NOVEL NEMATICIDAL BACILLUS STRAIN AND METABOLITE AND METHODS OF USE THEREOF

Abstract: The present invention relates to a newly discovered Bacillus sp. strain which exhibits pesticidal activity. The supernatant of cultures of this novel isolate are also effective pesticidal agents. Also provided are methods of using the novel strain or supernatant for treatment or prevention of disease in plants or animals.

Description: NOVEL NEMATICIDAL BACILLUS STRAIN AND METABOLITE AND METHODS OF USE THEREOF Technical Field Of The Invention This invention is in the field of biopesticides. More particularly, the present invention describes a novel, pesticidal strain of the bacterium Bacillus sp. which is active against nematodes. The bacterial strain of the present invention is also active against cockroach. This novel Bacillus sp. strain also produces a secondary metabolite in its supernatant which can be used as a biocontrol agent in the treatment and prevention of nematode infection in plants or animals and in the treatment of cockroach infestations. Background Of The Invention Every year 250 to 350 million dollars of chemical pesticides are used to control nematode infections on plants. Synthetic chemical pesticides are relatively expensive and, because of their toxicity to humans and wildlife, many have been banned from use. Many of the chemicals used for plant parasitic nematode control are toxic to humans, wildlife and other nontarget species. Also, some have leaked into the ground water. As a result, much research has been concentrated in the area of biopesticides which have the advantage of being cheaper to develop and safer for the environment. One commonly-used biopesticide is the gram-positive bacterium Bacillus thuringiensis. Pesticidal B. thuringiensis strains are known to produce crystal proteins during sporulation which are specifically toxic to certain orders and species of insects and nematodes. (See, e.g., U.S. Patent No. 4,999,192 and U.S. Patent No. 5,208,017). These proteinaceous endotoxins produced by B. thuringiensis also act as insecticidal agents against corn rootworm and other beetles. For instance, delta-endotoxin is synthesized by SUBSTITUTE SHEET (RULE 28) the B. thuringiensis sporulation cell and, upon ingestion by susceptible larvae, is transformed into a biologically-active moiety that destroys the gut epithelium of the insect. (See, e.g., U.S. Patent No. 5,427,786 to Payne et al). Although B. thuringiensis endotoxins have been shown to be effective pesticides as purified crystals, washed cell pellets and expressed proteins (e.g., U.S. Patents Nos. 5,093,120 to Edwards et al. and 5,151,363 to Payne), none of their supernatants have exhibited pesticidal activity with the exception of alpha-, gamma-, sigma- and beta-exotoxins. One B. thuringiensis thermostable metabolite, termed beta-exotoxin, has also been shown to have pesticidal properties. Burgjeron and Biache (1979), Entomophaga 11:279- 284 report a beta-exotoxin that is active against the Colorado potato beetle (Leptinotarsa decemlineata) . In addition, the known B. thuringiensis beta-exotoxin exhibits non-specific pesticidal activity; killing not only nematodes, but flies, armyworms and corn rootworms as well. Sigma exotoxin has a structure similar to beta-exotoxin and is active against Colorado potato beetle (Argauer et al. (1991) J. Entomol. Sci. 26:206-213). Alpha- exotoxin is toxic against larvae of Musca domestica