Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1998-01-26
2001-08-21
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C424S093461
Reexamination Certificate
active
06277624
ABSTRACT:
1. FIELD OF THE INVENTION
The invention is related to a mutant Bacillus strain which produces a factor which potentiates the pesticidal activity of a Bacillus related pesticide, a chemical pesticide and/or a virus with pesticidal properties, in which such a factor is obtained in larger amounts or has a greater potentiating activity compared to the parental strain, and methods for producing such mutant strains. The invention also relates to methods for obtaining the factor.
2. BACKGROUND OF THE INVENTION
Every year, pests detrimental to agriculture, forestry, and public health cause losses in the millions of dollars. Various strategies have been used to control such pests.
One strategy is the use of chemical pesticides with a broad range or spectrum of activity. However, there are a number of disadvantages with using chemical pesticides. Specifically, because of their broad spectrum of activity, these pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. Additionally, chemical pesticides are frequently toxic to animals and humans. Furthermore, targeted pests frequently develop resistance when repeatedly exposed to such substances.
Another strategy involves the use of biopesticides to control insect, fungal and weed infestations. Biopesticides are naturally occurring pathogens and/or the substances produced by these pathogens. The advantage of using biopesticides is that they are generally less harmful to non-target organisms and the environment as a whole compared to chemical pesticides.
2.1.
Bacillus thuringiensis
The most widely used biopesticide is
Bacillus thuringiensis. Bacillus thuringiensis
is a motile, rod-shaped, gram-positive bacterium that is widely distributed in nature, especially in soil and insect-rich environments. During sporulation,
Bacillus thuringiensis
produces a parasporal crystal inclusion(s) which is insecticidal upon ingestion to susceptible insect larvae of the orders Lepidoptera, Diptera, and Coleoptera. The inclusions may vary in shape, number, and composition. They are comprised of one or more proteins called delta-endotoxins, which may range in size from 27-140 kDa. The insecticidal delta-endotoxins are generally converted by proteases in the larval gut into smaller (truncated) toxic polypeptides, causing midgut destruction, and ultimately, death of the insect (Höfte and Whiteley, 1989,
Microbiological Reviews
53:242-255).
There are several
Bacillus thuringiensis
strains that are widely used as biopesticides in the forestry, agricultural, and public health areas.
Bacillus thuringiensis
subsp. kurstaki and
Bacillus thuringiensis
subsp. aizawai produce delta-endotoxins specific for Lepidoptera. A delta-endotoxin specific for Coleoptera is produced by
Bacillus thuringiensis
subsp. tenebrionis (Krieg et al., 1988, U.S. Pat. No. 4,766,203). Furthermore,
Bacillus thuringiensis
subsp. israelensis produces delta-endotoxins specific for Diptera (Goldberg, 1979, U.S. Pat. No. 4,166,112).
Other
Bacillus thuringiensis
strains specific for dipteran pests have also been described. A
Bacillus thuringiensis
isolate has been disclosed which is toxic to Diptera and Lepidoptera (Hodgman et al., 1993,
FEMS Microbiology Letters
114:17-22). SDS polyacrylamide gel electrophoresis of the purified crystal delta-endotoxin from this isolate revealed three protein species which are related to CryIA(b), CryIB, and CryIIA toxins. There has also been disclosed a
Bacillus thuringiensis
isolate which produces a dipteran-active crystal comprised of proteins with molecular weights of 140, 122, 76, 72, and 38 kDa (Payne, 1994, U.S. Pat. No. 5,275,815). EPO 480,762 discloses five B.t. strains which are each active against dipteran pests; each also have a unique crystal delta-endotoxin pattern.
