WO2015056080A1

WO2015056080A1 - Planting method

Info

Publication number: WO2015056080A1
Application number: PCT/IB2014/002122
Authority: WO
Inventors: Charles Lee FORESMAN; Anthony David BURD; Dirk Laris BENSON
Original assignee: Syngenta Participations Ag
Priority date: 2013-10-14
Filing date: 2014-10-13
Publication date: 2015-04-23
Also published as: AR097995A1

Abstract

Improved use of transgenic refuge in row crops can be achieved when at least 2 non-transgenic crop seeds are sown consecutively in a row of transgenic pest resistant crop seed.

Description

TITLE OF THE INVENTION

PLANTING METHOD

FIELD OF THE INVENTION

The present invention relates to reducing, preventing or delaying the development of resistance to insecticidal proteins or to oligonucleotides or polynucleotides with pesticidal effect. In particular, the invention relates to preventing or delaying the development of resistance to insecticidal proteins or oligonucleotides or polynucleotides which have pesticidal effect expressed by transgenic row plants. More specifically, the invention relates to the use of a non-transgenic refuge within a row of transgenic plants.

BACKGROUND AND SUMMARY OF THE INVENTION

Insects, nematodes, and related arthropods annually destroy an estimated 15 percent of agricultural crops in the United States and even more than that in developing countries. In addition, competition with weeds and parasitic and saprophytic plants account for even more potential yield losses.

Chemical pesticides are commonly used to keep pest numbers below economically damaging levels. For more than a decade insecticidal proteins expressed by transgenic plants have been used by growers to manage insects.

There is a long history of insects evolving resistance to chemicals stimulating the need for additional methods of controlling destructive insect pests. To better manage insect pests growers occasionally use diversity in deploying chemical pesticides as seed treatments, chemical sprays or direct application to the soil in combination with transgenic crops to avoid loss of crop yield.

There is a risk that species of insects with sustained solitary exposure to insecticidal proteins expressed by transgenic plants will likewise develop resistance to the transgenic traits.

There are various methods which can be used to reduce, prevent or delay the development of such resistance. One strategy for combating the development of resistance is to select a recombinant event which expresses levels of the insecticidal protein such that one or a few bites of a plant expressing the event by the pest would cause cessation of feeding and subsequent death of the pest. Another strategy would be to combine a second pest specific insecticidal protein in the form of a recombinant event in the same plant, preferably having a different mode of action to the first event. Still another strategy would combine a chemical pesticide with a pesticidal protein expressed in a transgenic plant. This could conceivably take the form of a chemical seed treatment of a recombinant seed which would allow for the dispersal into a zone around the root of a pesticidally efficacious amount of a chemical pesticide which would protect root tissues from target pest infestation so long as the chemical persisted or the root tissue remained within the zone of pesticide dispersed into the soil. So long as root tissue was within the zone of chemical pesticide protection, a target pest such as a corn rootworm would have to develop resistance to both forms of plant protection, i.e., to the chemical and to the recombinant protein, in the same generation in order to develop resistance to the combination of pesticidal agents. Development of resistance under this particular scenario is believed to be highly unlikely. Only root tissue which would grow beyond the zone of dispersal of the chemical pesticide treatment would be subject to only one form of protection.

A further approach has been to plant a non-transgenic refuge crop which provides a means for producing a steady and consistent population of adult insects which have never been exposed to the recombinant pesticide pressures and so have not had the opportunity to develop resistance as a result of the pesticide selection pressure when feeding on the recombinant plants. In theory, the adult insects which emerge from the refuge environment will disperse and breed with any insects which emerge from the recombinant fields, and if any of the insects which emerge from the recombinant fields have developed a level of resistance to the recombinant insecticidal proteins, the availability of that trait in the subsequent generations will be diluted, reducing or delaying the onset of the emergence of a race which will be totally resistant to the recombinant insecticidal corn plant.

In fact, the value of the refuge concept is even appreciated when using soil-applied insecticides. It is noted that the insecticides protect the roots during early plant development; once the plant is larger the roots grow outside the narrow treatment zone. These roots are available for example to corn rootworm larvae, meaning each plant has both a protected portion and an area of root mass available as refuge to the pests (Wright RJ et al, Larval susceptibility of an insecticide-resistant western corn rootworm (Coleoptera: Chrysomelidae) population to soil insecticides: laboratory bioassays, assays of detoxification enzymes, and field performance (2000), J Econ Entomol 93:7-13). Refuge use is particularly effective for insects with life stages which are limited in their ability to move through the soil any great distance. For example pests with a larval stage typically confined to immediately adjacent roots.

There are several strategies for planting non-transgenic refuges: planting blocks of non- transgenic crops adjacent to the transgenic crops; planting 'strips' consisting of multiple rows of non-transgenic crops; or by using a uniform seed mix of transgenic and non-transgenic seeds, for example as discussed in US6551962. Although it serves a valuable purpose, crop grown from non-transgenic seed may not provide equivalent properties such as yield, protein content, or other aspect which, in addition to its increased susceptibility to insect pests, brings down the overall productivity and/or profitability of the field.

Despite the options provided in the art, there remains a need to provide an optimised refuge planting strategy which offers greater efficiency in terms of the number of breeding insects without resistance to the insecticidal proteins produced per non-transgenic crop seed. This may allow the number of non-transgenic crop seeds to be planted to be reduced, or act to further reduce, delay or prevent the emergence of any resistant species of insect.

BRIEF DESCRIPTION OF THE INVENTION

It has been found that providing a non-transgenic refuge in a row of transgenic pest resistant crops by planting a mixture of transgenic pest resistant crop seeds and non-transgenic crop seeds wherein at least 2 non-transgenic crop seeds are consecutive and wherein 50-99.5% of the crops seeds are transgenic pest resistant crop seeds leads to a greater number of breeding insects per refuge seed than previous refuge strategies. Accordingly, the invention provides a method of sowing seeds in order to provide a non- transgenic refuge in a row of transgenic pest resistant crops comprising planting a mixture of transgenic pest resistant crop seeds and non-transgenic crop seeds wherein at least 2 non- transgenic crop seeds are consecutive and wherein 50-99.5% of the crops seeds are transgenic pest resistant crop seeds.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1. shows Bacillus thuringensis toxin (Bt) resistance evolution relative to the number of consecutively planted non-transgenic seeds according to a recessive resistance two- direction movement case.

Fig 2. shows Bacillus thuringensis toxin (Bt) resistance evolution relative to the number of consecutively planted non-transgenic seeds according to a recessive resistance single- direction case. Fig 3. shows Bacillus thuringensis toxin (Bt) resistance evolution relative to the number of consecutively planted non-transgenic seeds according to a non-recessive resistance single- direction movement case. DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "comprising" means "including but not limited to".

As used herein, the term "corn" means Zea mays or maize and includes all plant varieties that can be bred with corn, including wild maize species.

As used herein, the term "oil seed rape" includes canola.

