NZ596204A - Use of spinosyns for controlling earth mites and fleas affecting crops and pastures - Google Patents

Abstract

Disclosed is a method of controlling a pest selected blue oat mite (Penthaleus major) or from the family consisting of Sminthuridae, said method including applying to a locus where control is desired, after the emergence of seedlings from earth within the locus, an inhibitory or inactivating amount of a pesticidal compound selected from the group consisting of spinosyns, synthetically-modified spinosyns, and derivatives, salts and mixtures thereof.

Description

Patent No, 5 - Complete Specification No, Date: Methods of controlling pests We, Dow AgroSciences LLC. 9330 Zionsvilie Road, Indianapolis, Indiana 46268-1054, hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement Freehills M1782856 Page 1 11597773 1A Methods of controlling pests Field of the invention The present invention relates to methods of controlling pests, more particularly ground dwelling mites and springtails.

Background of the invention Earth mites and springtails are major pests affecting establishing crops and pastures across Australia and New Zealand, Such pests are commonly controlled with applications of organophosphate or synthetic pyrethroid insecticides during the crop establishment phase in autumn, or in spring pastures in preparation for the following 10 autumn. Both these groups of insecticides have been used so widely that there are now mite populations that are resistant to them. Furthermore, organophosphate and synthetic pyrethroid insecticides may not have good mammalian safety and are broad spectrum (non-selective) pesticides so they remove beneficial insects as well as the pests.

Spinosyns are a family of compounds produced from fermentation of Saccharopolyspora spinosa. Such fermentation products are described in US patent No. 5,362,634, which is incorporated herein by reference.

US patent no. 6,001,981, which is also incorporated herein by reference, describes chemical modifications of spinosyn compounds, Spinosad is a mixture of spinosyn A and spinosyn D and is registered by the United States Environmental Protection Agency (EPA) to control insects such as fruit flies, caterpillars, leaf miners, thrips, sawflies, spider mites, fire ants and leaf beetle larvae.

Spinetoram is an analogue of spinosad, and includes a mixture of 3-0-ethyi-5,6-dihydro Spinosyn J and 3-O-ethyl Spinosyn L (CAS Nos. 187166-40-1 and 187166-15-0, 25 respectively). 11597773 2 Spinosyns (including spinetoram) have a relatively narrow range of effectiveness i.e. they do not control all insects. While generally having activity against Lepidoptera (caterpillars, moths, butterflies), Thysanoptera (thrips), Hymenoptera (wasps and bees) and some Coleoptera (beetles), they are generally ineffective against most other insect-5 like organisms. For example, spinosyns have been tested against mites (phyophagous and predatory) in fruit trees and been shown to be ineffective at common use rates and use patterns.

It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

Summary of the invention In a first aspect the present invention provides a method of controlling a pest selected from the group consisting of ground dwelling mites and springtails, said method including applying to a locus where control is desired an inhibitory or inactivating amount of a pesticidal compound selected from the group consisting of spinosyns, 20 synthetically-modified spinosyns, and derivatives, salts and mixtures thereof.

The method of the present invention may be used to control a variety of ground dwelling mites and springtails. By the term a "ground dwelling mite" or "earth mite" as used herein in the description and claims is meant a mite that spends a substantial part of its time, for example between approximately 50% and approximately 95%, more preferably 25 between approximately 75% and 90% of its time on the soil surface, rather than on foliage of plants. Such ground dwelling or earth mites include those in the families Penthaleidae, Erythraeidae and Tetranychidae. For example, the method of the present invention is particularly applicable to control of red-legged earth mite (Halotydeus 11597773 3 destructor) and blue oat mite (Penthaleus major), Springtails include organisms from the families Collembola and Sminthuridae. The method of the present invention is particularly applicable to control of springtails such as the lucerne flea (Sminthurus viridus).

The pesticidal compounds may be applied to any locus inhabited by the ground-dwelling mite or springtail. The term "locus" as used herein in the description and claims refers to the environment in which the ground-dwelling mite or springtail lives or where its eggs are present, including the air or soil surrounding it, the food it eats or objects which it contacts. Typically the compounds are applied to the foliage of a plant which the 10 ground-dwelling mite or springtail might feed on and/or the soil surrounding such plants. For example, the method of the present invention may be applied to pastures or broad acre crops, pre- or post-emergence. In a particularly preferred embodiment, the compound is applied to very young seedlings or pre-emergence of the crop or pasture as a bare earth treatment. Crops or pastures to which the method of the present 15 invention may be applied include, but are not limited to, legumes such as chick peas, subterranean and other clovers, medics, lucerne, oilseeds such as canola, lupins, ryegrass, cereals such as oats and grains such as wheat.

A spinosyn or synthetically-modified spinosyn, or a derivatives, salt or mixture thereof may be used in the method of the present invention. Preferably, the spinosyn is a 20 compound as described in US patent nos. 5,382,634 or 6,001,981, the entire disclosures of which are incorporated herein by reference. The spinosyn compounds typically have the following structure. 11597773 (I) 11587773 Factor r1" K2' R.J'* o4 1 IV M5' r' * p7 * Spinosyn A H CH* o C2H5 ch3 CH i ch? Spinosyn b H chs c2ifc CH"( CHi -'H.? spinosyn c H CH, V^~-■"-*4 C CH chj ch; Spinosyn d CH, chi C2Hs CH-, CHk ch.;, Spinosyn E H ch:j \ ■■! ' ch.i CH? ch? ch? Spinosyn F H H o cihi CH.' chi CH- spinosyn g H ch3 ,0W"V-r\ V-- °<«A CH, C2Hs ch3 CH.

CH? Sp:nosyn H H ch;, c2h5 H chj CH? spmosyn J h ch". ■' >■ V'-T—' A c2h5 CH? H ch? Spinosyn K H CH;, c2Hs CH.i ch? H Spinosyn. L Oil CHx (cww»-^£*. 0 c2h5 ch3 H chj Spinosyn M h Of? (chi)nh-^^i, 0' \- \__ c2h5 ch;? H CH-, Spinosyn n ch". ch-, V-^- *»*\ c^hc ch3 H CH? Spinosyn 0 CH. ch. (chi)(n-^££. 0 \-^—-a c2hr, CH? CH? H Spinosyn p H ch3 c2h5 ch? H H Spinosyn, q ch? CH, c2h;, H ch3 ch? Spinosyn R H ch3 {CM,)NH-^^i. 0 C2Hr H ch, cm;.

Spinosyn s H CH-, ichiw-^ 0 \ —"-"-A ch;- H ch3 ch? Spinosyn t h ch? V—— C2hs h h ch? Spinosyn U h ch, c2h5 h c«3 h 11597773 6 Spinosyn v chj CH, c2h5 h CH.i h Spinosyn W CH i ch3 c.:hr, chj h h Spinosyn y h CJh \ \ \— CH,-, CH;< ch,; h spinosyn a 17-Psa h chi u n c2m ch;i ch i ch* Spinosyn d 17-Psa ch 1 ch;j u n C2Hs CH 1 ch;} ch3 Spinosyn e 17-Psa h chj H CBa ch3 c«3 chi Spinosyn F 17-Psa h h h CjHc chi chi ch.i Spinosyn h 17-Psa H ch1; h c2h5 h ch? ch_i Spinosyn J 17-Psa H CHj h C2Hs ch3 h CHj Spinosyn L 17-Psa ch.3 ch;i h c^.hr, CHj h ch3 and 11597773 7 Factor Rl' r2' r1'' I r4 r5* Spinosyn A 9-Psa H ch3 c2h5 H Spinosyn D 9-Psa CH.3 ch3 c2H5 h Spinosyn A Aalvcone H ch3 h c2h5 H Spinosyn D Aalvcone CH'b CH.i H c2h5 h The naturally produced spinosyn compounds may be produced via fermentation from cultures NRRL 18719, 185337, 18538, 18539, 18719, 1843 and 18823, These cultures have been deposited and made part of the stock culture collection of the Midwest Area 5 Northern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, IL 81604.

Preferably, the synthetically modified spinosyn is a compound as described in US patent no. 6,001.381. the entire disclosure of which is incorporated herein by reference.

By the term 'derivative* as used herein in the description and claims is meant an 10 organic compound obtained from, or regarded as derived from, a compound of the present invention. Examples of derivatives include compounds where the degree of saturation of one or more bonds has been changed (e.g., a single bond has been changed to a double or triple bond) or wherein one or more atoms are replaced with a different atom or functional group. Examples of different atoms and functional groups 15 may include, but are not limited to hydrogen, halogen, oxygen, nitrogen, sulphur, hydroxy, alkoxy, alkyf, alkenyl, alkynyl, amine, amide, ketone and aldehyde.