Cluthy (1980) FEMS Microbiol. Lett. 8:1-7. Gamma-exotoxins are various proteolytic enzymes, chitinases, and proteases. The toxic effects of gamma-exotoxins are only expressed in combination with beta- or delta- exotoxin. Forsberg et al. (1976) "Bacillus thuringiensis: Its Effects in Environmental Quality," National Research Council of Canada. Stonard et al. (1994) ACS Symposium Series 551:25 report a water soluble secondary metabolite active against corn rootworm in the supernatant of a Bacillus cereus strain. Thus, there remains a need for pesticidal Bacillus sp. strains which produce non- exotoxin, non-proteinaceous, active metabolites in their supernatant. There is also a need for a biocontrol agents which are specifically active against nematodes. Disclosure Of The Invention An isolated, pure culture of a novel Bacillus chitinosporus strain AQ746 and mutants and variants thereof exhibiting nematicidal activity are provided. Also provided is a nematicidal and insecticidal metabolite obtained from the supernatant of a culture of this novel strain. The present invention also provides methods of treating or protecting plants and animals from nematode infections using the novel strain or its metabolite. Methods of treating cockroach infestations are also provided. SUBSTITUTE SHEΣT (RULf 26) Brief Description of the Drawings Figures 1, 2, 3 and 4 show four gas chromatography plots of strain AQ746 (NRRL Accession No. B-2161) whole-cell cellular fatty acids, derivatized to methyl esters (FAMEs) using the MIDI chromatography. Corresponding tabular results are found in Tables 1, 2, 3 and 4 respectively. Modes For Carrying Out the Invention The present invention provides a novel isolate of a Bacillus chitinosporus strain which exhibits pesticidal activity. This strain is designated AQ746 and was isolated from plant roots and soil. This strain was deposited with the NRRL on August 28,1996 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure under Accession No. B-21618. Another aspect of the invention is a metabolite that is effective in killing nematodes. The metabolite produced by the bacterial strain of the subject invention is unique because it is active against nematodes and cockroaches, but inactive against flies, corn rootworm, beet armyworm. This novel strain and its insecticidal and nematicidal metabolite have many advantages over chemical pesticides, particularly with respect to cost and environmental safety. Definitions As used herein, the term "insects" includes all organisms included in the class "Insecta " "Pre-adult" insects refers to any form of an organism prior to the adult stage, including, for example, eggs, larvae and nymphs. "Insecticidal" refers to the ability of a substance to increase mortality or inhibit the growth rate of insects. "Nematicidal" means the ability of a substance to increase mortality or inhibit the growth rate of nematodes. "Pesticidal" refers to the ability of a substance to increase mortality or inhibit the growth rate of insects, nematodes and/or flies. "Stunting" refers to a decreased rate of growth or abnormal development. "Supernatant" refers to the liquid broth remaining when the cells grown in the broth are removed by centrifugation, filtration, sedimentation or any other means known in the SUBSTITUTE SHEET (RUtE 2b) art. The term "culturing" refers to the propagation of organisms on or in media of various kinds. The term "positive control" means a