|||||||||||||||III USOO5229292A United States Patent (19) 11 Patent Number: 5,229,292 Stock et al. 45 Date of Patent: Jul. 20, 1993

(54) BIOLOGICAL CONTROL OF INSECTS losphere, N.J. Fokkema and J. Van Den Heuvel (eds), USING PSEUDOMONAS STRANS Cambridge Univ Pres, Cambridge, pp. 312-334. TRANSFORMED WITH BACILLUS Lindow, S. E., 1986, In: Microbiology of the Phyllos THURINGIENSIS INSECT TOXINGENE phere, N. J. Fokkema and J. Van Den Heuvel (eds), Cambridge Univ. Press, Cambridge, pp. 293-311. 75 Inventors: Carolyn A. Stock, Monona; Thomas Kennell, D. E., 1985, In: Maximizing Gene Expression, J. McLoughlin, Cottage Grove; W. Reznikoff and L. Gold (eds), Butterworth Press, Janet A. Klein; Michael J. Adang, Boston, pp. 101-142. both of Madison, all of Wis. Goldberg et al., 1985, In: Maximizing Gene Expression, 73) Assignee: Stine Seed Farm, Inc., Adel, Iowa W. Reznikoff and L. Gold (eds), Butterworth Press, Boston, pp. 287-314. 21 Appl. No.: 891,305 Cullen et al., 1986, Tibtech, pp. 115-119. Kronstad et al. (1983) J. Bacteriol. 154:419–428. 22 Filed: Jul. 28, 1986 Whiteley et al.: (1982) In: Molecular Cloning and Gene 51) Int. Cl...... C12N 1/21: A01N 63/00 Regulation in Baciliganesan et al. (eds.) pp. 131-144. 52 U.S. C...... 435/252.34; 424/93 N Adang et al. (1985) Gene 36:289-300. 58 Field of Search ...... 435/93, 68, 172.3, 172.1, Thorne et al. (1986) J. Bacteriol. 166:801-811. 435/91, 252.74, 170, 171,253, 254, 320, 849, Held et al. (1982) Proc. Natl. Acad. Sci. USA 911, 69.1, 7.2, 172.5, 252.34, 320.1, 874; 79:6065-6069. 436/83; 935/6, 9, 10, 22, 32, 33, 59, 60, 61, 62, Watrud et al. (1985) In: Engineered Organism in the Environment: Scientific Issues Hafbvorson et al. (eds.) 849, 911 American Society of Microbiology Wash. D.C. (56) References Cited Palleroni and Holmes (1981) Int. J. System. Bacteriol. U.S. PATENT DOCUMENTS 31:479-48. 4,448,885 5/1984 Schnepf et al...... 435/252.33 Primary Examiner-Richard A. Schwartz 4,467,036 8/1984 Schnepf et al...... 435/320. Assistant Examiner-J. L. LeGuyader 4,588,584 5/1986 Lumsden et al...... 424/93 R Attorney, Agent, or Firm-Greenlee and Winner 4,652,628 3/1987 Walfield et al...... 530/324 4,695,455 9/1987 Barnes et al...... 424/93 D (57) ABSTRACT 4,771,31 9/1988 Herrnstadt ...... 536/27 Methods of biological control of agricultural insect pests are disclosed. These methods utilize biological FOREIGN PATENT DOCUMENTS control agents which are genetically altered strains of 0185.005 6/1986 European Pat. Off. . root-colonizing strains of members of the species P. cepacia. These strains are genetically altered by intro OTHER PUBLICATIONS duction of genes encoding insect toxic crystal proteins, Neilson J. of Bacteriol. 1983 pp. 559-566. and are thereby rendered insect toxic. Genetically al Bergey's Manual of Systematic Bacteriology, vol. 1 tered insect toxic bacterial strains are also provided. Eds. Krieg et al., pp. 141, 162, 165, 174, (1984). Panopoulos, N.J., 1986, In: Microbiology of the Phyl 17 Clains, 4 Drawing Sheets U.S. Patent July 20, 1993 Sheet 1 of 4 5,229,292

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5,229,292 1 2 have a major protein component of about 64 kid. No BIOLOGICAL CONTROL OF INSECTS USNG higher molecular weight precursor was identified in PSEUDOMONAS STRAINSTRANSFORMED gels of crystal components; however, an 83 kid protein is WITH BACILLUS THURINGIENSIS INSECT reported to be expressed by san diego CP genes cloned TOXINGENE in Escherichia coli. The coleopteran toxin does not cross react with antiserum raised to lepidopteran specific FIELD OF THE INVENTION strains kurstaki HD-1 or HD-73, and there is little pep The present invention relates in general to biological tide homology between the coleopteran toxin and insect control in plants. More particularly it relates to a HD-73 protein. It is not as yet known whether protox method of protecting plants from insect pests by inocu 10 ins are degraded to produce these toxic proteins in the lating plants or plant parts with strains of Pseudomonas cases of the dipteran and coleopteran specific toxins. cepacia, which are effective plant root or leaf coloniz ers, which have been genetically altered by introduc TABLE 1. tion of a gene encoding an insect toxic crystal protein Insects susceptible to B. thuringiensis insecticidal from a strain of Bacillus thuringiensis. Genetically al 15 protein tered strains of Pseudomonas cepacia and insecticidal COLEOPTERA compositions containing these strains are provided. Popilia japonia (Japanese beetle) BACKGROUND OF THE INVENTION Silophilus granarius (granary weevil) Gram-positive bacteria of the species Bacillus thurin 20 Anthononus grandis (boll weevil) giensis produce proteinaceous crystals that are lethal to Leptinotarsa decemlineata (Colorado potato beetle) a number of insects including agricultural insect pests Pyrrhalta luteola (elm leaf beetle) (Table 1). Reviews are available detailing the microbiol Diabiotica undecimpunctata (Western spotted cucum ogy, toxicology and molecular genetics of Bacillus thu ber beetle) ringiensis including: Sommerville (1973) Ann. N.Y. 25 Haltica tombacina Acad. Sci. 217:93-103; Rogoff and Yousten (1969) Ann. Otiorhynchus sulcatus (black vine weevil) Rev. Microbiol. 23:357-389; Bulla et al. (1975) Ann. Tenebrio molitor (yellow mealworm) Rev. Microbiol. 29:163-190; Dulmage (1981) in Micro Agelastica alni (blue alder leaf beetle) bial Control of Pests and Plant Diseases, Burges (ed.), DIPTERA Academic Press, London, pp. 193-222; Aronson et al. 30 Aedes aegypti (yellow-fever mosquito) (1986) Microbiol. Rev. 50:1-24. atlanticus A number of subspecies of Bacillus thuringiensis CatahS (Table 2) have been described which produce crystal capsius proteins toxic to lepidopteran, dipteran or coleopteran cinereus insects. The crystals are parasporal, forming during 35 communis sporulation, within the cell. Bacillus thuringiensis variet detrius ies display varying spectra of activity. Many subspecies, dorsalis for example kurstaki strains, are toxic primarily for dupreei lepidopterans. The crystals of these subspecies contain a melaninnon non-toxic protoxin of 130-140 kid molecular weight. nigromaculis (pature mosquito) This protoxin is solubilized and degraded to a toxic punctor polypeptide (about 68 kid molecular weight) in the mid sierrens (western treehole mosquito) gut of susceptible insects. The protoxin can also be ... solicitans (brown salt marsh mosquito) solubilized and activated by protease treatment, in vi Aedes sp. tro. Several subspecies, particularly israelensis strains, 45 A. taeniorhynchus (black salt marsh mosquito) display selective insecticidal activity toward Diptera A. tarsalis (i.e., mosquitoes or black flies). Dipteran toxic protein A. tornentor appears to be distinct from the kurastaki crystal protein, A. triseriatus having different overall amino acid composition and A. vexans (inland floodwater mosquito) being immunologically distinct. The major component 50 Anopheles crucians of crystals ofisraelensis strains has been reported to be A. freeborni a 26-28 kid protein, a 65 kd protein, or most recently a A. guadrinaculatus (common malaria mosquito) 130 kid protein (Hurley et al. (1985) Biochem. Biophys. A. sergenti Res. Comm. 126:961-965; Ward et al. (1984) FEBS A. strephensi Lett. 175:377-382; and Armstrong et al. (1985) J. Bac 55 Anopheles sp. teriol. 161:39-46; Visser et al. (-1986) FEMS Microbi Chironomus plunosus (Chironomus: midges, biting) ology Let. 33:211-214). The mosquitocidal toxin found Chirnomus sp. in some kurstaki subspecies is reported to be a 65 kd C. tumni protein (Yamamoto and McLoughlin (1981) Biochem. Culex erraticus Biophys. Res. Comm. 103:414–421) and is believed to be C. inornata distinct from the israelensis mosquitocidal protein. Two C. nigripalus Bacillus thuringiensis variants (Krieg et al. (1983) Z. C. peus Ang. Ent. 96:500-508; Krieg et al. (1984) Ang. Schad C. pipiens (northern house mosquito) lingskde. Pflanzenschutz, Univeltschutz 57:145-150; C quinquefasciatus (C. pipiens fatigans) (southern and Herrnstadt et al. (1986) Biotechnology 4:305-308) 65 house mosquito) have recently been described which display toxicity for C. rustuans. Coleoptera rather than Lepidoptera. Crystals of one of Culex sp. these varieties, designated san diego, are reported to C. tritaeniorhynchus 5,229,292 3 4. C. tarsalis (western encephalitis mosquito) A. pilosaria (pedaria) C. territans Aporia cateraegi (black-veined whitemoth) C, univittatus Archips argySopilus (ugly-nest caterpillar) Culiseta incidens (Culiseta: mosquitos) A. cerasivoranus C. inornata A. crataegana Diamessa sp. A. podana Dixa sp. (Dixa: midges) A. (Cacoecia) rosana Eusimulium (Simulium) latipes (Eusinnlium: gnats) A. xylosteana Goeldichironomus holoprasinus Articia caja Haematobia irritans (horn fly) O Argyrotaenia mariana (gray-banded leaf rollar) Hippelates collusor A. velutinana (red-banded leaf roller) Odagina ornata Ascia (Pieris) monuste oreis Pales pavida Ascotis selenaria Polpomyia sp. (Polpomyia: midges, biting) Atteva aurea (alianthus webworm) Polypediium sp. (Polypedilum: midges) 15 Autographa californica (alfalfa looper) Psorophora ciliata A. (Plusia) gamma P. columiae (confinnis) (Florida Glades mosquito, A. nigrisigna dark rice field mosquito) Autoplusia egena (bean lead skeletonizer) P. ferox Azochis gripusalis Simulium alcocki (Simulium: black flies) 20 Bissetia steniella S. argus Bombyx mori (silkworm) S. cervicornutum Brachionycha sphinx S. damnosum Bucculatrix thurberiala (cotton leaf perforator) S. jenningsi Bupolus piniarius (bupolus; looper) S. piperi 25 Cacoecimorpha pronubana teSCOrip Cactoblastis cactorum (cactus moth) tuberosum Caloptilia (gracillaria) in variabilis unicornutum C. (G) syringella (lilac leaf miner) yerStil C. (G) theiyora verecundum 30 Canephora asiatica vittatun Carponsia niponensis Uranotaenia inguiculata Ceramidia sp. U. lowii Cerapteryx graminis Wyeomyia mitchelli (Wyeomyia: mosquito) Chilo auricilius W. vanduzeei 35 C. sacchariphagus indicus HYMENOPTERA C. suppressalis (rice stem borer, Asiatic rice borer) Athalis rosae (as colibri) Choristoneura fumiferana (spruce budworm) Nematus (Pteronidea) ribesii (imported currantworm) C. murinana (fir-shoot roller) Neodiprion banksianae (jack-pine fly) Chrysodeixis (plusia) chalcites (green garden looper) Priophorus tristis Clepsos spectrana Pristiphora erichsonii (larch sawfly) Cnaphalocrocis medinalis LEPIDOPTERA Coleotechnites (Recryaris) milleri (lodgepole needle Achaea janta (croton caterpillar) miner) Achrois grisella (lesser wax moth) C. nanella Achyra rantalis (garden webworm) 45 Colias eurytheme (alfalfa caterpiller) Acleris variana (black-headed budworm) C. lesbia Acrobasis sp. Colotois pennaria Acrolepis aliella Crambus bonifatellus (fawn-colored lawn moth, sod Acrolepiopsis (Acrolepis) assectella (leek moth) webworm) Adoxohyes orana (apple leaf roller) 50 C. sper yellus Aegeria (Sanninoidea) exitiosa (peach tree borer) Crambus spp. Aglais urticae Cryptoblabes gnidiella (Christmas berry webworm) Agriopsis (Erannis) aurantiaria (Erannis: loopers) Cydia funebrana A. (E.) leucophaearia C (Grapholitha) molesta (oriental fruit moth) A. marginria 55 C. (Laspeyresta) pononella (codling moth) Agrotis ipsilon (as ypsilon) (black cutworm) Datana integerrina (walnut caterpillar) A. Segetum D. ministra (yellow-necked caterpillar) Alabama argillacea (cotton leafworm) Dendrolimus pini Alsophila