The salts of the pesticida! compounds of the present invention may be prepared using standard technology for preparing salts which are well known to those skilled in the art. Salts may be neutralized to form an acid addition salt. Acid addition salts that are 11597773 8 particularly useful include, but are not limited to, salts formed by standard reactions with both organic and inorganic acids such as sulphuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, laeic, fumaric, cholic, pamoic, mucic, glutamic, camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, stearic, salicylic, methanesulfonic, 5 benzenesuffonic, sorbic, picric, benzoic, cinnamic and like acids.

Mixtures of the pesticidal compounds may be used in the method of the present invention. For example, preferred mixtures include Spinosad, which is a mixture of Spinosyn A and Spinosyn D; and Spinetoram, which is a mixture of 3'-0-ethyl-5,6-dihydro Spinosyn J and 3"-0-ethyl Spinosyn L. 1 o More particularly, Spinosad comprises a mixture of approximately 50% to approximately 95% (2R,3aS,5aR,5bS,9S,13S,14R,16aS,16bR)-2-(6-deoxy-2,3»4-tri-0-methyl-a-l-mannopyranosyloxy)-13-(4-dimethylamino-2,3,4,6-tetradeoxy-{3-D-erythropyranosyloxy)-9-ethyl-2,3,3a, 5a, 5b, 6,7,9,10,11,12,13,14,15,16a, 16b-hexadecahydro-14-methyl-1 H-as-indaceno[3,2-d)oxacyclododecine-7,15-dione and approximately 50% to approximately 15 5% (2S,3aR,5aS,5bS,9S,13S,14R)16aS,16bS)-2-(6-deoxy-2,3,4-tri-0-methyl-a-L-mannopyranosyloxy)-13-(4-dimethylamino-2,3,4,6-tetradeoxy-{3-D-erythropyranosyloxy)-9-ethyl-2t3,3a,5a,5b,6,7,9,10,11,12,13,14,15,16a,16b-hexadecahydro-4,14-dimethyl-1 H-as»indaceno[3,2-d]oxacyclododecine-7,15-dione.

More particularly, Spinetoram comprises a mixture of approximately 50% to 20 approximately 90% {2/?,3aR,5aR,5bS,9S,13S,14R,16aS,16bR)-2-(6-deoxy-3-0-ethyl-2,4-di-0-methyl-a-L-mannopyranosyloxy)-13-[(2R,5S,6R)-5-(dimethylamino)tetrahydro-6-methylpyra n-2-yloxy]-9-ethyl-2,3, 3a ,4,5, 5a,5b,6,9,10,11,12,13,14,16a, 16b-hexadecahyd ro-14-methy I-1 H-as-indaceno[3,2-d]oxacyclododecine-7,15-dione and approximately 50% to approximately 10% (2Rl3aRl5aS,5bS,9S,13S,14R,16aS,16bS>-2-25 (6-deoxy-3-0-ethyl-2,4-di-0-methyl-a-L-mannopyranosyloxy)-13-[(2R,5S,6R)-5-(dimethylamino)tetrahydro-6-methylpyran-2-yloxy]-9-ethyl-2,3,3a,5a,5b,6,9,10,11,12,13,14,16a,16b-tetradecahydro-4,14-dimethyl-1 H-as-indaceno[3,2-d]oxacyclododecine-7,15-dione (bridged fused ring systems nomenclature); or 11597773 9 a mixture of approximately 50% to approximately 90% (1 S.2R5R7R.9R, 10S.14R, 15S.19S) •7--f6-deoxy-3-0-ethyl-2.4-di-0-methyl-a-L-mannopyranosyloxy)-15-[(2R5S,6f?)-5-(dimethylamino)tetrahydro-6-methylpyran-2-yloxy]-19-ethyl-14-methy l-20-oxatetracyclo[ 10.10.0.0210.05&]docos-11-erte-13,2.1 -dione 5 and approximately 50% to approximately 10% (1 S,2S,5R7R,9S, 10S, 14/?, 15S, 19S)-7-(0-deoxy-3-O-ethyl-2,4-di-O-methyt-a-L-mannopyranosyloxy)-15-[(2R5S,6R)-5-(dimethylamino)tetrahydro-6-methylpyran-2-yloxy]-19-ethyl-4.14-dimethyl-20-oxatetracyclo[10.10.0 0210 05 ^docosa-3,11 -diene-13,21 -dione (extended von Baeyer nomenclature) For further details on Spinosad and Spinetoram, see http1 //www alanwood.net/pesticides/iridex cn frame.html The pesticidal compound or mixture of compounds may be applied as part of a pesticidal composition. A pesticidal composition may be prepared according to the procedures and formulas which are conventional in the agricultural or pest control art. 15 The compositions may be concentrated and dispersed in water or may be used in the form of a dust, bait or granular formulation. The dispersions are typically aqueous suspensions or emulsions prepared from concentrated formulations of the compounds The water-soluble or water-suspension or emulsifiable formulations may be either solids, wettable powders or liquids, known as emulsifiable concentrates or aqueous 20 suspensions. Wettable powders may be agglomerated or compacted to form water dispersible granules. These granules may comprise mixtures of pesticidal compound, inert carriers and surfactants The concentration of the pesticidal compound is typically between about 0.1% to about 90% by weight. The inert carrier may be selected from one or more of the group consisting of attapulgite clays, montmorillonite clays and the 25 diatomaceous earths or purified silicates.

Surfactants comprise typically about 0.5% to about 10% by weight of the wettable powder, where the surfactants are typically sulfonated lignins, condensed naphthalene sulfonates, the naphthalene-sulfonates, alkyl-benenesulfonates, alkysulfonates or non-ionic surfactants such as ethylene oxide adducts of alkylphenols or mixtures thereof. 11597773 Emulsifiable concentrates of the compounds typically range from about 50 to 500 grams of compound per litre of liquid, equivalent to about 10% to about 50% by weight, dissolved in an inert carrier which is a mixture of a water immiscible solvent and emulsifiers Organic solvents include organics such as xylenes, and petroleum fractions 5 such as high-boiling napthlenic and olefinic portions of petroleum which include heavy and aromatic naptha Other organics may also be used such as terpenic solvents -rosin derivatives, aliphatic ketones such as cyclohexanone and complex alcohols. Emulsifiers for emulsifiable concentrates are typically mixed ionic and/or non-ionic surfactants such as those mentioned herein or their equivalents.

Aoueous suspensions may be prepared containing water-insoluble compounds, where the compounds are dispersed in an aqueous vehicle at a concentration typically in the range of between about 5% to about 50% by weight. The suspensions may be prepared by finely grinding the compound and vigorously mixing it into a vehicle of water, surfactants, and dispersants as discussed herein. Inert ingredients such as inorganic 15 salts and synthetic or natural gums may also be employed to increase the density and/or viscosity of the aqueous vehicle as is desired.

Precipitated flowables may be prepared by dissolving the active molecule in a water-miscible solvent and surfactants or surface active polymers. When these formulations are mixed with water, the active compound precipitates with the surfactant controlling 20 the size of the resulting micro-crystalline precipitate. The size of the crystal may be controlled through the selection of specific polymer and surfactant mixtures.

The compounds may also be applied as a granular composition that is applied to the soil. The granular composition typically contains from: about 0.5% to about 10% by weight of the compound. The compound may be dispersed in an inert carrier which is 25 typically clay or an equivalent substance Generally, granular compositions are prepared by dissolving the compound in a suitable solvent and applying it to a granular carrier which has been preformed, to a desirable particle size. The particle size is typically between about 0.5mm to 3mm. The granular compositions may also be prepared by forming a dough or paste of the carrier and compound, drying the 30 combined mixture, and crushing the dough or paste to the desired particle size. 11597773 11 The compounds may also be combined with an appropriate organic solvent. The organic solvent is typically a bland petroleum oil that is widely used in the agricultural industry. These combinations are typically used as a spray. More typically, the compounds are applied as a dispersion in a liquid carrier, where the liquid carrier is 5 water. The compounds may also be applied in the form of an aerosol composition. The compound is dissolved in an inert carrier, which is a pressure-generating propellant mixture. The aerosol composition is packaged in a container, where the mixture may be dispersed through an atomizing valve. Propellant mixtures contain either low-boiling halocarbons, which may be mixed with organic solvents or aqueous suspensions 10 pressurized with inert cases or gaseous hydrocarbons.

By the term an "inhibitory or inactivating amount" as used herein in the description and claims is meant a sufficient amount to result in a reduction in the number of ground-dwelling mrtes or springtails at the locus, or a reduction in their impact on the plants to which the method is applied, relative to an untreated control locus or untreated control plants.

