Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/64—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with three nitrogen atoms as the only ring hetero atoms
  • A01N43/647—Triazoles; Hydrogenated triazoles
  • A01N43/653—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles

Definitions

• the present invention relates generally to the use of the compound of the formula (I) for controlling nematodes in nematode resistant crops and to methods particularly useful for controlling nematodes
• the compound of the formula (I) is defined to be the compound of the formula (I)
• the compound of the formula (I) has a chiral sulphoxide group so that it forms two enantiomers having
• the separation may take place, for example, on a Daical Chiralpak AD-H 250 mm x 30 mm column using a mobile phase of n-heptane/ethanol/methanol 60:20:20 (v/v/V), a flow rate of 30 ml/min and UV detection at 220 nm.
• the two enantiomers can then be characterized by methods known from the literature, for example by X-ray structural analysis or by determining the optical rotation. Further, the two enantiomers can be synthesized in enantiomerically pure form as well as in enantiomerically enriched form by the process as described in WO 2011/006646.
• the present invention relates generally to the use of the racemate or the R enantiomer (compound (I-A)) or S enantiomer (compound (I-B)) , or a mixture of R- and S -enantiomer of the compound of the formula (I) for controlling nematodes in nematode resistant crops and to methods particularly useful for controlling nematodes and/or increasing crop yield in those crops. If a mixture of R- and S-enantiomer is present, the ratio of the two enantiomers can range from 50.5:49.5 to 99.5:0.5 (R):(S)enantiomer.
• the present invention further relates to the use of a mixture ranging from 50.5:49.5 to 99.5:0.5 (R) : ( S) enanti omer for controlling nematodes in nematode resistant crops.
• the compound of the formula (I) is already known (see WO 1999/055668, WO 2006/043635, WO 2011/006605, WO 2011/006603, WO 2011/006646, WO 2011/006646). Its insecticidal and acaricidai and nematicidal activity as well as its manufacturing process already have been described.
• Nematodes are tiny, worm-like, multicellular animals adapted to living in water. The number of nematode species is estimated at ha! f a million. An important part of the soil fauna, nematodes live in a maze of interconnected channels, called pores, that are formed by soil processes. They move in the films of water that cling to soil particles. Plant nematodes encompass plant parasitic nematodes and nematodes living in the soil. Plant-parasitic nematodes, a majority of which are root feeders, are found in association with most plants. Some are endoparasitic, living and feeding within the tissue of the roots, tubers, buds, seeds, etc. Others are ectoparasitic, feeding externally through plant walls.
• Endoparasitic root feeders include such economically important pests as the root-knot nematodes (Meloidogyne species), the reniform nematodes (Rotylenchuius species), the cyst nematodes ⁇ Heterodera species), and the root-lesion nematodes (Pratylenchus species).
• Direct feeding by nematodes can drastically decrease a plant's uptake of nutrients and water.
• Nematodes have the greatest impact on crop productivity when they attack the roots of seedlings immediately after seed germination.
• Nematode feeding also creates open wounds that provide entry to a wide variety of plant-pathogenic fungi and bacteria. These microbial infections are often more economically damaging than the direct effects of nematode feeding.
• nematode resistance is characterized by host plant cell death at or nearby the feeding site of the parasitic nematode. Particular resistance genes and nematode interaction influence the timing and localization of the resistance response. Williamson et al. (Trends in Genetics, Vol. 22, No.7, July 2006) describes the nature and mechanisms of plant-nematode interactions with respect to resistance in plants. Nematode-resistant plants can be related to three main approaches being nematode targets, nematode - crop interface and plant response.
• Antifeedant or nematicidal proteins, disruption of essential nematode gene expression by RNA interference, disruption of sensory function by RNA interference, peptides or plantibodies or nematicidal metabolites are examples for nematode targets; disruption of nematode pathogenicity factors regarding migration and invasion or regarding feeding site induction and maintenance by RNA interference, peptides or plantibodies, stealth or repellant plants; or the conversion of host plants to non-host plants are examples for nematode-crop interface while plant resistance gene or hypersensitive response activation by nematode invasion; Induced cell death or other site incompatibility by feeding site specific promoters or conversion of crops to tolerance are examples for plant response.
• nematode-resistant plants are described to be resistant towards specific nematodes, there is still some interactions between the nematode and the crop which, due to the different defense reactions of the plant, might lead to a partially impaired plant.
• One example of these defense reactions is the hypersensitive response.
