WO2007006079A1

WO2007006079A1 - Management of plant pathogens

Info

Publication number: WO2007006079A1
Application number: PCT/AU2006/000952
Authority: WO
Inventors: Robyn Louise Heath; Marilyn Anne Anderson
Original assignee: Hexima Limited
Priority date: 2005-07-08
Filing date: 2006-07-06
Publication date: 2007-01-18
Also published as: CA2614353A1; BRPI0612634A2; AU2006269808A1; EP1909559A1; EP1909559A4; CN101257792A

Abstract

The present invention relates generally to a multi-faceted approach to the control of plant pathogens including plant pests. More particularly, the present invention relates to plants such as crop plants genetically modified to produce at least two pesticidal or pestistatic agents which in at least one agent is a serine proteinase inhibitor or precursor thereof from the solanaceae family and at least one other is a non-serine proteinase inhibitor slected from the list a Bt protein, a member of the CRY family, a VIP and a defensin, and wherein said exhibits resistance or reduced susceptibility to a pest.

Description

MANAGEMENT OF PLANT PATHOGENS

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates generally to a multi-faceted approach to the control of plant pathogens including plant pests. More particularly, the present invention relates to plants such as crop plants genetically modified to produce at least two pesticidal or pestistatic agents which in combination provide the plant with enhanced resistance or reduced susceptibility to plant pests. The present invention further provides genetic constructs encoding the pesticidal or pestistatic agents and compositions comprising the agents. An integrated plant pest management strategy is also part of the present invention.

DESCRIPTION OF THE PRIOR ART Bibliographic details of the publications referred to in this specification are also collected at the end of the description.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Plant pathogens including insect pests and fungi are a major contributor to losses in agricultural crops. For example, the European corn borer alone is estimated to cost the corn growing industry up to a billion dollars a year in the United States in lost production and control measures. One particularly important agricultural crop is cotton. The cotton industry is a rapidly growing cash crop in many countries and in Australia, for example, approximately 200,000 bales of cotton are spun each year. Of these, 94% are sent for export. However, cotton plants are susceptible to a wide range of pests and the cost of controlling pest infestation in cotton in Australia alone is in the tens of millions of dollars. Traditionally, the primary method of controlling plant pests such as insects is the use of varying spectrum chemical insecticides and fungicides. However, for environmental, regulatory and health reasons, the use of chemicals on crops is being discouraged. In fact, many broad spectrum chemicals have been banned from use or their use severely limited to particular crops or in particular agricultural regions. There is an urgent need to develop alternative strategies to the control and management of crop pests including insects and fungi.

One approach is the use of more natural agents. One such agent is a serine proteinase inhibitor.

Several members of the families Solanaceae and Fabaceae accumulate serine proteinase inhibitors in their storage organs and in leaves in response to wounding (Brown and Ryan, Biochemistry 23:3418-3422, 1984; Richard Phytochemistry 7(5:159-169, 1977). The inhibitory activities of these proteins are directed against a wide range of proteinases of microbial and animal origin, but rarely against plant proteinases (Richardson, 1977 supra). It is believed that these inhibitors are involved in protection of the plants against pathogens and predators. In potato tubers and legume seeds, the inhibitors can comprise 10% or more of the stored proteins (Richardson, 1977 supra), while in leaves of tomato and potato (Green and Ryan, Science 116-111, 1972), and alfalfa (Brown and Ryan, 1984 supra), proteinase inhibitors can accumulate to levels of 2% of the soluble protein within 48 hours of insect attack, or other types of wounding (Brown and Ryan, 1984 supra; Graham et al, Plants 169:399-405, 1986). High levels of these inhibitors (up to 50% of total soluble protein) are also present in unripe fruits of the wild tomato, Lycopersicon peruvianum (Pearce et al, Planta 775:527-531, 1988).

There are two families of serine proteinase inhibitors in tomato and potato (Brown and Ryan, 1984 supra). Type I inhibitors are small proteins (monomer Mr 8100) which inhibit chymotrypsin at a single reactive site (Melville and Ryan, Archives of Biochemistry and Biophysics 138:100-102, 1970; Plunkett et al, Arch Biochem Biophys 213:463-412, 1982). Inhibitors of the type II family generally contain two reactive sites, one of which inhibits chymotrypsin and the other trypsin (Bryant et al, Biochemistry 75:3418-3424, 1976;

Plunkett et al, 1982 supra). The type II inhibitors have a monomer Mr of 12,300 (Plunkett et al, 1982 supra). Proteinase inhibitor I accumulates in etiolated tobacco (Nicotiana

■ tabacum) leaves (Kuo et al, Arch Biochem Biophys 230:504-510, 1984), and elicitors from Phytophthora parasitica var. nicotianae were found to induce proteinase inhibitor I accumulation in tobacco cell suspension cultures (Rickauer et al, Plant Physiol 9:1065-

1070, 1989).

The serine proteinase inhibitor precursor from tobacco, NaPI, has been particularly well characterized and used in pest control (See United States Patent Nos. 6,031,087, 6,440,727, 6,451,573 and 6,261,821 all of which are incorporated herein by reference).

Certain species of microorganisms are also known to possess pathogenocidal activity against a broad range of pests including Lepidoptera, Diptera, Coleptera, Hemiptera and others. Bacillus thuringiensis and Bacillus papilliae are two particularly useful biological control agents. Pesticidal activity is concentrated in parasporal crystalline protein inclusions although pesticidal proteins have also been isolated from the vegetative growth stage of Bacillus. Several genes encoding pesticidal proteins have been cloned and characterised (See United States Patent Nos. 5,366,982 and 5,840,868).

Corn and cotton plants have been genetically engineered to produce pesticidal δ- endotoxins and crytoxins from B. thuringiensis (Aronson Cell MoI Life Sci 59(3):4l7-425, 2002; Schnepfet α/, Microbiol MoI Biol Rev 62 (3):775-806, 1998).

Despite the success of biological control agents, there is always a danger of resistance developing. There is also some variability associated with the cytotoxicity of a single agent.

There is a need to develop alternative strategies for pest control in plants. SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

In accordance with the present invention, a multi-faceted approach is provided for pest control in plants, and in particular crop plants. In this approach, modules of pesticidal or pestistatic agents derived from biological sources are combined in plants or in compositions. At least one agent is a serine proteinase inhibitor or a precursor thereof, and at least one other agent acts in a manner different to a proteinase inhibitor. Generally, at least one agent is a Bt protein such as a Cry family protein a Vegetative Insecticidal Protein (VIP) or is a defensin like molecule. In a preferred embodiment, the serine proteinase inhibitor is from the Solanaceae family such as but not limited to NaPI from tobacco. Genetic material encoding the pesticidal or pestistatic agents is generally introduced into a desired plant which is then rendered genetically capable of producing the pesticidal or pestistatic agents. In combination, the two or more pesticidal or pestistatic modules confer enhanced, increased, more stable, more efficacious, greater pesticidal or pestistatic activity, broader spectrum and/or longer lasting resistance or reduced susceptibility to pest infestation. By "pest" includes any plant pathogen or insect or any agent which carriers a serine proteinase. Examples of plant pests include fungi, yeasts, and insects. Insects groups particular contemplated by the present invention include such as Helicoverpa, Leptiderpa, Coleoptera, Diptera, Orthoptera, Diatraea and Spocloptera.

Accordingly, one aspect of the present invention provides a plant genetically modified to produce at least two pesticidal or pestistatic agents wherein at least one agent is a serine proteinase inhibitor or precursor thereof wherein in precursor form, comprises at least three monomers wherein at least one monomer is a trypsin inhibitor and at least one other monomer is a chymotrypsin inhibitor and wherein at least one other agent is not a serine proteinase inhibitor. More particularly, the present invention provides a genetically modified plant or its progeny resulting from self-crossing, back-crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is a serine proteinase inhibitor or precursor thereof from the Solanaceae family wherein in precursor form, it comprises at least three monomers wherein at least one monomer is a trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and wherein at least one other agent is a non-serine proteinase inhibitor, wherein said plant exhibits resistance or reduced susceptibility to a pest.

In a preferred embodiment, the serine proteinase inhibitor or precursor is from tobacco and is referred to as NaPI. In another preferred embodiment, the other agent is an endotoxin such as a Bt protein including a Cry family protein, Cry, VIP or is a defensin or a component thereof. Reference to a defensin includes its N-terminal portion, acidic tail and/or a central portion alone or fused to a heterologous protein.

Accordingly, another aspect of the present invention provides a genetically modified plant or its progeny resulting from self-crossing, back-crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is the NaPI precursor or a monomer thereof or a recombinant or derivative thereof and wherein at least one other agent is a non-serine proteinase inhibitor wherein said plant exhibits resistance or reduced susceptibility to a pest.

Another aspect of the present invention contemplates a genetically modified plant or its progeny resulting from self-crossing, back-crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is a serine proteinase inhibitor or precursor thereof from the Solanaceae family wherein in precursor form, it comprises at least three monomers wherein at least one monomer is a trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and wherein at least one other agent is a non-serine proteinase inhibitor, wherein said plant exhibits resistance or reduced susceptibility to a pest except and wherein at least one other agent is a Bt family protein such as a Cry family protein or defensin. Examples of Cry proteins include Cry9, Cry2, Cry3, Cry IAc, VIP.

More particularly, the present invention is directed to a genetically modified plant or its progeny resulting from self crossing, back crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is NaPI precursor or a monomer thereof or a recombinant or derivative thereof and wherein at least one other agent is a non-serine proteinase inhibitor wherein said plant exhibits resistance or reduced susceptibility to a pest except wherein the least one other agent is selected from the list comprising a Bt protein and a defensin.

Examples of Bt crystal proteins as described in Hofte and Whiteley Microbial Reviews 53:242-255, 1989.

The present invention further provides two or more genetic constructs encoding the at least two pesticidal or pestistatic agents or a single construct encoding the at least two agents. In addition, compositions capable of being applied to crops, leaves, seeds, flowers or roots such as by spray, paint, powder or micronized vapour and comprising the at least two agents are contemplated by the present invention.

Parts, reproductive parts, fruits, seeds, flowers and harvested portions of crops genetically modified to produce the at least two pesticidal or pestistatic agents also form part of the present invention. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graphical representation of a mass of larvae surviving on a diet of cotton leaf discs coated with Cryl Ac. The control leaf discs were from cv Coker; the test leaf discs were from cv Coker homozygous for the NaPI gene. (Note: Leaves were selected to give only a small reduction in weight of larvae.)

Figure 2 is a graphical representation of a mass of larvae surviving on a diet of cotton leaf discs coated with Cryl Ac. The control leaf discs were from cv Coker; the test leaf discs were from cv Coker homozygous for the NaPI gene.

Figure 3 is a graphical representation of mortality of larvae after feeding on a diet of cotton leaves coated with Cry IAc. The control leaf discs were from cv Coker; the test leaf discs were from cv Coker expressing the NaPI gene.

Figure 4 is a graphical representation showing the average weight of larvae from day 11 to day 12 (approximately 20 hours). Error bars represent standard error of the mean.

