CA1259910A - Fatty acid enhancement of bacteria insecticide - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE The activity of the microbial insecticide, Bacillus thuringiensis (B.t.) Berliner has been found to be synergistically enhanced by the addition of unsaturated 18-carbon fatty acids. A synergistic effect with respect to increased larval mortality of the western spruce budworm, Choristoneura occidentalis Freeman was obtained in the C18 unsaturated fatty acid/B.t. treatments. Enhanced mortality was also observed in C18 fatty acid/B.t. treatments of the larval form of the silverspotted tiger moth, Halisidota argentata Packard. The increased insecticidal activity of the entomogenous bacteria, Bacillus thuringiensis (B.t.) Berliner by the addition of unsaturat-ed 18-carbon fatty acids represents a significant breakthrough for the expanded use of this microbial insecticide.

Description

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1. Field of Invention This invention is concerned with the synergistic enhancement of the microbial insecticide, Bacillus thuringiensis (B.t.) Berliner by the addition of unsaturated 18-carbon fatty acids.

2. Information Disclosure Statement Dissatisfaction with the dependence on chemical insecticides for protection of crops against pest insects has led to more serious consideration of biological agents for the regulation of insects. Along with the exploita-tion of parasitic and predaceous species of arthropods, the utility of disease producing micro-organisms as an alternate method for insect control is well documented.
Since its first appearance to the United States market in 1958 (L.A. ~alcon, 1971 Microbial control as a tool in integrated control programs in Biological Control. C.B. Iluffaker (ed.) Plenum Press, London and New York, pp. 346-362), many commercial preparations containing Bacillus thuringiensis (B.t.) have become available worldwide for the control of various lepidopterous and dipterous pests. The use of B.t. is attractive for management of pest insects because it is specific for target insects with little or no adverse impact on the beneficial or non-target arthropod fauna and is not hazardous to humans, mammals or other important animals. As a naturally occurring insect pathogen, B.t. does not pollute the eield habitat and insect pests are not as likely to develop resis-tance or tolerance to it (R.P. Jaques, 19~3 The potentlal of pathogens for pest control. Agric., Ecosystems and Envron., Vol. 10: pp. 101-126). The two major obstacles to grcater use of B.t., as cited in the Stanford Rescarch Institute Study (1977 New innovative pesticides:
an evaluation of incentives and disincentives for commercial development by .

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industry, prepared for United States Environmental Protection ~gency, Office of Pesticide Programs, pp. 174), are low or variable efficacy of con-trol and high cost of product.
c Research has shown that the unsaturated C18 fatty acids or their salts ~C18 y ) are highly to~ic to soft bodied insects including adelgids, aphids, mealybugs, whitefly, pe.lr psylla, rose slugs, etc. (G.S. Puritch, 1978 Symposium on the pharmacological effocts of lipids. AOCS monograph No.
5, 105-112).
~arious attempts have been made in order to improve the B.t. based inseetieide eomposition. United States Patent No. 3,113,066 to Emond teac}les use of a pesticidal oil in combination with B.t. United States Patent No.

3,911,110 to Smirnoff teaches the concurrent use of the enzyme chitinase and B.t. United States Patent No. 3,937,813 to Clark, Jr. discloses a cem-position containing B.t. and N'-(4-chloro-o-tolyl)-N,N-diinethyl formamidine.
United States Patent No. 3,944,664 to ~itagaki et al describes a synergistic aearieidal eomposition comprising the toxin of B.t. and an acaricide selected from l,l-bis-(p-chloropllellyl) ethanol, bis-(p-chlorophenyl) sulfide and bis-(p-chlorophenoxy) methane. United states Patent No. 4,107,294 to Chauthani teaches the eombination of B.t. and 1-(4-clllorophenyl)-3-~2,6-difluoro-benzoyl)-urea.
3. Summary of the Invention During recent investigations, it has been further discovered that eombinations of the unsaturated 18-carbon fatty acids and/or salts were synergistic with respect to insecticidal activity in combination with various formulationS of Bacillus thurillgiensis Berliner. TlliS WliCIUe filldillg pro-vides a solution to one of the major obstacles to the expanded use of B.t.;

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that of low or variable efficacy. By increasing the efficacy of insect control with the addition of a C10 unsaturated fatty acid synergist it ~ay also be possible to reduce the amount of B.t. needed for control and reduce the product cost. Therefore, this finding represents a significant breakthrough for the expanded use of the microbial insecticide, Bacillus thuringiensis (B.t.) Berliner.
The invention thus provides an insecticidal composition comprising:
(a) an insecticidally effective amount of an entomopathogenic bacterium Bacillus thuringiensis (B.t.) Berliner which is insecticidally active against sucking, blting and defoliating insects and (b) an unsaturated fatty acid having 18 carbon atoms or a salt thereof in an amount sufficient to enhance the efficacy of the microbial insecticide.
The invention also provides a method of protecting susceptible plants and animals against biting, sucking and defoliating inse&ts, which method comprises concurrently applying to the insects or habitat thereof, both (a) an entomopathogenic bacterium Bacillus thuringiensis (B.t.) Berliner which is insecticidally active against sucking, biting and defoliating insects (b) an unsaturated fatty acid having 18-carbon atoms or a salt thr~reof in an amount sufficient to enhance the efficacy of the microbial insecticide. Preferably (a) and (b) are applied together as a composition.

