ZA200408609B

ZA200408609B - Compositions and methods for preventing or reducing resistance of insects to insecticides

Info

Publication number: ZA200408609B
Application number: ZA2004/08609A
Authority: ZA
Inventors: David Moores Graham; Vera Gunning Robin
Original assignee: Rothamsted Res Ltd; Derpartment Of Primary Ind For And On Behalf Of The State Of New South Wales
Priority date: 2002-04-29
Filing date: 2004-10-25
Publication date: 2005-07-27

Description

COMPOSITIONS AND METHODS FOR PREVENTING OR REDUCING

RESISTANCE OF INSECTS TO INSECTICIDES

. The present invention relates to a method for preventing or reducing resistance of a - 5 pest to a pesticide and to formulations for use in such a method. In particular, the invention relates to insecticide resistance and to insecticidal compositions.

There are several definitions of insecticide resistance, often reflecting the interest of the scientist attempting the definition, rather than the phenomenon itself. The World

Health Organisation has defined resistance as “the development of an ability in a strain of insects to tolerate doses of toxicant that would prove lethal to the majority of individuals in a normal population of the same species”. Pesticide resistance is therefore to be similarly construed, although the main pests addressed herein are insects.

Insecticide resistance has become progressively more widespread since first being scientifically recorded in 1914. Over 500 insect and mite species now show tolerance to pesticides, and pesticide resistance has become a serious threat to the future success of pest control using chemicals.

There are three major mechanisms by which resistance can occur: reduced penetration of the pest by the pesticide; metabolism of the insecticide (resuiting in detoxification); and target-site insensitivity. These resistance mechanisms may exist individually in an insect, but are often found in combination where the overall resistance offered is substantially higher; this situation is referred to as ‘multi- factorial resistance’.

Many insects possess detoxification systems, which evolved originally to protect the insect from natural toxins in the environment. Metabolism of the insecticide may ) 30 occur before it reaches its target-site when it comes into contact with those detoxifying enzymes that render it either less toxic or more easily excreted, or both.

The most important enzyme systems involved in insecticide resistance include the groups a) mixed function oxidases, b) glutathione S-transferases and c) esterases.

Resistance resulting from enhanced activity of one or more of these enzyme groups has been found in several insect species.

Insect detoxifying enzyme systems can be studied either in vivo by conventionai . bioassays, or in vitro by biochemical assays. In conventional bioassays, there is . widespread employment of -synergists such as DEF (S,5,S-tributyi . phosphorothioate) and TPP (O,0,0-triphenyl phosphate). These are compounds ~ 5 that significantly enhance the toxicity of an insecticide, although they may be virtually non-toxic when used alone. Insecticide synergists act by inhibiting metabolic enzymes. Mortality differences in a bioassay, using a pesticide in the presence or absence of a synergist, should indicate whether a putative metabolic enzyme is involved in resistance. However, caution should be taken when using synergists; very often the chemical is not completely specific to the enzyme being examined, and it may be difficult to assess its possible effect upon other biological systems.

Esterases are enzymes that catalyse the hydrolysis of an ester bond. Organophosphate, carbamate and mast pyrethroid insecticides contain ester bonds and in some instances are sensitive to hydrolysis by esterases.

Esterases can act by either sequestering toxins to the insect or by hydrolysing the toxins. Therefore resistance to insecticides can result from either quantitative or qualitative changes in carboxylesterases, or a combination of the two. Qualitative changes could confer to the enzyme the ability to hydrolyse insecticidal esters at a significant rate, but may or may not affect the activity of the esterase towards the model substrates. Without a qualitative change, resistance can still occur by quantitative changes resulting from a process of gene amplification. This leads to the production of a greater amount of the same esterase, which sequesters the insecticide, resulting in resistance. Occasionally, the esterase may be both altered and amplified.

Piperonyl butoxide (PB or PBO) has been used extensively as a ‘tank mix’, both as an excipient due to its detergent/surfactant properties, and because of the wealth of literature describing its ability to inhibit oxidative metabolic enzymes (mixed function . oxidases). We have shown that certain non-specific esterases involved in pesticide resistance are partially inhibited by micromolar concentrations of piperonyl butoxide (IUPAC, London 1998).

In Australian Helicoverpa armigera (Hiibner), up to 70% of the activity of pyrethroid- ) resistance related esterases was inhibited by 10° M piperony! butoxide, both in . homogenates of resistant insects and in a partially-purified esterase extract . (Gunning et al in Piperonyl Butoxide, pp215-25, Academic Press (1998)).

Studies were also performed on esterases from the cotton aphid, Aphis gossypii (Glover) and the peach-potato aphid, Myzus persicae (Sulzer). Piperonyl butoxide was capable of inhibiting esterase activity from A. gossypii, but only when present at nominal concentrations of 10° M or greater. Total esterase activity was typically reduced by 50% in 30 minutes. This effect is not simply a consequence of a physico-chemical effect involving the substrate, since esterases present in M. persicae directly implicated in insecticide resistance were not inhibited when incubated with mM concentrations of piperony! butoxide for 40 minutes.

