversion agent, wherein the first conversion agent is a pyranosone dehydratase; and

(b) the second conversion agent, wherein the second conversion agent is a pyranose-2- oxidase, and the secondary precursor, wherein the secondary precursor is glucose.

51. A process according to claim 42 or use according to claim 43 wherein the material is a seed or a plant.

52. A process according to claim 42 or use according to claim 43 wherein the material is a seed.

53. A process or use according to claim 51 or 52 for preventing and/or inhibiting the growth of, and/or killing Botrytis, crown and root rot, or mildew.

54. A process or use according to claim 51 or 52 for preventing and/or inhibiting the growth of, and/or killing a plant pathogen selected from downey mildew, powdery mildew, Mycosphaerella, Paracercospora,, Ascomycetes, Leptosphaeria, Phoma, Xanthomonas, Pseudomonas, Fusarium, Rhizoctonia, Pythium, Phytophthora, Thielaviopsis, Aspergillus, Alternaria; Ascochyta; Botrytis; Cercospora; Colletotrichum; Diplodia; Erysiphe; Gaeumanomyces; Helminthosporium; Macrophomina; Nectria; Peronospora; Phoma; Phymatotrichum; Plasmopara; Podosphaera; Puccinia; Puthium; Pyrenophora; Pyricularia; Scerotium; Sckerotinia; Septoria; Uncinula; Venturia; Verticillium or Penicillium,

55. A process or use according to claim 51 or 52 for preventing and/or inhibiting the growth of, and/or killing a plant pathogen selected from Blumeria graminis, Erysiphe graminis, Botrytis cinerea, Peronospora, Bremia lactucae, Phytophthora, Puccinia, Uromyces, Alternaria, Bipolaris, Drechslerea, Helmintosporium, Exserohilum, Sclerotinia, Fusarium oxysporum, Fusarium, Cercospora, Septoria (tritici), Stagonospora (nodorum), Phoma (lingam), and Eutypa lata.

56. A process or use according to any one claims 51 to 55 wherein the seed or plant is one of a cereal, barley, wheat, maize, Triticale, rice, oats, rye, field beans, apple, pear, strawberry, pea, tomato, grape, Brassicas, tobacco, lettuce, sorghum, cotton, sugar cane, legumes, ornamentals, pot plants, turf grasses, sugar beet, celery, Crucifers, plantain, banana, grasses, oilseed rape, sunflower, soybean, and peanut.

57. A process or use according to any one claims 51 to 56 wherein the seed or plant material is sugar beet seeds or barley.

58. A process according to claim 42 or use according to claim 43 wherein the material is a foodstuff.

59. A process according to claim 42 or use according to claim 43 wherein the material is an animal feed.

60. A process or use according to any one of claims 42 to 59 for preventing and/or inhibiting the growth of mildew.

61. A process for preventing and/or inhibiting the growth of, and/or killing a microorganism in a material, comprising the steps of contacting the material either sequentially or simultaneously with

(i) an antimicrobial compound in a stabilised form (a "stabilised compound"), or (ii) (a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to the antimicrobial compound; and (b) (I) a primary precursor, or (II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and a secondary precursor; wherein the antimicrobial compound is selected from microthecin and derivatives thereof.

62. A kit for preparing an antimicrobial material as defined in any one of claims 1 to 41 , the kit comprising

(a) a first conversion agent capable of converting a precursor of an antimicrobial compound (a "primary precursor") to and antimicrobial compound, wherein the antimicrobial compound is selected from microthecin and derivatives thereof; and

(b) (I) a primary precursor, or

(II) a second conversion agent capable of converting a precursor of the primary antimicrobial precursor (a "secondary precursor") to the primary precursor; and a secondary precursor; in separate packages or containers; optionally with instructions for admixture and/or contacting and/or use.

63. A process for preventing and/or inhibiting the growth of, and/or killing a micro- organism in a material, comprising the steps of contacting the material with microthecin or a derivative thereof, wherein the micro-organism is selected from Alternaria spp. , Albugo spp., Amyloporia spp., Ascochyta spp., Aspergillus spp, Basidiophora spp, Bipolaris spp, Botrytis spp, Bremia spp, Cladosporium spp, Claviceps spp, Coniophora spp., Colletothchum spp, Diplodia spp, Dipiocarpon spp., Donkiopoha spp., Drechslera spp, Erysiphe spp, Eutypa spp, Fibropo a spp., Fusarium spp, Gaeumanomyces spp, Geotrichum spp, Guignardia spp, Gymnosporangium spp, Helmintosporium spp, Hemileia spp, Kabatiella spp, Leptosphaeria spp, Macrophomina spp, Marssonina spp., Monilinea spp, Merulus spp, Mycosphaerella spp, Nectria spp, Paracercospora spp, Penicillium spp, Peronophythora spp, Peronospora spp, Phellehas spp., Phoma spp, Phomopsis spp, Phymatotrichum spp, Phytophthora spp, Plasmophora spp, Podosphaera spp, Porai spp., Pseudocercosporella spp, Pseudoperonospora spp, Puccinia spp, Pyrenophora spp, Pyricularia spp, Rhizopus spp, Sclerophthora spp, Sclerotinia spp, Sclerotium spp, Septoria spp, Serpula spp., Sphaerotheca spp, Stagonospora spp, Taphπna spp, Thielaviopsis spp, Tilletia spp, Thchoderma spp, Uncinula spp, Ustilago spp, Venturia spp, and Verticillium spp

64. Use of an antimicrobial compound material for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material, wherein the antimicrobial compound is microthecin or a derivative thereof, and wherein the micro-organism is selected from Alternaria spp. , Albugo spp., Amyloporia spp., Ascochyta spp., Aspergillus spp, Basidiophora spp, Bipolaris spp, Botrytis spp, Bremia spp, Cladosporium spp, Claviceps spp, Coniophora spp., Colletothchum spp, Diplodia spp, Dipiocarpon spp., Donkiopoha spp., Drechslera spp, Erysiphe spp, Eutypa spp, Fibroporia spp., Fusarium spp, Gaeumanomyces spp, Geotrichum spp, Guignardia spp, Gymnosporangium spp, Helmintosporium spp, Hemileia spp, Kabatiella spp, Leptosphaeria spp, Macrophomina spp, Marssonina spp., Monilinea spp, Merulus spp, Mycosphaerella spp, Nectria spp, Paracercospora spp, Penicillium spp, Peronophythora spp, Peronospora spp, Phellehas spp., Phoma spp, Phomopsis spp, Phymatotrichum spp, Phytophthora spp, Plasmophora spp, Podosphaera spp, Porai spp., Pseudocercosporella spp, Pseudoperonospora spp, Puccinia spp, Pyrenophora spp, Pyricularia spp, Rhizopus spp, Sclerophthora spp, Sclerotinia spp, Sclerotium spp, Septoria spp, Serpuia spp., Sphaerotheca spp, Stagonospora spp, Taph na spp, Thielaviopsis spp, Tilletia spp, Trichoderma spp, Uncinula spp, Ustilago spp, Venturia spp, and Verticillium spp

65. A process according to claim 63 or use according to claim 64 characterised by the features of any one of claims 1 to 60.

66. A composition substantially as described herein and with reference to the accompanying Examples.

67. A process for preventing and/or inhibiting the growth of, and/or killing a microorganism in a material substantially as described herein and with reference to the accompanying Examples

68. A kit substantially as described herein and with reference to the accompanying Examples.

69. Use of an antimicrobial material for preventing and/or inhibiting the growth of, and/or killing a micro-organism in a material substantially as described herein and with reference to the accompanying Examples