• One consequence might result in impaired roots and loss of vigor of the affected plants .
• This invention now provides advantageous uses of the compound of the formula (I),formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) for controlling nematodes infesting nematode resistant crops and/or increasing yield. Accordingly, the present invention also relates to the use of compositions comprising the compound of the formula (I) for controlling nematodes infesting nematode resistant crops and/or increasing yield.
• the present invention also relates to the use of compositions comprising the compound of formula (I-A) for controlling nematodes infesting insect resistant crops. Accordingly, the present invention also relates to the use of compositions comprising the compound of formula (I-B) for controlling nematodes infesting insect resistant crops.
• the present invention also relates to the use of compositions comprising a mixture of the compounds of formula (I-A) and (I-B) for controlling nematodes infesting insect resistant crops ranging from 50.5:49.5 to 99.5:0.5. Accordingly, the present invention also relates to the use of compositions comprising
• An exemplary method of the invention comprises applying a the compound of the formula (I), formula (I-A), (I-B) or of a mixture of the compounds of formula (I-A) and (I-B) of the invention to either soil or a plant (e.g., seeds or foliarly) to control nematode damage and/or increase crop yield.
• a plant e.g., seeds or foliarly
• the present invention is drawn to compositions and methods for regulating pest resistance or tolerance in plants or plant cells.
• resistance is intended that the pest (e.g., insect or nematode) is killed upon ingestion or other contact with the plant or parts thereof.
• tolerance is intended an impairment or reduction in the movement, feeding, reproduction, or other functions of the pest.
• Methods for measuring pesticidal activity are well known in the art. See, for example, C zap I a and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Patent No. 5,743,477, all of which are herein incorporated by reference in their entirety.
• controlling denotes a preventive or curative reduction of the insect or nematode infestation in comparison to the untreated crop, more preferably the infestation is essentially repelled, most preferably the infestation is totally suppressed.
• pestally-effective amount an amount of the pesticide that is able to bring about death to at least one pest, or to noticeably reduce pest growth, feeding, or normal physiological development e.g.(retarding the growth or reproduction of nematodes, reducing a nematode population) and/or reducing damage to plants caused by nematodes.
• the present invention also relates to a method for the protection of seed and germinating plants, or plant from attack by pests, by selectively applying pesticidal agents to the seed of a transgenic plant.
• Pesticidal agents include chemical or biological control agents compositions applied to the seed of the transgenic plant, wherein the agent is intended to provide protection of the plant or seed thereof against damage caused by one or more plant pests. Furthermore, the invention relates to seed which has been treated with a pesticidal agent as described herein.
• a pesticidal agent to the seed of a transgenic plant results in an improved resistance or tolerance to one or more plant pests and/or improved yield r vigor compared to a transgenic plant cultivated from a seed not treated with a pesticidal agent as described herein, or a plant of the same species as the referenced transgenic plant that has been cultivated fr m a seed treated with a pesticidal agent as described herein but that lacks the transgene (either of which may be herein referred to as a "control" plant).
• treatment of the seed with these agents not only protects the seed itself, but also the resulting plants after emergence, from pests. In this manner, the immediate treatment of the crop at the time of sowing or shortly thereafter can be dispensed with.
• a mixture of compounds (I-A) and (I-B) can range from 50.5:49.5 to 99.5:0.5 (I-A): ( I-B). In a further preferred embodiment, a mixture of compounds (I-A) and (I-B) can range from 60:40 to 95:5 (I-A): (I-B). In an even further preferred embodiment, a mixture of compounds (I-A) and (I-B) can range from 75:25 to 90: 10 (I-A): (I-B).
• the methods according to the present invention have been found to provide a greater degree of plant vigor and yield in nematode, insect and fungal infested environments than would be expected from application of a biological or chemical control agent or the presence of an insect or nematode control gene alone. At least some of the insect control agents within the scope of the present invention have been shown to provide increased root mass even in the absence of insect pressure which increased root mass leads to improved establishment of the beneficial bacteria within the rhizosphere which, in turn, reduces overall losses in crop vigor and yields caused by either plant parasitic nematodes, insects or fungi.
• compositions of the present invention have been formulated to provide a stable environment for living biological control agents such as spore- forming, root-colonizing bacteria.
• Various additives may be added to each inventive composition depending on the desired properties for a final formulation which has the necessary physical and chemical stability to produce a commercially viable product.