4. Detailed Description of Invention The microbial insecticide (a) employed according to the present invention is an entomopathogenic bacterium Bacillus thuringiensis (B.t.) Berliner which is insecticidally active against sucking9 biting and defoliating insects. The microorganisms Bacillus thuringiensis form protein-containing parasporal endotoxin crystals. The entomopathogenic nature of Bacillus thuringiensis is mainly to be attributed to the effect of endotoxin. It is ! {!,~ 3 : `, ~!., .

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known, however, that some strains of Bacillus thuringiensis produce an extra-cellular water soluble exotoxin in addition to the intracellular endotoxin.
The exotoxin is toxic to mammals including human beings, therefore use of Bacillus thuringiensis microbial insecticide containing the exotoxin is banned in North America, Western Europe and in Japan. Where a strain which does not form exotoxin is used for producing the microbial insecticide, the microbial insecticide containing endotoxin crystals and spores may be employed without further purification for the formulation of the present invention. Where a strain which forms exotoxin is used, it is necessary to remove exotoxin pro-duced during the cultivation. Methods for the removal of exotoxin are wellknown in the art. The microbial insecticide used in the present invention ls accordingly free or ~ubstantially free from exotoxin of B.t. Commercially available formulated B.t. products containing crystalline endotoxin and free of exotoxin include Bactospeine ~ (product of Biochem Products, a division of Salsbury Laboratories, Inc., Montchanin, Delaware) which contains 8800 Inter-national Units per milligram (about 1.76% by weight) of the active ingredient of Bacillus thuringiensls Berliner, var. Kurstaki and Thuricide ~ (product of Sandoz, Inc.) which contains 4,000 International Units per milligram (about 0.8~ by weight) of the active ingredient of Bacillus thuringiensis Berliner, var. Kurstaki.
The fatty acid (b) is preferably selected from oleic acid, linoleic acid, their soaps (salts) and mixtures thereof. Linolenic acid or ricinoleic acid may also be used alone or be present in addition to oleic acid and/or linoleic acid. The cation forming the salt or soap with the fatty acid is not particularly critical, but usually is selected from sodium, potassium and ammonium. Other water-soluble soaps, for example, alkaline earth metal soaps and alkanolamine salts are also conceivable.

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. -- 4 ~.~S''`~910 One of the most convenient fatty acid forms is the sodium, potassium or ammonium salt of a Eatty acid mixture whose main ingredients are oleic acid and linoleic acid, because the acid mixture is easily available and the salt form is more soluble in an aqueous medium than the acid form.
In the composition for sale or in the ready-to-use composition, the proportions of the active ingredient of the microbial insecticide (a) to the fatty acid ~b) may range from about 1:1000 to about l:l, preferably from about 1:100 to about 1:1 by wt., more preferably about 1:20 to about 1:1.
The composition for sale may be in any suitable form.
Preferred forms include liquid suspensions, ~i.e., flowable concentr-ates), wettable powders and dry dusts. Ingredients required to form these formulations are well known in the art. In the composition for sale the total amount of the microbial insecticide ~a) and the fatty acid (b) may vary within a wide range. Practically the amount ranges from 1 to 95, preferably from 5 to 50% by wt. The ready-to-use composition may be in the form of a solution, emulsion or dispersion in suitable solvent. In one preferred embodiment an aqueous solution or dispersion is used in which the concentration of insecticide (a) ranges from about 0.01% to 2.0% (preferably 0.1 to 0.6) by wt. of formulated B.t. and the concentration of fatty acid or its salt (b) ranges from about 0.05 to about 5 ~preferably from about 0.25 to about 2%) by wt. The amount of B.t.
in this preferred embodiment, in terms of the active ingredient, may be from about 0.0001% to 0.1%, preferably 0.001 to 0.02%.
The ready-to-use composition may be applied to the insects directly or to habitat thereof, for example, to plants, to soil, to water in which the insects are expected to occur. One particularly preferred embodiment of the method is to apply an aqueous ready-to-use solution containing the essential ingredients of the present invention to plant surfaces on which the insects occur or expected to occur.

