CONTROLLING HEMIPTERAN INSECT PESTS WITH BACILLUS THURINGIENSIS

Abstract: The subject invention concerns the use of Bacillus thuringiensis (B.t.) endotoxins to control hemipteran insect pests. Specifically exemplified is B.t. strain 201T6, B.t. var. israelensis, and delta -endotoxins therefrom. These isolates and toxins can be administered to said pests, or to the environment of said pests, e.g., plants, to achieve control of the pests. A preferred embodiments is the transformation of plants to express the B.t. toxins.

Description: DESCRIPTION CONTROLLING HEMTPTERAN INSECT PESTS WITH BACILLUS THURINGIENSIS Cross-Reference to a Related Application This application is a continuation-in-part of co-pending application Serial No. 08/475,924, filed June 7, 1995. Field of the Invention The present invention relates to methods for controlling insect pests of the order Hemiptera. In particular, δ-endotoxins of Bacillus thuringiensis (B.t.) have been discovered, unexpectedly, to control hemipteran insect pests, e.g.,Lygus hesperus. 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 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. endotoxm delivery vehicles (Gaertner, F.H., L. Kim [1988] ΩBTECH 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 var. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis wax. kurstaki HD-1 produces δ -endotoxin crystals which are 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 B. t. var. israelensis and B.t. var. tenebrio is (a.ka. M-7, a.k.a. B.t. var. san diegό), 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 et al. Krieg, A., A.M. Huger, G.A. Langenbruch, W. Schnetter [1983] Z. ang. Ent. 96:500-508), describe Bacillus thuringiensis var. tenebrio is, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and the beetle Agelastica alni. Recently, new subspecies of B.t. have been identified, and genes responsible for active δ- endotoxin proteins have been isolated (Hδfte, H., H.R- Whiteley [1989] Microbiological Reviews 52(2):242-255). Hόfte and Whiteley classified B.t. crystal protein genes into 4 major classes. The classes were Cryl (Lepidoptera-specific), Cryll (Lepidoptera- and Diptera-specific), CryHJ (Coleoptera-specific), and CrylV (Diptera-specific). Prefontaine et al. (Prefontaine, G., P. Fast, P.C.K. Lau, M.A. Hefford, Z. Hanna, R. Brosseau [1987] Appl. Environ. Microbiol. 53(12):2808- 2814) describe probes useful in classifying lepidopteran-active genes. 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). B.t. crystalline toxins are generally recognized as being protoxins, requiring either particular physicochemical conditions (i.e., pH, redox, ionic strength), or the action of certain proteases, or both, to generate an active toxin (Hδfte and Whiteley, supra). In most cases, the insect supplies conditions for activation of the toxin; however, cases have been documented where pre-solubilization or pre-proteolysis have been necessary for optimum activity (Jacquet, J., R. Hutter, P. Luthy [1987] "Specificity of Bacillus thuringiensis delta-endotoxin,".4/?ρ/. Environ. Microbiol. 53:500-504) or detection of activity (Lambert, B., H.R. Hόfte, KL Annys, S. Jansens, P. Soetaert, M. Peferoen [1992] "Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae," Appl Environ. Microbiol. 58:2536-2542). 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. Patent 4,448,885 and U.S. Patent 4,467,036 both disclose the expression of B.t. crystal proteins in E. coli. U.S. Patents 4,797,276 and 4,853,331 disclose B. thuringiensis var. tenebrionis (a.k.a. B.t. san diego, a.k.a. M-7) which can be used to control coleopteran pests in various environments. U.S. Patent No. 4,918,006 discloses Bacillus thuringiensis var. israelensis toxins which are active against dipteran pests and reports that a protein of about 27 kDa, and fragments thereof, are responsible for the dipteran activity. U.S. Patent No. 4,849,217 discloses B.t. isolates which have activity against the alfalfa weevil. U.S. Patent No. 5,151,363 and U.S. Patent 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. Hemiptera represent a major group of insects that have not, to date, been effectively controlled by B.t. δ-endotoxins. Numerous hemipteran pest species, most notably Lygus species, cause considerable plant damage and economic loss each year. Hemiptera ranks among the most economically destructive orders. See Arnett, R.H. Jr. ([1985] American Insects, Van Nostrand Reinhold Co., Inc., NY). Of all Hemiptera, the mirids (Hemiptera: Miridae; includes Lygus) are the most notorious crop pests. Feeding causes injury that weakens plants and is a mode of plant disease transmission. In addition to L. hesperus, other pest Lygus include: L. lineolaris (Beauv.), L. pratensis (L.), L. rugulipennis Popp., and the common green capsid (Lygus pabulinus (L.)). Members of this genus are found variously on cotton, potato, sugar beet, celery, beans, peach, apple, alfalfa, pear, plum, quince and a variety of nursery stock, ornamental plants and vegetable crops. Specific Mirid pests include: the potato capsid Calocoris norvegicus (Gmel.), a pest of potato and brassicas (e.g., cabbage, cauliflower, broccoli, kale, Brussels sprout, turnip); the stack bug (also carrot plant bug) Orthops campestris (L.), a pest of carrot, celery, parsnip, parsley and dill; the apple capsid Plesiocoris rugicollis (Fall.), a pest of apple, currants and gooseberry; the tomato bug Cyrtopeltis modestus (Distant), a pest of tomatoes; the suckfly Cyrtopeltis notatus (Distant), a pest of tobacco; the white marked fleahopper Spanagonicus albofasciatus (Reuter), injurious to grass, with damage particularly noticeable on golf greens; the honeylocust plant bug Diaphnocoris chlorionis (Say), a pest of beets; the onion plant bug Labopidicola allii Knight, a pest of cultivated and wild onions; the cotton fleahopper Pseudatomoscelis seriatus (Reuter), found on cotton; the rapid plant bug Adelphocoris rapidus (Say), an occasional pest of cotton and legumes; the four-lined plant bug Poecilocapsus lineatus (Fabricius), often a pest of garden crops. Other Hemipteran pests include: Lygaeidae (seed bug family): chinch bug (Blissus leucopterus (Say)) is a pest of maize, sorghums, wheat, millets, rice, barley, rye and oats; false chinch bugs (Nysius spp., e.g.,N. ericae, N. raphanus Howard) are pests of brassicas, beets and potato. Pentatomidae (stink bug family): the brown stink bug, Euschistus servus (Say) is a pest of cotton. The green stink bug, Nezara viridula (L.) is a generalist pest of seedlings, especially vegetables and legumes; Eurygaster species, e.g., E. austriaca (Schr.) and is. integriceps (Put.) (wheat shield bug, Sunn pest, Senn bug) attack wheat and barley. Coreidae (squash bug family): Anasa tristis (DeGreer) (squash bug) is a local pest of squash; A. armigera (Say) (horned squash bug) is an occasional pest of cultivated cucumber. Pyrrhocoridae (red bug and cotton stainer family): the cotton stainer Dysdercus suturellus (Herrich-Schaeffer) is a pest of cotton. Tingidae (lace bug family): Corythucha arcuata (Say) often is a pest of roses, maple, apple and chestnut. Other Corythucha species include the cotton lace bug, chrysanthemum lace bug, elm lace bug and hawthorn lace bug. Belostomatidae (giant water bug family) : members of this family are known to attack and suck the blood of fishes and therefore can be a pest in fish hatcheries. Members of the Reduviidae and Cimicidae (including kissing bug and bed bugs, respectively) bite mammals and transmit diseases contractible by humans. The digestive system of hemipterans is unusual among the insects in several ways: certain hydrolytic digestive enzymes are absent such as trypsin; the midgut lacks a peritrophic membrane, and there is no crop. These features reflect the liquid diet and sucking mode of feeding, subject to evolutionary constraints. Because of the differences in diet, feeding mode, and digestive physiology and biochemistry, one would not necessarily expect that proteins having insecticidal activity against leaf-chewing insects would also have activity against fluid-feeding Hemiptera. Bacillus thuringiensis PS201T6, NRRL B-18750, deposited on January 9, 1991, or a δ- endotoxin therefrom, was previously found to have activity against certain pests. For example, see U.S. Patent Nos.5,273,746; 5,298,245; and 5,302,387, which disclose the use of B.t. PS201T6. B.t. isolates PS123D1 has been disclosed in EP 0409438. B.t. isolate PS71M3 has been disclosed in EP 0626 809 and in U.S. Patent No. 5,273,746. The above patents do not disclose or suggest the use of any B. t. isolate to control hemipteran insect pests. Practice of the present invention provides an alternative to control of hemipteran pests with chemical pesticides, thereby allowing more environmentally-friendly insect management and providing a tool for management of insecticide resistance. Brief Summary of the Invention The subject invention concerns the use of Bacillus thuringiensis (B.t.) δ-endotoxins to control hemipteran insect pests. Specifically exemplified herein is the use of the B.t. isolate PS201T6 and toxins therefrom to control the hemipteran insect pest Lygus hesperus. The use of B.t. var. israelensis isolates and toxins is also described. The subject invention also includes the use of variants of the exemplified B.t. isolates and toxins which have substantially the same pesticidal properties as the exemplified isolates and toxins. These variants