As used herein, the term non-transgenic crop seeds means crop seeds which have not been transformed with recombinant DNA techniques such that they are capable of expressing one or more selectively acting insecticidal proteins or one or more oligonucleotide or

polynucleotide which have a pesticidal effect, or a mixture of both, including RNA or DNA or RNA/DNA hybrids or chemically modified oligonucleotides or polynucleotides or mixtures of these. These non-transgenic pest resistant crops include crops which have been so transformed by the use of recombinant DNA techniques that they are capable of synthesising antipathogenic substances. The non-transgenic crop seeds also includes those that have been rendered tolerant to herbicides like bromoxynil or classes of herbicides (such as, for example, HPPD inhibitors, ALS inhibitors, for example primisulfuron, prosulfuron and trifloxysulfuron, EPSPS (5-enol-pyrovyl-shikimate-3-phosphate-synthase) inhibitors, GS {glutamine synthetase) inhibitors) as a result of conventional methods of breeding or genetic engineering.

As used herein, the terms pest, pesticide, and pesticidal are meant to be interchangeable and inclusive of the following terms: for example, insect, insecticide, and insecticidal when referring to an insect pest; or with the terms, for example, nematode, nematicide, and nematicidal when referring to a nematode pest; or with acaric, acaricide, and acaricidal when referring to an acaric pest; or with fungus or fungal, fungicide, and fungicidal or equivalent terms such as mycotic, and mycocidal when referring to fungal or related pests; or with plant or herb, planticide or herbicide, or planticidal or herbicidal when referring to a plant or a herb pest. As used herein, the term "non-transgenic refuge" refers to the use of a resistance management plan for reducing, eliminating or delaying the likelihood of development of resistance or slowing the development of resistance to one or more insecticides that are either present within a recombinant plant or present adjacent to one or more parts or tissues of a plant.

As used herein, the term 'low-mobility' means a pest, during the relevant stage(s) of their life cycle, are able to move 3-50m. However, under typical conditions, these pests would move less than 10m, and more typically less than 0.5m.

For exam le, insects from the order Cofeoptera would typically move less than 1 m, more typically less than 0.5m during the relevant stage(s) of their life cycle.

Insects from the order Lepidoptera would typically move less than 20m, more typically less than 10m during the relevant stage(s) of their life cycle.

The method according to the invention is particularly suitable for managing resistance development in low-mobility pests.

The method is especially suitable for managing resistance development in insects from the following orders:

Blattaria, e.g. Blatta orientalis, Blattella germanica, Leucophaea maderae and Perip!aneta Americana; Chilopoda, e.g. Geophilus carpophagus and Scutigera spp; Coleoptera, e.g., Acanthoscelides obtectus, Agelastica alni, Agriotes spp, Amphimallon solstitialis, Anobium punctatum, Anthonomus spp, Anthrenus spp, Atomaria spp, Attagenus spp, Bruchidius obtectus, Ceuthorrhynchus assimilis, Conoderus spp, Cosmopolites sordidus, Costelytra zealandica, Dermestes spp, Diabrotica spp, Epilachna varivestis, Gibbium psylloides, Hylotrupes bajulus, Hypera postica, Leptinotarsa decemlineata, Lyctus spp, Meligethes aeneus, Melolontha melolontha, Niptus hololeucus, Oryzaephilus surinamensis,

Otiorrhynchus sulcatus, Phaedon cochleariae, Psylliodes chrysocephala, Ptinus spp, Rhizopertha dominica, Sitophilus spp, Tenebrio molitor, Tribolium spp, and Trogoderma spp; Collembola, e.g., Onychiurus armatus; Dermaptera, e.g., Forficula auricularia;

Diplopoda, e.g., Blaniulus guttulatus; Hemiptera; Diptera, e.g., Aedes spp, Anopheles spp, Bibio hortulanus, Calliphora erythrocephaia, Ceratitis capitata, Chrysomyia spp, Culex spp, Cuterebra spp, Dacus oleae, Drosophila melanogaster, Fannia spp, Gastrophilus spp, Hylemyia spp, Hypoderma spp, Hyppobosca spp, Liriomyza spp, Lucilia spp, Musca spp, Oestrus spp, Oscinella frit, Pegomyia hyoscyami, Phorbia spp, Stomoxys spp, Tabanus spp, Tannia spp, Tipula paludosa; Heteroptera, e.g., Cimex lectularius, Dysdercus intermedius, Eurygaster spp, Piesma quadrata, Rhodnius prolixus and Triatoma spp; Homoptera, e.g., Aleurodes brassicae, Aonidiella aurantii, Aphis fabae, Aphis gossypii, Aphis pomi, Aspidiotus hederae, Bemisia tabaci, Brevicoryne brassicae, Cryptomyzus ribis, Empoasca spp,

Eriosoma !anigerum, Euscelis bilobatus, Hyalopterus arundinis, Laodelphax striatellus, Lecanium comi, acrosiphum avenae, yzus spp, Nephotettix cincticeps, Nilaparvata lugens, Pemphigus spp, Phorodon humuli, Phylloxera vastatrix, Pseudococcus spp, Psylla spp, Rhopalosiphum padi, Saissetia oleae and Trialeurodes vaporariorum; Hymenoptera, e.g., Diprion spp, Hoplocampa spp, Lasius spp, Monomorium pharaonis and Vespa spp; Isopoda, e.g., Armadillidium vulgare, Oniscus asellus and Porcellio scaber;

Isoptera, e.g., Reticulitermes spp;

Lepidoptera, e.g. Bucculatrix thurberiella, Bupalus piniarius, Cacoecia podana, Capua reticulana, Carpocapsa pomonella, Cheimatobia brumata, Chilo spp, Choristoneura fumiferana, Clysia ambiguella, Cnaphalocerus spp, Earias insulana, Euproctis chrysorrhoea, Hofmannophila pseudospretella, Homona magnanima, Hyponomeuta padella, Lithocolietis blancardella, Lymantria spp, Malacosoma neustria, Pectinophora gossypiella, Phyllocnistis citrella, Pieris spp, Plutel!a xylostella, Pyrausta nubilalis, Tineola bisselliella, Tinea pellionella, Tortrix viridana, Noctuidae(e,g. Agrotis spp, Euxoa spp, Feltia spp, Heliothis spp, Mamestra brassicae, Panolis flammea, Spodoptera spp and Trichoplusia ni) and Pyralidae (e.g.

Ephestia kuehniella and Galleria mellonella);

Orthoptera, e.g., Acheta domesticus, Gryllotalpa spp, Locusta migratoria migratorioides, Melanoplus spp and Schistocerca gregaria; Phthiraptera, e.g., Damalinia spp, Haematopinus spp, Linognathus spp, Pediculus humanus corporis, Trichodectes spp; Siphonaptera, e.g., Ceratophyllus spp and Xenopsylla cheopis; Symphyla, e.g., Scutigerella immaculata;

Thysanoptera, e.g., Frankliniella accidentalis, Hercinothrips femoralis, Thrips palmi and Thrips tabaci; Thysanura, e.g., Lepisma saccharina. The method according to the present invention is even more suitable for managing resistance development in insects from the following orders:

Coleoptera, e.g., Acanthoscelides obtectus, Agelastica alni, Agriotes spp, Amphimallon solstitialis, Anobium punctatum, Anthonomus spp, Anthrenus spp, Atomaria spp, Attagenus spp, Bruchidius obtectus, Ceuthorrhynchus assimilis, Conoderus spp, Cosmopolites sordidus, Costelytra zealandica, Dermestes spp, Diabrotica spp, Epilachna varivestis,