aescularis D. Sibiricus A. ponetaria (fall cankerworm) Depressaria narcella (a webworm) Amorbia essigana Desmia funeralis (grape leaf folder) Anadevidia (Plusia) peponis Diachrysia (Plusia) orichalcea (a semilooper) Anisota Senatoria (orange-striped oakworm) Diacrisia virginica (yellow wollybear) Anonis flava Diaphania (Margaronia) indica A. (Cosmophila) sabulifera 65 D. nitidalis (picklewormi) Antheraea pernyi Diaphora mendica Anticarsia gemmatalis (velvetbean caterpillar) Diatraea grandiosella (southwestern corn borer) Apocheina (Biston) hispedaria D. saccharalis (sugarcane borer) 5,229,292 5 6 Dichomeris marginella (juniper webworm) Loxostege commixtalis (alfalfa webworm) Drymonia ruficornis (as chaonia) L. Sticticalis (beet webworm) Drymonia sp. Lymantria (Porthetria) dispar (gypsy moth) (Ly Dryocampa rubicunda (greenstriped mapleworm) mantria: tussock moths) Earias insulana 5 L. monacha (nun-moth caterpillar) Ectropis (Boarmia) crepuscularia Malacosoma americana (eastern tent caterpillar) Ennomos subsignarius (elm spanworm) M. disstria (forest tent caterpillar) Ephestia (Cadra) cautella (almond moth) M. fragilis (=fragile) (Great Basin tent caterpillar) E. elutella (tobacco moth) M. neustria (tent caterpillar, lackey moth) E. (Anagasta) kuehniella (Mediterranean flour moth) 10 M. neustria var. testacea Epinotia tsugana (a skeletonizer) M. pluviale (western tent caterpillar) Epiphyas postvittana Mamerstra brassicae (cabbage moth) Erannis defoliaria (mottled umber moth) Manduca (Inotoparce) quinquemaculata (tomato horn E. tiliaria (linden looper) worm) Erinnysis elo 15 M. (I) sexta (tobacco hornworm) Erigaster henkei Maruca testulalis (bean pod borer) E. lanestris Melanolophia imitata Estigmene acrea (slat marsh caterpillar) Mesographe forficialis Eublenna annabilis Mocis repanda (Mocis: semilooper) Euphydryas chalcedonia 20 Molippa sabina Eupoecilia ambiguella Monema flavescens Euproctis chrysorrhoea (Nygmi phaeorrhoea) (brown Mythimna (pseudaletia) unipuncta (armyworm) tail moth) Nephantis serinopa E. fraterna Noctura (Triphaena) pronuba E. pseudoconspersa 25 Nomophila noctuella (lucerne moth) Eupterote fabia Nymphalis antiopa (morning-cloak butterfly) Eutromula (simaethis) pariana Oiketicus moyanoi Euxoa messoria (dark-sided cutworm) Onnatopteryx texana Galleria mellonella (greater wax moth) Operophtera brumata (winter moth) Gastropacha quercifolia 30 Opsophanes sp. Halisaota argentata O. fagata H. caryae (hickory tussock moth) Orgvis (Hemerocampa) antiqua (rusty tussock moth) Harrisina brillians (western grapeleaf skeletonizer) O. leucostigma (white-marked tussock moth) Heyda nubiferana (fruit tess totrix moth, green bud O. (H.) pseudotsugata (Douglas-fir tussock moth) worm) 35 O thyellina Heliothis (Helicoverpa) armigera (Heliothis = - Orthosia gothica Cloridea) (gram pod borer) Ostrinia (Pyrausta) nubilalis (European corn borer) H. (H.) assulta Palacrita vernata (spring cankerworm) Heliothis peltigera Pammene juliana H. virescens (tobacco budworm) 40 Pandemis dumetana H. viriplaca P. pyrusana H. zea (cotton bollworm, corn earworm, soybean Panilis flannea podworm, tomato fruitworm, sorghum headworm, Papilio cresphontes (orange dog) etc.) m P. denoleus Hellula undalis (cabbage webworm) 45 P. philenor Herpetogramma phaeopteralis (tropical sod webworm) Parallipsa (Aphemia) gularis Heterocampa guttivitta (saddled prominent) Paralobesia viteana H. manteo (variable oak leaf caterpillar) Paramyelois transitella Holcocera pulverea Parnara guttata Homoeosoma electellum (sunflower moth) 50 Pectinophora gossypiella (pink bollworm) Honona magnina Pericallis ricini Hyloicus pinastri Peridrina saucia (variegated vutworm) Hyphantria cunea (fall worm) Phalera bucephala Hypogymna norio Phlogophora meticulosa Itame (Thamnonona) wauaria (a spanworm) 55 Phryganidia californica (California oakworm) Junonia coenia (buckeye caterpillars) Phthorinaea (= Gnorimoschema) operculella (potato Kakivoria flavofasciata tuberworm) Keiferia (Gnorimoschema) lycopersicella (tomato pin Phylonorycter (Lithocolletis) blancardella (spotted ten worm) tiform leafminer) Lacanobia (Polia) olercea 60 Pieris brassicae (large white butterfly) Landina athasaria pellucidaria P. canidia sordida L. fiscellaria fiscellaria (hemlock looper) Prapae (imported cabbageworm, small white butter L. fiscellaria lugubrosa (western hemlock looper) fly) L. fiscellaria sonniaria (western oak looper) Plathypena scabra (green cloverworm) Lampides boeticus (bean butterfly) 65 Platynota sp. Leucona (Stilpnotia) salicis (satin moth) P. sultana L. wiltshirei Playptilia carduidactyla (artichoke plume moth) Lobesia (= Polychrosis) botrana Plodia interpunctella (Indian-meal moth) 5,229,292 7 8 Plutella xylostella as maculipennis (diamond-backed Potamophylax rotundipennis moth) Prays citri (citrus flower moth) TABLE 2 P. oleae (olive moth) Bacillus thuringiensis subspecies Pseudoplusia includens (soybean looper) 5 Gene Pygaera anastomosis Subspecies Location Toxicity? Availability. alesti P L USDA, ATCC Rachiplusia ou aizawaii P L., D. USDA Rhyacionia boliana (European pine shoot moth) canadensis USDA Sabulodes caberata (omonvorous looper) coineri USDA Samia cynthis (cynthis moth) 10 dakota L USDA darnstadiensis P D USDA Saturnia pavonia dendrolinus C USDA, ATCC Schirzura concinna (red-humped caterpillar) entonocidus C L USDA, ATCC Schoenobius bipunctifer finitimus P, C L USDA, ATCC Selenephera lunigera galleriae P L USDA, ATCC 5 indiana L USDA Sesamia inferens israelensis P D ATCC Sibine apicalis kenyae L., D USDA Sitotriga cerealella (Angoumois grain moth) kurstaki K-1 (HD-1) P L., D USDA, ATCC Sparganothis pilleriana kurstaki K-73 (HD73) P L USDA, ATCC kunaniotoensis USDA Spilonota (Tmetocera) ocellana (eye-spotted bud kyushuensis C D USDA moth) 20 norrisoni p L USDA Spilosoma lubricipeda (as methastri) ostriniae L USDA S. virginica (yellow woolybear) pakistani L USDA Spilosoma sp. san diego C 4. Soto P USDA, ATCC Spodoptera (Prodenia) eridania (southern armyworm) subtoxicus P, C USDA, ATCC S. exigua (beet armyworm, lucerne caterpillar) 25 tenebrionis C DSM S. frugiperda (fall leafworm) thompsoni P USDA thuringiensis P, C L., D. ATCC S. littoralis (cotton tochigiensis USDA S. litura tolworthi P L USDA S. mauritia (lawn armyworm) tohokuensis USDA S. (P) ornithogalli (yellow-striped armyworm) 30 toumanoffi P USDA S. (P.) praefica (western yellow-striped armyworm) wuhenensis P USDA Sylleptederogata Crystal protein gene located on a plasmid (P), on the chromosome (C) or both. See Aronson et al. (1986) Microbiol. Rev. 80:1-24. S. silicalis L = Lepidopteran; D = Dipteran; C = Coleopteran; not all strains of any one Symmerista canicosta variety may display the same toxicity range; the absence of a notation in this column does not indicate that the strain is nontoxic to insects. Thaumetopoea pityocampa (pine processionary cater 35 Strains of these varieties are publicly available from depositories: USDA = United pillar) States Department of Agriculture, Agricultural Research Service, Cotton Insect T processionea Research, P.O. Box 1033, Brownsville, Texas. ATCC = American Type Cuiture Collection, 12301 Parklawn Drive, Rockville, Maryland. DSM = Deutsche Sanrn T. wauaria (currant webworm) lung von Mikroorganismen, Schnitsphainstrasse Darmstadt, Federal Republic of T. wilkinsoni Germany. Strains of Bacilius thuringiensis varieties are also available from Northern Regional Research Laboratory, 1815 North University Street, Peoria, Illinois; and Thymelicus lineola (European skipper) from The Collection of Strains of Bacillus thuringiensis and Bacilius sphaericus, Thyridopteryx ephemeraeformis (bagworm) Laboratoire de Lutte Biologique (II), H. de Barjac, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France. Tineola bisselliella (webbing clothes moth) B. thuringiensis var. san diego is described in Herrnstadt et al. (1986) Biotechnology Trotrix viridana (oak tortricid) 4:305-309. Trichoplusia ni (cabbage looper) Udea profundalis (false celery leaftier) 45 A single strain of Bacillus thuringiensis may contain U. rubifallis (celery leaftier, greenhouse leaftier) more than one distinct crystal protein gene (Kron Vanessa cardui (painted lady) stad et al. (1983) J. Bacteriol. 154:419-428). In par V. io ticular, it was demonstrated that B. thuringiensis Xanthopastis timais HD-1 Dipel plasmids contain three distinct crystal Xestia (Anathes, Agrotis) c-nigrum (spotted cutworn) 50 protein genes. These distinct genes may encode Yonomeuta cognatella (= Y. evonymi) (Yponineuta = - toxic proteins with varying insect specificities Hyponomeuta) (Calabrese et al. (1980) Can. J. Microbiol. Y. evonynella 26:1006-1010; and Jarrett (1985).J. Appl. Bacteriol. Y. mahalebella 58:437-448). For example, the presence of multiple Y. malinella (small ermine moth) 55 genes encoding different toxic proteins may be Y. padella (small ermine moth) responsible for the combined lepidopteran and Y. Porrella dipteran toxicity of certain strains of Bacillus thu Zeiraphera diniana ringiensis (Yamamoto and McLoughlin (1981) Bio MALLPHAGA chem. Biophys. Res. Comm. 103:414-421). Bovicola boyis (cattle biting louse) Genes encoding crystal proteins have been identified B. crassipes (Angora goat biting louse) and cloned from several varieties of Bacillus thuringien B. limbata sis, including the protoxin genes from kurastaki strains B. ovis (sheep biting louse) HD-1 and HD-1-Dipel (Whiteley et al. (1982) in Molec Lipeurus caponis (wing louse) ular Cloning and Gene Regulation in Bacili, Ganesan et Menacnathis stramineus (chicken body louse) 65 al. (ed) pp. 131-144; Schnepf and Whiteley (1981) Proc. Menopon gallinae (shaft louse) Natl. Acad. Sci. USA 78:2893-2897 and U.S. Pat. Nos. TRICHOPTERA 4,448,885 and 4,467,036), and HD-73 (Schnepf and Hydropsyche pellucida Whiteley, 1981; Adang et al. (1985) Gene 36:289-300; 5,229,292 10 U.S. Pat. Nos. 4,448,885 and 4,467,036, Schnepf and growth of Agrobacterium and Rhizobium cells. Intro Whiteley), berliner 1715 (Klier et al. (1982) EMBO J. duction of partial protoxin genes proved to be less de 1:791-799), thuringiensis HD2 (Whiteley et al. (1985) in stabilizing and inhibitory while still rendering the cells Molecular Biology of Microbial Differentiation, Hoch and insect toxic. Rhizobium cells carrying partial protoxin Setlow (ed.), American Society of Microbiologists, genes formed root nodules that were toxic to THW. Washington, D.C., p. 225-229), sotto (Shibano et al. It has been reported that a gene encoding the 134 kid (1985) Gene 34:243-251), aizawa (Klier et al.(1985) in molecular weight crystal protein from Bacillus thuringi Molecular Biology of Microbial Differentiation, Hoch and ensis var. kurastaki HD-1 has been introduced into root Setlow (ed.), American Society for Microbiology, colonizing Pseudomonas fluorescens soil isolates. The Washington, D.C., p. 217-224), subtoxicus (Klier al., O crystal protein is reported to be expressed in these ge 1985), israelensis (Sekar (1985) Gene 33:151-158; Ward netically altered isolates, producing insect toxic Pseudo et al.(1984) FEBS Lett. 175:377-382; Thorne et monas fluorescens (Watrud, et al. 1985 supra). al.(1986) J. Bacteriol. 166:801-811) and san diego (Herrnstadt et al.(1986) Biotechnology 4:305-308). SUMMARY OF THE INVENTION The nucleotide sequence of the following crystal S It is an object of the present invention to provide a protein structural genes have been reported: several method for protecting plants from insect pests. genes from kurastaki HD-1-Dipel (Wong et al.