compound known to have pesticidal activity. "Positive controls" include, but are not limited to, commercially-available chemical pesticides. The term "negative control" means a compound known not to have pesticidal activity. Examples of negative controls include water or ethyl acetate. As used herein, an "agent" includes natural or synthetic products, microorganisms, plant extracts and chemicals. A "test compound" is the agent being assayed for nematicidal or insecticidal properties. "Treatment" is an approach for obtaining beneficial or desired results. For the purposes of this invention, beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of infection, stabilized (i.e. not worsening) state of infection, prevent spread of infection, delay or slowing of infection progression, amelioration or palliation of the infection. Thus, the present invention encompasses isolated, pure cultures of AQ746 which exhibit nematicidal and insecticidal activity. The B. chitinosporus strain of this invention may be grown in any conventional growth medium that supports Bacillus sp. Examples of suitable broth for culturing Bacillus sp., include but are not limited to, a broth composed of peptone, dextrose, yeast extract and malt extract and a broth using the same ingredients as well as proflo cottonseed extract and soy flour. Solid substrates are also suitable for growing Bacillus sp., strains. Growth procedures may also be readily scaled up to large fermentors by methods well known in the art. Unlike known insecticidal B. thuringiensis isolates, the novel strain of this invention produces a nematicidal and insecticidal agent which is found in the supernatant when this strain is grown in culture. The supernatant may be obtained by any conventional means including centrifugation, filtration, sedimentation or the like. The invention also encompasses methods of treating plants or animals infected with susceptible organisms with the novel Bacillus strain or its metabolite. The nematicidal and insecticidal metabolite of this invention may be formulated for use as a treatment by any method known in the art. For instance, the metabolite may be employed as a liquid or as a wettable powder, in granules or in dusts. Liquid formulations may be aqueous-based or non-aqueous-based and employed as foams, gels, suspensions, emulsifiable concentrates and the like. The formulations may be mixed with various materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical SUBSTITUTE SHEET P E 2b) materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants and the like. Another aspect of the invention provides mutants or variants of the AQ746 strain which retain nematicidal and insecticidal activity and produce a nematicidal and insecticidal metabolite in their supernatant. Such mutants or variants have altered genotypic or phenotypic characteristics, and may be produced by genetic manipulation techniques well known in the art. (See, e.g., U.S. Patent No. 4,190,495; U.S. Patent No. 4,968,619; Edward A. Birge, BACTERIAL AND BACTERIOPHAGE GENETICS (1981); and Brian B. Spear, BIOTECHNOLOGY IN AGRICULTURAL CHEMISTRY, 204-214 (1987)). Examples of genetic mutants include, but are not limited to, mutants selected for phenotype by classical genetic methods and mutants created using recombinant techniques. All patents and publications cited herein are incorporated by reference. The following examples are provided to illustrate the invention. These