The amount of compound applied to the loci of ground dwelling mites or springtails may be lower than the amount of compound applied to other pests such as Lepidoptera (caterpillars, moths, butterflies), Thysanoptera (thrips), Hymenoptera (wasps and bees) and some Coleoptera (beetles). The amount of compound applied may be 20 approximately 1 to 50% of the conventionally appled amount, more preferably approximately 5 to 40% more preferably approximately 10 to 30%, more preferably approximately 20%. For example, the compound may be applied at a rate of approximately 0.5 to 500g/ha, more preferably approximately 10 to 100g/ha, more preferably 1 to 10g/ha, more preferably approximately 1 to 5g/ha.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

Brief description of the figures Figure 1. Lucerne flea density 11597773 12 Figure 2. Lucerne flea control Figure 3. Redlegged earth mite density Figure 4. Spiny snout mite density Figure 5; Dose-response curves for H. destructor after 8 hrs (•) and 24 hrs (■) when 5 exposed to GF-1587, Figure 6: Dose-response curves for H. destructor after 8 hrs (•) and 24 hrs (■) when exposed to omethoate.

Figure 7: Average number of H. destructor per tub at different sampling dates post application of chemical treatments.

Figure 8: Average number of H. destructor in microcosms at sampling dates post application of treatment. Error bars represent standard error of the mean. Different letters above bars indicate significantly different means at each sampling date (at the P < 0.05 level, Tukey's-6 post hoc test).

Figure 9: Average number of P. major in microcosms at sampling dates post 15 application of treatment. Error bars represent standard error of the mean.

Different letters above bars indicate significantly different means at each sampling date (at the P < 0.05 level, Tukey's-Jb post hoc test).

Figure 10; Average number of 5. viridis in microcosms at sampling dates post application of treatment. Error bars represent standard error of the mean. 20 Different letters above bars indicate significantly different means at each sampling date (at the P < 0.05 level, Tukey's-6 post hoc test).

Figure 11: Average plant damage scores in microcosms at sampling dates post application of treatment. Error bars represent standard error of the mean. Different letters above bars indicate significantly different means at each 25 sampling date (at the P < 0.05 level, Tukey's-b post hoc test). 11597773 13 Figure 12: Average number of H. destructor in field plots at different sampling dates.

Error bars represent standard error of the mean. Different letters above bars indicate significantly different means at each sampling date (at the P < 0.05 level, Tukey's-b post hoc test).

Figure 13: Average number of Penthaleus spp. in field plots at different sampling dates. Error bars represent standard error of the mean. Different letters above bars indicate significantly different means at each sampling date (at the P< 0.05 level, Tukey's-6 post hoc test).

Figure 14: Average number of S: viridis in field plots at different sampling dates. Error 1 o bars represent standard error of the mean.

Figure 15: Average number of plants in field plots at different sampling dates. Error bars represent standard error of the mean. Different letters above bars indicate significantly different means at each sampling date (at the P < 0.05 level, Tukey's-b post hoc test).

Figure 16: Average plant damage scores in field plots at different sampling dates.

Error bars represent standard error of the mean. Different letters above bars indicate significantly different means at each sampling date (at the P < 0.05 level, Tukey's-b post hoc test).

Figure 17: Average crop vigour scores in field plots at different sampling dates. Error 20 bars represent standard error of the mean. Different letters above bars indicate significantly different means at each sampling date (at the P < 0.05 level, Tukey's-b post hoc test).

Detailed description of the embodiments The present invention will now be more fully described with reference to the 25 accompanying examples and figures. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above. 11587773 14 Example 1 Comparison of GF-2032 240 SC and GF-1587 120 SC with Lorsban 500 EC for the control of lucerne flea (Sm/nthurus viridis) in a mixed sward subterranean clover pasture Insecticide treatments were applied to a subterranean clover based pasture with a high infestation (4974/m2) of lucerne flea (Sminthurus viridis). The pasture also contained redlegged earth mite (Halotydeus destructor) (894/m2) and a beneficial predatory mite, the spiny snout mite (Neomolgus capiliatus) (131/m2). The chemical treatments applied were GF-2032 240 SC at 12, 24 and 96 g ai/ha, GF-1587 120 SC at 1, 2.5 and 10 g 10 ai/ha and Lorsban 500 EC at 35 g ai/ha, all applied at 100 L/ha total application volume. An unsprayed, untreated control was also in the trial By 10 to 14 days after application (DAA) the pasture was rapidly drying off and consequently no rating on clover damage/recovery was possible. The lucerne flea showed a natural decline from 4794 to 15 per square metre between 0 and 22 days 15 after application. Residual control from treatments was unable to be determined due to the absence of lucerne flea challenge All chemical treatments significantly reduced the number of lucerne flea at 3DAA and 7DAA with control ranging from 89.7% - 100% At 14DAA and 22DAA the population of lucerne flea had declined to low levels.

The 12 g ai/ha rate of GF-2032 240 SC was significantly less effective for lucerne flea control at 3DAA and 7DAA than the 24 and 96 g ai/ha rates.

The 1.0 g ai/ha rate of GF-1587 120 SC was significantly less effective than the 2.5 and 10 g at/ha rates for lucerne flea control at 3DAA and 7DAA.

GF-2032 at 24 and 96 g ai/ha rates was equivalent to GF-1587 at 2.5 and 10 g ai/ha ?5 rates for lucerne flea control.

GF-2032 at 24 and 96 g ai/ha and GF-1587 at 2.5 and 10.0 g ai/ha were equivalent to Lorsban 500 EC at 35 g ai/ha for the control of lucerne flea. 11597773 GF-2032 and GF-1587, at the rates applied, showed no significant effect on redlegged earth mite or spiny snout mite.

There was no adverse effect of GF-2032, GF-1587 or Lorsban when applied as a foliar spray to subterranean clover.

Introduction Aims • To evaluate three rates of GF-2032 240 SC for control of lucerne flea (Snunthurus viridis) in pasture.

• To evaluate three rates of GF-1587 120 SC for control of lucerne flea in pasture. 10 » To compare GF-2032 with GF-1587 for control of lucerne flea in pasture.

• To compare GF-2032 and GF-1587 with Lorsban 500 EC for control of lucerne flea in pasture • To evaluate the crop safety of GF-2032 and GF-1587 when applied as a foliar spray to subterranean clover in pasture.

Pests Lucerne flea {Sminthurus viridis) Redlegged earth mite (Halotydeus destructor) Beneficial Arthropods Spiny snout mite {Neomolgus capillatus) 11597773 16 Materials and Methods Product list Product name Active ingredient (ai) Concentration of active ingredient Formulation Batch number GF-2032 240 SC sulfoxaflor 240 g/L Suspension concentrate E2198-94 GF-1587 120 SC spinetoram 120 g/L Suspension concentrate Not provided Lorsban 500 EC chlorpyrifos 500 g/L Emulsifiable concentrate Not provided Treatment list Rate No.

Product Product (mL/ha) Active ingredient (g ai/ha) Application volume 1 Untreated control nil nil 2 GF-2032 240 SC 50 12.0 3 GF-2032 240 SC 100 240 4 GF-2032 240 SC 400 96.0 GF-1587 120 SC 8 3 1.0 100 L/ha 8 GF-1587 120 SC .8 2.5 7 GF-1587 120 SC 83.3 .0 8 Lorsban 500 EC 70 .0 11597773 17 Chronology of events Date Days after application (DAA) Pest stage Event 01/10/09 0 Adult & nymph Assessment 1 01/10/09 0 Adult & nymph Application 04/10/09 3 Adult & nymph Assessment 2 08/10/09 7 Adult & nymph Assessment 3 /10/09 | 14 Adult & nymph Assessment 4 23/10/09 ' 22 Adult & nymph Assessment 5 Insecticide treatments were applied to a subterranean clover based pasture with a high infestation (4974/m2) of lucerne flea (Sminthums viridis). The pasture also contained 5 redlegged earth mite (Halotydeus destructor) (894/m2) and spiny snout mite (.Neomoigus capillattis) (131/m2). By 10 T4DAA (days after application) the pasture was rapidly drying off and consequently no rating on clover damage/recovery was possible.