Figure 5 is a graphical representation showing the mean increase in larval mass from day 11 to day 12. Error bars represent standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a multi-faceted approach to pest control in plants, and in particular crop plants.

Reference to a "multi-faceted approach" means the use of two or more agents in combination to effect a pesticidal or pestistatic outcome on a targets pest or group of pests on commencement of or during its infestation or attempted infestation of a plant. The multi-faceted approach is modular in design in that each pesticidal or pestistatic agent is considered a modular component. Different modular components are used in the overall pest management system.

By "pestistatic" is meant the reduction in growth or reproduction or colonization ability of a pest without necessitating death of the pest. Whilst pesticidal activity is preferred, a pestistatic effect is acceptable and forms part of the present invention.

As used herein" pesticidal activity" includes "insecticidal activity" when the target pest is an insect or "fungicidal activity" when the target pest is a fungus or "bacteriocidal activity" when the target pest is a bacterium or "nematodicidal activity" when the target pest is a nematode. These terms refer to the activity of an agent, also referred to as a "pesticidal modular component" against a pest. The activity is conveniently measured in terms of the effect on pest mortality, pest weight loss, pest repellency and other behavioural, physiological and/or physical changes of the pest including ability to digest protein, the ability to grow, maintain itself and/or reproduce after exposure to the agent. Accordingly, the agent has an adverse effect on pest fitness as determined by at least one measurable parameter. In accordance with the present invention, at least two agents have an effect on pest fitness which may be additive or synergistic or otherwise complementary.

Accordingly, the at least two agents may exhibit the same effect on the measurable parameter of pest fitness or the combined effect may differ to the individual effects of each agent. In addition, at least one agent may exhibit pesticidal activity and another agent may exhibit pestistatic activity. Preferably, however, both agents or the combined effect of the agents is pesticidal.

The agents are proteinaceous in nature when they are produced by plant cells but may be chemically modified when incorporated into a pesticidal or pestistatic composition. The agents may, therefore, be peptides, polypeptides or proteins as described herein or chemical analogs or chemical mimetics or functional equivalents.

As indicated above, the agents exhibit an effect on pests which effect may be referred to as an "impact". Examples of impacts including changes in feeding ability or habits or capacity, ability to undergo development changes, cytotoxicity, growth retardation, reduction in reproductivity, effects on eggs or larvae, and mortality or morbidity of the pest. In addition, the efficacy of the pesticidal or pestistatic activity can be assess by the effect on the crop plant e.g. growth yeild, fewer pests, reduced pathology.

The subject agents are provided to or in a plant in a "pesticidal effective amount" or in a "pestistatic effective amount". These amounts connotes the quantity of agents, separately or in combination, to provide individual or overall pesticidal or pestistatic activity or other appropriate impact.

Reference herein to a plant includes a monocotyledonous plant or a dicotyledonous plant and the plant may be generated from genetically transformed callus or tissue or it may be a progeny of the transformed plant or a cross between the transformed plant or its progeny and a non-transformed plant. As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like.

Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. junceά), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativά), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italicά), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), cotton pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Car ica papaya), cashew (Anacardium occidental), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C sativus), cantaloupe (C. cantalupensis), and musk melon (C melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas- fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Plants of the present invention include crop plants (for example, cotton, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants. Turfgrasses include, but are not limited to: annual bluegrass (Poa annuά); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewings fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis glomerata); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (Phleum pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron smithii); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum); carpet grass (Axonopus affinis); centipede grass (Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum (Paspalum vaginatum); blue gramma (Bouteloua gracilis); buffalo grass (Buchloe dactyloids); sideoats gramma (Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

Preferred plants contemplated herein include cotton, sweet corn, tomato, tobacco, piniento, potato, sunflower, citrus, plums, sorghum, leeks, soybean, alfalfa, beans, pidgeon peas, chick peas, artichokes, curcurbits, lettuce, Dianthus, geraniums, cape gooseberry, maize, flax and linseed, lupins, broad beans, garden peas, peanuts, canola, snapdragons, cherry, sunflower, pot marigolds, Helichrysum, wheat, barley, oats, triticale, carrots, onions, orchids, roses and petunias. The embodiments of the present invention may be effective against a variety of pests. For purposes of the present invention, pests include, but are not limited to, insects, fungi, bacteria, nematodes, acarids, protozoan pathogens, animal-parasitic liver flukes, and the like. Pests of particular interest are insect pests, particularly insect pests that cause significant damage to agricultural plants. The term "insect pests" as used herein refers to insects and other similar pests such as, for example, those of the order Acari including, but not limited to, mites and ticks. Insect pests of the present invention include, but are not limited to, insects of the order Lepidoptera, e.g. Achoroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argittacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia Hneatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmiafeneralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia elutella, Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantia cunea, Keiferia lycopersicella, Lambdina βscellaria flscellaria, Lambdina βscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra conflgurata, Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes, Pectinophora gossypiella, Phryganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynota flouendana, Platynota stultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp., Thaurnstopoea pityocampa, Tinsola bisselliella, Trichoplusia M₁ Udea rubigalis, Xylomyges curtails, and Yponomeuta padella.

Also, the embodiments of the present invention may be effective against insect pests, including but not limited to insects selected from the orders Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, Coleoptera, etc., particularly Lepidoptera. Insect pests of the invention for the major crops include, but are not limited to: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zeae, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; western corn rootworm, e.g., Diabrotica virgifera virgifera; northern corn rootworm, e.g., Diabrotica longicornis barberi; southern corn rootworm, e.g., Diabrotica undecimpunctata howardi; Melanotus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis, corn blotch leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, two spotted spider mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, leser cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; chinch bug, e.g., Blissus leucopterus leucopterus; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, two-spotted spider mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, pale western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; southern corn rootworm, e.g., Diabrotica undecimpunctata howardi; Russian wheat aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ 'entialis, differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Acerϊa tulipae, wheat curl mite; Sunflower: Cylindrocupturus adspersus, sunflower stem weevil; Smicronyx fulus, red sunflower seed weevil; Smicronyx sordidus, gray sunflower seed weevil; Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; Zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis virescens, tobacco budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pink bollworm; boll weevil, e.g., Anthonomus grandis; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, two-spotted spider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhoper; chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum hilare, green stink bug; Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, tobacco budworm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, two-spotted spider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum hϊlare, green stink bug; Euschistus servus, brown stink bug; Jylemya platura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite; Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, crucifer flea beetle; Phyllotreta striolata, striped flea beetle; Phyllotreta nemorum, striped turnip flea beetle; Meligethes aeneus, rapeseed beetle; and the pollen beetles Meligethes rufimanus, Meligethes nigrescens, Meligethes canadianus, and Meligethes viridescens; Potato: Leptinotarsa decemlineata, Colorado potato beetle.

Furthermore, embodiments of the present invention may be effective against Hemiptera such as Lygus hesperus, Lygus lineolaris, Lygus pratensis, Lygus rugulipennis Popp, Lygus pabulinus, Calocoris norvegicus, Orthops compestris, Plesiocoris rugicollis, Cyrtopeltis modestus, Cyrtopeltis notatus, Spanagonicus albofasciatus, Diaphnocoris chlorinonis, Labopidicola allii, Pseudatomoscelis seriatus, Adelphocoris rapidus, Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae, Nysius raphanus, Euschistus servus, Nezara viridula, Eurygaster, Coreidae, Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, and Cimicidae. Pests of interest also include Araecerus fasciculatus, coffee bean weevil; Acanthoscelides obtectus, bean weevil; Bruchus rufmanus, broadbean weevil; Bruchus pisorum, pea weevil; Zabrotes subfasciatus, Mexican bean weevil; Diabrotica balteata, banded cucumber beetle; Cerotoma trifurcata, bean leaf beetle; Diabrotica virgifera, Mexican corn rootworm; Epitrix cucumeris, potato flea beetle; Chaetocnema conβnis, sweet potato flea beetle; Hypera postica, alfalfa weevil; Anthonomus quadrigibbus, apple curculio; Sternechus paludatus, bean stalk weevil; Hypera brunnipennis, Egyptian alfalfa weevil; Sitophilus granaries, granary weevil; Craponius inaequalis, grape curculio; Sitophilus zeamais, maize weevil; Conotrachelus nenuphar, plum curculio; Euscepes postfaciatus, West Indian sweet potato weevil; Maladera castanea, Asiatic garden beetle; Rhizotrogus majalis, European chafer; Macrodactylus subspinosus, rose chafer; Tribolium confusum, confused flour beetle; Tenebrio obscurus, dark mealworm; Tribolium castaneum, red flour beetle; Tenebrio molitor, yellow mealworm. Nematodes include parasitic nematodes such as root-knot, cyst, and lesion nematodes, including Heterodera spp., Meloidogyne spp., and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode); and Globodera rostochiensis and Globodera pallida (potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

The present invention is directed to a pest management system for plants which is multi- faceted in the sense that it involves two or more pesticidal and/or pestistatic agents generally designed to exhibit a combined impact on plant pests. At least one agent is a serine proteinase inhibitor and least one agent is a non-serine proteinase inhibitor (i.e. it acts via a mechanism different to the inhibition of serine proteinase activity). The multi- faceted system may employ both agents simultaneously or sequentially. In either event, this is described as the agents operating in "combination".

Accordingly, one aspect of the present invention provides a genetically modified plant or its progeny resulting from self-crossing, back-crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is a serine proteinase inhibitor or precursor thereof from the Solanaceae family wherein in precursor form, it comprises at least three monomers wherein at least one monomer is a trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and wherein at least one other agent is a non-serine proteinase inhibitor, wherein said plant exhibits resistance or reduced susceptibility to a pest.

The serine proteinase inhibitor is preferably from tobacco and may comprise in precursor form at least three monomers, or four monomers, or five monomers, or six monomers, or seven monomers, or eight monomers wherein at least one monomer inhibits trypsin and at least one monomer inhibits chymotrypsin. The term "precursor" as used herein is not intended to place any limitation on the utility of the precursor molecule itself or a requirement that the molecule first be processed into monomers before proteinase inhibitory activity is expressed. The precursor molecule has proteinase inhibitory activity and the present invention is directed to the precursor and to the individual monomers of the precursor.

In a most preferred embodiment, the proteinase inhibitor or its precursor is from Nicotiana alata (NaPI) as described in International Patent Application No. PCT/AU93/00659 [WO 94/13810] which is incorporated herein by reference.

The preferred at least one other agent is a toxin including an endotoxin such as derived from a microorganism or fungus. Particularly useful examples include toxins derived from

Bacillus species. Reference to a toxin includes a peptide, polypeptide or protein exhibiting pesticidal or pestistatic activity but which does not inhibit solely a serine proteinase. It may inhibit a serine proteinase only if it also exhibits another mode of pesticidal or pestistatic activity. Particularly useful toxins are Bt-related toxins from B. thuringiensis such as the Cry family of endotoxins. Examples of these endotxins include Cry9, Cry2s,

Cry3s or Cry IAc family of endotoxins.

