Control of resistant pests

Abstract

The present invention relates to methods of controlling pesticide resistant pests comprising exposing the pesticide resistant pests to a pest controlling amount of a triketone compound.

Claims

1. A method of controlling pesticide resistant pests, wherein the pesticide resistant pests are insects infesting an agricultural environment, said method comprising applying to the agricultural environment, a compound of formula (I) ##STR00019## wherein: R.sup.1 is selected from the group consisting of C(═O)R.sub.7, —OR.sub.8, —SR.sub.8, —C.sub.1-10hydroxyalkyl, —NR.sub.9R.sub.10, —C(═N—R.sub.9)R.sub.7, —C(═N—OH)R.sub.7, —NO, —NO.sub.2, —N(OR.sub.8)R.sub.7 and —OSO.sub.3R.sub.8; R.sub.2 is selected from the group consisting of hydrogen, —C.sub.1-10alkyl,—C.sub.2-10alkenyl, aryl and heteroaryl; R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are each independently selected from the group consisting of hydrogen, —C.sub.1-10alkyl, —C.sub.3-6cycloalkyl, —C.sub.2-10alkenyl, —C.sub.1-10haloalkyl, —C.sub.1-10dihaloalkyl, —C.sub.1-10trihaloalkyl, —OR.sub.8, —SR.sub.8, —NR.sub.9R.sub.10, —C(═N—R.sub.9)R.sub.7, —NO, —NO.sub.2, —NR.sub.9OR.sub.8, —OSO.sub.3R.sub.8, —C.sub.1-10alkylaryl and C(═O)R.sub.7; R.sub.7 is selected from the group consisting of hydrogen, —C.sub.1-10alkyl, —C.sub.2-10alkylaryl, C.sub.3-6cycloalkyl, —C.sub.2-10alkenyl, —C.sub.1-10alkylheteroaryl, —C.sub.1-10haloalkyl, —C.sub.1-10dihaloalkyl, —C.sub.1-10trihaloalkyl, —C.sub.1-10haloalkoxy, —C.sub.1-10hydroxyalkyl, —C.sub.1-10thioalkyl, —C.sub.1-10nitroalkyl, —C.sub.1-3alkylOC.sub.1-3alkyl, —C.sub.1-3alkylOC.sub.1-3haloalkyl, —C.sub.1-3alkylOC.sub.1-3dihaloalkyl, —C.sub.1-3alkylOC.sub.1-3trihaloalkyl, —OR.sub.8, —SR.sub.8 and —NR.sub.9R.sub.10; R.sub.8 is selected from the group consisting of hydrogen, —C.sub.1-10alkyl, —C.sub.2-10alkylaryl, —C.sub.3-6cycloalkyl, —C.sub.2-10alkenyl, —C.sub.1-10alkylheteroaryl, —C.sub.1-10haloalkyl, —C.sub.1-10dihaloalkyl, —C.sub.1-10trihaloalkyl, —C.sub.1-10haloalkoxy, —C.sub.1-10hydroxyalkyl, —C.sub.1-10thioalkyl and —C.sub.1-10nitroalkyl; R.sub.9 and R.sub.10 are independently selected from the group consisting of hydrogen, —C.sub.1-10alkyl, —C.sub.2-10alkylaryl, —C.sub.3-6cycloalkyl, —C.sub.2-10alkenyl, —C.sub.1-10alkylheteroaryl, —C.sub.1-10haloalkyl, —C.sub.1-10dihaloalkyl, —C.sub.1-10trihaloalkyl; or a tautomer thereof; wherein the agricultural environment is a plant selected from the group consisting of a crop, a tree, soil, an area around a plant as it grows, and an area where plants or parts of plants are stored.

2. The method according to claim 1 wherein the compound of formula (I) is a compound of formula (II): ##STR00020## wherein: R.sub.11 is selected from —CR.sub.12R.sub.13R.sub.14 or —NR.sub.15R.sub.16; one of R.sub.12 and R.sub.13 is hydrogen and the other is hydroxyl or —OCR.sub.17R.sub.18R.sub.19 or R.sub.12 and R.sub.13 together form an oxo group (═O) or a ═N—OH group; R.sub.14 is —CH(CH.sub.3)CR.sub.20R.sub.21R.sub.22, —CH.sub.2CH(CH.sub.3)CR.sub.20R.sub.21R.sub.22 or —CH(CH.sub.3)CH.sub.2CR.sub.20R.sub.21R.sub.22; R.sub.15 and R.sub.16 are independently selected from the group consisting of hydrogen and C.sub.1-10alkyl; R.sub.17, R.sub.18 and R.sub.19 are independently selected from the group consisting of hydrogen or halo; and R.sub.20, R.sub.21 and R.sub.22 are independently selected from hydrogen, hydroxyl, halo, NO.sub.2 and —OCR.sub.17R.sub.18R.sub.19; or a tautomer thereof.

3. The method according to claim 1 wherein the compound of formula (I) is a compound of formula (III): ##STR00021## wherein: one of R.sub.23 and R.sub.24 is hydrogen and the other is hydroxyl or —OCR.sub.27R.sub.28R.sub.29 or R.sub.23 and R.sub.24 together form an oxo group (═O); R.sub.25 is —CR.sub.30R.sub.31R.sub.32, —CH.sub.2CR.sub.30R.sub.31R.sub.32 or —CH(CH.sub.3)CR.sub.30R.sub.31R.sub.32; R.sub.26 is H or —CH.sub.3; wherein where R.sub.26 is H, R.sub.25 is —CH(CH.sub.3)CR.sub.30R.sub.31R.sub.32; R.sub.27, R.sub.28 and R.sub.29 are independently selected from hydrogen or halo; and R.sub.30, R.sub.31 and R.sub.32 are independently selected from hydrogen, hydroxyl, halo, NO.sub.2 and —OCR.sub.27R.sub.28R.sub.29; or a tautomer thereof.

4. The method according to claim 1 wherein the compound of formula (I) is selected from the group consisting of: ##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026## or a tautomer thereof.

5. The method according to claim 1 wherein the compound of formula (I) is selected from the group consisting of flavesone, leptospermone and isoleptospermone.

6. The method according to claim 5 wherein the compound of formula (I) is flavesone.

7. The method according to claim 1 wherein the pesticide resistant pests are insects resistant to one or more insecticides.

8. The method according to claim 1 wherein the pesticide resistant pest is exposed to the compounds of formula (I) in an amount in the range of about 200 ppm to about 800 ppm or about 300 ppm to about 600 ppm or about 800 ppm to about 2,500 ppm or about 900 ppm to about 2000 ppm.

9. The method according to claim 1 wherein the insects are selected from the group consisting of Rhyzopertha dominica, Sitophilus oryzae, Triobolium castaneum, Oryzaephilus surinamensis or Cryptolestes ferrugineus.

10. The method according to claim 9 wherein the insects are adults.

11. The method according to claim 1 wherein a pesticide resistant pest is exposed to a compound of formula (I) in combination with a second pesticide, wherein the second pesticide has a different mode of action from the compound of formula (I).

12. The method according to claim 11 wherein the second pesticide is selected from the group consisting of at least one of a sodium channel modulator, an acetylcholinesterase (AChE) inhibitor, a GABA-gated chloride channel antagonist, a nicotinergic acetylcholine receptor agonist, an allosteric acetylcholine receptor modulator, a chloride channel actuator, a juvenile hormone mimic, a homopteran feeding blocker, a mitochondrial ATP synthase inhibitor, an uncoupler of oxidative phosphorylation, a nicotinic acetylcholine receptor channel blocker, an inhibitor of chitin biosynthesis, a moulting disruptor, an ecdysone receptor agonist or disruptor, an octapamine receptor agonist, a mitochondrial complex I electron transport inhibitor, an acetyl CoA carboxylase inhibitor, a voltage-dependent sodium channel blocker, a mitochondrial complex IV electron inhibitor, a mitochondrial complex IV electron transport inhibitor and a ryanodine receptor modulator.