Gibbium psylloides, Hylotrupes bajulus, Hypera postica, Leptinotarsa decemlineata, Lyctus spp, Meligethes aeneus, Melolontha meloiontha, Niptus hololeucus, Oryzaephilus

surinamensis, Otiorrhynchus suicatus, Phaedon cochleariae, Psylliodes chrysocephala, Ptinus spp, Rhizopertha dominica, Sitophilus spp, Tenebrio molitor, Tribolium spp, and Trogoderma spp;

Lepidoptera, e.g. Bucculatrix thurberiella, Bupalus piniarius, Cacoecia podana, Capua reticulana, Carpocapsa pomonella, Cheimatobia brumata, Chilo spp, Choristoneura fumiferana, Clysia ambiguella, Cnaphalocerus spp, Earias insulana, Euproctis chrysorrhoea, Hofmannophila pseudospretella, Homona magnanima, Hyponomeuta padella, Lithocolletis blancardel!a, Lymantria spp, Malacosoma neustria, Pectinophora gossypiella, Phyllocnistis citrella, Pieris spp, Plutella xylostella, Pyrausta nubilalis, Tineola bissellielia, Tinea pellionella, Tortrix viridana, Noctuidae (e.g. Agrotis spp, Euxoa spp, Feltia spp, Heliothis spp, Mamestra brassicae, Panolis flammea, Spodoptera spp and Trichoplusia ni) and Pyralidae (e.g.

Ephestia kuehnielia and Galleria mellonella);

Hemiptera. The method according to the present invention is even more suitable for managing resistance development in insects from the following orders:

Gibbium psylloides, Hylotrupes bajulus, Hypera postica, Leptinotarsa decemlineata, Lyctus spp, Meligethes aeneus, Melolontha melolontha, Niptus hololeucus, Oryzaephilus

surinamensis, Otiorrhynchus sulcatus, Phaedon cochieariae, Psylliodes chrysocephala, Ptinus spp, Rhizopertha dominica, Sitophilus spp, Tenebrio molitor, Tribolium spp, and Trogoderma spp;

Lepidoptera, e.g. Bucculatrix thurberiella, Bupalus piniarius, Cacoecia podana, Capua reticulana, Carpocapsa pomonella, Cheimatobia brumata, Chilo spp, Choristoneura fumiferana, Clysia ambiguella, Cnaphalocerus spp, Earias insulana, Euproctis chrysorrhoea, Hofmannophila pseudospretella, Homona magnanima, Hyponomeuta padella, Lithocolletis blancardella, Lymantria spp, Malacosoma neustria, Pectinophora gossypiella, Phyllocnistis citrella, Pieris spp, Plutella xylostella, Pyrausta nubilalis, Tineola bissellielia, Tinea pellionella, Tortrix viridana, Noctuidae (e.g. Agrotis spp, Euxoa spp, Feltia spp, Heliothis spp, Mamestra brassicae, Panolis flammea, Spodoptera spp and Trichoplusia ni) and Pyralidae (e.g.

Ephestia kuehnielia and Galleria mellonella).

The method according to the present invention is even more suitable for managing resistance development in the following: Coleoptera, e.g. Diabrotica spp;

Lepidoptera, e.g. Bucculatrix thurberiella, Bupalus piniarius, Cacoecia podana, Capua reticulana, Carpocapsa pomonella, Cheimatobia brumata, Chilo spp, Choristoneura fumiferana, Clysia ambiguella, Cnaphalocerus spp, Earias insulana, Euproctis chrysorrhoea, Hofmannophila pseudospretella, Homona magnanima, Hyponomeuta padella, Lithocolletis blancardella, Lymantria spp, Malacosoma neustria, Pectinophora gossypiella, Phyllocnistis citrella, Pieris spp, Plutella xylostella, Pyrausta nubilalis, Tineoia bisselliella, Tinea pellionella, Tortrix viridana, Noctuidae (e.g. Agrotis spp, Euxoa spp, Feltia spp, Heliothis spp, Mamestra brassicae, Panolis flammea, Spodoptera spp and Trichoplusia ni) and Pyralidae (e.g.

Ephestia kuehniella and Galleria mellonella).

Lepidoptera, e.g. Noctuidae, (e.g. Agrotis spp, Euxoa spp, Feltia spp, Heliothis spp,

Mamestra brassicae, Panolis flammea, Spodoptera spp and Trichoplusia ni), and Pyralidae (e.g. Ephestia kuehniella and Galleria mellonella).

The method according to the present invention is most suitable for managing resistance development in the following Diabrotica species: Diabrotica barberi (northern corn rootworm), D. virgifera virgifera (western corn rootworm), D. undecimpunctata howardi (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa

(chrysanthemum beetle), D. virgifera zeae (Mexican corn rootworm), D. beniensis, D.

cristata, D. curvipustulata, D. dissimilis, D. elegantula, D, emorsitans, D. graminea, D.

hispanolae, D. iemniscata, D. linsleyi, D. milleri, D. nummularis, D. occlusa, D, porracea, D. scutellata, D, tibialis, D. trifasciata and D. viridula. In further embodiments, the Diabrotica insect is D. virgifera virgifera (western corn rootworm), D. undecimpunctata howardi

(southern corn rootworm) or D. barberi (northen corn rootworm).

In areas of high pest pressure, it may be desirable to treat the transgenic pest resistant crop seeds or both the non-transgenic crop seeds and the transgenic pest resistant crop seeds with a pesticidal agent.

Accordingly, the invention also provides the method wherein the transgenic pest resistant crop seed comprises a pesticidal agent other than that generated by the transgenic pest resistant crop seed; and the non-transgenic crop seed comprises a pesticidal agent other than that generated by the transgenic pest resistant crop seed. The pesticidal agent which the transgenic pest resistant crop seed comprises may be the same as or different to that which the non-transgenic pest resistant crop seed. The invention also provides the method wherein the transgenic pest resistant crop seed comprises a pesticidal agent other than that generated by the transgenic pest resistant crop seed; and the non-transgenic crop seed does not comprise a pesticidal agent. Conversely, it may be desirable to not treat either the non-transgenic crop seeds or the transgenic pest resistant crop seeds with a pesticidal agent.

Accordingly, the invention also provides the method wherein the transgenic pest resistant crop seed comprises a first pesticidal agent; and the non-transgenic crop seed does not comprise a pesticidal agent.

That is to say, that the invention provides a method wherein both the transgenic pest resistant crop seed and the non-transgenic crop seed have been treated with a pesticidal agent other than that generated by the transgenic pest resistant crop seed. The pesticidal agent used to treat the transgenic crop seed may be the same as or different to that which has been used to treat the non-transgenic pest resistant crop seed.

The invention also provides the method wherein the transgenic pest resistant crop seed has been treated with a pesticidal agent other than that generated by the transgenic pest resistant crop seed; and the non-transgenic crop seed has not been treated with any pesticidal agent.

The invention also provides the method wherein the transgenic crop seed has been treated with a pesticidal agent other than that generated by the transgenic pest resistant crop seed; and the non-transgenic crop seed has not been treated with any pesticidal agent.