The term "Cry9 family" is used herein to refer to nucleotide or amino acid sequences which share a high degree of sequence identity or similarity to previously described wild type Cry9 or Cry9D sequences. Bt toxin or endotoxin is intended to include the broader class of Cry toxins found in various status of B. thuringiensis and includes such toxins, as CrylAc, Cryls, Cry2s and Cry3s. Examples of suitable toxins can be found in US Patent Application Nos. 2005/0138685, 2004/0016020 and 2004/0221333 which are each incorporated herein by reference.

Other suitable toxins include vegetative insecticidal protein (VIP) and a defensin. Suitable defensins are described in International Patent Application No. PCT/AU2004/000524 which is incorporated herein by reference.

According, another aspect of the present invention contemplates a genetically modified plant or its progeny resulting from self-crossing, back-crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is a serine proteinase inhibitor or precursor thereof from the Solanaceae family wherein in precursor form, it comprises at least three monomers wherein at least one monomer is a trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and wherein at least one other agent is a non-serine proteinase inhibitor, wherein said plant exhibits resistance or reduced susceptibility to a pest except and wherein at least one other agent is selected from the list comprising a Bt protein, a Cry9 family protein, a Cry2 family protein or Cry3 family protein, Cry IAC, a VIP and a defensin.

More particularly, the present invention is directed to a genetically modified plant or its progeny resulting from self crossing, back crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is NaPI precursor or a monomer thereof or a recombinant or derivative thereof and wherein at least one other agent is a non-serine proteinase inhibitor wherein said plant exhibits resistance or reduced susceptibility to a pest except wherein the least one other agent is selected from the list comprising a Bt protein including a Cry family protein, a VIP and a defensin. Reference herein to NaPI, proteinase inhibitors, Bt, Cry9, CrylAc, Cryls, Cry2s, Cry3s, VIP and defensin and the like include derivatives and fragments and otherwise modified forms. Examples of modified forms include mutagenized forms such as mutants with altered substrate specificity or which exhibit greater stability or which direct the agent to a plant cell organelle such as a vacuole.

Serine Protease inhibitors such as NaPIs are known to act directly on the gut proteases of the digestive system of lepidopteran pests. A complex is formed between the specific inhibitor (either a trypsin or chymotrypsin inhibitor) and the protease (either a trypsin or chymotrypsin). These complexes are then excreted in the frass. The effect is to diminish the digestive capacity of the insect for protein and to deplete the protein reserves of the insect larvae.

Protease inhibitors may also affect the innate immunity system of insects. For this effect to occur, the inhibitors must move into the haemolymph where they can complex with the proteases in the cascades of the innate immunity system. Normally there is very restricted access of foreign molecules to this fundamental immune system of insects. An aspect of our invention is to provide conditions which allow enhanced access of PIs to the innate immunity system.

As indicated above, the present invention extends to parts of the genetically modified plants or, their progeny which include seeds, pollen, leaves, flowers, stigmas, stems, roots, root hairs, bark and apical meristems. Parts also include root stock or other form of commercially exploitable plant parts.

The present invention further provides nucleic acid molecules encoding the pesticidal or pestistatic agents. The nucleic acid molecules may be in a single construct, in multiple constructs or may be in situ in a plant cell genome. Conveniently, the nucleic acid molecules form a genetic composition. The nucleic acid molecules may also be part of beads such as beads used in biolistic DNA transfer. Accordingly, another aspect of the present invention provides a genetic composition comprising at least one construct encoding a serine proteinase inhibitor or a precursor form thereof wherein in precursor form it comprises at least three monomers wherein at least one monomer is a trypsin inhibitor and at least one other monomer inhibits chymotrypsin and at least one construct encodes a non-serine proteinase inhibitor.

Generally, the nucleic acid molecules are operably linked to one or more promoters.

The promoter may regulate the expression of the nucleotide sequence encoding the agent, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or pathogens, or metal ions, amongst others.

Preferably, the promoter is capable of regulating expression of a nucleic acid molecule in a plant cell, tissue or organ, at least during the period of time over which the nucleotide sequence encoding the agent is expressed therein.

Plant-operable promoters are particularly preferred for use in the constructs of the present invention. Examples of suitable promoters include pCaMV 35S (Fang et al, Plant Cell 7:141-150, 1989), PGELl (Hajdukiewicz et al, Plant MoI Biol 25:989-994, 1994), class III chitinase (Samac and Shah, Plant Cell 3:1063-1072, 1991), pin2 (Keil et al, EMBO J 5:1323-1330, 1989), PEP carboxylase (Pathirana et al, Plant J 72:293-304, 1997; MAP kinase (Schoenbeck et al, Molec Plant-Microbe Interact, 1999), MSV (Legavre et al, In: Vth International Congress of Plant Molecular Biology, Singapore, 1997), pltp (Hsu et al, Plant Sci 143:63-70, 1999), pmpi (Cordero et al, In: General Meeting of the International Program on Rice Biotechnology of the Rockefeller Foundation, Malacca, Malaysia, 1997) or glutamin synthase (Pujade-Renaud et al, Plant Physiol Biochem 55:85-93, 1997).

In the present context, the terms "in operable connection with" or "operably under the control" or similar shall be taken to indicate that expression of the nucleic acid molecule is under the control of the promoter sequence with which it is spatially connected; in a cell, tissue, organ or whole plant.

A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, inducible, or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al, Nature 373:810-812, 1985); rice actin McElroy et al, Plant Cell 2:163-171, 1990); ubiquitin (Christensen et al, Plant MoI Biol 72:619-632, 1989 and Christensen et al, Plant MoI Biol 18:675-689, 1992); pEMU (Last et al, Theor Appl Genet 87:581-588, 1991); MAS (Velten et al, EMBO J 3:2723-2730, 1984); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for regulating the expression of the nucleotide sequences of the present invention in plants are wound-inducible promoters. Such wound- inducible promoters, may respond to damage caused by insect feeding, and include potato proteinase inhibitor (pin II) gene (Ryan Ann Rev Phytopαth 25:425-449, 1990; Duan et αl, Nature Biotechnology 14:494-498, 1996); wunl and wun2, U.S. Pat. No. 5,428,148; winl and win2 (Stanford et al, MoI Gen Genet 275:200-208, 1989); systemin (McGurl et al, Science 225:1570-1573, 1992); WIPl (Rohmeier et al, Plant MoI Biol 22:783-792, 1993; Eckelkamp et al, FEBS Letters 323:73-76, 1993); MPI gene (Corderok et al, Plant J (5(2J:141-150, 1994); and the like, herein incorporated by reference.

Additionally, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the present invention. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-l,3-glucanase, chitinase, etc. See, for example, Redolfi et al, Meth J Plant Pathol 59:245-254, 1983; Uknes et al, Plant Cell 4:645-656, 1992; and Van Loon Plant MoI Virol 4:111-116, 1985. See also WO 99/43819, herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al, Plant MoI Biol 9:335-342, 1987; Matton et al, Molecular Plant-Microbe Interactions 2:325-331, 1989; Somsisch et al, Proc Natl Acad Sci USA 53:2427-2430, 1986; Somsisch et al, MoI Gen Genet 2:93-98, 1988; and Yang Proc Natl Acad Sci USA 95:14972-14977, 1996. See also, Chen et al, Plant J 10:955-966, 1996; Zhang et al, Proc Natl Acad Sci USA 97:2507-2511, 1994; Warner et al, Plant J 5:191-201, 1993; Siebertz et al, Plant Cell 7:961-968, 1989; U.S. Pat. No. 5,750,386 (nematode-inducible); and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium monϊliforme (see, for example, Cordero et al, Physiol MoI Plant Path 47:189- 200, 1992).

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- Ia promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al, Proc Natl Acad Sci USA 55:10421- 10425, 1991 and McNellis et al, Plant J 14(2) :2A1 -251, 1998 and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al, MoI Gen Genet 227:229-237, 1991, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced pesticidal protein expression within a particular plant tissue. Tissue-preferred promoters include those discussed in Yamamoto et al, Plant J 12 (2) -.255-265, 1997; Kawamata et al, Plant Cell Physiol 38(7):792-803, 1997; Hansen et al, MoI Gen Genet 254(3) :337-343, 1997; Russell et al, Transgenic Res 6(2):\57-\68, 1997; Rinehart et al, Plant Physiol 772(3J:1331-1341, 1996; Van Camp et al, Plant Physiol 112(2):525-535, 1996; Canevascini et al, Plant Physiol 112(2):5\3-524, 1996; Yamamoto et al, Plant Cell Physiol 35(5):773-778, 1994; Lam Results Probl Cell Differ 20:181-196, 1994; Orozco et al, Plant MoI Biol 23 |φ:l 129-1138, 1993; Matsuoka et al, Proc Natl Acad Sci USA 90(20):9586-9590, 1993; and Guevara- Garcia et al, Plant J 4(3):495-505, 1993. Such promoters can be modified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al, 1997 supra; Kwon et al, Plant Physiol 105:357-67, 1994; Yamamoto et al, 1994 supra; Gotor et al, Plant J 5:509-18, 1993; Orozco et al, 1993 supra and Matsuoka et al, 1993 supra.

Root-preferred or root-specific promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al, Plant MoI Biol 20(2) -.207-218, 1992 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner Plant Cell 3(7φ:1051-1061, 1991 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al, Plant MoI Biol

14 (3) -.433 -443, 1990 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al, Plant Cell 3(1) : 11-22, 1991 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al, Plant Cell 2 (7) -.633-641, 1990, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Promoters of rolC and rolD root-inducing genes of Agrobacterium rhizogenes may also be used (see Limerick Plant Science 79(l):69-76). Teeri et al, EMBO J 8(2):343-350, 1989) describe gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene. The TRl¹ gene fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al, Plant MoI Biol 29 (4) -.759-772, 1995); and rolb promoter (Capana et al, Plant MoI Biol 25(4):68l-691, 1994. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

"Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seed- germinating" promoters (those promoters active during seed germination). See Thompson et al, BioEssays 10:108, 1989, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19Bl (maize 19 kDa zein); and milps (myo-inositol-1 -phosphate synthase) (see U.S. Pat. No. 6,225,529, herein incorporated by reference). Gamma-zein and Glob-1 are endosperm- specific promoters. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where seed-preferred promoters from endl and end2 genes are disclosed; herein incorporated by reference. A promoter that has "preferred" expression in a particular tissue is expressed in that tissue to a greater degree than in at least one other plant tissue. Some tissue-preferred promoters show expression almost exclusively in the particular tissue.

Where low level expression is desired, weak promoters will be used. Generally, the term "weak promoter" as used herein refers to a promoter that drives expression of a coding sequence at a low level. By low level expression at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts is intended. Alternatively, it is recognized that the term "weak promoters" also encompasses promoters that drive expression in only a few cells and not in others to give a total low level of expression. Where a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the core ³⁵S CaMV promoter, and the like. Other constitutive promoters include, for example, those disclosed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; herein incorporated by reference.

The construct preferably contains additional regulatory elements for efficient transcription, for example, a transcription termination (or terminators) sequence.

The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3 '-non-translated DNA sequences generally containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3 '-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants or synthesized de novo.

As with promoter sequences, the terminator may be any termination sequence which is operable in the cells, tissues or organs in which it is intended to be used.