examples are not to be construed as limiting. Examples Example 1 Culturing of novel Bacillus strain A Bacillus strain isolated in 1993 from wheat roots in Saskatchewan, Canada and identified as TV8 was grown in two commonly used Bacillus culture media. Medium 1 contained peptone, dextrose, yeast extract and malt extract. Medium 2 contained the same ingredients and, in addition, proflo cottonseed extract and soy flour. One day old streaked cultures were used to inoculate either 250 mL baffled shake flasks or 15 mL culture tubes. Flasks or tubes were shaken at 200 rpm at 29°C for 5 days. To assay pesticidal activity, 35 mL of culture broth were centrifuged at 5,200 m for 20 minutes and the supernatant used in microassay described in Example 2. Identification and Characterization of the Bacillus strain Using the Biolog microplate panel for identification (Biolog, Inc., Hayward, CA) and Microbial Identification System (MIDI) (Microbial Identification System, Inc., Newark, DE), the strain was identified as Bacillus chitinosporus twice by MIDI (0.376 similarity index), but was also identified once as B. megaterium (0.298), B. alvei (0.364) and B. brevis (0.336). The MIDI profiles are shown in Tables 1 to 4 and in Figures 1 to 4. Isolates were identified based on whole-cell fatty acids derivatized to methyl esters and analyzed by gas chromatography using MIDI. Isolates were grown on TSA (BBL) petri plates at 28°C for 24 hours and the cells were then harvested. One mL of a methanolic NaOH (15% w/v) in 50% (v/v) methanol was added and the cells were saponified at 100°C for 30 minutes. Esterification of fatty acids was performed with 2 mL of 3.25 N HCl in 46% (v/v) methanol at 80°C for 10 minutes. The fatty acid methyl esters were extracted into 1.25 mL of 1 :1 (v/v) methyl-tert-butyl ether-hexane and the organic extract washed with 3 mL of 1.2% (w/v) NaOH before analysis by gas chromatography. The gas chromatograph (Hewlett-Packard 5890A) was equipped with a flame ionization detector and capillary column (Hewlett-Packard no. 1909 IB- 102, cross-linked 5% phenyl- methyl silicone; 25 m X 0.22 mmID; film thickness, 0.33 μm; phase ratio, 150) with hydrogen as the carrier gas. Fatty acid methyl ester peaks were automatically integrated by a Hewlett-Packard 3392 integrator and bacterial isolates named using the MIDI Microbial Identification Software (Sherlock TSBA Library Version 3.80). The fatty acid methyl ester profile of Xanthomonas maltophilia ATCC 13637 was used as a reference check for the MIDI determination. See MIDI (1990) "Identification of bacteria by gas chromatography of cellular fatty acids," Technical Note #101. MIDI, Inc., 115 Barksdale Professional Center, Newark, Delaware; Miller, L.T. (1982) "Single derivatization method for routine analysis of bacterial whole cell wall fatty acid methyl esters, including hydroxy acids," J. Clin. Microbiol 16:584-586. SUBSTITUTE SHEET (RIM 26) Table 1 Sherlock Version 1.06 DATA:C96905130A 05-SEP-96 13:38:44 ID 2607 WHEAT 3-TV8 (from slant) Date of run 05-SEP-96 13 13 47 Bottle 23 SAMPLE (AEROBE) RT Area Ar/Rt Respon ECL Name % Comment 1 Comment 2 1 494 337699200 0 023 7 023 SOLVENT PEAK < min rt 1 587 3018 0 019 7 238 < mm rt 2 272 474 0 030 8 821 < mm rt 5 989 2022 0 031 1 005 13 618 14 0 ISO 2 07 * deviates -0 000 Ref -0 001 6 466 11 10 0 032 0 990 14 000 14 0 1 12 ♦ deviates •0 000 Ret -0 001 7 354 12636 0 034 0 969 14 621 15 0 ISO 12 50 ♦ deviates 