Abstract:
A synergistic insecticidal composition comprises as a first ingredient the β-exotoxin of Bacillus thuringiensis or a metal salt thereof and has a second ingredient of one or more of the following chemical insecticides: 
     O,o-diethyl-O-(2-isopropyl-4-methyl-6-pyrimidyl) thionophosphate, 
     O,o-dimethyl, α,α,α-trichloro-1-hydroxyethyl phosphonate, 
     α-methoxy-4H-1,3,2-benzodioxaphosphorin-2-thione, 
     O,o,-dimethyl S-(α-(ethoxycarbonyl)benzyl) phosphorodithioate, 
     O,o-dimethyl S-(4-chlorophenyl) phosphorothioate 
     The first and second ingredients are used generally in a ratio of about 0.5:1 to 2.0:1 and may be dispersed in a major portion of an agronomically acceptable carrier.

Description:
BACKGROUND OF THE INVENTION 
     Organic phosphorous insecticides have appeared after the Second World War, playing the leading role in the eradication of hazardous insects. However, the disadvantages of these materials, such as development of insect resistance, the considerable toxicity of the material to humans and cattle, and simultaneous eradication of natural enemies, have been noticed in recent years. The most effective and widely used organic phosphorous insecticides are: 
     O,o-diethyl-O-(2-isopropyl-4-methyl-6-pyrimidyl)thionophosphate (referred to as Diazinon). 
     O,o-dimethyl, α,α,α-trichloro-1-hydroxyethyl phosphonate (referred to as DEP). 
     α-methoxy-4H-1,3,2-benzodioxaphosphorin-2-thione (referred to as Salithion. 
     O,o-dimethyl S-(α-(ethoxycarbonyl)benzyl)phosphorodithioate (referred to as PAP). 
     O,o-dimethyl S-(4-chlorophenyl) phosphorothioate (referred to as DMCP). 
     Bacillus thuringiensis, a spore forming microorganism with crystalline parasporal bodies, has been employed commercially as a microbial insecticide for the control of insects such as species of the order Lepidoptera and certain flies and mites. B. thuringiensis and its use as an insect pathogen is described inter alia, in C. L. Hannay and P. Fitz-James, &#34;The Protein Crystals of Bacillus thuringiensis Berliner&#34;, Can. J. Microb., I, 694-710 (1955); A. M. Heimpel, &#34;A Critical Review of Bacillus thuringiensis var. thuringiensis Berliner and Other Crystalliferous Bacteria&#34;, Ann. Rev. Entomology, 12, 287-322, (1967). B. thuringiensis insecticides are quite specific and are harmless to non-susceptible orders of insects, and relatively safe to animals and man. 
     &#34;Endotoxin&#34; is used by the art to define the toxicity associated with the water-insoluble crystals. &#34;Exotoxin&#34; denotes the so-called heat-stable, water-soluble fly toxin produced by Bacillus thuringiensis var. thuringiensis organisms. The water-soluble, heat-stable exotoxin was first reported in 1959 when its toxicity against the larvae of flies was noted. A review of the heat-stable exotoxin is contained in the previously mentioned article by A. M. Heimpel. This article summarizes the activity of the exotoxin (therein referred to as B.t. β-exotoxin) and concludes that exotoxin is effective against insects belonging to some species of the orders &#34;Lepidoptera, Diptera, Hymenoptera, Coleoptera, and Orthoptera&#34;. It is also reported that exotoxin affects insects only at molting or during metamorphosis. 
     The probable chemical structure of Bacillus thuringiensis exotoxin has been elucidated by Bond et al., &#34;A Purification and some Properties of an Insecticidal Exotoxin from Bacillus thuringiensis Berliner&#34;, R. P. M. Bond, C. B. C. Boyce and S. J. French, Biochem. J. (1969), 114, 477-488. 
     The proposed structure is: ##STR1## 
     Various processes are known for the production of exotoxin. All involve the fermentation of a Bacillus thuringiensis variety thuringiensis organism in a medium such as the following: 
     
         ______________________________________Ingredient            Weight (%)______________________________________Cane Molasses         0.5Beet Molasses         0.5Cottonseed Oil Meal   2.0Casein                1.0Corn Steep Liquor     3.33CaCO.sub.3            0.1______________________________________ 
    