• Nematode resistant / tolerant plants can be plants obtained by breeding and conventional propagation methods which can be assisted o supplemented e.g by one or more of the following methods use of double haploids, protoplast fusion, random and directed mutagenesis, molecular or genetic markers or by bioengineering and genetic engineering methods, including transgenic plants and including the plant varieties. Plants of the plant cultivar/vari eti e s or hybrids which are in each case commercially available or in use can be treated according to the invention.
• plant species and plant cultivars/varieties obtained by breeding, such as crossing or protoplast fusion or marker-assistant molecular breeding , and parts thereof are treated.
• Nematode resistance or tolerance can be introduced into plants by various technologies known to a person skilled in the art.
• An additional possibility is to support breeding by the use of markers, RAPDs (Randomly Amplified Polymorphi ON A ), AFLPs (Amplified Fragment Length Polymorphisms), or SSRs (Simple Sequence Repeats) that are associated with a fragment of DNA that co-segregates with the resistance trait in crossesof plants comprising a nematode resistance or tolerance.
• the mapped endogenous nematode resistant genes can be introgressed in other plants by e.g. crossing and back-crossing.
• a further possibility is to introduce nematode resistance or tolerance by genetic engineering leading to nematode resistant or tolerant transgenic plants and plant cultivars. If appropriate genetic engineering can be used in combination with conventional breeding methods.
• Genetically modified plants (or transgenic plants) are plants in which a foreign nucleic acid molecule or foreign nucleic acid molecules has/have been integrated into the genome.
• a foreign nucleic acid molecule means a nucleic acid molecule provided or assembled outside the plant and when introduced into the nuclear, chloroplastic or mitochondrial genome gives the transformed plant new properties by e.g.
• Methods for downregulating genes in a plant are known to a person skilled in the art and comprise but are not limited to antisense technology, cosuppression technology or RNA interference - RNAi - technology.
• the method of treatment according to the invention can be used in the treatment of transgenic plants or seeds.
• the transgenic plants or plant cultivars i.e. those obtained by genetic engineering
• which can be treated according to the invention can be any plant, preferably it is a cultivated plant, which can be cultivated for use as food, feed or industrial processes.
• RNA interference also referred to as gene silencing
• RNAi RNA interference
• a number of models have been proposed for the action of RNAi.
• 6,506,559 discloses that in nematodes, the length of the (ds) RNA corresponding to the target gene sequence may be at least 25, 50, 100, 200, 300, or 400 bases, and that even larger dsRNAs were effective at inducing R Ai in C. elegans.
• a dsRNA is expressed in a plant, a nematode feeding on this plant will incorporate the dsRNA. If this dsRNA will block the expression of an essential nematode gene then the nematode is expected to suffer and die.
• a plant expressing such dsRNA interfering with a nematode gene is also understood in connection with the present invention to be a nematode resistant or tolerant plant.
• Plants and plant cultivars which are preferably to be treated according to the invention include all plants which have genetic material which impart particularly advantageous, useful traits to these plants (whether obtained by breeding and/or biotechnological means). Plants and plant cultivars which are preferably to be treated according to the invention include all plants which have genetic material which impart particularly advantageous, useful traits to these plants (whether obtained by breeding and or biotechnological means).
• the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) is particularly useful in controlling plant-parasitic nematodes in plants carrying one or more of the genes listed in Table 1.
• the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) in combination with at least one agrochemically active compound is particularly useful in controlling plant-parasitic nematodes in plants carrying one or more of the genes listed in Table 1.
• the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) in combination with nematicidal biological control agent is particulaxly useful in controlling plant-parasitic nematodes in plants carrying one or more of the genes listed in Table 1.
• nucleotide and amino acid sequence information for each of these genes are represented by the SEQ ID NOs listed in columns 4 and 5 of Table 1 with respect to the United States Patent Application Serial No. listed in column 2 of Table 1.
• compound (I) is particularly useful in controlling plant-parasitic nematodes in plants carrying one or more of the genes listed in Table 1.
• compound (I-A) is particularly useful in controlling plant-parasitic nematodes in plants carrying one or more of the genes listed in Table 1.
• Compound (I-B) is particularly useful in controlling plant-parasitic nematodes in plants carrying one or more of the genes listed in Table 1.
• the compound of the formula (I) formula (I-A), ( I-B ) or a mixture of the compounds of formula (I-A) and (I-B) in combination with at least one agrochemically active compound is particularly useful in controlling plant-parasitic nematodes in plants carrying one or more of the genes listed in Table 1.
