INTRODUCTION

This invention relates to a stabilized liquid pesticide formulation comprising 0,S-dimethyl acetylphosphoramidothioate, commonly known as acephate, and the use of the undiluted formulation in a method of crop treatment, in particular, but not exclusively, in a method of treating citrus and other tree crops.

BACKGROUND

Acephate is an insecticidal active compound described in, for example, The Pesticide Manual, 8 ^th edition, page 1 (1987, The British Crop Protection Council). Acephate has the ula:

US Patent Nos. 3,716,600 and 3,645,172 discloses the preparation and activity of acephate, and the disclosures contained in these references are incorporated herein by reference. Acephate is widely used as an active ingredient in agricultural insecticides due to its efficacy in killing and controlling a variety of pests and insects.

However, acephate is not very stable in conventional non-solid pesticidal formulations, such that a vigorous decomposition of acephate in these conventional formulations takes place depending on storage conditions and, as a result of this instability, the insecticidal activity of acephate cannot be fully utilized.

Furthermore, the primary metabolite for acephate is 0,S-dimethyl phosphoramidothioate, commonly known as methamidophos. Plants and crops grown with the use of methamidophos include ornamentals, potatoes, pome fruit, stone fruit, citrus fruit, subtropical tree crops, vines, hops, brassicas, beet, cotton, maize, tobacco and others.

Methamidophos is classified as a WHO Toxicity Class "Class 1b, Highly Hazardous" compound, while acephate is classified as "Class III, Slightly Hazardous". Methamidophos is regarded as toxic and the high mammalian toxicity necessitates extra precautions and care by the operators in the handling and application procedures. It is rapidly absorbed through the stomach, lungs, and skin in humans. The acute oral LD ₅₀ for methamidophos in male rats has been reported as 15.6 mg/kg, while the LD ₅ o for acephate in male rats has been reported as 1447 mg/kg.

Many nations use methamidophos on crops, including developed nations such as Spain, United States, Japan, and Australia. However, due to its toxicity and general undesirability, the use of pesticides that contain methamidophos is currently being phased out in some of these countries. The maximum residue limit ("MRL") for methamidophos in any edible commodity in the European Union is currently set at 0.01 ppm. Therefore, producers and exporters of fruit and other commodities are exposed to serious risk when using methamidophos on their crops.

It is therefore clear that it is desirable, when selecting to work with acephate rather than methamidophos, that the acephate active ingredient in the pesticidal formulation be stabilized, such that the breakdown of acephate to methamidophos, and the risks associated therewith, is avoided.

US Patent no. 5,698,540 to Sumitomo Chemical Company discloses a stabilized pesticidal composition comprising acephate and one or more N- alkyl-2-pyrrolidones. It is reported here that the storage stability of acephate bulk or acephate in acephate containing liquid formulations is improved by the addition of N-alkyl-2-pyrrolidone(s). The liquid pesticidal compositions of this invention may further include an emulsifier, a solvent, and/or other pesticidal ingredients.

US Patent no. 5,488,043 also to Sumitomo Chemical Company discloses a method for stabilizing acephate by adding a sodium salt of condensed phosphate and/or a modified synthetic silicic acid to a dry pesticidal formulation. The dry formulation may further include a solid carrier, selected from a list given in the specification of the patent. The dry formulation may further comprise certain other auxiliaries, for example, surface active agents, colouring agents, perfume compounds, and other known stabilizers.

US Patent no. 6,335,026 also to Sumitomo Chemical Company discloses stabilized solid pesticidal compositions containing acephate and at least one of a further stabilizer compound selected from the group consisting of boron oxide, metaboric acid, aluminium oxide, and the calcined product of synthetic silicon dioxide. The composition may further comprise certain other auxiliaries, for example, surface active agents, colouring agents, perfume compounds, and other known stabilizers.

