For nearly forty years, agriculture, public health agencies and the forestry industry have come to rely more and more on synthetic chemicals for protection against wide variety of peats. However, the use of such broad-spectrum chemical pesticides posed several problems, such as environmental contamination, adverse effects on non-target organisms, residues in food and water, accumulation of chemicals in the food chain and development of resistance in insects due to the repeated use of chemical insecticides. Public concern for environmental quality has led to increased emphasis on alternative safe pest management strategies.
For many years there has been considerable interest in using pathogens, particularly viruses and bacteria that cause disease and show toxicity in insects, as alternatives to synthetic chemicals for controlling insect pests and vectors of various animal and human diseases. At present, probably the best understood, and certainly the commercially most successful biological control agent is a gram-positive soil bacterium, Bacillus thuringiensis (B. thuringiensis, Bt)
Many B. thuringiensis strains with different insect host spectra have been identified. Most of them are active against larvae of certain members of the Lepidoptera, but some show toxicity against a few dipteran or coleopteran species. B. thuringiensis has proved to be effective and harmless to the environment owing to its specificity, and therefore, is considered as an alternative to conventional insecticides. Formulations of B. thuringiensis sporecrystal mixtures are commercially available. However, the practical use of B. thuringiensis as a biological insect control agent is greatly limited by its limited host-range and poor efficacy against many pests.
Several agricultural biotechnology companies are currently devoting their major resources to screening soil samples from different environments all over the world in the hope of getting new isolates of B. thuringiensis with increased toxicity and broader or different host range, with very limited success.
Attempts to improve efficacy and host range have failed primarily because so far little effort has been devoted to understanding the molecular basis for the specificity of B. thuringiensis.
B. thuringiensis is known to produce crystalline inclusions during sporulation. When ingested by the larvae of target insects, these crystalline inclusions are solubilized in the larval midgut, releasing one or more crystal proteins (Cry proteins, also referred to as .delta.-endotoxins) of 27 to 140 kD exhibiting highly specific insecticidal activity. Experimental evidence shows that most of these crystal proteins are protoxins that are proteolytically converted into smaller toxic polypeptides in the insect midgut. The "activated" toxin interacts with the midgut epithelium cells of susceptible insects. According to a recent model, the toxins induce the formation of small, non-specific pores (0.5 to 1 nm) in the membrane of susceptible cells, resulting in a net influx of ions and an accompanying inflow of water. As a consequence, the cells swell and lyse.
Many insecticidal proteins, such as those of the Cry type produced by B. thuringiensis, are limited in their usefulness as insecticides because their host range is rather narrow, typically limited to species of the same genus. According to a recent review by H. Hofte and H. R. Whiteley [Microbiological Reviews 53, 242-255 (1989)], nucleotide sequences have been reported in the art for 42 B. thuringiensis crystal protein-encoding genes, of which 14 are clearly distinctly different, whereas the rest are identical or only slightly different and thus represent the same gene including its different variants. Hofte and Whiteley provide a detailed characterization of these genes, and propose a nomenclature and classification scheme for the genes and the encoded crystal proteins based on their structure (nucleotide and deduced amino acid sequences) as well as their host range. The specificity of the crystal proteins of B. thuringiensis is considered to be determined by hypervariable amino acid regions of these proteins, whereas the toxic moiety of these same proteins is thought to be largely the same and defined in highly conserved regions. For example, Hofte and Whiteley, Supra, compared the deduced amino acid sequence of a wide range of B. thuringiensis Cry proteins and show the existence of five highly conserved regions, i.e. regions similar and in many cases of identical amino acid sequence that occur in almost all Cry proteins. Ge et al., Proc. Natl. Acad. Sci. U.S.A. 86, 4037-4041 (1989) in studies aimed at defining the specificity domain of B. thuringiensis Cry proteins, used recombinant DNA techniques to exchange certain sets of conserved and variable regions between two B. thuringiensis Cry protein molecules with markedly different toxicities for larvae of Bombyx mori . Exchanging the toxic moiety (conserved regions between amino acids 90 and 332) of the highly toxic protein for the same region from the protein with low toxicity still yielded a protein of high toxicity.
The hypervariable amino acid regions responsible for the host range/specificity of these insecticidal proteins are thought to bind the proteins to specific protein receptors on the microvillar membrane of insect gut epithelia [Hofmann et al., Proc. Nat Sci. U.S.A. 85, 7844-7848 (1988) Van Rie et al., Science 247, 72-74 (1990)]. Binding the insecticidal protein to the membrane enables the toxic domain of the protein to come in contact with a target protein or form a pore, either of which leads to cell death [Hoefte and Whiteley, Supra]. However, the precise mode-of-action of B. thuringiensis Cry toxins at the molecular level is not yet fully understood.
The insecticidal activity of B. thuringiensis crystal proteins has traditionally been investigated by using crude preparations of spore-crystal mixtures [Burges, H. D. (ed.): "Microbial control of pests and plant diseases 1970-1980" Academic Press, Inc. (London), Ltd., London]. However, the results of these studies are difficult to interpret, especially because many B. thuringiensis strains produce more than one crystal protein simultaneously, therefore, it is difficult to determine the toxicity spectrum and other properties of the individual proteins. Single crystal proteins have so far been obtained by purification from B. thuringiensis [Yamamoto and McLaughlin, Biochem. Biophys, Res. Commun. 103, 414-421 (1982)], or through the introduction and expression of the corresponding genes in heterologous hosts [see references cited in Table 2 of the review article by Hofte and Whatley, Supra].
Attempts have been made to improve the host range of B. thuringiensis strains through the introduction of new crystal protein genes, for example by conjugation of plasmids from other B. thuringiensis strains or through direct transformation of crystal protein genes cloned in a B. thuringiensis replicon [Heierson et al., J. Bacteriol. 169, 1147-1152 (1987)]. Carlton, B. C. [Proceedings of the Conference "Biotechnology, Biological Pesticides and Novel Plant-Pest Resistance for Insect Pest Management" held Jul. 18-20, 1988, organized by Insect Pathology Resource Center, Boyce Thompson Institute for Plant Research at Cornell University, Ithaca, N.Y., U.S.A., pp. 38-431 report the construction of a bifunctional B. thuringiensis strain having activity against Colorado potato beetle and caterpillar insects. The potato beetle activity was generated from a natural isolate of Bt. A strain known to be active against caterpillars was mated with the strain showing potato beetle activity to produce a transconjugant having the desired properties.
Data from in vitro experiments strongly suggest that activated toxins recognize high-affinity binding sites (putative receptors) on the midgut epithelium of susceptible insects only, and that the presence, absence, or modification of these receptors may be an important factor in determining the host range of B. thuringiensis. However, so far this observation has not been utilized in any way in the development of novel bioinsecticides with improved properties and broader host-range. The scarce attempts to improve the host range of B. thuringiensis by using techniques of recombinant DNA technology concentrated on the construction of new B. thuringiensis strains harboring a combination of genes encoding toxins showing activity against different insect hosts.