• WO201 1023571A1 WO201 1/082217A2, WO2010060162A1, WO2010/027809A1,
• WO2010147879A1 WO2010/027805 A2, WO2010/027805A3, WO2010/027804 A2,
• the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) - or the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) - in combination with at least one agrochemically active compound and/or a nematicidal biological control agent is particularly useful in controlling plant-parasitic nematodes in plants carrying one or more of the following genes Hsl pro ⁇ !
• SYV46 cry5 cry6 cryl2, cryl3, cryl4, cry21 cry5B, cry6A, cry 12 A, cry 14 A, cry21A, Cry55, GmBAG6, GmAP2, CLAVAT A 3/E SR .
• the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) - or the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) - in combination with at least one agrochemically active compound and/or a nematicidal biological control agent is particularly useful in controlling plant-parasitic nematodes in plants containing natural nematode resistant/tolerant genes. Examples for such plant are varieties of soybean have been bred to express a characteristic in the plant which reduces damage due to the soybean cyst nematode (SCN).
• Soybean genetic resistance to SCN have been found in various resistant sources, for example, Plant Introduction (PI) lines PI88788, PI548402, PI437654, PI90763, PI209332, PI89882 and PI548316. These indictor lines are suitable for use as the source of resistance in breeding programs against SCN. Further example are varieties of soybean expressing characteristics associated with resistance to Southern Root Knot Nematode (SRKN, US2009064354) or are cotton plants comprising root knot nematode resistance as described in US2011173713.
• PI Plant Introduction
• a preferred embodiment comprises the nematode-resistant plant as described above treated with the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and
• a preferred embodiment comprises the nematode-resistant plant as described above treated with the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) in combination with at least one agrochemically active compound and/or nematicidal biological control agent.
• compositions and methods of the present invention comprise treatment of a transgenic plant comprising one or more of the genes listed in Table 1 with the compound of the formula (I), formula (I-A), (I-B) or of a mixture of the compounds of formula (I-A) and (I-B) - or the compound of the formula (I) formula (I-A), (I-B) or of a mixture of the compounds of formula (I-A) and (I-B) in combination with at least one agrochemically active compound and/or nematicidal biological control agent.
• the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) - or the compound of the formula (I) formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and ( I-B) in combination with at least one agrochemically active compound andor nematicidal biological control agent - is applied to the seed of the transgenic plant comprising one or more of the genes listed in Table 1 , including biologically-active variants and fragments thereof.
• An exemplary method of the invention comprises applying the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) - or the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and ( I-B) in combination with at least one agrochemically active compound and/or nematicidal biological control agent of the invention to propagation material (e.g seeds) of plants to combat nematode damage and/or increase crop yield.
• agrochemically active compound and/or nematicidal biological control agent of the invention to propagation material (e.g seeds) of plants to combat nematode damage and/or increase crop yield.
• a further exemplary method o the invention comprises applying the compound of the formula (I) formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) - or the compound of the formula (I) formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) in combination with at least one agrochemically active compound and/or nematicidal biological control agent to either soil or a plant (e.g. foliarly) to combat nematode damage and/or increase crop yield.
• agrochemically active compound and/or nematicidal biological control agent to either soil or a plant (e.g. foliarly) to combat nematode damage and/or increase crop yield.
• the nematicidal active ingredient is the compound of the formula (I), formula (I-A), (I-B) or a mixture of the compounds of formula (I-A) and (I-B) and the nematode resistant crop comprises a transgenic plant comprising Axmi031 or Axn2 (Table 1).
• the transgenic plant is homozyguous with respect to the exogeneous gene of Table 1.
• the transgenic plant is hemizyguous with respect to the exogeneous gene o Table 1.
• the nucleotide and amino acid SEQ ID NOs listed in Table 1 are exemplary sequences and do not limit the scope of the invention.
• the invention encompasses plants and plant parts, including plant cells and seed, comprising one or more of the genes listed in column 1 of Table 1.
• the invention encompasses plants and plant parts, including plant cells and seed, comprising one or more nucleotide sequences listed in column 4 of Table 1.
• the invention encompasses plants and plant parts, including plant cells and seed, comprising one or more nucleotide sequences encoding one or more of the polypeptides listed in column 5 of Table 1.
• the invention encompasses plants and plant parts, including plant cells and seed, comprising one or more nucleotide sequences encoding a biologically-active variant or fragment of the amino acid sequenc e(s) listed in column 5 of Table 1.
