Patent Publication Number: US-11033025-B2

Title: Coating compositions for pathogen control in monocotyledonous plants

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This is a National Stage of International Application No. PCT/GB2012/000356, filed Apr. 19, 2012, claiming priority from British Patent Application No. 1106741.0, filed Apr. 20, 2011, the contents of all of which are incorporated herein by reference in their entirety. 
     The present invention relates to coating compositions including an organic component and a biological agent for applying to plant structures of monocotyledonous plants, such as seeds, from which roots and shoots are capable of growing, uses of coating compositions on monocotyledonous plant structures, such as seeds, methods of producing such coating compositions and monocotyledonous plant structures such as seeds coated with such coating compositions. In particular, the invention relates to monocotyledonous plant structure coating compositions that comprise an organic carrying material and biological agents selected from chemicals and biological agents active against one of more plant pathogens selected from bacterial, fungal and arthropod pathogens that infest plant structures of monocotyledonous plants, such as seeds and bulbs. 
     Large losses in monocotyledonous crops are recorded annually and come about as a result of plant infestations due to pathogens such as bacteria, fungi and arthropods which can infest the plant at various stages of development, such as at the seed stage. Agronomic losses due to pathogen infestations remain high despite many defensive measures that have been devised by man to combat such infestations. Such defensive measures include the use of synthetic chemicals; the employment of genetic engineering in plants; and the use of live biological agents that are applied in the form of coatings, sprays and washes to monocotyledonous seeds. 
     Pesticides in the form of chemical agents such as fungicides, bactericides and arthropodicides, typically in the form of insecticides and/or acaricides may be applied to monocotyledonous crops in the form of soil drenches, liquid seed treatments and the like. Such kinds of chemical treatments tend to be indiscriminate and may adversely affect beneficial bacteria, fungi and arthropods as well as the plant pathogens at which such treatments are targeted. 
     When conventional pesticides are used, for example, as seed treatments the seeds are coated with pesticide directly or the pesticide is applied to the seed in the presence of an inorganic carrier. Such seed treatments are typically applied in liquid form or as wet slurry and subsequently the seeds are dried. Such treatments are mostly aimed at providing direct protection against pathogens such as arthropods and/or seed borne microorganisms and/or soil borne microorganisms that attack the seed. The high level of chemicals that are typically used introduces a chemical load to the environment that may give rise to ecological concerns. 
     One problem in applying a biological agent that is a chemical agent in conventional seed coating procedures is that the chemical agent is typically applied as slurry and this may give rise to an uneven application of the coating whereby the seeds are not fully coated or a percentage of the seeds, up to 20% depending on seed type and coating procedure do not get substantially coated. Furthermore, the seed coatings may not be uniform and this gives rise to physical weaknesses in the seed coat and the coating may flake off. 
     A further problem arises when using biological agents that are selected from beneficial live bacterial and fungal species that may be applied conventionally to plant structures, for example as spores in conjunction with an inorganic carrier in the form of particulate compositions or in the form of liquid compositions which may then be dried back, is that the applied biological agents rapidly lose viability. Without the intention of being bound by theory it is thought that as the seeds or storage organs are dried back, the micro-environment alters and the viability of applied live biological agents may be seen to decrease sharply and almost as soon as the applied composition is dried. The loss of viability of the biological agent is typically associated with the splitting of the fungal or bacterial spores which renders them non-viable. 
     It has now been found that by using an organic carrier material in conjunction with a biological agent, the viability of the biological agent is improved on monocotyledonous seeds, relative to the viability of biological agents applied to such seeds conventionally. Furthermore, the coating of the plant structure is less susceptible to flaking. 
     It is an object of the present invention to supply improved coatings comprising biological agents for monocotyledonous plant structures such as seeds. Furthermore, it is an object of the invention to supply seed coatings that utilise fewer chemical additives and/or lesser amounts thereof for protecting seed and/or young plantlets from pathogens than conventional seed coatings. 
     These and other objects of the invention will become apparent from the following description and examples. 
     According to the present invention there is provided a monocotyledonous plant structure coating composition, wherein the said coating composition comprises at least one organic carrier material in the form of particles wherein the carrier material is selected from waxes having a melting point of ≥50° Centigrade and one or more biological agents that possess an activity against one or more pathogens of a monocotyledonous plant. 
     Farmers crops are typically destined for many diverse markets such as food derived from cereals e.g. bread, breakfast cereals, starch production, brewing and in the production of fodder for domesticated livestock, such as chickens and cattle. For the purposes of the present invention “seeds” and “seed” are to be construed as being in the plural or the singular depending on context. Reference to “seed” and “seeds” is used interchangeably and means seeds, typically viable seeds, to which compositions of the invention may be applied. Monocotyledonous seed as provided herein means seeds that are capable of germinating to at least conventional levels of germination typical of monocotyledonous plant seeds. Monocotyledonous seeds includes those that may be used for the planting of monocotyledonous plants such as varieties of  Oryza  spp. such as  Oryza sativa  (rice),  Triticum  spp. such as  T. aestivum  (wheat: Spring and Winter varieties),  Secale  spp. such as  Secale cereale  (rye),  Avena  spp. such as  Avena sativa  (oats),  Zea  spp. such as  Zea mays  [corn (maize)],  Sorghum  spp. such as  Sorghum bicolor  (sorghum),  Hordeum  spp. such as  Hordeum vulgare  (barley) and hybrid crosses of monotcotyledonous plants such as x  Triticosecale  (triticale: cross between wheat and rye). 
     The organic carrier material is selected from organic materials that can be applied to monocotyledon seeds preferably as a dry powder wherein the powder particles are of a pre-determined volume mean diameter or the powder particles are presented in liquid form, such as an oleaginous formulation or as an aqueous formulation. 
     Generally, the organic carrier material of use in the invention is present in the form of particles in a composition of the invention and which composition has a volume mean diameter of a certain size as defined herein. To obtain particles of organic materials of a volume mean diameter applicable for use in the invention, organic materials in the form of, for example, 1 to 5 kilogram blocks or tablets may be broken up or kibbled into small millimeter-sized pieces (such as from 2 mm-8 mm approximate diameter in size, for example from 4 mm to 6 mm) in a kibbling machine. The millimeter-sized pieces can then be passed through a comminuting means such as a standard mill, e.g. an Apex Comminuting mill, and milled or comminuted into particles having an approximate diameter in the range from 100 μm-500 μm, for example from 250 μm-300 μm. The micron-sized comminuted particles can then be passed through a micronising apparatus, such as an AFG micronising air mill to obtain particles of a desired VMD range, such as from 15 μm-20 μm, that is of use in the present invention. The skilled addressee will appreciate that such procedures for obtaining small particles are well known in the art. Preferably, dry powder compositions of the invention comprise composite particles having a volume mean diameter of ≥5 μm, for example of 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm up to 40 μm or any value thereinbetween. As stated herein, the volume mean diameter of the composite particles is typically ≥10 μm or ≥12 μm and may lie in the range from 10 μm to 200 μm and may have a value that lies anywhere there inbetween. for example from ≥10 μm to 100 μm; or from ≥10 μm to 40 μm; or from ≥10 μm to 30 μm or any desired volume mean diameter value in between. Preferably, dry powder compositions of the invention comprise particles having a volume mean diameter of ≥8 μm, for example of 8 μm, 9 μm, 9.7 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm and the like up to any volume mean diameter of choice, such as up to 200 μm or any volume mean diameter in between for example 40 μm or 30 μm. Particles of the invention that possess a volume mean diameter ≥10 μm are considered to be less of a thoracic hazard to humans and are not thought to be allergenic. 