In summary, the transgenic pest resistant crop seeds and non-transgenic crop seeds may be treated with a pesticidal agent in the following combinations:

wherein 'treated' means been treated with a pesticidal agent other than that generated by the transgenic pest resistant crop seed

Pesticides that may be used in this method include those known in the art as exemplified, for example, in "The Pesticide Manual", 15th Ed., British Crop Protection Council 2009. In particular insecticides may be used. Many suitable options are available depending on the crop and pest to be controlled. Examples of insecticide classes which can be used in the practice of the current invention include benzoylureas, carbamates, chloronicotinyls, diacylhydrazines, diamides, fiproles, macrolides, nitroimines, nitromethylenes, organochlorines, organophosphat.es, organosilicons, organotins, phenylpyrazoles, phosphoric esters, pyrethroids, spinosyns, tetramic acid derivatives and tetronic acid derivatives. Nematicides and/or fungicides may also be preferred for use with the current invention. Pesticides that may be used in this method also include biological pesticides, for example Actinomycetes spp., Agrobacterium spp., Alcaligenes spp., Arthrobacter spp., Arthrobotrys spp. including A. dactyloides, A, oligospora, and A. superb, Aureobacterium spp., Azobacter spp., Bacillus spp. including B. agri, B. aizawai, B. albolactis, B.

amyloliquefaciens, B. cereus, B. firmus, B. coagulans, B. endoparasiticus, B. endorhythmos, B. firmus, B. kurstaki, B. !acticola, B. ladimorbus, B. lactis, B. laterosporus, B. ientimorbus, B. licheniformis, B. megaterium, B. medusa, B. metiens, B. natto, B. nigrificans, B. popillae,

B. pumilus, B. siamensis, B. sphaericus, B. subtilis, B. thuringiensis, and B. uniflagellates, Beijerinckia spp., Brevibacillus spp. including B. laterosporus, Burkholderia spp. including B. cepacia, Chaetomium spp. including C. globosum, Chromobacterium spp., C/aw^'bacter spp., Clostridium spp., Comomonas spp., Corynebacterium spp. including C. paurometabolu and

C. pauronietabolum, Curtobacterium spp., Cylindrocarpon spp. including C. heteronema, Dactylaria spp. including D. Candida, Desulforibtio spp., Enterobacter spp., Exophilia spp. including E. jeanselmei and E. pisciphila, Flavobacterium spp., Fusarium spp. including F. aspergilus and F. solani, Gliocladium spp. including 6. catenuiatum, G. roseum and G.

wrens, G I uconobacter spp., Harposporium spp. including H. anguillulae, Hirsutella spp. including H. rhossiliensis and H. minnesotensis, Hydrogenophage spp., Klebsiella spp., Lecanicillium spp. including L. lecanii, Meristacrum spp. including M. asterospermum, Methylobacterium spp., Monacrosporium spp. including M. cionopagum, M. drechsleri, and M. gephyropagum, Myrothecium spp. including M. verrucaria, Nematoctonus spp. including N. geogenius, N. leiosporus, Neocosmospora spp. including N. vasinfecta, Paecilomyces spp. including P. Iilacinus, Paenibacillus spp. including P. macerans, Pasteuria spp. including P. nishlzawae, P. penetrans, P. ramosa, P. thornei, and P. usgae , Phylfobacterium spp., Phingobacterium spp., Photorhabdus spp., Pochonia spp. including P. chlamydosporia, Pseudomonas spp. including P. fluorescens, Rhizobium spp., Serratia spp., Stagonospora spp. including S. heteroderae and S. phaseoli, Stenotrotrophomonas spp., Xenorhadbus spp., afrovorax spp.

Pesticides that may be used in this method further include polynucleotide compositions which could be used for treating seeds. These include compositions comprising oligonucleotides or polynucleotides or a mixture of both, including RNA or DNA or RNA/DNA hybrids or chemically modified oligonucleotides or polynucleotides or mixtures of these. These polynucleotides may be a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic polynucleotides consisting mainly of ribonucleotides (with one or more terminal deoxyribonucleotides) or synthetic polynucleotides consisting mainly of deoxyribonucleotides (with one or more terminal dideoxyribonucleotides). The polynucleotide may include, but is not limited to, non-canonical nucleotides such as inosine, thiouridine,,or pseudouridine.

These polynucleotides may include chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are known in the art and are commercially available (for example from Thermo Scientific, PromoKine, Sigma, and Amgen). By way of example, a naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide can be partially or completely modified with phosphorothioate, phosphorodithioate or methylphosphonate internucleotide linkage modifications, modified nucleoside bases. Modified sugars can be used in oligonucleotide or polynucleotide synthesis and oligonucleotides or polynucleotides can be labelled with a fluorescent moiety (for example fluorescein or rhodamine) or other label (for example biotin).

The polynucleotides can be single- or double-stranded RNA or single- or double-stranded DNA or double-stranded DNA/RNA hybrids or modified analogues thereof, of suitable oligonucleotide length.

The polynucleotides that provide single-stranded RNA may be selected from the group consisting of: a single-stranded RNA molecule, a single- stranded RNA molecule that self- hybridizes to form a double- stranded RNA molecule, a double-stranded RNA molecule, a single-stranded DNA molecule, a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, a single-stranded DNA molecule including a modified Pol Ell gene that is transcribed to an RNA molecule, a double-stranded DNA molecule, a double- stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, and a double-stranded, hybridized RNA DNA molecule, or combinations thereof. The polynucleotides may include chemically modified nucleotides or non-canonical nucleotides. The polynucleotides can include double-stranded DNA formed by intramolecular hybridization, double-stranded DNA formed by intermolecular hybridization, double-stranded RNA formed by intramolecular hybridization, or double-stranded RNA formed by intermolecular hybridization.

The polynucleotides include single-stranded DNA or single-stranded RNA that self-hybridizes to form a hairpin structure having at least a partially double-stranded structure including at least one segment that will hybridize under physiological conditions in a plant, a tissue or root of a plant or a pest of the plant, a pest of the tissue or a pest of the root of the plant, to RNA transcribed from the gene targeted for suppression. Without any intention of being bound by theory or any physicochemical mechanisms, it is believed that such polynucleotides are or will produce single-stranded RNA molecules wherein at least one segment of such molecules would hybridize under physiological conditions in a plant or root cell to RNA species transcribed (for example mRNA) from the gene that has been targeted for suppression.

The polynucleotides may further include a promoter, such as a promoter functional in a plant, a tissue or root of a plant or a pest of the plant, a pest of the tissue or a pest of the root of the plant for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter which are plant specific, tissue specific, root specific or have transcriptional activity in a pest of the plant, a pest of the tissue or a pest of the root of the plant.

Preferred pesticides that may be used in this method include, but are not limited to:

imidacloprid, metalaxyl, vitavax, permethrin, carboxin, thiamethoxam, clothianidin, permethrin, permethrin, esfenvalerate, tebupirim-phos, cyfluthrin, Bacillus thurin-gensis, beta cyfluthrin, bifenthrin, chlorpyrifos, gamma cyhalothrin, propargite, terbufos, metaldehyde, dimethoate, bifenthrin, spinosad, tefluthrin, chlor-ethoxyfosmethoxy- fenozide, lambda- cyhalothrin, methomyl, chlor- pyrifos, chlor-pyrifos, malathion, zeta-cypermethrin, permethrin, permethrin, gamma-cyhalothrin, spinetoram, fipronil, carbaryl, lambda-cyhalothrin, metaldehyde, phorate and lambda-cyhalothrin

Especially preferred pesticides for this method are:

tebupirim-phos, cyfluthrin, bifenthrin, chlorpyrifos, gamma-cyhalothrin, terbufos, tefluthrin, chlorethoxyfos, lambda-cyhalothrin, and fipronil.