The stabilized acephate compositions disclosed in the prior art each suffers from certain disadvantages including the increased cost of manufacture, the necessity of further processing steps for certain applications, and the suitability of the compositions for use in certain applications.

Therefore, there remains a need for improved stabilized acephate formulations that addresses at least some of the shortcomings of known compositions.

SUMMARY OF THE INVENTION

According to a first aspect to the present invention there is provided a stabilized liquid pesticidal formulation, suitable for use as an undiluted concentrate stem application pesticide, the formulation comprising a pesticidally effective amount of 0,S-dimethyl acetyiphosphoramidothioate in methyl alcohol.

Preferably, formulation comprises 0,S-dimethyl acetyiphosphoramidothioate at a concentration of between about 100 mg/mL to about 600 mg/mL.

More preferably, the formulation comprises 0,S-dimethyl acetyiphosphoramidothioate at a concentration of between about 250 mg/mL to about 450 mg/mL.

Even more preferably, the formulation comprises 0,S-dimethyl acetyiphosphoramidothioate at a concentration of between about 300 mg/mL to about 400 mg/mL.

Most preferably, the formulation comprises 0,S-dimethyl acetyiphosphoramidothioate at a concentration of about 350 mg/mL.

In one embodiment of the invention, the formulation comprises no further auxiliary agents including surface active agents and stabilizers.

According to a second aspect to the present invention there is provided a method of treating trees or plants against pests including the step of applying an undiluted concentrate stabilized liquid pesticidal formulation comprising a pesticidally effective amount of 0,S-dimethyl acetylphosphoramidothioate in methyl alcohol directly to the trunk or stem of the tree or plant.

In one embodiment, more than one application of the undiluted concentrate pesticide formulation is made to the tree or plant.

In a preferred embodiment, the applications are made at intervals ranging between 7 days and 28 days.

In another preferred embodiment, the applications are made at intervals ranging between 14 days and 28 days.

In a particularly preferred embodiment, the applications are made at 21 or 28 day intervals.

Preferably, the formulation comprises 0,S-dimethyl acetylphosphoramidothioate at a concentration of between about 100 mg/mL to about 600 mg/mL.

More preferably, the formulation comprises 0,S-dimethyl acetylphosphoramidothioate at a concentration of between about 250 mg/mL to about 450 mg/mL.

Even more preferably, the formulation comprises 0,S-dimethyl acetylphosphoramidothioate at a concentration of between about 300 mg/mL to about 400 mg/mL.

Most preferably, the formulation comprises 0,S-dimethyl acetylphosphoramidothioate at a concentration of about 350 mg/mL.

The plants or trees may be citrus varieties, deciduous fruit including stone fruit and pome fruit, vines, subtropical crops including avocado, mango and nut crops including macadamia and pecans. The pests may be selected from the group consisting of Aphid (Toxoptera citricidus), Psylla {Trioza erytreae), Thrip (Scirtothrips aurantii), Orange dog (Papillio spp.), and bud mite (Aceria sheldoni).

However, it will be appreciated by those persons skilled in the art that the use of the pesticide formulation is not limited to the aforementioned species. It is envisaged for use against not only those species, but may also include all representatives of that particular group of insect, for example: fam: Psyllidae, (i) Genus: Diaphorina, Species D. citri ; (ii) Genus: Trioza, Species: T erytreae; and

fam: Aphididae (i) Genus: Aphis, Species: gossypii; (ii) Genus: Toxoptera Species: T. citricidus.

Preferably, the undiluted concentrate pesticide formulation is applied at an application rate of between about 0.1 mL per 10 mm trunk or stem diameter to 25 mL per 150 mm trunk or stem diameter.

More preferably, the undiluted concentrate pesticide formulation is applied at an application rate of between about 0.1 mL per 10 mm trunk or stem diameter to 1 mL per 50 mm mm trunk or stem diameter.

In one embodiment, residue analysis results for 0,S-dimethyl phosphoramidothioate is below 0.05 ppm after 7 days, 14 days, 21 days, and 28 days after application.