The initial population of lucerne flea, prior to spraying was 4794/m2, which decreased to 15/m2 between 0 and 22DAA, making residual control of any treatment impossible to 10 determine. By 3DAA, all chemical treatments had significantly reduced the number of lucerne flea, with Lorsban 500 EC showing the greatest reduction of 99.5%. GF-2032 showed a significant reduction in lucerne flea numbers between 12 and 24 g ai/ha, (80 down to 6,3/0.1 m2), but did not further decrease the number of lucerne flea by increasing the rate to 96 g ai/ha. GF-1587 showed a significant reduction in lucerne flea 15 numbers between 1.0 and 2.5 g ai/ha, (82,5 down to 10/0.1 m2). but did not further decrease the number of lucerne flea by increasing the rate to 10 g ai/ha. At 7DAA, 24 g ai/ha of GF-2032 and 2.5 g ai/ha of GF-1587 were giving equivalent control of lucerne flea to 35 g ai/ha of Lorsban, 99.3% -100%. By 14DAA, lucerne flea densities were low (12.5/0.1 m2) and there was no significant reduction by any treatment, which again was Z0 the same observation occurring at 22DAA.

Redlegged earth mite (RLFM) density showed no significant reduction during the trial period, with the only apparent trend being the increase of RLE.M numbers following the 11507773 18 decline in lucerne fiea density in the untreated control plots.

The spiny snout mite showed no significant reduction in density until 22DAA when all treatments showed a reduction compared with the untreated control, this however does not appear to be directly related to treatment effect and is more likely to be a result of 5 population dynamics following the rise in RLEM numbers for the untreated control.

There were no signs of phytotoxicity or crop effects from any treatment in this trial. Results Table 1. Lucerne flea density Rate (g ai/ha) Lucerne flea density (mean no/0.1 m2) No.

Treatment Days after application 0 3 7 14 22 1 Untreated control 0.0 3713 a 158.8 a 12,5 1 5 be 2 GF-2032 240 SC 120 80.0 b 14 4 b 19 41 : 3 GF-2032 240 SC 24.0 6.3 cd 0.6 c 18 1 1", : : .j 4 GF-2032 240' SC 96.0 479 4 13 8 c 1.9 c 0.0 ■1 - GF-1587 120 SC 1 0 82.5 b 13 8 b 41 9 100 a 6 GF-1587 120 SC 2.5 .0 c 0.0 c 8.8 ' "■ i 7 GF-1587 120 SC 0 .0 cd 1.9 c 0.6 0.0 c 8 ' Lorsban 500 EC i ,0 13 d 0.0 c 00 O.C P-value 0.0001 0,0001 0.5629 0.0462 CV 80,4 86.0 298.2 152.6 LSD (P=0.05) t t nsd t Means within columns followed by the same letter are not significantly different at the 5% level according to feast significant difference (LSD) test. t: LSD mean comparison based on log{x+1} transformation, nsd: riO' significant treatment effect. 11597773 19 Table 2, Lucerne flea control Rate (g ai/ha) Lucerne flea control (mean % of UTC) No.

Treatment Days after application 0 3 7 1 Untreated control 0.0 0 0 c d 2 GF-2032 240 SC 12..0 82 7 b 89.7 c 3 GF-2032 240 SC 24,0 97 8 a 993 a 4 GF-2032 240 SC 38.0 0.0 97,0 a 98.7 a OF 1587 120 SC 1 0 75 3 b 91.5 be 8 GF-1587 120 SC 2.5 90 4 a 100.0 a 7 GF-1587 120 SC .0 97,1 a 97 9 ab 8 Lorsban 500 EC .0 99.5 a 100.0 a P-value 0,0001 0.0001 CV 6.8 .2 LSD (P«0.05) | 8.11 6.45 Means within columns followed by the same tetter arc not significantly different'at the 5% level* according to least significant difference (L SDi test. 11597773 Table 3, Redlegged earth mite density Ho. . _ - . Treatment Rate (g ai/ha) Redlegged earth mite density (mean no./0.1 rrt1) Days after application 0 3 7 14 22 1 Untreated control 0,0 ftfl 4 131.9 126 6 211,3 253.5 2 GF-2032 240 SC 120 1144 73.8 75.6 168.0 3 GF-2032 240 SC 24,0 112 5 108.1 108.1 99.0 4 GF-2032 240 SC 960 79.4 33.1 103 1 34 5 GF-1587 120 SC 1 0 138.8 150 0 190 6 225.0 8 GF-1587 120 SC 2.5 85.0 161.9 89 4 63 0 7 GF-1587 120 SC 100 108,1 125.0 131 3 173,0 8 Lorsban 500 EC .0 .0 85.6 12.5 21.0 P-va!ue 0.2683 0.3040 0.1508 0.2091 * CV 59.5 69.2 83.1 110.2 i LSD (P=0.05) nsd nsd nsd nsd Means wilbin columns followed by the same letter are not significantly different at the 5% towel according to least significant difference (LSU) test, nsd no significant treatment effect. 11597773 21 Table 4, Spiny snout mite density Rate (g ai/ha) Spiny snout mite density (mean noi'0.1 m2) No.

Treatment Days after application 0 3 7 14 22 1 Untreated control 0.0 13.1 16.3 6 9 31.5 a 2 GF-2032 240 SC 12.0 21.3 8.8 8.8 8.5 b | 3 GF-2032 240 SC 24.0 113 .0 0 145 b I 4 GF-2032 240 SC 96.0 13 1 18.1 138 6.3 8 5 b i — GF-1587 120 SC 1.0 0 16 3 6 1' . : 6 GF-1587 120 SC 2.5 .6 6 Q 4 v# „T 9.5 b 7 GF-1587 120 SC .0 18.8 19.4 8.1 18.0 b 8 Lorsban 500 EC .0 0 .0 8.1 110 b P-value 0.4088 0.0988 0.2469 0.0036 CV 49.4 38.7 63.0 49.6 LSD (P=OJO^f nsd nsd nsd .71 Means within columns followed by the same letter are not significantly different at the 5% level according to least significant difference (LSD} test, nsd no significant treatment effect 11597773 22 Table 5. Spiny snout mite control Rate Spiny snout mite control (mean "A, of UTC) No.

Treatment (g ai/ha) Days after application 22 1 Untreated control 0.0 o : b Q. 2 GF-2032 ?40 SC 12.0 71.9 a 3 GF-2032 240 SC 24.0 54 3 a 4 GF-2032 240 SC 96.0 72 7 a GF-1587 120 SC 1.0 47.9 a 6 GF-1587 120 SC 2 5 68,5 a 7 GF-1587 120 SC 100 38.4 a 8 Lorsban 500 EC .0 64.0 a P-value 0.0040 CV 44.7 I LSD (P=0.05) 34.30 "Hans within columns followed by the same letter are not Significantly different at the 5* level according to least signfficant difference (LSD] test.

Conclusions • All chemical treatments significantly reduced the number of lucerne flea at 3DAA and 7DAA with control ranging from 83.7% - 100%. At 14DAA and 22DAA the population of lucerne fiea had declined to low levels.

• The 12 g ai/ha rate of GF-2032 240 SC was significantly less effective for lucerne flea control at 3DAA and 7DAA than the 24 and 96 g ai/ha rates.

• The 1.0 g ai/ha rate of GF-1587 120 SC was significantly less effective than the 2,5 and 10 g ai/ha rates for lucerne flea control at 3DAA and 7DAA.

• GF-2032 at 24 and 98 g ai/ha rates was equivalent to GF-1587 at 2.5 and 10 g ai/ha rates for lucerne flea control. 11597773 23 • GF-2032 at 24 and 96 g ai/ha and GF-1587 at 2,5 and 10.0 g ai/ha were equivalent to Lorsban 500 EC at 35 g ai/ha for the control of lucerne flea.

• GF-2032 and GF-1587, at the rates applied, showed no significant effect on redlegged earth mite or spiny snout mite, • There was no adverse crop effect of GF-2032, GF-1587 or Lorsban when applied as a foliar spray to subterranean clover.

Example 2 Efficacy of GF-1587 against the redlegged earth mite (Halotydeus destructor) The redlegged earth mite (Halotydeus destructor) is one of the most important 10 establishment pests of grain crops and pastures in Australia. Currently, this species is largely controlled using broad-spectrum pesticides that target the active phase of their lifecycle. However, with reports of resistance to some conventional pesticides there is increased pressure to develop more sustainable options for the control of this pest. The aim of this experiment is to conduct direct contact bioassays to determine the efficacy of 15 GF-1587 against the redlegged earth mite and compare this to a conventional pesticide.