Gunning et al (1998) therefore proposed the use of a synergist or esterase inhibitor such as PBO simultaneously in a tank mix with an insecticide such as pyrethroid to improve efficacy of the insecticide in the field. Furthermore, data obtained by

Gunning et al (Pest Biochem & Physiol 63 50-62 (1999)) reveal significant pyrethroid synergism by organo-phosphates; in earlier studies, workers in the field did not observe this effect, doubtless because the pre-treatment period used in such studies (profenofos and DEF) never exceeded 30 minutes, which is too short a period for such an effect to become evident.

Thus, in some cases where resistance is conferred by esteratic enzymes, PBO or similarly acting analogue of PBO, such as a UV stable variant thereof, could be added to inhibit the esterases for a period of time prior to the addition of a conventional insecticide. This would normally necessitate a second insecticide application, je a pre-treatment with a metabolic enzyme inhibitor prior to insecticide spray, which is not an economic proposition compared to a single application e.g. of the tank mix. } The present invention overcomes the problem of multiple application by proposing that, if an insecticide were microencapsulated or otherwise administered in a non- immediate release formulation and the PBO or other esterase inhibitor not so, then a single application would suffice. The PBO would immediately begin to act on the esterases and, after a given period, the micro-encapsulation would break down and release the conventional insecticide. By this time, the resistance-associated . enzymes would be inhibited, and thus the resistance mechanism overcome. . Various formulations involving both a synergist, such as PBO, and an insecticide, - 5 such as a pyrethroid are known.

European patent specification no. 238 184 relates to the use of a microencapsulated pesticide and a non-micro-encapsulated pesticide, wherein the two pesticides are preferably the same, eg permethrin. European patent specification no. 427 991 discloses a mixture of a microencapsulated organophosphorous and/or carbamate pesticide with a flowable phase comprising a pyrethroid pesticide. Both of these specifications suggest the use of the formulation for kill-knock down combined action, as does the German patent specification no. 2411 373, which discloses a partly micro encapsulated formulation of a pyrethroid, optionally containing a synergist. The entire text of all three of these earlier patent applications is hereby incorporated by reference. However, none of these formulations relates to one suitable for the purposes of this invention, namely to reduce or prevent pesticide resistance by enabling an esterase inhibitor to come into contact with the pest first, followed by the pesticide, in a single application.

Accordingly, the present invention provides a method for preventing or reducing resistance to a pesticide by a pest, which method comprises the administration to the crop, other substrate or the pest of a composition comprising: (a) a rapid-release formulation of an inhibitor of a factor causing or contributing to the resistance of the pest to the pesticide; and, substantially simultaneously, (b) a non-rapid-release formulation of the pesticide.

Furthermore, the present invention provides a composition, suitable for use in such a method, which composition comprises: oo 30 (a) a rapid-release formulation of an inhibitor of a factor causing or contributing to the resistance of the pest to the pesticide; and, substantially simultaneously, i (b) a non-rapid-refease formulation of the pesticide.

Preferably, the rapid release formulation and the sustained release formulation are comprised in the composition in physical admixture. However, the formulations (a) and (b) may be administered separately. By "substantially simultaneously’ herein is meant that the formulations are brought into contact with the substrate and/or the } pest at about the same time, avoiding the need to revisit the site of the substrate and/or pest in order to apply the second of the two formulations. Both formulations } would thereby come into contact with the substrate and/or the pest within the order - 5 of seconds, preferably within 10 seconds and more preferably, within one or two seconds, of each other rather than in the order of minutes or longer. Preferably, the formulations (a) and (b) are administered simultaneously.

The rapid release formulation is suitably any standard pesticide formulation known to those skilled in the art or yet to be discovered and suitable for the purpose. Such formulations include, for example, wettable powders, granulates, emulsifiable concentrates and ultra-low volume formulations to which water can be added to form an emuision, a suspension and the like. Preferably, the rapid release formulation, comprising PBO or other metabolic enzyme inhibitor, is in the form of an emulsifiable concentrate. It will be appreciated that the preferred enzyme inhibitor, or combination of enzyme inhibitors, will be selected on the basis of which pesticide compound or compounds are being employed against a specific pest.

The non-rapid release formulation is suitably any non-immediate release formulation known in the art or yet to be discovered, such as sustained, controlled or slow release formulations suitable for the purpose. Preferably, the non-rapid release formulation is one that prevents an effective dose of the pesticide from being released or coming into effective contact with the pest or its target in the pest until the esterase inhibitor, or inhibitor of another factor causing or contributing to pesticide resistance, has at least begun its inhibiting effect on its target in the pest.

