Abstract:
The subject invention concerns a novel and useful insecticide with activity against insect pests of the order Coleoptera. Pests in this order do heavy damage to crops, e.g., corn. The insecticide of the subject invention is a novel B. thuringiensis microbe given the specie designation M-7. The spores or crystal of this microbe are useful to control Coleoptera pests in various environments.

Description:
DESCRIPTION 
     BACKGROUND OF THE INVENTION 
     The spore-forming microorganism Bacillus thuringiensis (Bt) produces the best-known insect toxin. The toxin is a protein, designated as δ-endotoxin. It is synthesized by the Bt sporulating cell. The toxin, upon being ingested in its crystalline form by susceptible insect larvae, is transformed into biologically active moieties by the insect gut juice proteases. The primary target is insect cells of the gut epithelium, which are rapidly destroyed. Experience has shown that the activity of the Bt toxin is so high that only nanogram amounts are required to kill suseptible insect larvae. 
     The reported activity spectrum of Bt covers insect species within the order Lepidoptera, which is a major insect problem in agriculture and forestry. The activity spectrum also includes the insect order Diptera, wherein reside some species of mosquitoes and blackflies. See Couch, T. L., (1980) &#34;Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis,&#34; Developments in Industrial Microbiology 22, 61-67; Beegle, C. C., (1978) &#34;Use of Entomogenous Bacteria in Agroecosystems,&#34; Developments in Industrial Microbiology, 20, 97-104. 
     Krieg et al., Z. ang. Ent. (1983) 96: 500-508, describe a Bt isolate named Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles of the order Coleoptera. These are Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni. This is the only known Bt isolate reported to contain such activity; all previously identified Bt strains have had activity against caterpillars (order Lepidoptera) or larvae of certain files (order Diptera). 
     The Krieg et al. Bt isolate is not available for sice-by-side comparison with the Bt isolate of the subject invention. Therefore, since the Krieg et al. Bt isolate is not available to the public, the Krieg et al. publication is not a valid patent law reference under U.S. law. 
     BRIEF SUMMARY OF THE INVENTION 
     Disclosed and claimed is a novel Bacillus thuringiensis isolate which , surprisingly, has activity against beetles of the order Coleoptera but not against Trichoplusia ni, Spodoptera exigua or Aedes aegypti. Included in the Coleoptera are various Diabrotica species (family Chrysomelidae) that are responsible for large agricultural losses. For example, D. undecimpunctata (western spotted cucumber beetle), D. longicornis (northern corn rootworm), D. virgitera (western corn rootworm), and D. undecimpunctata howardi (southern corn rootworm). 
     DETAILED DISCLOSURE OF THE INVENTION 
     The novel Bacillus thuringiensis isolate of the subject invention, designated &#34;M-7,&#34; is unusual in having a unique parasporal body (crystal) which under phase contrast microscopy is dark in appearance with a flat, square configuration. 
     A subculture of B thuringiensis M-7 has been deposited in the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., USA on Feb. 27, 1985. The culture was assigned the accession number NRRL B-15939 by the repository. This deposit is available to the public upon the grant of a patent disclosing it. The deposit is also 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 a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. 
     B. thuringiensis M-7, NRRL B-15939, can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle, the bacteria can be harvested by fist separating the Bt spores and crystals from the fermentation broth by means well known in the art. The recovered Bt spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers and other components to facilitate handling and application for particular target pests. These formulation and application procedures are all well known in the art and are used with commercial strains of B. thuringiensis (HD-1) active against Lepidoptera, e.g., caterpillars. 
     Formulated products can be sprayed or applied onto foliage to control phytophagous beetles, or formulated bait granules containing an attractant and spores and crystals of Bt M-7 can be applied to the soil for control of soil-inhabiting Coleoptera. Formulated Bt M-7 can also be applied as a seed-coating or root treatment or total plant treatment. 
     Against Diabrotica larvae, which feed on the roots of corn, a bait granule containing cucurbitacin (a phagostimulant for diabroticite beetles derived from cucurbits) or other insect attractants and spores and crystals of Bt M-7 can be formulated. These granules can be planted in the row at planting. Also, a formulated product (wettable powder, etc.) can be sprayed directly on foliage to control susceptible adult beetles. 
     Following are examples which illustrate procedures, including the best mode, for practicing the 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. thuringiensis M-7 NRRL B-15939 
     A subculture of B. thuringiensis M-7 NRRL B-15939 can be used to inoculate the following medium known as LB broth: 
     Tryptone: 10 gm 
     Yeast extract: 5 gm 
     NaCl: 5 gm 
     5N NaOH: 0.6 ml 
     Water: 1000 ml 
     As per standard microbiological techniques, the above medium would be sterilized prior to inoculation and the inoculation would be done using aseptic procedures. 
     A procedure that has produced good results is as follows: 
     A series of 150 ml Erlenmeyer flasks containing sterile PWYE medium (peptone 5.0%; yeast extract 0.1%; NaCl, 0.5% in 1 liter of water; adjust pH to 7.5) are inoculated from a petri plate culture of B. thuringiensis M-7, NRRL B-15939. The flasks are incubated at 30° C. on a rotary shaker (200 rpm) overnight. From this starter culture, 300 ml of LB broth in a 2 liter flask is inoculated using 7.5 ml of the starter. The LB-broth flasks are incubated under the same conditions as the starter, but are harvested after 4 days. 
     The above procedure can be readily scaled up to large fermentors by procedures well known in the art. 
     The Bt 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 techniqes, e.g., centrifugation. 
     EXAMPLE 2 
     Testing of B. thuringiensis M-7 NRRL B-15939 Sprores and Crystal 
     B. thuringiensis M-7 NRRL B-15939 spores and crystal, obtained as described above, were tested against various insects. The insect species tested and a summary of the results are listed below in Table 1. 
     