Examples of terminators particularly suitable for use in the synthetic genes of the present invention include the SV40 polyadenylation signal, the HSV TK polyadenylation signal, the CYCl terminator, ADH terminator, SPA terminator, nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the Rubisco small subunit gene (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, any r/zo-independent E. coli terminator, or the lacZ alpha terminator, amongst others.

In a particularly preferred embodiment, the terminator is the SV40 polyadenylation signal or the HSV TK polyadenylation signal which are operable in animal cells, tissues and organs, octopine synthase (OCS) or nopaline synthase (NOS) terminator active in plant cells, tissue or organs, or the lacZ alpha terminator which is active in prokaryotic cells.

Those skilled in the art will be aware of additional terminator sequences which may be suitable for use in performing the subject invention. Such sequences may readily be used without any undue experimentation.

Means for introducing (i.e. transfecting or transforming) cells with the constructs are well- known to those skilled in the art.

The constructs described supra are capable of being modified further, for example, by the inclusion of marker nucleotide sequences encoding a detectable marker enzyme or a functional analogue or derivative thereof, to facilitate detection of the synthetic gene in a cell, tissue or organ in which it is expressed. According to this embodiment, the marker nucleotide sequences will be present in a translatable format and be expressed. In addition, transport sequences may be included to direct one or more agents to particular plant organnelles.

Those skilled in the art will be aware of how to produce the constructs described herein and of the requirements for obtaining the expression thereof, when so desired, in a specific cell or cell-type under the conditions desired. In particular, it will be known to those skilled in the art that the genetic manipulations required to perform the present invention may require the propagation of a genetic construct described herein or a derivative thereof in a prokaryotic cell such as an E. coli cell or a plant cell or an animal cell. The constructs of the present invention may be introduced to a suitable cell, tissue or organ without modification as linear DNA, optionally contained within a suitable carrier, such as a cell, virus particle or liposome, amongst others. To produce a genetic construct, a nucleic acid is inserted into a suitable vector or episome molecule, such as a bacteriophage vector, viral vector or a plasmid, cosmid or artificial chromosome vector which is capable of being maintained and/or replicated and/or expressed in the host cell, tissue or organ into which it is subsequently introduced.

Accordingly, a further aspect of the present invention provides a genetic construct which at least comprises a genetic element encoding one or both pesticidal or pestistatic agents as herein described and one or more origins of replication and/or selectable marker gene sequences.

Usually, an origin of replication or a selectable marker gene suitable for use in bacteria is physically-separated from those genetic sequences contained in the genetic construct which are intended to be expressed or transferred to a plant cell, or integrated into the genome of a plant cell.

As used herein, the term "selectable marker gene" includes any gene which confers a phenotype on a cell on which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof.

Suitable selectable marker genes contemplated herein include the ampicillin-resistance gene (Amp¹), tetracycline-resistance gene (Tc¹), bacterial kanamycin-resistance gene

(Kan¹), the zeocin resistance gene (Zeocin is a drug of the bleomycin family which is trade mark of InVitrogen Corporation), the AURI-C gene which confers resistance to the antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin phosphotransferase gen (nptlϊ), hygromycin-resistance gene, β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein-encoding gene or the luciferase gene, amongst others. Preferably, the selectable marker gene is the nptll gene or Kan^r gene or green fluorescent protein (GFP)-encoding gene.

Those skilled in the art will be aware of other selectable marker genes useful in the performance of the present invention and the subject invention is not limited by the nature of the selectable marker gene.

The present invention extends to all genetic constructs essentially as described herein, which include further genetic sequences intended for the maintenance and/or replication of said genetic construct in prokaryotes or eukaryotes and/or the integration of said genetic construct or a part thereof into the genome of a eukaryotic cell or organism.

Standard methods may be used to introduce the constructs into the cell, tissue or organ, for example, liposome-mediated transfection or transformation, transformation of cells with attenuated virus particles or bacterial cells, cell mating, transformation or transfection procedures known to those skilled in the art.

Additional means for introducing recombinant DNA into plant tissue or cells include, but are not limited to, transformation using CaCl₂ and variations thereof, direct DNA uptake into protoplasts, PEG-mediated uptake to protoplasts, microparticle bombardment, electroporation, microinjection of DNA, microparticle bombardment of tissue explant or cells, vacuum-infiltration of tissue with nucleic acid, or in the case of plants, T-DNA- mediated transfer from Agrobacterium to the plant tissue.

For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al, (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed. Examples of microparticles suitable for use in such systems include 1 to 5 μm gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

The methods of the invention involve introducing a polypeptide or polynucleotide into a plant. "Introducing" is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

"Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al, Biotechniques 4:320-334, 1986), electroporation (Riggs et al, Proc Natl Acad Sci USA 83:5602-5606, 1986), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al, EMBO J 3:2717- 2722, 1984), and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al, Plant Cell, Tissue, and Organ Culture: Fundamental Methods, 1995; and McCabe et al, Biotechnology (5:923-926, 1988); and Lecl transformation (WO 00/28058). For potato transformation see Tu et al, Plant Molecular Biology 57:829-838, 1998 and Chong et al, Transgenic Research P:71-78, 2000. Additional transformation procedures can be found in Weissinger et al, Ann Rev Genet 22:421-477, 1988; Sanford et al, Particulate Science and Technology 5:27-37, 1987 (onion); Christou et al, Plant Physiol 87:671-674, 1988 (soybean); McCabe et al, Bio/Technology 6:923-926, 1988 (soybean); Finer and McMullen In Vitro Cell Dev Biol 27PΛ75-1S2, 1991 (soybean); Singh et al, Theor Appl Genet 96:319-324, 1998 (soybean); Datta et al, Biotechnology 8:736-740, 1990 (rice); Klein et al, Proc Natl Acad Sci USA £5:4305-4309, 1988 (maize); Klein et al, Biotechnology 6:559-563, 1988 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al, Plant Physiol 97:440-444, 1988 (maize); Fromm et al, Biotechnology 5:833-839, 1990 (maize); Hooykaas-Van Slogteren et al, Nature (London) 311:763-764, 1984; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al, Proc Natl Acad Sci USA 54:5345-5349, 1987 (Liliaceae); De Wet et al, The Experimental Manipulation of Ovule Tissues, 197-209, 1985 (pollen); Kaeppler et al, Plant Cell Reports P:415-418, 1990 and Kaeppler et al, Theor Appl Genet 84:560-566, 1992 (whisker- mediated transformation); DΗalluin et al, Plant Cell 4: 1495- 1505, 1992 (electroporation); Li et al, Plant Cell Reports 72:250-255, 1993 and Christou and Ford Annals of Botany 75:407-413, 1995 (rice); Osjoda et al, Nature Biotechnology 14:745-750, 1996 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

In a further embodiment of the present invention, the genetic constructs described herein are adapted for integration into the genome of a cell in which it is expressed. Those skilled in the art will be aware that, in order to achieve integration of a genetic sequence or genetic construct into the genome of a host cell, certain additional genetic sequences may be required. In the case of plants, left and right border sequences from the T-DNA of the Agrobacterium tumefaciens Ti plasmid will generally be required.

The present invention further extends to an isolated cell, tissue or organ comprising the constructs or parts thereof. The present invention extends further to regenerated tissues, organs and whole organisms derived from said cells, tissues and organs and to propagules and progeny thereof as well as seeds and other reproductive material. For example, plants may be regenerated from transformed plant cells or tissues or organs on hormone-containing media and the regenerated plants may take a variety of forms, such as chimeras of transformed cells and non-transformed cells; clonal transformants (e.g. all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissue (e.g. a transformed root stock grafted to an untransformed scion in citrus species). Transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, first generation (or Tl) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques.

The present invention contemplates any other DNA sequence differing in its codon usage but encoding the same protein or a similar protein with substantially the same pesticidal or pestistatic activity, can be constructed, depending on the particular purpose. It has been described in some prokaryotic and eucaryotic expression systems that changing the codon usage to that of the host cell is desired for gene expression in foreign hosts (Bennetzen & Hall, J Biol Chem 257:3026, 1982; Itakura, Science 198:1056-1063, 1977). Codon usage tables are available in the literature (Wada et al, Nucl Acids Res 18:2367-1411, 1990; Murray et al, Nucleic Acids Research 77:477-498, 1989) and in the major DNA sequence databases. Accordingly, synthetic DNA sequences can be constructed so that the same or substantially the same proteins are produced. It is evident that several DNA sequences can be devised once the amino acid sequence of the instant agents proteins of this invention. Such other DNA sequences include synthetic or semi-synthetic DNA sequences that have been changed in order to inactivate certain sites in the gene, e.g. by selectively inactivating certain cryptic regulatory or processing elements present in the native sequence, or by adapting the overall codon usage to that of a more related host organism, preferably that of the host organism in which expression is desired. Synthetic DNA sequences could also be made following the procedures described in EP 0 385 962, EP 0 618 967, or EP 0 682 115.

Small modifications to a DNA sequence such as described above can be routinely made by PCR-mediated mutagenesis (Ho et al, Gene 77:51-59, 1989; White et al, Trends in Genet 5:185-189, 1989). New synthetic or semi-synthetic genes can be made by automated DNA synthesis and ligation of the resulting DNA fragments.

To prevent or delay the development of resistance by pests, it is preferred to also express in the same plant host, preferably a transgenic plant, the other protein or protein complex, which has a different mode of action, and a high toxicity to the same pest targeted by the first agent (e.g. serine proteinase inhibitor) when produced in a transgenic host, preferably a plant. Suitable candidates to be combined with the serine proteinase inhibitor include the mature VIPlAa protein when combined with the mature VIP2Aa or VIP2Ab protein of PCT publication WO 96/10083 in case these VIP proteins have a different mode of action compared to the serine proteinase inhibitors; the corn rootworm toxins of Photorhabdus or Xenorhabdus spp., e.g., the insecticidal proteins of Photorhabdus luminescens W-14 (Guo et al, J Biol Chem 274:9836-9842, 1999); the CryET70 protein of WO 00/26378; the insecticidal proteins produced by Bt strains PS80JJ1, PS149B1 and PS167H2 as described in WO 97/40162, particularly the about 14 kD and about 44 kD proteins of Bt strain PS149B1; the Cry3Bb protein of U.S. Pat. No. 6,023,013; protease inhibitors such as the N2 and Rl cysteine proteinase inhibitors of soybean (Zhao et al, Plant Physiol 777:1299- 1306, 1996) or oryzastatine such as rice cystatin (Genbank entry S49967), corn cystatin (Genbank entries D38130, D 10622, D63342) such as the corn cystatin expressed in plants as described by Me et al, Plant MoI Biol 30: 149- 157, 1996). Also included herein are all equivalents and variants, such as truncated proteins retaining insecticidal activity, of any of the above proteins.

DNA of the encoding serine proteinase inhibitor genes of the subject invention, can be ligated in suitable expression vectors and transformed in E. coli, and the clones can then be screened by conventional colony immunoprobing methods (French et al, Anal Biochem 156:417-423, 1986) for expression of the toxin with monoclonal or polyclonal antibodies raised against proteinase inhibitor. Also, the DNA can be ligated in suitable Bt shuttle vectors (Lereclus et al, Bio/Technology 70:418, 1992) and transformed in a crystal minus Bt-mutant. The clones are then screened for production of ISP proteins (by SDS-PAGE, Western blot and/or insect assay).

