Toxins active against pests

Details Toxins active against pests Toxins active against pests

2002-03-15

2004-06-22

Graser, Jennifer E. (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C424S185100, C424S190100, C424S236100, C530S350000

Reexamination Certificate

active

06752992

ABSTRACT:

BACKGROUND OF THE INVENTION
The soil microbe
Bacillus thuringiensis
(
B.t.
) is a Gram-positive, spore-forming bacterium. Most strains of
B.t
. do not exhibit pesticidal activity. Some
B.t.
strains produce, and can be characterized by, parasporal crystalline protein inclusions. These “&dgr;-endotoxins” are different from exotoxins, which have a non-specific host range. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain
B.t.
toxin genes have been isolated and sequenced, and recombinant DNA-based
B.t.
products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering
B.t.
toxins to agricultural environments are under development, including the use of plants genetically engineered with
B.t.
toxin genes for insect resistance and the use of stabilized intact microbial cells as
B.t.
toxin delivery vehicles (Gaertner, F. H., L. Kim [1988
] TIBTECH
6:S4-S7). Thus, isolated
B.t.
endotoxin genes are becoming commercially valuable.
Until the last fifteen years, commercial use of
B.t.
pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of
B. thuringiensis
subsp.
kurstaki
have been used for many years as commercial insecticides for lepidopteran pests. For example,
B. thuringiensis
var.
kurstaki
HD-1 produces a crystalline &dgr;-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, investigators have discovered
B.t.
pesticides with specificities for a much broader range of pests. For example, other species of
B.t.,
namely
israelensis
and
morrisoni
(a.k.a.
tenebrionis,
a.k.a.
B.t
. M-7, a.k.a.
B.t. san diego
), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] “Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms,” in
Controlled Delivery of Crop Protection Agents,
R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255.). See also Couch, T. L. (1980) “Mosquito Pathogenicity of
Bacillus thuringiensis
var.
israelensis,” Developments in Industrial Microbiology
22:61-76; and Beegle, C. C. (1978) “Use of Entomogenous Bacteria in Agroecosystems,”
Developments in Industrial Microbiology
20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983)
Z. ang. Ent.
96:500-508 describe
Bacillus thuringiensis
var.
tenebrionis,
which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle,
Leptinotarsa decemlineata,
and
Agelastica alni.
Recently, new subspecies of
B.t.
have been identified, and genes responsible for active &dgr;-endotoxin proteins have been isolated (Höfte, H., H. R. Whiteley [1989
] Microbiological Reviews
52(2):242-255). Höfte and Whiteley classified
B.t.
crystal protein genes into four major classes. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992
] Bio/Technology
10:271-275). CryV has been proposed to designate a class of toxin genes that are nematode-specific. Lambert et al. (Lambert, B., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996
] Appl. Environ. Microbiol
62(1):80-86) and Shevelev et al. ([1993
] FEBS Lett.
336:79-82) describe the characterization of Cry9 toxins active against lepidopterans. Published PCT applications WO 94/05771 and WO 94/24264 also describe
B.t.
isolates active against lepidopteran pests. Gleave et al. ([1991
] JGM
138:55-62) and Smulevitch et al. ([1991
] FEBS Lett.
293:25-26) also describe
B.t.
toxins. A number of other classes of
B.t.
genes have now been identified.
The cloning and expression of a
B.t.
crystal protein gene in
Escherichia coli
has been described in the published literature (Schnepf, H. E., H. R. Whiteley [1981
] Proc. Natl. Acad. Sci. USA
78:2893-2897.). U.S. Pat. Nos. 4,448,885 and 4,467,036 both disclose the expression of
B.t.
crystal protein in
E. coli.
U.S. Pat. Nos. 4,990,332; 5,039,523; 5,126,133; 5,164,180; and 5,169,629 are among those which disclose
B.t.
toxins having activity against lepidopterans. PCT application WO96/05314 discloses PS86W1, PS86V1, and other
B.t.
isolates active against lepidopteran pests. The PCT patent applications published as WO94/24264 and WO94/05771 describe
B.t.
isolates and toxins active against lepidopteran pests.
B.t.
proteins with activity against members of the family Noctuidae are described by Lambert et al., supra. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose
B. thuringiensis
strain
tenebrionis
which can be used to control coleopteran pests in various environments. U.S. Pat. No. 4,918,006 discloses
B.t.
toxins having activity against dipterans. U.S. Pat. Nos. 5,151,363 and 4,948,734 disclose certain isolates of
B.t.
which have activity against nematodes. Other U.S. patents which disclose activity against nematodes include U.S. Pat. Nos. 5,093,120; 5,236,843; 5,262,399; 5,270,448; 5,281,530; 5,322,932; 5,350,577; 5,426,049; and 5,439,881. As a result of extensive research and investment of resources, other patents have issued for new
B.t.
isolates and new uses of
B.t.
isolates. See Feitelson et al., supra, for a review. However, the discovery of new
B.t.
isolates and new uses of known
B.t.
isolates remains an empirical, unpredictable art.
Isolating responsible toxin genes has been a slow empirical process. Carozzi et al. (Carozzi, N. B., V. C. Kramer, G. W. Warren, S. Evola, G. Koziel (1991)
Appl. Env. Microbiol.
57(11):3057-3061) describe methods for identifying novel
B.t.
isolates. This report does not disclose or suggest the specific primers, probes, toxins, and genes of the subject invention for lepidopteran-active toxin genes. U.S. Pat. No. 5,204,237 describes specific and universal probes for the isolation of
B.t.
toxin genes. This patent, however, does not describe the probes, primers, toxins, and genes of the subject invention.
WO 94/21795 and Estruch, J. J. et al. ([1996
] PNAS
93:5389-5394) describe toxins obtained from Bacillus microbes. These toxins are reported to be produced during vegetative cell growth and were thus termed vegetative insecticidal proteins (VIP). These toxins were reported to be distinct from crystal-forming &dgr;-endotoxins. Activity of these toxins against lepidopteran pests was reported.
Black cutworm (
Agrotis ipsilon
(Hufnagel); Lepidoptera: Noctuidae) is a serious pest of many crops including maize, cotton, cole crops (Brassica, broccoli, cabbages, Chinese cabbages), and turf. Secondary host plants include beetroots, Capsicum (peppers), chickpeas, faba beans, lettuces, lucerne, onions, potatoes, radishes, rape (canola), rice, soybeans, strawberries, sugarbeet, tobacco, tomatoes, and forest trees. In North America, pests of the genus Agrotis feed on clover, corn, tobacco, hemp, onion, strawberries, blackberries, raspberries, alfalfa, barley, beans, cabbage, oats, peas, potatoes, sweetpotatoes, tomato, garden flowers, grasses, luceme, maize, asparagus, grapes, almost any kind of leaf, weeds, and many other crops and garden plants. Other cutworms in the Tribe Agrotini are pests, in particular those in the genus Feltia (e.g.,
F. jaculifera
(Guenée); equivalent to
ducens subgothica
) and Euxoa (e.g.,
E. messoria
(Harris),
E. scandens
(Riley),
E. auxiliaris
Smith,
E. detersa
(Walker),
E. tessellata
(Harris),
E. ochrogaster
(Guenée). Host plants include various crops, including rape.
Cutworms are also pests outside North America, and the more e

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