Suitably, the non-rapid release formulation prevents release of the pesticide or contact thereof with the pest or the substrate for at least 30 after application of the composition. Such formulations include, for example, the pesticide encapsulated in a degradable capsule and preferably comprise micro-encapsulation technology. ] 30 One such example of a surface spray encapsulating a pyrethroid insecticide is

Karate Zeon [trademark] (lambda-cyhalothrin). The optimal time delay for release of the pesticide will be determined by a number of factors and will require experimentation to determine the time/response profile of the inhibitor(s) selected.

A non-release formulation which corresponds with this profile will then be selected/developed. :

Suitable micro-encapsulation formulations include those analogous to those . described in the aforementioned European and German patent specifications but : adapted so as to microencapsulate the insecticide (eg pyrethroid) and not the - . metabolic enzyme inhibitor (eg esterase inhibitor, eg PBO). - 5

The pesticide itself is suitably any that is capable of acting as such and to which resistance has been identified amongst the, or some of the, pest(s) against which it is otherwise active. Examples of suitable pesticides that may comprise the active ingredients of component (b) of the composition therefore include pyrethroids, organo-phosphates and carbamates. Preferably, the pesticide is a pyrethroid, such as fenvalerate, s-fenvalerate, cypermethrin (both alpha and zeta forms), bifenthrin, deltamethin and beta-cyfluthrin. It will be appreciated that new pesticides and new classes of pesticides are discovered from time to time, and that resistance to pesticides can develop over time. It is intended that the principles of this invention, and the inventive concepts therein, can be applied to a wide range of pesticides, both known and those yet to be discovered, as and when resistance is identified.

Component (b) therefore preferably comprises an amount equivalent to a standard dosage of the pesticide. For example, in the case of beta-cyfluthrin for pesticidal activity against Helicoverpa, a typical dose comprises 8g/L of an ultra-low volume or 25g/L of an emulsifiable formulation; and for alpha-cypermethrin a typical dose comprises 16g/L of an ultra-low volume or 100g/L of an emulsifiable formulation.

The inhibitor is suitably any that is capable of preventing or reducing resistance of the pest(s) to the pesticide. Suitable inhibitors therefore include esterase inhibitors, microsomal oxidase inhibitors and glutathione S-transferase inhibitors. Preferably, the inhibitor is an esterase inhibitor, such as PBO, ethion, profenofos and dimethoate.

Co . 30 Component (a) preferably comprises an amount of the inhibitor sufficient to prevent or reduce the resistance of the pest(s) to the pesticide and will depend on pest size (eg a white fly needs a lot less inhibitor than an H. armigera grub), degree of ester- mediated resistance efc, but is determinable by those skilled in the art.

The pest(s) against which the composition of the invention is/are directed can be any which are known to offer at least some resistance to a pesticide and which it is considered necessary to disable and/or kill. Examples include those that attack or . damage or otherwise reduce the commercial or other value of a substrate, such as : crops, particularly arable crops, such as food-and material crops including cotton. } Other pests include those that are a nuisance to or an adversary of other living : S organisms, including mammals, such as humans.

Accordingly, the pest(s) may include one or more of Helicoverpa armigera,

Helicoverpa punctigera, Heliothis virescens, Aphis gossypii, | Myzus persicae, P. includens, W. cervinata, Bemisia tabaci and mosquito species.

By way of example, the cotton bollworm Helicoverpa armigera and B-biotype B. tabaci (Poinsettia or silverleaf whitefly) are major crop pests worldwide. Extreme insecticide resistance exacerbates the pest status of these insects. Pyrethroid and other resistances in Australian H. armigera and B-biotype B. tabaci are caused by an over production of esterase isoenzymes which sequester and metabolise insecticides.

For administration to the substrate, any method known in the art for application of a pesticide or the like to a substrate may be used and may depend upon factors such as the particular substrate (eg crop), target pest stage of the crop and the like.

Examples of such methods include spraying by ground or aerial application. For administration to crops, particularly over vast areas such as the Australian cotton fields, it is preferred to spray a composition comprising a suspension or emulsion of the components (a) and (b) in water, optionally also comprising a surfactant or other excipients, (although PBO itself can act as a surfactant) or an ultra-low volume (omitting the water) composition, supplied in a tank, such as one adapted to be transported by aircraft or, for example as in the case of whitefly sprays, by ground rig (such as tractor, tank or boom spray).

The rate of administration of the compositions according to the invention will accord with known or approved (registered) rates of the active ingredients of each of the formulations (a) and (b). For example, for H. armigera, the registered rate in

Australia for PBO is in the range of from 250 -~360 g a.i./ha and for a pyrethroid rates is in the range of from about 12 - 80 g a.i./ha.

The present invention therefore further provides:

(a) the use of a composition according to the invention in the treatment or : prevention of pesticide resistance; : (b) the use of a composition according to the invention in the treatment or . : prevention of damage to or destruction of a substrate by a pest; ’ 5 (c) the use of a composition according to the invention in pest control; and (d) a method for preparing a composition according to the invention, which method comprises bringing the components (a) and (b) into physical admixture.

The present invention will now be illustrated by the following Examples.