     The medium is adjusted to a pH of about 7.6 with ammonium hydroxide and then sterilized at about 120° C. for about 15 minutes. The medium is inoculated with Bacillus thuringiensis var. thuringiensis and the fermentation is conducted for about 24 hours at about 30° C. At the termination of the fermentation the cells in the broth are in the prespore stage of development and not more than about 1% of the total population contained spores. 
     The final whole culture is screened through a 200 mesh screen and the resulting mixture of cells and liquor is concentrated at about 125° F. with a vacuum of about 25 inches of mercury. Final drying and micropulverizing produced a 200 mesh powder which is characterized by a LD 50  of 2.9 mg%. 
     Another process for the production of both the exotoxin and endotoxin of Bacillus thuringiensis is proposed by Drake et al. U.S. Pat. No. 3,087,865. Drake et al. further disclose the precipitation of exotoxin from aqueous supernatant fermentation liquor by addition of calcium chloride. The calcium salt thus produced, as well as corresponding magnesium and barium salts, are disclosed to possess insecticidal activity. 
     Other salts of β-exotoxin which evidence insecticidal activity and may also be used in accordance with this invention are the copper, cadmium, manganese, tin, zinc, lead, cobalt, aluminum and iron salts. 
     DESCRIPTION OF THE INVENTION 
     The present invention proposes to minimize the untoward side effects of organophosphorous insecticides and reserving their prominent insecticidal activity. The present invention is based on unexpected findings of marked synergistic insecticidal activity obtained by the combination of β-exotoxin originated from Bacillus thuringiensis with the organophosphorous chemicals, Diazinon, DEP, Salithion, PAP and DMCP. The combination exerts its pesticidal activity at far smaller concentrations as compared with the dosage required in sole applications of the respective ingredients. In addition, the combined composition thus obtained is effective against insects which have acquired resistance to the organophosphorous insecticidal compounds. 
     The influence on the natural enemies of the target insects is substantially reduced. The synergistic insecticidal composition according to the present invention provides a unique means of eradicating hazardous insects in view of marked insecticidal activity combined with secondary factors such as low toxicity to the man and cattle, and less influence on the natural enemies. 
     Briefly, the compositions of this invention comprise a first ingredient of the group consisting of β-exotoxin and metal salts thereof and a second ingredient of the group consisting of O,O-diethyl-O-(2-isopropyl-4-methyl-6-pyrimidyl)-thionophosphate; O,O-dimethyl, α,α,α-trichloro-1-hydroxyethyl phosphonate; α-methoxy-4H-1,3,2-benzodioxaphosphorin-2-thione; O,O-dimethyl S-(α-(ethoxycarbonyl)benzyl) phosphorodithioate; and O,O-dimethyl S-(4-chlorophenyl) phosphorothioate. The invention also contemplates admixtures of the defined ingredients with an agronomically acceptable carrier, the total active ingredients comprising preferably about 1% to 40% of the admixture of active ingredients and carrier. 
     Exotoxin is known to possess insecticidal activity against the larvae of fly. However, the activity is quite weak or nil against certain other hazardous insects, and it is practically of no use in eradication of parasitic insects. Nevertheless, blending exotoxin with Diazinon, DEP, Salithion, PAP or DMCP results in a tremendous potential and prolonged action of the insecticidal activity. 
     The dosage and concentration of the insecticidal composition vary depending on the modes of application. In the case of powdery moth, green caterpillar, cabbage armyworm, and aphid, 200 to 800 liters per acre of a liquid preparation containing 50-250 ppm of the organophosphorous insecticide, and 100-500 ppm of β-exotoxin gives excellent protection which persists for 1-3 months after spraying. Sole application of Diazinon and the like requires spraying 200-800 liters per acre in a concentration of more than 500 ppm. In the compositions of this invention the proportion of organophosphorous insecticide to exotoxin is in the range of 0.1:1 to 10:1, and most preferably 0.5:1 to 2:1. 
     The insecticidal compositions according to the present invention exert a marked eradicating effect against a variety of hazardous insects and are particularly useful for eradication of those insects which are difficult to treat by organophosphorous insecticides alone. 
     For example, the powdery moth has been observed to increase in vegetable fields. Quick development of insecticide resistance and short life cycle render eradication extremely difficult. However, application of the insecticidal composition according to the present invention provides satisfactory and persisting results. 
     For the eradication of sanitary hazardous insects, a typical example is shown by the cockroach. This insect is found to increase in the urban districts and exhibit marked resistance to the organophosphorous insecticides. Though it is sensitive to chlorine-containing insecticides such as Dieldrin, use of these chlorine-containing insecticides may be limited in the near future in view of the residual toxicity problem. 
     The synergistic insecticidal compositions of this invention provide a key to the solution of this problem. For example, a poison diet containing 0.1% of Diazinon and 0.5% of exotoxin gives rise to an extremely high eradication effect on cockroach. The insecticidal effect is far superior to the diet containing only 0.1% of Diazinon. Furthermore, the insecticidal effect of the preparation containing 0.5% of exotoxin alone is nil. 
     The toxicity of exotoxin against warm blooded animals and fish is extremely low. In addition, the toxicity to insects is obtained via the oral route. Accordingly, the effects on the natural enemies of the hazardous insects are small compared with contact poisons such as BHC. 
     The synergistic effect of this invention does not exist in the case of other types of organophosphorous compounds, including EPN, Parathion, Methyl parathion, and the like. Chlorine-containing insecticides such as DDT, BHC, Endrin, and the like, are also unaffected by exotoxin. 
    