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The invsntion includes enhanced and synergistic activity from the combination of insecticidal bacterium (a) with the fatty acid (b).
The following examples are illustrative.
Example 1 Test of unsaturated Clg potassium salt (Salt A = oleate/linoleateJ
54.0%:43.0% the balance being other fatty acids) in combination with the micro-bial insecticide, Bacillus thuringiensis BIerliner for the control of the western spruce budworm, Choristoneura occidentalis Freeman.
Host plant Douglas-fir, Pseudotsuga menziesii (Mirb.) Sample Unit and Procedure -Western spruce budworm larvae, Choristoneura occidentalis Freeman in the 5th and 6th instars were separated into groups containing five larvae per replicate, 5 replicates per treatment and randomly assigned to the following treatments:
(a) Tap water Control (= diluent for treatments) (b) 0.25% Salt A
(c) 0.1% Bactospeine ~ (= 0.00176% active ingredient or 1 ml/l of Bactospeine containing 8,800 International Units of B.t. per milligram [1.76%
active ingredient])*
(d) 0.3% Thuricide ~ (=0.0024% active ingredient or 3 ml/l of Thuricide containing 4,000 International Units of B.t. per milligram 10.8% active ingre-dient])*

(f) 0.25~ + 0.3% Thuricide ~ *
; * Both formulations of Bacillus thuringiensis Berliner were used at 1/2 of the recommended label rates.

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.'1.;~9~3~(3 These solutions were made up fresh before use. The unsaturated fatty acid salts were mad~ by neutralizing a fatty acid mixture of oleic and linoleic (54.0%:43.0~) with potassium hydroxide. The Bacillus thuringiensis (B.t.) were two standard retail formulations; ~actospeine ~ produced by Sa:Lsbury Laboratories Inc. and Thuricide ~ produced by Sandoz Inc.
Caged (20x20x24 cm) larvae and foilage (Douglas-fir) were sprayed with 25 ml of treatment solution applied with 10 ml plastic syringe with a furnace-burner-tip nozzle (Monarch 0.75 GPH: 45AR). Mortality was assessed by counting living and dead larvae 3 and lO days after treatment.

Results Table ]: Observed and corrected mean percent mortality(assessed 10 days post-treatment) of W. spruce budworm, Choristoneura occidentalis Freeman to the treatment solutions, and comparison of means between expected additive values with observed values.
Treatment Observed Abbotts Expected Mean % Corrected Additive Mort. Mean % Mort. Values Tap water 32.0 0 control (=diluent) 0.25~o Salt A 40.0 11.8 0.1% Bactospeine ~ 79.2 69.4 (B-t-) 0.3% Thuricide ~ 75.0 63.2 (_.t.) 0.25% Salt A~+ 0.1% 96.0 94.1 81.2 Bactospeine 0.25% Sal~A + 0.3% 85.7 79.0 75.0 Thuricide~

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The synergistic interaction of Salt A with the B.t. is clearly evident by referring to the data listed in Table 1. The Salt A/B.t. combina-tion gave higher mortality than the sum of the mortalities obtained for Salt A
or B.t. alone, thus they acted synergistically. These combinations (0.25% Salt A ~ 0.1% Bactospeine ~ 0.25% Salt A ~ 0.3% Thuricide ~ provided good control of W. spruce budworm larvae at one~half of the recommended rate of the B.t. alone.
In addition to the above described synergistic activity~ a reduction in the lag time between treatment and effect was observed in the Salt A/B.t.
combinations. Results at the three day post-treatment assessment show enhanced (0.25 Salt A/O.l Bactospeine ~ and synergistic (0.25%/0~3% Thuricide ~
activity with respect to budworm mortality in the Salt A/B.t. combinations versus the sum of Salt A and B.t. mortalities alone (Table 2). Enhanced and synergistic activity at the 3 day post-treatment assessment indicates a reduc-tion in time between treatment and effect in the Salt A/B.t. combinations.
_ _ Table 2: Observed, corrected and expected additive mean percent mortality of W. spruce budworm, Choristoneura occidentalis Freeman 3 days post-treatment.
Treatment 3 Day Assessment ObservedAbbott's Expected Mean % Mort. Corrected Additive Mean % Mort. Values Tap water Control (=diluent) 4.0 0 0.25% Salt A 12.5 8.9 0.1% Bactospeine ~ (B.t.) 39.1 36.6 0.3% Thuricide ~ (_.t.) 13.6 10.0 0.25% Salt A + 0.1% Bactospeine ~ 45.8 43.5 45.5 0.25% Salt A ~ 0.3% Thuricide ~ 36.0 33.3 18.9 . _ _ . _ _ _ _ . . _ _ _ ~ ~ ~ - 8 -~.~5~3~L~

