Pesticidal toxins

Details Pesticidal toxins Pesticidal toxins

2000-04-12

2003-04-15

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C536S023100, C536S023710, C435S254200, C435S412000, C435S418000, C435S419000, C435S320100, C800S302000

Reexamination Certificate

active

06548291

ABSTRACT:

BACKGROUND OF THE INVENTION
The soil microbe
Bacillus thuringiensis
(
B.t
.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. 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 these
B.t
. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as
B.t
. endotoxin 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 ten 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 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; 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 4 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).
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. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of
B.t
. crystal protein in
E. coli
. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose
B. thuringiensis
strain
tenebrionis
(a.k.a. M-7, a.k.a.
B.t
. san diego) 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. No. 4,849,217 discloses
B.t
. isolates which have activity against the alfalfa weevil. U.S. Pat. No. 5,208,077 discloses coleopteran-active
Bacillus thuringiensis
isolates. U.S. Pat. No. 5,151,363 and U.S. Pat. No. 4,948,734 disclose certain isolates of
B.t
. which have activity against nematodes. 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. However, the discovery of new
B.t
. isolates and new uses of known
B.t
. isolates remains an empirical, unpredictable art.
Coleopterans are an important group of agricultural pests which cause a very large amount of damage each year. Examples of coleopteran pests include alfalfa weevils and corn rootworm.
The alfalfa weevil,
Hypera postica
, and the closely related Egyptian alfalfa weevil,
Hypera brunneipennis
, are the most important insect pests of alfalfa grown in the United States, with 2.9 million acres infested in 1984. An annual sum of 20 million dollars is spent to control these pests. The Egyptian alfalfa weevil is the predominant species in the southwestern U.S., where it undergoes aestivation (i.e., hibernation) during the hot summer months. In all other respects, it is identical to the alfalfa weevil, which predominates throughout the rest of the U.S.
The larval stage is the most damaging in the weevil life cycle. By feeding at the alfalfa plant's growing tips, the larvae cause skeletonization of leaves, stunting, reduced plant growth, and, ultimately, reductions in yield. Severe infestations can ruin an entire cutting of hay. The adults, also foliar feeders, cause additional, but less significant, damage.
Approximately 9.3 million acres of U.S. corn are infested with corn rootworm species complex each year. The corn rootworm species complex includes the northern corn rootworm;
Diabrotica barberi
, the southern corn rootworm,
D. undecimpunctata howardi
, and the western corn rootworm,
D. virgifera virgifera
. The soil-dwelling larvae of these Diabrotica species feed on the root of the corn plant, causing lodging. Lodging eventually reduces corn yield and often results in death of the plant. By feeding on cornsilks, the adult beetles reduce pollination and, therefore, detrimentally effect the yield of corn per plant. In addition, adults and larvae of the genus Diabrotica attack cucurbit crops (cucumbers, melons, squash, etc.) and many vegetable and field crops in commercial production as well as those being grown in home gardens.
Control of corn rootworm has been partially addressed by cultivation methods, such as crop rotation and the application of high nitrogen levels to stimulate the growth of an adventitious root system. However, chemical insecticides are relied upon most heavily to guarantee the desired level of control. Insecticides are either banded onto or incorporated into the soil. The major problem associated with the use of chemical insecticides is the development of resistance among the treated insect populations.
BRIEF SUMMARY OF THE INVENTION
The subject invention concerns novel materials and methods for controlling non-mammalian pests. In a preferred embodiment, the subject invention provides materials and methods for the control of coleopteran pests. In specific embodiments, the materials and methods described herein are used to control alfalfa weevil and/or corn rootworm.
The subject invention advantageously provides two new classes of polynucleotide sequences which encode corresponding novel classes of pesticidal proteins. One novel class of polynucleotide sequences as described herein encodes toxins which have a full-length molecular weight of approximately 40-50 kDa. In a specific embodiment, these toxins have a molecular weight of about 43-47 kDa. A second class of polynucleotides, which encodes pesticidal proteins of about 10-15 kDa, is also provided according to the subject invention. In a specific embodiment, these toxins have a molecular weight of about 13-14 kDa. The subject invention concerns polynucleotides which encode the 40-50 kDa and 10-15 kDa toxins, polynucleotides which encode pesticidal fragments of the full length toxins, and polynucleotide sequences which encode longer forms of these

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