include mutant microbes. Procedures for making mutants are well known in the microbiological art. Ultraviolet Ught and chemical mutagens such as nitrosoguamdine are used extensively toward this end. Recombinant hosts which have been transformed to express B.t. toxins can also be used according to the subject invention. These recombinant hosts may be, for example, microorganisms or plants. According to the subject invention, hemipteran insect pests can be controlled using the B.t. isolate itself, variants of the B.t. isolates, δ-endotoxins obtained from said isolates, commercial preparations made from cultures of the isolates, or toxins produced by DNA of this isolate. In one embodiment, the toxins may be produced by DNA which has been transformed into another host. In a preferred embodiment, the transformed host is a plant. Brief Description of the Sequences SEQ ID NO. 1 is the nucleotide sequence encoding an approximately 30 kDa toxin of PS201T6. SEQ ID NO. 2 is the deduced amino acid sequence of an approximately 30 kDa toxin of PS201T6. SEQ ID NO.3 is the amino acid sequence of a truncated 201T6 toxin of about 25 kDa. Detailed Disclosure of the Invention The subject invention concerns the discovery of a new method for controlling hemipteran insect pests which involves contacting said pests with a Bacillus thuringiensis (B.t.) δ -endotoxin in a manner wherein the hemipteran insect ingests the δ -endotoxin. Specifically exemplified is the use of toxins from the B.t. isolate known as PS201T6. Also exemplified is the use of toxins from Bacillus thuringiensis var. israelensis (B.t.i.) isolates to control hemipteran pests. The Bacillus thuringiensis isolate PS201T6 has the following characteristics in its biologically pure form and as compared to B.t. HD-1: Table 1. Comparison of B.t. PS201T6, and B.t. HD-1 B.t. PS201T6 B.t. HD-1 Inclusions: elliptical bipyramid & bipyramid Approximate 133,000 130,000 molecular wt. of 31,000 68,000 proteins by SDS-PAGE Host range Cockroaches Lepidoptera Diptera Corn Rootworm The israelensis variety of B.t. is well known and readily recognized by those skilled in this art. Characteristics generally associated with the israelensis category of B.t. include dipteran activity, H14 serotype, and a protein pattern which includes an approximately 28 to 33 kD protein and, generally, additional proteins of about 70 kD and 130 kD. Other B.t. varieties which express israelensis-t pe toxins can also be used according to the subject invention. Such toxins would have a size similar to the toxins produced by B.t.i. and a similar activity profile, including dipteran activity. B.t. var. morrisoni serotype 8a, 8b have, for example, been reported to express B. t.i. -type toxins. An example of such an isolate is PS71M3. As used herein, the term "Bacillus thuringiensis var. israelensis toxin" includes toxins which are similar or related to toxins expressed by B.t.i. but which happen to be expressed by a different variety of B.t. The isolate designated PS123D1 is specifically exemplified as a B.t.i. isolate useful according to the subject invention. Of particular interest in controlling hemipteran pests are toxins of approximately 28 to 33 kD of a B.t.i. isolate or, preferably, modified forms of this toxin. One embodiment of the subject invention involves the use of truncated forms of the 28 kDa PS123D1 toxin to control hemipterans. A specific example is the truncated toxin which has amino acids removed from the N-terminus and is about 25 kDa. As described herein, the truncated forms of the toxins can be obtained through treatment of B. t. culture supernatants and/or by growing B. t. cultures under appropriate conditions to result in the production of activated toxins as a result of the advantageous effects of endogenous proteases. Other modifications which cause, for example, solubilization of the B.t.i. toxin can also be utilized to obtain highly active toxins. The Bacillus thuringiensis isolate PS123D1 has the following characteristics in its biologically pure form: Table 2. Characteristics of B. t. PS123D1 B.t. PS123D1 Inclusions: amorphic Approximate molecular wt. of 133,000 proteins by SDS-PAGE 127,000 72,000 28,000 The cultures disclosed in this application have been deposited in the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA. Culture Repository No. Deposit date Bacillus thuringiensis PS201T6 NRRL B-l 8750 January 9, 1991 E. coli NM522 (pMYC2362) NRRL B-21018 December 2, 1992 E. coli NM522 (pMYC2357) NRRL B-21017 December 2, 1992 Bacillus thuringiensis PS123D1 NRRL B-21011 October 13, 1992 Bacillus thuringiensis PS71M3 NRRL B-18930 December 27, 1991 The subject cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of the deposits