Bacillus thuringiensis toxins and genes for controlling...

Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives

Reexamination Certificate

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C435S252300, C435S419000

Reexamination Certificate




Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Insects of the Order Coleoptera (coleopterans) are an important group of agricultural pests which cause extensive damage to crops each year. There are a number of beetles that cause significant economic damage; examples include Chrysomelid beetles (such as flea beetles and corn rootworms) and Curculionids (such as alfalfa weevils).
Flea beetles include a large number of genera (e.g., Altica, Apphthona, Argopistes, Disonycha, Epitrix, Longitarsus, Prodagricomela, Systena, Psylliodes, and Phyllotreta).
Phyllotreta striolata
includes the striped flea beetle.
Phyllotreta cruciferae
includes the canola flea beetle, the rape flea beetle, and the crucifer flea beetle. Canola, also known as rape, is an oil seed brassica (e.g.,
Brassica campestris, Brassica rapa, Brassica napus
, and
Brassica juncea
Flea beetles include a large number of beetles that feed on the leaves of a number of grasses, cereals, and herbs.
Phyllotreta cruciferae, Phyllotreta striolata
, and
Phyllotreta undulata
, are particularly destructive annual pests that attack the leaves, stems, pods, and root tissues of susceptible plants.
Psylliodes chrysocephala
, a flea beetle, is also a destructive, biennial pest that attacks the stems and leaves of susceptible plants.
Chemical pesticides have provided effective pest control; however, the public has become concerned about contamination of food with residual chemicals and of the environment, including soil, surface water, and ground water. Working with pesticides may also pose hazards to the persons applying them. Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides form the marketplace could limit economical and effective options for controlling costly pests.
In addition, the regular use of pesticides for the control of unwanted organisms can select for resistant strains. This has occurred in many species of economically important insects and other pests. The development of pesticide resistance necessitates a continuing search for new control agents having different modes of action.
Thus, there is an urgent need to identify new methods and compositions for controlling pests, such as the many different types of coleopterans that cause considerable damage to susceptible plants.
Certain strains of the soil microbe
Bacillus thuringiensis
), a Gram-positive, spore-forming bacterinum, can be characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and are specific in their toxic activity. These &dgr;-endotoxins, which are produced by certain
. strains, are synthesized by sporulating cells. Certain types of
. toxins, upon being ingested by a susceptible insect, are transformed into biologically active moieties by the insect gut juice proteases. The primary target is cells of the insect gut epithelium, which are rapidly destroyed by the toxin.
Certain Bacillus toxin genes have been isolated and sequenced. The cloning and expression of a
. crystal protein gene in
Escherichia coli
has been described in the published literature. In addition, with the use of genetic engineering techniques, new approaches for delivering these Bacillus toxins to agricultural environments are under development, including the use of plants genetically engineered with toxin genes for insect resistance and the use of stabilized intact microbial cells as
. endotoxin delivery vehicles. Recombinant DNA-based
. products have been produced and approved for use. Thus, isolated Bacillus toxin genes are becoming commercially valuable.
Until fairly recently, commercial use of
. pesticides has been largely restricted to a narrow range of lepidopteran(caterpillar)pests. Preparations of the spores and crystals of
B. thuringiensis
have been used for many years as commercial insecticides for lepidopteran pests. For example,
B. thuringiensis
HD-1 produces a crystalline &dgr;-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, new subspecies of
. have been identified, and investigators have discovered
. pesticides with specificities for a much broader range of pests. For example, other species of
., namely
, a.k.a.
. M-7, a.k.a.
B.t. san diego
), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively.
Höfte and Whiteley (Höfte, H., H. R. Whiteley [1989
] Microbiological Reviews
52(2):242-255) classified
. crystal protein genes into four major classes. The classes were Cryl (Lepidoptera-specific),CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). CryV and CryVI were proposed to designate a class of toxin genes that are nematode-specific. Other classes of
. genes have now been identified.
The 1989 nomenclature and classification scheme of Höfte and Whiteley for crystal proteins was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover 14 different types of toxin genes which were divided into five major classes. As more toxin genes were discovered, that system started to become unworkable, as genes with similar sequences were found to have significantly different insecticidal specificities. A revised nomenclature scheme has been proposed which is based solely on amino acid identity (Crickmore et al. [1996] Society for Invertebrate Pathology, 29th Annual Meeting, 3rd International Colloquium on
Bacillus thuringiensis
, University of Cordoba, Cordoba, Spain, September 1-6, abstract). The mnemonic “cry” has been retained for all of the toxin genes except cytA and cytB, which remain a separate class. Roman numerals have been exchanged for Arabic numerals in the primary rank, and the parentheses in the tertiary rank have been removed. Many of the original names have been retained, with the noted exceptions, although a number have been reclassified. See also “Revisions of the Nomenclature for the
Bacillus thuringiensis
Pesticidal Crystal Proteins,” N. Crickmore, D. R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean,
Microbiology and Molecular Biology Reviews
(1998) Vol. 62:807-813; and Crickmore, Zeigler, Feitelson, Schnepf, Van Rie, Lereclus, Baum, and Dean, “
Bacillus thuringiensis
toxin nomenclature” (1999) That system uses the freely available software applications CLUSTAL W and PHYLIP. The NEIGHBOR application within the PHYLIP package uses an arithmetic averages (UPGMA) algorithm.
. isolate PS86A1 is disclosed in the following U.S. Pat. No. 4,849,217 (activity against alfalfa weevil); U.S. Pat. No. 5,208,017 (activity against corn rootworm); U.S. Pat. No. 5,286,485 (activity against lepidopterans); and U.S. Pat. No. 5,427,786 (activity against Phyllotreta genera). A gene from PS86A1 was cloned into
. MR506, which is disclosed in U.S. Pat. No. 5,670,365 (activity against nematodes) and PCT international patent application publication No. WO93/04587 (activity against lepidopterans). The sequences of a gene and a Cry6A (CryVIA) toxin from PS86A1 are disclosed in the following U.S. Pat. No. 5,186,934 (activity against Hypera genera); U.S. Pat. No. 5,273,746 (lice); U.S. Pat. Nos. 5,262,158 and 5,424,410 (activity against mites); as well as in PCT international patent application publication No. WO94/23036 (activity against wireworms). U.S. Pat. Nos. 5,262,159 and 5,468,636, disclose PS86A1, the sequence of a gene and toxin therefrom, and a g


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