In a particularly preferred embodiment, residue analysis results for 0,S- dimethyl phosphoramidothioate is below 0.01 ppm after 7 days, 14 days, 21 days, and 28 days after application.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in more detail with reference to the following non-limiting embodiments and figures in which: shows a graphical representation of the rate of uptake in citrus through its efficacy against orange dog (Papillio spp.) and citrus aphid (Toxoptera citricidus); shows a graphical representation of the efficacy of the pesticide formulation according to the present invention compared to Citrimet 500 AL against citrus aphid (Toxoptera citricidus); shows a graphical representation of the efficacy of the pesticide formulation according to the present invention compared to Citrimet 500 AL against citrus thrip {Scirtothrips aurantii); shows a graphical representation of the efficacy of the pesticide formulation according to the present invention compared to Citrimet 500 AL against citrus bud mite (Aceria sheldoni); shows a graphical representation of the efficacy of the pesticide formulation according to the present invention compared to Citrimet 500 AL against citrus psylla (Trioza erytreae); shows a graphical summary of the mean % efficacy of the pesticide formulation according to the present invention compared to Citrimet 500 AL for all pests tested; and shows a graphical representation of residual activity of a stem application of the pesticide formulation according to the present invention on Asian Citrus Psylla (ACP) in non-bearing citrus as depicted by mean % mortality.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter with reference to the accompanying figures, in which some of the non-limiting embodiments of the invention are shown. In the accompanying figures like features are indicated by like reference numerals. The invention as described hereinafter should not be construed to be limited to the specific embodiments disclosed, with slight modifications and other embodiments intended to be included within the scope of the invention.

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

As used herein, throughout this specification and in the claims which follow, the singular forms "a", "an" and "the" include the plural form, unless the context clearly indicates otherwise.

The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms "comprising", "containing", "having", "including", and variations thereof used herein, are meant to encompass the items listed thereafter, and equivalents thereof as well as additional items.

In its broadest form, this invention relates to a stabilized liquid pesticide formulation including O.S-dimethyl acetylphosphoramidothioate, commonly known as acephate, and the use of the undiluted formulation in a method of crop treatment, in particular, but not exclusively, in a method of treating citrus crops.

It is envisaged that the stabilized liquid pesticide formulation comprises the active ingredient 0,S-dimethyI acetylphosphoramidothioate (acephate) at a concentration of between about 100 mg/mL to about 600 mg/mL, preferably at a concentration of between about 250 mg/mL to about 450 mg/mL, more preferably at a concentration of between about 300 mg/mL to about 400 mg/mL, and most preferably at a concentration of about 350 mg/mL of methyl alcohol (methanol).

It is accepted that acephate breaks down to methamidophos as the major metabolite of acephate. According to common knowledge, in conducting residue analysis testing or breakdown product testing, one would expect methamidophos residues as a metabolite of degraded acephate for formulations where the acephate is not stabilized by an auxiliary or additional stabilizers. According to the testing conducting in development of the present invention this did not occur. The results indicate that residues remained in the plant system as acephate.

Without thereby wishing to be bound by the confines of any particular theory, it is believed that residues remain as acephate in the plant system and following translocation to the distal plant extremities (growth points), it is then rapidly dissipated via the methamidophos metabolite stage to basic building blocks of and acceptable nature. The test results show that the residue levels of acephate for the formulation of the current invention to be below the limit of quantification 28 days after application.

It is envisaged that the method of the present invention for treating trees or plants against pests includes the step of applying an undiluted concentrate stabilized liquid pesticide formulation comprising the active ingredient 0,S- dimethyl acetylphosphoramidothioate (acephate) in methyl alcohol directly to the trunk or stem of a tree or plant.