does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of a deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them. In one preferred embodiment of the subject invention, a toxin from B. t. isolate PS201 T6 is used to achieve control of hemipteran insect pests. In another preferred embodiment, the PS201T6 toxin is activated. Toxins can be activated by, for example, c turing the microbe, under conditions which facilitate the activation of said toxin by the action of compounds which exist naturally or are produced in said culture. Activation may also be achieved by adding a compound to a Bacillus thuringiensis strain PS201 T6 culture, or a supernatant thereof, wherein said compound participates in the activation of said toxin either through direct action on said toxin or by facilitating the action of a second compound. The additional compound may be, for example, a protease, or a compound which raises the pH of the culture or supernatant. Bacillus thuringiensis PS201T6 is now available to the public, without restriction, by virtue of the issuance of U.S. Patent No. 5,273,746. Plasmid pMYC2362 harbors a gene encoding a 130 kDa toxin from PS201T6. Plasmid pMYC2357 harbors a gene encoding a 30 kDa toxin from PS201T6. Further information regarding examples of cloning genes from PS201T6 can be found in EP-A-0 708 830 and WO 95/02694. Further examples of cloning genes from PS71M3 are described in WO 95/02694 and EP-A-0457498. Genes and toxins. In one embodiment of the subject invention, genes which encode B. t. toxins active against hemipteran insect pests are used to transform a suitable host. The genes and toxins useful according to the subject invention include not only the full length sequences but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified. As used herein, the terms "variants" or "variations" of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having similar pesticidal activity. As used herein, the term "equivalent toxins" refers to toxins having the same or essentially the same biological activity against the target pests as the claimed toxins. It would be apparent to a person skilled in this art, having the benefit of this disclosure, that genes encoding hemipteran-active toxins can be identified and obtained through several means. The genes can be obtained from the isolate deposited at a culture depository as described above. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as BaBl or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins. Equivalent toxins and/or genes encoding these equivalent toxins can be derived from B. t. isolates and/or DNA libraries using the teachings provided herein. There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example, antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other B. t. toxins. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or Western blotting. Antibodies to the toxins disclosed herein, or to equivalent toxins, or fragments of these toxins, can readily be prepared using standard procedures in this art. The genes which encode these toxins can then be obtained from the microorganism. Fragments and equivalents which retain the pesticidal activity of the exemplified toxins would be within the scope of the subject invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to "essentially the same" sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect the pesticidal activity of the protein. A further method for identifying the toxins and genes of the subject invention is through the use of oligonucleotide probes. These probes are nucleotide sequences having a means for detection. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample have substantial homology. The probe's means of detection provides a means for deteπnining in a known manner whether hybridization has occurred. Such a probe analysis provides a rapid method for identifying toxin-encoding genes of the subject invention. The nucleotide segments which are used as probes according to the invention can be synthesized by use of DNA synthesizers using standard procedures. These nucleotide sequences can also be used as PCR primers to amplify genes of the subject invention. Recombinant hosts. The toxm-encoding genes harbored by the isolates disclosed herein can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the production of the toxin. With suitable microbial hosts, e.g., Pseudomonas, the microbes can be apphed to the situs of the pest, where they will proliferate and be ingested by the pest, resulting in control of the pest. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell which retains the toxic activity, then can be applied to the environment of the target pest. Methods for treatment of microbial cells are disclosed in United States Patent Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference. A wide variety of ways are available for introducing a B.t. gene encoding a toxin into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in United States Patent No. 5,135,867, which is incorporated herein by reference. Furthermore, materials and methods for introducing B. t. genes into plants in order to confer upon such plants the ability to produce insecticidal toxins are well known in the art. In a preferred embodiment, the B.t. genes are modified to facilitate optimal stability and expression in the selected plant host. In this regard, U.S. Patent No. 5,380,831 is incorporated herein by reference. Mutants. Mutants of the isolate described herein can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate. The mutants can be made using ultraviolet light and nitrosoguamdine by procedures well known in the art. Following are examples which illustrate procedures for practicing the present invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. Example 1 - Culturing B.t. Strain PS201T6 A subculture of B.t. isolate strain PS201T6 can be used to inoculate the following peptone, glucose, and salts medium. Bacto Peptone 7.5 g 1 Glucose 1.0 g/1 KH 2 P0 4 3.4 g/1 K 2 HP0 4 4.35 g/1 Salt Solution 5.0 ml/1 CaCL Solution 5.0 ml/1 Salts Solution (100 ml) MgS0 4 -7H 2 0 2.46 g MnS0 4 -H 2 0 0.04 g ZnS0 4 -7H 2 0 0.28 g FeS0 4 -7H 2 0 0.40 g CaCl 2 Solution (100 ml) CaCl 2 -2H 2 0 3.66 g pH 7.2 The salts solution and CaCl 2 solution are filter-steriUzed and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30 ° C on a rotary shaker at 200 rpm for 64 hr. The above procedure can be readily scaled up to large fermentors by procedures well known in the art. The B.t. spores and crystals, obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation. Example 2 - Production of Activated 201T6 Toxin (201T6-D) Activated 201T6 toxin can be produced by a variety of methods which result in truncation of the 201T6 toxin. In this regard, reference can be made to WO95/02693. In one such method, cultures of PS201T6 were harvested by centrifugation and resuspended to l/9th to l/25th of their original culture volume in 0.1 Na 2 C0 3 /NaHC0 3 pH 11.0 containing 0.5 mg/ml pronase E (Sigma Chemical Company, P-5147 Type XTV bacterial protease from Streptomyces griseus). The suspension was incubated at 37 °C overnight with mixing. The suspensions were dialyzed against 2 changes of 50 to 100 volumes each of either distilled water or 0.1 M Na 2 C0 3 /NaHC0 3 pH 9.5 to yield "dialyzed suspensions." The suspension resulting from 0.1 M Na 2 C0 3 /NaHC0 3 pH 9.5 dialysis was centrifuged to remove cells, spores, and debris. Additional purification from spores and debris can be accomplished by filtration through a Whatman glass microfibre filter, a 0.8 micron cellulose acetate filter, and a 0.2 micron cellulose acetate filter to yield a "filtered supernatant." Dried preparations of the processed toxin were prepared either before or after filtration by dialyzing against 2 changes of 50 to 100 volumes distilled water, followed by lyophilization (lyophilized, pronase-treated toxin). Example 3 - Alternative Method for Production of Activated 201T6 Toxin Cultures of PS201T6 were harvested by centrifugation and resuspended to l/9th to l/25th of their original culture volume in 0.1 M Na 2 C0 3 /NaHC0 3 , 0.5% 2-mercaptoethanol, pH 11.0. The suspension was incubated for about 2 hours at room temperature. The suspension was centrifuged to remove cells, spores, and debris. Additional purification from spores and debris can be accomplished by filtration through a Whatman glass microfibre filter, a 0.8 micron cellulose acetate filter, and a 0.2 micron cellulose acetate filter to yield a "filtered supernatant." The suspensions were dialyzed against 2 changes of 50 to 100 volumes each of either distilled water or 0.1 M pH 9.5 to yield "dialyzed suspensions." Dried preparations of the processed toxin were prepared either before or after filtration by dialyzing against two changes of 50 to 100 volumes distilled water, followed by lyophilization. Material prepared according to this procedure is referred to herein as 201T6-D. Example 4 - Activity of B.t. Isolates Against the Hemipteran Insect Lvsus hesperus Five newly-emerged (≤ one week old) L. hesperus adults were placed in a 1.0 oz. plastic portion cup (Fabrikal Corp.) with two pinholes in the side to allow gas exchange. A small piece of Scott MICROWΓPES tissue was placed in the bottom of the cup to absorb liquid excreta. In place of a lid, two pieces of PARAFTLM were stretched across the top, sandwiching in between them 200 μL of test solution. "Autoprocessed" B.t. PS201T6 cell culture broth (activated by resident proteases as described in Example 3) was presented to L. hesperus adults. The autoprocessed broth was estimated to contain approximately 4.5 mg/ml of toxin. 