13. A method of protecting stored plant parts from pest infestation by pesticide resistant pests, wherein the pest infestation is caused by a population of pests comprising pesticide resistant pests, said method comprising contacting the plant part with a compound of formula (I) ##STR00027## wherein: R.sub.1 is selected from the group consisting of —C(═O)R.sub.7, —OR.sub.8, —SR.sub.8, —C.sub.1-10hydroxyalkyl, —NR.sub.9R.sub.10, —C(═N—R.sub.9)R.sub.7, —C(═N—OH)R.sub.7, —NO, —NO.sub.2, —N(OR.sub.8)R.sub.7 and —OSO.sub.3R.sub.8; R.sub.2 is selected from the group consisting of hydrogen, —C.sub.1-10alkyl, —C.sub.2-10alkenyl, aryl and heteroaryl; R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are each independently selected from the group consisting of hydrogen, —C.sub.1-10alkyl, —C.sub.3-6cycloalkyl, —C.sub.2-10alkenyl, —C.sub.1-10haloalkyl, —C.sub.1-10dihaloalkyl, —C.sub.1-10trihaloalkyl, —OR.sub.8, —SR.sub.8, —NR.sub.9R.sub.10, —C(═N—R.sub.9)R.sub.7, —NO, —NO.sub.2, —NR.sub.9OR.sub.8, —OSO.sub.3R.sub.8, —C.sub.1-10alkylaryl and —C(═O)R.sub.7; R.sub.7 is selected from the group consisting of hydrogen, —C.sub.1-10alkyl, —C.sub.2-10alkylaryl, C.sub.3-6cycloalkyl, —C.sub.2-10alkenyl, —C.sub.1-10alkylheteroaryl, —C.sub.1-10haloalkyl, —C.sub.1-10dihaloalkyl, —C.sub.1-10trihaloalkyl, —C.sub.1-10haloalkoxy, —C.sub.1-10hydroxyalkyl, —C.sub.1-10thioalkyl, —C.sub.1-10nitroalkyl, —C.sub.1-3alkylOC.sub.1-3alkyl, —C.sub.1-3alkylOC.sub.1-3haloalkyl, —C.sub.1-3alkylOC.sub.1-3dihaloalkyl, —C.sub.1-3alkylOC.sub.1-3trihaloalkyl, —OR.sub.8, —SR.sub.8 and —NR.sub.9R.sub.10; R.sub.8 is selected from the group consisting of hydrogen, —C.sub.1-10alkyl, —C.sub.2-10alkylaryl, —C.sub.3-6cycloalkyl, —C.sub.2-10alkenyl, —C.sub.1-10alkylheteroaryl, —C.sub.1-10haloalkyl, —C.sub.1-10dihaloalkyl, —C.sub.1-10trihaloalkyl, —C.sub.1-10haloalkoxy, —C.sub.1-10hydroxyalkyl, —C.sub.1-10thioalkyl and —C.sub.1-10nitroalkyl; R.sub.9 and R.sub.10 are independently selected from the group consisting of hydrogen, —C.sub.1-10alkyl, —C.sub.2-10alkylaryl, —C.sub.3-6cycloalkyl, —C.sub.2-10alkenyl, —C.sub.1-10alkylheteroaryl, —C.sub.1-10haloalkyl, —C.sub.1-10dihaloalkyl, —C.sub.1-10trihaloalkyl; or a tautomer thereof.

14. The method according to claim 13 wherein the plant part is grain.

15. The method according to claim 13 where the pest population comprising pesticide resistance pests is selected from the group consisting of Rhyzopertha dominica, Sitophilus oryzae, Triobolium castaneum, Oryzaephilus surinamensis and Cryptolestes ferrugineus.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a graphical representation of dose-response curves of a susceptible population of H. destructor when exposed to flavesone at concentrations of 0, 3, 10, 30, 100, 300, 1000, 3000 and 10000 mg a.i./L (ppm) at 4, 6, 8 and 24 hours.

(2) FIG. 2 is a graphical representation of dose-response curves of susceptible and resistant populations of H. destructor when exposed to flavesone at concentrations of 0, 3, 10, 30, 100, 300, 1000, 3000 and 10000 mg a.i./L (ppm) at 24 hours.

(3) FIG. 3 is a graphical representation of dose-response curves of susceptible and resistant populations of H. destructor when exposed to bifenthrin at concentrations of 0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1.0, 10, 100, 1000 and 10000 mg a.i./L (ppm) at 24 hours.

(4) FIG. 4 is a graphical representation of dose-response curves of susceptible and resistant populations of H. destructor when exposed to chlorpyrifos at concentrations of 0, 0.7, 7.0, 70 and 700 mg a.i./L (ppm) at 24 hours.

(5) FIG. 5 is a graphical representation of dose-response curves of susceptible and resistant populations of green peach aphid exposed to flavesone at concentrations of 0, 10, 100, 300, 1000, 2000, 5000, 10000, 30000 and 100000 mg a.i./L and 48 hours exposure.

(6) FIG. 6 is a graphical representation of dose-response curves of susceptible and resistant populations of green peach aphid exposed to flavesone at concentrations of 0, 10, 100, 300, 1000, 2000, 5000, 10000, 30000 and 100000 mg a.i./L and 96 hours exposure.

(7) FIG. 7 is a graphical representation of dose-response curves of susceptible and resistant populations of green peach aphid exposed to pirimicarb at concentrations of 0.025, 0.25, 2.50, 25.0, 250.0 and 2500.0 mg a.i./L and 48 hours exposure.

(8) FIG. 8 is a graphical representation of dose-response curves of susceptible and resistant populations of green peach aphid exposed to pirimicarb at concentrations of 0.025, 0.25, 2.50, 25.0, 250.0 and 2500.0 mg a.i./L and 96 hours exposure.

EXAMPLES

Example 1: Larval Packet Test—Cattle Tick

(9) The larval packet test (LPT) is a modification of that first described by Stone and Haydock (1962, Bull. Entomol. Res., 563-578, http://dx.doi.org/10.1017/5000748530004832X) for evaluation of field resistance in cattle tick (Rhipicephalus microplus) larvae.

(10) The first LPT assay was conducted to identify the potential range of flavesone acaricidal activity against larvae of a susceptible non-resistant field strain (NRFS) of R. microplus as a reference strain using a wide range of concentrations (1 in 10 in series).

(11) The test compound, flavesone, 6-isobutyryl-2,2,4,4-tetramethylcyclohexane-1,3,5-trione, 96.7%, was used. As its volatility/evaporative properties were unknown, the LPT method was modified to incorporate the use of suitably-sized polyethylene plastic sheets to envelop the larval packets, minimizing exposure of the test active to the atmosphere.

(12) In addition, the use of the solvent trichloroethylene (TCE) was removed to avoid the time required for evaporation in the preparation of test papers. The test solutions were prepared in olive oil only at the diluent and the papers immediately enveloped in the plastic sheets and sealed with bulldog clips to minimize evaporation.

(13) A stock solution with a concentration of 100,000 ppm (10%) flavesone was prepared in olive oil as the diluent (1.035 mL flavesone (96.7%) to 8.965 mL olive oil) and then further diluted 1 in 10 in series to also give 10,000 ppm, 1,000 ppm, 100 ppm, 10 ppm and 1 ppm concentration. The negative control was olive oil only. No positive control was included in this experiment. Due to the viscosity of the olive oil, all solutions were prepared using reverse pipetting technique.

(14) Filter papers (75 mm×85 mm Whatman® No. 541), with grid patterns, were impregnated on ½ of the paper with 225 μL of each solution using a micro-pipette and immediately folded in half, enveloped in polyethylene plastic and sealed with 3 bulldog clips. The impregnated papers were kept at room temperature on aluminium trays for a minimum of 60 minutes to allow for dispersal across the grid pattern of the paper prior to aliquoting of larvae. The packets were prepared in duplicate for each concentration, including the negative control.

(15) An 8 dram vial containing approximately 20,000 hatched 7-21 day old NRFS larvae (about 1 g eggs) was opened and set up on a moated tray with a small amount of detergenated water, about 15 to 30 minute before use. Only larvae that migrated to the top of the vial were used in the assay.

(16) Aliquots of approximately 100 larvae were placed into each packet using plastic disposable forceps and the packets resealed and incubated and 27° C. and 85% relative humidity (RH).

(17) After 24 hours, the larval packets were opened and the numbers of dead and live larvae were counted under a magnification lamp. Percentage mortality was calculated and, where applicable, corrected using Abbott's Formula (Abbott, 1925, J. Economic Entomology, 18:256-257):

(18) $\frac{\left(\mathrm{Treated}\%\mathrm{mortality}\right)-\left(\mathrm{control}\%\mathrm{mortality}\right)}{100-\left(\mathrm{control}\%\mathrm{mortality}\right)}\times 100$

(19) LC.sub.50 and LC.sub.99 values were determined by Probit mortality vs log concentration analysis. Probit values were derived from “Transformation of Percentages to Probit's Tables” published by Fisher R. A. and Yates F. (1938).