    
     The invention is illustrated by the following examples: 
     EXAMPLE 1 
     Test solutions in an amount of 30 ml. and having the concentrations specified in Table 1 were sprayed onto cabbage cultivated in porcelain pots 12 cm in diameter. After 24 hours, the leaf was cut and placed in a petri dish having a diameter of 12 cm and 10 tobacco cutworm larvae (third stage) were allowed to eat the treated cabbage leaf for three days. This was followed by feeding with a fresh untreated leaf. The mortality after 1, 3, 5, and 9 days was determined. The results of Table 1 are the average of three such experiments. 
     
                                           Table 1__________________________________________________________________________      Concen-      tration      (Active             Mortality after release      ingredient)             of the insectTest Preparation      ppm    1 day                  3 days                      5 days  9 days__________________________________________________________________________Exotoxin alone      1000   0  % 5   % 20  % 65  %       500   0    5     20    60       100   0    0     16    20       50    0    0     5     20Diazinon alone      100    28   44    47    47Diazinon + exotoxin      100 + 100             100  100   100   100Diazinon + exotoxin      100 + 200             100  100   100   100DEP alone  100    9    14    18    32DEP + exotoxin      100 + 100             31   73    95    100DEP + exotoxin      100 + 200             68   90    100   100DEP + exotoxin      100 + 500             100  100   100   100Salithion alone      100    33   33    35    35Salithion + exotoxin      100 + 100             65   95    100   100Salithion + exotoxin      100 + 200             100  100   100   100PAP alone  100    42   53    58    63PAP + exotoxin      100 + 100             100  100   100   100DMCP alone 100    20   20    20    25DMCP + exotoxin      100 + 500             100  100   100   100__________________________________________________________________________ 
    
     EXAMPLE 2 
     Thirty ml. of each test solution at the specified concentrations was sprayed onto cabbage cultivated in a pot. The cabbage leaves were cut off periodically and placed in petri dishes of 9 cm in diameter. Ten young larvae of powdery moth were placed in each dish and the mortality determined after 24 hours. The results of Table 2 are the average of two replications. 
     
                                           Table 2__________________________________________________________________________      Concen-             Mortality      tration      4 days                         8 days                               16 daysTest Preparation      (ppm)  1st day                   after after after__________________________________________________________________________Exotoxin alone      200    10  % 3   % 0   % 0   %Diazinon alone      100    36    5     0     0Diazinon + exotoxin      100 + 200             100   100   100   95DEP alone  100    20    0     0     0DEP + exotoxin      100 + 200             100   100   76    43Salithion alone      100    40    10    0     0Salithion + exotoxin      100 + 200             100   100   100   63PAP alone  100    65    20    0     0PAP + exotoxin      100 + 200             100   100   100   100DMCP alone 100    40    0     0     0DMCP + exotoxin      100 + 200             100   100   100   20ControlEPN        100    50    40    20    0EPN + exotoxin      100 + 200             62    55    43    0Parathion  100    73    30    25    0Parathion + exotoxin      100 + 200             50    60    20    0BHC        500    80    40    45    20BHC + exotoxin      500 + 500             88    50    30    0Non-treatment      --     0     0     0     0__________________________________________________________________________ 
    
     Diazinon, DEP, Salithion, PAP, and DMCP were found to be synergized by blending with exotoxin. A marked potentiation and improvement in residual effect with respect to the insecticidal activity against the young larvae of powdery moth are shown. Noticeable synergistic effect could not be observed upon blending exotoxin with EPN, parathion, BHC and the like. 
     EXAMPLE 3 
     Cabbage fields infested with powdery moth larvae were divided into separate areas of 3.3 square meters. Wettable powders containing 20% of exotoxin, emulsions containing 40% of Diazinon, and wettable powders containing 10% of exotoxin and 20% of Diazinon were diluted with water and sprayed in an amount of 300 ml. per area. The degree of dilution for each insecticide is shown in the table. The test was carried out in 3 replications, and the number of surviving insects was determined immediately prior to spraying and at 2, 6, 11, and 18 days after the spraying. The results are shown in Table 3. 
     
