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). PA1 O,o-dimethyl, .alpha.,.alpha.,.alpha.-trichloro-1-hydroxyethyl phosphonate (referred to as DEP). PA1 .alpha.-methoxy-4H-1,3,2-benzodioxaphosphorin-2-thione (referred to as Salithion. PA1 O,o-dimethyl S-(.alpha.-(ethoxycarbonyl)benzyl)phosphorodithioate (referred to as PAP). PA1 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, "The Protein Crystals of Bacillus thuringiensis Berliner", Can. J. Microb., I, 694-710 (1955); A. M. Heimpel, "A Critical Review of Bacillus thuringiensis var. thuringiensis Berliner and Other Crystalliferous Bacteria", 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.
"Endotoxin" is used by the art to define the toxicity associated with the water-insoluble crystals. "Exotoxin" 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. .beta.-exotoxin) and concludes that exotoxin is effective against insects belonging to some species of the orders "Lepidoptera, Diptera, Hymenoptera, Coleoptera, and Orthoptera". 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., "A Purification and some Properties of an Insecticidal Exotoxin from Bacillus thuringiensis Berliner", 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 0.5 Beet Molasses 0.5 Cottonseed Oil Meal 2.0 Casein 1.0 Corn Steep Liquor 3.33 CaCO.sub.3 0.1 ______________________________________
The medium is adjusted to a pH of about 7.6 with ammonium hydroxide and then sterilized at about 120.degree. 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.degree. 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.degree. 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.sub.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 .beta.-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.