Brief Description of the Figures

The invention will now be described, by way of example only, with reference to the following Figures wherein:-

Figure 1 shows percentage H. armigera esterase activity (expressed as % of control, + standard deviation) remaining at fixed periods following topical application of 1ul of 1% PBO;

Figure 2 shows percentage B-type B. tabaci (Australian) esterase activity (expressed as % of control, + standard deviation) remaining at fixed periods following exposure to 0.1% PBO;

Figure 3 & 4 show comparison of percentage esterase inhibition by H. armigera larvae using PBO pre-treatment, fenvalerate and zeta-cypermethrin;

Figure 5 shows rate of onset of symptoms of pyrethroid poisoning (in daylight and } night) in 3" instar pyrethroid susceptible H.armigera larvae. Larvae were treated, by topical application, with a discriminating dose of lambdacyhalothrin using either

Co 30 Karate or Karate Zeon; x Figure 6 shows toxicity, using a topical application bioassay, of Karate EC and

Karate Zeon and mixtures of piperonyl butoxide (1%) and Karate EC and Karate

Zeon to pyrethroid susceptible and resistant (80 fold resistant to lambdacyhalothrin) 3" instar H.armigera. Insecticides were applied to H.armigera at night (under red light) and bioassays were left overnight in darkness;

g . . Figure 7 shows toxicity, using a leaf dip bioassay, of Karate EC and Karate Zeon : and mixtures of piperonyl butoxide (1%) and Karate EC and Karate Zeon to aduit . : pyrethroid susceptible native B.tabaci and resistant (2000 fold resistant to ’ 5 lambdacyhalothrin) B-biotype B.tabaci. Insecticides were applied to B.tabaci at night (under red light) and bioassays were left overnight in darkness;

Figure 8 shows field control on cotton of pyrethroid resistant, second instar

H.armigera (80 fold to lambdacyhalothrin), using registered rates of delayed release

Karate Zeon and immediate release Karate EC, and mixtures of Karate Zeon and

Karate EC with PBO. Error bars represent standard deviations. (Rates of insecticide applied were: PBO 320g a.i./ha, and lambdacyhalothrin 15g a.i./ha);

Figure 9 shows field control on cotton of pyrethroid resistant, B-biotype B.tabaci adults, using sprays of registered rates of delayed release Karate Zeon and immediate release Karate EC, and mixtures of Karate Zeon and Karate EC with piperonyl butoxide). Sprays were applied to cotton under heavily overcast light conditions. Error bars represent standard deviations. (Rates of insecticide applied were: PBO 320g a.i./ha, and lambdacyhalothrin 15g a.i./ha).

EXAMPLES

General Methods & Materials

Esterase activity was determined by measuring the rate of hydrolysis of the model substrate, 1-naphthyl acetate, by carboxylesterases present in organo-phosphate resistant insects such as H. armigera, or the hydrolysis of 1-naphthyl buturate for B. } tabacii. Such hydrolysis will result in a characteristic yellow/brown colour after complex with FBRR (fast blue RR salt) with absorbance at 450nm, which is . 30 measured to determine the reaction rate. FBRR (0.6% of final solution), was dissolved in pH6.0, 0.2M phosphate buffer (0.5L), then 1.86% 1-naphthyl acetate or 1-naphthyl butyrate was added.

Kinetic assays were performed using a Bio-Rad 3550 micro plate reader (Bio-Rad

Laboratories, UK using Kinetic Collector 2.0 software run on a Mackintosh SE micro- computer), taking absorbance readings at 450nm automatically at 14-second intervals for 10 minutes. The rate was calculated by the online computer as the } slope of the fitted regression line, using an absorbance limit of 2.0; readings are : given in milli-OD (unit of optical density). : 5 Insecticides used were technical grade: fenvalerate (98%, Shell) (R-(R*,S*))-4- chioro-a(1-methylethyl)benzene acetic acid, cyano (3-phenoxyphenyl) methyl ester); cypermethrin ((R.S)-alpha-cyano-3-phenoxybenzyl-(1RS)-cis,trans-3-(2,2-dichloro- vinyl)-S,S-dimethylcyclopropane-carboxylate); and zeta-cypermethrin ((S)-cyano(3- phenoxyphenyl)-methyl(+)-cis-trans-3-(2,2,-dichloroethenyl)-2,2-dimethylcyclo- 10 propanecarboxylate) (85%, FMC). The insecticide synergist, piperony! butoxide (96% pure, technical grade), and an 800g/l emulsifiable concentrate formulation of this chemical (PBEC80) were supplied by Endura Spa, Bologna, Italy.

Example 1: _PBO inhibits H. armigera esterases

Kinetic assays confirmed that esterase activity was inhibited by the insecticide synergist, PBO, over a 24-hour period (Figure 1), providing evidence that PBO inhibits H. armigera esterases. In addition, kinetic assays illustrate that esterase inhibition by PBO does not occur immediately after dosage, but occurs with maximum enzyme inhibition from 3 to 4 hours after (70 to 72% esterase activity inhibition). Generally, esterases begin to gradually recover until full esterase activity is present at 24hrs. However, it should be noted that percentage esterase of control remains at less than 50 % between 2 and 11hrs.

