AU680282B2 - Synergistic enhancement of microbial pesticides - Google Patents

Description

i i ;i Regulation 3.2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

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iccirobioal Pyoduc*s P+ L-d Name of Applicant: LUMINIS PTY -LT Actual Inventor: DUDLEY EDWIN PINNOCK Address for Service: Invention title: R K MADDERN ASSOCIATES, 345 King William Street, Adelaide, South Australia, Australia SYNERGISTIC ENHANCEMENT OF MICROBIAL

PESTICIDES

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;r Details of Associated Provisional Application No: PM 1299 dated 17th September 1993 The following statement is a full description of this invention, including the best method of performing it known to us.

1 Microbial pesticides are pesticides which comprise or are derived from microorganisms. Microbial pesticides, such as those based on Bacillus thuringiensis, B. sphaericus or baculoviruses, are well-known. However, some species of pests are not sufficiently susceptible to the pesticides to enable them to be used for effective control. Even within a pest species, some varieties may be relatively susceptible and others relatively resistant to control by a microbial pesticide.

The present invention provides a means whereby the performance of these pesticides may be enhanced, by synergism, with an enzyme inducer. The enzyme inducer should be an inducer of an enzyme of the pest to be controlled, and in particular a metabolic enzyme of that pest. The net effect of the combination of microbial pesticide and synergist is to make the pesticide more potent, or conversely, the pest more susceptible to this means of control.

The synergist may be applied separately or in combination with the pesticide in a variety of ways, e.g. by spraying, dosing, I C dusting, or in baits or granules, such that an effective amount is delivered to the target pest, such as a pest insect (e.g.

L; 'moth, beetle, fly or louse), pest acarid mite or tick) or 6* v pest nematode. In particular, the pest may be a moth, such as Helicoverpa armiaera, a beetle, such as Leptinotarsa i O decemlineata, a fly, such as the blowfly (Lucilia cuprina) or C°Oo"" the housefly (Musca domestica), a mosquito, such as Aedes aegypti, or a louse, such as the sheep body louse (Bovicola iDamalinia) ovis).

These pesticidal compositions comprise: an effective amount of a combination of a microbial pesticide and an enzyme inducer, such as a monooxygenase inducer, e.g. phenobarbital, sodium phenobarbital or other enzyme inducer. The ratio of the microbial pesticide to the enzyme inducer is from about 100,000:1 to 1:1 by weight.

la The synergistic enzyme inducers may affect oxygenases such as cytochrome P450 monooxygenase, oxidases such as mixed-function oxidases, hydrolases, esterases, proteases and other hydrolytic enzymes. The inducers may comprise, for example, butylated hydroxytoluenes, xanthines, phenobarbital or its salts, for example its sodium salt, alkylated benzenes, alkylbenzenes, substituted phenyl-2-propynyl esters, methylene dioxyphenyls, juvenile hormone analogues or polyvinylpyrrolidones.

As an example, the susceptibility of Bovicola (Damalinia) ovis to Bacillus thuringiensis is increased by the addition of a synergistic amount of the cytochrome P450 monooxygenase inducer, sodium phenobarbital. As described in more detail in the following Example, the LC 50 of 9.4,$g/ing diet (8.0-11.0, J fiducial limits) with Bacillus thuringiensis is reduced to 4.9kg/mg diet 95% fiducial limits) when combined with the synergist.

The following Example is presented as illustrative but not restrictive of the present invention.

EXAMPLE

Materials and Methods Preparation of B. thuringiensis. B. thuringiensis (var.

kurstaki) strain WB3S16 was grown in a 130 litre capacity LH fermenter (LH Engineering Pty, Ltd, UK) containing 80 litres of culture medium comprising potato starch (Difco) 10 g/l; nutrient broth (Difco) 8 g/l; and besan flour (Sungrown Products) 5 g/l.

The culture medium was supplemented with MnSO 4 at 10 M concentration and 0.1% Lanquel antifoam (Diamond Shamrock). A 1% inoculum was prepared from a 5 h pre-culture of cells grown in tryptose-phosphate broth (Oxoid) at 300C and shaken at 200 rpm. The seed inoculum was prepared from the K 2 j\ j \j I transfer of spores grown on nutrient agar for 72 h at 30 0 C in the dark. Controlled fermentation conditions of 30°C and pH were maintained constantly throughout growth. The culture was harvested following cell lysis using a Sharples super centrifuge (Sharples Corporation, USA) and the sedimented product was lyophilised and ground into a fine powder.

Bovicola (Damalinia) ovis strain. B. ovis (Manildra strain) were collected from infested sheep by vacuum suction into a collecting vial. Collected lice were maintained at 34 0 C and relative humidity Bioassays. Dose-response assays were carried out using sheep skin diet as described by Drummond et al (1992). Bioassays were 15 performed in Falcon® multi-welled plates using five serial dilutions ranging from 0.016-0.5 mg of B. thuringiensis powder incorporated in 10 mg of diet. Fifty B. ovis were used per B.

thuringiensis concentration. Control assays also used 50 lice which were fed diet only. Mortality was assessed after 24 h at 34°C and 70% R.H.

To determine the effect of monooxygenase induction on the susceptibility of B. ovis to B. thuringiensis, the diet was pretreated with 0.002% sodium phenobarbital. Solutions of sodium phenobarbital (PB) in 70% ethanol were applied to the diet which was then air-dried. Serial dilutions of B.

thuringiensis powder were added to PB-treated diet as described above. Control assays used 50 lice maintained on PB-treated diet without B. thurinciensis.