(1983) J. It is also an object of the present invention to provide Biol. Chem. 258:1960-1967; Schnepf and Whiteley biological control agents and insecticidal compositions (1985) J. Biol. Chem 260:6264-6272; Thorne et al., containing these agents which are useful in methods for 1986); kurastaki HD-73 (Adang et al. 1985) supra; israe 20 protecting plants from insect pests. lensis mosquitocidal protein gene (Thorne et al., (1986) It is a further object of the present invention to pro supra; dendrolimus crystal protein gene N-terminal vide methods and inoculating compositions for protect (Nagamatsu et al. (1984) Agri. Biol. Chem 48:611-619) ing plants from insect pests. and sotto crystal protein toxic fragment (Shibano et al. It is yet another object of the present invention to (1985) supra. 25 provide genetically altered bacteria which contain and Bacillus thuringiensis crystal proteins are useful as express crystal protein genes of B. thuringiensis. insecticides because they are highly specific in their In one embodiment, the present invention provides a toxicity, being totally nontoxic toward most nontarget method of protecting plants from insect pests which organisms. Since the protoxin crystals must be ingested comprises genetic alteration of a plant root and leaf for toxicity, crystals must be located where they will be 30 colonizing strain of bacterium of the species Pseudomo eaten by target insect larvae. The utility of Bacillus nas cepacia by introducing a gene encoding a crystal thuringiensis insecticidal compositions in limited by the protein of a strain of Bacillus thuringiensis into the plant fact that they must be applied repeatedly to plants to colonizing strain and inoculating plants with an insecti afford protection, as Bacillus thuringiensis is subject to cidally effective concentration of the resultant geneti UV inactivation and washoff by rainfall. A solution to 35 cally altered insect toxic strain of P. cepacia, this problem is the use of other bacteria which have an In another embodiment, the present invention pro affinity for plants (i.e. colonize, proliferate or persist on vides an insecticidal plant protective composition con plants) as vectors for the delivery of insect toxic pro taining an insecticidally effective concentration of a tein. This can be accomplished by the introduction of genetically altered, insect toxic, plant colonizing bacte crystal protein genes into and the expression of crystal 40 rium of the species P. cepacia. protein toxin by plant colonizing bacteria. In yet another embodiment, the present invention In several cases, cloned Bacillus thuringiensis crystal provides genetically altered plant-colonizing strains of protein genes have been expressed in heterologous the species Pseudomonas cepacia into which a gene en strains, including E. coli (Schnepf and Whiteley (1982) coding a crystal protein of a strain of B. thuringiens has supra; Adang et al. (1985) supra; Held et al. (1982) Proc. 45 been introduced thereby rendering the plant-colonizing Natl. Acad. Sci. USA 79:6065-6069; Thorne et al. strains toxic to insects of the orders Lepidoptera, Dip (1986) supra), B. subtilis (Held et al. (1985) supra; tera or Coleoptera. Thorne et al. (1986) supra, Rhizobium and Agrobacte In a preferred embodiment, the present invention rium strains (U.S. patent application Ser. No. 756,355 provides genetically altered strains of plant colonizing (Adang et al.), filed Jul. 18, 1985) and Pseudomonas 50 Pseudomonas cepacia type Wisconsin into which a gene fluorescens (Watrud et al. (1985) in Engineered Organism encoding a crystal protein of a B. thuringiensis strain has in the Environment, Scientific Issues, Halvorson et al. been introduced. Such genetically altered strains of P. (eds.), American Society for Microbiology, Washing cepacia type Wisconsin are particularly useful in meth ton, D.C.). Crystal protein genes have been expressed in ods of biological control of agricultural pests and plant heterologous bacteria under the control of the homolo 55 protection because in addition to being insect toxic they gous CP gene promoters or under the control of heter are good plant colonizers, broad spectrum fungal antag ologous promoters. onists and are non-phytopathogenic. U.S. patent application Ser. No. 756,355 (Adang et As an example of the preferred embodiment, the al.), filed Jul. 18, 1985, describes the introduction of present invention provides genetically altered insect complete and partial Bacillus thuringiensis HD73 crystal 60 toxic strains of the plant root and leaf colonizing bacte protein protoxin genes into strains of Agrobacterium rium P. cepacia 526. and Rhizobium. The complete and partial crystal pro tein coding sequences were cloned into the broad host BRIEF DESCRIPTION OF THE FIGURES range vector prx290 and introduced into these strains. FIG. 1 is a restriction endonuclease map of the rele The crystal protein genes were expressed in these bac 65 vant portion of DNA fragment 208/43-487. This frag teria and cells containing the genes were toxic to Tab ment contains the HD73-like crystal protein gene cod baco Hornworm (THW). Plasmids containing the crys ing region isolated from B. thuringiensis var. kurstaki tal protein genes were unstable in and inhibited the HD-1 Dipel. Restriction sites are indicated using con 5,229,292 11 12 ventional abbreviations. The 5.1 kb SphI fragment is HD73, etc.) for which it has been demonstrated that used in the construction of pSUP487 (vide infra). This such partial protoxins and altered protoxins are toxic to 5.1 kb SphI fragment contains the full-length coding insects on ingestion. region of the HD73-like gene and approximately 900 The term crystal protein (CP) gene or CP coding base pairs of 5'-flanking and 500 base pairs of 3'-flanking sequence refers to the DNA sequence encoding the sequences. insect toxic crystal protein. In cases where the toxin FIG. 2 is a restriction endonuclease map of the rele protein is formed from a precursor protoxin, the term vant portion of DNA fragment 158/51-16. This 10.58 kb describes the sequence encoding the full-length pro fragment contains the HD73 crystal protein gene cod toxin. ing region isolated from B. thuringiensis var. kurstaki O A gene contains a structural portion extending from a HD73. Restriction sites are indicated using conven 5'-start codon (usually ATG) to a 3'-stop codon (TAG, tional abbreviations. This DNA fragment is the source TGA or TAA), which portion encodes the amino acid of crystal protein structural gene sequences used in the sequence of a polypeptide. A gene also contains regula construction of puC5'Bt and subsequently pSKP/Bt. tory sequences, usually external to the structural gene FIG. 3 is a diagram of the construction of plasmid 15 which affect regulation of expression of the structural pSUP487. The SphI fragment containing HD73-like gene. The promoter is the nucleotide sequence adjacent crystal protein sequences is indicated. Restriction sites to the 5'- end of a structural gene which is involved in are indicated as follows: S=SphI; B=BamHI; the initiation of transcription. Promoters contain se E=EcoRI. Antibiotic markers carried by the various quences which insure proper binding and activation of constructions are indicated. The direction of transcrip 20 tion of the crystal protein gene in the construct RNA polymerase, influence where transcription will pSUP487 is indicated by an arrow. start and affect the level of transcription. Promoter FIG. 4 is a diagram of the construction of plasmid sequences can extend several hundred base pairs 5’ to pSKP/Bt. Restriction sites are indicated as follows: the structural gene. B=BamHI; E=EcoRI; H = HindIII; Bg=BglII. Anti 25 It may be desirable to construct chimaeric genes in biotic markers carried by the various constructs are which a structural gene is placed under the regulatory indicated. The HindIII-BamHI fragment that carries control of a heterologous promoter from another gene. the nptI gene promoter region from Tn5 is indicated in The majority of promoters control initiation of tran the pSKP and pSKP/Bt by the letter P. The approxi scription in only one direction, so in order to be placed mately 4.05 kb fragment of pSKP/Bt which carries the 30 under the control of a promoter, a structural gene must nptII promoter/crystal protein structural gene chi be located downstream (3'-direction) of the promoter. maera is indicated. The direction of transcription of the The distance between the promoter and the structural crystal protein sequences in pSKP/Bt is indicated by an gene is believed to be important as well. These con OW. straints are understood in the art. The choice of heterol 35 ogous promoter used in any such chimaeric construc DETAILED DESCRIPTION OF THE tion is dependent on level of gene expression and on the INVENTION kind of selective expression, if any, that is desired. The The following definitions are provided for clarity and chosen promoter must be active in the bacterium in apply throughout the specification and in the claims. which the construction is to be introduced. Crystal protein is used to refer to protein of the paras The term recombinant DNA molecule is used herein poral crystals formed in strains of B. thuringiensis. Crys to distinguish DNA molecules in which heterologous tal protein is the active agent in insect toxic strains of B. DNA sequences have been artificially cleaved from thuringiensis. Crystal protein (CP), also often referred to their natural source or ligated together by the tech as the 7t-endotoxin, may have two forms, a toxin and a niques of genetic engineering, for example by in vitro precursor, higher molecular weight protoxin. The tern 45 use of restriction enzymes or ligation using DNA ligase. crystal protein includes both forms. The toxin is formed The process of cloning a DNA fragment involves from the protoxin by specific cleavage of the carboxy excision and isolation of a DNA fragment from its natu terminal end of the protoxin. In kurstaki strains the ral source, insertion of that DNA fragment into a re protoxin is a 130 kid protein which is activated (cleaved) combinant vector and incorporation of the vector into a in the insect gut to form a 68kd toxin. A protoxin then SO microorganism or cell where the vector and its inserted is composed of an N-terminal portion in which insect DNA fragment are replicated. The term cloned DNA is toxicity resides and a dispensable (for toxicity) carboxy used to designate a DNA fragment or molecule pro terminal portion. Dipteran and coleopteran insect toxic duced by the process of cloning and copies (or clones) crystal proteins may also have toxin and protoxin forms. of the DNA fragment or molecule replicated therefrom. The term partial protoxin refers to any truncated 55 A microorganism that is isolated from a particular protoxin molecule lacking a portion of the carboxy-ter environment is considered to colonize that environ minus of the protoxin but retaining the portion of the ment. Thus, bacteria isolated from plant roots are root protein essential for toxicity, particularly the carboxy colonizing bacteria. The ability of a bacterium to colo terminal region of the toxin. A partial protoxin may or nize a particular environment can also be assessed as the may not have a deletion at the amino