WO2014198729A1 - Procédés et compositions de lutte contre les nématodes - Google Patents

Procédés et compositions de lutte contre les nématodes Download PDF

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Publication number
WO2014198729A1
WO2014198729A1 PCT/EP2014/062038 EP2014062038W WO2014198729A1 WO 2014198729 A1 WO2014198729 A1 WO 2014198729A1 EP 2014062038 W EP2014062038 W EP 2014062038W WO 2014198729 A1 WO2014198729 A1 WO 2014198729A1
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Prior art keywords
bus
test substance
expression
tkt
group
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PCT/EP2014/062038
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English (en)
Inventor
Jonathan Ewbank
Olivier ZUGASTI
Nathalie PUJOL
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Centre National De La Recherche Scientifique (Cnrs)
Université D'aix Marseille
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Publication of WO2014198729A1 publication Critical patent/WO2014198729A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to methods and compositions for controlling nematodes.
  • Nematodes are typical soil-dwelling, microscopic, un-segmented, round worms that often parasitic for plant, cattle and human.
  • the free-living nematode Caenorhabditis elegans is an important pathogen in fungi and straw mushrooms.
  • plant parasitic nematodes infect many major food and fiber crops, as well as turf grasses and ornamentals, and cause significant reduction in crop yield and quality.
  • Nematodes have been estimated to cause annual crop losses of about $78 billion worldwide (from the report "Plant & Soil Nematodes: Societal Impact and Focus for the Future", sponsored by USDA-CSRS and The Society of Nematologists).
  • nematodes feed on the roots, but a few species attack above-ground plant parts. Damage from nematodes is primarily due to feeding and wounding, which in turn affects water and nutrient absorption, and can lead to secondary infections by bacterial and fungal pathogens. In addition, root- feeding nematodes have been implicated in the transmission of viral diseases. Plant-parasitic nematodes seldom kill plants outright. A loss of yield or quality can occur without specific above-ground symptoms. Sometimes these losses are attributed to other pests, fertility problems, or improper water management. The symptoms of nematode damage may vary among crops and are generally manifested as root galling, lesions, premature wilting, chlorosis and overall reduction in plant vigor.
  • nematicides have been developed.
  • the most popular means of controlling nematodes include the use of soil fumigants such as methyl bromide, or non-fumigant nematicides such as organophosphates and carbamates.
  • soil fumigants such as methyl bromide
  • non-fumigant nematicides such as organophosphates and carbamates.
  • These and most other conventionally used nematicides are toxic to mammals and other non-target organisms, pose a potential threat to the environment and ground water, and may leave chemical residues in food products.
  • the present invention relates to methods and compositions for controlling nematodes.
  • the present invention relates to an agent capable of inhibiting the expression or activity of at least one gene selected from the group consisting of bus-12, bus-2, C14H10.3, C26B9.3, cpt-6, dpy-20, F35H12.5, K06A9.1, K08E3.5, sdc-2, tkt-1, Y38C1AB.5, and ykt-6 for use in a method for controlling nematodes.
  • the present invention relates to an agent capable of inhibiting the expression or activity of at least one gene selected from the group consisting of bus-12, bus-2, C14H10.3, C26B9.3, cpt-6, dpy-20, F35H12.5, K06A9.1, K08E3.5, sdc-2, tkt-1, Y38C1AB.5, and ykt-6 for use in a method for controlling nematodes.
  • Controlling nematodes means preventing nematodes to develop or to grow.
  • the agents described herein may be used to keep an organism healthy (e.g. a plant) and may be used curatively, preventively or systematically to control nematodes.
  • the agent of the invention will render the nematodes more susceptible to infection by microorganism such as fungus that are present in their environment (i.e. by increasing the adhesion of said microorganisms to the worm's cuticle) and thus will finally lead to the killing of nematodes.
  • the "organisms” as mentioned above refer to plants, cattle or humans.
  • the controlling of nematodes as used herein encompasses the reduction of damage to plants and encompasses increased yield.
  • Nematodes encompass all species of the order Nematoda. In particular the present invention focuses on species that are parasitic or cause health problems to plants.