(20) The results are shown in Tables 1 and 2.

(21) TABLE-US-00001 TABLE 1 mortality Concentration ppm NRFS (%) 100,000 100 10,000 100 1,000 96.97 100 1.82 10 0.62 1 0.0

(22) LC.sub.50 and LC.sub.99 values were determined and are shown in Table 2.

(23) TABLE-US-00002 TABLE 2 Strain LC.sub.50 (ppm) LC.sub.99 (ppm) NRFS 171 3346

Example 2: LPT Assay With Resistant Larvae

(24) The LPT Assay of Example 1 was repeated using a narrow range of concentrations, 1 in 2 series, of flavesone to determine LC.sub.50 and LC.sub.99 values against the susceptible NRFS and the multi-resistant Tiaro reference strain.

(25) The Tiaro strain of R. microplus comprises of about 30% fluazuron, 60.6% cypermethrin (SP), 57.6% flumethrin (SP), 16.2% amitraz (amidine), 11.3% DDT, 9.3% chlorpyrifos (OP) and 2.4% dieldrin resistance [2014 acaricide resistance profiling].

(26) The synthetic pyrethroid (SP) cypermethrin was included in the assay as a positive control.

(27) The stock solution of flavesone (100,000 ppm) was diluted 1 in 10 with olive oil (1 mL to 9 mL diluent) to give 10,000 ppm, which was then further diluted 1 in 2 in series (5 mL to 5 mL diluent) to give 5,000 ppm, 2,500 ppm, 1,250 ppm, 625 ppm, 312.5 ppm and 156.25 ppm concentrations.

(28) For the preparation of the positive control, cypermethrin, a stock solution of a concentration of 10,000 ppm was prepared in 2:1 trichloroethylene (TCE)/olive oil as the solvent (0.0352 g cypermethrin, 94.8% purity, to 10 mL solvent) and then further diluted 1 in 2 in series (5 mL to 5 mL solvent) to also give 5,000 ppm, 2,500 ppm, 1,250 ppm, 625 ppm, 312.5 ppm and 156.25 ppm.

(29) The flavesone papers were prepared as in Example 1.

(30) The cypermethrin papers were impregnated with 670 μL of each solution using a micro-pipette and hung on a rack in a fume-hood to allow papers to dry (evaporation of TCE) for a minimum of 60 minutes. The papers were then folded in half and sealed with three bulldog clips, placed on aluminium trays prior to aliquoting the larvae. All packets were prepared in duplicate.

(31) The negative control papers were prepared for both flavesone (olive oil only) and the positive control, cypermethrin (2:1 TCE/olive oil).

(32) Mortality was assessed at 24 hours and LC.sub.50 and LC.sub.99 values determined by Probit mortality vs log concentration analysis. A dose-response relationship was determined at 24 hours contact exposure. The results are shown in Table 3.

(33) TABLE-US-00003 TABLE 3 Flavesone Cypermethrin Conc (ppm) NRFS Tiaro Conc (ppm) NRFS Tiaro 10,000 100 100 10,000 100 66.84 5,000 100 100 5,000 100 62.22 2.500 100 100 2.500 100 59.72 1,250 100 100 1,250 100 20.90 625 11.76 12.83 625 99.56 21.92 312.5 0 0.13 312.5 89.01 20.36 156.25 0 0 156.25 67.33 0.56

(34) At 1,250 ppm flavesone for both strains, some larvae were still waving their legs in the air; however, did not take a step to indicate survival (flaccid paralysis). At 2,500 ppm flavesone for both strains, no movement was noted with 100% mortality observed.

(35) There was no evidence of cross-resistance to flavesone when comparing LC.sub.50 and LC.sub.99 data between the NRFS and Tiaro strains.

(36) Negative control mortality ranged from 0% to 0.51% and where applicable was corrected using Abbott's formula.

(37) LC.sub.50 and LC.sub.99 values were determined using Probit mortality vs log concentration analysis and are shown in Table 4.

(38) TABLE-US-00004 TABLE 4 Flavesone Cypermethrin Strain LC.sub.50 (ppm) LC.sub.99 (ppm) Strain LC.sub.50 (ppm) LC.sub.99 (ppm) NRFS 690 1109 NRFS  128 540 Tiaro 647 1039 Tiaro 3082 >10,000

Example 3

(39) The experiment of Example 2 was repeated only on the NRFS strain with dilutions of 1 to 2 in series and cypermethrin as a positive control. The concentrations of flavesone were 5,000 ppm, 2,500 ppm, 1,250 ppm, 625 ppm and 312.5 ppm. The concentrations of cypermethrin were 1,250 ppm, 652 ppm, 312.5 ppm, 156.25 ppm, 78.125 ppm and 36.0625 ppm.

(40) Mortality was assessed at 24 hours and LC.sub.50 and LC.sub.99 values determined by probit mortality vs log concentration analysis.

(41) As with Example 2 at 1,250 ppm flavesone, some larvae were still waving their legs in the air; however did not take a step to indicate survival (flaccid paralysis). At 2,500 ppm flavesone, no movement was noted with 100% mortality observed.

(42) Negative control mortality ranged between 0%-1.02% and where applicable was corrected using Abbott's formula.

(43) The results are shown in Table 5:

(44) TABLE-US-00005 TABLE 5 Flavesone Cypermethrin Conc (ppm) NRFS Conc (ppm) NRFS 5,000 100 1250 100 2.500 100 625 100 1,250 100 312.5 99.16 625 11.76 156.25 79.56 312.5 1.28 78.125 20.57 39.0625 0

(45) The LC.sub.50 and LC.sub.99 values are shown in Table 6:

(46) TABLE-US-00006 TABLE 6 Flavesone Cypermethrin Strain LC.sub.50 (ppm) LC.sub.99 (ppm) Strain LC.sub.50 (ppm) LC.sub.99 (ppm) NRFS 610 1050 NRFS 112 317

Example 4

(47) It was noted in Example 2 and Example 3 that although 100% mortality was recorded at 1,250 ppm flavesone concentration after 24 hours contact exposure, some larvae at this concentration, for both the NRFS and Tiaro strains, were still waving the legs in the air but unable to take a step to indicate survival (flaccid paralysis), therefore moribund. This experiment was conducted to confirm whether to not these larvae died within a further 24 hours contact exposure, with mortality assessed at 48 hours.

(48) Concentrations of 1,250 ppm, 625 ppm and 312.5 ppm flavesone concentrations prepared for Example 3 were used on the same day of preparation. Duplicate papers were prepared for both the NRFS and Tiaro strains (including negative controls) as described in Example 2.

(49) Mortality was assessed at 48 hours and LC.sub.50 and LC.sub.99 values were determined by Probit mortality vs log concentration analysis. The results are shown in Tables 7 and 8.

(50) Negative control mortality ranged between 0.79% and 3.91% and corrections were made by using Abbott's formula.

(51) TABLE-US-00007 TABLE 7 Flavesone Conc (ppm) NRFS Tiaro 1250 100 100 625 66.83 84.13 312.5 2.17 0.97

(52) TABLE-US-00008 TABLE 8 Flavesone Strain LC.sub.50 (ppm) LC.sub.99 (ppm) NRFS 526 923 Tiaro 521 887

Example 5: Evaluation of Flavesone as a Grain Protectant

(53) A laboratory established insect population of Rhyzopertha dominica (QRD1440) with a history of resistance to organophosphates and pyrethroids were used in this study.

(54) Residue free organically produced wheat grain was used in the study. The moisture content of the wheat was kept at 11%.

(55) Test solutions of flavesone (25 ppm), deltamethrin (K-Obiol®, 1 ppm) and chlorpyrifos (Reldan®, 5 ppm and 10 ppm) in water were prepared. Water was used as the control sample. Five lots of 240 g of wheat was weighed into glass 1 L capacity jars, one jar per treatment and control.

(56) The test solutions and control solutions were pipetted onto the inside of one of the jars (one jar per sample) immediately above the grain surface at a rate equivalent to 10 mL of solution per kg of wheat. The jars were sealed, briefly shaken and tumbled by hand, then tumbled mechanically for 10 minutes. The moisture content was 12%, reflecting the upper limit accepted by Australian bulk handling companies. The day after treatment, each 240 g wheat sample was divided into three replicates of 80 g and placed into glass jars 250 mL capacity.