terminus of the ability of the bacterium to proliferate or persist in that protein. A partial protoxin may also include altered environment after it is introduced. Thus a bacterium amino acid sequence not in the protoxin, particularly that becomes established and persists on leaf tissue after amino acid sequences appended to the carboxy-terminal inoculation, is considered to colonize leaf tissue. The end of the toxin protein moiety. The primary distinction relative ability of bacterial strains to colonize a particu of a partial protoxin is that it lacks amino acid sequences 65 lar environment can be measured as in Example 5 present in the complete protoxin but that it is toxic to (Table 6). For comparative purposes in this application, some insects. The term partial protoxin is used particu tested isolates that were recoverable from plant root or larly in reference to the lepidopteran toxins (HD-1, rhizosphere at a level of greater than or equal to about 5,229,292 13 14 106 bacteria/g root or soil were considered to be good provides reproducible methods for isolation of P. root and/or rhizosphere colonizers. cepacia type Wisconsin strains. Representative examples The present invention is based on applicants' discov of P. cepacia type Wisconsin, P. cepacia 406 and P. ery that genes encoding insect toxic crystal proteins of cepacia 526 have been placed on deposit with the Amer B. thuringiensis can be introduced into plant colonizing 5 ican Type Culture Collection, 12301 Parklawn Drive, strains of bacteria of the species Pseudomonas cepacia Rockville, Md., with the accession numbers ATCC and that these genes are successfully expressed, render 53266 and ATCC 53267, respectively. P. cepacia type ing the genetically altered strains of P. cepacia toxic to Wisconsin strains are distinguished in that they are good insects. Plant colonizing insect toxic strains of P. cepacia colonizers of plant roots and rhizosphere, as well as are useful as biological control agents for the protection 10 colonizers of plant leaves, are broad spectrum fungal of plants from insect pests. antagonists, particularly of fungi of the genus Fusarium, Members of the species Pseudomonas cepacia are are non-phytopathogenic, and are effective for the pro non-fluorescent, non-denitrifying, nutritionally versatile tection of plants against fungal infection and invasion. P. bacteria, which are further distinguishable from other cepacia type Wisconsin strains are particularly effective species of Pseudomonas by a number of biochemical 15 for the protection of corn plants from the pathogen and nutritional criteria that are summarized in Palleroni Fusarium moniliforme. Although initially isolated from and Holmes (1981) Int. J. System. Bacteriol. 31:479-481 corn root, P. cepacia type Wisconsin strains were found and Palleroni (1983) "Pseudomonadaceae" in Bergey's to colonize the roots of diverse plants including corn, Manual of Systematic Bacteriology, Vol. 1, Krieg (ed), sorghum, rape, sunflower, wheat, alfalfa, cotton, soy Williams and Wilkins, Baltimore, Md p. 140-219. Mem- 20 bean, peas, tomato and French bean. These bacteria bers of the species P. cepacia are ubiquitous to soil envi were also found to colonize the leaves of several differ ronments but do not normally predominate in soil since ent plants, including tobacco, cotton and Cape Primrose they are outgrown by fluorescent pseudomonads. (Streptocarpus). Strains of P. cepacia have also been isolated from rotten P. cepacia type Wisconsin strains have broad spec onions and clinical samples. At one time the designation 25 trun anti-fungal activity against fungi of the classes P. cepacia was limited to phytopathogenic onion iso Ascomycetes (i.e., Sclerotinia spp.), Phycomycetes (i.e., lates. Now, soil isolates, earlier designated P. multivo Pythium spp.), Basidomycetes (i.e., Rhizoctonia spp.) rans, and clinical isolates, earlier designated P. kingii, and Fungi Imperfecti (i.e., Fusarium spp.). are included in the species P. cepacia. U.S. Pat. No. 4,798,723, filed Jul. 28, 1986) reports 30 TABLE 3 the identification of root colonizing strains of non Pseudomonas cepacia type Wisconsin fluorescent, non-denitrifying, nutritionally versatile Strain Source? Pseudomonas strains from unsterile corn root macer 406 site a, Jacques corn parental line 1 isolated oiginally on nutrient agar ates. These corn root isolates have been identified by 526 site a, Jacques corn parental line 86 isolated on classical biochemical and nutritional criteria (see Table 35 King's B medium 4 for examples of assays used) as belonging to the spe 462 site a, Jacques corn parental line 13 isolated on cies P. cepacia. Some of these isolates, those obtained combined carbon medium 531 site b, hybrid corn line 7780 isolated on King's B from corn roots and cornfield soil originating from sites medium near Prescott, Wis., USA, can be grouped and distin 504 site b, hybrid corn line 7780, isolated on guished from other P. cepacia strains. This novel group 40 combined carbon medium of P. cepacia strains has been designated P. cepacia type All typed to P. cepacia using conventional criteria. See Palleroni and Holmes, 1981, and Bergey's Manual of Systematic Bacteriology VI (1984). Wisconsin. Table 3 is a list of several P. cepacia strains Original root material taken from test fields of Jacques Seed Company, Prescott, that have the distinguishing characteristics of P. cepacia Wisconsin. Site a is the field of the experimental station which has been in continu type Wisconsin. All of the strains in Table 3 were iso ous corn cultivation for 40 years. Site b is the demonstration planting field at the seed lated from Wisconsin cornfield samples. U.S. Pat. No. 45 processing plant in Prescott, Wisconsin. Site a and site b are several kilometers apart. 4,798,723, which is hereby incorporated by reference, TABLE 4 Characterization and comparison of P. cepacia root isolates and culture collection strains ATCC ATCC ATCC ATCC ATCC 64-22 REACTIONS/ENZYMES 526 406 64 65 29424 17460 25416 0856 17616 NS/NK reduction of nitrates -- -- O O -- O O 0. -- to nitrites reduction of nitrates O O O 0 O -- O O O to nitrogen indole production 0 0 O 0 O O O acidification with glucose 0. O O O O O O arginine dihydrolase O O O O O O O lease O O O O O O O hydrolysis (R-glucosidase) -- -- O ------O hydrolysis (protease) -- -- O r -- O O R-galactosidase ------glucose assimilation ------arabinose assimilation ------mannose assimilation ------nannitol assimilation ------N-acetyl-glucosamine ------assimilation natose assimilation O O O 0 O 0 O gluconate assimilation ------caprate assimilation ------adipate assimilation ------5,229,292 15 16 TABLE 4-continued Characterization and comparison of P. cepacia root isolates and culture collection strains ATCC ATCC ATCC ATCC ATCC 64-22 REACTIONS/ENZYMES 526 406 64 65 29424 17460 25416 0856 1766 NS/NK malate assimilation -- -- n------citrate assiniation ------phenyl-acetate assimilation ------cytochrone oxidate O O O O O 0 O -- -- Motility ------Gram staining - m ------Pigmentation yellow pale white white - yellow yellow Plate inhibition (in vitro) of: Fusarium moniliforme 4. 4. 4. 2 3 s 5 O 0 4. Sclerotinia sp. 4. 4. 5 2 0 4. 1 O O ND Macrophomina sp. 3 3 2 1 2 2 0 O ND Plant bioassay - % reduction 62 8. 24 O O O 4.36 5.96 33.7 0-2.52 in seedling infection Onion pathogenicity test O 3 3 4. 1. 3 0 4. Corn root colonization at 6.60 ND ND ND 6.86 6.0) 5.60 6.06 6.72 6.73-7.10 2 weeks cfu/g dry wt root log 10 units crystal protein genes (i.e., partial protoxin genes) pro In principle, any plant colonizing strain of P. cepacia duces partial protoxins, which are precursors of toxins. can be used in the present invention as a "vector' for In an exemplary embodiment of the present inven delivering to that plant an insect toxic crystal protein. P. 25 tion, genes encoding crystal protein insect toxins were cepacia type Wisconsin strains are preferred “vectors' introduced into a strain of P. cepacia type Wisconsin. because they are not pathogenic to plants and colonize Specifically the genes encoding the HD73 and HD73 diverse plants. Since these strains are found to colonize like genes derived from B. thuringiensis var. kurstaki and persist on both plant roots and leaves, they are strains HD73 and HD-1 Dipel, respectively, were intro useful for carrying insect toxic proteins to both of these 30 duced into P. cepacia 526 by cloning the crystal protein plant environments to protect a plant from root and encoding sequences into incompatibility group Q foliar insect damage. (IncC) plasmids and introducing these plasmids into the The activity spectrum of crystal toxins varies among P. cepacia type Wisconsin 526 strain. varieties and strains of B. thuringiensis and is correlated In one genetic construction of the present invention, with the presence of distinct crystal proteins and genes 35 designated pSUP487, the CP HD73-like gene derived encoding them. Different crystal proteins are responsi from the HD-1 Dipel B. thuringiensis strain remained ble for the toxicity displayed by B. thuringiensis varieties under the regulatory control of its homologous B. thu against insects belonging to different families. For ex ringiensis promoter sequences. In another construction, ample, kurstakistrains contain CP specific for Lepidop designated pSKP/Bt the HD73 structural gene from B. terans, israelensis strains contain CP specific for Dipter thuringiensis HD73 was placed under the regulatory ans and tenebrionis and san diego strains contain CP control of a heterologous promoter, the nptI gene specific for Coleopterans. The activity of some kurstaki promoter from the transposon Tn5, which was known strains against Lepidopterans and Dipterans is due to to be active in P. cepacia strains. Both of these plasmids the presence of at least two different crystal proteins. are reproducibly prepared from readily available start Activity range toward insects within a particular family 45 ing materials, as described in examples 1 and 2 and is also correlated with the presence of multiple CP and diagrammed in FIGS. 3 and 4. genes encoding them. It is not yet known what struc The recombinant plasmids containing the CP sequen tural features of the CP genes are associated with the ces were introduced into P. cepacia 526 using conven varying insect specificities of the toxins. tional triparental mating techniques. Both P. cepacia The methods of the present invention can utilize any 50 transconjugants, P. cepacia 526(pSUP487) and P. cepacia of the crystal protein genes of B. thuringiensis. Partial 526(pSKP/Bt), expressed several proteins that reacted protoxin genes can also be employed i& desired. The with crystal protein antibody. The proteins detected on specific crystal protein genes or coding sequences intro western blots ranged in size from about 70 to 92 kid, but duced into root colonizing P. cepacia are chosen to no protein bands corresponding in size to the HD73 obtain the desired toxicity range for the resultant geneti 55 protoxin (130 kid) or the toxin (68kd) were detected. cally altered bacterium. If it is desirable, more than one The proteins expressed using either construction were CP gene can be introduced into a single strain of P. similar, but the level of expression appeared to be cepacia. It is also contemplated that any P. cepacia active higher from the nptI promoter. The proteins detected promoter sequences can be used in the construction of are believed to represent the products of incomplete CP chimaeric genes. 