Example 2: PBO inhibits B-type B. tabaci esterases

Kinetic assays also showed that PBO inhibits B-type B. fabaci esterases over a 26- hour period. After an initial rapid inhibition of esterases (by 1 hour), there is a gradual decrease to maximum esterase inhibition (36% of control at 11 hours), prior . 30 to a gradual recovery in esterase activity with full esterase activity witnessed, 30 hours after initial PBO exposure (Figure 2). Percentage activity of the control remains at less than 50 % between 7.5 and 17 hours and, overall, esterases suffer some degree of inhibition between 1 and 26 hours.

: Example 3: PBO increases pyrethroid mortality ) Synergism studies confirmed that PBO increases pyrethroid mortality (Figures 3 & 4). These involved a comparison of esterase inhibition (expressed as % of control, + standard deviation) incurred by H. armigera larvae over time following topical application of 1pi PBO (1%), and the effect on mortality of pyrethroid-resistant larvae when exposed to increasing PBO (1ul,1% PBO/larva) pre-treatment intervals before fenvalerate (1ul, 0.125% fenvalerate/larva, Figure 3) and zeta-cypermethrin (1ul, 0.01% zeta-cypermethrin/larva, Figure 4) exposure. Effects were more pronounced with zeta-cypermethrin. There is a highly significant (p<0.01) increase in mortality, until a plateau (100% mortality) is reached (4 — 5hrs for fenvalerate, and 4 — 10hrs for zeta-cypermehrin); thereafter, the synergistic effects decline. This trend corresponds with earlier findings, where esterases are inhibited to a high degree (more than 50% reduction in esterase activity) between 4 and 18 hours.

Example 4: Composition

The following ingredients may be mixed together in water to form a composition suitable for application from ground or air at standard rates for the pyrethroid:

Formulation (a): 800g/L. PBO (PBO EC formulation)

Formulation (b): 250g/L Lambda-cyhalothrin (in the form of Karate-Zeon (trademark); (Karate-Zeon is 250g/L a.i.)

Example 5: Laboratory studies with H.armigera

Introduction ] 30 : Laboratory bioassays on pyrethroid resistant and susceptible H. armigera were conducted in darkness to delay the release of pyrethroid from microencapsulation using Karate Zeon®.

Karate Zeon® is a microencapsulated formulation of the pyrethroid } lambdacyhalothrin and is the only encapsulated insecticide on the Australian field crop market. Developed to increase operator safety, this formulation provides a : delayed lambdacyhalothrin release (in sunlight after mixing with water), of approximately 30 minutes, Release of the microcapsule contents is partially triggered by sunlight. A 30-minute delay -in pyrethroid release is, however, insufficient to allow the maximal synergist action needed for control of resistant insects. Nonetheless, pyrethroid release in Karate Zeon®, can be delayed beyond 30 min by reducing light conditions.

To demonstrate proof of the concept of control of insecticide resistance, using a simultaneous application of a synergist and a delayed release insecticide, we used

Karate Zeon® and artificially delayed pyrethroid release from encapsulation by using the insecticide in darkness. However, the technology required to prepare delayed release insecticide formulations with a longer time delay to release is known to those skilled in the art. Thus, microencapsulation techniques may be applied and adapted to give the desired time delay with a specific insecticide.

General Methods

H. armigera populations used were: pyrethroid susceptible strain and a pyrethroid selected, resistant strain (approximately 80 fold resistant to lambdacyhalothrin).

Third instar pyrethroid resistant. and susceptible H. armigera larvae were treated with the insecticide synergist piperonyl butoxide (PBO) and two formulations of lambdacyhalthrin Insecticides used were: piperonyl butoxide (800g/L ai), non- encapsulated Karate EC® (50g/L ai). microencapsulated Karate Zeon® (250 g/L ai).

Insecticides were serially diluted in water. Insecticides were applied topically to larvae, using a standard, Helicoverpa bioassay procedure (Gunning et al, 1984).

Experiments were conducted at 25°C. Mortality was assesses after 24 h . Control groups were treated with water or PBO and there was no control mortality. Full dosage mortality curves were plotted. Data were analysed by probit analysis.

Proof of delay of pyrethroid release in darkness

Pyrethroids are neurotoxins affecting the insect peripheral nervous system and symptoms of poisoning in H. armigera are well known Gunning, R.V. (Bioassay for detecting pyrethroid nerve insensitivity in Australian Helicoverpa armigera, Journal of Economic Entomology, 89:816 — 819, 1996). Time of delay of pyrethroid release .. was estimated (using treatments of Karate EC and Zeon Karate), by recording time to first onset of pyrethroid ‘poisoning symptoms in pyrethroid susceptible H. - armigera. Larvae were treated with a dose known to kill 100% of susceptible H. armigera larvae, both in strong daylight and in darkness. Three replicates of 30 insects were dosed for each treatment. Night observations of larvae were made under red light (insects cannot see red light).