Three replicate assays were carried out for each B.

thuringiensis concentration and control. LC 50 values were derived by probit analysis of pooled data as described by Finney (1971) after correction for control mortality with Abbott's (1925) formula.

Results Previous experimental work has indicated that B. thuringiensis is significantly more toxic against pyrethroid-resistant B. ovis compared to pyrethroid-susceptible B.ovis. Treatment with PB resulted in a significant increase in the susceptibility of pyrethroid-susceptible B. ovis to B. thurinaiensis (see Figure with a resulting dose response similar to that of pyrethroid-resistant B. ovis not treated with PB (Table 1).

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5 0S 55 0 5 5 55 *S Table 1. Toxicity of B. thurincjiensis against pyrethroid-susceptible and resistant B. ovis a. ovis Dose-response to B. thuringiensis (aLg/mg diet) Treatment and strain LC 50 (95% fiducial limits) Slope(S.E.) Chi 2 Control Pyrethroid-susceptible Pyrethroid-res istant Sodium phenobarbi tal Pyrethroid-susceptible 9.4 (8.0 11.0) 3.3 (2.7 4.1) 4.9 (4.1 5.9) 1.5 (0.18) 1.2 (0.12) 1.3 (0.04) 5.8 3.2 0.4 i References Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18:265-267.

Drummond, J. and Pinnock, D.E. 1992. Toxicity of Bacillus thuringiensis against Damalinia ovis (Phthiraptera: Mallophaga).

J. Invert. Pathol. 60:102-103.

Finney, D.J. 1971. Probit Analysis. Cambridge University Press, Cambridge.

*l While the present invention has been described in terms of preferred embodiments in order to facilitate better understanding of the invention, it should be appreciated that various modifications can be made without departing from the principles of the invention. Therefore, the invention should be understood to include all such modifications within its scope.

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Claims (18)

1. A pesticidal composition comprising an effective.ampunt of a &^nerisf~c combination of a microbial pesticide and a inducer of an enzyme of the pest to be controlled.

2. A pesticidal composition according to Claim 1, wherein the pest is an insect, an acarid or a nematode.

3. A pesticidal composition according to Claim 1 or Cla.m 2, wherein the ratio of the microbial pesticide to the enzyme i inducer is from about 100,000:1 to 1:1 by weight.

4. A pesticidal composition according to any one of Claims 1 to 3, wherein the enzyme inducer is an inducer of an oxygenase, an oxidase, a hydrolase, an esterase, a protease or other S hydrolytic enzyme. A pesticidal composition according to Claim 4, wherein the enzyme inducer is a cytochrome P450 monooxygenase inducer.

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6. A pesticidal composition according to any one of Claims 1 to 3, wherein the enzyme inducer is selected from butylated hydroxytoluenes, xanthines, phenobarbital and its salts, alkylated benzenes, alkylbenzenes, substituted phenyl-2-propynyl esters, methylene dioxyphenyls, juvenile hormone analogues and I polyvinylpyrrdlidones.

7. A pesticidal composition according to any one of Claims 1 to 6, wherein the microbial pesticide is based on Bacillus thurinaiensis, Bacillus sphaericus or a baculovirus.

8. A pesticidal composition according to any one of Claims 1 to 7, substantially as described herein.

9. A method of controlling a pest organism comprising S, S 7 Ace application of an effective amount of a microbial pesticide S r3 cc and a: inducer of an enzyme of the pest to be controlled to the pest organism or the locus thereof.

10. A method according to Claim 9, wherein the pest is an insect, an acarid or a nematode.

11. A method according to Claim 9 or Claim 10, wherein the microbial pesticide and the enzyme inducer are applied in a ratio of from about 100,000:1 to 1:1 by weight.

12. A method according to any one of Claims 9 to 11, wherein the enzyme inducer is an inducer of an oxygenase, an oxidase, a hydrolase, an esterase, a protease or other hydrolytic enzyme.

13. A method according to Claim 12, wherein the enzyme inducer is a cytochrome P450 monooxygenase inducer.

14. A method according to any one of Claims 9 to 11, wherein the enzyme inducer is selected from butylated hydroxytoluenes, xanthines, phenobarbital and its salts, alkylated benzenes, alkylbenzenes, substituted phenyl-2-propynyl esters, methylene dioxyphenyls, juvenile hormone analogues and polyvinylpyrrolidones. S:

15. A method according to any one of Claims 9 to 14, wherein the microbial pesticide is based on Bacillus thuringiensis, Bacillus sphaericus or a baculovirus. S 30

16. A method according to any one of Claims 9 to 15, wherein the t microbial pesticide and the enzyme inducer are applied separately.

17. A method of controlling a pest organism comprising application of a composition according to any one of Claims 1 to 8 toithe pest organism or the locus thereof. 8 K A~

18. A method according to any one of Claims 9 to 17, substantially as described herein. Dated this 16th day of September 1994 LUlninif rPty Ltd- 0)cvobicl Prioc; s By its Patent Attorneys R K MADDERN ASSOCIATES Pfy L-+d) ft-s.. ftft~S ft* ft ft 4 *.ft. *ft St ft*ft* ft St 9* 6956 ft er 65 t ft ft ft. ft ft *Sftft **Oftftft ft 41 4 I ABSTRACT The invention provides a means whereby the performance of microbial pesticides are enhanced by synergism with an enzyme inducer. The invention composition comprises an effective amount of a combination of a microbial pesticide and an inducer of an enzyme of the pest to be controlled. The net effect of the combination of microbial pesticide and synergist is to make the pesticide more potent or conversely, the pest more susceptible to this means of control. 4* *o *6 S jl