  • Plant nematodes encompass plant parasitic nematodes and nematodes living in the soil. Plant parasitic nematodes include, but are not limited to, ectoparasites such as Xiphinema spp., Longidorus spp., and Trichodorus spp.; semiparasites such as Tylenchulus spp.; migratory endoparasites such as Pratylenchus spp., Radopholus spp., and Scutellonema.
  • ectoparasites such as Xiphinema spp., Longidorus spp., and Trichodorus spp.
  • semiparasites such as Tylenchulus spp.
  • migratory endoparasites such as Pratylenchus spp
  • sedentary parasites such as Heterodera spp., Globodera spp., and Meloidogyne spp.
  • stem and leaf endoparasites such as Ditylenchus spp., Aphelenchoides spp., and Hirshmaniella spp.
  • the agents described herein are particularly suitable for protecting plants against nematodes.
  • a "plant” as used herein encompasses a plant cell, plant tissue (including callus), plant part, whole plant, ancestors and progeny.
  • the term “plant' also encompasses all plants and plant populations such as desired and undesired wild plants or crop plants (inclusive of naturally occurring crop plants).
  • Crop plants can be plants which can be obtained by conventional plant breeding and optimization methods or by biotechnological and recombinant methods or by combinations of these methods, inclusive of the transgenic plants and inclusive of the plant cultivars protectable or not protectable by plant breeders' rights.
  • a plant part may be any part or organ of the plant and include for example a seed, fruit, fruit body, stem, leaf, needle, stalk, shoot, flower, anther, root, tuber or rhizomes.
  • plant' also encompasses vegetative and generative material for example cuttings, tubers, rhizomes, offsets, seeds, suspension cultures, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, and microspores.
  • the agents described herein are particularly suitable for protecting plants against nematodes, wherein said plant is chosen from the group of known nematode host plants, consisting of (corn, rice, barley, wheat, millet) Acer, Betula, Prunus and other fruit trees, Fraxinus, Ulmus cotton, coffee, tea, citrus, Vinca, tobacco tomato, cowpea, lettuce, rape, potato, bean, celery, cucurbits, pepper, carrots, aubergines, Allium, eggplant, strawberry, garlic, cabbage, soybean, banana Rosaceae, Liliaceae, Azalea, Rhododendron.
  • nematode host plants consisting of (corn, rice, barley, wheat, millet) Acer, Betula, Prunus and other fruit trees, Fraxinus, Ulmus cotton, coffee, tea, citrus, Vinca, tobacco tomato, cowpea, lettuce, rape, potato, bean, celery, cucurbits, pepper, carrots, aubergines, Allium, eggplant,
  • the agents described herein are used to protect plants against nematodes, wherein said plant is selected from the group comprising rice, soybean, cotton, potato, banana, strawberry, garlic, eggplant, carrot, tobacco, tomato, cucumber, lettuce, celery and lilies.
  • bus- 12 has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGeneOOO 10442.
  • bus- 12 encodes a predicted nucleotide-sugar transporter and is most similar to sqv-7 within the elegans genome, which encodes a protein that can transport UDP-glucuronic acid, UDP-N acetylgalactosamine, and UDP-galactose, when expressed in yeast cells, and UDP-galactose when expressed in canine cells.
  • An exemplary amino acid sequence is SEQ ID NO: l .
  • SEQ ID NO: 1 MIVFVAASVFCSLMSKIMVTRYFFDYPVVILMMQSAATLFVIEVTRVLGILKV APYCFEKGRHIVIPSILYTISQWITVASFEGIAMPNFDSVKRLTPILILVGLAAR RQQRVDQNKTFIIIGLSIASAFAVNLDLSVDRYSLMYGLVGAILQAAAFVLFEE HLQNYNYTEVLYMHSFNSLVFYLLADMVRDELRDAFMYMITSAHPLFIIVFA VSMFAGVLFHFTTFSCLEK GPLNMQIVSNVRAVVETFLAYYLSVYLFYDVY PGVLNWAFLAVTFVAARALVNRDCEPEIVKGPWMSKA
  • bus-2 has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGene00044618.
  • bus-2 encodes a predicted glycosyltransferase that contains conserved domains of core-1 betal,3 galactosyltransferases.