60 transcription of the complete CP structural gene, but It has been demonstrated, at least with the Lepidop such aberrant size products could also result from CP teran specific crystal proteins, like HD73 CP, that the gene sequence deletions. full-length crystal protein coding region need not be Genetically altered P. cepacia 526 transconjugants expressed by a cell to produce insect toxic proteins. containing B. thuringiensis CP sequences were found to U.S. patent application Ser. No. 617,321, (M.J. Adang), 65 be insect toxic using diet assays (Table 5) and assays on filed Jun. 4, 1984, teaches the use of DNA plasmids excised leaf (Table 7) and whole plants (Table 8). Cell carrying partial crystal protein genes to produce insect suspensions of P. cepacia 526 containing either pSUP487 toxic proteins and bacterial cells. Expression of partial or pSKP/BT applied to the surface (about 4x 105 bac 5,229,292 17 18 teria/cm2) of an artificial diet were found to be toxic to inoculants are known in the art. Indirect seed inocula Tobacco hornworm (THW, Manduca sexta) neonate tion involves application of an inoculating material into larvae (Table 5). In this experiment, a ten-fold dilution the vicinity of the seed at the time of planting. of the bacterial suspension decreased larvae mortality. In order to establish leaf colonizing strains on plant Excised tobacco leaves were protected from THW leaves, it is only necessary to inoculate leaves with an damage by inoculation with P. cepacia 526(pSUP487) or appropriate composition containing the desired strain. 526(pSKP/Bt). Tobacco leaves were sprayed with Foliar inoculants can be applied, in principle, at any seruspensions of both P. cepacia 526 transconjugants time during growth of the plant. Inoculation by spray (about 1 x 108 bacteria/ml) and neonate THW larvae ing of liquid or particulate inoculating compositions is were placed on leaves. Significant larval killing was O particularly useful. noted on treated leaves which showed no sign of insect damage. Uninoculated and control leaves were de The concentration of insect toxic bacteria that will be voured. required to produce insecticidally effective inoculating Young tobacco plants were sprayed with bacterial compositions will depend on the strain of P. cepacia suspensions of P. cepacia transconjugants, P. cepacia 15 utilized, the exact CP gene construction used, and the 526(pSUP487) and 526(pSKP/BT) (about 5x 1010 bac formulation of the composition. The concentration of teria/ plant). After bacterial inoculation, THW larvae cells required for effective insecticidal activity can be were placed on plant leaves. As shown in Table 9, larval determined by protection assays, for example those mortality on plants inoculated with P. cepacia 526 described in Examples 6 and 7. (pSUP487) and 526 (pSKP/Bt). Inoculation with genet Inoculating compositions must be suitable for agricul ically altered insect toxic P. cepacia protected plants tural use and dispersal in fields. Generally, components from insect damage. of the composition must be non-phytotoxic, non-bac In an exemplary embodiment of the present inven teriostatic and non-bacteriocidal. Foliar applications tion, P. cepacia strains were genetically altered by intro must not damage or injure plant leaves. In addition to duction of a recombinant vector which carried the de 25 appropriate liquid or solid carriers, inoculating compo sired crystal protein gene construction. While this sitions may include sticking and adhesive agents, emul method rendered the transformed bacteria insect toxic sifying and wetting agents, and bacterial nutrients or and capable of protecting plants on which they were other agents to enhance growth or stabilize bacterial inoculated from insect pests, it was found that the intro cells. Inoculating compositions for insect pest control duced recombinant vectors were not stably maintained 30 may also include agents which stimulate insect feeding. in the bacteria without application of selective pressure. Reviews describing methods of application of biolog Thus, after a number of generations, the insect toxic ical insect control agents and agricultural inoculation phenotype was lost. It may be desirable to increase the are available. See, for example, Couch and Ignoffo stability of insect toxic phenotype in cells. As is well (1981) in Microbial Control of Pests and Plant Disease known to those skilled in the art, stabilization can be 35 1970-1980, Burges (ed.), chapter 34, pp. 621-634; Corke achieved in several ways including vector stabilization and Rishbeth, ibid, chapter 39, pp. 717-732; Brockwell or preferably by integration of the CP gene construc (1980) in Methods for Evaluating Nitrogen-Fixation, tions into the bacterial chromosome. A DNA vector Bergersen (ed.) pp. 417-488; Burton (1982) in Biological can be stabilized in a bacterial cell by incorporating into Nitrogen Fixation Technology for Tropical Agriculture, the DNA vector a gene that is required for cell survival. Graham and Harris (eds.) pp. 105-114; and Roughley This can be done, for example, by preparing an auxotro (1982) ibid, pp. 115-127. phic mutant of the bacterium. This mutant must not be Except as noted hereafter, standard techniques for capable of survival without complementation of the cloning, DNA isolation, amplification and purification, auxotrophy. A gene or genes that complement the le for enzymatic reactions involving DNA ligase, DNA thal auxotrophic mutation are then placed on the DNA 45 vector along with the CP sequences. Retention of the polymerase, restriction endonucleases and the like, and vector is then required for survival of the cell and the various separation techniques are those known and CP sequences are stabilized. commonly employed by those skilled in the art. A num It may not be desirable for environmental reasons to ber of standard techniques are described in: Maniatis et introduce recombinant genes on vectors that may be 50 al. (1982) Molecular Cloning, Cold Spring Harbor Lab transmitted in the natural population. For this reason it oratory, Cold Spring Harbor, New York; Wu (ed.) may be preferred to integrate the CP gene constructions (1979) Meth. Enzymol. 68; Wu et al. (eds.) (1983) Meth. into the bacterial chromosome. Example 8 provides one Enzymol. 100 and 101: Grossman and Moldave (eds.) method of integrating CP sequences into the chromo Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in some using homologous recombination. Other methods 55 Molecular Genetics, Cold Spring Harbor Laboratory, and modifications of these methods are well known in Cold Spring Harbor, N.Y.; Old and Primrose (1981) the art. For example Barry (1986) Biotechnology Principles of Gene Manipulation, University of California 4:446-449 describes a method for stable chronosomal Press, Berkeley; Schleif and Wensink (1982) Practical integration of foreign genes which employs a defective Methods in Molecular Biology; Glover (ed.) (1985) DNA transposon. 60 Cloning Vol. I and II, IRL Press, Oxford, UK; Hames A primary use of the genetically altered P. cepacia of and Higgins (eds.) (1985) Nucleic Acid Hybridization, the present invention is the inoculation of plants or their IRL Press, Oxford, UK; Sellow and Hollaender (1979) roots to provide protection from insect pests. Root Genetic Engineering Principles and Methods, Vols. 1-4, and/or rhizosphere colonization by these P. cepacia Plenum Press, New York, which are expressly incorpo strains can be accomplished by direct or indirect inocu 65 rated by reference herein. Abbreviations and nomencla lation of seeds at the time of planting. Direct seed inocu ture, where employed, are deemed standard in the field lation involves application of an inoculant to the seed and commonly used in professional journals such as prior to sowing. Many variations of preparing such seed those cited herein. 5,229,292 19 20 1984). A plasmid, pBt73-16, containing the full-length EXAMPLE 1.1 coding region of the HD73 CP gene was described and Isolation of an HD73 CP insect toxin gene from B. has been placed on deposit with The Northern Regional thuringiensis var. Kurstaki HD-1 Dipel Research Laboratory in strain E. coli HB101 (pBt73-16) DNA hybridization experiments (Kronstad, 1983) 5 with accession no. NRRL B-15759. The plasmid pBt73 showed that B. thuringiensis var. kurstaki HD-1 Dipel 16 is designated clone 158/51-16, herein (FIG. 2). This (NRRL deposit, NRRL B-3792) harbors three homolo clone (158/51-16) contains the full-length HD73 CP gous but distinct crystal protein genes, which are found coding region. to be carried on 6.6, 5.3 and 4.5 kb HindIII DNA frag The plasmid pUC5'Bt contains the HD73 CP sequen ments, respectively. The gene carried on the 4.5 kb O ces from clone 158/51-16 in which a Ban.HI restriction fragment, also called the HD-1 crystal protein gene, has site was created by oligo-directed site specific mutagen been cloned by Schnepf and Whiteley (1981) supra. The esis 7 base pairs 5' to the ATG start codon of the CP gene carried on the 6.6 kb fragment is similar to the structural gene. This construction allows the removal of crystal protein gene of B. thuringiensis var. kurstaki the 5'-promoter sequences of the CP gene to produce a HD73 (Adang et al. (1985) supra, and is designated the 5 HD73-like gene herein. The third crystal protein gene "promoterless' CP gene. The plasmid puC5'Bt was (5.3 type) appears to be unique. constructed from 158/51-16 as follows: The isolation and cloning of the HD73-like CP gene The 3.7 kb Nde fragment from 158/51-16 (FIG. 2) from B, thuringiensis var. kurstaki HD-1 Dipel is de was blunt-ended with DNA polymerase (Klenow frag scribed in Adang et al. (1986) submitted to Biotechnol 20 ment) and cloned into the SmaI site of M13mpl9 (Nor ogy Advances in Invertebrate Pathology and Cell Cul rander et al., 1983). After several plaque purifications, ture, Academic Press, N.Y. (issued in 1987 at pages one clone was identified and designated 1.6.4. The crys 85-99) Briefly, B. thuringiensis plasmid DNA, 30 MD tal protein sequences in this clone were oriented so that and larger, was prepared by the procedure of Kronstad the BamHI site of the M13 polylinker was at the 3' end et al. (1983) supra. A Sau3A partial digest of the plasmid 25 of the CP sequences. This is important for the subse DNA was ligated into the vector puC18 (Norrander, J. quent use of the mutagenized clone in the construction (1983) Gene 26:101-106) and transformed into E. coli of puC5'BT and pSKP/Bt. MC1061 (Casadaban and Cohen (1980) J. Mol. Biol. Next, single stranded DNA from 1.6.4 was used as a 138:179-207). Transformant colonies were screen by template for oligo-directed site-specific mutagenesis hybridization of the 3.7 kb BamHI fragment of 30 using methods described in Norrander et al. (1983) pUC5'BT which contains the HD73 CP gene sequen Gene 26:101-106. The oligonucleotide used for muta ces. Hybridizing colonies were further screened for genesis was a 25-mer having the following sequence: crystal protein expression by colony immunoblot 5'-GAGATGGAG'GATCCTTATGGATAAC-3'. method using MAb-1, a monoclonal antibody raised to This oligonucleotide spans the CP gene sequence from HD73 CP (Adang et al. (1985) supra, and rabbit poly 35 bases - 16 to --9 (to the ATG start codon). The restric clonal antibody raised to solubilized HD-73 crystals. tion enzyme cutting site is indicated by". Nucleotides Bacterial colonies on nitrocellulose filters were lysed in 11-13 of the oligonucleotide are mismatched to the CP CHCl3 vapor followed by DNAase and lysozyme treat gene sequence and provide the BamHI restriction (5'- ment (Helfman et al. (1983) Proc. Natl. Acad. Sci. USA G'GATCC-3) site. Mutagenized clones were selected 80:31-35). Filters were treated with rabbit polyclonal by probing plaque lifts with radioactively-labeled 25 antiserum or MAb-1, followed by detection using an oligomer. Blots were washed at first at room tempera alkaline phosphatase ELISA system (Blake et al. (1984) ture and autoradiographed. Blots were then rewashed Anal. Biochem. 