Methodology Redlegged earth mites were tested for their response to the chemical GF-1587. The active ingredient of GF-1587 is 120 g/L spinetoram and the recommended field rate is 100 g a.i./hectare. Based on an application rate of 50 L water per hectare this equates 20 to 2 g a.i./L, Redlegged earth mites were also tested for their response to omethoate (Le Mat; Bayer Crop Sciences; 290 g/L), The recommended field rate is 100 mL diluted in 100 L of water per hectare. The dose representing the field rate therefore contained 0.29 g a.i./L Redlegged earth mites used for pesticide bioassays were collected from a Victorian site 25 that had no known history of pesticide application in the previous five years. Following collection, samples were stored in small plastic containers with leaf material and paper towelling to absorb excess moisture. Containers were kept at 4°C for two days prior to 11597773 24 testing, which does not influence pesticide tolerance of earth mites (James 1987; Hoffmann etal. 1997), The toxicology bioassay used to examine these pesticides followed Hoffmann et at, (1997). Ten concentrations were tested ranging from 0.0001 to 2 times the 5 recommended rate, along with a control of water. For each concentration about 15 ml was poured into a 40 mL glass vial and swirled to ensure a complete coating, with excess solution poured off. Approximately 15-20 vials per concentration were coated in this manner, then inverted and left to dry overnight. Control vials were treated in the same way except water was used in place of the pesticide. A single adult mite was then 10 placed in each vial along with a leaf of common vetch (Vicia sativa) The leaf was added to provide food and increase humidity The vials were then sealed with laboratory parafilm and placed at 18°C. After eight hours mites were scored as being alive (moving freely), incapacitated (inhibited movement) or dead (no movement over a five second period). Incapacitated mites were pooled with dead individuals for analysis as they 15 invariably die and therefore do not contribute to the next generation (Urnina & Hoffmann 1999). Due to the low level of mortality in GF-1587 treatments, mites were also scored after exposure for 24 hours Bioassays were used to generate dose-response curves by plotting percentage mortality against pesticide concentration, Logtt regression analysts following Robertson 20 & Preisier (1992) was implemented in SPSS (version 16 0) and used to estimate LDS0 values (along with 957. confidence intervals) LD50 values refer to the concentration at which 50% mortality occurs Findings Control mortality was low in all bioassays performed (always less than 5%). Dose-25 response curves generated for GF-1587 are shown in Figure 5. After 8 hours exposure mite mortality was low, indicating this chemical has relatively low efficacy against H. destructor. Even after 24 hours exposure, 100% mortality was not reached at the highest chemical concentration, which represents twice the recommended field rate (4 g/L). Dose-response curves generated for omethoate are shown in Figure 6. As 30 expected, omethoate had a high level of toxicity against H. destructor. After 8 hours 11537773 exposure, 100% mortality was reached at 0,01 times the recommended field rate (0,03 g/L).

L.D50 values and their 95% confidence intervals are shown in Table 6. The nature of the dose-response curve for GF-1587 after exposure for 8 hours precluded log it analysis 5 and computation of LD50 values. It is therefore difficult to determine the difference in tolerance levels between chemicals, although omethoate is clearly more effective against H. destructor. After 24 hours exposure, LD50 estimates for H. destructor indicate significant differences between chemicals. Based on the concentration of active ingredients, omethoate had an LD50 estimate that is >1800 times lower than GF-1587.

Table 6: LD50 estimates and 15% confidence intervals for H. destructor computed from logit models for responses to GF-1587 and omethoate.

! Chemical Exposure LC50 value 95% CIs (g/L) Lower (g/L) CI Upper CI GF-1587 8 hrs 24 hrs -0.27974 -0,19741 -1.58181 0.00050 0.00015 0.00122 omethoate 8 hrs 24 hrs 0.00017 0.00006 0.00043 Conclusions The dose-response curve and LD50 values estimated for omethoate are consistent to those found in previous studies (e.g. Umina 2007), This confirms that the population of mites used was suitable and the bioassay method robust. The results for GF-1587 and omethoate indicate significant differences in efficacy between chemicals. Omethoate is considerably more toxic to H. destructor than GF-1587. Only after exposure to GF-1587 for 24 hours was a high level of mortality reached; and this was only at the highest 20 concentration tested, which was 'twice the recommended field rate.

The active ingredient of GF-1587 is spinetoram, which works by contact and ingestion,. Control via ingestion is thought to be 5-10 times more effective than through direct contact. Therefore, the level of toxicity against H. destructor may have been 26 underestimated using the bioassay method used here. An assay that encompasses treated leaves may be more realistic of the situation in the field.

Example 3 Microcosm trial examining the efficacy of GF-1587 against the redlegged earth mite (Halotydeus destructoi) The redlegged earth mite {Halotydeus destructor) is an important pest of crops and pastures in Australia, and is largely controlled using broad-spectrum pesticides. However, with reports of resistance to some conventional pesticides there is increased pressure to develop more sustainable options for the control of this pest The aim of this experiment is to examine the efficacy of GF-1587 against the redlegged earth mite using microcosm trials and to compare this with a conventional pesticide.

Methodology The efficacy of differing rates of GF-1587 and omethoate were tested using a microcosm method (under shade-house conditions) routinely used by researchers to determine aspects of invertebrate biology, including response to chemicals (e.g. Umina and Hoffmann 2003).

A mixture of pasture seeds: phalaris (Phalaris aquatica cv. Holdfast), rye grass (Lolium multifiorum cv. Winter star II), white clover (Trifolium repens cv. Winter white) and subterranean clover {Trifolium subterraneum cv. Gosse); were sown into clear plastic tubs (approximately 45 cm long, 35 cm wide and 25 cm deep) using sterilised sandy loam (3:1) soil. These tubs were enclosed with a clear plastic lid that had a large gauze w.ndow for ventilation and to prevent the movement of mites. The tubs were placed in a shade-house in a randomised block arrangement and watered to germinate seedlings.

After 16 weeks, H. destructor were collected from a Victorian site that had no known history of pesticide application in the previous five years, and added to each tub to represent approximately 6,000 mites per m2, which is characteristic of population densities in the field (Ridsdill-Smith 1997). One day after the introduction of mites, tubs were sprayed with one of five treatments (i) GF-1587 (Dow AgroSciences; 120 g/L) at 1 t1687773 27 g a.i./L: (ii) GF-1587 at 2 g a.i./L; (I) GF-1587 at 4 g a.i./L; (iv) omethoate (Le Mat; Bayer Crop Sciences; 290 g/L) at 0.29 g a.i./L; or (v) water only (which acted as a control). Four plastic tubs were assigned to each treatment (20 tubs in total).

Treatments were applied using an Inter® 16L knapsack with hand-held lance and 5 Goizper flat fan nozzle (02 - Fine) Pressure was maintained at 300kPa and a total volume of 15 mL was applied per tub. Tubs were scored 1, 2 and 5 days after application of treatments. This was performed by taking six random suctions per tub and directly counting all mites alive.

Mite numbers at the three sampling dates were analysed using a repeated measures 10 AN OVA to determine differences between treatments. Differences for each sampling period were also determined between tieatments using an ANOVA and Tukey's-b post hoc tests All analyses were conducted with the statistical program SPSS v16 0.

Findings The repeated measures ANOVA showed an overall effect of treatment on the number of 15 H. destructor in microcosms over the three sampling periods (F4.15 = 179.72, P < 0.001). For each sampling period, the three rates of GF-1587 and omethoate all had significantly fewer mites than the control microcosms (Figure 7). There were no significant differences between the GF-1587 treatments and omethoate at all three sampling dates. The percentage reduction in mite numbers relative to the unsprayed 20 tubs is given in Table 7. 1-DAT the reduction in mite numbers compared with the controls was > 90% for all rates of GF-1587, and > 99% for omethoate. 2 DAT and 5-DAT the reduction in mite numbers relative to the control tubs for all rates of GF-1587 was > 95%, and for omethoate was 100%.

The decline in H. destructor numbers in the control tubs is likely due to the timing this 25 experiment was performed. Mites were not added to microcosms until towards the end of the active mite season in Victoria (Umina and Hoffmann 2003), and therefore numbers would be expected to decrease naturally. 11597773 28 Table 7; Reductions in H. destructor numbers (expressed as percentage) relative to unsprayed control tubs at different sampling dates post application of chemical treatments.

T reatment 1-OAT 2-DAT -DAT GF 1537 1g a.i./L 92 2 95.2 100 GF-1587 2g a.i./L 93.2 96.8 985 GF-1587 4ga.i./L 94.5 98.9 100 omethoate 99.7 100 100 Conclusions The results indicate GF-1587 provides a high level of control of H. destructor under semi-field conditions, although this is sightly lower than the conventional insecticide, omethoate. Even at the lowest rate tested (1g a.i./L), > 95% control was achieved after only two days post application of treatment. These findings are somewhat contradictory 10 to previous work, which showed GF-1587 was considerably less toxic to H. destructor using a contact laboratory bioassay (Example 2). The active ingredient of GF-1587 is spinetoram; and control via ingestion is thought to be 5-10 times more effective than through direct contact. It is likely that the level of toxicity against H. destructor was underestimated using the bioassay method, GF-1587 may therefore be a useful chemical for the control of H. destructor, although assessment under field conditions is needed to accurately determine this.