  • An exemplary amino acid sequence is SEQ ID NO:2.
  • 'C14H10.3 has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGene00007593.
  • An exemplary amino acid sequence is SEQ ID NO:3.
  • 'C26B9.3 has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGene00016133.
  • An exemplary amino acid sequence is SEQ ID N0:4.
  • cpt-6 has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGene00020911.
  • cpt-6 encodes for a Carnitine Palmitoyl Transferase.
  • An exemplary amino acid sequence is SEQ ID N05.
  • dpy has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGene00001079.
  • dpy-20 encodes a BED zinc finger protein, with no known homo logs outside of nematodes, that is required for normal body morphology.
  • An exemplary amino acid sequence is SEQ ID NO:6.
  • F35H12.5 has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGeneOOO 18077.
  • An exemplary amino acid sequence is SEQ ID N0:7.
  • K08E3.5 has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGeneOOO 10665.
  • An exemplary amino acid sequence is SEQ ID N0:9.
  • GTVIIIANHGDRIDIPPGSILENKIVSGNLRILEH As used herein the term "sdc-2" has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGene00004746.
  • the sdc-2 gene encodes a protein that represses transcription of X chromosomes to achieve dosage compensation, and that also represses the male sex- determination gene her-1 to elicit hermaphrodite differentiation.
  • An exemplary amino acid sequence is SEQ ID NO: 10.
  • tkt-1 has its general meaning in the art and refers to the ene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGene00008506.
  • tkt-1 encodes an ortholog of the human gene TRANSKETOLASE (TKT; OMIM:606781), which when mutated leads to Wernicke- Korsakoff syndrome (OMIM:277730).
  • An exemplary amino acid sequence is SEQ ID NO: l 1.
  • 'Y38C1AB.5" has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGene00021407.
  • An exemplary amino acid sequence is SEQ ID NO: 12.
  • ykt-6 has its general meaning in the art and refers to the gene referenced in the Wormbase (http://www.wormbase.Org/#01-23-6) under the access number WBGene00015164.
  • An exemplary amino acid sequence is SEQ ID NO: 13.
  • SEQ ID NO: 13 MKLYSILVFHK VDTSDVKLFKSECDLSSFSFFQRGSVQEFMTFTAKLLVERS GLGARSSVKENEYLVHCYVR DGLSAVCVTDAEYQQRVAMSFLGRVLDDFT TRVPATQWPGIRSDKDCSYTGLKDLLEKWQNPREADPMTRVQEEVEETKMV MHNTIQSVLDRGEKLDDLVK SENLSDQSKMFYTSARKMNKCCNYV
  • the agent is a low molecular weight antagonist, e. g. a small organic molecule that inhibits the activity of the gene.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • the agent is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • the agent is an inhibitor of expression.
  • An "inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
  • said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme.
  • the present invention also relates to a nematicidal composition, comprising an effective amount of at least one agent capable of inhibiting the expression or activity of at least one gene selected from the group consisting of bus-12, bus-2, C14H10.3, C26B9.3, cpt- 6, dpy-20, F35H12.5, K06A9.1, K08E3.5, sdc-2, tkt-1, Y38C1AB.5, and ykt-6.
  • the composition further comprises at least one other compound selected from the group consisting of surfactant, solid or liquid diluent,
  • the present invention relates to the use of nematicidal composition in agriculture or horticulture.
  • compositions may be prepared in a manner known per se.
  • the agent can be converted into the customary formulations, such as solutions, emulsions, wettable powders, water dispersible granules, suspensions, powders, dusting agents, foaming agents, pastes, soluble powders, granules, suspo-emulsion concentrates, microcapsules, fumigants, natural and synthetic materials impregnated with active compound and very fine capsules and polymeric substances.
  • formulations can be prepared in a known manner, for example by mixing the active compounds with surfactant or diluent normally used in nematicidal compositions such as for example mixing with extenders, that is liquid solvents, liquefied gas and/or solid diluents or carriers, if appropriate with the use of surface-active agents, that is emulsifiers and/or dispersants and/or foam-formers.