     In liquid formulations, particles of a pre-determined volume mean diameter are suspended therein in a suspension formulation and applied to the seeds which are then dried using conventional drying procedures. Where the organic carrier material is applied to monocotyledonous plant seeds in a dry powder form, the particles of the organic powder material may have a volume mean diameter as herein described. The “one or more biological agents” that may be added to dry powders of the invention include chemicals of use against pathogens such as arthropods such as insects, arachnids or if appropriate, their larvae, eggs, or pupae; chemicals of use against bacterial pathogens; and chemicals of use against fungal pathogens. Additionally, beneficial live biological agents may be added to such dry powders of use in the present invention, the live biological agents being able to target bacterial pathogens of the monocotyledonous plant and/or to target fungal pathogens of the monocotyledonous plant. Spores of choice of beneficial live biological agents such as fungal conidia or hyphae or mycelia of fungi that do not form spores or conidia-like structures may be added to dry powders of use in the present invention. Suitable organic carrier materials of use in the invention are typically made up of waxes having a melting point of ≥50° C., more preferably of ≥60° C., and most preferably are made up of hard waxes having a melting point of ≥70° C. 
     Natural waxes of use in the present invention include carnauba wax, beeswax, Chinese wax, shellac wax, spermaceti wax, myricyl palmitate, cetyl palmitate, candelilla wax, castor wax, ouricury wax, wool wax, sugar cane wax, retamo wax, rice bran wax and the like. 
     Synthetic waxes of use in the present invention include suitable waxes selected from paraffin wax, microcrystalline wax, Polyethylene waxes, Fischer-Tropsch waxes, substituted amide waxes, polymerized α-olefins and the like. 
     Mineral waxes of use in the invention include montan wax (e.g. Lumax® Bayer) ceresin wax, ozocerite, peat wax and the like. 
     Suitable organic carrier particles may be selected from waxes such as carnauba wax, beeswax, montan wax, Chinese wax, shellac wax, spermaceti wax, myricyl palmitate, cetyl palmitate, candelilla wax, castor wax, ouricury wax, wool wax, sugar cane wax, retamo wax, and rice bran wax or a mixture of two or more thereof. Such waxes typically display a high enthalpy of lattice energy during melt. Preferably the organic carrier material is carnauba wax which may be applied in liquid form, typically in the form of a suspension, or more preferably in powder form as discrete particles. Generally, the particles of use in the invention possess a volume mean diameter as herein described. 
     Additionally, the organic carrier particles of use in compositions of the invention may contain other components such as additives selected from UV blockers such as beta-carotene or p-amino benzoic acid, colouring agents such as optical brighteners and commercially available colouring agents such as food colouring agents, plasticisers such as glycerine or soy oil, antimicrobials such as potassium sorbate, nitrates, nitrites, propylene oxide and the like, antioxidants such as vitamin E, butylated hydroxyl anisole (BHA), butylated hydroxytoluene (BHT), and other antioxidants that may be present, or mixtures thereof. The skilled addressee will appreciate that the selection of such commonly included additives will be made depending on end purpose, and perceived need. 
     Liquid formulations of the invention may be formulated as an aqueous formulation or as an oleaginous formulation, depending on design. Aqueous formulations may include surfactants selected from commercially available surfactants such as Libsorb, Silwet L77, Tween 80, Torpedo II, Newmans T80, Fortune, Guard, Rhino, Biopower, and the like. 
     Oleaginous formulations, that is to say oil-based formulations, may contain any oil suitable for use in the present invention which may be selected from petroleum oils, such as paraffin oil, and vegetable oils such as rapeseed oil, soybean oil, sunflower oil, palm oil and the like. Oil formulations of use in the invention contain organic carrier particles as described herein and these in turn may be admixed with flow agents such as hydrophilic precipitated silicas, for example Sipernat 383 DS, Sipernat 320, EXP 4350, and Sipernat D-17 and the like. Such free-flowing agents may be dispersed in oils, for example, for anti-foaming purposes. 
     The skilled addressee will appreciate that where an aqueous or an oil formulation may be used to apply biological agents of use in the invention, the liquid element should be removed from the coated plant structure after coating is achieved, for example by drying off using conventional drying processes, leaving a seed coating composition in dry particulate form, wherein the seed coating composition is made up of the organic carrier as herein described and the at least one biological agent, also as herein described. 
     A biological agent for the purposes of the present invention is one that can be used to control the population of a plant pathogen of a monocotyledonous plant, and may be selected from chemical fungicides, arthropodicides such as insecticides and acaricides, bactericides and from live biological agents that are able to control the population of one or more seed or soil borne pathogens of a monocotyledonous seed. Preferably, the population of the soil borne pathogen on or in the immediate proximity of the monocotyledonous seed is reduced either by the biological agent rendering it unable to reproduce or by killing it. Examples of biological agents of use in the present invention that are chemicals of use on monocotyledonous seeds include those chemical agents most commonly used on stored grain seeds that are effective against arthropods such as rice weevil,  Sitophilus oryza ; granary weevil,  Sitophilus granaries ; lesser grain borer,  Rhyzopertha dominica; Angoumois  grain moth,  Sitotroga cerealella ; cadelle,  Tenebroides mauritanicus ; saw-toothed grain beetle,  Oryzaephilus surinamensis ; flat grain beetle,  Cryptolestes pusillus ; flour beetles,  Tribolium  species; dermestids,  Trogoderma  species; bruchids, several bean and cowpea weevils; Indian-meal moth,  Plodia interpunctella ; and almond moth,  Ephestia cautella . Examples of suitable chemicals of use in the invention may be selected from the pyrethroids, such as α-cypermethrin, λ-cyhalothrin, [cyano-(3-phenoxyphenyl)-methyl]3-(2,2-dibromoethenyl)-2,2-dimethyl-cyclopropane-1-carboxylate (deltamethrin), and  T -fluvalinate, the organophosphates such as chlorpyriphos (diethoxy-sulfanylidene-(3,5,6-trichloropyridin-2-yl)oxy-I{circumflex over ( )}{5}-phosphane), malathion (diethyl 2 dimethoxyphosphino-thioyl-sulfanylbutanedioate), coumaphos (3-chloro-7-diethoxyphosphinothioyloxy-4-methylcoumarin), and stirifos ([(E)-2-chloro-1-(2,4,5-trichlorophenyl)ethenyl]dimethyl phosphate) the carbamates such as amitraz (N-(2,4-dimethylphenyl)-N-[(2,4-dimethylphenyl)iminomethyl]-N-methylmethanimidamide), the spinosans such as spinosad (Dow Agrichemical, France), the gamma amino butyric acid (GABA) inhibitors such as fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4 (trifluoromethylsulfinyl)pyrazole-3-carbonitrile), the neonicotinoids such as imidacloprid (N-[1-[(6-Chloro-3-pyridyl)methyl]-4,5-dihydroimidazol-2-yl]nitramide), the anthranilamides, the formononetins such as 7-Hydroxy-3-(4-methoxyphenyl)chromone, the essential oils such as tea tree oil, thyme oil (also known as thymol), citronella oil, and menthol, and the insect growth regulators such as methoxyfenozide (N-tert-butyl-N′-(3-methoxy-o-toluoyl)-3,5-xylohydrazide) and the like. 
     Examples of live biological agents (also known as biocontrol organisms or biocontrol agents) that are commonly referred to in the art as “biological antagonists” that may be used in coating compositions of the present invention include  Pseudomonas  spp. such as  P. Chlororaphis  for use on barley and oats and other monocotyledonous plants (available from BioAgri AB, Uppsala, Sweden),  Burkholderia  spp. such as  B. cepaciatype  Wisconsin for use on barley, sorghum, and wheat (available as “Deny” from Stine Microbial Products, Memphis, USA; and for use on Maize  B. cepaciatype  available as “Intercept” from Soil Technologies Corp., Fairfield, USA). 
     The skilled addressee will appreciate that compositions of the invention may also be added direct to the soil or growing medium into which plant structures as herein defined are to be planted. Such compositions may be added as powders and mixed with the soil or applied as liquid suspensions using conventional procedures. 
     Soil borne pathogens for the purposes of the present invention are ones that are able to colonise the seed cuticle and/or ones that populate the soil and which are capable of acting on monocotyledon seeds. Such soil borne pathogens are typically bacteria and/or fungi. Examples of soil borne bacterial and fungal pathogens that attack monocotyledonous plants include  Rhizoctonia  spp. (e.g.  R. microsclerotia  active against maize; and rice; sorghum; wheat; barley; oats; and rye),  Aspergillus  spp. such as  A. flavus  and  A. niger  (e.g. active against maize),  Tilletia  spp. such as  T. tritici , and  T. laevis  (e.g. active against wheat)  Sclerophthora  spp. such as  S. rayssiae , and  S. graminicola  (e.g. active against maize),  Peronosclerospora  spp. such as  P. sorghi  and  P. spontanea  (e.g. active against maize).  Pythium  spp. (e.g. active against maize; rice; sorghum; wheat; barley; oats; rye),  Fusarium  spp. (e.g. active against maize; rice; sorghum; wheat; barley; oats; rye),  Claviceps  spp. such as  C. purpurea  (e.g. active against rye; triticale; wheat; and barley),  C. africana  (e.g. active against sorghum),  C. gigantea  (e.g. active against maize),  Gibberella  spp. such as  G. Avenacea  (e.g. active against maize),  Burkholderia glumae  (e.g. active against rice)  Pseudomonas fuscovaginae  (e.g. active against rice),  Sclerophthora  spp. such as  S. macrospora  (e.g. active against rice),  Cochliobolus  spp. such as  C. miyabeanus  (e.g. active against rice),  Fusarium  spp. (active against rice, oats, wheat; maize), and the like. 