This method is particularly suited to crops wherein the seeds are planted no more than 1-100 cm apart in a row, more typically 3-40 cm apart and even more typically 5-20 cm apart. It is preferred in the method that from 2 to 000 non-transgenic crop seeds be planted consecutively. More preferably, from 5 to 200 non-transgenic crop seeds should be planted consecutively.

In another embodiment of the invention, it is preferred in the method that from 2 to 1000 non- transgenic crop seeds be planted in the equivalent position of adjacent row(s). More preferably, from 5 to 200 non-transgenic crop seeds should be planted in the equivalent position of adjacent rows. In this method, each row should not contain consecutively planted non-transgenic crop seeds.

This method is particularly suited to crops wherein the rows are planted no more than 1-100 cm apart, more typically 3-40 cm apart and even more typically 5-20 cm apart.

Preferably, from 60 to 99% of the crops seeds are transgenic pest resistant crop seeds. More preferably, from 70 to 99% of the crops seeds are transgenic pest resistant crop seeds. Even more preferably, from 80 to 99% of the crops seeds are transgenic pest resistant crop seeds. Most preferably, from 90 to 99% of the crops seeds are transgenic pest resistant crop seeds. The method according to the invention is suitable for use with crops, for example, selected from: cereals, such as corn, wheat, barley, rye, rice, hops or oats; beet, such as sugar beet or fodder beet; oil plants, such as oil seed rape, mustard, poppy, sunflowers, castor oil plants, soybean, or groundnuts; cucumber plants, such as marrows, cucumbers or melons; fibre plants, such as cotton, flax, hemp or jute; vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, lettuce, cucurbits or paprika; tobacco; nuts; sugar cane. This list does not represent any limitation.

Crops of elevated interest in connection with present invention are corn; cotton; soybean; rice; oil seed rape; groundnuts; and vegetables, such as tomatoes, potatoes, cucurbits, lettuce and sugar cane.

Of even greater interest for use with the present invention are corn, cotton, sugar cane and soybean.

Of greater interest again for use with the present invention is corn.

One group of transgenic crops that are preferred for the invention are those which have been transformed with recombinant DNA techniques such that they are capable of expressing one or more selectively acting insecticidal proteins. Such proteins can include those produced by bacteria, such as those proteins produced by the genus Bacillus.

Insecticidal proteins that may be expressed by such transgenic plants include, for example, insecticidal proteins from Bacillus cereus or Bacillus popilliae; or insecticidal proteins from Bacillus thuringiensis,, such as the insecticidal crystal proteins listed by Crickmore et al.

(1998, Microbiology and Molecular Biology Reviews, 62: 807-813), updated by Crickmore et al. (2013) at the Bacillus thuringiensis toxin nomenclature, online at: http://vvww.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/), or insecticidal portions thereof, for example without limitation, proteins of the Cry protein classes CrylA (e.g. CrylAa, CrylAb and CrylAc), CrylB (e.g. Cryl Ba, CryIBb and Cryl Bc), CrylC (e.g. CryICa and CrylCb), Cryl D, Cryl E, CrylF, Cryl G, Cryl H, Cryl l (e.g. Cryl la to Cryl lg), CryU, Cry2 (e.g.

Cry2Aa, Cry2Ab and Cry2Ac), Cry3 (e.g. Cry3A, Cry3B and Cry3C), Cry4, Cry5, Cry6, Cry7, Cry8 (e.g. Cry8A, Cry8B, Cry8C, Cry8D, Cry8E, Cry8F, Cry8G, Cry8H, Cry8l, Cry8J, Cry8K, Cry8L, Cry8M, Cry*n, Cry80, Cry8P, Cry8Q, Cry8R, Cry8S and Cry8T), Cry9 (e.g. Cry9A, CryB and Cry9C), or the hybrid Cry proteins Cry34/Cry35, Cry23/Cry37, Cry48/Cry49, or the cytotoxic proteins Cyt1 , Cyt2, and Cyt3, or the vegetative insecticidal proteins (VlP)listed online at: http://www.lifesci.sussex.ac.uk Home/Neil_Crickmore/Bt/), for example without limitation, VIP1, VIP2, VIP3(e.g. VIP3A, VIP3B, VIP3C) and VIP4; or insecticidal proteins of bacteria colonising nematodes, for example Photorhabdus spp. or Xenorhabdus spp., such as Photorhabdus luminescens, Xenorhabdus nematophilus; toxins produced by animals, such as scorpion toxins, arachnid toxins, wasp toxins and other insect-specific neurotoxins; toxins produced by fungi, such as Streptomycetes toxins, plant lectins, such as pea lectins, barley lectins or snowdrop lectins; agglutinins; proteinase inhibitors, such as trypsine inhibitors, serine protease inhibitors, patatin, cystatin, papain inhibitors; ribosome-inactivating proteins (RIP), such as ricin, maize-RIP, abrin, luffin, saporin or bryodin; steroid metabolism enzymes, such as 3-hydroxysteroidoxidase, ecdysteroid-UDP-glycosyl-transferase, cholesterol oxidases, ecdysone inhibitors, HMG-COA-reductase, ion channel blockers, such as blockers of sodium or calcium channels, juvenile hormone esterase, diuretic hormone receptors, stilbene synthase, bibenzyl synthase, chitinases and glucanases.

In the context of the present invention there are to be understood by Cry proteins, for example CrylAb, CrylAc, Cry1 F, Cry2Ab, Cry3A, Cry3Bb or Cry9c and the like, or vegetative insecticidal proteins (VIP), for example VIP1 , VIP2, VIP3A or VIP3B and the like, expressly also hybrid proteins, truncated proteins and modified proteins. Hybrid proteins are produced by combining different domains of Cry proteins, for example without limitation, Cry1 Ac/Cry 1Ab hybrid (US Patent 5,128,130); Cry1 E/Cry1C hybrid (WO95/30752);

Cry1 Ac/Cry 1 F (US Patent 6,713,063) or hybrid VIP proteins, for example without limitation, Vip3A/Vip3B (US Patent 7,244,820) and Vip3A/Vip3C (US Patent 7,378,493). An example for a truncated insecticidal protein is a truncated CrylAb or truncated CrylB (US Patent 5,625, 136), or truncated CrylF (US Patent 6,218,188),. In the case of modified insecticidal proteins, one or more amino acids of a naturally occurring protein are replaced. Such modified insecticidal proteins include without limitation, Cry3A055, in which a cathepsin-D- recognition sequence is inserted into a Cry3A toxin (see US Patent 8,247,369). The processes for the preparation of such transgenic plants are generally known to the person skilled in the art and are described, for example, in the publications mentioned above as well as for example, US Patents 6,114,608, 6,720,488, 7,276,583, 8,247,369, 8,455,720, The insecticidal proteins expressed by transgenic plants impart to the plants tolerance to harmful insects. Such insects may come from any taxonomic group of insects, but are especially commonly found in the orders Coleoptera (beetles), Lepidoptera (butterflies and moths) and Diptera (flies). Transgenic plants that express one or more insecticidal proteins are known and some of them are commercially available. Examples of such plants include, without limitation the maize events: Bt11 , comprising a CrylAb gene (US Patent 6,114,608; AgrisureCB); MIR604, comprising a Cry3A055 gene (US Patent 7,361 ,813, AgrisureRW); MIR 62, comprising a Vip3A gene (US Patent 8,232,456; Agrisure Viptera); Event 5307, comprising a eCry3.1Ab gene (US Patent 8,466,346; Agrisure Duracade), TC1507, comprising a Cry1 Fa2 gene (US Patent 7,288,643; Herculex I); DAS59122, comprising a Cry34/Cry35 gene (US Patent 7,323556; HerculexRW); MON810, comprising a CrylAb gene (US Patent 6,713,259;