  • extenders that is liquid solvents, liquefied gas and/or solid diluents or carriers, if appropriate with the use of surface-active agents, that is emulsifiers and/or dispersants and/or foam-formers.
  • agents for use according to the invention can also be used in a mixture with other known active compounds, such as herbicides, fertilizers and/or growth regulators.
  • the present invention also encompasses the use of solid formulations for slow- releasing the agents as described herein.
  • the formulations release the agent as described herein into the environment (soil, aqueous medium, plants) in a controlled and slow fashion (complete release within several days up to a few months).
  • the solid slow-release formulation is extruded and comprises a thermoplastic, and/or a water-insoluble polymer.
  • suitable thermoplastic water-insoluble polymers from the group of the polylactides are based on polycondensates of lactic acid and are described, for example, in WO 97/41836 , WO 96/18591 , WO 94/05484 , U.S. Pat. No. 5,310,865 , U.S.
  • the present invention also relates to the use of surfactant-diatomaceous earth compositions for nematicidal use in the form of dry spreadable granules comprising at least one agent as described herein.
  • the granules comprises in addition to the diatomaceous earth, a surfactant composition designed to provide binding, rewetting and disintegration properties to the granules.
  • diatomaceous earth is meant a silica material characterized by a large surface area per unit volume.
  • Diatomaceous earth is a naturally occurring material and consists mainly of accumulated shells or frustules of intricately structured amorphous hydrous silica secreted by diatoms.
  • Suitable disintegration aids include water-soluble salts of alkylnaphthalenesulfonate-formaldehyde condensates; sodium lignosulfonate, diphenyloxide, ethoxylated tristyrylphenols, ethoxylated tristyrylphenol phosphates, ethylene oxide/propylene oxide block copolymers, and acid, salts and copolymers of the polyacrylates.
  • Suitable rewetting agents include alkylnaphthalenesulfonates, sodium methyloleoyl taurate, sulfosuccinates, carboxylates, alkylarylsulfonates, ethoxylated alkyl phenols and ethoxylated alcohols.
  • Suitable binders can be traditional binders well known in the art such as the starches, the sugars, etc., but preferably the granules use residuals or the secondary characteristics of the disintegration aids and/or rewetting agents described herein as the binding agents.
  • the nematicidal granules can be broadcast, that is, applied with a dry spreader to a target area and, when exposed to water via, for example, rain or irrigation, readily decompose or disintegrate and actively spread.
  • the dry spreadable granules show hardness and an ability to maintain integrity upon normal, commercial handling in a dry spreading operation and yet be capable of quickly disintegrating or scattering upon what may be a minimal exposure to water, such as, for example, a light rain.
  • the highly active disintegration process allows the nematicide to be delivered over a larger surface area than that immediately covered by the original granules resulting in a much more effective delivery of the nematicide to the targeted area.
  • the application of the agent or compositions as described herein to the plant may be in the usual way, such as for example by spraying on the plants.
  • the agent or compositions as described herein may be applied to the seed grains (coating) by either soaking the grains in a liquid preparation of the agent or coating them with a solid preparation of the compound.
  • the agents or compositions as described herein can be applied to a seed that has been harvested, cleaned and dried to a moisture content below 15%.
  • the seed can be one that has been dried and then prepared with water and/or another material and then re-dried before or during the application with the agents or compositions as described herein.
  • the agents or compositions as described herein can be applied to the seed at any time between harvest of the seed and sowing of the seed.
  • the agents as described herein can be applied to the seed pure, that is, without any diluting or additional components present.
  • the compounds or compositions as described herein can be applied to the seeds in the form of a nematicide formulation as described above.
  • Other methods of application of the compound or compositions as described herein to the plant are possible, such as, for example, the direct treatment of particular parts or organs of the organism, such as treatment of the plant stems, buds or leaves.
  • the compound or composition as described herein is applied to the roots of the plant.