Results (Figure 5) show that poisoning symptoms developed in H. armigera treated with non-encapsuiated Karate EC in approximately 30 minutes, both in daylight and darkness. Using encapsulated Karate Zeon, poisoning symptoms developed in approximately one hour in daylight, while dark conditions delayed the onset of poisoning symptoms until 4.5 h. Thus, use of Karate Zeon in darkness delayed pyrethroid release from its microencapsulation by approximately 3.5h.

Night Bioassays with pyrethroid resistant H. armigera

Karate EC and Karate Zeon were serially diluted in water to form a range of concentrations for bioassay (0.005 — 10 pg lambdacyhalothrin/pl). Groups of pyrethroid resistant or susceptible larvae (n=30) received the following insecticide treatments under red light and were held in darkness:

Susceptible strain

Karate EC, Karate Zeon .

Resistant strain

Karate, Karate EC + PBO, Karate Zeon. Karate Zeon + PBO. Each insect received a dose of 10 ug of PBO

Treatment : LDsp (ng/larva) Resistance

DN hs il nl P=

ELT CN LC I LL CE

EC EL I Le CER

LC RE LC RN L.A LA

Rasen [1 ow |os-om le

Table 1 Probit analysis of response of pyrethroid resistant and susceptible H. armigera to night bioassays of formulations of lambdacyhalothrin and piperonyl butoxide

Bioassay results are shown in Table 1 and Figure 6. The toxicities of Karate EC and

Karate Zeon to susceptible larvae were not significantly different. Toxicities of

Karate EC and Karate Zeon to resistant H. armigera were also indistinguishable (46 fold resistance factor). PBO and delayed release Karate Zeon completely overcame resistance (RF =1), while a PBO and Karate EC reduced the level of resistance to 25 fold.

Conclusions

H. armigera treated with PBO and delayed release Karate Zeon became effectively - susceptible to fambdacyhalothrin with complete suppression of resistance. Night use i of Karate EC + PBO incompletely suppressed resistance, further emphasising that, in order to control resistant insects, a delay between PBO application and pyrethroid release is necessary for optimal esterase inhibition by PBO.

Example 6: Laboratory Studies with B-biotype bemisla tabaci

Introduction

Laboratory bioassays on pyrethroid resistant and susceptible B-biotype B. tabaci were conducted in darkness to defay the release of pyrethroid from microencapsulation using Karate Zeon®.

General Methods

Pyrethroid susceptible (Northern Australian native B. tabaci) and resistant B-biotype

B. tabaci adults (~2000 resistant fold to lambdacyhalothrin) were treated with formulated insecticide synergist piperonyl butoxide and two formulations of lambdacyhalothrin (non-encapsulated Karate EC® and microencapsulated Karate

Zeon®). Insecticides used were piperonyl butoxide 800g/L EC, Karate EC (50g/L

EC) and Karate Zeon (250g/L.). . Formulated lambdacyhalothrin was serially diluted in water to form a number of test concentrations (0.1 ~ 10000 ppm lambdacyhalothrin). A standard leaf dip bioassay technique for adult whiteflies was used (Cahill 1995). Cotton leaf discs were dipped in lambdacyhalothrin concentrations in a mixture containing 1% PBO. The leaves were dried and placed on an agar bed in petri dishes. Adult whiteflies were added } and the peri dishes sealed. Bioassays were conducted at night at 25°C. Water dipped and PBO controls were performed. Mortality was assessed and corrected : for control mortality (which did not exceed 5%) Full dose response curves were plotted and data analysed by probit analysis.

Results

Dosage mortality data are shown in Table 2 and Figure 7. Data from susceptible B. tabaci show no differences between the toxicity of Karate EC and Karate Zeon.

There were also no differences between the toxicity of Karate EC and Karate Zeon to resistant B. tabaci (RF ~ 140). Pyrethroid resistant B.tabaci treated with PBO and delayed release Karate Zeon were indistinguishable from susceptible strains in response to lambdacyhalothrin (RF = 1), while treatment with PBO and Karate EC reduced resistance to lambdacyhalothrin somewhat (RF = 52). [Trstocat [slope | Uo ppm | Filial lis | Resim fcr _

Seiemec Joo [ow fom-em

Sefermezen [30 [om oman

R¥adelC Jom Jers wows we

R. Karate Zeon + | 2.96 0.48 -0.70 1

EE in

Table 2 Probit analysis of the response of pyrethroid resistant and susceptible H. armigera to night bioassays of formulations of lambdacyhalothrin and piperonyi butoxide

Conclusions i B. tabaci treated with PBO and delayed release Karate Zeon became effectively susceptible to lambdacyhalothrin with complete suppression of resistance. Night use of Karate EC + PBO incompletely suppressed resistance, further emphasising that, in order to control resistant insects, a delay between PBO application pyrethroid release is necessary for optimal esterase inhibition by PBO.