1 ml of the broth was diluted to 100 ml with a 15% sucrose solution. 200 μl of the resulting 45 μg/ml solution was used as the test solution. Insects were allowed to feed for three days. Mortality after three days was compared with that of starved insects and control insects provided a "blank" of 15% sucrose solution that contained no toxin. After establishing activity of 45 μg/mL B.t. PS201T6 after three days, a subsequent experiment was conducted to establish activity of 10 μg/mL after two days. Representative results are shown in Tables 3 and 4. Table 3. Three-day mortality of Lygus hesperus provided 45 μgB.t. PS201T6/mL Initial number of Number of insects Percent of insects Treatment live insects dead after 3 days dead after 3 days 15% sucrose solution 25 7 28 45 μg B.t. 25 17 68 PS201T6/mL of l5% sucrose solution Starved (no food, 25 25 100 water) Table 4. Two-day I nortality of Lygus hesperus provided 4.5 μg B.t. PS201T6/mL Initial number of Number of insects Percent of insects Treatment live insects dead after 2 days dead after 2 days 15% sucrose solution 50 29 58 4.5 μgB.t. 50 41 82 PS201T6/mL of l5% sucrose solution Starved (no food, 50 50 100 water " ) Example 5 - Activity of B.t. Isolate PS201T6 Against the Hemipteran InsectZygitf lineolaris As an alternative to the filter paper bioassay method, an insect is presented with "SCOTCH BRITE" or similar material saturated with the test mixture. The "SCOTCH BRITE" mesh material is cut from BioRad "MINITRANS-BLOT" cell fiber pads (8 x 11 cm) into cubes approximately 0.5 cm per side. The cubes are then saturated with test solution and a single cube is placed into a 1.25 oz "SOLO" Cup Company plastic souffle cup along with one L. lineolaris adult. The container is sealed with a souffle cup lid, and several pinholes are made in the cup or Ud to provide adequate air exchange. The insect is allowed to feed ad libitum, and mortality is measured after four days. Test solutions were prepared by feeding protein samples or blanks with sterile-filtered (0.2 μm) sucrose solution in amounts needed to obtain desired protein concentrations and 10% sucrose in the final test solution. Proteins can be obtained as described in Example 3. Prior to sterile- filtering the sucrose solution, green food-grade vegetable dye (Iris Co.) was added to provide a visual stimulus to encourage feeding. Feeding was readily observed; within minutes of presentation with a cube containing dyed test solution, insects could be seen inserting the stylet into the cube mesh. Dyed excreta was visible shortly thereafter, generally within minutes of initiation of feeding. Results are shown in Table 5. Dose is expressed as μg toxin/mL diet. A buffer blank control and a BSA control were used as the negative controls. The buffer blank was run at a concentration equivalent to the background in the 1000 μg/mL treatment. The BSA control was run at 500 μg BSA/mL diet. Table 5. Summary of mortatity data (including summation across buffer and BSA negative controls) Dose Number dead/total Percentage dead 0 (controls) 21/240 9 500 60/119 50 1000 62/90 69 Example 6 - Activated Toxins from B.t.i. The removal of a portion of the N-terminus of the 123D1 28 kDa toxin results in an advantageous activation of this toxin which increases the potency of its activity. Removal of amino acids can be accompUshed by treatment with trypsin or other appropriate enzyme, or enzyme mixture, such as pronase or endogenous proteins in B.t. culture broths. Other biologicaUy active fragments could be obtained by those skilled in the art using the teachings provided herein. As those skilled in the art having the benefit of this disclosure would readily recognize, the specific media used to grow the B.t. culture can be modified to achieve optimum activation of the B. t. toxin. For example, the cell density can be modulated by adjusting or changing the culture medium. Also, media having proteases therein can be used to enhance the activation of the B.t. toxins. Example 7 - Insertion of Toxin Genes Into Plants One aspect of the subject invention is the transformation of plants with genes encoding a toxin active against hemipteran pests. The transformed plants are resistant to attack by hemipterans. Genes encoding pesticidal toxins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are weU known in the art. For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the B.t. toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli ceUs are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant ceUs has been intensively researched and sufficiently described in EP 120516; Hoekema (1985) In: The Binary Plant Vector System, Offset- durkkerij Kanters B.V, Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4: 1-46; and An et al. (\9%5) EMBOJ. 