                       TABLE 1______________________________________Summary of Effects of Bacillus thuringiensisStrain M-7 on Larvae of Several Insects                               Feed-   Common                Stage ing In-                                     Mor-Species Name       Order      Tested                               hibition                                     tality______________________________________Trichoplusia   Cabbage    Lepidoptera                         L1    -     -ni      LooperSpodoptera   Beet army- Lepidoptera                         L1    -     -exigua  wormDiabrotica   Western spot-              Coleoptera L2    +     +undecim-   ted cucum-punctata   ber beetleAedes   Yellow fever              Diptera    L1    -     -aegypti mosquito______________________________________ 
    
     The method used to test for D. undecimpunctata (WSCB) activity consisted of spraying a spore/crystal suspension onto leaf discs of lettuce in a spray tower apparatus. (The larvae of this species are reared on lettuce leaves.) The spray is dried in a laminar flow hood and placed in a container on moist filter paper. Ten larvae of WSCB are added and the containers are incubated at 25° C. and 14 hr photoperiod. Fresh treated discs are added as needed. Inhibition of feeding is noted and mortality is recorded at 5 and 7 days. Results of 2 bioassays are given in Table 2. 
     
                       TABLE 2______________________________________Results of 2 Bioassays of Bacillus thuringiensis M-7Against Second InstarDiabrotica undecimpunctata U. at 7 Days Post-Inoculation         Avg. no. leafTreatment     discs consumed/rep.                       % Mortality______________________________________Exp 1Control       3              7.5 ± 15.04.3 × 10.sup.7 spores/ml         &lt;1            27.5 ± 9.64.3 × 10.sup.8 spores/ml         0             62.5 ± 26.3Exp 2Control       1             12.5 ± 12.61 × 10.sup.6 spores/ml         &lt;1            30.0 ± 8.21 × 10.sup.7 spores/ml         0             50.0 ± 21.6______________________________________ 
    
     Recent tests have shown that B. thuringiensis M-7 NRRL B-15939 is also active against the yellow mealworm, Tenebrio molitor. This beetle is a specie of the family Tenebriondae, which is in the order Coleoptera.