Example 7: Field Studies with a Synergist and delayed release pyrethroid on . cotton against h.armigera — night sprays : Introduction ) 5

The H. armigera laboratory studies described above were followed up with a small- scale replicated field trial on conventional cotton at the Australian Cotton Research

Institute at Narrabri, NSW, February 2003. :

Trial method

In the lack of H. armigera pressure on cotton, pyrethroid resistant second instar H. armigera larvae, which were the progeny a field strain originating from Queensland, were placed on cotton plant. The strain was 20 fold resistant to lambdacyhalothrin.

Insecticides used were piperonyl butoxide 800g/L a.i EC, Karate EC (50g/L EC a.) and Karate Zeon (250g/L a.i). Insecticides were mixed with water. Insecticides were sprayed at registered rates on cofton of PBO 320g a.i. /ha, and lambdacyhalthrin 15g a.ifha, using a calibrated hand held boom spray Treatments were: an untreated control, PBO control, Karate EC, Karate Zeon and Karate EC and Karate Zeon mixed with PBO.

The trial was conducted on replicated, 1 row x 2m plots. Each plot contained from 13 — 17 mature cotton plants. There was an unsprayed buffer of two rows between each plot. Just prior to sunset, ten second instar H. armigera larvae were placed onto the terminals of each plant in the test plots and the plots sprayed. H. armigera numbers per plant were assessed one day after treatment. Temperature ranged from 24 — 26°C. The mean percentage mortality and standard deviation were calculated for each treatment. There was no mortality in the control.

Results

Results (Figure 8) show that lambdacyhalothrin controlled ~ 40% of H. armigera irrespective of formulation, treatment with a PBO + Karate EC mix did not give significantly increase mortality. However, PBO mixed with Karate Zeon gave greater than 90% mortality of resistant insects, indicating almost complete suppression of resistance. These consistent with the results of the laboratory bioassays.

i Conclusions

These field data demonstrate that the synergist piperonyl butoxide, when applied simultaneously with a delayed release pyrethroid, provided effective field control of pyrethroid resistant H. armigera. Delayed release of pyrethroid allowed time for the synergist to inhibit resistance-associated esterases, providing 100% greater control of pyrethroid resistant H. armigera than PBO mixed with a non-encapsulated Karate

EC.

Example 8: Field Studies with a Synergist and delayed release pyrethroid on : cotton against B-biotype b.tabaci — reduced light conditions

Introduction

The B. tabaci laboratory studies described above, were followed up with a small- scale, replicated field trial on conventional, commercial cotton at Emerald, Qid,

February 2003. Since rain and high wind prevented any night spraying, insecticides were applied in the morning and the trial conducted under greatly reduced light (heavily overcast, low cloud and rain showers), compared to normal daylight conditions.

Trial method

The frial was conducted on mature cotton with low B. tabaci pressure (~ 2 whitflies/terminal). Whiteflies were approximately 100 fold resistant to lambdacyhalothrin .

Insecticides used were piperonyl butoxide 800g/L a.i EC, Karate EC (50g/L EC a.i) and Karate Zeon (250g/L a.i). Insecticides were mixed with water and sprayed at registered rates on cotton (PBO 320g a.i. /ha, and lambdacyhaltthrin 15g a.i/ha) using a calibrated hand held boom spray. Treatments were: an unsprayed control,

PBO control, and both Karate EC and Karate Zeon mixed with PBO.

The trial was conducted on replicated, 1 row x 10m plots. There was an unsprayed buffer of two rows between each plot. Whitefly numbers were assessed, by counting adults on each terminal, prior to spraying. The plots were sprayed under reduced ambient light conditions and adult whitefly numbers were assessed one day : after treatment. Temperature ranged from 28 — 34°C and relative humidity was 80- 100%. Mean numbers of whiteflies/ terminal and standard deviations were - calculated for each treatment. . 5

Results :

Trial data are shown in Figure 9. One day after treatment, there was no detectable mortality in untreated, or PBO controls. Differences between whitefly control and lambdacyhalothrin formulation were highly significant. Karate EC mixed with PBO provided some 25% control of whiteflies, while Karate Zeon + PBO gave virtually complete control indicating complete suppression of resistance. Results are consistent with the laboratory bioassay data.

Conclusions

Trial results indicate that very subdued daylight delayed the release of lambdacyhalothrin from encapsulation. There was a sufficient delay between the application of PBO and pyrethroid reiease from microencapsuiation, to aflow adequate inhibition of esterases by PBO prior to pyrethroid release. Therefore application of a synergist and a delayed release insecticide controlled highly pyrethroid resistant B-biotype B. tabaci in the field.

Claims

19 RY } Claims: )

. 1. A method for preventing or reducing resistance to a pesticide of a substrate . pest, which method comprises the administration to the substrate or the pest of a ] 5 composition comprising: (a) a rapid-release formulation of an inhibitor of a factor causing or contributing to the resistance of the pest to the pesticide; and, substantially simultaneously, (b) a non-rapid release formulation of the pesticide.