The genes encoding the invention can be sequenced in a conventional manner (Maxam and

Gilbert Methods in Enzymol 65:499-560, 1980; Sanger Proc Natl Acad Sci USA 74:5463- 5467, 1977) to obtain the DNA sequence. Sequence comparisons indicated that the genes are different from previously described genes encoding proteins secreted during the vegetative growth phase of Bacillus or other bacterial species and Bacillus thuringiensis crystal proteins with activity against Coleoptera (Crickmore, et al, Microbiology and

Molecular Biology Reviews (52:807-813, 1998; WO 98/44137, WO 94/21795, WO 96/10083, WO 00/09697, WO 9957282, and WO 9746105).

A pesticidal or pestistatic composition of the subject invention can also be formulated in a conventional manner using the microorganisms transformed with the genes, or preferably their respective proteins or pesticidally or pestistatically effective portions thereof as an active ingredient, together with suitable carriers, diluents, emulsifiers and/or dispersants (e.g., as described by Bernhard and Utz, An Environmental Biopesticide: Theory and Practice 255-267, 1993). This pesticidal or pestistatic composition can be formulated as a wettable powder, pellets, granules or dust or as a liquid formulation with aqueous or nonaqueous solvents as a foam, gel, suspension, concentrate, etc. Known microorganisms include cells of Pseudomonas or other bacteria that serve to encapsulate the proteins in a stable environment prior to application to the insects. Also included in the invention is a product comprising the agents described herein as a combined preparation for simultaneous, separate or sequential use to protect corn plants against corn rootworms, particularly such product is an insecticidal composition or a transgenic corn plant.

A method for controlling pests in accordance with the subject invention can comprise applying (e.g., spraying), to a locus (area) to be protected, an pesticidal or pestistatic effective amount of the agents or host cells transformed with the gene of the subject invention. The locus to be protected can include, for example, the habitat of the insect pests or growing vegetation or an area where vegetation is to be grown. To obtain the agents , cells of the recombinant hosts expressing the agents can be grown in a conventional manner on a suitable culture medium and the protein can then be obtained from the medium using conventional means. The agent can then be separated and purified by standard techniques such as chromatography, extraction, electrophoresis, or the like. The protease-resistant toxin form can then be obtained by protease, e.g. cysteine or serine protease, digestion of the protein.

While the invention does not depend on a particular biological mechanism for increasing the resistance of a plant to a plant pest, expression of the nucleotide sequences of the invention in a plant can result in the production of the pesticidal or pestistatic proteins of the invention and in an increase in the resistance of the plant to a plant pest. The plants of the invention find use in agriculture in methods for impacting plant pests. Certain embodiments of the invention provide transformed crop plants, such as, for example, cotton plants, which find use in methods for impacting insect pests of the plant.

A "subject plant or plant cell" is one in which genetic alteration, such as transformation, has been effected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in phenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed. As indicated above, one of skill in the art will readily acknowledge that advances in the field of molecular biology such as site-specific and random mutagenesis, polymerase chain reaction methodologies, and protein engineering techniques provide an extensive collection of tools and protocols suitable for use to alter or engineer both the amino acid sequence and underlying genetic sequences of proteins of agricultural interest.

Thus, the Cry9 family proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the pesticidal or pestistatic proteins can be prepared by introducing mutations into a synthetic nucleic acid (e.g., DNA molecule). Methods for mutagenesis and nucleic acid alterations are well known in the art. For example, designed changes can be introduced using an oligonucleotide-mediated site-directed mutagenesis technique. See, for example, Kunkel Proc Natl Acad Sd USA §2:488-492, 1985; Kunkel et al, Methods in Enzymol 154:367-382, 1987; U.S. Pat. No. 4,873,192; Walker and Gaastra, Techniques in Molecular Biology 1983, and the references cited therein.

The mutagenized Cry9 family nucleotide sequences of the invention may be modified so as to change about 1, 2, 3, 4, 5, 6, 8, 10, 12 or more of the amino acids present in the primary sequence of the encoded polypeptide. Alternatively, even more changes from the native sequence may be introduced such that the encoded protein may have at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more of the codons altered, or otherwise modified compared to the corresponding wild- type protein. In the same manner, the encoded protein may have at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more additional codons compared to the corresponding wild-type protein. It should be understood that the mutagenized Cry9 family nucleotide sequences of the present invention are intended to encompass biologically functional, equivalent peptides which have pesticidal activity, such as an improved pesticidal activity as determined by antifeedant properties against fall armyworm larvae. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.

One of skill in the art would recognize that amino acid additions and/or substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, charge, size, and the like. Exemplary amino acid substitution groups that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

In certain embodiments the nucleic acid sequences of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. For example, the polynucleotides of the present invention may be stacked with any other polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as other B. thuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al, Gene 45:109, 1986), pentin (described in U.S. Pat. No. 5,981,722) and the like. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including but not limited to traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,049); barley high lysine (Williamson et al, Eur J Biochem 7(55:99-106,1987; and WO 98/20122) and high methionine proteins (Pedersen et al, J Biol Chem 261:6279, 1986; Kirihara et al, Gene 77:359, 1988; and Musumura et al, Plant MoI Biol 12:123, 1989); increased digestibility (e.g., modified storage proteins (U.S. Application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. Application Ser. No. 10/005,429, filed Dec. 3, 2001)), the disclosures of which are herein incorporated by reference.

The polynucleotides of the present invention can also be stacked with traits desirable for disease or herbicide resistance (e.g., fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al, Science 266:789, 1994; Martin et al, Science 262:1432, 1993; and Mindrinos et al, Cell 7S:1089, 1994); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene and GAT gene as disclosed in U.S. application Ser. No. 10/004,357; and 10/427,692); and traits desirable for processing or process products such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al, J Bacteriol 770:5837-5847, 1988) facilitate expression of polyhydroxyalkanoates (PHAs)), the disclosures of which are herein incorporated by reference. One could also combine the polynucleotides of the present invention with polynucleotides providing agronomic traits such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are herein incorporated by reference.

These stacked combinations can be created by any method including but not limited to cross breeding plants by any conventional or TopCrosse methodology, or genetic transformation. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference.

Compositions of the invention find use in protecting plants, seeds, and plant products in a variety of ways. For example, the compositions can be used in a method that involves placing an effective amount of the pesticidal or pestistatic composition in the environment of the pest by a procedure selected from the group consisting of spraying, dusting, broadcasting, or seed coating.

Before plant propagation material (fruit, tuber, bulb, corm, grains, seed), but especially seed, is sold as a commercial product, it is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures of several of these preparations, if desired together with further carriers, surfactants, or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal, or animal pests. In order to treat the seed, the protectant coating may be applied to the seeds either by impregnating the tubers or grains with a liquid formulation or by coating them with a combined wet or dry formulation. In addition, in special cases, other methods of application to plants are possible, e.g., treatment directed at the buds or the fruit.

The plant seed of the invention comprising the nucleotide sequences encoding the agents may be treated with a seed protectant coating comprising a seed treatment compound, such as, for example, captan, carboxin, thiram, methalaxyl, pirimiphos-methyl, and others that are commonly used in seed treatment. In one embodiment within the scope of the invention, a seed protectant coating comprising a pesticidal composition of the invention is used alone or in combination with one of the seed protectant coatings customarily used in seed treatment.

In the present invention, a composition includes whole organisms, cells, spore(s), pesticidal or pestistatic protein(s), pesticidal or pestistatic component(s), pest-impacting component(s), mutant(s), living or dead cells and cell components, including mixtures of living and dead cells and cell components, and including broken cells and cell components or an isolated pesticidal or pestistatic protein can be formulated with an acceptable carrier into a pesticidal composition(s) that is, for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder, and an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste, and also encapsulations in, for example, polymer substances.

Such compositions disclosed above may be obtained by the addition of a surface-active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protectant, a buffer, a flow agent or fertilizers, micronutrient donors, or other preparations that influence plant growth. One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaracides, plant growth regulators, harvest aids, and fertilizers, can be combined with carriers, surfactants or adjuvants customarily employed in the art of formulation or other components to facilitate product handling and application for particular target pests. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifϊers, binders, or fertilizers. The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area, plant, or seed to be treated. For example, the compositions of the present invention may be applied to grain in preparation for or during storage in a grain bin or silo, etc. The compositions of the present invention may be applied simultaneously or in succession with other compounds. Methods of applying an active ingredient of the present invention or an agrochemical composition of the present invention that contains at least one of the pesticidal proteins produced by the bacterial strains of the present invention include, but are not limited to, foliar application, seed coating, and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.

Suitable surface-active agents include, but are not limited to, anionic compounds such as a carboxylate of, for example, a metal; a carboxylate of a long chain fatty acid; an N- acylsarcosinate; mono or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates such as alkyl- benzene sulfonates or lower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol- formaldehyde condensates; more complex sulfonates such as the amide sulfonates, e.g., the sulfonated condensation product of oleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g., the sodium sulfonate of dioctyl succinate. Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols. Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamine such as an acetate, naphthenate or oleate; or oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.

Examples of inert materials include but are not limited to inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates, or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells. The compositions of the present invention can be in a suitable form for direct application or as a concentrate of primary composition that requires dilution with a suitable quantity of water or other diluant before application. The pesticidal concentration will vary depending upon the nature of the particular formulation, specifically, whether it is a concentrate or to be used directly. The composition contains 1 to 98% of a solid or liquid inert carrier, and 0 to 50% or 0.1 to 50% of a surfactant. These compositions will be administered at the labeled rate for the commercial product, for example, about 0.01 lb-5.0 Ib. per acre when in dry form and at about 0.01 pts.-10 pts. per acre when in liquid form.

Insect pests may be tested for pesticidal or pestistatic activity of compositions of the instant invention in early developmental stages, e.g., as larvae or other immature forms. The insects may be reared in total darkness at from about 2O⁰C. to about 3O⁰C. and from about 30% to about 70% relative humidity. Bioassays may be performed as described in Czapla and Lang J Econ Entomol &?f<5J:2480-2485, 1990. Methods of rearing insect larvae and performing bioassays are well known to one of ordinary skill in the art.

A wide variety of bioassay techniques are known to one skilled in the art. General procedures include addition of the experimental compound or organism to the diet source in an enclosed container. Pesticidal activity can be measured by, but is not limited to, changes in mortality, weight loss, attraction, repellency and other behavioral and physical changes after feeding and exposure for an appropriate length of time. Bioassays described herein can be used with any feeding insect pest in the larval or adult stage.

The present invention is further described by the following non-limiting Examples. EXAMPLE 1 NaPI

A bioassay was developed based on feeding larvae of H. armigera on cotton leaves homozygous for NaPI and coated with varying concentrations of CrylAc. The NaPI leaves were specifically selected to give a small reduction in weight of the larvae. (In previous bioassays, without CrylAc, leaves have been used which give a greater effect on larval growth and development.)