  • the present invention also relates to a method for screening a nematicide comprising the step consisting of i) providing a test substance and ii) determining whether the test substance is able to inhibit the expression or the activity of at least one gene selected from the group consisting of bus-12, bus-2, C14H10.3, C26B9.3, cpt-6, dpy-20, F35H12.5, K06A9.1, K08E3.5, sdc-2, tkt-1, Y38C1AB.5, and ykt-6 in a nematode cell and iii) positively selecting the test substance that is able to inhibit the expression or the activity of at least one gene selected from the group consisting of bus-12, bus-2, C14H10.3, C26B9.3, cpt-6, dpy-20, F35H12.5, K06A9.1, K08E3.5, sdc-2, tkt-1, Y38C1AB.5, and ykt-6.
  • nematicide as used herein means that the compound is capable of controlling nematodes. Determination of the expression level of a gene can be performed by a variety of techniques. Generally, the expression level as determined is a relative expression level. More preferably, the determination comprises contacting the sample with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of polypeptide or nucleic acids of interest originally in the sample.
  • Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth
  • the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array.
  • the substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like.
  • the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
  • the contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody-antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the sample.
  • the expression level may be determined by determining the quantity of mR A. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the subject) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.
  • RNA and/or amplification e.g., Northern blot analysis
  • amplification e.g., RT-PCR
  • quantitative or semi-quantitative RT-PCR is preferred.
  • Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.
  • Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
  • LCR ligase chain reaction
  • TMA transcription-mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers.
  • nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical.
  • appropriate means such as a detectable label
  • a wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 %> formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the method may further comprises the steps consisting of i) providing a nematode and a funguns ii) determining the adhesion level of the fungus to the nematode's cuticle in presence of the selected test substance, iii) comparing the adhesion level determined at step ii) with the level determined in the absence of the test substance and iv) positively selecting the test substance when the adhesion level determined in the presence of the test substance is higher than the adhesion level determined in the absence of the test substance.
  • the nematode is Caenorhabditis elegans and the fungus is
  • C. elegans is a nematode worm which occurs naturally in the soil but can easily be grown in the laboratory on nutrient agar seeded with a food source such as E. coli bacteria. These characteristics of C. elegans make it an extremely useful tool in the drug discovery process.
  • C . elegans may be used in the development of high-throughput test substance screens, useful in the identification of potential candidate nematicides, in which worms are exposed to the test substance and any resultant phenotypic are recorded. Such screening methods are critically dependent upon the ability to expose C. elegans to the test substance under test.
  • nematode worms are grown on a solid growth medium which has been seeded with a nematode food source to which has been added a sample of a test substance.
  • the worms take up the substance as they feed on the food source.
  • the method preferably uses E. coli as a food source for the nematodes but other food sources known for use with nematode worms could also be used. These include other bacterial species, yeasts and slime moulds (e.g. Dictyostelium discoideum).
  • the food source can be living organisms, dead organisms, synthetic food sources or extracts from living organisms.
  • the food source can even be beads of any particular material on which nematodes such as C.
  • elegans can feed.
  • Suitable solid growth media include NGM agar which is routinely used for the culture of C. elegans in the laboratory.
  • NGM agar which is routinely used for the culture of C. elegans in the laboratory.
  • other polymer materials known to support growth of nematodes can be used. These polymer materials can be chosen in such a way that they give more or less adherence to the walls of microtiter plates.
  • the method of the invention can be performed in various formats, including single small agar plates (e.g. 3.5 cm diameter) as well as microtiter plates (e.g. 12, 24, 96 and 384 well plates).
  • agar plates e.g. 3.5 cm diameter
  • microtiter plates e.g. 12, 24, 96 and 384 well plates
  • additional problems have to be overcome, such as the formation of a meniscus by the agar.
  • the problem of the formation of an optically distracting meniscus can be overcome with the use of microtiter plates manufactured or coated with a specific wall material. These plates, described as WIM plates for Without Interfering Meniscus, are provided by Swiss Aircraft and Systems Company. Other plates designed to prevent disturbing meniscus formation can also be used with equivalent effect.
  • the polymer solution may be modified to achieve adhesive properties resulting in a decrease or absence of the optically distracting meniscus.
  • Most of the other problems arising from using microtiter plates can be solved by applying microtiter plates in specific materials or coated with specific materials. It is an essential feature of the method of the invention that an amount of the test substance to be tested is added to the nematode food source rather than to the solid growth medium. There are several different ways in which this can be achieved. In a first method a layer of food source is seeded onto the solid growth medium and an appropriate amount of the test substance under test is then spread on top of the food source layer. For example, when E .