0 000 Ref -0 000 7 483 60318 0 035 0 966 14 71 1 15 0 ANTEISO 59 48 ♦ deviates 0 000 Ref -0 000 7 897 516 0 034 0 958 15 001 15 0 0 50 ♦ deviates 0 001 Ref 0 001 8 501 1518 0 042 0 947 15 386 16 1 w7c alcohol 1 47 ♦ deviates -0 000 8 876 2946 0 036 0 941 15 625 16 0 ISO 2 83 ♦ deviates -0 001 Ref -0 002 9 083 3858 0 039 0 938 15 757 16 1 wile 3 69 ♦ deviates -0 000 9 463 6840 0 038 0 933 15 999 16 0 6 51 ♦ deviates -0 001 Ref -0 002 10 104 672 0 037 0 925 16 387 ISO 17 1 wlOc 0 63 ♦ deviates -0 000 10 254 1044 0 044 0 923 16 477 Sum In Feature 5 0 98 ♦ deviates 0 001 17 1 ISO I/ANTEI B 10 506 3228 0 041 0 920 16 630 17 0 ISO 3 03 ♦ deviates 0 001 Ref -0 001 10 658 5514 0 041 0 919 16 722 17 0 ANTEISO 5 17 ♦ deviates -0 000 Ref -0 002 ****** 1044 Summed Feature 5 0 98 17 1 ISO I/ANTEI B 17 1 ANTEISO BΛ I *ECL Solvent Ar Total Area Named Area % Named Total Amnt Nbr Rev ECL Deviation Ref ECL Shift 337699200 10222 10222 100 00 97981 9 0 001 0 001 TSBA IRev 3 80)] Bacillus 0 298 B megaterium 0 298 B m GC subgroup B* 0 298 B m GC subgroup A* 0 204 B brevis* 0 296 B alvei* 0 270 CLΓN [Rev 3 80)] Micrococcus 0 189 M leuteus 0 189 M 1 GC subgroup A 0 189 RHIZ-1 [Rev 1 0] * NO MATCH * SUBSTITUTE SHEET (RULE 28) Table 2 Sherlock Version 1.06 DATA:C96822290A 22-AUG-96 09:28:17 ID 2340 WHEAT 3-TV8 (from slant) Date of run 22-AUG-96 09 03 24 Bottle 6 SAMPLE (AEROBE) RT Area Ar/Rt Respon ECL Name % Comment 1 Comment 2 1 491 348839400 0 024 7 025 SOLVENT PEAK < mm rt 2 268 468 0 025 8 826 < mm rt 5 988 1800 0 035 1 030 13 618 14 0 ISO 2 39 *devιates -0 000 Ref 0 010 6 465 972 0 034 1 014 13 999 14 0 1 27 ♦ deviates -0 001 Ref 0 009 7 353 10686 0 034 0 989 14 621 15 0 ISO 13 63 ♦ deviates 0 000 Ref 0 010 7 483 44604 0 035 0 985 14 712 15 0 ANTEISO 56 69 ♦ deviates 0 001 Ref 0 010 7 895 480 0 031 0 975 15 000 15 0 0 60 ♦ deviates -0 000 Ref 0 009 8 500 888 0 037 0 961 15 386 16 1 w7c alcohol 1 10 ♦ deviates -0 000 8 876 2724 0 038 0 953 15 625 16 0 ISO 3 35 *devιates -0 001 Ret 0 007 9 082 2688 0 038 0 948 15 757 16 1 wile 3 29 ♦ deviates -0 000 9 463 6228 0 038 0 941 15 999 16 0 7 56 ♦ deviates -0 001 Ref 0 007 10 106 498 0 037 0 929 16 388 ISO 17 I wlOc 0 60 ♦ deviates 0 001 10 252 816 0 044 0 926 16 476 Sum In Feature 5 0 97 ♦ deviates 0 000 17 1 ISO I/ANTEI B 10 505 2886 0 040 0 922 16 630 17 0 ISO 3 43 ♦ deviates 0 001 Ref 0 006 10 658 4314 0 041 0 919 16 722 17 0 ANTEISO 5 11 ♦ deviates 0 000 Ref 0 006 18 263 168 0 067 21 210 > max rt * ***** 816 Summed Feature 5 0 97 17 1 ISO I/ANTEI B 17 1 ANTEISO B/i I ♦ ECL Solvent Ar Total Area Named Area % Named Total Amnt Nbr Rev ECL Deviation Ref ECL Shift 348839400 79584 79584 100 00 77519 9 0 001 0 008 TSBA [Rev 3 80)] Bacillus 0 364 B alvei* 0 364 B brevis* 0 339 B lentus 0 220 CLΓN [Rev 3 80)] Micrococcus 0 215 M leuteus 0 215 M 1 GC subgroup A 0 215 SUBSTITUTE SHEET (RULE 28) Table 3 Sherlock Version 1.06 DATA:C96905130A 05-SEP-96 14:29:18 ID 2608 WHEAT 3-TV8 (from frozen) Date of run 05-SEP-96 14 04 30 Bottle 24 SAMPLE (AEROBE) RT Area Ar/Rt Respon ECL Name % Comment 1 Comment 2 1 494 336586200 0 023 7 025 SOLVENT PEAK < mm rt 2 272 576 0 025 8 822 < min rt 5 989 1824 0 032 1 002 13 618 14 0 ISO 2 21 ♦ deviates 0 000 Ref 0 000 6 465 1 1 16 0 040 0 988 13 999 14 0 1 33 ♦ deviates -0 001 Ref 0 000 7 354 10464 0 034 0 969 14 621 15 0 ISO 12 23 ♦ deviates 0 000 Ret 0 001 7 483 50760 0 035 0 966 14 712 15 0 ANTEISO 59 17 ♦ deviates 0 001 Ref 0 001 8 501 1344 0 