Preparation of Leaves

The leaves were cut into discs using a hole puncher and coated with CrylAc solutions by immersing in the test solution for 5 minutes. The discs were removed from the solution with tweezers and then air dried on plastic mesh (approx. 30 min). When there was no visible moisture on the leaf disc surface, the discs were transferred to wells of a 24 well plate.

The wells contained filter paper moistened with water. Untransformed leaf material of cv Coker was used in control experiments.

Handling of Eggs

H. armigera eggs (supplied by Department of Primary Industry & Fisheries, Indooroopilly, Queensland, Australia) were delivered by air freight to the laboratory and incubated (18- 2O⁰C depending on developmental stage) to the brown egg stage (pre hatching). Eggs were suspended in polyacrylate (Aquakeep polyacrylate, 1 mg/ml H₂O) and one egg placed in each well containing a leaf disc. Single eggs were then transferred to individual wells.

The wells were covered with perforated Mylar film and incubated at 25° in a controlled temperature cabinet with lights (16 hr light, 8 hr dark). The larvae were removed at day 6 or 7 and individually weighed. Preparation of Cry IAc solution

Cryl Ac crystal protein/spore mix (10 mg from CSIRO, Canberra, Australia) was dissolved in 0.9 ml of 5OmM Na₂ CO₃, (pH 10.5). Bovine Pancreatic Trypsin (Sigma T-8642 Type 8) stock 0.75 mg/ml was prepared in the same buffer and 0.1 ml of stock added to the crystal protein spore mix and incubated overnight at room temperature. The solution was centrifuged (14,000 rpm) for 5 minutes and the supernatant collected and stored at -7O⁰C.

The concentration of activated Cry IAc protein was estimated as 2.5 mg/ml on the basis of 50% starting material being Cry IAc, and 50% of this being the final activated material. Estimated molecular weights protoxin 133 kD: activated toxin 66 kD. Dilutions were prepared in 0.03% v/v Triton Ag 98 as wetting agent.

A suitable assay for Cryl Ac is described in Liao et al, J Invertebrate Pathol §0:55-63.

Experiment 1 - Examination of the effect on mass of larvae

In this experiment, sub-lethal concentrations of Cryl Ac were selected. The focus was to examine the effect on mass of the larvae. The second fully expanded leaf (from growing tip) of glasshouse grown plants was used for disc preparation. Experiment duration was 7 days.

Results

The egg hatch was 70-100%; 12 eggs per test (1 egg/well). The mass of surviving larvae is shown in Figure 1. There was insignificant mortality.

It appears that both Cryl Ac and NaPI contribute to the decrease in weight of the larvae with increasing concentrations of Cryl Ac. (The result for cv Coker at 10 ug Cryl Ac seems anomalous. Experiment 2 - Effects of high concentration of CryIAc

This experiment was designed to achieve mortality by using higher concentrations of CryIAc. Higher numbers of eggs were used. For Day 1, the third fully expanded leaf was used. For Day 4, additions of leaf material of the second fully expanded leaf was used. Experiment duration was 6 days.

Results

The egg hatch was 50-83%; 40 eggs per test (1 egg/well). The mass of surviving larvae is shown in Figure 2. The mortality is shown in Figure 3.

This experiment is essentially the same as experiment 1 except that the concentrations of CryIAc are increased over the range 50 to 250 ug/ml. These concentrations result in significant mortality, and thus the data presented refers to a subset of the original sample that have survived at each CryIAc concentration.

The difference in mass between the larvae feeding on Coker or NaPI leaves is essentially the same at each concentration of CryIAc over the range 50-250 ug/ml.

With no added CryIAc, there is not significant difference in mortality. At each concentration of CryIAc there is a significant enhancement of mortality in leaves expressing NaPI over that obtained with Coker control material.

In no case, in any of the experiments, did expression of the NaPI gene diminish the effect of CryIAc. Experiment 3 - Bioassay with third instar larvae

The aim of this experiment was to determine the effect of Bt toxin (Cry IAc) and NaPI on 3^rd to 4^th instar H. armigera larvae.

Larvae (48) were grown for 11 days on cotton leaves cv Coker before being transferred to new plates containing fresh leaf discs. The 48 larvae were separated into 4 treatments of 12 larvae.

The four treatments were as follows:

- Untransformed Coker 315 leaves

- Transgenic NaPI leaves

- Untransformed Coker leaves coated with 100 ug/mL Cry IAc - Transgenic NaPI leaves coated with 100 ug/mL CrylAc

The larvae were grown for a further 20 hours at 25⁰C. Larval weight was recorded at days 7, 11 and 12.

Experimental details

Two, 24 well plates were used. Single H. armigera eggs, suspended in Polyacrylate (lg/L), were placed in each well. Initially one leaf disc was placed in each well. Further discs were added when required.

Coker leaves at positions 3, 4 and 5 were used for the first 11 days. The leaves were harvested, cut into discs and pooled before they were added to the wells. Only leaves from position 2 or 3 were used for the treatments. Results

All H. armigera eggs used in the assay hatched (Table l).At day 11 the larvae were weighed and then fed the test diets. About 20 hours later (day 12) the larvae were weighed again (Table 1, Figure 4).

Table 1. Results of H. armigera bioassay using 3^rd instar larvae

CrylAc concentration 0 ug/mL lOOug/mL

C N C N

No eggs 12 12 12 12

Hatched larvae 12 12 12 12

Surviving larvae 11 11 12 12

Average weight (mg) day 11 64.6 65.3 67.1 69.0

Average weight (mg) day 12 95.2 91.0 81.4 74.5

% increase in mass day 11-12 47% 39% 21% 8%

C- untransformed Coker leaves, N- transgenic leaves expressing NaPI

There was a small difference in average weight gained between the larvae fed on control Coker leaves and the transgenic NaPI expressing leaves over the 20 hours. The control larvae increased their weight by an average of 48% while the NaPI fed larvae increased their weight by an average of 41% (Table 1, Figure 5). Note that the average weight of larvae at day 11 was slightly different for each test group (Table 1) and this must be taken into account in the calculations. Since the larvae were all fed the same diet, this difference is due to natural variation of the larvae.

There was a greater difference in weight when the larvae were fed CrylAc. The CrylAc fed larvae only increased their weight by an average of 21% (Table 1, Figure 5). Finally, larvae fed transgenic leaves coated with Cry IAc only increased their weight by an average of 7%. It is interesting to note that 3 out of the 12 larvae actually lost weight or did not increase in weight (Table 1, Figure 5).

These results suggest that the combination of NaPI and Cry IAc increases the effect in diminishing larval growth. These results are in line with those obtained from Examples 1 and 2 in which freshly hatched larvae were allowed to feed for 7 days on test leaves.

EXAMPLE 2 Properties of midgut membrane

When H armigera larvae are fed a diet containing NaPI or Bt proteins, such as Cry IAc₅ there are different effects on the gut epithelial cells.

The presence of Bt in the diet of lepidopteran pests results in a swelling of the columnar cells and a proliferation of stem cells at the base of the epithelial layer. Ultimately cells in the epithelial layer burst when exposed to Bt and related bacterial toxins. In contrast, the presence of NaPIs in the diet causes a limited swelling of cells in the epithelial layer without bursting. There is some associated movement of material into the intercellular spaces. When the diet contains both Bt and NaPI, there is an extensive and more rapid breakdown of the epithelial cell structures. The intercellular spaces are more distended and there is enhanced access of material to the haemolymph. Material which penetrates through to the haemolymph may then come into contact with proteases involved in the innate immunity response. In particular, when PIs gain access to the haemolymph they can interfere with the protein cascades essential to this innate immunity response. The ability of the insect gut to regenerate after damage from either toxin is reduced.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY

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Claims

1. A genetically modified plant or its progeny resulting from self-crossing, back- crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is a serine proteinase inhibitor or precursor thereof from the Solanaceae family wherein in precursor form, it comprises at least three monomers wherein at least one monomer is a trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and wherein at least one other agent is a non-serine proteinase inhibitor, wherein said plant exhibits resistance or reduced susceptibility to a pest.

2. The genetically modified plant of Claim 1 wherein the serine proteinase inhibitor is provided in precursor form.

3. The genetically modified plant of Claim 2 wherein the precursor comprises at least four monomers.

4. The genetically modified plant of Claim 2 wherein the precursor comprises at least five monomers.

5. The genetically modified plant of Claim 2 wherein the precursor comprises at least six monomers.

6. The genetically modified plant of Claim 5 wherein the serine proteinase inhibitor is NaPI from Nicotiana alata.

7. The genetically modified plant of any one of Claims 1 to 6 wherein the non-serine proteinase inhibitor is an endotoxin.

8. The genetically modified plant of Claim 7 wherein the non-serine proteinase inhibitor is a Bt protein.

9. The genetically modified plant of Claim 7 or 8 wherein the endotoxin is selected from the list conistsing of a member of the Cry family and VIP.

10. The genetically modified plant of any one of Claims 1 to 6 wherein the non-serine proteinase inhibitor is a defensin molecule or a pest toxic part thereof.

11. A genetically modified plant or its progeny resulting from self-crossing, back- crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is the NaPI precursor or a monomer thereof or a recombinant or derivative thereof and wherein at least one other agent is a non-serine proteinase inhibitor wherein said plant exhibits resistance or reduced susceptibility to a pest.

12. The genetically modified plant of any one of Claim 11 wherein the non-serine proteinase inhibitor is an endotoxin.

13. The genetically modified plant of Claim 12 wherein the non-serine proteinase inhibitor is a Bt protein.

14. The genetically modified plant of Claim 12 or 13 wherein the endotoxin is selected from the list conistsing of a member of the Cry familyand VIP.

15. The genetically modified plant of Claim 11 wherein the non-serine proteinase inhibitor is a defensin molecule or a pest toxic part thereof.

16. A genetically modified plant or its progeny resulting from self-crossing, back- crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is a serine proteinase inhibitor or precursor thereof W

- 59 -

from the Solanaceae family wherein in precursor form, it comprises at least three monomers wherein at least one monomer is a trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and wherein at least one other agent is a non-serine proteinase inhibitor, wherein said plant exhibits resistance or reduced susceptibility to a pest except and wherein at least one other agent is selected from the list comprising a Bt protein, a Cry family protein, a VIP and a defensin.

17. A genetically modified plant or its progeny resulting from self crossing, back crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is NaPI precursor or a monomer thereof or a recombinant or modular derivative thereof and wherein at least one other agent is a non- serine proteinase inhibitor wherein said plant exhibits resistance or reduced susceptibility to a pest except wherein the least one other agent is selected from the list comprising a Bt protein, a member of the Cry family, a VIP and a defensin.