     According to a further aspect of the invention there is provided use of organic carrier particles of wax in the manufacture of a coating composition as defined herein that includes a biological agent as defined herein above. In a preferment of this aspect of the invention, the coating composition is a seed coating composition. In a further preferment of this aspect of the invention the coating composition is a storage organ coating composition wherein the storage organ is selected from tubers, tuberous roots, corms, bulbs and rhizomes. The organic carrier particles are selected from natural waxes, synthetic waxes, and mineral waxes having a melting point of ≥50° C., more preferably of ≥60° C., and most preferably are made up of hard waxes having a melting point of ≥70° C. Suitable waxes of use in this aspect of the invention may be selected from waxes such as carnauba wax, beeswax, montan wax, Chinese wax, shellac wax, spermaceti wax, myricyl palmitate, cetyl palmitate, candelilla wax, castor wax, ouricury wax, wool wax, sugar cane wax, retamo wax, and rice bran wax or a mixture of two or more thereof. Preferably, the seed coating that is employed in this aspect of the invention includes carnauba wax as the organic carrier. Preferably, in this aspect of the invention, the organic carrier particles have a mean volume diameter ≥5 μm, such as in the range ≥8 μm to 200 μm, as herein described. 
     In a third aspect of the invention there is provided use of wax as an organic carrier in particulate form in a monocotyledonous seed coating composition as described herein. The organic carrier particles in this aspect of the invention are selected from natural waxes, synthetic waxes, and mineral waxes having a melting point of ≥50° C., more preferably of ≥60° C., and most preferably are made up of hard waxes having a melting point of ≥70° C. Suitable organic carrier particles of use in this aspect of the invention may be selected from carnauba wax, beeswax, montan wax, Chinese wax, shellac wax, spermaceti wax, myricyl palmitate, cetyl palmitate, candelilla wax, castor wax, ouricury wax, wool wax, sugar cane wax, retamo wax, and rice bran wax or a mixture of two or more thereof. Preferably, the wax carrier particles of use in this aspect of the invention comprise organic carrier particles of carnauba wax. Preferably still, the organic carrier particles of use in this aspect of the invention have a mean volume diameter ≥8 μm, such as in the range of ≥10 μm to 200 μm. 
     In a fourth aspect of the invention there is provided a method of manufacturing seed coating composition as herein described that comprises 
     1) selecting an organic carrier material wherein the carrier material is selected from waxes having a melting point of ≥50° Centigrade; 
     2) comminuting said organic carrier material into particles of a desired mean volume diameter ≥5 μm, such as in the range ≥8 μm to 200 μm; and 
     3) adding biological agent to the product particles of step 2). 
     The biological agent of use in this aspect of the invention is selected from a chemical agent which is an arthropodicide such as an insecticide or an acaricide or a mixture thereof, or a chemical fungicide or a fungus species and/or a bacterium species or a mixture of one or more thereof. Suitable fungus species and bacterium species are known and may be selected from  Trichoderma  spp., such as  Trichoderma harzanium  for use on wheat and  Bacillus  spp. such as  Bacillus subtilis  for use on wheat, and  Pseudomonas  species such as  P. fluorescens  for use on wheat and  P. Chlororaphis  for use on barley and oats and other monocotyledonous plants (available from BioAgri AB, Uppsala, Sweden),  Burkholderia  spp. such as  B. cepaciatype  Wisconsin for use on barley, sorghum, and wheat (available as “Deny” from Stine Microbial Products, Memphis, USA; and for use on Maize  B. cepaciatype  available as “Intercept” from Soil Technologies Corp., Fairfield, USA), and the like. 
     Suitable fungicides are known for use in monocotyledonous seed treatments for maize include fludioxonil[4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile], mefenoxam[methyl N-(methoxyacetyl)-N-(2,6-xylyl)-D-alaninate], azoxystrobin[methyl (2E)-2-{2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate], captan[(3aR,7aS)-2-[(trichloromethyl)sulfanyl]-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione], carboxin[5,6-dihydro-2-methyl-1,4-oxathiine-3-carboxanilide], maneb[manganese ethylenebis(dithiocarbamate) (polymeric)], metalaxyl[methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate], oxadixyl[2-methoxy-N-(2-oxo-1,3-oxazolidin-3-yl)acet-2′,6′-xylidide], PCNB [pentachloronitrobenzene] and Thiram[tetramethylthiuram disulfide or bis(dimethylthiocarbamoyl)disulfide]; for rice carboxin[5,6-dihydro-2-methyl-1,4-oxathiine-3-carboxanilide], mancozeb [manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt], metalaxyl [methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate], and PCNB [pentachloronitrobenzene] and Thiram [tetramethylthiuram disulfide or bis(dimethylthiocarbamoyl)disulfide]; for sorghum captan [(3aR,7aS)-2-[(trichloromethyl)sulfanyl]-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione], mancozeb [manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt], metalaxyl [methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate], oxadixyl[2-methoxy-N-(2-oxo-1,3-oxazolidin-3-yl)acet-2′,6′-xylidide], and PCNB[pentachloronitrobenzene]; for wheat captan[(3aR,7aS)-2-[(trichloromethyl)sulfanyl]-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione], tiabendazole (also known as TBZ)[2-(thiazol-4-yl)benzimidazole or 2-(1,3-thiazol-4-yl) benzimidazole, metalaxyl[methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate], oxadixyl[2-methoxy-N-(2-oxo-1,3-oxazolidin-3-yl)acet-2′,6′-xylidide] and triadimenol [(1RS,2RS;1 RS,2SR)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol]; and for barley, oats, and rye imazolil (RS)-1-(β-allyloxy-2,4 dichlorophenethyl) imidazole or allyl (RS)-1-(2,4-dichlorophenyl)-2-imidazol-1-ylethyl ether, mancozeb [manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt], maneb [manganese ethylenebis (dithio-carbamate) (polymeric)], PCNB [pentachloronitrobenzene], Thiram [tetramethylthiuram disulfide or bis(dimethylthiocarbamoyl)disulfide], Triadimenol (1RS,2RS;1 RS,2SR)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol, and difenoconazole 3-chloro-4-[(2RS,4RS;2RS,4SR)-4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-2-yl]phenyl 4-chlorophenyl ether. 
     Suitable insecticides are also known for use on monocotyledonous crops as seed treatments such as thiamethoxam[(EZ)-3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene(nitro)amine] for rice and maize; imidacloprid[(E)-1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine], methiocarb[4-methylthio-3,5-xylyl methylcarbamate], and thiodicarb[(3EZ,12EZ)-3,7,9,13-tetramethyl-5,11-dioxa-2,8,14-trithia-4,7,9,12-tetraazapentadeca-3,12-diene-6,10-dione] for maize, and cereals crops (rye, wheat, oats, and triticale), and clothianidin[(E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine] for maize and cereals (rye, oats, wheat and triticale), cipermethrin[(RS)-α-cyano-3-phenoxybenzyl(1RS,3RS;1 RS,3SR)-3-(2,2-dichlorovinyl)-2,2dimethylcyclopropanecarboxylate or (RS)-α-cyano-3-phenoxybenzyl (1RS)-cis-trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate] for wheat. 
     The organic carrier material in this aspect of the invention may be selected from waxes such as from those waxes as hereinbefore described. Suitable waxes may be selected from waxes such as carnauba wax, beeswax, montan wax, Chinese wax, shellac wax, spermaceti wax, myricyl palmitate, cetyl palmitate, candelilla wax, castor wax, ouricury wax, wool wax, sugar cane wax, retamo wax, and rice bran wax or a mixture of two or more thereof. Preferably, the wax carrier particles of use in this aspect of the invention comprise dry particles of carnauba wax, ouricury wax, and rice bran wax or a mixture of two or more thereof. Preferably, the selected carrier material is carnauba wax. 
     In a further aspect of the invention, there is provided a seed coating composition produced by the method as described herein. 
     In a further aspect of the invention there is provided a coating composition as described herein for use on monocotyledonous seeds. 
     In a further aspect of the invention there is provided a method of coating a monocotyledonous seed with a coating composition that comprises an organic carrier material and a biological antagonist to one or more fungal pathogens, bacterial pathogens and arthropod pathogens so as to limit damage by the said pathogens to the said monocotyledonous seed, the method comprising adding a biological antagonist to an organic carrier material wherein the organic carrier material is in dry particulate form, mixing the two components together and applying the resulting composition in dry particulate form to monocotyledonous seeds. Thus, the seed coating composition of use in the invention is applied in dry particulate form. Naturally, the skilled addressee will appreciate that the organic carrier material may also contain added pigments, plasticisers and other minor components as herein described. In an alternative, the seed coating may be applied in liquid form as herein described and then the seeds dried, leaving a coating composition that is in dry particulate form when on the seed. However, it is preferred that the coating composition is applied in dry, particulate form for ease of application and production costs are kept low. The organic carrier material in this aspect of the invention may be selected from carnauba wax, beeswax, montan wax, Chinese wax, shellac wax, spermaceti wax, myricyl palmitate, cetyl palmitate, candelilla wax, castor wax, ouricury wax, wool wax, sugar cane wax, retamo wax, and rice bran wax or a mixture of two or more thereof. Preferably, the organic carrier material is carnauba wax in dry particulate form. 
     The treatment composition in this aspect of the invention includes one or more biological agents selected from chemical arthropodicides such as insecticides and acaricides, fungicides, bactericides and live biological agents as herein before described. 
     There now follow examples that illustrate the invention. It is to be understood that the examples are not to be construed as limiting the invention in any way. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 : Spore loadings of  Trichoderma  on wheat 
     