136:175-179). at 48 C. and autoradiographed. Mutagenized clones Several colonies were selected by immunological containing the sequence of the 25-mer give a darker screening; one of these, designated E. coli (pBTI-89A) 45 was chosen for further study. The plasmid pBT1-89A, hybridization signal than clones containing wild-type herein designated 208/43-487 (FIG. 1) contained the sequence after the 48 C. wash (Norrander et al. 1983) HD73-like (6.6 kb type) CP gene from B. thuringiensis supra. Presumptive mutant clone selections were fur var. kurstaki HD-1 Dipel. This recombinant was ther characterized by restriction analysis using BamHI mapped using restriction enzyme analysis. The restric endonuclease, which generates an approximately 3.7 kb tion enzyme digests of the HD73-like gene are similar if 50 BamHI fragment not found in the wild-type DNA. This not identical to comparable digests of the HD73 gene. fragment contains the "promoterless' CP sequences. The open reading frame of the HD73-like gene contain The 5'- BamHI site is from the mutagenesis and the ing the same EcoRI, Xbal, EcoRV, BglII, SacI, PstI, 3'-BamHI site is derived from M13 sequences. This 3.7 Aval, XhoI, Kipni, HindIII and Pvul sites contained in kb BamHI fragment was inserted into the vector puC19 the HD73 gene. 55 to give plasmid puC5'BT. EXAMPLE 1.2 EXAMPLE 2 Isolation of crystal protein insect toxin genes from Introduction of B.t, insecticidal toxin genes into Bacillus thuringiensis var. kurstaki HD73 Pseudomonas cepacia 526 The crystal protein (CP) gene in B, thuringiensis var. Two plasmids were constructed to facilitate intro kurstaki HD73 (NRRL B-4488) is located on a 75 kb duction of the insect toxic crystal protein into P. cepacia plasmid. The molecular cloning of full-length and par 526. Both of these constructions are derivatives of in tial protoxin genes of HD73 CP has been described compatibility group Q (IncO) plasmids which were previously (Adang et al. (1985) Gene 36:289-300; and in 65 found to conjugate into P. cepacia 526 at high frequen U.S. patent application Ser. Nos. 535,354 (Kemp cies and also to be stably maintained. Incompatibility /Adang), filed Sep. 24, 1983; 848,733 (Kemp/Adang), group P plasmids like pRK290 would not conjugate filed Apr. 4, 1986; and 617,321 (Adang), filed Jun. 4, into P. cepacia 526. 5,229,292 21 22 The first construction, designated pSUP487, is a de nated pSKP/Bt. In this construction (FIG. 4), the 5' end rivative of the Incq plasmid pSUP204 (Priefer et al. of the CP structural gene is adjacent to the 3' end of the (1985) J. Bacteriol. 163:324-330) into which the pro nptII promoter sequences so that the CP gene is under moter and coding regions of the HD73 CP gene from B. the regulatory control of the nptII promoter. The com thuringiensis var. kurstaki HD-1 Dipel was cloned. A posite or chimaeric mpt I promoter/CP structural gene 5.1 kb SphI fragment of clone 208/43-487 (FIG. 2) can be removed from the pSKP/Bt plasmid on an ap containing the CP gene coding region and approxi proximately 4.05 kb HindIII-BamHI fragment. mately 0.9 kb of 5' and 0.5 kb of 3' sequences flanking Recombinant plasmids pSUP487 or pSKP/Bt were the coding region was cloned into the single Sphl site of introduced into P. cepacia 526 using conventional meth pSUP204, as diagrammed in FIG. 3. The resultant plas O ods of triparental matings employing the plasmid mid DNA was isolated and introduced into E. coli pRK2013 (Ditta et al. (1980) Proc. Natl. Acad. Sci. HB101, after which chloramphenicol-resistant, tetracy USA 77.7347-7357)as a mobilizer. The presence of cline-sensitive transformants were selected. Plasmid pSUP487 or pSKP/Bt in P. cepacia 526 transconjugants DNA from these selections was isolated and subjected was confirmed by restriction enzyme analysis and to restriction enzyme analysis to confirm the presence 15 Southern blot analysis using the 3.7kb BanhI fragment of the CP containing fragment. One of the selections of puC5'BT as a hybridization probe. The P. cepacia that was shown to contain the CP fragment was chosen 526 strains which carry the plasmids pSUP487 and and the isolated plasmid was designated pSUP487. pSKP/Bt are designated conventionally as P. cepacia The second construction, designated pSKP/Bt, is a 526(pSUP487) and P. cepacia 526(pSKP/Bt). derivative of the IncQ plasmid pSUP104 (Priefer et al., 20 1985) supra), which carries a "promoterless' CP gene EXAMPLE 3 derived from B. thuringiensis var. kurstaki HD73 under Expression of Bt crystal protein by P. cepacia the regulation of the nptII gene promoter sequences 526-Immunodetection of crystal protein derived from the transposon Tn5. The plasmid pKS4 was the source of the nptII pro 25 Standard western blot analyses (see, for example, noter sequences. This plasmid is a derivative of Adang et al. (1985) supra), were performed to examine pBR322 which contains the nptII kanamycin resistance expression of Bt crystal protein in P. cepacia 526 gene from transposon Tn5. This plasmid can be ob (pSUP487) and P. cepacia 526(pSKP/Bt). tained from E. coli C600 (pKS4) which is on deposit Total protein obtained from mid-logarithmic phase with NRRL with accession no. NRRL B-15394. The 30 cultures grown in nutrient broth was separated by SDS plasmid pKS4.6 was constructed from pKS4 as de electrophoresis, transferred to nitrocellulose and was scribed in U.S. patent application Ser. No. 788,984 filed first reacted with a rabbit polyclonal antibody raised to Oct. 19, 1985, which is hereby incorporated by refer Bt crystal protein and then with 125I-protein A. Poly ence. The feature of pKS4.6 that is important for the clonal antibodies to Bt crystal protein were prepared construction of pSKP/Bt is that it has been engineered 35 essentially as described in U.S. patent application Ser. to contain a BamHI restriction site 6 base pairs 5' to the No. 617,321 (Adang), filed Jun. 4, 1984. Briefly, crystals ATG start codon of the nptII structural gene. This were isolated from both B. thuringiensis var. kurstaki restriction site was created by use of oligo-directed site strains HD-1 Dipel and HD73, the crystals were solubi specific mutagenesis (Norrander, 1983) supra). The lized and the crystal proteins were separated by gel presence of this restriction site allows the nptII gene electrophoresis. The 130 kid CP protoxin protein band promoter to be removed on an approximately 350 bp was excised from the gel, lyophilized and ground to a HindIII-BamHI fragment of pKS4.6. As diagrammed in powder. Rabbits were injected subcutaneously with 50 FIG. 4, a small HindIII-BamHI fragment was removed ng crystal protein powder suspended in complete from pSUP104 and replaced with the nptII promoter Freunds' adjuvant followed by two injections (50 ng fragment to produce plasmid pSKP. Transformants 45 crystal protein each injection) in incomplete adjuvant containing pSKP are chloramphenicol-resistant and over a four week period. For use in western blots, rabbit tetracycline-sensitive. The pSKP construction is distin antibody was cleared by reaction to E. coli HB101 total guishable by the presence of a BglII restriction site, not protein and P. cepacia 526 total protein. present in pSUP104. Protein from the parent P. cepacia 526, the two P. Promoterless HD73 CP gene sequences were derived 50 cepacia 526 transformants, as well as E. coli HB101 from plasmid pUC5'Bt, the construction of which is strains transformed with pSUP487 and pSKP/Bt, were described above. This plasmid contains a BamHI re examined. Several proteins bands not found in P. cepacia striction site 7 base pairs 5' to the CP gene start codon. 526 which reacted with CP antibody were identified in The promoterless CP gene can then be removed from both transformed P. cepacia 526 strains. None of the pUC5'Bt as an approximately 3.7 kb BamHI fragment 55 protein bands of the P. cepacia transconjugants corre (FIG. 4). sponded in size to the protoxin (130 kid) or toxin (68kd) The 3.7 kb BanhI fragment from puC5'BT was bands found in B. thuringiensis HD73. In contrast, E. cloned into the single BamHI site of pSKP. The resul coli HB101 (pSUP487) produces two protein bands of tant ligation mixture was transformed into E. coli about 130 and 68 kid which react with CP antibody. E. HB101 and chloramphenicol-resistant transformants 60 coli HB101 (pSKP/Bt) also produces CP antibody reac were selected. Plasmid DNA from these selections was tive proteins of about 130 and 68kd, but in addition, this examined by miniprep techniques (Maniatis et al. (1982) strain produces at least 3 other CP antibody reactive supra), and the orientation of insertion of the CP se proteins having molecular weights intermediate be quences with respect to the nptII promoter was deter tween 68-130 kid. The CP antibody reactive proteins mined by restriction enzyme analysis using HindIII 65 found in the P. cepacia 526 transformants appear to digests. One of the selected transformants which con correspond to some of the intermediate size proteins tained a plasmid in which the CP sequences were in observed in E. coli HB101 (pSKP/Bt). There are sev serted in the desired orientation was chosen and desig eral potential explanations for the presence of aberrant 5,229,292 23 24 sized CP proteins in the P. cepacia 526 transconjugants. zation assays. This antibiotic resistance marker is car For example, transcription of the CP gene may be pre- ried on the bacterial chromosome so that rif resistance maturely terminated, the full-length protoxin may be can be used as a strain marker. Experiments to deter degraded or a portion of the plasmid construction may mine the effect of the presence of CP sequences on the have been deleted. 5 ability of P. cepacia 526 to colonize plants were per formed using a rif-resistant P. cepacia 526(pSUP487) EXAMPLE 4 transconjugant. Plasmid stability was also determined in Expression of crystal protein in P. cepacia 526: Insect this experiment by measuring the retention of the plas bioassays mid-borne chloramphenicol-resistance marker. 526The psuP487 insecticidal and activitypsKP/B of thetransconiugants Pseudomonas cepaciaagainst 10 greenhouseColonization experinents. assays y Experientsperformed wereO designed toin Tobacco Hornworm (Manduca sexta) was determined prevent release of P. cepacia 526 transconjugants into on standard diet assays (for example, Schesser et al. the environment. Nested Magenta boxes were used for results(1972) ofAppl. one suchEnviron. assay Microbiol.are presented 33.3780). in Table 5.The In- 5 wE.E; partsconting soil, k E.a soil peat, play al REmix sects were obtained from commercial sources. Test E. u E.T.: i it. 1. 