18. The genetically modified plant of Claim 1 or 11 or 16 or 17 wherein the plant is selected from the list consisting of corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. junceά), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solatium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), cotton pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane {Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers, vegetables include tomatoes {Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C sativus), cantaloupe (C. cantalupensis), and musk melon (C meld). Ornamentals include azalea {Rhododendron spp.), hydrangea {Macrophylla hydrangea), hibiscus {Hibiscus rosasanensis), roses {Rosa spp.), tulips {Tulipa spp.), daffodils {Narcissus spp.), petunias {Petunia hybrida), carnation {Dianthus caryophyllus), poinsettia {Euphorbia pulcherrima), and chrysanthemum. Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine {Pinus taeda), slash pine {Pinus elliotii), ponderosa pine {Pinus ponderosa), lodgepole pine {Pinus contorta), and Monterey pine {Pinus radiata); Douglas- fir {Pseudotsuga menziesii); Western hemlock {Tsuga canadensis); Sitka spruce {Picea glauca); redwood {Sequoia sempervirens); true firs such as silver fir {Abies amabilis) and balsam fir {Abies balsamea); and cedars such as Western red cedar {Thuja plicata) and Alaska yellow-cedar {Chamaecyparis nootkatensis). Plants of the present invention include crop plants (for example, cotton, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants, turfgrasses include, but are not limited to: annual bluegrass {Poa annua); annual ryegrass {Lolium multiflorum); Canada bluegrass {Poa compressa); Chewings fescue {Festuca rubra); colonial bentgrass {Agrostis tenuis); creeping bentgrass {Agrostis palustris); crested wheatgrass {Agropyron desertorum); fairway wheatgrass {Agropyron cristatum); hard fescue {Festuca longifolia); Kentucky bluegrass {Poa pratensis); orchardgrass {Dactylis glomerata); perennial ryegrass {Lolium perenne); red fescue {Festuca rubra); redtop {Agrostis alba); rough bluegrass {Poa trivialis); sheep fescue {Festuca ovina); smooth bromegrass {Bromus inermis); tall fescue {Festuca arundinacea); timothy {Phleum pratense); velvet bentgrass {Agrostis canina); weeping alkaligrass {Puccinellia distans); western wheatgrass {Agropyron smithii); Bermuda grass {Cynodon spp.); St. Augustine grass {Stenotaphrum secundatum); zoysia grass {Zoysia spp.); Bahia grass {Paspalum notatum); carpet grass {Axonopus affinis); centipede grass {Eremochloa ophiuroides); kikuyu grass {Pennisetum clandesinum); seashore paspalum {Paspalum vaginatum); blue gramma {Bouteloua gracilis); buffalo grass {Buchloe dactyloids); sideoats gramma (Bouteloua curtipenduld), oil-seed plants, leguminous plants, corn, wheat, barley, rice, sorghum, rye, millet, cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive beans, peas, guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils and chickpea.

19. The genetically modified plant of Claim 18 wherein the plant is a cotton, sweet corn, tomato, tobacco, piniento, potato, sunflower, citrus, plums, sorghum, leeks, soybean, alfalfa, beans, pidgeon peas, chick peas, artichokes, curcurbits, lettuce, Dianthus, geraniums, cape gooseberry, maize, flax and linseed, lupins, broad beans, garden peas, peanuts, canola, snapdragons, cherry, sunflower, pot marigolds, Helichrysum, wheat, barley, oats, triticale, carrots, onions, orchids, roses or petunias.

20. The genetically modified plant of Claim 1 or 11 or 16 or 17 wherein the plant is cotton.

21. The genetically modified plant of Claim 1 or 11 or 16 or 17 wherein the plant pest is selected from the listing consisting of insects, fungi, bacteria, nematodes, acarids, protozoan pathogens, animal-parasitic liver flukes, and the like.

22. The genetically modified plant of Claim 21 wherein the pest is an insect.

23. The genetically modified plant of Claim 22 wherein the insect is selected from the list consisting of Helicoverpa, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, Coleoptera.

24. The genetically modified plant of Claim 22 wherein the pest is selected from the list consisting of Achoroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integetrima, Dendrolimus sibericus, Desmiafeneralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos sύbsignaria, Eoreuma loftini, Esphestia elutella, Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantia cunea, Keiferia lycopersicella, Lambdina flscellaria flscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes, Pectinophora gossypiella, Phryganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynota flouendana, Platynota stultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp., Thaurnstopoea pityocampa, Tinsola bisselliella, Trichoplusia hi, Udea rubigalis, Xylomyges curiails, and Yponomeuta padella, Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zeae, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; western com rootworm, e.g., Diabrotica virgifera virgifera; northern corn rootworm, e.g., Diabrotica longicornis barberi; southern corn rootworm, e.g., Diabrotica undecimpunctata howardi; Melanotus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis, corn blotch leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, two spotted spider mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, leser cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; chinch bug, e.g., Blissus leucopterus leucopterus; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, two-spotted spider mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, pale western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; southern corn rootworm, e.g., Diabrotica undecimpunctata howardi; Russian wheat aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower: Cylindrocupturus adspersus, sunflower stem weevil; Smicronyx fulus, red sunflower seed weevil; Smicronyx sordidus, gray sunflower seed weevil; Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; Zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis virescens, tobacco budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pink bollworm; boll weevil, e.g., Anthonomus grandis; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, two-spotted spider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhoper; chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum hilare, green stink bug; Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, tobacco budworm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ entiάlis, differential grasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, two-spotted spider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Jylemya platura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite; Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, crucifer flea beetle; Phyllotreta striolata, striped flea beetle; Phyllotreta nemorum, striped turnip flea beetle; Meligethes aeneus, rapeseed beetle; and the pollen beetles Meligethes ruflmanus, Meligethes nigrescens, Meligethes canadianus, and Meligethes viridescens; Potato: Leptinotarsa decemlineata, Colorado potato beetle, Hemiptera such as Lygus hesperus, Lygus lineolaris, Lygus pratensis, Lygus rugulipennis Popp, Lygus pabulinus, Calocoris norvegicus, Orthops compestris, Plesiocoris rugicollis, Cyrtopeltis modestus, Cyrtopeltis notatus, Spanagonicus albofasciatus, Diaphnocoris chlorinonis, Labopidicola allii, Pseudatomoscelis seriatus, Adelphocoris rapidus, Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae, Nysius raphanus, Euschistus servus, Nezara viridula, Eurygaster, Coreidae, Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, and Cimicidae. Pests of interest also include Araecerus fasciculatus, coffee bean weevil; Acanthoscelides obtectus, bean weevil; Bruchus rufmanus, broadbean weevil; Bruchus pisorum, pea weevil; Zabrotes subfasciatus, Mexican bean weevil; Diabrotica balteata, banded cucumber beetle; Cerotoma trifurcata, bean leaf beetle; Diabrotica virgifera, Mexican corn rootworm; Epitrix cucumeris, potato flea beetle; Chaetocnema conβnis, sweet potato flea beetle; Hypera postica, alfalfa weevil; Anthonomus quadrigibbus, apple curculio; Sternechus paludatus, bean stalk weevil; Hypera brunnipennis, Egyptian alfalfa weevil; Sitophilus granaries, granary weevil; Craponius inaequalis, grape curculio; Sitophilus zeamais, maize weevil; Conotrachelus nenuphar, plum curculio; Euscepes postfaciatus, West Indian sweet potato weevil; Maladera castanea, Asiatic garden beetle; Rhizotrogus majalis, European chafer; Macrodactylus subspinosus, rose chafer; Tribolium confusum, confused flour beetle; Tenebrio obscurus, dark mealworm; Tribolium castaneum, red flour beetle; Tenebrio molitor and yellow mealworm.

25. The genetically modified plant of Claim 22 wherein the pest is a nematode selected from the list consisting of Heterodera spp., Meloidogyne spp., and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode); and Globodera rostochiensis and Globodera pallida (potato cyst nematodes) and lesion nematodes including Pratylenchus spp.

26. A pest management control system comprising generating a genetically modified plant or its progeny resulting from self-crossing, back-crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is a serine proteinase inhibitor or precursor thereof from the Solanaceae family wherein in precursor form, it comprises at least three monomers wherein at least one monomer is a trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and wherein at least one other agent is a non-serine proteinase inhibitor, wherein said plant exhibits resistance or reduced susceptibility to a pest.

27. The pest management control system of Claim 26 wherein the serine proteinase inhibitor is provided in precursor form.

28. The pest management control system of Claim 27 wherein the precursor comprises at least four monomers.

29. The pest management control system of Claim 27 wherein the precursor comprises at least five monomers.

30. The pest management control system of Claim 27 wherein the precursor comprises at least six monomers.

31. The pest management control system of Claim 30 wherein the serine proteinase inhibitor is NaPI from Nicotiana alata.

32. The pest management control system of any one of Claims 26 to 31 wherein the non-serine proteinase ibhibitor is an endotoxin.

33. The pest management control system of Claim 32 wherein the non-serine proteinase inhibitor is a Bt protein.

34. The pest management control system of Claim 32 or 33 wherein the endotoxin is selected from the list conistsing of a member of Cry family and VIP.

35. The pest management control system of any one of Claims 26 to 31 wherein the non-serine proteinase inhibitor is a defensin molecule or a pest toxic part thereof.

36. A pest management control system comprising generating a genetically modified plant or its progeny resulting from self-crossing, back-crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is the NaPI precursor or a monomer thereof or a recombinant or modular derivative thereof and wherein at least one other agent is a non-serine proteinase inhibitor wherein said plant exhibits resistance or reduced susceptibility to a pest.

37. The pest management control system of Claim 36 wherein the non-serine proteinase inhibitor is an endotoxin.

38. The pest management control system of Claim 37 wherein the non-serine proteinase inhibitor is a Bt protein.

39. The pest management control system of Claim 37 or 38 wherein the endotoxin is selected from the list conistsing of a member of the Cry family and VIP.

40. The pest management control system of Claim 36 wherein the non-serine proteinase inhibitor is a defensin molecule or a pest toxic part thereof.

41. A pest management control system comprising generating a genetically modified plant or its progeny resulting from self-crossing, back-crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is a serine proteinase inhibitor or precursor thereof from the Solanaceae family wherein in precursor form, it comprises at least three monomers wherein at least one monomer is a trypsin inhibitor and at least one monomer is a chymotrypsin inhibitor and wherein at least one other agent is a non-serine proteinase inhibitor, wherein said plant exhibits resistance or reduced susceptibility to a pest except and wherein at least one other agent is selected from the list comprising a Bt protein, a Cry family protein, a VIP and a defensin.

42. A pest management control system comprising generating a genetically modified plant or its progeny resulting from self crossing, back crossing or crossing with another plant, said plant comprising at least two pesticidal and/or pestistatic agents wherein at least one of said agents results from the genetic modification or crossing wherein at least one agent is NaPI precursor or a monomer thereof or a recombinant or modular derivative thereof and wherein at least one other agent is a non-serine proteinase inhibitor wherein said plant exhibits resistance or reduced susceptibility to a pest except wherein the least one other agent is selected from the list comprising a Bt protein, a Cry family protein, a VIP and a defensin.