    
    
     EXAMPLES SECTION 
     Control of  Alternaria  sp. of wheat ( Triticum aestivum ) [available from the United Kingdom National Culture Collection (UKNCC)] by means of seed treatments using examples of the antagonists  Trichoderma harzianum, Pseudomonas fluorescens  and  Bacillus subtilis  [available from United Kingdom National Culture Collection (UKNCC)] 
       Alternaria  Leaf Blight 
     Symptoms 
     Lower most leaves are always the first to show the sign of infection, which gradually spreads to the upper leaves. The disease first makes it appearance as small, oval, discoloured lesions, irregularly scattered on the leaves. The spots became irregular in shape as these enlarge and take up dark brown to grey colour. As the disease progresses, several spots come closer and cover large leaf areas, eventually resulting In death of the entire leaf. A bright yellow marginal zone is sometimes seen around the spots. In case of severe attack, leaf sheaths, awns and glumes are also infected. 
     Black powdery spores of the fungus cover the lesions at this stage under moist conditions. These spores are disseminated by wind and cause disease on healthy leaves and plants. The disease spreads very fast under warm and humid conditions. Heavily infected fields present a burnt appearance. 
     Disadvantages of Conventional Seed Treatment 
     
         
         
           
             i) Limited dose capacity—The amount of pesticide that can be applied is limited by how much will actually stick to the seed. 
             ii) Limited duration of protection—The duration is often short due to the relatively small amount of biological agent (e.g. chemical) applied to the seed, dilution of the biological agent as the plant grows, and breakdown of the biological agent. 
             iii) Limited shelf life of treated seed—Producing excess treated seed is undesirable because the shelf life of treated seed may be limited. Surplus treated seed cannot be sold for grain. 
           
         
       
    
     All three of these limitations may be overcome or significantly reduced through the inclusion of carnauba wax particles as a carrier for a biological agent, in this case dormant microorganisms that are applied to seeds. Under favourable conditions, the microorganisms grow and colonize the exterior of the developing seed or seedling. Biological agents may help in reducing seed decay, seedling diseases, or root rot. 
     The following tests are performed to examine the potential effect of the inclusion of carnauba wax particles. 
     Phase One—Isolate Cultures 
     1. Culture Maintenance 
     Records are kept with each isolate sub-culture being assigned an accession number. All plates and slides relating to that sub-culture are labelled with an accession number. 
     In addition, permanent lactophenol (LP) mount slide are made from each of the original cultures and file for reference purposes 
     No more than three generations of sub-culture occur before passaging through a living host and re-isolating in order to maintain the fitness of the organism. 
     Sub-cultures are stored for future use on Potato Dextrose Agar (PDA) at 4° c. 
     Each isolate is assigned an accession number and sub-cultures are labelled with that number. 
     DNA is extracted for identity verifications and stored at −20° C. A reference sample of the pure culture is stored on glycerol at −20° C. Upon completion of the experiment DNA identification of the culture is repeated to confirm that the organism has not mutated during the course of the work. 
     2. Culturing of the Causal Agent 
     Isolation of a pathogenic fungus from diseased tissue into pure culture is one of the standard techniques in identifying and describing a disease. It is an essential step in proving the pathogenicity of previously un-encountered organisms. 
     Techniques Commonly Involve: 
     
         
         a. Surface-sterilisation treatment 
         b. Plating (possibly on selective medium) of samples of diseased tissue, with appropriate precautions. 
         c. Sub-culturing to get pure cultures.
 
3. Purification of Cultures
 
       
    