4:277-287. Once the inserted DNA has been integrated in the genome, it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than ceUs that do not contain the inserted DNA. A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as weU as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot repUcate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can repUcate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polyUnker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen. Genet. 163:181-187). The Agrobacterium used as host ceU is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives. The transformed cells grow inside the plants in the usual manner. They can form germ ceUs and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties. In a preferred embodiment of the subject invention, plants will be transformed with genes wherein the codon usage has been optimized for plants. See, for example, U.S. Patent No. 5,380,831. Also, advantageously, plants encoding a truncated toxin will be used. The truncated toxin typically will encode about 55% to about 80% of the full length toxin. Methods for creating synthetic B.t. genes for use in plants are known in the art. Example 8 - Cloning of Novel B.t. Genes Into Insect Viruses A number of viruses are known to infect insects. These viruses include, for example, baculoviruses and entomopoxviruses. In one embodiment of the subject invention, hemipteran-active genes, as described herein, can be placed within the genome of the insect virus, thus enhancing the pathogenicity of the virus. Methods for constructing insect viruses which comprise B.t. toxin genes are well known and readily practiced by those skiUed in the art. These procedures are described, for example, in Meπyweather etal. (Merryweather, A.T., U. Weyer, M.P.G. Harris, M. Hirst, T. Booth, R.D. Possee [1990] J. Gen. Virol. 71 : 1535-1544) and Martens et al. (Martens, J.W.M., G. Honee, D. Zuidema, J.W.M. van Lent, B. Visser, J.M. Vlak [1990] Appl. Environmental Microbiol. 5r5(9):2764-2770). It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this appUcation and the scope of the appended claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT INFORMATION: Applicant Name(s): MYCOGEN CORPORATION Street address: 5501 Oberlin Drive City: San Diego State/Province: California Country: US Postal code/zip: 92121 Phone number: (619) 453-8030 Fax number: (619)453-6991 Telex number: (ii) TITLE OF INVENTION: CONTROLLING HEMIPTERAN INSECT PESTS WITH BACILLUS THURINGIENSIS (iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Saliwanchik & Saliwanchik (B) STREET: 2421 N.W. 41st Street, Suite A-1 (C) CITY: Gainesville (D) STATE: FL (E) COUNTRY: USA (F) ZIP: 32606 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (VU) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/475,924 (B) FILING DATE: 07-JUN-1995 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Sanders, Jay M. (B) REGISTRATION NUMBER: 39,355 (C) REFERENCE/DOCKET NUMBER: MA96.C1 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 352-375-8100 (B) TELEFAX: 352-372-5800 (2) INFORMATION FOR SEQ ID Nθ:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 795 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus thuringiensis (B) STRAIN: neoleoensis (C) INDIVIDUAL ISOLATE: PS201T6 (vii) IMMEDIATE SOURCE: (A) LIBRARY: LambdaGem (TM)-ll library of Kenneth E. Narva (B) CLONE: 201T635 (Xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: ATGAAAGAGT CAATTTACTA CAATGAAGAA AATGAAATAC AAATTTCACA AGGAAACTGT 60 TTCCCAGAAG AATTAGGACA TAATCCTTGG AGACAACCTC AATCCACAGC AAGAGTTATT 120 TATTTAAAAG TAAAAGATCC TATTGATACT ACTCAATTAT TAGAAATAAC AGAAATCGAA 180 AATCCCAATT ATGTATTACA AGCTATTCAA CTAGCTGCTG CCTTCCAAGA TGCATTAGTA 240 CCAACTGAAA CAGAATTTGG AGAAGCCATT AGATTTAGTA TGCCTAAAGG ATTAGAAGTT 300 GCAAAAACTA TTCAACCTAA GGGTGCTGTT GTTGCTTACA CAGATCAAAC TCTGTCACAA 360 AGCAACAACC AAGTTAGTGT TATGATTGAT AGAGTTATTA GTGTTTTAAA AACTGTAATG 420 GGAGTAGCTC TTAGTGGTTC CATTATAACT CAATTAACAG CTGCTATCAC TGATACTTTT 480 ACAAACCTTA ATACACAAAA AGATTCTGCT TGGGTTTTTT GGGGAAAAGA AACTTCACAT 540 CAAACAAATT ACACATATAA TGTCATGTTT GCAATTCAAA ATGAAACAAC TGGACGCGTA 600 ATGATGTGTG TACCTATTGG ATTTGAAATT AGAGTATTTA CTGATAAAAG AACAGTTTTA 660 TTTTTAACAA CTAAAGATTA CGCTAATTAT AGTGTGAATA TTCAAACCCT AAGGTTTGCT 720 CAACCACTTA TTGATAGCAG AGCACTTTCA ATTAATGATT TATCAGAAGC ACTTAGATCT 780 TCTAAATATT TATAC 795 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 265 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (Vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus