TITLE

SEED DRESSING FIELD OF INVENTION

This invention is concerned with an antifungal seed dressing. BACKGROUND OF THE INVENTION

Fungal diseases cause extensive and destructive damage to crops, such as barley and wheat causing significant economic loss to the agricultural community.

Such fungal diseases may be controlled by the application of antifungal seed dressing compositions to the seeds of the crops comprising an effective amount of an antifungal compound. However, some species of fungi, for example, fungus Tilletia, have developed resistance to the antifungal compounds on the market.

Aromatic thiosemicarbazones are effective antifungal agents against bunt (Tilletia spp.), smut (Ustilago spp.), and rice blast (Magnaporthe spp.) and resistant species of the fungus Tilletia. However, most aromatic thiosemicarbazones are unsuitable for use in seed dressings because the compounds have an extremely unpleasant taste and can act as an irritant if inhaled by a user. Australian Patent No. 498467 describes thiosemicarbazone compounds which have high fungicidal activity, and seed dressing formulations comprising such thiosemicarbazones.

Thiosemicarbazone seed dressing formulations of 498467 and the prior art in general are formulated as a powder or suspension concentrate, or

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as a water dispersible powder or granule. In each of these forms, the thiosemicarbazone ultimately ends up in the form of a fine crystal on the surface of the cereal grain, which can become dislodged during handling and become air-borne as a dust. The disadvantage of such powder and suspension concentrate formulations is that, in use, the formulations generate a large quantity of dust which has a disagreeable taste and therefore acts as an irritant to people who come into contact with the formulations.

SUMMARY OF INVENTION The inventor has discovered a method of formulating a thiosemicarbazone in a liquid formulation for use as a seed dressing or mixing a suspension concentration of a thiosemicarbazone with an emulsified oil for use as a seed dressing. In each case the liquid formulation or mixture does not generate dust nor act as an irritant to persons in contact with the formulation and therefore alleviates the problems of the prior art.

Previously, it has not been possible to produce an effective liquid formulation comprising a thiosemicarbazone because the solvents used in the thiosemicarbazone liquid formulation either are reactive with thiosemicarbazones or do not have a high solvency for thiosemicarbazones. The invention therefore provides an antifungal seed dressing (i) in the form of a liquid formulation including a thiosemicarbazone of general formula 2 herein and a solvent having the following general formula (Formula 1):

HO-[CH(R ₄ )-CH(R ₅ )-O] _n -R wherein R = Ci to C ₆ alkyl or substituted alkyl;

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R ₄ and R ₅ = H or CH ₃ ; and n = 1 to 4. or (ii) alternatively a thiosemicarbazone of general formula 2 herein in the form of a suspension concentrate which is combined with an emulsifiable oil.

It is intended to describe the first embodiment of this invention i.e. the antifungal seed dressing in the form of a liquid formulation in detail before describing the second embodiment concerning the suspension concentrate of the thiosemicarbazone combined with the emulsified oil. DETAILED DESCRIPTION OF THE FIRST EMBODIMENT

Thus having regard to this embodiment, the inventor has devised a new liquid thiosemicarbazone formulation that can be used as a seed dressing. The new liquid formulation reduces or eliminates dust generation from treated seeds, thus making the seed dressing commercially acceptable. The liquid formulation is a true solution and comprises a thiosemicarbazone dissolved in the solvent referred to above.

Through experimentation, the inventor has discovered that only selective solvents are suitable for use in a liquid thiosemicarbazone formulation. The solvents must have the following properties: (i) be non reactive with thiosemicarbazones;

(ii) be non phytotoxic; and (iii) have a high solubility for thiosemicarbazones. The inventor has also determined that some solvents are not effective solvents across a range of temperature and the solvency of the solvent may

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decrease with a decrease in temperature. For example, at 20 degrees Celsius a thiosemicarbazone may be soluble in a solvent but at 2 degrees Celsius it is far less soluble. This is an important finding for the development of an effective liquid thiosemicarbazone formulation since the formulation must be stable across a wide range of temperatures.

Preferably, the formulation is stable in temperatures that range from -5 degrees to 50 degrees Celsius.

Experiments have been carried out to assess whether the liquid thiosemicarbazone formulation of the invention generates dust in use. The results demonstrate that the thiosemicarbazone liquid formulation generates very little dust and does not act as an irritant to a user of the formulation.