(57) 50 adult R. dominica QRD1440 (1 to 3 weeks post-emergence) were added to each jar of treated or control wheat. Each jar was covered with filter paper as a lid and stored at 25° C. and 55% R.H. for 14 days, after which the wheat sample was sieved to retrieve the adult insects. Mortality was recorded. All adults, dead and alive, were discarded. The jars of wheat were incubated for a further 6 weeks and the number of progeny recorded. The results are shown in Table 9:

(58) TABLE-US-00009 TABLE 9 14 day mortality of QRD1440 R. dominica 14 Day adult mortality F1 Progeny Treatment (ppm) No. dead Count and Replicate (total) No. dead/total Control A 0/50 1/204 B 0/50 0/133 C 0/50 0/151 Flavesone (25 ppm) A 19/50  0/37  B 11/50  0/62  C 14/50  0/90  Deltamethrin (1 ppm) A 1/50 0/142 B 0/50 0/79  C 1/50 2/292 Chlorpyrifos (5 ppm) A 0/50 2/470 B 1/50 3/172 C 0/50 3/355 Chlorpyrifos (10 ppm) A 0/50 1/350 B 0/50 0/317 C 0/50 2/232
At 25 ppm of flavesone the QDR1140 R. dominica resistant strain had higher mortality than the control and other pesticides used. The flavesone treatment also resulted in less F1 progeny being produced.

Example 6: Concentrations of Flavesone

(59) The experiment of Example 5 was repeated with flavesone at concentrations of 25 ppm, 50 ppm and 75 ppm. Water was the control.

(60) The results are shown in Table 10:

(61) TABLE-US-00010 TABLE 10 14 day mortality QRD1440 R. dominica at different flavesone concentrations 14 day Adult Mortality F1 Progeny Treatment (ppm) No. dead/ count and Replicate total No. dead/total Control A  0/50  5/515 B  1/50  2/471 C  1/50  3/341 Flavesone (25 ppm) A 22/50  0/20 B 21/50 0/5 C 30/50  0/20 Flavesone (50 ppm) A 47/50 0/0 B 49/50 0/0 C 47/50 0/0 Flavesone (75 ppm) A 50/50 0/0 B 50/50 0/0 C 50/50 0/0

Example 7: Control of Resistant Strain of Lesser Grain Borer QRD1440 R. dominica

(62) The experiment of Example 5 was repeated with flavesone at a concentration of 60 ppm. Water was used as the control.

(63) The results are shown in Table 11.

(64) TABLE-US-00011 TABLE 11 Response of resistant strain of lesser grain borer (R. dominica, QRD1440) to flavesone at a rate of 60 ppm 14 day Adult Mortality F1 Progeny Treatment (ppm) No. dead/ count and Replicate total No. dead/total Control A  0/50 2/589 B  0/50 2/204 C  0/50 2/575 Flavesone (60 ppm) A 50/50 0/0  B 50/50 0/0  C 50/50 0/0 

Example 8: Comparative Studies With a Susceptible Strain of Lesser Grain Borer QRD14 R. dominica

(65) The experiment of Example 5 was repeated using a laboratory reared susceptible strain QQRD14 of R. dominica and different concentrations of flavesone to determine efficacy.

(66) The results are shown in Table 12.

(67) TABLE-US-00012 TABLE 12 Response of susceptible strain of lesser grain borer (R. dominica, QQRD14) to flavesone at a broad application range. 14 day Adult Mortality F1 Progeny Treatment (ppm) No. dead/ count and Replicate total No. dead/total Control A  2/50 4/499 B  0/50 9/604 C  0/50 3/801 Flavesone (5 ppm) A  0/50 4/930 B  0/50 4/999 C  0/50 1/866 Flavesone (10 ppm) A  6/50 3/446 B  8/50 0/444 C  6/50 3/487 Flavesone (25 ppm) A 50/50 0/0  B 50/50 0/0  C 50/50 0/0  Flavesone (50 ppm) A 50/50 0/0  B 50/50 0/0  C 50/50 0/0  Flavesone (100 ppm) A 50/50 0/0  B 50/50 0/0  C 50/50 0/0 

Example 9: Control of Halotydeus destructor (Redlegged Earth Mite)—Dose Response in Susceptible Populations

(68) The efficacy of flavesone against H. destructor was assessed using the glass vial technique developed by Hoffmann et al. (1997, Exp. Appl. Acarol., 21:151-162), adapted for plastic vials. A susceptible population of mites was collected from capeweed (Arctotheca calendula) at a Victorian site (37° 40′33″S, 145° 07′ 45″E) that had no known history of insecticide application. Following collection, samples were stored in small plastic containers with leaf material and paper towel to absorb excess moisture. Containers were kept at 4° C. prior to testing.

(69) Serial dilutions of each insecticide were prepared from the compositions shown in Table 13:

(70) TABLE-US-00013 TABLE 13 Insecticide Active ingredient Field rate Concentration Flavocide 500EW Flavesone 500 g/L 2000 mL/100 L 10000 mg a.i./L Talstar ® 250EC Bifenthrin 250 g/L  40 mL/100 L  100 mg a.i./L Lorsban ™ 500EC Chlorpyrifos 500 g/L  140 mL/100 L  700 mg a.i./L

(71) The test compositions included 0.1% Tween 20 non-ionic surfactant to aid the spread of insecticides when coating plastic vials. This concentration of Tween 20 has been previously shown to have no toxic effect on H. destructor. For each insecticide concentration tested, 3, 10, 30, 100, 300, 1000, 3000 and 10000 mg a.i./L (ppm), approximately 10 mL of solution was poured into a 15 mL plastic vial and swirled to ensure a complete coating, with excess liquid removed. Eight vials were coated per concentration and were left to dry overnight. Control vials were treated in the same way but with water used in place of test composition.

(72) Eight susceptible H. destructor mites were then placed into each vial along with a leaf of common vetch (Vicia sativa). The leaf was added to provide food and increase humidity. Vials were then sealed with a lid and placed at 18° C. After 4, 6, 8 and 24 hours of exposure, the mites were scored as alive (moving freely), incapacitated (inhibited movement), or dead (no movement over a 5 second period). Incapacitated individuals were pooled with dead individuals for analysis as they invariably died and therefore did not contribute to the next generation.

(73) The results for flavesone are shown in FIG. 1. Mortality of H. destructor increased dramatically between 100 and 300 mg a.i./L and mortality increased with duration of exposure. At 4 hours of exposure, flavesone at 300 mg a.i./L had caused an average of 55% mortality, while at application rates below this, little mortality was observed. All application rates above 300 mg a.i./L resulted in 100% mortality by 4 hours exposure. By 8 hours of exposure, mortality at 300 mg a.i./L had risen to 100%. At lower application rates, increased mortality was observed at 24 hours.

Example 10: Control of Halotydeus destructor (Redlegged Earth Mite)—Dose Response in Susceptible and Resistant Populations

(74) The experiment set out in Example 9 was repeated with susceptible and resistant populations of H. destructor, with the exception that the mites were observed at 6 and 24 hours. The resistant population of H. destructor was collected from a lucerne paddock in the Upper South-East district of South Australia, where resistance to synthetic pyrethroids was confirmed in late 2016.

(75) The data produced in the assays were assessed for concentrations that caused 50, 90 and 99% mortality (lethal concentration, LC), together with 95% confidence intervals (CIs) and were estimated from observations of mortality after 24 hours exposure using a binomial logistic regression (Robertson & Preisler, 1992, Pesticide Bioassays with Arthropods. CRC: Boca Raton; Venables & Ripley, 2002, Modern Applied Statistics with S. Springer: New York). Population differences were tested by comparing the change in model deviance with and without the population factor (different regression intercepts for each population). Differences in regression slopes between populations were tested by comparing the change in model deviance with and without the population×dose interaction term. The resistance ratio of the insecticide-resistant population was estimated as the ratio of its LC.sub.50 to that of the susceptible population. All analyses were performed using R 3.3. (R Core Team 2017, R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org).

(76) Dose response curves showing the effects of flavesone on susceptible and resistant H. destructor populations after 24 hours exposure are shown in FIG. 2. Flavesone was equally effective against both insecticide-resistant and susceptible populations as evidenced by closely aligning dose-response curves for both populations (χ.sup.2=1.40, df=1, p=0.24). LC.sub.50 values (and 95% CIs) were calculated as 40.6 (33.3−49.6) mg a.i./L and 34.2 (27.9−41.9) mg a.i./L for the susceptible and resistant populations, respectively, for flavesone as shown in Table 14. There was no evidence that the regression slopes for concentration were significantly different between populations (χ.sup.2=0.01, df=1, p 0.91).