Several
Bacillus thuringiensis
strains have been described which have pesticidal activity against pests other then Lepidoptera, Coleoptera, and Diptera. Five
Bacillus thuringiensis
strains have been disclosed which produce delta-endotoxins that are toxic against nematodes (Edwards, Payne, and Soares, 1988, Eur. Pat. Appl. No. 0 303 426 B1). There has also been disclosed a
Bacillus thuringiensis
strain, PS81F, which can be used to treat humans and animals hosting parasitic protozoans (Thompson and Gaertner, 1991, Eur. Pat. Appl. No. 0 461 799 A2). Several
Bacillus thuringiensis
isolates have also been disclosed with activity against acaride pests. These isolates produce crystals comprised of proteins with molecular weights in the (wide) range of 35 kDa to 155 kDa (Payne, Cannon, and Bagley, 1992, PCT Application No. WO 92/19106). There have also been disclosed
Bacillus thuringiensis
strains with activity against pests of the order Hymenoptera (Payne, Kennedy, Randall, Meier, and Uick, 1992, Eur. Pat. Appl. No. 0 516 306 A2); with activity against pests of the order Hemiptera (Payne and Cannon, 1993, U.S. Pat. No. 5,262,159); with activity against fluke pests (Hickle, Sick, Schwab, Narva, and Payne, 1993, U.S. Pat. No. U.S. 5,262,399; and with activity against pests of the order Phthiraptera (Payne and Hickle, 1993, U.S. Pat. No. 5,273,746). Furthermore, another strain of
Bacillus thuringiensis
subsp. kurstaki, WB3S-16, isolated from Australian sheep wool clippings, has been disclosed that is toxic to the biting louse
Damalinia ovis
, a Phthiraptera pest (Drummond, Miller, and Pinnock, 1992, J. Invert. Path. 60:102-103).
The delta-endotoxins are encoded by cry (crystal protein) genes which are generally located on plasmids. The cry genes have been divided into six classes and several subclasses based on relative amino acid homology and pesticidal specificity. The major classes are Lepidoptera-specific (cryI); Lepidoptera-and Diptera-specific (cryII); Coleoptera-specific (cryIII); Diptera-specific (cryIV) (Höfte and Whiteley, 1989,
Microbiological Reviews
53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cryV genes by Tailor et al., 1992,
Molecular Microbiology
6:1211-1217); and Nematode-specific (referred to as cryV and cryVI genes by Feitelson et al., 1992,
Bio/Technology
10:271-275).
Delta-endotoxins have been produced by recombinant DNA methods. The delta-endotoxins produced by recombinant DNA methods may or may not be in crystal form.
Some strains of
Bacillus thuringiensis
have been shown to produce a heat-stable pesticidal adenine-nucleotide analog, known as &bgr;-exotoxin type I or thuringiensin, which is pesticidal alone (Sebesta et al., in H. D. Burges (ed.),
Microbial Control of Pests and Plant Diseases,
Academic Press, New York, 1980, pp. 249-281). &bgr;-exotoxin type I has been found in the supernatant of some
Bacillus thuringiensis
cultures. It has a molecular weight of 701 and is comprised of adenosine, glucose, and allaric acid (Farkas et al., 1977,
Coll. Czechosslovak Chem. Comm.
42:909-929; Lüthy et al., in Kurstak (ed.),
Microbial and Viral Pesticides,
Marcel Dekker, New York, 1982, pp. 35-72). Its host range includes, but is not limited to,
Musca domestica, Mamestra configurata
Walker,
Tetranychus urticae, Drosophila melanogaster,
and
Tetranychus cinnabarinus.
The toxicity of &bgr;-exotoxin type I is thought to be due to inhibition of DNA-directed RNA polymerase by competition with ATP. It has been shown that &bgr;-exotoxin type I is encoded by a cry plasmid in five
Bacillus thuringiensis
strains (Levinson et al., 1990,
J. Bacteriol.
172:3172-3179). &bgr;-exotoxin type I was found to be produced by
Bacillus thuringiensis
subsp. thuringiensis serotype 1,
Bacillus thuringiensis
subsp. tolworthi serotype 9, and
Bacillus thuringiensis
subsp. darmstadiensis serotype 10.
Another &bgr;-exotoxin classified as &bgr;-exotoxin type II has been described (Levinson et al., 1990,
J. Bacteriol.
172:3172-3179). &bgr;-exotoxin type II was found to be produced by
Bacillus thuringiensis
subsp. morrisoni serotype 8ab and is active against
Leptinotarsa decemlineata.
The structure of &bgr;-exotoxin type II is not completely known, but is significantly diffe
Lidster William D.
MacIntosh Susan C.
Manker Denise Carol
Outtrup Helle
Starnes Robert L.
Marx Irene
Rockey Milnamow & Katz Ltd.
Valent BioSciences, Inc.
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