For the purposes of this invention, by "thiosemicarbazone" is meant any compound with the general formula (Formula 2):

wherein R ¹ , R ² and R ³ , which may be the same or different, are selected from the group consisting of hydrogen, iodo, bromo, chloro, fluoro, cyano, alkyl or substituted alkyl group containing 1 to 5 carbons, alkoxy group containing 1 to 15 carbons, phenyl or substituted phenyl and naphthyl or substituted naphthyl. Examples of suitable substituents for alkyl in the case of Formula 1 and 2, phenyl and naphthyl are given in Australian Patent 498467 which is herein incorporated into the specification by reference.

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Preferably, the thiosemicarbazone used in the liquid formulation of the invention is selected from the group consisting of para-tolualdehyde thiosemicarbazone (PTT), benzaldehyde thiosemicarbazone, 4- ethylbenzaldehyde thiosemicarbazone, 4-chlorobenzaIdehyde thiosemicarbazone, 4-iso-propylbenzaldehyde thiosemicarbazone, ortho- tolualdehyde thiosemicarbazone, 4-fluorobenzaldehyde thiosemicarbazone.

The amount of thiosemicarbazone included in the formulation and required for effective control of fungi depends on the nature of the thiosemicarbazone, the solubility of the thiosemicarbazone, the strain of fungi to be treated, the degree of control required, the type of medium infected and the conditions under which the formulation is applied to seeds.

Preferably, the amount of thiosemicarbazone included in the liquid formulation falls within the range of 10 to 500 g/L. More preferably, the amount falls within the range 50 to 300 g/L. More preferably, the solvent is diethylene glycol methyl ether.

Diethylene glycol methyl ether has the formula set forth in Formula 1 wherein R ₄ = R ₅ = H; R = CH ₃ ; and n = 2.

Solvency of the solvent to thiosemicarbazone decreases as the alkyl group R increases in size and also when R ₄ or R ₅ is CH ₃ . High solvency of the solvent to thiosemicarbazone is important, particularly when formulations are stored commercially in a low temperature environment (for example, less than 5 ⁰ C).

The liquid formulation may further comprise additives. The additives may be selected from the group consisting of pigments or dyes, insecticides,

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co-solvents, adhesion agents and fungicides.

The insecticide protects treated seeds and grain from insect attack. Suitable insecticides are selected from, but not limited to, the group consisting of cypermethrin, triflumuron, permethrin, bio-resmethrin, maldison, bifenthrin, imidacloprid, fipronil, alpha-cypermethrin, lambda-cyhalothrin, pirimiphos-methyl and organo-chlorines, such as lindane. More than one insecticide may be included in the formulation to protect the treated seeds or grain from attack by different insect species.

The amount of insecticide used in the formulation will depend on the efficacy of the insecticide. Preferably, the amount of insecticide added to the formulation falls within the range of 0.1 to 20 g/L. More preferably, the amount falls within the range of 1 to 10 g/L.

The type of pigment or dye included in the formulation depends on the colour and intensity of colour required on the grain or seeds. Suitable dyes and pigments include tartrazine, blue FCF, rhodamine B, phthalocyanine blue, phthalocyanine green, pigment red 57.1 and methyl lake violet.

Preferably, the amount of dye included in the formulation falls within the range 5 to 50 g/L which relates to approximately a 30% solution of dye, or 1.5 to 15 g/L dye solids. Preferably, the amount of pigments included in the formulation falls within the range 1 to 50 g/L pigment solids.

Adhesion agents help improve adhesion of the seed dressing to the grain or seeds. Suitable adhesion agents include, but are not limited to, soluble polymers and/or low vapour pressure solvents, for example, polyvinyl

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pyrrolidone (PVP), carboxymethyl cellulose (CMC), glycerol and polyethylene glycol (PEG).