  • test substance can be mixed directly with the food source prior to seeding onto the solid growth medium.
  • the final concentration of the test substance added to the food source can be optimised by performing test screens with serial dilutions of the test substance, as described the examples given herein.
  • a final concentration of the test substance should be chosen which is sufficient to have an effect on the worms but which does not cause any non-specific effects e.g. which is not lethal to the food source organisms to avoid worm starvation.
  • care must also be taken to ensure that the final concentration of the solvent is kept low, typically below -0.5% (equivalent to ⁇ total volume of solvent per 2ml agar) . Higher concentrations of DMSO, especially more than 1%, are not tolerated well by C. elegans .
  • any changes in the phenotype of the worms (i.e. adhesion of the fungus to the worm's cuticle) following exposure to the test substance can be readily observed over time under dissecting scopes, or any other method of visual detection.
  • exposure to the test substance can be readily observed over time under dissecting microscopes, by any other method of visual detection, or through the use of appropriate automated apparatus, for example, but not limited to, the COPAS Biosort (TM) manufactured by Union Biometrica.
  • EXAMPLE From fungal spore adhesion to effector gene transcription.
  • bus-2 and bus- 12 that were originally isolated by Jonathan Hodgkin and encode proteins predicted to act in surface glycosylation.
  • mutations in bus-2 and bus- 12 reduce the adhesion of the bacterial pathogen Microbacterium nematophilum.
  • bus-2 mutants there is an increased exposure on the cuticle of fucosyl glycan compared with N2 worms [PMID: 17339204, 20385555].
  • coniospora would therefore be that fucosyl glycans are a target for spores binding.
  • transcription of antimicrobial peptide genes increases markedly.

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Abstract

La présente invention concerne des procédés et des compositions destinés lutter contre les nématodes. En particulier, la présente invention se rapporte à un agent capable d'inhiber l'expression ou l'activité d'au moins un gène sélectionné dans le groupe constitué de bus-12, bus-2, C14H10.3, C26B9.3, cpt-6, dpy-20, F35H12.5, K06A9.1, K08E3.5, sdc-2, tkt-1, Y38C1AB.5, et ykt-6, pour une utilisation dans un procédé de lutte contre les nématodes.
PCT/EP2014/062038 2013-06-10 2014-06-10 Procédés et compositions de lutte contre les nématodes WO2014198729A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007089632A2 (fr) * 2006-01-27 2007-08-09 The University Of North Carolina At Chapel Hill Vecteurs chimères de liaison à l'héparine et au sulfate d'héparane
WO2011082217A2 (fr) * 2009-12-30 2011-07-07 Divergence, Inc. Compositions et méthodes de lutte contre le nématode des lésions racinaires compositions and methods for the control of root lesion nematode
EP2385129A1 (fr) * 2010-05-03 2011-11-09 BASF Plant Science Company GmbH Procédés améliorés pour la régulation génétique chez les plantes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007089632A2 (fr) * 2006-01-27 2007-08-09 The University Of North Carolina At Chapel Hill Vecteurs chimères de liaison à l'héparine et au sulfate d'héparane
WO2011082217A2 (fr) * 2009-12-30 2011-07-07 Divergence, Inc. Compositions et méthodes de lutte contre le nématode des lésions racinaires compositions and methods for the control of root lesion nematode
EP2385129A1 (fr) * 2010-05-03 2011-11-09 BASF Plant Science Company GmbH Procédés améliorés pour la régulation génétique chez les plantes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHRISTINA M. TAYLOR ET AL: "Targeting Protein-Protein Interactions for Parasite Control", PLOS ONE, vol. 6, no. 4, 27 April 2011 (2011-04-27), pages e18381, XP055083202, DOI: 10.1371/journal.pone.0018381 *
OLA BILLING ET AL: "A Directed RNAi Screen Based on Larval Growth Arrest Reveals New Modifiers of C. elegans Insulin Signaling", PLOS ONE, vol. 7, no. 4, 12 April 2012 (2012-04-12), US, pages e34507, XP055048840, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0034507 *

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