thuringiensis (B) STRAIN: neoleoensis (C) INDIVIDUAL ISOLATE: PS201T6 (Vii) IMMEDIATE SOURCE: (A) LIBRARY: LambdaGem (TM)-ll Library of Kenneth E. Narva (B) CLONE: 201T635 ( i) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Lys Glu Ser He Tyr Tyr Asn Glu Glu Asn Glu He Gin He Ser 1 5 10 15 Gin Gly Asn cys Phe Pro Glu Glu Leu Gly His Asn Pro Trp Arg Gin 20 25 30 Pro Gin Ser Thr Ala Arg Val He Tyr Leu Lys Val Lys Asp Pro He 35 40 45 Asp Thr Thr Gin Leu Leu Glu He Thr Glu He Glu Asn Pro Asn Tyr 50 55 60 Val Leu Gin Ala He Gin Leu Ala Ala Ala Phe Gin Asp Ala Leu Val 65 70 75 80 Pro Thr Glu Thr Glu Phe Gly Glu Ala He Arg Phe Ser Met Pro Lys 85 90 95 Gly Leu Glu Val Ala Lys Thr He Gin Pro Lys Gly Ala Val Val Ala 100 105 110 Tyr Thr Asp Gin Thr Leu Ser Gin Ser Asn Asn Gin Val ser Val Met 115 120 125 He Asp Arg Val He Ser Val Leu Lys Thr Val Met Gly Val Ala Leu 130 135 140 Ser Gly ser He He Thr Gin Leu Thr Ala Ala He Thr Asp Thr Phe 145 150 155 160 Thr Asn Leu Asn Thr Gin Lys Asp Ser Ala Trp Val Phe Trp Gly Lys 165 170 175 Glu Thr Ser His Gin Thr Asn Tyr Thr Tyr Asn Val Met Phe Ala He 180 185 190 Gin Asn Glu Thr Thr Gly Arg Val Met Met Cys Val Pro He Gly Phe 195 200 205 Glu He Arg Val Phe Thr Asp Lys Arg Thr Val Leu Phe Leu Thr Thr 210 215 220 Lys Asp Tyr Ala Asn Tyr ser Val Asn He Gin Thr Leu Arg Phe Ala 225 230 235 240 Gin Pro Leu He Asp Ser Arg Ala Leu Ser He Asn Asp Leu Ser Glu 245 250 255 Ala Leu Arg Ser Ser Lys Tyr Leu Tyr 260 265 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 222 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus thuringiensis (B) STRAIN: neoleoensis (C) INDIVIDUAL ISOLATE: PS201T6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Val Lys Asp Pro He Asp Thr Thr Gin Leu Leu Glu He Thr Glu He 1 5 10 15 Glu Asn Pro Asn Tyr Val Leu Gin Ala He Gin Leu Ala Ala Ala Phe 20 25 30 Gin Asp Ala Leu Val Pro Thr Glu Thr Glu Phe Gly Glu Ala He Arg 35 40 45 Phe Ser Met Pro Lys Gly Leu Glu Val Ala Lys Thr He Gin Pro Lys 50 55 60 Gly Ala Val Val Ala Tyr Thr Asp Gin Thr Leu Ser Gin Ser Asn Asn 65 70 75 80 Gin Val Ser Val Met He Asp Arg Val He Ser Val Leu Lys Thr Val 85 90 95 Met Gly Val Ala Leu Ser Gly Ser He He Thr Gin Leu Thr Ala Ala 100 105 110 He Thr Asp Thr Phe Thr Asn Leu Asn Thr Gin Lys Asp ser Ala Trp 115 120 125 Val Phe Trp Gly Lys Glu Thr Ser His Gin Thr Asn Tyr Thr Tyr Asn 130 135 140 Val Met Phe Ala He Gin Asn Glu Thr Thr Gly Arg Val Met Met Cys 145 150 155 160 Val Pro He Gly Phe Glu He Arg Val Phe Thr Asp Lys Arg Thr Val 165 170 175 Leu Phe Leu Thr Thr Lys Asp Tyr Ala Asn Tyr Ser Val Asn He Gin 180 185 190 Thr Leu Arg Phe Ala Gin Pro Leu He Asp Ser Arg Ala Leu Ser He 195 200 205 Asn Asp Leu Ser Glu Ala Leu Arg Ser Ser Lys Tyr Leu Tyr 210 215 220

Claims: Claims A method of controlling hemipteran insect pests which comprises administering to said pests, or the environment of said pests, an effective hemipteran insect pest-controlling amount of a Bacillus thuringiensis (B.t.) δ -endotoxin. 2. The method, according to claim 1, which comprises contacting said hemipteran insect pest with a δ -endotoxin from a Bacillus thuringiensis var. israelensis isolate. 3. The method, according to claim 1, which comprises contacting said hemipteran insect pests with a δ -endotoxin from a Bacillus thuringiensis isolate selected from the group consisting of PS201T6, PS123D1, PS71M3, and mutants thereof. 4. The method, according to claim 1 , wherein the Bacillus thuringiensis δ -endotoxin is a full-length toxin, or a hemipteran-active fragment thereof. 5. The method, according to claim 3, wherein said Bacillus thuringiensis toxin is an activated toxin. 6. The method, according to claim 4, wherein said activation is effected by the truncation of said Bacillus thuringiensis strain PS201T6 toxin. 7. The method, according to claim 1, wherein said B.t. toxin is from PS201T6. 8. The method, according to claim 1, wherein said hemipteran pest is Lygus hesperus. 9. The method, according to claim 1, wherein said hemipteran pests is Lygus lineolaris. 10. The method, according to claim 1, wherein the Bacillus thuringiensis δ -endotoxin is administered to the hemipteran insect pests in planta by inserting a B. t. insecticidal structural gene into the plant genome such that the B.t. δ -endotoxin is expressed in the plant tissue which the hemipteran insect pest ingests. 11. The method, according to claim 10, wherein said B.t. gene encodes a PS201T6 toxin ipteran-active fragment or variant thereof.

Inventors: CONLAN CHRISTOPHER, STOCKHOFF BRIAN, CONLAN, CHRISTOPHER, STOCKHOFF, BRIAN
Applicants: MYCOGEN CORP, MYCOGEN CORPORATION
Application year: 1996
Main classification: A01N63
IPC codes: A01M1, A01N63, C07K14, C12N5, C12N7, C12N15, C12R1