Additional fungicide(s) may be used to control fungal pathogens not affected by thiosemicarbazones. The amount and type of fungicide used depends on the solubility, stability and activity of the fungicide and their effect on thiosemicarbazone solubility and stability. Suitable fungicides may be selected from, but not limited to, the group consisting of thiram, fluazinam, mancozeb, flutriafol, carbendazim, thiabendazole, triadimefon, captan, imazalil, difenoconazole. Co-solvents may increase solubility of the thiosemicarbazone to create a more concentrated formulation and to reduce raw material costs. The type of co-solvent included in the formulation depends on the solubility and efficacy of the thiosemicarbazone included in the formulation. Suitable co-solvents may be selected from, but not limited to, the group consisting of aromatic hydrocarbons (Solvesso®), cyclohexanol, ethylene glycol, propylene glycol, alkylene carbonates (Jeffsol®), glycerol and aliphatic alcohols (n-hexanol). SECOND EMBODIMENT

The inventor has also discovered, however, that if a suspension concentrate of a thiosemicarbazone of formula 2 herein is combined with an emulsified oil as a single product, the effect is to suppress or eliminate dust that would otherwise have been produced by the thiosemicarbazone if used alone. This type of formulation is known as a "suspo-emulsion". These formulations may be prepared by mixing. Reference to preparation of

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suspension concentrates of the thiosemicarbazone are given in Australian Patent 498467.

Suitably the formulation of the second embodiment of the invention can be prepared using the following detailed procedure. A. THIOSEMICARBAZONE SUSPENSION CONCENTRATE

An amount of thiosemicarbazone is slurried in water, using a suitable dispersing agent. The slurry is then milled to the required particle size using standard milling techniques such as ball-milling or bead-milling. A preferred particle size as determined by laser particle size analyzer (e.g. Malvern Mastersizer) is that 90% of particles are below 30 micrometers and more preferably 90% of particles are below 10 micrometres.

The amount of thiosemicarbazone in the slurry is from 10 to 60% by weight, preferably 30 to 50% by weight, but sufficient to enable the desired amount of thiosemicarbazone to be formulated into the final product. The dispersant may be any suitable dispersant known to the art. Such dispersants may be anionic, non-ionic or cationic, such as, but not limited to, lignin sulphonates, naphthalene formaldehyde condensates, alkylphenol alkoxylates, tristyrylphenol alkoxylates, tristyrylphenol alkoxylate phosphates and their salts, fatty alcohol alkoxylates and fatty amine alkoxylates. The amount of dispersant used may be from 1 -10% by weight of the slurry and more preferably 4% by weight of the slurry.

A preferred formulation is:

PTT 50%

Dispersant 4%

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Water to 100 %

B. EMULSIFIABLE OIL

Into a suitable water-immiscible oil is blended sufficient emulsifier to enable the oil to emulsify spontaneously on addition to water. Oils suitable for this purpose may be petroleum derived. They may be paraffinic (Recosol®) or isoparaffinic (Isopar®) and have low vapour pressure.

Alternatively, suitable oils may be vegetable oils such as, but not limited to, canola, soy and cotton seed. Suitable emulsifiers are known to the art and may be anionic, non- ionic or cationic and are added in sufficient amount to enable formulation of a fine emulsion when oil is added to water. The amount of emulsifier used may be from 2-10% of the weight of the oil and more preferably 5.0% of the weight of oil. Examples of suitable emulsifiers are alkyl-aryl alkoxylates, fatty alcohol alkoxylates, fatty amine alkoxylates, vegetable oil alkoxylates, alkyl- aryl sulphonates, sorbitan esters, alkoxylated sorbitan esters. An especially preferred emulsifier is a mixture of ceto/stearyl alcohol ethoxylate and calcium alkylbenzene sulphonate. A preferred formulation is: Recosol P212 95%

Emulsifier 5%

C. OPTIONAL INGREDIENTS

These may be the same as described in the first embodiment. However in the second embodiment pH buffers, protective biocides,

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clays and stabilizers may also be used. Example of pH buffers are ortho- phosphoric acid, potassium dihydrogen phosphate and ammonium dihydrogen phosphate. Examples of protective biocides are benzisothiazolone solution (e.g. Proxel® GXL20 and triazine solution (e.g. Glokill® 77). Examples of clays are bentonite, attapulgite, diatomaceous earth, and examples of stabilizers arexanthan gum, carboxymethyl cellulose, polyvinyl pyrrolidone, polyethylene glycol and acrylic polymers.

A preferred combined formulation:

50% thiosemicarbazone suspension concentrate 600g/L Recosol P212 emulsifiable oil 312g/L

Red pigment (solids) 15g/L pH buffer, stabilizers and water to required volume

The amount of thiosemicarbazone suspension concentrate in the combined formulation may be varied according to the amount of thiosemicarbazone in the suspension concentrate and the desired amount of thiosemicarbazone in the combined formulation. The preferred amount of thiosemicarbazone in the suspension concentrate is from 10 to 60% by weight, more preferably 30 to 50% by weight.