                                           Table 3__________________________________________________________________________             Surviving powdery moth             (mean value for 3 districts) Test Dilution       Concen-             Immediately                     2   6   11  18prepara- degree       tration             before  days                         days                             days                                 days tion times (ppm) spraying                     after                         after                             after                                 after__________________________________________________________________________Exotoxin20%   1,000 200   34.7    20.3                         14.7                             13.6                                 41.4wettablepowderDiazinon40%   2,000 200   24.0    0   18.3                             28.0                                 67.3emulsionExotoxin10%diazinon 1,000 100 + 200             40.3    0   0   1.1 020%emulsionNon-spraying --    --    23.6    18.8                         26.7                             37.0                                 50.4__________________________________________________________________________ 
    
     as seen from the above Table, the combination of exotoxin and Diazinon shows a very noticeable activity for preventing powdery moth larvae and such a combination apparently provides the synergistic activity as compared with the activity in the concentrations of the single uses. 
     EXAMPLE 4 
     In petri dishes of 9 cm diameter and 7 cm height were placed 200 g. of soybean cake medium mixed with the specified concentrations of test insecticide. Thirty third-stage fly larvae were placed in each dish and each dish was covered with gauze. After about 2 weeks, the total number of ecdyzed adult fly insects was determined. Each treatment was performed in 5 replications. The average of the results is shown in Table 4. 
     
                       Table 4______________________________________        Concentration         Number of        in the medium                   Number of  ecdyzedTest Insecticide        (ppm)      larvae tested                              adult insect______________________________________Exotoxin     20         150        61&#34;            10         150        79Diazinon     20         150        63&#34;            10         150        72Diazinon + exotoxin        10 + 10    150         0&#34;            5 + 5      150         0DEP          20         150        43&#34;            10         150        81DEP + exotoxin        10 + 10    150         0&#34;            5 + 5      150         0Salithion    20         150        34&#34;            10         150        58Salithion + exotoxin        10 + 10    150         0&#34;            5 + 5      150         0PAP          20         150        24&#34;            10         150        50PAP + exotoxin        10 + 10    150         0&#34;            5 + 5      150         0DMCP         20         150        43&#34;            10         150        87DMCP + exotoxin        10 + 10    150         0&#34;            5 + 5      150         0ControlParathion    20         150        60&#34;            10         150        101Parathion + exotoxin        10 + 10    150        111&#34;            5 + 5      150        93Methyl parathion        20         150        41&#34;            10         150        93Methyl parathion        10 + 10    150        70+ exotoxin&#34;            5 + 5      150        94EPN          20         150        23&#34;            10         150        64EPN + exotoxin        10 + 10    150        44&#34;            5 + 5      150        58Non-treatment        --         150        99______________________________________ 
    
     EXAMPLE 5 
     The bottom of petri dishes of 9 cm diameter, 7 cm height, was covered with filter paper. Smaller petri dishes of 3 cm diameter and containing 10 ml of test solutions diluted with milk to specified concentration, were placed on the large dishes. Five adult brown winged aphids were released in each dish which was then covered with a cover glass having an opening for air and allowed to stand at 25° C. for five days. Mortality was then determined. Each treatment was done with four replications. Average results are set forth in Table 5. 
     
                       Table 5______________________________________      Concentraton               Number of  Mortality      (%)      insects tested                          after 5 days______________________________________Exotoxin     1.0        20         0    %Diazinon     0.1        20         25Diazinon + exotoxin        0.1 + 0.5  20         95&#34;            0.2 + 0.5  20         100&#34;            0.2 + 0.1  20         100DEP          0.1        20         10DEP + exotoxin        0.1 + 0.5  20         85&#34;            0.2 + 0.5  20         100&#34;            0.2 + 1.0  20         100______________________________________ 
    
     The insecticidal composition of this invention can be prepared in the form of dust, wettable powder, emulsion, granule, or aqueous solution by blending the active ingredients with a suitable carried and, if desired, adding a surfactant, dispersing agent, spreader, or the like. 
     The present invention is further illustrated in detail by the following examples. 
     EXAMPLE 6 
     A. Exotoxin 15%, Diazinon 25%, white carbon 10%, clay 46.5%, sodium lignin sulfonate 1.5%, and sodium alkyl aryl sulfonate 1.5%, were mixed uniformly and pulverized to provide a wettable powder. 
     B. Exotoxin 20%, DEP 10%, methanol 30%, and dimethyl sulfoxide 30% were dissolved in polyoxy-ethylene alkyl ether. 
     C. Exotoxin 2%, Salithion 1%, clay (-300 mesh) 96%, and white carbon 1% were blended uniformly and subsequently pulverized to provide a dust. 
     D. Commercial cornstarch 44.4%, carboxymethyl cellulose 5%, starch 50%, exotoxin 0.5%, and Diazinon 0.1% were blended uniformly, kneaded after addition of water to make a dough, extruded through 5 cm.-holes, and dried and cut into 5 - 10 cm. lengths.