• a fragment of a nucleotide sequence that encodes a biologically active portion of a pesticidal protein of the invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 contiguous amino acids, or up to the total number of amino acids present in a full-length pesticidal protein listed in Table 2 herein.
• Such biologically active portions can be prepared by recombinant techniques and evaluated for pesticidal activity.
• the fragment is a proteolytic cleavage fragment.
• the proteolytic cleavage fragment may have an N-terminal or a C -terminal truncation of at least about 100 amino acids, about 120, about 130, about 140, about 150, or about 160 amino acids relative to the amino acid sequence listed in Table 2.
• the fragments encompassed herein result from the removal of the ( ' -terminal crystallization domain, e.g., by proteolysis or by insertion of a stop codon in the coding sequence.
• Preferred pesticidal proteins of the present invention are encoded by a nucleotide sequence sufficiently identical to the nucleotide sequence(s) listed in Table 2, or are pesticidal proteins that are sufficiently identical to the amino acid sequence(s) listed in Table 2.
• “sufficiently identical” is intended an amino acid or nucleotide sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%o, 98%, 99%) or greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters.
• these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
• the sequences are aligned for optimal comparison purposes.
• the two sequences are the same length.
• the percent identity is calculated across the entirety of the reference sequence (e.g.., a sequence listed in Table 2).
• the percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
• a gap i.e. a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues.
• the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
• a nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karl in and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karl in and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
• Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.
• Gapped BLAST in BLAST 2.0
• PS I -Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra.
• the default parameters of the respective programs e.g., BLASTX and BLASTN
• Alignment may also be performed manually by inspection.
• ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence.
• the ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed.
• GENEDOCTM A non-limiting example of a software program useful for analysis of ClustalW alignments.
• GENEDOCTM (Karl Nicholas) allows assessment of amino acid (or DNA) similarity and identity between multiple proteins.
• Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4: 11 -17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA).
• ALIGN program version 2.0
• a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
• GAP Version 10 which uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used to determine sequence identity or similarity using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity or % similarity for an amino acid sequence using GAP weight of 8 and length weight of 2, and the BLOSUM62 scoring program. Equivalent programs may also be used.
• Equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
• “Variants” of the amino acid sequences listed in Table 2 include those sequences that encode the pesticidal proteins disclosed herein but that differ conservatively because of the degeneracy of the genetic code as well as those that are sufficiently identical as discussed above.
• Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below.
• Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site -directed mutagenesis but which still encode the pesticidal proteins disclosed in the present invention as discussed below.
• variant isolated nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.
• conservative amino acid substitutions may be made at one or more, predicted, nonessential amino acid residues.
• a “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a pesticidal protein without altering the biological activity, whereas an "essential” amino acid residue is required for biological activity.
• a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
• amino acids with basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
• nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
• beta-branched side chains e.g., threonine, valine, isoleucine
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
• amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related toxins to the sequences of the invention (e.g., residues that are identical in an alignment of homologous proteins).
• residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the invention (e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins).
• residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the invention e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins.
• residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the invention e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins.
• residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the invention e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins.
• one of skill in the art
• variant nucleotide sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ability to confer pesticidal activity to identify mutants that retain activity.
• the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques. Using methods such as PC R. hybridization, and the like corresponding pesticidal sequences can be identified, such sequences having substantial identity to the sequences of the invention. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY).
• Variant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenic and recombinogenic procedures such as ON A shuffling. With such a procedure, one or more different pesticidal protein coding regions can be used to create a new pesticidal protein possessing the desired properties, in this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
• sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene of the invention and other known pesticidal genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity.
• Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751 ; Stemmer (1994) Nature 370:389-391 ; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc.
• Domain swapping or shuffling is another mechanism for generating altered pesticidal proteins. Domains may be swapped between pesticidal proteins, resulting in hybrid or chimeric toxins with improved pesticidal activity or target spectrum. Methods for generating recombinant proteins and testing them for pesticidal activity are well known in the art (see, for example, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd et al.
• Variants and fragments of the proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, pesticidal activity.
• By "retains activity” is intended that the variant will have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the pesticidal activity of the native protein.
• Methods for measuring pesticidal activity are well known in the art. See, for example, C/apla and Lang (1990) J. Econ. Entomol. 83 : 2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J.
• agrochemically active compounds are to be understood as meaning all substances which are or may be customarily used for treating plants.