Example 4 Efficacy of GF-1587 against mite pests and lucerne flea under semi-field conditions The redlegged earth mite (Halotydeus destructor), blue oat mite (Penthaleus major) and lucerne flea (Sminthurus viridis) are among the most important establishment pests of 11587773 29 grain crops and pastures in Australia Currently, these pests are largely controlled using broad-spectrum pesticides that target the active phase of their life-cycle. 1 he aim of this experiment is to examine the efficacy of multiple rates of GF-1587 against these pests using microcosm trials and to compare these with a conventional pesticide.

Methodology The efficacy of differing rates of GF-1587 and omethoate were tested using a microcosm method {under shade-house conditions) routinely used by researchers to determine aspects of invertebrate biology, including response to chemicals (e.g. Umina and Hoffmann 2003) In May 2009, a mixture of pasture seeds: phalaris {Phalaris aquatica cv. Holdfast), rye grass (Lolium multifiorum cv, Winter star II), white clover (Trifolium repens cv. Winter white) and subterranean clover (Trifolium subterraneum cv. Gosse); were sown into clear plastic tubs (approximately 45 cm long, 35 cm wide and 25 cm deep) using sandy loam (3:1) soil. These tubs were enclosed with a clear plastic lid that had a large gauze 15 window for ventilation and to prevent the movement of mites and lucerne flea. The tubs were randomly placed in a shade-house and watered to germinate seedlings.

After 10 weeks, mites and lucerne flea were collected from Victorian sites that had no known history of pesticide application in the previous five years Approximately 200 H. destructor. 200 S. vindis and 100 P major were added to each tub Three days after the 20 introduction of these species, tubs were sprayed with one of six treatments, For full treatment details see Table 8. Six plastic tubs were assigned to each treatment (36 tubs in total). Treatments were applied using an Inter® 161 knapsack with hand-held lance and Goizper" flat fan nozzle (02 - Fine), Pressure was maintained at 300kPa and a total volume of 10 ml was applied per tub. 11597773 Table 8, Names, active ingredients, formulation concentrations and rates of treatments applied.

Treatment Active ingredient Application rate (g a.UL) Field rate (g aJJha) Control GF-1587 (0 025g) spinetoram (120 g/L) 0.025 2.5 GF-1587 (0.05g) spinetoram (120 g/L) 0 05 GF-1587 (0 1 g) spinetoram (120 g/L) 0.1 GF-1587 (1 g) spinetoram (120 g/L) 1 100 Le-mat© (Bayer CropSciences) omethoate (290 g/L) 0.29 29 Tubs were scored 1, 3, 7 and 14 days after the application of treatments (DAT) by 5 taking nine random' suctions per tub and directly counting the number of each species alive. Plant damage scores were recorded for each tub at 3-DAT, 7-DAT and 14-DAT, using a 0 - 10 scale, where 0 indicates no visible damage, 5 indicates 50% of the leaves damaged and 10 indicates all plants dead or dying. Phytotoxicity was assessed visually in all tubs at 7-DAT and 14-DAT, however no phytotoxic effects were seen in 10 any treatments at either sampling date, Data analysis Prior to analysts, count data for each pest species was log transformed (log n+1) and plant damage scores were arcsine square root transformed. Data was checked for normality using the Kolomogorov-Smimov test (normal distribution) and Levene's test (homogeneity of variances) following Sokal & Rohlf (1995), To maintain biological meaning, however, all figures were plotted using untransformed data.

Overall effects of treatment on the number of lucerne flea and mite species were assessed using a repeated measures analysis of variance (ANOVA) Differences in numbers of each species between treatments at each sampling date (1-DAT. 3-DAT, 7- ^ ^ i > 31 DAT and 14DAT) were then determined using an ANOVA and Tukey's 7; post hoc tests Plant damage scores were analysed in the same way, overall effects of treatment were assessed using a repeated measures ANOVA, and differences between treatments at each sampling point were obtained using an ANOVA and Tukey's-d post hoc tests, All 5 analyses were conducted in PASW Statistics (version 18).

Findings The repeated measures ANOVA showed an overall significant effect of treatment on the number of H. destructor over the four sampling periods (F5.30 = 35 15. P < 0.001). For each sampling period, omethoate had significantly fewer H destructor than the control 10 microcosms (Figure 8) There were no significant differences between any GF-1587 treatments and the controls at 1-DAT and 3-DAT. There were significantly fewer mites m GF-1587 (0 1g a.i./L) and GF-1587 (1g a i /L) treatments compared with the controls at 7-DAT. However, the number of mites in these treatments was still greater than the number of mites in the microcosms sprayed with omethoate. At 14-DAT, all GF-1587 15 treatments had significantly fewer H, destructor than the controls; only GF-1587 (0.025g a.i./L) had significantly more mites than the omethoate tubs.

The repeated measures ANOVA showed an overall significant effect of treatment on the number of P. major over the four sampling periods (F5.30 = 29.73, P < 0.001). For each sampling period, omethoate had significantly fewer P. major than the control 20 microcosms (Figure 9j There were no significant differences between any GF-1587 treatments and the controls at 1-DAT. At 7-DAT there were significantly fewer mites in all GF-1587 treatments and the controls, although the microcosms treated with GF-1587 still had greater numbers of mites compared with omethoate tubs. At 14-DAT, the number of P. major had declined in all treatments, including the controls, only GF-1587 25 (1g a.i./L) and omethoate had significantly fewer P. major than the control microcosms.

The repeated measures ANOVA showed a significant effect of treatment on the number of S. viridis over the four sampling periods (F5.30 = 168 22, P < 0.001). There were significantly fewer S. viridis in all chemical treatments at each sampling point compared with the control microcosms. (Figure 10). GF-1587 (0 025g a.i./L) had a significantly 30 greater number of S. viridis compared with GF-1587 (0.1g a.i/L), GF-1587 (1g a.i./L) 11.597773 32 and omethoate at 1-OAT arid 3-DAT, At 7-DAT and 14-DAT, no S, viridis were detected in any of the microcosms sprayed with chemical treatments.

The repeated measures ANOVA showed treatment had a significant overall effect on plant damage scores (F5.30 = 64,57, P < 0.001). Control microcosms had significantly 5 higher average plant damage than all other treatments (Figure 11). Plant damage scores continued to increase over the three sampling periods in the control microcosms while plant damage remained relatively stable for all other treatments. Microcosms sprayed with omethoate had lower plant damage scores than all GF-1587 treatments.

Conclusions These findings indicate GF-1587 provides a high level of control against S. viridis and a lower -but still significant -level of control against H, destructor and P. major under semi-field conditions.

GF-1587 did not significantly reduce H. destructor numbers 1 and 3 days post spraying, however there was a reduction in numbers observed 7 and 14 days after spraying. In 15 particular, the two highest rates examined (GF-1587 0.1 g a.i./L and GF-1587 1 g a.i./L) provided a high level of control beyond 7-DAT. For P. major, the findings are very similar to those for H. destructor. There is some level of control achieved at 3-DAT, but the biggest differences between numbers of P. major in the controls and GF-1587 treatments is evident 7-DAT and 14-DAT. The reason(s) for this delayed response are 20 unknown, but could be related to sub-lethal and/or indirect effects of GF-1587, such as anti-feeding properties or reduced mite fitness. Omethoate, a conventional pesticide commonly applied to control earth mites in southern Australia, provided an immediate and high level of control against both H. destructor and P. major.

GF-1587 was highly effective against S. viridis. Even at the lowest rates examined, 25 GF1587 provided a high level of control. The differences in efficacy between S, viridis and earth mites could be explained by the differences in feeding mechanisms. The active ingredient of GF-1587 is spinetoram, which is most toxic to pests when ingested.. Earth mites feed by penetrating the epidermal cells of plants and sucking out the cellular contents using a pharyngeal pump (Ridsdill-Smith 1997; Umina et al., 2004). S. viridis 11597773 33 feeding occurs through a chiselling action, where the leaf epidermis is stripped off before the underlying mesophyll is eaten (Davidson 1934, Swan 1940} All rates of GF-1587 examined here resulted in low plant damage scores within tubs, although these were higher than the damage scores from omethoate treatments at each 5 sampling date. The level of protection provided to pasture seedlings in tubs sprayed with GF-1587 was mostly due to the successful control of S, viridis despite each microcosm initially containing a combination of pest species. The lower numbers of H destructor and P. major in the GF-1587 treatments would have contributed but to a lessee extent, particularly at the first two sampling dates. This is supported by direct 10 observations of the feeding damage symptoms, which is distinguishable between earth mites and S. viridis.