YieldGard®CB), MON863, comprising a Cry3Bb1 gene (US Patent 7,705,216; Yieldgard Rootworm), ON88017, comprising a Cry3Bb1 gene (US Patent 8,212, 13; Yieldgard VTRW), MON89034, comprising a Cry1A.105 gene and a Cry2Ab2 gene (US Patent

8,062,840; Yieldgard VT PRO), the cotton events: COT102, comprising a Vip3 gene (US Patent 8,133,678), CE43-67B, comprising a CrylAb gene (US Patent 7,834,254), ON531 , comprising a CrylAc gene (US Patent 7,964,348; Bollgard), Event 15985, comprising a CrylAc gene and a Cry2Ab2 gene (US Patent 7,858,764; Bollgardll), Other transgenic insect tolerant crops include the maize events:CBH-351 , DAS-06275-8, DBT418, MON80100, MIR152V, 3210M, and 3243M, and the cotton events: MON15985, MON757, MON1076, 281 , 281-24-236, 3006-210-23, DAS21023, DAS24236and Event-1.

Transgenic plants that express one or more herbicide tolerance genes are also known and some of them are commercially available. Examples of such plants include, without limitation the maize events: GA21, comprising dmEPSPS gene (US Patent 6,040,497; Agrisure GT), NK603, comprising a EPSPS gene (US Patent 8,273,959; Roundup Ready), the

phosphinothricin tolerant events B16 T14 orT25, or the cotton events: MON88913, comprising a EPSPS gene (US Patent 8,435,743). Transgenic plants created by making breeding stacks using individual transgenic events are also known. These may result multiple proteins being expressed from a single event or a combination of different events. Examples of such breeding stacks include without limitation: Bt11 X IR604 X GA21 (Agrisure 300GT), Bt 1 X MIR162 X IR604 X GA21 (AgrusureViptera 3111), Bt11 X IR604 X TC1507 X 5307 (Agrisure Duracade 5122), TC1507 X DAS59122 (Herculex Xtra), Mon810 X MON863 (Yieldgard Plus CL), Mon810 X MON88017 (Yieldgard VT Triple), Mon89034 X MON880 (Yieldgard VT Triple Pro), MON88017 X MON89034 X TC1507 X DAS59122 (SmartStax) and COT102 X COT67B (VIPCOT).

5. IPC 531 Cotton from Monsanto Europe S A 270-272 Avenue de Tervuren, B-1 150 Brussels, Belgium, registration number C/ES/96/02. Accordingly, transgenic crop containing multiple transgenic events can be used in the method according to the invention.

Another group of transgenic crops that are preferred for the invention are those which have been transformed with recombinant DNA techniques such that they are capable of expressing one or more oligonucleotide or polynucleotide which have a pesticidal effect, or a mixture of both, including RNA or DNA or RNA DNA hybrids or chemically modified oligonucleotides or polynucleotides or mixtures of these.

These polynucleotides may be a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic polynucleotides consisting mainly of ribonucleotides (with one or more terminal deoxyribonucleotides) or synthetic polynucleotides consisting mainly of deoxyribonucleotides (with one or more terminal dideoxyribonucleotides). In some of the embodiments described here, the polynucleotide includes, but is not limited to, non-canonical nucleotides such as inosine, thiouridine, or pseudouridine.

The polynucleotides can be single- or double-stranded RNA or single- or double-stranded DNA or double-stranded DNA RNA hybrids or modified analogues thereof, of suitable oligonucleotide length.

The polynucleotides that provide single-stranded RNA may be selected from the group consisting of: a single-stranded RNA molecule, a single- stranded RNA molecule that self- hybridizes to form a double- stranded RNA molecule, a double-stranded RNA molecule, a single-stranded DNA molecule, a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, a single-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, a double-stranded DNA molecule, a double- stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, and a double-stranded, hybridized RNA/DNA molecule, or combinations thereof. The polynucleotides may include chemically modified nucleotides or non-canonical nucleotides. The polynucleotides can include double-stranded DNA formed by intramolecular hybridization, double-stranded DNA formed by intermolecular hybridization, double-stranded RNA formed by intramolecular hybridization, or double-stranded RNA formed by intermolecular hybridization.

The polynucleotides may include single-stranded DNA or single-stranded RNA that self- hybridizes to form a hairpin structure having at least a partially double-stranded structure including at least one segment that will hybridize under physiological conditions in a plant, a tissue or root of a plant or a pest of the plant, a pest of the tissue or a pest of the root of the plant, to RNA transcribed from the gene targeted for suppression. Without any intention of being bound by theory or any physicochemical mechanisms, it is believed that such polynucleotides are or will produce single-stranded RNA molecules wherein at least one segment of such molecules would hybridize under physiological conditions in a plant or root cell to RNA species transcribed (for example mRNA) from the gene that has been targeted for suppression.

The transgenic crops may also include those which, in addition having been so transformed by the use of recombinant DNA techniques that they are capable of synthesising one or more selectively acting toxins, have been rendered tolerant to herbicides like bromoxynil or classes of herbicides (such as, for example, HPPD inhibitors, ALS inhibitors, for example

primisulfuron, prosulfuron and trifloxysulfuron, EPSPS (5-enol-pyrovyl-shikimate-3- phosphate-synthase) inhibitors, GS (glutamine synthetase) inhibitors) as a result of conventional methods of breeding or genetic engineering. Further, the transgenic crops may also include those which, in addition having been so transformed by the use of recombinant DNA techniques that they are capable of synthesising one or more selectively acting insecticidal proteins, have been so transformed by the use of recombinant DNA techniques that they are capable of synthesising antipathogenic substances having a selective action, such as, for example, the so-called "pathogenesis- related proteins" (PRPs, see e.g. EP-A-0 392 225). Examples of such antipathogenic substances and transgenic plants capable of synthesising such antipathogenic substances are known, for example, from EP-A-0 392 225, WO 95/33818, and EP-A-0 353 19 . The methods of producing such transgenic plants are generally known to the person skilled in the art and are described, for example, in the publications mentioned above.

Antipathogenic substances which can be expressed by such transgenic plants include, for example, ion channel blockers, such as blockers for sodium and calcium channels, for example the viral KP1 , KP4 or KP6 toxins; stilbene synthases; bibenzyl synthases;

chitinases; glucanases; the so-called "pathogenesis-related proteins" (PRPs; see e.g. EP-A- 0 392 225); antipathogenic substances produced by microorganisms, for example peptide antibiotics or heterocyclic antibiotics (see e.g. WO 95/33818) or protein or polypeptide factors involved in plant pathogen defence (so-called "plant disease resistance genes", as described in WO 03/000906).

The invention also encompasses a method for providing a non-transgenic refuge in a field of transgenic pest resistant crops comprising planting a plurality of rows of transgenic pest resistant crop seeds wherein at least one row in the field is planted according to the method described herein.