     Small disinfected root pieces of an artificially inoculated plant are cultured on water agar. The fungal colonies that appear most frequently are likely the target pathogen. Several saprophytes may also be present in infected plant tissues and they may grow into the medium with the principal pathogen. Routine surface-sterilisation consists of wiping the tissue with (or immersing in) 0.1% solution of sodium hypochlorite (NaOCl or sometimes referred to as “NaClO”) followed by rinsing with sterile distilled water. To obtain a pure culture of the pathogen, a small sample is taken from the growing edge of a colony with a flamed loop or scalpel and streaked over the surface of a pre-poured plate of PDA. The inclusion of chloramphenicol (a bacteriostatic anti-microbial) at 30 mg/l reduces the risk of bacterial contamination. As the streak progresses over the agar, fungal spores are separated until single spores are obtained from which separate colonies will grow. 
     Repeat this procedure until pure cultures are obtained. 
     4. Single Spore Isolation 
     Single spore isolations are important to investigate pathogenic variability. An inoculum of spores is placed in a tube containing 10 ml of sterile water. This spore suspension is streaked along a marked line on the surface of a thin tap water agar medium, and incubated at 22° C. After 24 hr incubation, select germinated spores using a stereoscopic microscope and transferred one spore at a time to another agar plate. 
     5. Slide Preparation for Microscopic Examination and Reference 
     Identification of the pathogen: the tissue may be sectioned or surface scraped and then mounted in water/lactophenol. Fungal structures seen macroscopically may be separated from the host tissue to be examined and identified. Identification depends on spore formation and therefore infected material will be incubated in a moist environment overnight prior to examination in order to encourage sporulation. Cotton blue stain will be added to the lactophenol in order to highlight fungal structure. The specimen will be placed in a drop of satin on a glass slide and gently warmed by passing through a low flame for a few seconds before mounting in lactophenol. 
     Whole mount sections can be cleared and stained for ease of identification using the following method: 
     Leaf disks are made clear by heating them in tubes in lactophenol until clear (up to 20 minutes), without boiling. Stain by heating in 0.5% cotton blue in lactophenol on a slide for 5-10 minutes. Rinse thoroughly in lactophenol and mount in the same. 
     6. Growth and Media 
     Sub-cultures are assessed for growth and germination at a range of temperatures, 13.5° C., 18° C. and 22.5° C. A range of media is examined for suitability. Whilst PDA is generally suitable for most fungal species it has been found that use of a low nutrient agar, such as tap-water agar, reduce prolific growth and can encourage sporulation. Therefore PDA, tap-water agar, and a selective media from literature, Czapek&#39;s Dox agar (Dawson (1962) Saboutaudia 1. 214-219), are included within the assessment trials. 
     A 5 mm diameter disk is cut from the margin of an actively growing culture using a flamed cork borer. This is placed upside down in the centre of the pre-poured media plates. Five replicates are made for each media type and temperature (45 plates in total). Complete randomisation will be applied to plates in each incubator. Plates are observed until one culture succeeds in completely covering the plate in any one media. At this point the following measurements are taken: fungus colony diameter, colour and margin. In addition, the level of sporulation is recorded. 
     Five 5 mm disks are cut from each plate using a flamed cork borer and suspended in 20 ml of distilled water (+0.05% Tween 20®). The sample is then sonicated for 2 minutes to release the spores and then vortexed to aid the formation of a uniform spore suspension. Samples are assessed for spore concentration using an Improved Neubauer haemocytometer using standard counting methodology. 
     The mean for each media type is calculated and ANOVA is applied to examine the results for significant differences. 
     Phase Two—In Vitro Studies: 
     1. Screen Microorganisms and Carnauba Wax to Determine Interactions 
     In order to explain effects observed the microorganisms, pathogens and antagonists, are screened against carnauba wax to identify any carrier only effect. This will enable the determination of treatment effect as well as any synergy occurring as a result of the use of using an antagonist with carnauba wax particles.
         a. Plates of appropriate media are used based on the findings of the experiment above. Air-milled carnauba wax is sterilised using the autoclave and then ground using a twin blade mill, producing particles with an approximate VMD of The sterilised media is then cooled to 50° C. (molten stage). The carnauba wax is then incorporated into the media. Two concentrations of carnauba wax are tested; 1 g/l and 10 g/l. A 5 mm diameter disk is cut from the margin of an actively growing culture using a flamed cork borer. This is placed upside down in the centre of the pre-poured media/carnauba wax plates. Five replicates will be made for each concentration and incubated at the optimum temperature for growth/sporulation (as determined in previous experiment). Growth rates and characteristics are compared to the controls using data from the Growth and Media experiment above.
 
Differences Will be Analysed Using ANOVA.
   b. Disks of the pathogen and antagonists are dusted with different carnauba wax treatments and put on appropriate media. The carnauba wax particles need to be free of microorganisms to be able to carry out this experiment. Growth of treated and untreated organisms are compared.
 
2. Investigate Antagonist Action Against Pathogens
 
i. Effect of Antagonists on Viability of  Alternaria  sp. Mycelium (In Vitro Assay I)
       

     All antagonistic isolates are tested in a dual culture assay against pathogenic fungi on PDA or alternative pre-defined media. Agar plugs of  Alternaria  sp. and the antagonist isolate to be tested are arranged 7 cm apart on 9 cm agar plates. Inhibition zones and zones of overlapping are assessed after 7 days incubation at 13.5° C., 18° C. and 22.58° C. Where an antagonist overgrows the mycelium of  Alternaria  sp., the zone of hyphal interaction between both is investigated microscopically (100×). Fungal strains without a microscopically visible effect on mycelium of  Alternaria  sp. are excluded from further experiments. Furthermore, the viability of  Alternaria  sp. in the region of interaction is tested by transfer of mycelial discs onto water agar plates 5 days after first contact. The  Alternaria  sp. mycelium is assessed as viable when the growth of typical hyphae is observed microscopically (100×). Each experiment is repeated three times with three samples per replicate. 
     ii. Effect of Antagonists on Germination of  Alternaria  sp. Sclerotia Produced In Vitro (in vitro assay II) 
     Sclerotia of  Alternaria  sp. of uniform size are placed on a 6 day old culture (PDA, 20° C.) of the fungal antagonist. After incubation for 14, 28 and 35 days at 20° C., eight sclerotia per replicate (three replicates per antagonist) are transferred from the agar plate onto water agar. Mycelial growth from these sclerotia is assessed under a light microscope (100×). 
     3. Confirmation of Pathogenicity 
     Steps to perform Koch&#39;s postulates (Koch 1890, criteria designed to establish a causal relationship between a causative microbe and a disease)
         a) Describe the symptoms expressed by the diseased crop plants.   b) Isolate the suspected pathogen—the same cultures should be isolated from plants with similar symptoms   c) Obtain a pure culture and use it to inoculate healthy plant material.   d) Observe the symptoms expressed by the inoculated plants—symptoms should be the same as those observed originally in the crop plants.   e) Re-isolate the pathogen from the newly diseased material. The culture should be the same as the original purified culture.
 
i. Indirect Application—Plant
   Using healthy plants—soil can be inoculated directly using a spore suspension made from a pure agar culture or from a culture grown in flasks. A fungal spore or bacterial suspension can be added post-emergence so that the root system is drenched by the suspension. Plants are then observed over 7 days and symptoms recorded. Koch&#39;s Postulates are applied in order to confirm that the symptoms relate to the inoculated pathogen.
 
ii. Direct Application—Seed
   Inoculum for preparing spore suspensions is grown on water agar containing sterile seeds. Fungal spores and hyphae or bacterial spore and vegetative growth are scraped from the colony and transfer to sterile water. This spore suspension is then applied to seeds and mixed to ensure a uniform distribution. Seeds are then:
           Placed on moist filter paper and incubated at optimum growth temperature for 5 days.   sown in heat sterilised potting compost and incubated in a propagator at optimum growth temperature for 7 days   
           Symptom expression and germination is recorded for both sets of experiments and Koch&#39;s postulates applied
 
4. Carnauba Wax/Antagonist Co-Location Analysis
       

     A dry powder formulation of spores is produced using a spore separator. Moisture content of the formulation is reduced to below 5% using a dehumidifier and silica beads. Spore concentration is determined using a Neubauer haemocytometer and standardised counting methodology. 
     Steps in Air Milling in Boyes Micronisation Process (for carnauba wax particles with a VMD of approx. 25 μm and 75 μm) 
     1. 2 kg carnauba wax blocks are first kibbled into approximately 4 to 6 mm pieces in a KT Handling Ltd Model 04 kibbler (serial no. 729/C) following the manufacturer&#39;s instructions. 
     2. The kibbled pieces are then passed through an Apex Construction Ltd Model 314.2 Comminuting Mill (serial no. A21306) and reduced further in size to a range of 250 to 300 um. 
     3. The comminuted particles are then passed through a Hosokawa Micron Ltd Alpine 100AFG jet mill (serial no. 168092) following the manufacturer&#39;s instructions, setting the mill at a suitable speed (a speed of 8000 rpm for particles having a VMD of 15 μm or at a speed of 2500 rpm for particles having a VMD of 75 μm), with a positive system pressure of 0.03 bar.
 