This experiment was conducted under semi-field (shade house) conditions, which allows known pest numbers to be added to each replicate tub, increases statistical power and allows for greater control over environmental factors compared with 15 conventional field trials. Large-scale trials are desirable to accurately determine the efficacy of GF-1587 against S. viridis (and other pest species) on various crop types and to understand the impacts/practicalities of applying GF-1587 to different, plant stages in the field.

Example 5 Efficacy of GF-1587 against the redlegged earth mite and blue oat mites in canola field trial Blue oat mites (Penthaluus spp.), the redlegged earth mite (Halotydeus destructor) and lucerne flea (Sminthurus viridis) are among the most important establishment pests of grain crops and pastures in Austraiia. Currently, these pests are largely controlled using broad-spectrum pesticides that target the active phase of their life-cycle The aim of this experiment is to examine the efficacy of GF-1587 against these pests when applied as a foliar application to an emerging canola crop.

T 1 34 Methodology A field experiment to examine the impacts of GF-1587 (provided by Dow AgroSciences) was established in a long-term pasture paddock near Inverleigh, Victoria (38°09"08"5, 144°00'36"E). Twelve plots (20 m x 20 m) were pegged out within the paddock in a 5 randomized block design and a 5 m buffer was left between plots. Canola seed used in all plots was treated with Gaucho'v (400 ml/100kg seed) less than one week prior to sowing using a Cimbria CC-Lab 2 rotary seed treater.

One week post-sowing, all plots were sampled for pest numbers via suction using a Stihl Blowervac (model BG55). A defined area within a 0 09 square metre frame was 10 vacuumed and the contents were transferred to a vial containing 70% (v/v) ethanol. All pest species were then identified and counted in the laboratory under a microscope. This was repeated randomly four times within each plot. Following this pre-spray' sampling, plots were randomly allocated one of 3 treatments (see Table 9), and each treatment was assigned to 4 replicate plots, A bare-earth spray (bifenthrin) was then 15 applied to the conventional plots, and no sprays were applied to the control plots or the GF-1587 plots.

Pest numbers and plant growth were then monitored regularly in all plots. After approximately three weeks, canola plants were at the cotyledon - early 1st true leaf stage, and pests were sampled again in all plots using the methods described above 20 ("0-DAT" sample). Omethoate was then applied to the conventional plots, and GF-1587 was applied to the GF-1587 plots. Post-spraying, pest numbers within each plot were sampled at 3 DAT, 7DAT, 14-DAT and 28-DAT. Sampling and sorting followed the methods above.

Table 9. Names, active ingredients and rates of treatments applied.

Treatment Product Active ingredient Field rate (tnL/ha) Rate (g a.iiha) Control 0 0 1t597773 Conventional Bare-earth spray Foliar spray Talstar® 250EC Le-mat® 290SL bifenthrin (250 g/L) omethoate (290 g/L) 40 100 29 GF-1587 Foliar spray GF-1587 spinetoram (120 g/L) 83.3 Canola. plant counts were conducted at 0-DAT, 3-DAT, 7-DAT, 14-DAT and 28-DAT. Four 0,5 square metre quadrants were randomly pegged out in each plot and the number of plants within each of these quadrants was recorded at each sampling date, 5 Canola plants were also scored for pest feeding damage at 0-DAT, 3-DAT, 7-DAT, 14-DAT and 28-DAT, based on visual assessments using a 0-10 scale (0 = no damage, 5 = damage to 50% of leaf surface, etc), A 0 09 square metre frame was placed randomly five times within each plot, and scores were based on plants within this frame. This method of assessing plant damage has been validated for earth mites previously (Liu & 10 Rsdsdill-Smith 2000; Umina & Hoffmann 2004), Crop vigour scores were recorded at 3-DAT, 7-DAT, 14-DAT and 28-DAT, by visually assessing the density and growth stage of plants within each plot using a percentage scale (0-100%) in comparison to the best plot. Phytotoxicity was also assessed visually in all plots at 7-DAE and 14-DAE, however no phytotoxic effects were seen in any 15 treatment at either sampling date.

Data analysis Prior to analysis, plant damage scores and crop vigour scores were arcsine square root transformed, while count data for each pest species was log transformed (log n+1). Data was checked for normality using the Kolomogorov-Smirnov test (normal 20 distribution) and Levene's test (homogeneity of variances) following Sokal & Rohlf (1995).. To maintain biological meaning, however, all figures were plotted using untransformed data. Mite numbers and plant counts were converted to number per m2 for graphical representation.

Overall effects of treatment on the number of mite species and lucerne flea were 11597773 36 assessed using a repeated measures analysis of variance {ANOVA}, with pre-spray numbers from each plot included as a covariate in the analysis. Differences in numbers of each species between treatments at each sampling date (0-DAT, 3-DAT, 7-DAT, 14-DAT and 28DAT) were then determined using one-way ANOVAs and Tukey's-6 post 5 hoc tests. Again, pre-spray numbers from each plot were included as a covariate in the analyses. Due to the differences in timing of pesticide applications between GF-1587 and the conventional treatment (which make it difficult to directly compare the two), all above-mentioned analyses were repeated considering only GF-1587 and the 'untreated' controls. For these analyses, 0-DAT numbers were included as a covariate in the 10 analysis and pre-spray numbers were excluded.

Repeated measures ANOVAs were performed against plant counts, plant damage scores and crop vigour scores. Differences between, treatments at each sampling point were obtained using one-way ANOVAs and Tukey's-6 post hoc tests. These analyses were repeated considering only GF-1587 and the 'untreated' controls. For plant counts 15 and plant damage scores, 0-DAT data was included as a covariate in the analysis.

All analyses were conducted in PASW Statistics (version 18).

Findings Treatment effects on pests The repeated measures ANOVA showed a significant effect of treatment on the number 20 of H. destructor over the five sampling periods (F2.45 = 249,402., P < 0.001), which indicates responses to treatments varied over time. For each sampling period (except the pre-spray), the conventional treatment had significantly fewer mites than both the controls and GF-1587 (Figure 12). When the conventional treatment was removed from the analysis, one-way ANOVAs indicate no significant reduction in H. destructor 25 numbers in GF-1587 compared with the controls. The repeated measures ANOVA also showed no significant overall effect of treatment on the number of H. destructor in plots over the four sampling periods (F1,30 = 0.029, P = 0,867).

Beyond the pre-spray samples, only low numbers of Penthaleus spp. were collected in 11597773 37 this experiment. Despite this, the repeated measures ANOVA showed a significant effect of treatment on the number of Penthaieus spp over the five sampling periods (F2.45 = / 693, P < 0.01), which indicates that responses to treatments varied over time. There was a significant difference in mite numbers at 14-DAT; significantly more 5 mites were found in the controls and GF-1587 plots compared with the conventional plots (Figure 13). When the conventional treatment was removed from the analysis, there was no significant difference in numbers between GF-1587 and the controls. The repeated measures ANOVA also showed no overall significant effect of treatment on the number of Penthaieus spp. over the four sampling periods (F1,30 = 0,056, P = 0,815), For S, viridis, the repeated measures ANOVA showed no overall effect of treatment on numbers over the five sampling periods (F2.45 = 0.191, P = 0.827), There were also no significant differences in S viridis numbers between treatments at any sampling date (Figure 14). When the conventional treatment was removed from the analysis, there was still no significant difference in numbers between GF-1587 and the controls at any 15 sampling date, even 14-DAT and 28-DAT. The repeated measures ANOVA also showed no overall significant effect of treatment on the number of S, viridis over the four sampling periods (F1.30 = 0.029, P = 0 867) after the conventional treatment was removed from the analysis.