The preferred amount of thiosemicarbazone in the combined formulation is from 50 to 500g/L, more preferably 100 to 400g/L.

The preferred amount of oil in the combined formulation is 50 to 400g/L, more preferably 150 to 350g/L.

It will also be appreciated that the seeds which have the dressings of the invention applied thereto may be selected from wheat, barley, oats,

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triticale and rice.

BRIEF DESCRIPTION QF THE FIGURE

FIG 1 shows a schematic representation of a dust measurement apparatus as described herein and FIG 2 shows a plan view of the apparatus shown in FIG 1.

EXAMPLES FIRST EMBODIMENT Method of preparation of formulation

Formulations according to this invention are prepared by mixing the required quantity of solvent, thiosemicarbazone and optional ingredients in a glass beaker or other suitable container and stirring at ambient temperature until all ingredients are dissolved. Dressing of seeds

In the laboratory, seeds may be dressed in several ways: A quantity of seed is placed in a glass jar such that the jar contains approximately 50% bulk volume of seed.

If a powder seed dressing is used, the required amount of seed dressing is added directly to the jar, which is then capped and shaken or tumbled until there is an even coating of seed dressing on the seed. If a liquid seed dressing is used, the liquid dressing is dribbled down the side of the jar, which is immediately capped and shaken or tumbled until there is an even coating of dressing on the seeds.

Instead of a glass jar, a partially inflated and sealed plastic bag may be used.

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The grain or seeds may be tumbled in an apparatus similar to a small concrete mixer.

The required quantity of dressing is added to the tumbling grain. The tumbling continues for approximately 5 minutes or until the coating on the seeds or grain is visibly even. Seed dressing formulation A preferred formulation is:

PTT 150g/L

Red dye 20g/L Insecticide 4g/L

Suitable solvent(s) to required volume

Although thiosemicarbazones are soluble to some extent in many solvents, in most, the solubility is too low for commercial application. Some solvents also react with thiosemicarbazones and are therefore unacceptable. The inventor discovered that a class of solvents known as "glycol ethers" exhibit good solvency characteristics and can be suitably used for preparation of thiosemicarbazone liquid seed dressings as described previously having the general Formula 1. Solubility of thiosemicarbazones The solubility of the following aromatic aldehyde thiosemicarbazones was determined (R3 = H in all compounds): benzaldehyde thiosemicarbazone (R1 = R2 = H)

4-ethyl benzaldehyde thiosemicarbazone (R1 = H, R2 = ethyl)

4-methoxybenzaldehyde (p-anisaldehyde) thiosemicarbazone (R1 =

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H, R2 = methoxy)

2-chlorobenzaldehyde thiosemicarbazone (R1 = Cl, R2 = H) 4-chlorobenzaldehyde thiosemicarbazone (R1 = H, R2 = Cl) 2,4-dichlorbenzaldehyde thiosemicarbazone R1 = R2 = Cl) 4-cyanobenzaldehyde thiosemicarbazone (R1 = H, R2 = CN)

4-bromobenzaldehyde thiosemicarbazone (R1 = H, R2 = Br) o-tolualdehyde thiosemicarbazone (R1 = methyl, R2 = H) in the following solvents: diethyleneglycol methyl ether; diethyleneglycol ethyl ether; diethyleneglycol butyl ether; ethyleneglycol ethyl ether; dipropyleneglycol methyl ether; ethylene glycol; and aromatic solvent (Solvesso 200).

Results

• Glycol ether solvents are the most effective solvents for thiosemicarbazones

• Diethyleneglycol methyl ether was the best solvent. Ethyl ether is less effective and butyl ether is less still.

• Solubility of thiosemicarbazones is influenced by the type of substituent on the benzene ring. For example, halogen (Cl, Br) and CN reduce solubility, and ethyl and methoxy increase solubility of thiosemicarbazones.

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Measurement of dust emission from seeds

The purpose of this invention was to develop a formulation which would overcome the problems directly associated with the bitter taste of dust emitted from treated grain during commercial use. The inventor designed a device which collects and measures dust emitted from treated grain. A preferred embodiment of the device is shown in FIGS 1 and 2.