1a bacteria were grown overnight in Luria broth with era E. E. ME St. 3. part appropriate antibiotics. Culture samples (10 ml) were rice hulls. A bottom Magenta box was used as a water centrifuged (5,000 rpm, 10 min), washed once with reservoir, which could be replenished when necessary. phosphate buffered saline (PBS) to remove residual 20 A wick connected the top and bottom boxes allowing antibiotics and resuspended in 10 ml of PBS. The num- transfer of moisture to the closed top box. Corn seeds ber of bacterial colony forming units (cfu/ml) in these inocular with 1 ml of a stationary phase culture cultures ranged from 1.6-4.6x108cfu/ml (Table 5). For (approx. 4X 10 cells/seed) of the test strain. Inoculated insect bioassays, 10 ul of each bacterial culture (undi- seeds were then planted in the top Magenta box (l luted) was uniformly applied to the surface (1.3 cm2 25 seed/box, 14 replicas/treatment), covered with soil and Mothsurface diet) area) (1 of ml gelled in a artificial 4 ml vial) diet and (cummercial the treated Gypsy diet thennation. covered Uninoculated E. E. controls were also included.yet gami samples were allowed to dry. In some trials, 10x or Four plants from each treatment were harvested at 7, 100X dilutions of the cultures were used to treat the 13 and 20 days after planting. Roots of the harvested diet samples. One THw larva was placed in each via 30 plants were washed thoughly underunningtap (lovials/assay) and all vials were allowed to incubate at Water and then macerated (5 Sec) in a blender in 100m 26 c. for 4 days. Neonate larvae are used in bioassays of PBS. Rootwash was collected into a stoppered sink As shown in Table 5, P. cepacia 526 containing either "8 few gallons of bleach to insure contain pSUP487 or pSKP/Bt is toxic to THW larvae. No ment of recombinant strains. Viable cell counts were toxicity was displayed by the parent P. cepacia 526, P. 35 determined by plating 10, 10 and 105 dilutions of the cepaciaa 526(pSUP204)526 P or PBS medium controls - antibioticsroot macerate added, solution with 100on nutrientg/ml rifampicin agar plates added with and no TABLE 5 with 100 g/ml rifampicin plus 50pg/ml chlorampheni Insect Diet Bioassays col (cam). The results of one such colonization assay on THW Larvae Mortality 40 corn roots is shown in Table 6. Percent colonization - (no. alive/total)' - was measured as the percent viable counts of rif-resist - Dilutions - ant bacteria to total bacteria. Plasmid stability, deter P. capacia Strain CFU/ml Undiluted 10x 100X mined as the percent retention of plasmid drug marker, 526 1.6 x 108 10/0 ND ND was measured as the percent viable counts of rif/cam. :SE:E. : X E. 10/10E. NDE. NR, 45 resistantmide (150 bacteria ug/ml) to was rif addedresistant to bacteria.all plates Cyclohexi to inhibit PBS 4.6 x 108 10/10 ND ND fungal growth. TABLE 6 Colonization of Corn Roots by Pseudomonas cepacia 526 and Transconivgants CFU/g Roots (Fresh Wt.) % Colonization % Plasmid Retention (time after planting) (time after planting) (time after planting) Treatment 7 Days 13 Days 20 Days 7 Days 13 Days 20 Days 7 Days 13 Days 20 Days 526 Rif 6 x 106 1 x 106 8 x 105 60 45 42 m - 526 (pSUP204) 5 x 106 5 x 106 1 x 106 90 45 25 100 120 90 526 (pSUP487) 4 x 106 4 x 106 2 x 106 73 70 28 40 38 7 Colony forming units; mean of 4 replicas

NDBacterial = not counts done in undiluted sample; CFU = colony forming units. The stability of the Bt CP gene containing construct Those laye live were of reduced size compared to controls. 60 pSUP487 in Pseudomonas cepacia 526 was further exam Larvae cither missing or killed by cap. ined by determining the loss of plasmid drug resistance marker chloramphenicol in liquid culture in the absence EXAMPLE 5 of selective pressure Test cultures including P. cepacia 526 and the P. cepacia 526 transconjugants containing Colonization Assay 65 pSUP487 and pSUP204 were grown overnight in Luria Pseudomonas cepacia 526 has been shown to be a good broth with appropriate antibiotics (100 g/ml rif, 50 plant colonizer A spontaneous rifampicin (rif) -resistant ug/ml cam). Fresh Luria broth cultures were then inoc mutant of P. cepacia 526 was isolated and used in coloni- ulated from each of the overnight cultures of the test 5,229,292 25 26 strains so that the initial inoculum level was about 100 bacteria/ml. These cultures were grown to stationary EXAMPLE 7 phase (about 24 hr) at 28 C. in the absence of antibiot Protection of tobacco plants by P. cepacia 526 pSUP487 ics, after which viable cell counts on nutrient agar plates and pSKP/Bt transconjugants with appropriate antibiotic were determined. This Test bacterial samples were prepared as in diet assays growth period represented approximately 20-24 gener (vide supra), except that PBS bacterial samples con ations. The pSUP487 plasmid proved to be relatively tained from about 1-5x 1010 bacteria/ml. Tobacco unstable being retained in only 5.3% of the total P. plants (Nicotiana tabacum var. Kentucky 17) were cepacia 526 compared to the parent pSUP204 plasmid propagated from nodal sections in MS medium without which was retained in 97% of the P. cepacia 526 over O hormone supplements (Murashige and Skoog (1962) this time period. Physiol. Plant. 15:473) in Magenta boxes under sterile EXAMPLE 6 conditions. After plants had rooted, they were removed from their boxes, sprayed with about 1 ml of bacterial Protection of tobacco leaves by P. cepacia 526 test sample and returned to their boxes. Ten THW lar (pSUP487) and P. cepacia 526 (pSKP/Bt) 15 vae were place on each test plant. Four replicas of each The insecticidal activity of the P. cepacia 526 treatment were prepared. Treatments included the P. pSUP487 and pSKP/Bt transconjugants and their plant cepacia 526 pSUP487 and pSKP/Bt transconjugants protection ability were examined by spraying bacterial and P. cepacia 526 and P. cepacia 526 (pSUP204) con cultures on tobacco leaves. These and all experiments trols. using P. cepacia 526 transconjugants were performed so 20 On the third day after inoculation, leaf samples were that release of the organisms to the environment was taken to assay colonization and plasmid retention. Sam prevented. Test bacterial samples were prepared as in ples were weighed and placed in 10 ml of PBS which diet assays (vide supra). PBS bacterial cultures con contained 0.1% peptone. These leaf samples were tained from about 1-5X108 bacteria/ml. Excised to shaken at room temperature for two hours, after which bacco leaves from young plants that had been grown in 25 appropriate dilutions were plated on nutrient agar with growth chambers were sprayed with about 200 ul sam out antibiotics, with antibiotics either 100 ug/ml rif or ples of either P. cepacia 526 pSUP487 or pSKP/Bt 100 g/ml rif +50 g/ml cam. All plates contained 150 transformants or P. cepacia 526, P. cepacia 526 ug/ml cycloheximide. Plates were incubated at 28 C. (pSUP204) or PBS controls. Treated leaves were al for 24-36 hours and counted. The leaf/PBS sample was lowed to dry and then placed in petri dishes on moist 30 considered as the 10X dilution. Results of colonization ened filter paper (one leaf/dish). Five THW neonate and plasmid stability assays are given in Table 8. TABLE 8 Tobacco Plant Colonization; Spray Treatment Colonization and Plasmid Stability Bacterial Leaf Population Ave. wt. of % 3 days Post-Inoculation? Leaf Samples 9% Plasmid P. cepacia Strain CFU/ml NA NA(rif) NA(rif -- can) (gm) Colonization Stability 526 : 2 X 1010 5.2 x 108 5.6 x 108 2 x 103 .86 107 - 526 (pSUP204) 1.9 x 1010 3.6 x 108 3.5 x 108 3.9 x 108 .85 97 11 526 (pSUP487) 5.9 x 1010 4.8 x 108 4.7 x 108 3.9 x 106 .88 97 0.83 526 (pSKF/BT) 1.6 x 100 4 x 108 3.8 x 108 3.7 x 108 .83 95 97 Bacterial counts in undiluted test sample CFU/ml, mean of 4 replicas On the sixth day after inoculation, larvae were recov- . ered from each test plant box. Larvae were weighed and mortality was recorded. The results of this experi larvae were then placed on each treated leaf. Four ment are given in Table 9. Treatment with P. cepacia leaves were treated with each bacterial sample. After 526 (pSUP487) and P. cepacia 526 (pSKP/Bt) induced three days, leaves were scored for protection and death 50 significant larvae mortality compared to controls. The of larvae. As shown in Table 7, the samples of P. cepacia average weight of larvae which remained alive on P. 526 containing the plasmids pSUP487 and pSKP/Bt cepacia 526 (pSKP/Bt) treated plants was significantly were toxic to the larvae, while the controls were not. lower than in controls. Furthermore, all leaves treated with these transconju It was noted that stability of pSKP/Bt plasmid was gant strains were protected from the larvae, while the 55 significantly higher, in this experiment, than expected control leaves were devoured. based on previous experiments. Insect toxicity of 526 TABLE 7 strains containing pSKP/Bt is also lower than expected. Excised Leaf Protection; and Insect Mortality; It is believed that a spontaneous deletion of part of the Leaf Spray Experiment CP sequences in the pSKP/Bt has occurred in this ex THW Larvae Mortality periment. P. cepacia Strain (no. alive/total) TABLE 9 PBS 20/20 526 20/20 Tobacco Plant Protection; Spray Treatment 526 (pSUP204) 20/20 THW Larval Mortality 526 (pSUP487) 2/20 65 P. cepacia Strain No. Alive/Total Average Larval Wt. (ng) 526 (pSKP/Bt) 5/20 526 37/38 16.5 5 THW larvae/leaf, 4 leaves/treatment 526 (pSUP204) 39/39 7.5 526 (pSUP487) 0/40 NDb 5,229,292 27 28 identify clones from the wild-type library which con TABLE 9-continued tain the analogous genomic sequences and map the Tobacco Plant Protection: Spray Treatment chromosome region of interest. It is into these regions THW Larval Mortality of the chromosome that the CP gene constructs will be P. cepacia Strain No. Alive/Total Average Larval Wt. (mg) inserted. Once a restriction enzyme map of the cloned 526 (pSKP/B) 19/39 7.3 genomic region of interest is obtained, a convenient For some treatments, not all larvae were recovered from boxes. cloning site, usually a unique restriction enzyme site, is ND = not determined. chosen for introduction of the CP sequences. The 5.1 SphI fragment (example 1.2, FIG. 2) which contains the EXAMPLE 8 O HD73-like gene from.B, thuringiensis HD-1 Dipel and its homologous promoter sequences or the approxi Integration of Bt CP gene sequences into the P. cepacia mately 8.3 kb HindIII-BamHI fragment from pSKP/Bt 526 chromosome containing the HD73 CP gene chimaera in which the The following represents an example of one way in structural gene is under the control of the npt gene which CP gene sequences can be stably integrated into 15 promoter can be used as sources of CP gene sequences the P. cepacia 526 chromosome via homologous recom for introduction into the P. cepacia 526 genome. In bination. order to follow the recombination event that will inte It is first important to identify chromosomal sequen grate the cloned CP sequences into the chromosome, a ces in P. cepacia 526 that are not essential to the growth marker gene (for example, encoding an antibiotic resis or other desirable properties, for example colonizing 20 tance) must also be introduced into the genomic clone ability, of the strain. It is into these locations in the along with and closely-linked to the introduced CP chromosome that the CP sequences can be inserted sequences. It is also required that sufficient wild-type without affecting