(77) A large difference was seen in sensitivity to bifenthrin between susceptible and resistant populations (χ.sup.2=167.57, df=1, p=0.0001) as shown in FIG. 3. Comparing the LC50 values between populations showed that the resistant population requires about 3,500 times the dose of bifenthrin compared to the susceptible population to achieve 50% mortality after 24 hours. This population likely included a mixture of resistant and susceptible individuals. The LC50 value for the susceptible population of 0.04 (0.03−0.08) mg a.i./L is consistent with previous studies using H. destructor collected from this location. The regression coefficients were also significantly different between populations (χ.sup.2=28.76, df=1, p=0.0001).

(78) For chlorpyrifos, dose-responses were also significantly different between the insecticide-resistant and susceptible populations (χ.sup.2=44.13, df=1, p=, 0.0001). The resistant population was 6.5 times more resistant to chlorpyrifos than the susceptible population. This is comparable to the organophosphate resistance seen in South Australia. There is no evidence that the regression slopes for concentration were different between populations. (χ.sup.2=1.77, df=1, p=0.18).

(79) LD.sub.50, LD.sub.90 and LD.sub.99 values and confidence intervals for 24 hours exposure are shown in Table 14.

(80) TABLE-US-00014 TABLE 14 Regression slope LC Lower - upper 95 coeffeicient LC (mg % CIs Insecticide Population (±SE) quantile a.i./L) (mg a.i./L) Flavesone Susceptible 1.75 (0.17) LC.sub.50 40.6 33.25-49.58 LC.sub.90 142.55 105.76-192.15 LC.sub.99 561.35 335.01-940.61 Resistant 1.78 (0.17) LC.sub.50 34.22 27.93-41.91 LC.sub.90 117.89  86.60-160.47 LC.sub.99 454.76 267.01-774.52 Bifenthrin Susceptible 0.54 (0.05) LC.sub.50 0.04 0.03-0.08 LC.sub.90 2.66 1.03-6.86 LC.sub.99 233.10  44.58-1218.91 Resistant 0.27 (0.05) LC.sub.50 143.79  59.03-350.21 LC.sub.90 5.4 × 10.sup.5 6.8 × 10.sup.5-4.2 × 10.sup.6 LC.sub.99 4.3 × 10.sup.9 .sup. 1.1 × 10.sup.8-1.7 × 10.sup.12 Chlorpyrifos Susceptible 1.68 (0.26) LC.sub.50 0.21 0.14-0.30 LC.sub.90 0.76 0.44-1.33 LC.sub.99 3.18 1.25-8.10 Resistant 1.27 (0.18) LC.sub.50 1.37 0.93-2.01 LC.sub.90 7.76  4.15-14.50 LC.sub.99 51.56  17.49-152.04

(81) These results show that flavesone is efficacious against H. destructor in both susceptible and insecticide-resistant populations and caused 50% mortality at 24 hours with a concentration between 34−40 mg a.i./L.

Example 11: Efficacy of Flavesone Against Susceptible and Resistant Populations of Green Peach Aphid

(82) Colonies of M. persicae (green peach aphid) were established from long-term laboratory cultures of a known insecticide-susceptible population, and a population which has previously been shown to be resistant to carbamates and synthetic pyrethroids. Each colony was maintained separately on bok choi plants (Brassica napus chinensis) within an exclusion cage in a constant temperature room at 24° C. with a photoperiod of 16:8 LD.

(83) Laboratory bioassays were used to determine the efficacy of flavesone against M. persicae following the leaf dip method described in Moores et al. (1994, Pesticide Biochemistry and Physiology, 49, 114-120). A pilot study was first conducted which confirmed that the leaf dip method was appropriate to elicit a clear dose response for Flavocide 500EW against M. persicae, and to determine the appropriate rate range and timing of mortality assessments (scored at 24, 48, 72 & 96 hours) (data not shown).

(84) Bioassays were then conducted to assess the efficacy of flavesone against a susceptible and resistant population of M. persicae, and to calculate LC values. The efficacy of a conventional insecticide, pirimicarb, was tested for comparison. Nine concentrations of flavesone ranging from 1×10.sup.−3 to 10 times the proposed field rate (Table 15), and six concentrations of pirimicarb were serially diluted and tested, along with a water control, against the susceptible and resistant aphid populations. Leaf discs (25 mm diameter) cut from bok choi leaves were submerged for 1 second in the insecticide solutions, or in the water control, and placed adaxial side up on 10 g/L agar in 35 mm petri dishes. Six replicate leaf discs were prepared per treatment. Once leaves were air-dry, eight M. persicae nymphs were transferred to each insecticide-dipped leaf disc using a fine-haired paintbrush.

(85) After aphid introduction, each petri dish was inverted onto a lid containing a 25 mm diameter filter paper to control humidity. All petri dishes were then placed into an incubator held at 18° C.±2° C. with a photoperiod of 16:8 LD cycle. At 48 & 96 hours, aphids were scored as alive (vibrant and moving freely), dead (not moving over a 5 second period), or incapacitated (inhibited movement). Incapacitated individuals were pooled with dead individuals for analysis as they invariably die and therefore do not contribute to the next generation.

(86) TABLE-US-00015 TABLE 15 Chemical treatments used in this study. Insecticide Active Ingredient Field Rate Concentration Flavocide Flavesone 500 g/L 2000 mL/100 L* 10,000 mg a.i./L 500EW Pirimor Pirimicarb 500 g/kg 500 g/ha  2,500 mg a.i./L 500WG *Suggested field rate of flavesone 500EW provided by the client (1% flavesone v/v).

Data Analysis

(87) Dose-response curves were generated by plotting percentage mortality against log concentration. Mortality data was analysed using a logistic regression model with random effects. Logistic regression is suited for the analysis of binary response data (i.e. dead/alive) with the random effect component of the model controlling for the non-independence of mortality scores within replicates. Concentrations that resulted in 50, 90, and 99% mortality (lethal concentration, LC) (along with 95% confidence intervals, CIs) were calculated using a binomial logistic regression (Robertson & Preisler 1992, Pesticide Bioassays with Arthropods, CRC: Boca Ratan; Venables & Ripley 2002, Modern Applied Statistics with S, Springer, New York, http://www.stats.ox.ac.uk/pub/MASS4). Population differences were tested by comparing the change in model deviance with and without the population factor (different regression intercepts for each population). Differences in regression slopes between populations were tested by comparing the change in model deviance with and without the population×dose interaction term.

(88) Analyses were conducted using R version 3.3.1 (R Development Core Team 2017. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria, http://R-project.org).

Results

(89) While the dose-response curves for the susceptible and resistant populations exposed to flavesone appeared similar at 48 hours (FIG. 5), a significant difference between populations was detected (c2=8.09, df=1, p<0.01). However, by 96 hours, the dose-response curves for the susceptible and resistant M. persicae populations exposed to flavesone had become more closely aligned and were not significantly different (c2=0.78, df=1, p=0.38) (FIG. 6). LC50 values (and 95% CIs) after 96 hours exposure were estimated as 2,731 (2,259−3,303) mg a.i./L for the susceptible population, and 3,151 (2,568−3,865) mg a.i./L for the resistant population (Table 16). The regression slopes between populations did not differ significantly at 48 hours (c2=0.49, df=1, p=0.48) or 96 hours (c2=0.72, df=1, p=0.40).

(90) TABLE-US-00016 TABLE 16 LD50, LC90, and LC99 values (and 95% confidence intervals) and regression coefficients for M. persicae, computed from logit models for responses to insecticides after 96 hours exposure. Regression slope LC Active coefficients LC (mg Lower - 95% Ingredient Population (±SE) quantile a.i./L) CIs (mg a.i./L) Flavesone Susceptible 2.77 LC.sub.50 2,731 (2,259-3,303) LC.sub.90 6,034 (4,488-8,113) LC.sub.99 14,330  (8,826-23,267) Resistant 2.38 LC.sub.50 3,151 (2,568-3,865) LC.sub.90 7,929  (5,700-11,028) LC.sub.99 21,708 (12,551-37,544) Pirimicarb Susceptible 1.52 LC.sub.50 18.5 (13.4-25.6) LC.sub.90 78.9 (47.2-132)  LC.sub.99 384 (154.9-952.2) Resistant −0.13 LC.sub.50 N/A N/A LC.sub.90 N/A N/A LC.sub.99 N/A N/A

(91) At 48 and 96 hours, there were clear differences in the dose-response curves between the susceptible and resistant populations after exposure to pirimicarb (48 hr: c2=269.9, df=1, p<0.0001; 96 hr: c2=257.5, df=1, p<0.0001) (FIGS. 7 & 8). The regression slopes were also significantly different between populations (48 hr: c2=66.6, df=1, p<0.0001; 96 hr: c2=107.2, df=1, p<0.0001). The estimated LC50 for the susceptible population after 96 hours of 18.5 mg a.i./L is consistent with previous laboratory studies using M. persicae (Umina et al. 2014, Journal of Economic Entomology, 107(4), 1626 1638). Very low mortality observed for the resistant population when exposed to pirimicarb prevented the calculation of meaningful LC values (Table 16).