Example 2 Test of unsaturated Clg salts (oleate/linoleate = 54.0%/43.0% for Salt A and 77%/7% for Salt B, the balance in each case being other fatty acids) in combination with the microbial insecticide, Bacillus thuringiensis Berliner for the control of the silverspotted tiger moth, Halisodota argentata packard.
Host Plant Douglas-fir, Pseudotsuga menziesii (Mirb.) Sample Unit and Procedure Silverspotted tiger moth larvae (4th and 5th instars) were placed 50 10 larvae per 1 year old Douglas-fir seedling into cages (23x23x27 cm), with 3 replicates (each containing 50 larvae) per treatment. These were randomly assigned to the following six treatments:
(a) Tap water control (=diluent for treatments) (b) 0.25% Salt B
(c) 0.25% Salt A
(d) 0.6% Thuricide ~ (0.0048% active ingredient or 6 ml/l of Thuricide con-taining 4,000 International Units of B.t. per milligram 10.8% active ingre-dient]) (e) 0.25% Salt B ~ 0.6% Thuricide (f) 0.25% Salt A ~ 0.6% Thuricide ~
Approximately 20 mls of fresh treatment solution was applied per replicate to larvae and foilage using a 10 cc plastic syringe with a furnace-burner-tip sprayer (Monarch 0.75 GPH; 45A~). Mortality was assessed 7 days post-treatment.

` 9 ~L2599~0 able 3: Mortality assessment of silverspotted tier moth larvae, Halisidota ar~entata Packard to treatment solutions. Assessments made 7 days post-treatment. (Treated May 3/83).
Treatments Replicates % Mort. Abbott's ~lort. = X/50 Corrected Mort.

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Tap water control 4 2 6 8 0 0.25 Salt B 35 39 36 73.3 71 0.25 Salt A 30 35 31 64 61 0.6% Thuricide ~ 20 24 13 38 33 0.25 Salt + 0.6%
Thuricide ~ 40 39 46 83.3 81.9 0.25 Salt ~ + 0.6%
Thuricide 40 48 44 88 87 Enhanced insecticidal activity was obtained in the Salt B/B.t.
(81,9%) and Salt A/B,t. (87.0%j co~binations over the mortality obtained from the Salt B (71.0%), Salt A ~61%) and B.t. (33%) alone (Table 3).

Claims (14)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An insecticidal composition comprising, (a) an insecticidally effective amount of an entomopathogenic bacterium Bacillus thuringiensis (B.t.) Berliner which is insecticidally active against sucking, biting and defoliating insects, and (b) an unsaturated fatty acid having 18 carbon atoms or a salt thereof in an amount sufficient to enhance the efficacy of the microbial insecticide.

2. The composition according to claim 1, wherein the componetn (b) is selected from the group consisting of oleic acid, linoleic acid, their soaps and mixtures thereof.

3. The composition according to claim 1, wherein the component (b) is the sodium, potassium or ammonium salt of the fatty acid.

4. The composition according to claim 1, wherein the component (b) is the sodium, potassium or ammonium salt of a fatty acid mixture whose main ingredients are oleic acid and linoleic acid.

5. The composition according to claim 1, 2 or 3, wherein the proportion of (a):(b) by wt. ranges from about 1:100 to about 1:1.

6. The composition according to claim 1, 2 or 3, wherein the proportion of (a):(b) by wt. ranges from about 1:20 to about 1:1.

7. The composition according to claim 1, 2 or 3, which is in the form of an aqueous solution in which the concentration of the insecticide (a) ranges from about 0.01% to 2.0% by wt. or 0.88 to 78 billion viable B.t. spores per gram (BU) and the concentration of the fatty acid or its salt (b) rangtes from 0.05 to about 5% by wt.

8. A method of protecting susceptible plants and animals against biting, sucking and defoliating insects, which method comprises concurrently applying to the insects or habitat thereof both (a) an entomopathogenic bacterium Bacillus thuringiensis (B.t.) Berliner which is insecticidally active against sucking, biting and efoliating insects and (b) an unsaturated fatty acid having 18 carbon atoms or a salt thereof in an amount sufficient to enhance the efficacy of the microbial insecticide.

9. The method according to claim 8, wherein the components (a) and (b) are appoied together as a composition.

10. The method according to claim 8 or 9, wherein the component (b) from the group consisting of oleic acid, linoleic acid, their soaps and mixtures thereof.

11. The method according to claim 8 or 9, wherein the component (b) is the sodium, potassium or ammonium salt of the fatty acid.

12. The method according to claim 8 or 9, wherein the component (b) is the sodium, potassium or ammonium salt of a fatty acid mixture whose main ingredients are oleic acid and linoleic acid.

13. The method according to claim 8 or 9, wherein the proportion of (a):(b) by wt. ranges from about 1:100 to about 1:1.

14. The method according to claim 8 or 9, wherein the proportion of (a):(b) by wt. ranges from about 1:20 to about 1:1.

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