desired properties. One way in which genomic sequences flanking (1 kb on either side is usu potential insertion sites can be identified is by use of ally sufficient) the introduced CP and marker sequences transposon mutagenesis. Transposon Tn5 insertions into 25 remain to drive recombination. The suicide shuttle vec the chromosome of P. cepacia 526 can be obtained using torpSUP205 derivative that contains the genomic clone the suicide vector pSUP1011 (Simon et al. (1983) Bio of interest into which the CP construct and marker gene technology 9:784-791). P. cepacia 526 is mated with E. are incorporated is then introduced into an appropriate coli SM10 which carries vector pSUP1011 and selection carrier strain (for example, E. coli HB101) and the sui is made for kanamycin resistant P. cepacia 526 which 30 cide vector derivative is mated into P. cepacia . The must carry Tn5 insertions, designated P. cepacia double recombination event that results in integration of 526:Tn5. It is preferred to do this selection using mini the CP and marker sequences into the P. cepacia 526 mal medium to exclude auxotrophic mutants. These chromosome is selected by screening for P. cepacia 526 Tn5 insertions are then screened using Eckhardt gels which have the CP-linked marker phenotype and which (Eckhardt (1978) Plasmid 1:584-585) to distinguish 35 have lost the marker phenotype of the shuttle vector those mutants with chromosomal Tn5 insertions. The (chloramphenicol-resistance with pSUP205). It is desir Tn5 chromosomal insertion mutants are then screened able to confirm that the desired sequences have been in colonization assays (as in Example 5) to select mu incorporated in the resultant P. cepacia 526 selections tants unaffected in colonization. The location of Tn5 in using restriction analysis of genomic DNA followed by these mutants represent potential sites for insertion of 40 Southern blotting using radiolabelled CP sequence as a CP sequences. - hybridization probe. It is also desirable to assay plant The location of Tn5 insertions are then mapped and colonization ability and insect toxicity of any confirmed the CP sequences are inserted in their vicinity. These selections. steps are achieved by preparing genomic libraries of Those skilled in the art will appreciate that the inven both P. cepacia 526 and of P. cepacia 526:Tn5 (chromo 45 tion described herein and the methods of isolation and some insert). Genomic libraries of these strains can be identification specifically described are susceptible to prepared, for exampie, by partial digestion of genomic variations and modifications other than as specifically DNA with a restriction enzyme such as Sau3A. It is described. It is to be understood that the invention in preferred that the digestion conditions be adjusted so cludes all such variations and modifications which fall that the resultant fragments are in the 30 kb range, to SO within its spirit and scope. facilitate DNA packaging. The digested DNA is then We claim: cloned into an appropriate vector, such as into the 1. A genetically altered strain of Pseudomonas cepacia BamHI site of the cosmid pSUP205 (Simon et al., 1983), type Wisconsin which strain colonizes plant roots or the ligation mix is introduced into an appropriate strain, leaves, said strain being genetically altered by introduc like E. coli HB101, and transformants having the appro 55 tion of a cloned insect toxic protein gene or partial priate antibiotic markers chloramphen resistant, tetra protoxin gene of a strain of Bacillus thuringiensis such cycline-sensitive with pSUP205) are selected. The that said strain of Pseudomonas cepacia type Wisconsin choice of vector in the preparation of the library of the is thereby rendered toxic to insects. Tn5 mutant strain is not critical; however, the use of a 2. The genetically altered strain of Pseudomonas plasmid that will act as a suicide shuttle vector in the cepacia type Wisconsin of claim wherein said insect preparation of the library of the wild-type strain is pre toxic protein gene or partial protoxin gene is carried on ferred. The P. cepacia 526:Tn5 mutant strain clone a plasmid. library is then screened using hybridization techniques 3. The genetically altered strain of Pseudomonas with a Tn5-labelled probe to identify clones containing cepacia type Wisconsin of claim 1 wherein said insect Tn5 sequences, thereby identifying regions of the chro 65 toxic protein gene or partial protoxin gene is integrated mosome into which CP gene constructions can be in into the chromosome of said strain. serted. Inserts in these Tn5 containing clones are then 4. The genetically altered strain of Pseudomonas used as probes in similar hybridization experiments to cepacia type Wisconsin of claim 1 wherein said strain is 5,229,292 29 30 obtained by genetic alteration of Pseudomonas cepacia an insect toxic protein gene, which insect toxic partial 526. protoxin gene is the partial protoxin HD-73-like gene of 5. The genetically altered strain of Pseudomonas Bacillus thuringiensis var. kurstaki HD-1-Dipel. cepacia type Wisconsin of claim 1 wherein said strain is 12. An insecticidal plant protective composition obtained by genetic alteration of Pseudomonas cepacia which comprises 406. (a) an insecticidally effective concentration of an 6. A genetically altered strain of Pseudomonas cepacia insect toxic, non-phytopathogenic strain of Pseudo type Wisconsin, which strain colonizes plant roots or monas cepacia which colonizes roots or leaves of a leaves, said strain being genetically altered by the intro plant, which strain has been genetically altered by duction of an insect toxic protein gene or partial pro O toxin gene of a strain of Bacillus thuringiensis such that the introduction of a cloned structural gene of a said string of Pseudonnanas cepacia type Wisconsin ex strain of Bacillus thuringiensis, which gene encodes presses said insect toxic protein gene or partial protoxin a crystal protein or a partial protoxin and which gene, and is thereby rendered toxic to insects, wherein gene is expressed by said Pseudomonas cepacia; and said genetically altered insect toxic strain is Pseudomo 15 (b) an inert carrier. nas cepacia 526 (pSUP487). 13. An insecticidal plant protective composition 7. A genetically altered strain of Pseudomonas cepacia which comprises p1 (a) an insecticidally effective con type Wisconsin, which strain colonizes plant roots or centration of an insect toxic strain of Pseudomonas leaves, said strain being genetically altered by the intro cepacia type Wisconsin, which insect toxic strain has duction of an insect toxic protein gene or partial pro 20 been produced by genetically altering a strain of Pseu toxin gene of a string of Bacillus thuringiensis such that domonas cepacia type Wisconsin by the introduction of said strain of Pseudomonas cepacia type Wisconsin ex a cloned Bacillus thuringiensis structural gene encoding presses said insect toxic protein gene or partial protoxin an insect toxic crystal protein or a partial protoxin so gene, and is thereby rendered toxic to insects, wherein that said structural gene is expressed by said strain of said genetically altered insect toxic strain is Pseudomo 25 Pseudomonas cepacia type Wisconsin; and nas cepacia 526 (pSKP/Bt). (b) an inert carrier. 8. The genetically altered strain of claim 1 wherein 14. The insecticidal plant protective composition of said strain is genetically altered by the introduction of claim 13 wherein said insect toxic strain of Pseudomonas an insect toxic protein gene, which insect toxic protein cepacia type Wisconsin is obtained by genetic alteration gene is the crystal protein gene of Bacillus thuringiensis 30 of Pseudomonas cepacia 526. var. kurstaki HD-73. 15. The insecticidal plant protective composition of 9. The genetically altered strain of claim 1 wherein claim 13 wherein said insect toxic strain of Pseudomonas said strain is genetically altered by the introduction of cepacia is obtained by genetic alteration of Pseudomonas an insect toxic protein gene, which insect toxic protein cepacia 406. gene is the HD-73 like crystal protein gene of Bacillus 35 16. An insecticidal plant protective composition com thuringiensis var. kurstaki HD-1-Dipel. prising an insecticidally effective concentration of the 10. The genetically altered strain of claim 1 wherein insect toxic strain of Pseudomonas cepacia type Wiscon said strain is genetically altered by the introduction of sin of claim 6 and an inert carrier. an insect toxic protein gene, which insect toxic partial 17. An insecticidal plant protective composition com protoxin gene is the partial protoxin HD-73 gene of 40 prising an insecticidally effective concentration of the Bacillus thuringiensis var. kurstaki HD-73. insect toxic strain of Pseudomonas cepacia type Wiscon 11. The genetically altered strain of claim 1 wherein sin of claim 7 and an inert carrier. said strain is genetically altered by the introduction of k k k

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65 UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 5,229, 292 Page 1 of 2 DATED Jul. 20, 1993 INVENTOR (S) : Carolyn A. Stock; Thomas J. McLoughlin; Janet A. Klein; Michael J. Adang it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

At column 1, line 56, please rewrite "" (t1986) " as -- (1986)--. At column 2, line 40, please rewrite " (pature . . ." as -- (pasture . . . --. At column 2, line 57, please rewrite "Chirnomus" as -- Chironomus--. At column 4, line 18, please rewrite "lead" as -- leaf--. At column 4, line 40, please rewrite "clepsos" as -- clepsis--. At column 4, line 42, please rewrite " (Recrvaris)" as -- (Recurvaria) --. At column 6 line 52, please rewrite "vutworm)" as --cutworm--. At column 7 line 9, please rewrite " (omonvorous" as --omnivorous--. At column 7 line 26, please rewrite "leafworm")" as --armyworm--. At column 7 line 27, please rewrite " (Cotton" as -- (Cotton leafworm)--. At column 7, line 32, please rewrite "Sylleptederogata" as --Syllepte derogata--. At column 9, line 5, please rewrite "Society of Microbiolgists" as --Society for Microbiology--. At column 9, line 51, please rewrite "Organism" as --Organisms--. At column 15, line 52, please rewrite "is" as --if--. At column 21, line 57, please rewrite "BanHI" as --BamHI--. in Table 5, please rewrite "capacia". as --At cepacia--. column 23, At line column 43, 23, line 48, please move the following from lines 58-61 to line 48, as a footnote to Table 5. :

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. 5,1: 7 229 292 Page 2 of 2 DATED Jul. 20, 1993 INVENTOR(S)Carolyn A. Stock; Thomas J. McLoughlin; Janet A. Klein; Michael J. Adang it is Certified that error appears in the above-indentified patent and that said Letters Patent is hereby Corrected as shown below:

Bacterial counts in undiluted sample; CFU colony forming units. D = not done Those larvae alive were of reduced size compared to controls. Larvae either missing or killed by cap. t column 24, line 49, please rewrite "Transconivgants" as -- Transconjugants--. At column 27, line 56, please rewrite "chloramphen" as -- (chloramphenicol--. At column 29 line 12, please rewrite "string" as --strain--. At column 29, line 21, please rewrite "string" as --strain--. At column 30, line 17, please delete "pl".

Signed and Sealed this Thirtieth Day of May, 1995

BRUCELEHMAN Attesting Officer Commissioner of Patents and Trademarks