(92) This study demonstrates that flavesone is efficacious against M. persicae. The LC50 of flavesone after 96 hours exposure was between 2,731-3,151 mg a.i./L. The efficacy of pirimicarb was very high against the susceptible population and matched closely with previously published bioassay data. Resistance to pirimicarb in the insecticide-resistant population was confirmed. This population is also resistant to synthetic pyrethroids, as demonstrated by pesticide bioassay results and genetic screening (see Umina et al. 2014)

(93) The dose-response curves for flavesone for the susceptible and resistant populations were closely aligned at 96 hours after exposure. This shows that flavesone is effective against M. persicae populations with resistance to carbamates, and that flavesone has a different mode of action to this class of insecticide. There was some evidence for population differences in responses after 48 hours. The reason(s) for this remains unclear but could reflect natural differences between the populations such as colony health, general hardiness, or bacterial endosymbionts.

Example 12: Toxicity of Flavesone to Aedes aegypti LVP (Insecticide Susceptible and PRS Insecticide Resistant Strains L3 Larvae

(94) Mosquito larval topical assay techniques were conducted.

(95) A dose response assay was used to determine the lethal concentration (LC.sub.50) value, performed with a minimum 5-point dose of flavesone diluted in sterile ddH.sub.2O, minimum of 4 technical replicates (5 mosquitos per replicate) per dose.

(96) Two species of mosquito were used. Aedes aegypti (yellow fever mosquito) Liverpool strain (insecticide susceptible, LVP) and PRS Puerto Rican strain (synthetic pyrethroid resistant) at L3 stage larvae.

(97) Negative control: vehicle

(98) Positive control: technical grade synthetic pyrethroid (SP) and organophosphate (OP): deltamethrin (SP), permethrin (SP), and malathion (OP).

(99) Phenotypic endpoint: scored for death/paralysis at 24, 48 and 72 hours.

(100) Dose points (selected from pilot assays which are not shown):

(101) Flavesone: 6.25 μg/mL, 25.0 μg/mL, 50 μg/mL, 75 μg/mL & 100 μg/mL; H.sub.2O control

(102) Deltamethrin: 1.56 ng/mL, 6.25 ng/mL, 12.5 ng/mL, 25 ng/mL, 50 ng/mL; 0.625% DMSO negative control

(103) Permethrin: 6.25 ng/mL, 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL; 0.625% DMSO negative control

(104) Malathion: 0.0156 μg/mL, 0.0625 μg/mL, 0.125 g/mL, 0.25 μg/mL, 1 μg/mL; 0.5% EtOH negative control

(105) Larvae were transferred to a 24 well tissue plate using a wide-bore plastic transfer pipette, 5 larvae per well. The water was gently removed from the well with a 1 mL pipette and an equivalent amount of ddH.sub.2O was added. The appropriate volume of test compound was added to each of the four replicate wells per treatment and the plate gently swirled to ensure uniform mixing. The plate was placed in a test or growth chamber under constant conditions of 22-25° C. and about 75-85% relative humidity on a 12 h light/12 hr dark cycle. Assessment of dead and non-responsive larvae was undertaken at 24, 48 and 72 hours.

(106) The results are shown in Table 17.

(107) TABLE-US-00017 TABLE 17 Time post Positive Positive Positive exposure Test Control Control Control to formulation Deltamethrin Permethrin Malathion chemistry Flavesone (SP) (SP) (OP) Lethal Concentration (LC50) LVP Strain (insecticide susceptible) 24 hours 40.9 μg/mL 11.9 ng/mL  21.9 ng/mL 110.3 ng/mL 48 hours 40.5 μg/mL  4.9 ng/mL  16.2 ng/mL  38.9 ng/mL 72 hours 39.7 μg/mL  3.7 ng/mL  16.6 ng/mL 29.75 ng/mL Lethal Concentration (LC50) PRS Strain (insecticide resistant) 24 hours 40.9 μg/mL 52.2 ng/mL 164.2 ng/mL 290.6 ng/mL (N = 2) 48 hours 40.5 μg/mL 50.1 ng/mL 150.5 ng/mL 176.7 ng/mL (N = 2) 72 hours 39.7 μg/mL 41.2 ng/mL 128.0 ng/mL 126.7 ng/mL (N = 2)

Example 12: Evaluation of Flavesone as a Grain Protectant Against Major Stored Grain Pests Having Resistance to Commonly Used Pesticides

Insects

(108) Laboratory established strains (both susceptible and resistant) of five species were considered for this stage of experiments. The resistant strains listed below represent the grain protectant-resistant genotypes that are commonly encountered in grain storages in Australia, particularly in the eastern grain belt: R. dominica strain QRD1440 is resistant to OP protectants and pyrethroids. T. castaneum strain QTC279 is resistant to malathion and bioresmethrin C. ferrugineus strain QCF73 is resistant to phosphine O. surinamensis strain QOS302 is resistant to fenitrothion & chlorpyrifos-methyl S. oryzae strain QSO393 is resistant to fenitrothion

Testing Program

Grain Treatment and Bioassays

(109) Residue and insect-free organically produced wheat was used in this study. Moisture content of the wheat before treatment was kept at 11%. Chemicals for use in these experiments: flavesone, K-Obiol EC Combi (50 g/L Deltamethrin, 400 g/L PBO) and Reldan (500 g/L Chlorpyrifos-methyl) were obtained from Bio-Gene Technology, Bayer Crop Science, and Dow AgroSciences respectively. Two rates (25 and 60 ppm) were considered for the stand alone flavesone experiments.

(110) For each strain of the borers (internal feeders), R. dominica and S. oryzae, three lots of 160 g of wheat was weighed into glass jars (500 mL capacity), i.e. one jar per treatment and another for the control (distilled water only). The solutions of each treatment (prepared at the predetermined dilution rates as alone and in combinations) were pipetted separately onto the inside of glass jars immediately above the grain surface at the rate equivalent to 10 mL of solution per kilogram of wheat. Distilled water was applied to control grain at the same rate as the treatment. All jars were sealed, briefly shaken and tumbled by hand, and then tumbled mechanically for 1 hour. Moisture content after treatment was 12%, reflecting the upper limit accepted by Australian bulk handling companies. One day after treatment, each 240 g lot of wheat was divided into three replicates of 80 g, which were placed into separate glass jars (250 mL capacity). The procedure for T. castaneum, C. ferrugineus and O. surinamensis was kept the same except that three lots of 600 g of wheat was treated per strain. One day after treatment each 600 g lot of wheat was divided into three replicates of 190 g which was then placed into glass jars (500 mL, capacity). The remaining 30 g of wheat was grounded to flour, divided into three lots of 10 g and added to the relevant replicates of whole wheat so that each replicate weighed a total of 200 g. The aim of grinding 5% of each replicate to flour was to improve the reproduction of these three pest species, which are external feeders. The above activity was repeated twice over the following two days for making a total of three replicates for each treatment.

(111) Bioassays were initiated by adding 50 adults (1-3 weeks post-emergence) to each jar of treated or control wheat. Each jar was covered with a filter paper lid and stored in a constant environment room at 25° C. and 55% r.h. for 2 weeks, after which the adults were sieved from the wheat and mortality recorded. Thereafter, all adults (dead and alive) were discarded and the jars of wheat were incubated for a further 6 weeks when the number of adult progeny were recorded. To synchronise progeny emergence, jars containing S. oryzae and O. surinamensis were incubated at 25° C. and 55% r.h., and jars containing the other species were incubated at 30° C. and 55% r.h.