To put the invention into practice, a grower is provided with transgenic and non-transgenic crop seeds in an appropriate ratio, for example a ratio of transgenic pest resistant crop seeds to non-transgenic crop seeds of 95:5. These may be provided separately or in a mixture wherein the different seeds can be readily recognised, for example by using different colours for the transgenic versus non-transgenic crop seeds. Other identifiers such as bar codes and RFID tags are contemplated. The grower sows the seeds appropriately such that in at least one row of seeds there are at least two consecutive non-transgenic crop seeds. The skilled person would be aware of techniques that can be used to achieve this, for example, by planting the seeds by hand. The same result could be achieved by using a planting system comprising two hoppers, one of which contains the transgenic pest resistant crop seed and one of which contains the non- transgenic crop seed. Dual hopper or other suitable planters are well known in the art as evidenced by US867074 and US7418908. In a more modern approach the planting system could be programmed to sow the seeds such that a row contained at least two consecutive non-transgenic crop seeds, for example by allowing the seed to be sown from the hopper containing non-transgenic crop seed for a set amount of time. Alternatively the planting scheme for the field could be planned in advance and suitable equipment loaded, see for example US6474500.

Such techniques would lead to the formation of a non-transgenic refuge as described herein. As an improvement on the known method of large tracts or blocks of non-transgenic refuge, or randomly interspersing refuge plants within a transgenic crop, a non-transgenic refuge of the invention would lead to a per seed greater number of viable breeding insects which have never been exposed to the recombinant pesticide pressures and so have not had the opportunity to develop resistance as a result of the pesticide pressure when feeding on the recombinant plants. EXAMPLES

Resistance evolution model

A model of Bacillus thuringensis toxin (Bt) resistance evolution using the present invention under three different sets of assumptions is shown below. Methods

Population genetics

The model assumes a panmictic population of insects, initially at Hardy-Weinberg

equilibrium, controlled by a single autosomal di-allelic locus conferring complete resistance with no cost.

In the recessive cases, Bt mortality is additionally assumed to be complete and

instantaneous. Therefore, SS and SR individuals incur 100% mortality on plants expressing Bacillus thuringensis toxin (Bt plants), resistant (RR) individuals incur no mortality on Bt plants, and all individuals incur no mortality on non-transgenic (refuge) plants. In the non-recessive case, other values are used for SS and SR mortality on Bt plants.

Generations occur as discrete synchronized pulses such that allele frequency, q, can be tracked as:

„ „ number of resistance alleles after selection r;

"t+ϊ total number of alleles after selection

Note that it is equivalent to multiply all fitness values (w) by a constant background rate. In the recessive case, with fitness of RR standardized at 1 , it can be rewritten as: a ~ ⁽HX¾ c 2

The fitnesses of SR and RR need to be determined accounting for the distribution of exposure resulting from movement. In all cases shown, an initial allele frequency of 0.001 is used and durability is taken to be the number of generations until the allele frequency first exceeds 0.5.

Movement

Individuals are initially placed on plants independent of insect and plant type. In an infinite field, this averages to r individuals on refuge plants and (1-r) individuals on Bt plants.

Individuals are then capable of moving to either adjacent plant in a row, but not across rows. Movement is independent of insect and plant type and occurs continuously according to a constant instantaneous rate, μ, but can be scaled as, m, the proportion of individuals that move at least once in the absence of mortality:

m = l-e^~" Eq. 3

Plant sequence

The field is modeled as an infinite and edgeless repeating sequence of k refuge plants followed by / Bt plants, satisfying an overall proportion of refuge plants, r. That is, the number of Bt plants following a run of refuge plants is:

Note that r must be a value such that / is an integer. A 5% seed blend refuge is used in all cases below.

For the random case, the lengths of runs of refuge plants follow the distribution:

Eq. 5

That is, given that the first plant is a refuge plant to begin the run, the probability of a run of length k is the product of the probabilities that each subsequent plant is a refuge plant (each having probability r in the random case) multiplied by the probability that the final plant is a Bt plant (1-r) to end the run.

To determine the probability that an individual is initially located on a plant in a run of a given length, the probability of each run must be weighted with the number of plants in that run: Fitness

Recessive resistance two-direction movement case

For the recessive case, fitness of SS and SR individuals is the probability of initially being placed on a refuge plant multiplied by the probability of never moving to a Bt plant. This is modeled using a continuous transition matrix. For a run of length k, the transition matrix is of size k by k, with movement gains on the subdiagonals (each with rate μ/2, since movement can be to either adjacent plant) and movement losses (each with rate -μ) on the main diagonal. Because individuals that move out of the run end up on Bt, those individuals die and are removed from the system. The transition matrix is therefore:

With random independent placement, the initial vector is of length k, with all entries of value r/k:

The multiplication by /"accounts for the probability that an individual begins in a run of refuge plants in the first place (as opposed to a Bt plant).

Fitnesses of SS and SR are then the total proportion remaining in the system:

w = y._e ^M Eq. 9 Since V multiplied by e^M gives the vector of the proportion of individuals found on each plant in the run of refuge plants, the sum of this vector is the proportion of individuals that remained in the run of refuge plants. For the random case, the average fitness is determined by weighting the fitness in each length of run of refuge plants by the probability of starting in a run of refuge plants of that length:

Eq. 10

Although k can be from 1 to infinity, numerical approximation was done by summing to runs of length 100 for the cases below, since the series converges and runs of that length are sufficiently rare for a refuge of 0.05. This averaged fitness is then used in equation 2 to determine durability across a range of values for movement (μ) and sequence distributions.

Results

With no movement (/r?=0), all plant sequences are equivalent with a durability of 59 generations. This is because no individuals move off of refuge plants, so the adjacent plant types become irrelevant. Similarly, when movement is 1 , individuals are moving so much that they are guaranteed to eventually move to a Bt plant and die; therefore, only RR individuals survive and allele frequency becomes 1 after a single generation. For intermediate values of movement, longer runs of refuge plants improve durability. Random runs of refuge plants are 95% runs of single refuge plants, with 4.75% runs of 2 refuge plants, and 0.25% runs of more than 2 refuge plants (for an average of 20/19»1.053). Therefore, durability of random sequences is improved over runs of single refuge plants, but less than for runs of 2 refuge plants. This is seen in Fig. 1.

Recessive resistance single-direction movement case

Durability was also modeled using single direction movement with recessive resistance. If individuals do not return to plants after having left, movement becomes unidirectional and can be modeled by modifying the movement matrix. In this case, the initial vector is still equation 8 but the transition matrix becomes:

Because of symmetry, the answer is the same for the opposite direction and therefore does not need to be considered separately.

For the random case with movement in a single direction and complete Bt mortality, survival becomes: _{w = r} . _e-M<l-r) _{Eq 1 2}

This simplification occurs because the type of plant an individual moves onto is now independent of past moves. Results

Durability of all plant sequences is again 59 generations with no movement and 1 generation with complete movement. Longer runs of refuge plants still improve durability at intermediate levels of movement with random runs slightly better than runs of single refuge plants, but worse than runs of 2 refuge plants. Although the not identical, the unidirectional and bidirectional models of movement produce very similar results. This is seen in Fig. 2.