4. The grinding air is to be kept to 6 bar, the system rinsing air flow and Classifying Wheel gap rinsing air are both to be set at a minimum of 0.5 bar and no more than 0.75 bar, the cleaning air filter is to register a delta of no more than 5 bar to achieve a final particle size with a VMD of 15 um or 75 μm as required.
 
     Entostat was combined with wheat seed at three loadings (see below). 
     Two sizes of carnauba wax particle having VMDs of 15 μm and 75 μm, respectively, are examined in combination with the spore formulation at two different ratios (1:3, 2:2). Samples of the carnauba wax/spore mixture are analysed using electron photomicroscopy to determine the co-location effect. Any variation observed is recorded. 
     In addition, both sizes of carnauba wax referred to, are mixed with a homogenised sample of mycelium and examined as described above. 
     5. Carnauba Wax Particle Loading 
     Carnauba wax particle adhesion to seeds is approximated through the use of photomicroscopy (qualitative) and fluorometric analysis (quantitative). Two sizes of carnauba wax particles (with 1% glo-brite) are used having a VMD of 15 μm and 75 μm, respectively. Four combinations: Two ratios of carnauba wax/spore formulation, together with one mycelial and a vehicle control (carnauba wax only), makes a total of eight treatments. Treatments are applied to 10 g of seed and replicated three times. Three subsamples are taken from each replicate and the mean used in analysis. 
     For fluorometric analysis three 1 g samples are each added to 5 ml of ethanol and sonicated to aid the release of the carnauba wax particles from the seeds. Samples are analysed using a Perkin Elmer L55 Fluorometer (Perkin Elmer, Ma, USA). Statistical analysis of variation between treatments is performed using ANOVA. 
     Seed size and architecture varies greatly between crop species and this influences application rates and method. A homogeneous mix is attained through tumbling seed and carnauba wax formulation in a cylinder, adapted to produce lateral mixing/tumbling through the inclusion of angled interior vanes, placed on a Wheaton roller for 5 minutes. 
     Phase Three—In vivo: 
       Alternaria  sp., together with the most successful antagonist model are used in a series of in vivo experiments. The basic design is a split-plot experiment with temperature being the main plot factor (13.5° C., 18° C. and 22.5° C.) and carnauba wax/antagonist ratio (3 treatments:2× spore, 1× mycelial) being the sub-plot. Four homogeneous mixes of each treatment are prepared using the method described above and these represent the replicates. 
     Treatments: 
     
         
         
           
             1) Application rate 1—7.5×10 6  conidia kg −1    
             2) Application rate 2—7.5×10 8  conidia kg −1    
             3) Application 3—Mycelia 
             4) Control 1—Vehicle control (Carnauba wax only) 
             5) Control 2—no treatment
 
Mixes (true replicates): A, B, C, D
 
Subsamples of each mix: α, β, γ
 
Mixes and treatments are arranged according to a Randomised Block design.
 
Pot Studies
 
           
         
       
    
     Each temperature (growth chamber) contains 60 plant pots. 
     Treated seed is sown in accordance with supplier&#39;s recommendation. Soil/compost (1:1 John Innes No. 2 and peat compost) is heat sterilised prior to inoculation with 10 ml of  Alternaria  sp. spore suspension and thoroughly mixed before sowing. 
     Plants are placed in the growth chambers for a period of 21 days with observations of symptom expression made every 48 hours post emergence. Water is applied through capillary matting twice daily. 
     After 21 days plants are removed from their pots and the following assessment measurements taken: 
     
         
         
           
             % germination 
             % pre-emergence damping off 
             % post-emergence damping off 
             Root weight 
             Shoot weight 
           
         
       
    
     In addition, symptom expression is assessed based on a damage scale. 
     Means of the measurements taken from the subsamples α, β, γ are compared for each treatment using ANOVA. 
     Samples are taken from 5 plants exhibiting symptoms and Koch&#39;s Postulates applied to confirm the causal organism (by comparison to the reference slide of the master culture). The experiment is repeated. 
     Second Example 
     Control of  Pythium graminicola  [United Kingdom National Culture Collection (UKNCC)] on wheat ( Triticum aestivum ) by means of seed treatments using fludioxonil. 
     Experimental Design—as per the Pot Study in Example 1, above 
     Carnauba wax is melted using copper pans. During cooling fludinoxonil is added at 1% of the mass of the carnauba. This mixture is allowed to solidify before chipping and processing through an air mill as described above, with the exception that the speed is set at 6000 rpm to produce particles with a VMD of 25 μm. 
     Treatments for the Pot Study 
     Control 1—Vehicle control (Carnauba wax only) 
     Control 2—no treatment 
     Treatment 1—1% fludinoxonil carnauba wax at 10 g per kg of seed 
     Treatment 2—1% fludinoxonil carnauba wax at 3.2 g per kg of seed 
     Assessment and analysis as with previous Pot Study 
     Third Example 
     Relating to: 
     Control of  Agriotes Mancus  spp. (Coleoptera: Elateridae), or Wheat Wireworm, (the larval form of the click beetle) that preys on wheat ( Triticum aestivum ), by means of seed treatments using thiamethoxam. 
     Early-season wireworm damage consists of hollowed-out seeds where larvae have entered during germination. Seedling plants also can be injured or killed by larvae tunneling into the plant below the soil line. Occasionally, wireworms bore into the stalks of larger plants and tunnel in a few inches, but the damage is not significant. 
     Experimental Design—as Pot Study Above 
     Carnauba wax is melted using copper pans. During cooling thiamethoxam is added at 1% of the mass of the carnauba. This mixture is allowed to solidify before chipping and processing through a mill as described above (speed set at 6000 rpm) to produce particles with a VMD of 25 μm. 
     Treatments for the Pot Study 
     Control 1—Vehicle control (Carnauba wax only) 
     Control 2—no treatment 
     Treatment 1—1% thiamethoxam carnauba wax at 4.2 g per kg of seed 
     Treatment 2—1% thiamethoxam carnauba wax at 1.3 g per kg of seed 
     Empty pots are lined with a nylon mesh screening material before filling with potting soil. A wire frame is constructed and the nylon meshed tied off over the frame to provide a caged experimental arena designed so that the insect cannot escape the treated area. 
     Seeds are allowed to germinate for three days before adding five 3 rd  instar larvae to the soil surface of each pot before resealing the mesh cage. 
     Observations are made over 21 days. 
     Plants are assessed for: 
     
         
         
           
             % germination 
             Damage 
             Root weight 
             Shoot weight 
           
         
       