Data Analysis

(112) Each data set is presented in simple tables with percentage adult mortality and number of live adult F1 progeny (mean±standard error of 3 replicates) of each species as well the percentage progeny reduction calculated from the mean numbers of F1 progeny in the treated wheat and untreated control.

Results

Effectiveness of Flavesone

(113) Control mortality in both susceptible and resistant strains of all 5 species was negligible (0-1.3%) (Tables 18-22). The number of adult progeny produced in controls of R. dominica were 234 and 211 for the susceptible (QRD14) and resistant strain (QRD1440), respectively (Table 18); 118 (QTC4) and 321 (QTC279) for T. castaneum (Table 19), 360 (QCF31) and 344 (QCF73) for C. ferrugineus (Table 20), 348 (VOS48) and 412 (QOS302) for O. surinamensis (Table 21) and 716 (LS2) and 610 (QSO393) for the susceptible and resistant strains, respectively, of S. oryzae (Table 22).

(114) As expected, 25 ppm of flavesone failed to achieve complete mortality of adults in both the susceptible (QRD14) and resistant (QRD1440) strains of R. dominica, but achieved 100 and 88% progeny reduction in the respective strains (Table 18). However, the higher rate of 60 ppm of flavesone achieved complete control of adults and progeny of both strains (Table 1) validating the Phase I results.

(115) Against the strains of other four species, however, both the proposed rates (25 and 60 ppm) of flavesone failed to achieve complete mortality of adults (Tables 19-22); although complete progeny reduction was achieved at 60 ppm in C. ferrugineus and O. surinamensis (Tables 20 and 22). Both rates of flavesone under performed against the susceptible (QTC4) and resistant (QTC279) strains of T. castaneum with no adult mortality achieved and a maximum of 45% progeny reduction yielded against the former and a 36% against the latter at the higher rate of 60 ppm (Table 19). Against C. ferrugineus, adult mortality reached 90 and 62% in the susceptible (QCF31) and resistant (QCF73) strains, respectively, at the highest dose of 60 ppm (Table 20). At the lower dose of 25 ppm, progeny reduction in this species was recorded at the similar level of 75% for both strains and a 100% progeny reduction was recorded at the 60 ppm level (Table 20). In the case of O. surinamensis, flavesone at 25 ppm achieved adult mortalities of 22 and 0.7% in the susceptible (VOS48) and resistant (QOS302) strains, respectively; and a maximum of 91% in the former and 14% in the latter at the higher dose of 60 ppm (Table 21). Both rates of flavesone, however, produced very high percentage of progeny reduction (61-99%) at 25 ppm and complete reduction of progeny (100%) at 60 ppm in both strains of this species (Table 21). The effectiveness of flavesone against S. oryzae was similar to that observed against T. castaneum (Table 18 and 22). Both rates failed to achieve any significant mortality against the adults of both strains (Table 22). At 60 ppm, however, flavesone achieved 29 and 50% progeny reduction, in the resistant (QSO393) and susceptible (LS2) strains, respectively (Table 22).

(116) TABLE-US-00018 TABLE 18 Effectiveness of Flavesone against adults and progeny production of Rhyzopertha dominica in treated wheat. Adult Progeny Treatment mortality Live adult reduction Strain (mg/kg) (%)* progeny* (%) QRD14 Control  0.7 ± 0.7 234.7 ± 101.5 — Flavesone 25 98.0 ± 1.2 0.0 ± 0.0 100 Flavesone 60  100 ± 0.0 0.0 ± 0.0 100 QRD1440 Control  0.0 ± 0.0 211.0 ± 69.4  — Flavesone 25 56.7 ± 4.1 23.7 ± 9.0  88.8 Flavesone 60  100 ± 0.0 0.0 ± 0.0 100 *Mean ± standard error

(117) TABLE-US-00019 TABLE 19 Effectiveness of Flavesone against adults and progeny production of Tribolium castaneum in treated wheat. Adult Progeny Treatment mortality Live adult reduction Strain (mg/kg) (%)* progeny* (%) QTC4 Control 1.3 ± 1.3 118.0 ± 41.2 — Flavesone 25 0.0 ± 0.0 120.0 ± 16.1 — Flavesone 60 0.0 ± 0.0 65.0 ± 3.2 45.2 QTC279 Control 0.7 ± 0.7 321.3 ± 35.0 — Flavesone 25 0.0 ± 0.0 265.3 ± 27.7 17.4 Flavesone 60 0.0 ± 0.0 204.3 ± 27.8 36.4 *Mean ± standard error

(118) TABLE-US-00020 TABLE 20 Effectiveness of Flavesone against adults and progeny production of Cryptolestes ferrugineus in treated wheat. Adult Progeny Treatment mortality Live adult reduction Strain (mg/kg) (%)* progeny* (%) QCF31 Control 1.3 ± 0.7 306.0 ± 2.5  — Flavesone 25 27.5 ± 17.5 72.0 ± 20.3 76.5 Flavesone 60 90.0 ± 2.0  0.0 ± 0.0 100 QCF73 Control 0.0 ± 0.0 344.3 ± 18.4  — Flavesone 25 3.3 ± 1.3 83.7 ± 6.5  75.7 Flavesone 60 62.0 ± 6.1  0.0 ± 0.0 100 *Mean ± standard error

(119) TABLE-US-00021 TABLE 21 Effectiveness of Flavesone against adults and progeny production of Oryzaephilus surinamensis in treated wheat. Adult Progeny Treatment mortality Live adult reduction Strain (mg/kg) (%)* progeny* (%) VOS48 Control  0.7 ± 0.7 348.3 ± 32.3 — Flavesone 25 22.7 ± 2.4  1.3 ± 0.7 99.6 Flavesone 60 91.3 ± 5.9  0.0 ± 0.0 100 QOS302 Control  0.0 ± 0.0 412.3 ± 10.1 — Flavesone 25  0.7 ± 0.7 160.3 ± 15.7 61.2 Flavesone 60 14.7 ± 8.7  0.0 ± 0.0 100 *Mean ± standard error

(120) TABLE-US-00022 TABLE 22 Effectiveness of Flavesone against adults and progeny production of Sitophilus oryzae in treated wheat. Adult Progeny Treatment mortality Live adult reduction Strain (mg/kg) (%)* progeny* (%) LS2 Control 0.0 ± 0.0 716.0 ± 75.8 — Flavesone 25 1.3 ± 0.7 735.3 ± 60.6 — Flavesone 60 4.0 ± 1.2 355.3 ± 64.7 50.3 QSO393 Control 0.0 ± 0.0 610.0 ± 81.5 — Flavesone 25 0.7 ± 0.7  572.7 ± 100.9  6.1 Flavesone 60 0.7 ± 0.7 430.0 ± 51.0 29.5 *Mean ± standard error

Example 13: Evaluation of Combination of Flavesone and Chlorpyrifos-Methyl (Reldan) Against Major Stored Grain Pests Having Resistance to Commonly Used Pesticides

(121) The experiments of Example 12 were repeated using a combination of flavesone and chlorpyrifos-methyl.

(122) Across all the combined treatment experiments, control mortality in both susceptible and resistant strains of all 5 species was negligible (0-3%) (Tables 23-27). The number of adult progeny produced in R. dominica controls were 186 for the susceptible (QRD14) and resistant (QRD1440) strains (Table 23), 59 (QTC4) and 480 (QTC279) for T. castaneum (Table 24), 467 (QCF31) and 188 (QCF73) for C. ferrugineus (Table 25), 526 (VOS48) and 429 (QOS302) for O. surinamensis (Table 26) and 720 (LS2) and 565 (QSO393) for the susceptible and resistant strains, respectively, of S. oryzae (Table 27).

(123) All experimental combinations of flavesone and chlorpyrifos-methyl applied both at the higher and lower rates were highly successful against the susceptible strains of all 5 test species, with of 100% adult mortality and progeny reduction (Tables 23-27). The effectiveness of all these combinations was greatest against the resistant strain of C. ferrugineus, where complete control of adults and progeny were achieved (Table 26). Moreover, with the exceptions of 99% progeny reduction in a couple of combinations, all these treatments achieved 100% control of progeny in resistant strains of T. castaneum (QTC279), O. surinamensis (QOS302) and S. oryzae (QSO393) (Tables 24, 26 and 27). Against the resistant strain of R. dominica (QRD1440), however, complete adult mortality was achieved only at the combination of flavesone 60+chlorpyrifos-methyl 5 and complete progeny reduction was achieved in grain treated with the combinations of flavesone 30+ chlorpyrifos-methyl 10, flavesone 60+chlorpyrifos-methyl 5, flavesone 60+ chlorpyrifos-methyl 10 (Table 23).