040 0 949 15 386 16 1 w7c alcohol 1 54 ♦ deviates 0 000 8 876 2676 0 038 0 943 15 625 16 0 ISO 3 05 ♦ deviates -0 001 Ref -0 000 9 082 3336 0 039 0 940 15 757 16 1 wile 3 78 ♦ deviates -0 000 9 463 5490 0 039 0 935 16 000 16 0 6 20 ♦ deviates -0 000 Ref 0 000 10 105 1002 0 063 0 928 16 388 ISO 17 1 wlOc 1 12 ♦ deviates 0 001 10 251 1014 0 042 0 926 16 476 Sum In Feature 5 1 13 ♦ deviates 0 000 17 1 ISO I/ANTEI B 10 505 2538 0 042 0 924 16 630 17 0 ISO 2 83 ♦ deviates 0 001 Ref 0 000 10 657 4872 0 040 0 922 16 722 17 0 ANTEISO 5 42 ♦ deviates -0 000 Ref -0 001 ****** 1014 Summed Feature 1 13 17 1 ISO I/ANTEI B 17 1 ANTEISO BΛ I *ECL Solvent Ar Total Area Named Area % Named Total Amnt Nbr Rev ECL Deviation Ref ECL Shift 336586200 86436 86436 100 00 82875 8 0 000 0 001 TSBA [Rev 3 80)] Bacillus 0 336 B brevis* 0 336 B megaterium 0 290 B m GC subgroup B* 0 290 B m GC subgroup A* 0 214 B alvei* 0 259 CLIN [Rev 3 80)] Micrococcus 0 176 M leuteus 0 176 M 1 GC subgroup A. 0 176 RHIZ-1 [Rev 1 0] * NO MATCH * Table 4 Sherlock Version 1.06 DATA:C94922552A 22-SEP-94 14:54:30 ID 208 176 TV8 Date of run 22-SEP-94 14 29 33 Bottle 24 SAMPLE (AEROBE) RT Area Ar/Rt Respon ECL Name % Comment 1 Comment 2 1 518 169261200 0 021 7 038 SOLVENT PEAK < min rt 6 154 1152 0 034 0 973 13 618 14 0 ISO 240 ♦ deviates -0 000 Ret 0 003 6 642 570 0 042 0 969 14 000 14 0 1 18 ♦ deviates -0 000 Ref 0 003 7 546 4722 0 034 0 965 14 621 15 0 ISO 9 74 ♦ deviates 0 000 Ret 0 004 7 678 28926 0 035 0 964 14 712 15 0 ANTEISO 59 62 ♦ deviates 0 001 Ret 0 004 8 712 1230 0 037 0 963 15 387 16 1 w7c alcohol 2 53 ♦ deviates 0 001 9 092 1530 0 041 0 963 15 625 16 0 ISO 3 15 ♦ deviates -0 001 Ref 0 002 9 303 2574 0 045 0 963 15 758 16 1 wile 5 30 ♦ deviates 0 001 9 686 2034 0 037 0 963 15 999 16 0 4 19 ♦ deviates -0 001 Ref 0 001 10 336 618 0 046 0 964 16 388 ISO 17 1 wlOc 1 27 ♦ deviates 0 001 10 485 1164 0 045 0 964 16 477 Sum In Feature 5 2 40 ♦ deviates 0 001 17 1 ISO I/ANTEI B 10 741 1278 0 041 0 964 16 630 17 0 ISO 2 63 ♦ deviates 0 001 Ref 0 002 10 895 2712 0 041 0 964 16 722 17 0 ANTEISO 5 59 ♦ deviates -0 000 Ret 0 001 16 636 2238 0 1 10 20 087 >max rt 18 012 1488 0 129 20 896 >max rt 19 467 540 0 033 21 751 >max rt 19 529 408 0 047 21 787 >max rt 19 932 1404 0 092 22 024 >max rt ****** 1 164 Summed Feature 5 2 40 17 1 ISO I/ANTEI B 17 1 ANTEISO B/i I ♦ ECL Solvent Ar Total Area Named Area % Named Total Amnt Nbr Rev ECL Deviation Ref ECL Shift 169261200 48510 48510 100 00 46780 8 0 001 0 003 TSBA [Rev 3 80)] Bacillus 0 37f i (not an approved name) B chitinosporus 0 37( ) (not an approved name) B brevis^ 0 31 - ! B megaterium 0 24 ' B m GC subgroup B** 0 24" 1 B m GC subgroup A* 0 20C 1 CLIN [Rev 3 80] ♦ NO MATCH ♦ Comparison with TSBA [Rev 3 80] Bacillus-chitinosporus (not an approved name) Distance 4523 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 13 0 ANTIESO x+ 14 0 ISO x+ 14 0 x + 15 0 ISO +x 15 0 ANTIESO +x 15 0 x+ 16 1 w/c alcohol +x 16 0 ISO +x 16 00 wile x+ 16 0 *_ ISO 17 1 wlOC x+ 17 0 ISO +x 17 0 ANTIESO +x SUMMED +x FEATURE 5 In addition to being characterized by MIDI, isolates were assessed for carbon substrate utilization using the Biolog microplate panel (Biolog, Inc., Hayward, CA). The Biolog microplate is comprised of prefilled and dried panel wells with 95 different carbon substrates. Isolates were grown in liquid medium at 28°C for 24 hours and then the cell suspensions were washed in 0.85% saline. The washed