The concentration of 0,S-dimethyl acetylphosphoramidothioate (acephate) in methyl alcohol is between about 100 mg/mL to about 600 mg/mL, preferably at a concentration of between about 250 mg/mL to about 450 mg/mL, more preferably at a concentration of between about 300 mg/mL to about 400 mg/mL, and most preferably at a concentration of about 350 mg/mL of methyl alcohol (methanol).

The stabilized liquid pesticide formulation of the present invention is a systemic insecticide formulated as an undiluted concentrate stem paint-on or stem spray-on treatment, for the control of insect pests.

Research and testing was conducted towards acquiring data in support of an application for registration for use on citrus for the control of and protection of the crop from damage caused by insect pests. The insect pests positively shown to be controlled by stabilized liquid pesticide formulation of the present invention include: Orange dog (Papillio spp.), Citrus aphid (Toxoptera citricidus), Citrus thrip (Scirtothrips aurantii), Citrus bud mite (/Acer/a sheldoni) and Citrus psylla (Trioza erytreae).

However, it will be appreciated by those persons skilled in the art that the use of the pesticide formulation is not limited to the aforementioned species. It is envisaged for use against not only those species, but may also include all representatives of that particular group of insect, for example: fern: Psyllidae, (i) Genus: Diaphorina, Species D. citri ; (ii) Genus: Trioza, Species: T erytreae; and

fam: Aphididae (i) Genus: Aphis, Species: gossypii; (ii) Genus: Toxoptera Species: T. citricidus.

Efficacy and crop safety trials

Efficacy and phytotoxicity field trials were conducted in three different citrus producing areas, on three different citrus types (oranges, lemons, soft citrus), four different varieties, on four different rootstocks.

A stabilized liquid pesticide formulation according to the present invention, comprising 350 g/L of the active ingredient 0,S-dimethyl acetylphosphoramidothioate in methyl alcohol, was used in the trials shown below.

A summary of the trail data is provided below in Table 1 , while physical and chemical analysis information for the stabilized liquid pesticide formulation of the present invention is shown in Table 2. Table 1. Summary of trial data obtained for the stabilized liquid pesticide formulation of the present invention. J. Terblanche Citrus Stem application - (50-55 mm diameter)

Citrus 06 Orchard J137, Navelina i) Vertical band spray application(one side of stem)

Freudenhof Boerdery,

Marble Hall, Limpopo Rootstock: Dosage rates:

Carrizo citrange i) 4 mi per tree-vertical band spray (one side).

8.m£ per tree- vertical band spray (both sides.)

Ns of applications : 4

J. Terblanche Navel Stem application - (55-60 mm diameter)

Citrus 07 Orchard J 137, i) Vertical band spray application

Freudenhof Boerdery, Rootstock: ii) Horizontal band paint application Marble Hall, Limpopo Carrizo (width equal to stem diameter)

Dosage rates:

i) 1.5-1.8 m£ per tree-vertical band spray application.

3.5 m£ per tree-vertical band spray application ii) 1.5-1.8 m{ per tree-horizontal band paint application.

N° of applications : 4

H. Le Roux Lemons, Stem application : (100-110 mm diameter)

Citrus 08 Britz, North West Lisbon i) Vertical band spray application

Province

Rootstock: Dosage rates:

Swingel i) 4 mi per tree-vertical band spray application. citrumelo 8 mi per tree-vertical band spray application

N5 of applications : 4

J. Louwrens Citrus Stem application: (64-70mm diameter)

10114 BTE Dralta Boerdery, Sweet Pera i) Horizontal band paint application

Bokfontein, North West Orange (width equal to stem diameter)

Province

Dosage rates:

1 mi per tree- horizontal band application (1/2 width)

2 m£ per tree- horizontal band application (1x width) 4 mi per tree- horizontal band application (2x width)

Ns of applications : 1

The results obtained in the above tabulated trails are graphically represented, and will be discussion at the hand of, the graphs shown in Figures 1 to 6. The results from the trials will be discussed per insect pest tested, and depicted as overall mean % efficacy obtained from all trials. Table 2. Physical and chemical analysis results for the

pesticide formulation according to the present invention.