Treatment effects on plants The repeated measures ANOVA showed a significant effect of treatment on the number of plants per metre squared over the five sampling periods (F2.45 = 97.594, P < 0.001), which indicates responses to treatments varied over time For each sampling period, the conventional treatment had significantly higher numbers of plants than both the controls and GF-1587 plots (Figure 15). When the conventional treatment was removed from the 25 analysis, one-way ANOVAs indicate no significant difference in plant numbers between GF-1587 and the controls. The repeated measures ANOVA also showed no overall significant effect of treatment on the number of plants over the four sampling periods (F1.30 = 0.430, P = 0.517), For plant damage scores, the repeated measures ANOVA showed a significant effect of ' treatment over the five sampling periods (F2.45 = 188,587, P < 0.001), which indicates 11597773 38 responses to treatments varied over time. For each sampling period, the conventional treatment had significantly less plant feeding damage than the controls and GF-1587 plots (Figure 16). At 7-DAT, GF-1587 had significantly less plant damage than the untreated controls. When the conventional treatment was removed from the analysis, 5 one-way ANOVAs indicate no significant difference in plant damage scores between GF-1587 and the controls, even at 7-DAT. The repeated measures ANOVA also showed no overall significant effect of treatment on plant damage over the four sampling periods (F1,30 - 1.460, P~ 0.238), For crop vigour, the repeated measures ANOVA showed a significant effect of treatment 10 over the five sampling periods (F2.9 = 47.288, P < 0.001), indicating responses to treatments varied over time. For each sampling period, the conventional treatment had significantly higher crop vigour scores compared with the controls and GF-1587 (Figure 17). GF-1587 typically had higher crop vigour scores than the controls, but these were not significantly different. When the conventional treatment was removed from the 15 analysis. one-way ANOVAs indicate no significant difference in crop vigour between GF-1587 and the controls. The repeated measures ANOVA aiso showed no overall significant, effect of treatment on crop vigour over the four sampling periods (F1,6 = 0.087, P =0,778).

Conclusions These findings indicate GF-1587 does not provide a high level of control against H. destructor when applied as a foliar application to a canola crop at the cotyledon - early 1&; true-leaf stage. "I he conventional treatment, which involved a single bare-earth application of bifenthrin and a single post-emergent application of omethoate, provided excellent control of H. destructor and good protection to canola seedlings.

High numbers of H destructor were present within the field piots, while Penthaieus spp. and S viridis numbers were generally low. The results for H. destructor clearly show that the bare-earth application in the conventional plots greatly reduced mite numbers. At the time of the second application (0-DAT), which coincided with the application of GF1587, H. destructor numbers were below 20 per m2 GF-1587 did not significantly 30 reduce numbers of H. destructor. In the previous examples, GF-1587 was shown to 11597773 39 have some efficacy against this species. The reason(s) for these differences could be numerous. It is likely that the limited plant foliage at the time of application in the field trial contributed to the poor efficacy of GF1587 against mites. The active ingredient of GF-1587 is spinetoram; and control via ingestion is thought to be 5-10 times more 5 effective than through direct contact. In the field trial, the majority of product' landed on bare ground and was effectively wasted. However, a large proportion of GF-1587 applied in the microcosm trials would have landed on plant foliage and have subsequently been taken up by the pasture seedlings. it is difficult to assess the efficacy of GF-1587 against Penthaieus spp. and S. viridis 10 due to the low numbers present at the field site. From an earlier microcosm study, it appears GF-1587 has similar toxicity to P. major as H. destructor. Although Penthaieus spp. were reasonably abundant at the beginning of the trial, numbers declined rapidly, even within the untreated control plots. This is probably because the majority of these mites were Penthaieus major. Penthaieus major is the most abundant Penthaieus spp., 15 but does not survive on, and very rarely attacks canola. Sub-samples of Penthaieus spp. collected within all plots were identified and > 90% were P. major. Less than 10% were Penthaieus falcatus, which is the only Penthaieus spp. that can survive on and commonly attacks canola (Umina and Hoffmann 2004). The results against S, viridis support findings from an earlier microcosm trial (Example 4), indicating GF-1587 will 20 provide control of this pest under certain conditions.

Most conventional insecticides used to control mites and S. viridis are applied at crop emergence. In many instances this occurs when there is little plant foliage present, although this depends considerably on weather conditions and previous paddock history (which directly impacts the densities of mite and lucerne flea populations). Because 25 GF1587 exhibits strong trans-laminar activity, efficacy will be optimised if applied when crop plants are beyond the early cotyledon stage. This will be most likely when used in conjunction with insecticide-treated seed. GF-1587 appears effective in controlling S. viridis and may have sub-lethal and/or indirect effects on earth mites (Umina and Roberts 2009). GF-1587 may also prove beneficial as a foliar spray in spring when S. 30 viridis and earth mite numbers are at their highest, and these pests are often found 40 attacking lucerne, clover and other late-sown crops, REFERENCES Davidson J. 1934. The Lucerne Ffea Sminthurus viridis L, (Collemboia) in Australia, Bulletin of the Council for Scientific and Industrial Reseatch No. 79.

Hoffmann, A. A.,. Porter, S. & Kovacs, I. 1997. The response of the major crop and pasture pest, the red-legged earth mite (Halotydeus destructor) to pesticides: Dose-response curves and evidence for tolerance. Experimental and Applied Acarology, 21, 151-162. http //www.alanwood.net/pesticides/index cn frame.html James, D. G. 1987. Toxicity of pesticides to the redlegged earth mite, Halotydeus destructor. Plant Protection Quarterly, 2, 156-157.

Ridsdill-Smith TJ. 1997. Biology and control of Halotydeus destructor (Tucker) (Acarina. Penthaleidae) a review. Experimental and Applied Acarology 21: 195-224.

Robertson, J. L. & Preisier, H. K. 1992, Pesticide Bioassays with Arthropods. CRC: 15 Boca Raton.

Sokal RR and Rohlf FJ 1995. Biometry. The principles and practice of statistics in biological research, 3rd edition. New York" W, H Freeman. 887 p.

Swan DC, 1940, The lucerne flea: its life history and control in South Australia. Journal of the Department of Agriculture of South Australia 43:462-471.

Umina PA and Hoffmann .AA. 2003, Diapause and implications for control of Penthaieus species and Halotydeus destructor (Acari: Penthaleidae) in south-eastern Australia. Experimental and Applied Acarology 31: 209-223, Umina PA, Hoffmann AA and Weeks AR. 2004. Biology, ecology and control of the 41 Penthaieus species complex: (Acari: Penthaleidae). Experimental and Applied Acarology 34: 211-237, Umina, P. A. & Hoffmann, A. A. 1999. Tolerance of cryptic species of blue oat mites (.Penthaieus spp.) and the redlegged earth mite (Halotydeus destructor) to pesticides. Australian Journal of Experimental Agriculture. 39, 621-628, Umina, P.A. 2007, Pyrethroid resistance discovered in a major agricultural pest in southern Australia: the redlegged earth mite Halotydeus destructor (Acari: Penthaleidae), Pest Management Science 63, 1185-1190.

Claims (14)

12243403 RECEIVED at IPONZ on 21 December 2011 42 The claims defining the invention are as follows:

1. A method of controlling a pest selected from the family consisting of Sminthuridae, said method including applying to a locus where control is desired, after the emergence of seedlings from earth within the locus, an inhibitory or inactivating 5 amount of a pesticidal compound selected from the group consisting of spinosyns, synthetically-modified spinosyns, and derivatives, salts and mixtures thereof.

2. A method according to claim 1 wherein said pest is a lucerne flea (Sminthurus viridis).

3. A method of controlling a blue oat mite (Penthaieus major), said method including 10 applying to a locus where control is desired, after the emergence of seedlings from earth within the locus, an inhibitory or inactivating amount of a pesticidal compound selected from the group consisting of spinosyns, synthetically-modified spinosyns, and derivatives, salts and mixtures thereof.

4. A method according to any one of claims 1 to 3 wherein said locus includes a 15 pasture or a broad acre crop.

5. A method according to claim 4, wherein the pesticidal compound is applied to foliage of a plant.

6. A method according to claim 4, wherein the pesticidal compound is applied to soil.

7. A method according to any one of claims 1 to 6 wherein said pesticidal compound 20 comprises Spinetoram or Spinosad.

8. A method according to claim 7 wherein said pesticidal compound comprises > ; . RECEIVED at IPONZ on 21 December 2011 12243403 43 Spinotoram.

9. A method according to any one of claims 1 to 8 wherein the pesticidal compound is provided in a pesticidal composition selected from the group consisting of water-soluble, water-suspension, and emulsifiable formulations, and water-dispersible 5 granules.

10. A method according to any one of claims 1 to 9 wherein said pesticidal compound is applied at a rate of approximately 0.5 to 500g/ha.

11. A method according to claim 9 wherein said pesticidal compound is applied at a rate of approximately 10 to 10Og/ha. 10

12. A method according to claim 9 wherein said pesticidal compound is applied at a rate of approximately 1 to 10g/ha.

13. A method according to claim 9 wherein said pesticidal compound is applied at a rate of approximately 1 to 5g/ha.

14. A method according to claim 1 or 3 substantially as hereinbefore described. 15