2. A method according to Claim 1 wherein component (a) and component (b) are comprised in the composition as an admixture.

3. A method according to Claim 1 wherein component (a) and component (b) are administered separately.

4. A method according to Claim 3 wherein both components of the composition are administered to the substrate or the pest within 10 seconds of each other.

5. A method according to Claim 3 or Claim 4 wherein both components are administered to the substrate or the pest within one or two seconds of each other.

6. A method according to-any previous claim wherein the rapid release formulation of component (a) is a standard pesticide formulation which includes at least one formulation selected from the group consisting of:- wettable powders; granules; emulsifiable concentrates; and uitra-low volume formulations to which water can be added to form an emulsion, a suspension and the like.

30 .

7. A method according to any preceding claim wherein the rapid release formulation of component (a) comprises a metabolic enzyme inhibitor.

8. A method according to any preceding claim wherein the inhibitor of component (a) includes at least one inhibitor selected from the group comprising:-

esterase inhibitors; mixed function oxidase inhibitors; and glutathione S-transferase . inhibitors. . 9. A method according to any preceding claim wherein the inhibitor of ) 5 component (a) comprises an esterase inhibitor.

10. A method according to Claim 9 wherein the esterase inhibitor includes at least one compound selected from the group consisting of:- §,S,S-tributyl phosphorothionate; O,0,0-tripheny! phosphate; piperonyl butoxide (PBOY); profenofos; ethion; and dimethoate.

11. A method according to Claim 10 wherein the inhibitor of component (a) is ‘piperonyl butoxide (PBO).

12. A method according to any preceding claim wherein the non-rapid release formulation of component (b) comprises a formulation which prevents an effective dose of pesticide from being released or coming into effective contact with the pest or the substrate until the inhibitor of component (a) has begun its inhibiting effect on its target in the pest.

13. A method according to any preceding claim wherein the non-rapid release formulation of component (b) includes at least one of the formulations selected from the group comprising:- sustained release formulations; controlled release formulations; and slow release formulations. . 30

14. A method according to any preceding claim wherein the non-rapid release formulation of component (b) comprises one or more pesticides encapsulated in a degradable capsule.

15. A method according to Claim 14 wherein one or more pesticides are microencapsulated within a degradable capsule.

16. A method according to any preceding claim wherein the pesticide of : component (b) includes at least one compound selected from the group comprising:- : pyrethroids; organo-phosphates; and carbamates. ) . 5

17. A method according to Claim 16 wherein the pesticide of component (b)- includes at least one pyrethroid selected from the group comprising:- fenvalerate: S-fenvalerate; cypermethrin (both alpha and zeta forms); bifenthrin; deltamethin; and beta-cyfluthin.

18. A method according to any preceding claim wherein the inhibitor of component (a) is piperonyl butoxide and the pesticide of component (b) is a pyrethroid.

19. A method of preventing or reducing resistance to a pesticide substantially as herein described.

20. A pesticide composition suitable for use in the method as defined in any of Claims 1 to 19 inclusive, said composition comprising:- (a) a rapid release formulation of an inhibitor of a factor causing or contributing’ : to the resistance of a pest'to a pesticide; and (b) a non-rapid release formulation of the pesticide.

21. A pesticide composition wherein component (a) and component (b) are . formulated as an admixture. : - 30

22. A pesticide composition as claimed in Claim 20 or Claim 21 wherein the rapid release formuiation of component (a) is a standard pesticide formulation ) selected from the group consisting of:- wetitable powders; granules; emulsifiable concentrates; and ultra-low volume formulations to which water can be added to form an emulsion, a suspension and the like.

23. A pesticide composition as claimed in any of Claims 20 to 22 inclusive . wherein the inhibitor of component (a) includes at least one inhibitor selected from : the group comprising:- . 5 esterase inhibitors; mixed function oxidase inhibitors; and glutathione S-transferase inhibitors.

24. A pesticide composition as claimed in Claim 23 wherein the inhibitor of component (a) is an esterase inhibitor.

25. A pesticide composition as claimed in any of Claims 20 to 24 inclusive wherein the pesticide of component (b) includes at least one compound selected from the group comprising:- pyrethuroids; organo-phosphates; and carbamates.

26. A pesticide composition as claimed in any of claims 20 to 26 inclusive

. wherein the inhibitor of component (a) is piperonyl butoxide and the pesticide of component (b) is a pyrethroid.

27. A pesticide composition substantially as herein described.

28. The use of a composition or method as claimed in any of Claims 1 to 27 inclusive in the treatment or prevention of pesticide resistance.

29. The use of a composition or method as claimed in any of Claims 1 to 27 inclusive in the treatment or prevention of damage to or destruction of a substrate by a pest.

30. A method of preparing a pesticide composition according to any of Claims 20 to 27 inclusive, said method comprising bringing component (a) and component (b) into physical admixture.