cell suspension was used to inoculate each panel well of a Gram Position Microplate (Biolog, Inc., Hayward, CA). After incubating the Microplate at 28°c for 24 hours, carbon utilization reactions were assessed. Substrate utilization profiles were then compared to the Biolog Gram-Positive Data Base (release 3.50) and matched to the closest specie. See, Bochner, B.R. (1991) "Identification of over 500 Gram-negative species by a single test panel," Amer. Clin. Lab. April, p.14; Bochner, B.R. (1989) "Sleuthing out bacterial identities," Nature 339:157-158. AQ746 gave a score of 0.445 in the Biolog test (Bacillus pasteuriϊ). Example 2 Assaying pesticidal activity of Bacillus strain Assays were performed in 96-well microplates. Each well contained a solid agar substrate, a test organism suspension and either a positive control, a negative control or supernatant obtained as described in Example 1 from the Bacillus sp. strain. Test organisms used were either pre-adult corn rootworms (e.g., Diabrotica virgifera, D. longicornis or D. undecimpundata), pre-adult beet armyworms (Spodoptera exigua), pre-adult flies (Drosophila melanogaster), the N2 strain of the nematode Caenorhabditis elegans or pre-adult German cockroach (Blatella germanica). Test organisms were diluted in 0.1% agar to a concentration of approximately 5 organisms/25 uL. To assay nematicidal activity, an agar substrate was prepared for the wells of the microplate by pouring 975 mLs of deionized water into a 1 liter bottle. Three grams of sodium chloride (NaCl), 2.5 grams of peptone, 17.0 grams of bactoagar and 1.0 mL of a 5 mg/mL solution of cholesterol in ethanol were added to the bottle. The contents were sterilized at a minimum of 15 psi, 250F° for 30 minutes. After sterilization, 1.0 mL of a filter-sterilized IM CaCl 2 solution, 1.0 mL of a filter sterilized IM MgSO 4 solution, and 25.0 mL of a filter sterilized, IM, pH 6.0 solution of KH 2 HPO 4 were added. 200 uL was dispensed into each well of a 96-well microplate, and the plates allowed to cool. For SUBSTITUTE SHEET (RULE 28) testing the other insects, the same procedure was used, except a wheat-germ-based medium was dispensed into the 96-well microplate. The formulation for the wheat-germ-based medium is prepared as follows. 1 L of wheat germ diet is prepared with 14.5 g agar, 13.8 g alphacel (celufil), 38.5 g sucrose, 32.25 g casein, 27.48 g wheat germ, 9.25 g Wesson salt mix, 1.0 g methyl paraben, 1.0 g sorbic acid, 0.4 mL linseed oil, 0.064 g streptomycin, 0.064 g chlorotetracycline, 9.0 g Vanderzants vitamin mix, 3.0 mL of propionic acid/phosphoric acid solution (418 mL propionic acid and 42 mL of phosphoric acid and 540 mL water), and 5.0 mL of 190 proof EtOH and 840 mL of water. The components are dissolved and then sterilized. A 1 ppm solution of Avid® was used as a positive control. Deionized water was used as a negative control. Two replicates of test sample or control were used for each assay. 