The efficacy of the stabilized liquid pesticide formulation of the present invention was compared to that of commercially available Citrimet 500 AL (Reg. No. L4361 ) which is registered for use in inter alia citrus and mango crops. Citrimet 500 AL comprises the undesirable active ingredient methamidophos.

In Figures 1 to 6 the term "UTC" refers to an untreated control, while the term "UTC infested flushes" refers to the percentage of insect pest infested flushes, giving an indication of the level of infestation and confirming that the levels of pest infestation are significant. Further, the terms "Spectra Stem 350 AL" and "SS" refers to the pesticide formulation according to the present invention.

Figure 1 shows the rate of uptake in citrus through its efficacy against Orange dog (PapHlio spp.) and Citrus aphid (Toxoptera citricidus). As can be seen from Figure 1 the rate of uptake of the pesticide formulation as a horizontal band stem treatment in citrus occurred between 48 and 72 hours following the application, as determined by mortality in orange dog larvae. Figure 2 shows the efficacy of the pesticide formulation against citrus aphid (Toxoptera citricidus). As can be seen from Figure 2, the efficacy of the pesticide formulation with horizontal band application and vertical spray application compared well with the standard Citrimet 500 AL horizontal band application on citrus aphid. Figure 2 further shows that the difference in efficacy on citrus aphid was achieved irrespective of the application method used (vertical spray application or horizontal band application) in the 4 trails conducted.

Figure 3 shows the efficacy of the pesticide formulation against citrus thrip (Scirtothrips aurantii). It is clear from Figure 3 that the efficacy of the pesticide formulation with vertical spray, vertical band, and horizontal band compares well with the standard Citrimet 500 AL horizontal band application on citrus thrip. Figure 3 further shows that the difference in efficacy on citrus thrip was achieved irrespective of the application method used in the 7 trials conducted.

Figure 4 shows the efficacy of the pesticide formulation against citrus bud mite (Aceria sheldoni). The efficacy of the pesticide formulation with vertical spray, vertical band, and horizontal band compares well with the standard Citrimet 500 AL horizontal band application on citrus bud mite. Again, similar to the results obtained for citrus thrip and citrus aphid, Figure 4 shows that there was no application dependent statistical difference in efficacy on citrus bud mite for the various application methods tested in the 4 trials conducted.

Figure 5 shows the efficacy of the pesticide formulation against citrus psylla (Trioza erytreae). The efficacy of the pesticide formulation with vertical spray, vertical band, and horizontal band compares well with the standard Citrimet 500 AL horizontal band application on citrus psylla. Again, similar to the results shown in Figures 1 to 4, Figure 5 shows that there was no application dependent statistical difference in efficacy on citrus bud mite for the various application methods tested in the 6 trials conducted.

As can be seen from Figure 6, and indeed from each of Figures 2 - 5 discussed above, all the methods of application compared well with the registered Citrimet 500 AL pesticide product. From a review of the test results, it may prove slightly beneficial to apply the pesticide formulation of the present invention as a horizontal band, according to the methods known in the art.

Crop safety of the products used as stem treatments on tree crops is largely dependent on the formulation, as these treatments are usually undiluted applications directly onto the bark of the tree trunk. Instances have been cited in the literature of incompatible formulations that have been used as undiluted stem applications in the past that have resulted in severe phytotoxicity. Cases have been documented where insecticide formulations containing similar active ingredients, but registered for use as diluted spray applications on other types of agricultural crops, have caused widespread phytotoxicity in the form of bark split, seedling mortality and branch die back when applied as undiluted stem applications in citrus. There have also been cases where certain rootstocks used on commercial varieties of citrus have shown incompatibility to certain insecticide formulations used as stem applications.