    
     The procedures detailed within Example One are followed to examine the antagonistic effect of  Trichoderma harzianum  [United Kingdom National Culture Collection (UKNCC)],  Pseudomonas fluorescens  [UKNCC] and  Bacillus subtilis  [UKNCC] on  Fusarium  sp., a fungal pathogen of Rice ( Oryza sativa ). 
     The procedures detailed within Example One are followed to examine the antagonistic effect of  Trichoderma harzianum  [United Kingdom National Culture Collection (UKNCC)],  Pseudomonas fluorescens  [UKNCC] and  Bacillus subtilis  [UKNCC] on  Colletotrichum graminicola , a fungal pathogen of  Sorghum  ( Sorghum bicolor ). 
     The procedures detailed within Example Two are followed to examine the effect of metalaxyl on  Pythium  sp., a fungal pathogen of Rice ( Oryza sativa ). 
     The procedures detailed within Example Two are followed to examine the effect of prochloraz on  Rhizoctonia  sp., a fungal pathogen of sorghum ( Sorghum bicolor ). 
     The procedures detailed within Example Three are followed to examine the effect of thiamethoxam on the White Grub ( Phyllophaga crinite ), an insect pest of  Sorghum  ( Sorghum bicolor ). 
     The procedures detailed within Example Three are followed to examine the effect of imidacloprid/beta-cyfluthrin on Rice Seed Midges ( Cricotopus sylvestris ), an insect pest of Rice ( Oryza sativa ). 
     Suppression of Causal Agents of Fungal Disease in Wheat ( Triticum aestivum ) Using a Seed Coating Comprised of  Trichoderma  sp. and Carnauba Wax Particles 
     The potential for  Trichoderma  sp. (Ascomycota) as a biocontrol agent in the defence against plant pathogens is known. 
       Trichoderma  hyphae are capable of penetrating the hyphae of other fungi and extracting nutrients from within, resulting in the suppression and eventual death of the host.  Trichoderma  exhibits rapid mycelial growth and is capable of out-competing other fungi for nutrients. 
     There are several commercially available formulations of  Trichoderma  marketed as crop protection products. These are commonly supplied as a wettable powder formulation and applied to the area of cultivation as a drench. The disadvantage of this form of application is that it is necessary to treat the entire cultivation area, whereas it is the region immediately surrounding the seed or plant that requires the treatment. The larger the number of conidia delivered to this area the greater the level of control they are able to impart. Therefore a targeted application system able to deliver sufficient conidia to the required area offers a distinct advantage in the use of  Trichoderma  over conventional applications. 
     Experimental Aim: To Assess the Potential Use of Entostat as a Seed-Coating Technology for the Delivery of Beneficial Microbes 
     Methods 
     Steps in Air Milling in Boyes Micronisation Process (for carnauba wax particles with a VMD of approx. 10 μm) 
     1. 2 kg carnauba wax blocks are first kibbled into approximately 4 to 6 mm pieces in a KT Handling Ltd Model 04 kibbler (serial no. 729/C) following the manufacturer&#39;s instructions. 
     2. The kibbled pieces are then passed through an Apex Construction Ltd Model 314.2 Comminuting Mill (serial no. A21306) and reduced further in size to a range of 250 to 300 um. 
     3. The comminuted particles are then passed through a Hosokawa Micron Ltd Alpine 100AFG jet mill (serial no. 168092) following the manufacturer&#39;s instructions, setting the mill at a speed of 12500 rpm, with a positive system pressure of 0.03 bar. 
     4. The grinding air is to be kept to 6 bar, the system rinsing air flow and Classifying Wheel gap rinsing air are both to be set at a minimum of 0.5 bar and no more than 0.75 bar, the cleaning air filter is to register a delta of no more than 5 bar to achieve a final particle size with a VMD of 9.7 μm. 
     Entostat was combined with wheat seed at three loadings (see below).
     1. Baseline data: seed coating techniques   1.1. Seed Coating.  Trichoderma harzianum  (containing 7.75×10 9  colony forming units g −1  Sylvan Bio, Loches, France) with a germination percentage of 95% was applied to wheat (var. Hereward, Herbiseeds, Twyford, UK) using carnauba wax particles with a VMD of 9.7 μm. A target loading was set at 10 5  conidia per seed based on information obtained from literature.
       Carnauba particles were mixed with the dry conidia powder at different ratios and applied 0.01 g (0.2% by mass) directly to dry seed, 5 g of seeds per concentration. For each concentration, four batches of 10 seeds were used for evaluation of conidia loading.   Conidia to carnauba ratios used were:   100% Conidia, 50% Conidia, 25% Conidia and 9% Conidia with the remainder in each case being made up of carnauba wax particles.   
       1.2. Enumeration. Direct enumeration to determine conidia loading of seeds was done through the use of a haemocytometer (Improved Neubauer, Hawksley, Lancing, UK).
       Inoculum: Preparation of suspension.   Propagules are usually formulated in a water carrier, although those with hydrophobic cell walls (such as  Trichoderma ) are not readily suspended in water. To uniformly suspend hydrophobic propagules in water it is necessary to sonicate and/or use mechanical suspension methods. Mechanical suspension of propagules using micropestles provides good suspension of conidia in water without causing damage to cells. A surfactant may also facilitate suspension of propagules (Tween20 at 0.05%). To suspend hydrophobic conidia, harvested conidia are placed in a 1.5 ml microcentrifuge tube, ≈0.5 ml of sterile water is added to the tube, the micropestle is inserted into the tube, and the conidial mass is gently agitated with the micropestle by hand (prevents liberation of conidia into air). The micropestle is the attached to the motor (e.g. Kontes, Argos pellet pestle motor) and the suspension is vigorously agitated while moving the pestle in and up and down, and side to side motion, circa. 30 seconds. Since the haemocytometer method does not distinguish between viable and non-viable propagules, it is necessary to determine spore viability so that doses can be prepared on the basis of viable propagules.   Seed washes and enumeration of  Trichoderma  loadings were done on 4 batches of seeds per treatment. Inoculum was washed from seeds by placing into 1 ml sterile 0.05% Tween 20  (or substitute—similar non-ionic surfactant/dispersal agent) in a Eppendorf tube and vortexing for 30 seconds to remove conidia from the seed surface. Samples were then sonicated for two minutes to break up any conidial clumping. Counts obtained were used to calculate the mean conidia loading of seed coated with the various treatments. Results obtained using 100% conidia powder were used as a benchmark and the conidia/carnauba combination powders compared against it as a determination of efficiency of loading.   Confirmation of conidial viability was achieved by dilution plating on  Trichoderma  Specific Media (TSM) (see below). A dilution series was set up and duplicate plates inoculated from the series. Colony Forming Units (CFU) counts were made after 7 days, allowing inoculum levels on seeds to be quantified. In addition, fresh, unused conidia were plated to provide a comparison of before and after seed application.   Germination percentage was also measured. A satisfactory density of conidia was obtained by spreading approximately 10 6  conidia in 100 μl on the media in a 9 cm petri dish. Conidia were incubated in the dark at 25° C. for five days, and the area to be observed was then fixed using lactophenol. Phase contrast microscopy using an inverted compound microscope enabled sufficient examination of the conidia.   Conidia were considered viable if germtube lengths were two times the diameter of the propagule in question. Numbers of germinated and non-germinated conidia in arbitrarily-selected fields of view or in parallel transects, defined with an ocular micrometer, were counted. A minimum of 300 conidia were counted to provide an accurate estimate. It is desirable to determine the viability of propagules on replicate cultures and at various positions on the same plate.   This allowed calibration of the seed-coating techniques to obtain similar levels of  Trichoderma  loadings on the seeds for each coating method.   
       1.3. Seed Germination. One batch (5 seeds) of seeds from each treatment was placed on seed test paper (Whatman 181) in a 9 cm Petri dish. Dishes were sealed with Parafilm and held at 20° C. for 7-10 days and germination rate determined. This was repeated with untreated seed.
 
 Trichoderma  Selective Media (adapted from Williams, Clarkson et al 2003) was prepared as follows:
 
For 1000 ml
 
Basal Medium Ingredients:
   

                                            0.2 g MgSO 4     3.0 g glucose           0.9 g K 2 HPO 4     0.15 g rose bengal           0.15 g KCl   20 g agar           1.0 g NH 4 NO 3     950 ml distilled water                    
Basal Medium Process
 
     Mix liquid ingredients with all solid ingredients, except the agar in a 1 L Erlenmeyer flask. Add the 20 g agar and stir or shake. Plug with cotton wool and cover with foil. Autoclave. 
                             Biocidal Medium (per liter)                                            0.25 g crystallized chloramphenicol           0.2 g quintozene           0.2 g captan           1.2 ml propamocarb (Previcur)           50 ml sterile distilled water                    
Seed Weight
 
     Used as a measure of the homogeneity of the seed batch. Eight replicates of 25 seeds are weighed and the coefficient of variation (Cv) recorded. This coefficient should not exceed a value of 5. If it does then the procedure is repeated and the mean of all 16 samples used to calculate the number of seeds per gram. 
                                                     Crop   Mean Weight (g)   SD   Cv   TGW (g)                      Wheat   1.258   0.059   4.678   50.305                    
Results
 
Direct Enumeration Counts Using Haemocytometer
 
     Initial Spore Density of  Trichoderma harzianum  dry spore preparation (at 5% moisture content), determined using haemocytometer, was 7.75×10 9  spores g −1  (n=4,±2.6×10 7  95% CL). 
     Spore Counting of Seed Wash 
     
       
         
           
               
               
               
               
               
               
             
               
                   
               
               
                   
                 Variable 
                 Spore % 
                 N 
                 Mean 
                 SE Mean 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SporeCount 
                 9 
                 4 
                 300750 
                 11499 
               
               
                   
                   
                 25 
                 4 
                 757750 
                 21453 
               
               
                   
                   
                 50 
                 4 
                 1062500 
                 18875 
               
               
                   
                   
                 100 
                 4 
                 2145000 
                 109278 
               
               
                   
               
               
                 *10 5  target spores per seed 
               
            
           
         
       
     
     There was a clear and statistically significant difference between the mean spore counts per seed achieved by the different treatments as determined by one-way ANOVA (F(3,12)=190.83, p=&lt;0.001). All treatments exceeded the target of 10 5  spores seed −1 . 
     