40 uL of supernatant sample prepared as described in Example 1 was dispensed onto the agar surface of microplate wells. A 25 uL aliquot of the pre-adult organism suspension was dispensed into each sample well. The plates were then placed in a fume hood to dry for approximately 2 to 3 hours until the agar solution was dried, but the eggs were not overdried. The microplate was sealed with an airtight substance such as Mylar®, and each well ventilated with a pin press. The plates were incubated at 27 C° for up to 7 days. After incubation, wells were scored by noting neonate mortality or the degree of larval development. Sample wells containing all dead or stunted larvae were given a score of 1, wells containing some dead and other severely stunted larvae were given a score of 2, live but stunted larvae were scored as 3 and sample wells containing no dead larvae were given a score of 4. Scores were averaged among replicates within each sample. Results are summarized in Table 5. SU8ST1TUTE SHEET (RULE 26) Table 5 Pesticidal Activity of AQ746 Supernatant Score Rating These results show that the novel B. chitinosporus strain produces a metabolite in its supernatant which is effective against nematodes and cockroaches, but not against corn rootworm, armyworm or flies. Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameter, concentrations and conditions without departing from the spirit and scope of the invention and without undue experimentation. While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows the scope of the appended claims. SUBSTITUTE SHEW 26)

Claims: CLAIMS What is claimed is: 1. An isolated, pure culture of a novel strain of Bacillus chitinosporus, designated AQ746, NRRL Accession No. B-21618 and mutants and variants thereof. 2. The culture of claim 1 wherein the novel strain of Bacillus chitinosporus or the mutants or variants thereof exhibit nematicidal and insecticidal activity but do not exhibit pesticidal activity against corn rootworm, beet armyworm or fruit fly. 3. A non-exotoxin, non-proteinaceous metabolite obtained from the supernatant of a culture of a novel strain of Bacillus chitinosporus, AQ746, NRRL Accession No. B- 21618 that exhibits nematicidal and insecticidal activity. 4. A method of treating or preventing nematode infection in a plant or an animal comprising administering an effective amount of the culture of claim 1 to a plant or an animal in need of such treatment or protection. 5. A method of treating or preventing nematode infection in a plant or an animal comprising administering an effective amount of the culture of claim 2 to a plant or an animal in need of such treatment or protection. 6. A method of treating or preventing nematode infection in a plant or an animal comprising administering an effective amount of the metabolite of claim 3 to a plant or an animal in need of such treatment or protection. 7. A method of treating or preventing cockroach infestations comprising administering an effective amount of the culture of claim 1. SUBSTITUTE SHEET (RULE 28) 8. A method of treating or preventing cockroach infestations comprising administering an effective amount of the culture of claim 2. 9. A method of treating or preventing cockroach infestations comprising administering an effective amount of the culture of claim 3. SUBSTITUTE SHEET (RULE 28)

Inventors: GERMIDA JAMES J, HEINS SHERRY D, JIMENEZ DESMOND R, MANKER DENISE C, MARRONE PAMELA GAIL, GERMIDA, JAMES, J., HEINS, SHERRY, D., JIMENEZ, DESMOND, R., MANKER, DENISE, C., MARRONE, PAMELA, GAIL
Applicants: AGRAQUEST INC, GERMIDA JAMES J, HEINS SHERRY D, JIMENEZ DESMOND R, MANKER DENISE C, MARRONE PAMELA GAIL, UNIV SASKATCHEWAN, AGRAQUEST, INC., GERMIDA, JAMES, J., HEINS, SHERRY, D., JIMENEZ, DESMOND, R., MANKER, DENISE, C., MARRONE, PAMELA, GAIL, UNIVERSITY OF SASKATCHEWAN
Application year: 1997
Main classification: A01N63
IPC codes: A01N63, A61K9, A61K35, A61P33, C12N1, C12R1