(124) TABLE-US-00023 TABLE 23 Effectiveness of Flavesone in combination of Chlorpyrifos-methyl (OP) against adults and progeny of Rhyzopertha dominica in treated wheat. Adult Progeny mortality Live adult reduction Strain Treatment (mg/kg) (%)* progeny* (%) QRD14 Control  0.0 ± 0.0 186.7 ± 62.7  — Flavesone 30 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 5 Flave sone 30 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 5 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 QRD1440 Control  0.7 ± 0.7 186.7 ± 62.7  — Flavesone 30 + 81.3 ± 7.7  4.3 ± 3.0 97.7 chlorpyrifos-methyl 5 Flavesone 30 + 96.0 ± 0.0  0.0 ± 0.0 100 chlorpyrifos-methyl 10 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 5 Flavesone 60 + 99.3 ± 0.7  0.0 ± 0.0 100 chlorpyrifos-methyl 10 *Mean ± standard error

(125) TABLE-US-00024 Table 24 . Effectiveness of Flavesone in combination of Chlorpyrifos-methyl (OP) against adults and progeny of Tribolium castaneum in treated wheat. Adult Progeny mortality Live adult reduction Strain Treatment (mg/kg) (%)* progeny* (%) QTC4 Control  2.0 ± 0.0 59.3 ± 26.1 — Flavesone 30 + chlorpyrifos-methyl 5 100 ± 0.0 0.0 ± 0.0 100 Flavesone 30 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 5 Flavesone 60 + chlorpyrifos-methyl 10 100 ± 0.0 0.0 ± 0.0 100 QTC279 Control  0.0 ± 0.0 480.7 ± 25.6  — Flavesone 30 + chlorpyrifos-methyl 5 99.3 ± 0.7  0.0 ± 0.0 100 Flavesone 30 + chlorpyrifos-methyl 10 100 ± 0.0 0.0 ± 0.0 100 Flavesone 60 + chlorpyrifos-methyl 5 100 ± 0.0 0.0 ± 0.0 100 Flavesone 60 + chlorpyrifos-methyl 10 100 ± 0.0 0.0 ± 0.0 100 *Mean ± standard error

(126) TABLE-US-00025 TABLE 25 Effectiveness of Flavesone in combination of Chlorpyrifos-methyl (OP) against adults and progeny of Cryptolestes ferrugineus in treated wheat. Adult Progeny mortality Live adult reduction Strain Treatment (mg/kg) (%)* progeny* (%) QCF31 Control  2.7 ± 1.8 467.0 ± 21.2  — Flavesone 30 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 5 Flavesone 30 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 5 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 QCF73 Control  2.7 ± 0.7 188.7 ± 30.9  — Flavesone 30 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 5 Flavesone 30 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 5 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 *Mean ± standard error

(127) TABLE-US-00026 TABLE 26 Effectiveness of Flavesone in combination of Chlorpyrifos-methyl (OP) against adults and progeny of Oryzaephilus surinamensis in treated wheat. Adult Progeny mortality Live adult reduction Strain Treatment (mg/kg) (%)* progeny* (%) QVOS48 Control  1.3 ± 0.7 526.3 ± 24.4  — Flavesone 30 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 5 Flavesone 30 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 5 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 QOS302 Control  0.7 ± 0.7 429.0 ± 28.2  — Flavesone 30 + 164.0 ± 41.0  4.0 ± 2.0 61.8 chlorpyrifos-methyl 5 Flavesone 30 + 116.3 ± 37.2  8.7 ± 4.1 72.9 chlorpyrifos-methyl 10 Flavesone 60 + 27.3 ± 3.7  0.7 ± 1.6 99.8 chlorpyrifos-methyl 5 Flavesone 60 + 30.7 ± 1.8  0.0 ± 0.0 100 chlorpyrifos-methyl 10 *Mean ± standard error

(128) TABLE-US-00027 TABLE 27 Effectiveness of Flavesone in combination of Chlorpyrifos-methyl (OP) against adults and progeny of Sitophilus oryzae in treated wheat. Adult Progeny mortality Live adult reduction Strain Treatment (mg/kg) (%)* progeny* (%) LS2 Control  2.7 ± 2.7 720.3 ± 112.3 — Flavesone 30 + 100 ± 0.0 100 ± 0.0  100 chlorpyrifos-methyl 5 Flavesone 30 + 100 ± 0.0 100 ± 0.0  100 chlorpyrifos-methyl 10 Flavesone 60 + 100 ± 0.0 100 ± 0.0  100 chlorpyrifos-methyl 5 Flavesone 60 + 100 ± 0.0 100 ± 0.0  100 chlorpyrifos-methyl 10 QSO393 Control  0.0 ± 0.0 565.7 ± 35.0  — Flavesone 30 + 100 ± 0.0 0.3 ± 0.3 99.9 chlorpyrifos-methyl 5 Flavesone 30 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 Flavesone 60 + 100 ± 0.0 0.7 ± 1.3 99.9 chlorpyrifos-methyl 5 Flavesone 60 + 100 ± 0.0 0.0 ± 0.0 100 chlorpyrifos-methyl 10 *Mean ± standard error

(129) Table 28 provides an overview of the effectiveness of the combination of chlorpyrifos-methyl and flavesone.

(130) TABLE-US-00028 TABLE 28 Overview of effectiveness of Flavesone in combination with Chlorpyrifos- methyl (CM) at different rates against five major stored grain pests. 30 ppm flavesone plus 60 ppm flavesone plus 5 ppm CM 10 ppm CM 5 ppm CM 10 ppm CM Pest species Strain Adults F1 Adults F1 Adults F1 Adults F1 R. dominica Susceptible  ✓* ✓ ✓ ✓ ✓ ✓ ✓ ✓ Resistant X X X ✓ ✓ ✓ 99.3✓ ✓ T. castaneum Susceptible ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Resistant 99.3✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ C. ferrugineus Susceptible ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Resistant ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ O. surinamensis Susceptible ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Resistant X X X X X 99.8✓ X ✓ S. oryzae Susceptible ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Resistant ✓ 99.9✓ ✓ ✓ ✓ 99.9✓ ✓ ✓

Example 14: Evaluation of the Combination of Flavesone and Deltamethrin (K-Obiol) Against R. dominica Susceptible and Resistant Strains

(131) The Experiment of Example 12 was repeated using a combination of flavesone and deltamethrin with R. dominica susceptible QRD14 and resistant QRD1440 strains.

(132) In these experiments, the control mortality remained below 1% in both the susceptible and resistant strains of this species and similar number of live adult progeny (126 and 125) were emerged (Table 29). In all combinations, complete control of both adults and progeny was achieved against the susceptible strain (QRD14), and a high level of control was achieved against the resistant strain (QRD1440) (Table 29). Against adults of the resistant strain, all combinations yielded percentage mortality of 93-100%. Similarly, all combinations yielded 99-100% reduction of progeny of the resistant strain QRD1440 (Table 29).

(133) The results are shown in Table 29.

(134) TABLE-US-00029 TABLE 29 Effectiveness of Flavesone in combination of Deltamethrin against adults and progeny of Rhyzopertha dominica in treated wheat. Adult Progeny Treatment mortality Live adult reduction Strain (mg/kg) % progeny* (%) QRD14 Control  0.7 ± 0.7 126.3 ± 29.9  — Flavesone 30 +  100 ± 0.0 0.0 ± 0.0 100 deltamethrin 0.5 Flavesone 30 +  100 ± 0.0 0.0 ± 0.0 100 deltamethrin 1 Flavesone 60 +  100 ± 0.0 0.0 ± 0.0 100 deltamethrin 0.5 Flavesone 60 +  100 ± 0.0 0.0 ± 0.0 100 deltamethrin 1 QRD1440 Control  0.7 ± 0.7 125.0 ± 47.7  — Flavesone 30 + 93.3 ± 3.5 1.0 ± 1.0 99.2 deltamethrin 0.5 Flavesone 30 + 97.3 ± 0.7 0.0 ± 0.0 100 deltamethrin 1 Flavesone 60 +  100 ± 0.0 0.0 ± 0.0 100 deltamethrin 0.5 Flavesone 60 + 99.3 ± 0.7 0.0 ± 0.0 100 deltamethrin 1 *Mean ± standard error