The apparatus 1 comprises a stainless steel tube 2, which is 1300 mm in length and 100 mm in diameter. One end 3 of the tube may be sealed during use (the bottom end) with a removable, air-tight closure, such as a rubber gasket 4. Internally, the tube has four horizontal baffles 5, each in the shape of an inverted "V", placed at 400, 500, 600 and 700 mm from the top of the tube and at 60° angles to each other. Located approximately 350 mm from the bottom of the tube is a 30 mm diameter dust extraction tube 7, leading outside the apparatus, from which air, containing dust, may be extracted and collected by means of a dust filter 8 and pump (not shown).

The inlet of the dust extraction tube 9 is centrally placed and angled down to prevent grain from entering the inlet. The type of dust filter used is a commercially available "pro2000" dust mask filter cartridge manufactured by

Scott Health and Safety Oy, Finland. However, other suitable filters may be used for dust collection.

During use, air is drawn through the filter using a standard electrical vacuum pump at a velocity of approximately 15 litres per minute.

Dust emission measurement of the seed dressings of the invention was carried out as follows.

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The typical commercial treatment rate for seed grain is 1 gram or millilitre per 1 kilogram of grain. A quantity of grain is carefully mixed with the required amount of formulation and blended thoroughly to ensure even coating of the grain. A filter cartridge is weighed and then attached to the dust extraction tube and vacuum pump. The pump is started.

A weighed quantity of treated grain or seeds is poured centrally into the top 11 of the steel tube of the apparatus, hitting the baffles on the way down and creating dust. After a set time, the vacuum pump is stopped, the filter cartridge is removed and re-weighed, yielding a weight of dust collected.

It may be appreciated that some dust may remain on the internal surfaces of the apparatus. This is removed using a blast of compressed air between each measurement.

Samples of wheat were treated and tested as follows: (1) no treatment

(2) 1 ml/Kg of liquid seed dressing

(3) 1 g/Kg of RaxiKDT powder seed dressing

RaxilθT is a powder seed dressing manufactured by Bayer Cropscience, incorporating the fungicide "tebuconazole". It was used here for comparison purposes.

The combined amount of dust collected from 3 times 1 Kg of grain or seeds from each treatment was

(1) 0.032g

(2) 0.014g

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(3) 0.207g

The results show that the new formulation actually reduces the dust emitted in comparison to the untreated grain. The amount of dust emitted from Raxil T treated grain is far greater than the dust emitted from grain or seeds treated with the new formulation. Phytotoxicitv experiments

Germination trials were conducted of seeds treated with a formulation of the invention. The seeds were treated with standard (1 ml/Kg) and double (2 ml/Kg) amounts of formulation. Part of each group of treated seeds was stored at 4O ⁰ C for 2 weeks.

To test germination, 20 grains of wheat were placed on a pad of wet cotton wool and allowed to remain at ambient temperature for 4 days. The number of germinated seeds was counted and compared with a similar test on untreated seed. There was no statistical difference in germination between seeds of either of the above treatments.

All seeds dressed with a formulation of the invention germinated satisfactorily suggesting the formulation is not phytotoxic. SECOND EMBODIMENT

Formulations in accordance with the second embodiment are prepared by mixing a suspension concentrate of the thiosemicarbazone and an emulsified oil together with any optional ingredients in a suitable container and stirring at ambient temperature until the mixture is homogeneous. Dressing of seeds is carried out as described in the example concerning the first embodiment. The measurement of dust emission from the seeds is also

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carried out as described in the example concerning the first embodiment. Samples of wheat were treated and tested as follows:

(1) no treatment;

(2) 1 ml/kg of seed dressing comprising 50% thiosemicarbazone suspension concentrate at a concentration of 600g/L; Recosol

P212 emulsifiable oil at a concentration of 312g/L; Red pigment (solids) and pH buffer, stabilizers and water to required volume; and

(3) 1 g/kg of Raxil T seed dressing. The results obtained were:

(1) 0.019 g

(2) 0.013 g

(3) 0.188 g.

The results described above are similar to those obtained in the example of the first embodiment and show that the new formulation (2) reduces the dust omitted in comparison to the untreated grain. The amount of dust emitted from Raxil T treated grain is far greater than the dust emitted from grain or seeds coated with formulation (2) above.

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