Trial results were obtained for the pesticide formulation according to the present invention at between 0.5 - 3 times a proposed recommended rate, to ascertain any phytotoxicity. The results obtained were for tests conducted on 6 citrus varieties: 4 orange, and 2 lemon, with the sample set including the common navel (2), soft citrus (2) and Turkey citrus varieties, and on 4 of the most commonly used rootstocks.

The parameters used to determine phytotoxicity were those typically used in such tests and included: bark split, branch discoloration, branch die back. No visual symptoms of phytotoxicity were observed in the treated trees from the single, double (8 trials) or triple dosage rates (2 trials) applied, supporting the position of an absence of any subsequent phytotoxicity from the treatment of the trees with the pesticide formulation of the present invention.

The pesticide formulation of the present invention used as an undiluted stem treatment formulation exhibited consistently high levels of efficacy on all target insect species, with good commercial control, and comparable efficacy obtained to that recorded for the commercially available and registered product Citrimet 500 AL.

From the data presented the pesticide formulation of the present invention applied in the undiluted form, either as a stem band application or as a directed spray treatment to the tree trunk or stem in citrus, was shown to be safe for use on both oranges and lemons on the widely grown and common varieties of citrus when applied at the tested 0.5 - 3 times the recommended dosage rates, with no phytotoxicity noted during the evaluations.

Residues in citrus fruit were tested for the Minneola, Turkey and Lemon cultivars. Residue sampling methods were conducted according to Good Agricultural Practice (GAP), where representative fruit samples were collected from the treated trees and placed directly into impermeable bags, avoiding any physical contact or contamination, at the specified intervals following application (0 days; 7 days; 14 days; 21 days and 28 days after application).

The impermeable plastic bags specifically used for the storage of residue samples) containing the fruit residue samples were then sealed and placed in frozen storage within two hours of collection. The frozen samples were then delivered to the accredited laboratory (South African Bureau of Standards) by suitably qualified personnel, within 2 months of collection, for subsequent residue analysis according to the methods as described.

The results of the residue analysis testing are shown in Table 3 below.

Table 3. Residues in citrus fruit.

Ace = Acephate, Meth = Methamidophos, DAA = days after application, LOQ = 0.01 mg/kg Figure 7 shows the results of tests conducted at the University of Florida to determine the level and residual activity of a stem application of a pesticide formulation according to the present invention ("Spectra Stem") on young non- bearing citrus plants on the Asian Citrus Psylla, Diaphorina citri.

Materials & Methods

CropA ariety: Sweet Orange cv "Hamlin"

Rootstock: Swingel

Age/size: 1 m tall plants grown in 4L pots

Application method: Paint brush used to apply required amount in a horizontal band encircling the stem

Five (5) trees were treated with the pesticide formulation by applying 1 ml of the formulation with a paint brush at the highest branch point. Five (5) trees were treated with water only for the control experiment.

A 20 cm diameter paper bowl was placed around the trunks for collection of the psyllids. A fabric cover was placed over the plants extending below the base of the bowl.

Fifty (50) adult Asian citrus psyllids were introduced and the cloth cover was tied beneath the bowl to prevent the psyllids from escaping. The plants remained outside in ambient conditions and watered as needed.

Mortality was evaluated at 3, 7, 14, 21 , 28, 35 and 46 days post-treatment. At each evaluation period, the covers were removed from the plants and the number of live and dead psyllids counted and removed. Another set of 50 psyllids were then introduced.

As can be seen from the results presented in Figure 7, the formulation according to the present invention is effective as undiluted concentrate stem application pesticide against Asian Citrus Psylla, Diaphorina citri. The above description of some of the illustrative embodiments of the invention is to indicate how the invention can be made and carried out. Those of ordinary skill in the art will know that various details may be modified thereby arriving at further embodiments, but that many of these embodiments will remain within the scope of the invention. For example, it will be appreciated that the stabilized liquid pesticide formulation described may be altered by including minor auxiliary compounds such perfumes, colouring agents, alternatively the formulation may be altered by including a further pesticidal agent.