       
         
           
               
               
               
               
               
               
               
             
               
                   
               
               
                   
                 Mean 
                   
                   
                   
                   
                   
               
               
                   
                 Spore 
                 *Expected 
                 As a % 
                 **As a % 
               
               
                 % 
                 Count 
                 Spore 
                 of 100% 
                 of 
                 t 
                 p 
               
               
                 Spores 
                 Seed −1   
                 Count 
                 Treatment 
                 Expected 
                 value 
                 value 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 100%  
                 905250 
                 n/a 
                 n/a 
                 n/a 
                 n/a 
                 n/a 
               
               
                 50%  
                 1062500 
                 1072500 
                 50% 
                  99% 
                 −0.53 
                 0.633 
               
               
                 25%* 
                 757750 
                 536250 
                 35% 
                 141% 
                 10.32 
                 0.002 
               
               
                 9% 
                 300750 
                 193050 
                 14% 
                 156% 
                 9.37 
                 0.003 
               
               
                   
               
               
                 *Expected Spore Count is calculated from the mean spore count achieved by the 100% Treatment, assuming a perfect distribution. Therefore the 50% Treatment would be expected to result in half the spores of the 100% Treatment, and so on. 
               
               
                 **Essentially a measure of improvement in spore adhesion efficiency. 
               
            
           
         
       
     
     The addition of Entostat above 50% appears to improve the efficiency of spore adhesion to seed as the actual mean counts significantly exceed the expected results based on the 100% spore treatment (t-test). 
     Germination Determination 
     Mean conidia germination (from a sample of 300) 
     Fresh conidia—276.50±10.85, n=4 
     Seed wash conidia—275.25±6.02, n=4 
     There was no statistically significant difference between the viability of fresh conidia and those washed from seeds as determined by one-way ANOVA (F(1,6)=0.04, p=0.847). 
     Enumeration Estimate from CFU Counts 
     Comparison of Haemocytometer and CFU (corrected for dilution) counts 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                   
                   
                   
                 Grouping 
               
               
                 Treatment 
                 N 
                 Mean 
                 SE Mean 
                 (using Tukey method) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 100CFU 
                 4 
                 2150000 
                 83964 
                 A 
               
               
                 100Haemo 
                 4 
                 2145000 
                 109278 
                 A 
               
               
                 50CFU 
                 4 
                 1060000 
                 12910 
                 B 
               
               
                 50Haemo 
                 4 
                 1062500 
                 18875 
                 B 
               
               
                 25CFU 
                 4 
                 787500 
                 15058 
                 C 
               
               
                 25Haemo 
                 4 
                 757750 
                 21453 
                 C 
               
               
                 9CFU 
                 4 
                 303250 
                 9059 
                 D 
               
               
                 9Haemo 
                 4 
                 300750 
                 11499 
                 D 
               
               
                   
               
               
                 Mean that do not share a letter are significantly different. 
               
            
           
         
       
     
     There was a statistically significant difference between groups as determined by one-way ANOVA (F(7,24)=205.95, p=&lt;0.001). A Tukey post-hoc test revealed significance was as a result of differences in the spore % rather than the counting method applied. 
     SUMMARY 
     Wheat seed can be coated with  Trichoderma  spores in excess the target 10 5  spores seed −1  for all treatments. 
     Use of Entostat at a ratio greater than 1:1 increases the efficiency of spore delivery as a result of a reduction in wasted or lost spores. 
     The germination viability of the spores was unaffected by their use as a seed coating. 
     Enumeration through direct counting of spores using a haemocytometer or through the use of CFU counting gives statistically similar results and therefore either method may be used once germination viability has been proved unaffected by the treatment. 
     The described method for wheat as provided above is used to assess the delivery efficiency of spores by Entostat to seeds of barley, rye, oats, maize and rye grass. Similar or better results are obtained, depending on loading and seed size. 
     Effects of Seed Coating on Disease Suppression 
     Seeds are coated with  Trichoderma  using water or Entostat to achieve loadings of ca. 10 5  and 10 6  CFUs seed −1 . Water treatments are suspensions of spores in sterile water in which the seed samples are soaked for one hour. Seeds are then dried back, a likely commercial scenario, or sown wet coated. Entostat is applied at ratios of 3:1, and 9:1, Entostat to spores respectively. Seed treatment methods are then compared for their ability to protect germinating wheat seedlings from  Gaeumannomyces graminis , the causal agent of take-all disease in wheat. 
     Inoculation of seeds with  Trichoderma . Wheat seed cv. Hereward is inoculated as follows (target concentration per seed): 
     
         
         
           
             1)  Trichoderma  at 10 5 /seed using a water suspension (wet coating) 
             2)  Trichoderma  at 10 6 /seed using a water suspension (wet coating) 
             3)  Trichoderma  at 10 5 /seed using a water suspension (dry coating) 
             4)  Trichoderma  at 10 6 /seed using a water suspension (dry coating) 
             5)  Trichoderma  at 10 5 /seed using Entostat at 3:1 
             6)  Trichoderma  at 10 6 /seed using Entostat at 3:1 
             7)  Trichoderma  at 10 5 /seed using Entostat at 9:1 
             8)  Trichoderma  at 10 6 /seed using Entostat at 9:1 
             9) No  Trichoderma , water only 
             10) No  Trichoderma , Entostat only 
             11) Seed only 
           
         
       
    
     Enumeration.  Trichoderma  is quantified using standard dilution plating methods on  Trichoderma  specific media. This confirms CFU loadings per seed for treatments 1-8. Dilution platings are carried out in duplicate. 
       Gaeumannomyces  Bioassay 
     Inoculum preparation— Gaeumannomyces  sp., known to be pathogenic on wheat, barley, rye, oats and turf grass, is grown on PDA plates from stock cultures, and incubated at 20° C. to produce actively growing colonies. Agar plugs are removed from the plates and used to inoculate sterilised (autoclaved at 121° C. for 20 mins) John Innes No. 2 potting mix (80% moisture content; 60 g) mixed with potato cubes (2 mm 2 , 25 g) in 500 ml Erlenmeyer flasks. Flasks are incubated at 20° C. for 14 days. Inoculum levels in the medium are quantified using a dilution plating method. 
     Effectiveness of seed treatment on  Gaeumannomyces . Seeds are sown into individual cells of seed trays containing  Gaeumannomyces -inoculated medium (approx. 15 ml/cell). Four replicate batches of ten seeds per treatment are planted into the cells. Once sown, the trays are placed in a plant growth chamber (Weiss Gallenkamp Fitotron SG120) at 20° C. with ca. 16 h lighting. Cells are bottom watered. The number of seedlings surviving are recorded every 3 days for 21 days. 
     Time to emergence, percentage successful emergence and percentage plants expressing symptoms are recorded and the results analysed. Differences in Entostat treated seed and untreated seed are observed.