Non-recessive resistance single-direction movement case

When Bt mortality is not instantaneous for both susceptible (SS) or heterozygous (SR) individual insects, the state space must be extended to include Bt plants, such that V is a vector of length k+l with multiplying by r no longer necessary:

M is now a k+l by k+l matrix, with the diagonal for Bt plants also including a death rate (cQ, and transitions allowed between the first and last plant:

- μ_Άί~ά for i = j, i > k

M = Eq. 14 μ_κ for j - i = \, i > k

μ_β! for j = 1, i = k + 1

0 otherwise k+l xk

Fitness is still determined according to equation 9.

It is straightforward to calculate different matrices using different movement or survival rates for different genotypes to obtain a different average fitness for each. For example, SS and SR individuals may have a different value for d if it is not completely recessive. Furthermore, movement from Bt plants becomes relevant once mortality is less than complete, hence the subscripts on μ. For the present case, movement rates for Bt and refuge plants are assumed to be the same. However, to model asymmetric movement, one simply needs to assign different movement rates for Bt and refuge plants. It is also possible to allow these to interact with genotype. For example SS individuals may have higher movement from Bt plants than refuge plants, while RR individuals may not distinguish between plant types.

For the random case with movement in a single direction and less than complete Bt mortality, survival becomes:

Because fitness is different for SR and RR individuals, durability is determined according to equation 1. Results

Death rates used for SS and SR are d = 6 and 3, corresponding to survival on pure Bt of approximately 0.0025 and 0.0498. All plant sequences are still the equivalent when there is no movement or 100% movement with 9 and 3 generations of durability. The former is lower than in the recessive case since the advantage of the R allele manifests in both

heterozygotes and homozygotes. The latter is higher because the recessive case also includes complete Bt mortality, while the current case allows some susceptible individuals to survive even when exposed to Bt. Despite these differences, the benefit of longer runs of refuge plants is still apparent for intermediate levels of movement. This is seen in Fig. 2.

Biological Example: Emergence of western corn rootworm larvae IPiabrotica viraifera viraifera LeContel

Experimental Design and Methods

The experimental design was a randomized complete block with three replications. Previous crop was a "trap crop" consisting of late planted pumpkins and corn designed to attract adult corn rootworm beetles ensuring high populations of corn rootworm larvae the following spring. The plot size for each treatment was 10 ft. (four rows) ^χ 10 ft. Prior to enclosing each plot with a 12 ft. x 12 ft. x 7 ft. tent approximately 6 weeks after planting, plants were cut to approximately 2 ft. in height to facilitate beetle collection. Beetles were collected every Monday, Wednesday, and Friday from the seventh week after planting until the end of the thirteenth week after planting.

In both examples 1 and 2, 95% of seeds were transgenic pest resistant seeds. The transgenic pest resistant crop seed used was NK N69Z-5222, which expresses the mCry3A and eCry3.1Ab corn rootworm-active insecticidal proteins. The non-transgenic crop seed used was NK N69Z-GT. All seed was treated with Cruiser (0.5 milligrams of active ingredient per seed).

Planting and Insecticide Application

Seeds were planted in 30-inch (approx. 76 cm) rows at an approximate depth of 1.75 inches (4.45 cm) with approximately 5 inches ( 3 cm) between each seed. Force CS (tefluthrin) was applied in furrow at a spray volume of 5 gallons per acre (approx. 46.8 litres per hectare) using a C0₂ system. In example 1 , the non-transgenic seed was planted randomly. In example 2, four non- transgenic seeds were planted consecutively in one row of each plot.

Results and Discussion

Mean values for cumulative emergence are presented in Table 1.

These results are indicative that more com root worm adults emerge in non-transgenic refuge planted according to the present invention than in randomly planted non-transgenic refuge.

Table 1. Emergence of western corn rootworm larvae

Example Mean cumulative

emergence

(beetles/plot)

1 33

Claims

1. A method of sowing rows of crops comprising planting a mixture of transgenic pest resistant crop seeds and non-transgenic crop seeds wherein at least 2 non-transgenic crop seeds are planted consecutively and wherein 50-99.5% of the mixture is transgenic pest resistant crop seeds.

2. The method according to claim 1 wherein the non-transgenic crop seeds comprise a pesticidal agent which is different to that produced by a crop grown from the transgenic pest resistant crop seeds.

3. The method according to claim 1 wherein the transgenic pest resistant crop seeds and the non-transgenic crop seeds comprise a pesticidal agent which is different to that produced by the transgenic crop.

4. The method according to either claim 2 or claim 3 wherein the pesticidal agent is selected from imidacloprid, metalaxyl, vitavax, permethrin, carboxin, thiamethoxam, clothianidin, permethrin, permethrin, esfenvalerate, tebupirim-phos, cyfluthrin, Bacillus thurin- gensis, beta cyfluthrin, bifenthrin, chlorpyrifos, gamma cyhalothrin, propargite, terbufos, metaldehyde, dimethoate, bifenthrin, spinosad, tefluthrin, chlor-ethoxyfosmethoxy- fenozide, lambda-cyhalothrin, methomyl, chlor-pyrifos, malathion, zeta-cypermethrin, permethrin, permethrin, gamma cyhalothrin, spinetoram, fiproni!, carbaryl, lambda-cyhalothrin, metaldehyde, phorate or lambda-cyhalothrin.

5. The method according to any one of claims 1 -4 wherein from 2 to 20 non-transgenic crop seeds are planted consecutively.

6. The method according to any one of claims 1-5 wherein the crop is selected from cereal; cotton; soybean; rice; oil seed rape; groundnuts; and vegetables.

7. The method according to any one of claims 1-6 wherein the crop is selected from a cereal, sugar cane, cotton or soybean.

8. The method according to any one of claims 1-7 wherein the crop is selected from a cereal, cotton or soybean.

9. The method according to any one of claims 1-8 wherein the crop is selected from a corn, cotton or soybean.

10. The method according to any one of claims 1-9 wherein the crop is corn.

11. The method according to any one of claims 1-9 wherein the transgenic crop is capable of synthesising one or more insecticidal proteins.

12. The method according to any one of claims 1-11 wherein the transgenic crop is capable of synthesising one or more insecticidal proteins produced by the genus Bacillus.

13. The method according to any one of claims 1-12 wherein the transgenic crop is capable of synthesising one or more δ-endotoxins or vegetative insecticidal proteins (VIP).

14. The method according to any one of claims 1 -13 wherein the transgenic crop is capable of synthesising one or more toxins selected from CrylA(b), CrylA(c), CrylF, CrylF(a2), CryllA{b), CrylllA, CrylllB(bl), Cry9c, VIP1 , VIP2, VIP3 or VIP3A.

15. The method according to anyone of claims 1-14 wherein the transgenic crop comprises multiple transgenic events.

16. A method for providing a non-transgenic refuge in a field of transgenic pest resistant crops comprising planting a plurality of rows of transgenic pest resistant crop seeds wherein at least one row in the field is planted according to the method of any one of claims 2-15.

17. A method of managing resistance development in low-mobility pests comprising sowing seeds according to any one of claims 1-16.

18. A method according to claim 17 wherein the pests are selected from the order Coleoptera, the order Lepidoptera, the order Diptera or the order Hemiptera.

19. A method according to either claim 17 or claim 18 wherein the pests are selected from the order Coleoptera or the order Lepidoptera.

20. A method according to any one of claims 18-20 wherein the pests are selected from the family Noctuidae, the family Pyralidae or the genus Diabrotica.

21. A method according to any one of claims 18-20 wherein the pest is Diabrotica spp.