43. A pest management control system of Claim 26 or 36 or 41 or 42 wherein the plant is selected from the list consisting of corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. junceά), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Ofγza sativa), rye (Secale cereale), sorghum {Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), cotton pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidental), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers, vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C sativus), cantaloupe (C. cantalupensis), and musk melon (C melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas- fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsameά); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Plants of the present invention include crop plants (for example, cotton, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants, turfgrasses include, but are not limited to: annual bluegrass (P Oa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewings fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis glomerata); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (Phleum pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron smithii); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum); carpet grass (Axonopus affinis); centipede grass (Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum (Paspalum vaginatum); blue gramma (Bouteloua gracilis); buffalo grass (Buchloe dactyloids); sideoats gramma (Bouteloua curtipendula), oil-seed plants, leguminous plants, corn, wheat, barley, rice, sorghum, rye, millet, cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive beans, peas, guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils and chickpea.

44. The pest management control system of Claim 43 wherein the plant is a cotton, sweet corn, tomato, tobacco, piniento, potato, sunflower, citrus, plums, sorghum, leeks, soybean, alfalfa, beans, pidgeon peas, chick peas, artichokes, curcurbits, lettuce, Dianthus, geraniums, cape gooseberry, maize, flax and linseed, lupins, broad beans, garden peas, peanuts, canola, snapdragons, cherry, sunflower, pot marigolds, Helichrysum, wheat, barley, oats, triticale, carrots, onions, orchids, roses and petunias.

45. The pest management control system of Claim 26 or 36 or 41 or 42 wherein the plant is cotton.

46. The pest management control system of Claim 26 or 36 or 41 or 42 wherein the plant pest is selected from the listing consisting of insects, fungi, bacteria, nematodes, acarids, protozoan pathogens, animal-parasitic liver flukes, and the like.

47. The pest management control system of Claim 46 wherein the pest is an insect.

48. The pest management control system of Claim 47 wherein the insect is selected from the list consisting of Helicoverpa, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, Coleoptera.

49. The pest management control system of Claim 47 wherein the pest is selected from the list consisting of Lepidoptera, e.g. Achoroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmiafeneralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia elutella, Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantia cunea, Keiferia lycopersicella, Lambdina fiscellaria flscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra conflgurata, Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes, Pectinophora gossypiella, Phryganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynota flouendana, Platynota stultana, Platyptilia carduϊdactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp., Thaurnstopoea pityocampa, Tinsola bisselliella, Trichoplusia hi, Udea rubigalis, Xylomyges curiails, and Yponomeuta padella, Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zeae, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; western corn rootworm, e.g., Diabrotica virgifera virgifera; northern corn rootworm, e.g., Diabrotica longicornis barberi; southern corn rootworm, e.g., Diabrotica undecimpunctata howardi; Melanotus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis, corn blotch leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, two spotted spider mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, leser cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; chinch bug, e.g., Blissus leucopterus leucopterus; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, two-spotted spider mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, pale western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; southern corn rootworm, e.g., Diabrotica undecimpunctata howardi; Russian wheat aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus dijferentialis, differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower: Cylindrocupturus adspersus, sunflower stem weevil; Smicronyx fulus, red sunflower seed weevil; Smicronyx sordidus, gray sunflower seed weevil; Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; Zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis virescens, tobacco budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pink bollworm; boll weevil, e.g., Anthonomus grandis; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, two-spotted spider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhoper; chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum hilare, green stink bug; Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, tobacco budworm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, two-spotted spider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Jylemya platura, seedcorn maggot; Mayetiola destructor', Hessian fly; Petrobia latens, brown wheat mite; Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, crucifer flea beetle; Phyllotreta striolata, striped flea beetle; Phyllotreta nemorum, striped turnip flea beetle; Meligethes aeneus, rapeseed beetle; and the pollen beetles Meligethes rufimanus, Meligethes nigrescens, Meligethes canadianus, and Meligethes viridescens; Potato: Leptinotarsa decemlineata, Colorado potato beetle, Hemiptera such as Lygus hesperus, Lygus lineolaris, Lygus pratensis, Lygus rugulipennis Popp, Lygus pabulinus, Calocoris norvegicus, Orthops compestris, Plesiocoris rugicollis, Cyrtopeltis modestus, Cyrtopeltis notatus, Spanagonicus albofasciatus, Diaphnocoris chlorinonis, Labopidicola allii, Pseudatomoscelis sehatus, Adelphocoris rapidus, Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae, Nysius raphanus, Euschistus servus, Nezara viridula, Eurygaster, Coreidae, Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, and Cimicidae. Pests of interest also include Araecerus fasciculatus, coffee bean weevil; Acanthoscelides obtectus, bean weevil; Bruchus ruβnanus, broadbean weevil; Bruchus pisorum, pea weevil; Zabrotes subfasciatus, Mexican bean weevil; Diabrotica balteata, banded cucumber beetle; Cerotoma trifurcata, bean leaf beetle; Diabrotica virgifera, Mexican corn rootworm; Epitrix cucumeris, potato flea beetle; Chaetocnema conβnis, sweet potato flea beetle; Hypera postica, alfalfa weevil; Anthonomus quadrigibbus, apple curculio; Sternechus paludatus, bean stalk weevil; Hypera brunnipennis, Egyptian alfalfa weevil; Sitophilus granaries, granary weevil; Craponius inaequalis, grape curculio; Sitophilus zeamais, maize weevil; Conotrachelus nenuphar, plum curculio; Euscepes postfaciatus, West Indian sweet potato weevil; Maladera castanea, Asiatic garden beetle; Rhizotrogus majalis, European chafer; Macrodactylus subspinosus, rose chafer; Tribolium confusum, confused flour beetle; Tenebrio obscurus, dark mealworm; Tribolium castaneum, red flour beetle; Tenebrio molitor and yellow mealworm.

50. The pest management control system of Claim 47 wherein the pest is a nematode selected from the list consisting of Heterodera spp., Meloidogyne spp., and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode); and Globodera rostochiensis and Globodera pallida (potato cyst nematodes) and lesion nematodes including Pratylenchus spp.

51. A pesticidal or pestistatic composition comprising a serine proteinase inhibitor and at least one non-serine proteinase inhibitor.

52. The pesticidal or pestistatic composition of Claim 51 wherein the serine proteinase inhibitor is NaPI from Nicotiana alata.

53. The pesticidal or pestistatic composition of Claim 51 or 52 wherein the non-serine proteinase ibhibitor is an endotoxin.

54. The pesticidal or pestistatic composition of Claim 53 wherein the non-serine proteinase inhibitor is a Bt protein.

55. The pesticidal or pestistatic composition of Claim 53 or 54 wherein the endotoxin is selected from the list conistsing of a member of the Cry family and VIP.

56. The pesticidal or pestistatic composition of Claim 51 wherein the non-serine proteinase inhibitor is a defensin molecule or a pest toxic part thereof.

57. The pesticidal or pestistatic composition of Claim 51 wherein the plant pest is selected from the listing consisting of insects, fungi, bacteria, nematodes, acarids, protozoan pathogens, animal-parasitic liver flukes, and the like.

58. The pesticidal or pestistatic composition of Claim 57 wherein the pest is an insect.

59. The pesticidal or pestistatic composition of Claim 58 wherein the insect is selected from the list consisting of Helicoverpa, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera and Coleoptera.

60. The pesticidal or pestistatic composition of Claim 58 wherein the pest is selected from the list consisting of Lepidoptera, e.g. Achoroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonelϊa, Datana integerrima, Dendrolimus sibericus, Desmiafeneralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia elutella, Erannis tilaria, Estigmene acrea, Eulia salubrϊcola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantia cunea, Keiferia lycopersicella, Lambdina flscellaria fiscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes, Pectinophora gossypiella, Phryganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynota flouendana, Platynota stultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp., Thaurnstopoea pityocampa, Tinsola bisselliella, Trichoplusia hi, Udea rubigalis, Xylomyges curiails, and Yponomeuta padella, Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zeae, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; western corn rootworm, e.g., Diabrotica virgifera virgifera; northern corn rootworm, e.g., Diabrotica longicornis barberi; southern corn rootworm, e.g., Diabrotica undecimpunctata howardi; Melanotus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus lencopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis, corn blotch leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, two spotted spider mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, leser cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, white grub; Eleodes, Conoderus, end Aeolus spp., wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; chinch bug, e.g., Blissus leucopterus leucopterus; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, two-spotted spider mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, pale western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; southern corn rootworm, e.g., Diabrotica undecimpunctata howardi; Russian wheat aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower: Cylindrocupturus adspersus, sunflower stem weevil; Smicronyx fulus, red sunflower seed weevil; Smicronyx sordidus, gray sunflower seed weevil; Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; Zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis virescens, tobacco budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pink bollworm; boll weevil, e.g., Anthonomus grandis; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ 'entialis, differential grasshopper; Thrips tabaci, onion thrips; Fraήkliήkiella fusca, tobacco thrips; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, two-spotted spider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhoper; chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum hilare, green stink bug; Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, tobacco budworm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, two-spotted spider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Jylemya platura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite; Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, crucifer flea beetle; Phyllotreta striolata, striped flea beetle; Phyllotreta nemorum, striped turnip flea beetle; Meligethes aeneus, rapeseed beetle; and the pollen beetles Meligethes rufimanus, Meligethes nigrescens, Meligethes canadianus, and Meligethes viridescens; Potato: Leptinotarsa decemlineata, Colorado potato beetle, Hemiptera such as Lygus hesperus, Lygus lineolaris, Lygus pratensis, Lygus rugulipennis Popp, Lygus pabulinus, Calocoήs norvegicus, Orthops compestris, Plesiocoris rugicollis, Cyrtopeltis modestus, Cyrtopeltis notatus, Spanagonicus albofasciatus, Diaphnocoris chlorinonis, Labopidicola allii, Pseudatomoscelis seriatus, Adelphocoris rapidus, Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae, Nysius raphanus, Euschistus servus, Nezara viridula, Eurygaster, Coreidae, Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, and Cimicidae. Pests of interest also include Araecerus fasciculatus, coffee bean weevil; Acanthoscelides obtectus, bean weevil; Bruchus rufmanus, broadbean weevil; Bruchus pisorum, pea weevil; Zabrotes subfasciatus, Mexican bean weevil; Diabrotica balteata, banded cucumber beetle; Cerotoma trifurcata, bean leaf beetle; Diabrotica virgifera, Mexican corn rootworm; Epitrix cucumeris, potato flea beetle; Chaetocnema conβnis, sweet potato flea beetle; Hypera postica, alfalfa weevil; Anthonomus quadrigibbus, apple curculio; Sternechus paludatus, bean stalk weevil; Hypera brunnipennis, Egyptian alfalfa weevil; Sitophilus granaries, granary weevil; Craponius inaequalis, grape curculio; Sitophilus zeamais, maize weevil; Conotrachelus nenuphar, plum curculio; Euscepes postfaciatus, West Indian sweet potato weevil; Maladera castanea, Asiatic garden beetle; Rhizotrogus majalis,, European chafer; Macrodactylus subspinosus, rose chafer; Tribolium confusum, confused flour beetle; Tenebrio obscurus, dark mealworm; Tribolium castaneum, red flour beetle; Tenebrio molitor and yellow mealworm.

61. The pesticidal and pestistatic composition of Claim 58 wherein the pest is a nematode selected from the list consisting of Heterodera spp., Meloidogyne spp., and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode); and Globodera rostochiensis and Globodera pallida (potato cyst nematodes) and lesion nematodes including Pratylenchus spp.