• Fungicides, bactericides, insecticides, acaricides, nematicides, moUuscicides, safeners, plant growth regulators and plant nutrients as well as biological control agents may be mentioned as being preferred.
• plants and plant parts can be treated.
• plants are meant all plants and plant populations such as desirable and undesirable wild plants, cultivars and plant varieties (whether or not protectable by plant variety or plant breeder's rights).
• Cultivars and plant varieties can be plants obtained by conventional propagation and breeding methods which can be assisted or supplemented by one or more biotechnological methods such as by use of double haploids, protoplast fusion, random and directed mutagenesis, molecular or genetic markers or by bioengineering and genetic engineering methods.
• plant parts are meant all above ground and below ground parts and organs of plants such as shoot, leaf, blossom and root, whereby for example leaves, needles, stems, branches, blossoms, fruiting bodies, fruits and seed as well as roots, tubers, corms and rhizomes are listed.
• Crops and vegetative and generative propagating material for example cuttings, corms, rhizomes, tubers, runners and seeds also belong to plant parts.
• the transgenic plants or plant cultivars i.e. those obtained by genetic engineering
• which can be treated according to the invention include also all plants which - besides nematode resistant / tolerant traits contain other genetic modifications, received genetic material which imparted particularly other advantageous useful traits to these plants.
• the treatment according to the invention may also result in superadditive (“synergistic") effects.
• superadditive for example, reduced application rates and/or a widening of the activity spectrum and/or an increase in the activity of the active compounds and compositions which can be used according to the invention, better plant growth, higher harvest yields, bigger fruits, larger plant height, greener leaf color, higher quality higher sugar concentration within the fruits, better storage stability and/or processability of the harvested products are possible, which exceed the effects which were actually to be expected.
• the active compound combinations according to the invention may also have a strengthening effect in plants. Accordingly, they are also suitable for mobilizing the defense system of the plant against attack by unwanted microorganisms. This may, if appropriate, be one of the reasons of the enhanced activity of the combinations according to the invention, for example against fungi.
• Plant-strengthening (resistance-inducing) substances are to be understood as meaning, in the present context, those substances or combinations of substances which are capable of stimulating the defense system of plants in such a way that, when subsequently inoculated with unwanted microorganisms, the treated plants display a substantial degree of resistance to these microorganisms, in the present case, unwanted microorganisms are to be understood as meaning phytopathogenic fungi, bacteria and viruses.
• the substances according to the invention can be employed for protecting plants against attack by the abovementioned pathogens within a certain period of time after the treatment.
• the period of time within which protection is effected generally extends from 1 to 10 days, preferably 1 to 7 days, after the treatment of the plants with the active compounds.
• Plants and plant cultivars which are preferably to be treated according to the invention include all plants which have genetic material which impart particularly advantageous, useful traits to these plants (whether obtained by breeding and/or biotechnological means).
• Plants and plant cultivars which are also preferably to be treated according to the invention are - besides nematode resistance / tolerance - resistant against one or more biotic stresses, i.e. said plants show a better defense against animal and microbial pests, such as against insects, mites, phytopathogenic fungi, bacteria, viruses and/or viroids.
• Plants and plant cultivars which may also be treated according to the invention are those plants whichare - besides nematode resistance / tolerance - resistant to one or more abiotic stresses.
• Abiotic stress conditions may include, for example, drought, cold temperature exposure, heat exposure, osmotic stress, flooding, increased soil salinity, increased mineral exposure, ozone exposure, high light exposure, limited availability of nitrogen nutrients, limited availability of phosphorus nutrients, shade avoidance.
• Plants and plant cultivars which may also be treated according to the invention are those plants characterized - besides nematode resistance / tolerance - by enhanced yield characteristics. Increased yield in said plants can be the result of, for example, improved plant physiology, growth and development, such as water use efficiency, water retention efficiency, improved nitrogen use, enhanced carbon assimilation, improved photosynthesis, increased germination efficiency and accelerated maturation.
• Yield can furthermore be affected by improved plant architecture (under stress and non- stress conditions), including but not limited to, early flowering, flowering control for hybrid seed production, seedling vigor, plant size, internode number and distance, root growth, seed size, fruit size, pod size, pod or ear number, seed number per pod or ear, seed mass, enhanced seed filling, reduced seed dispersal, reduced pod dehiscence and lodging resistance.
• Further yield traits include seed composition, such as carbohydrate content, protein content, oil content and composition, nutritional value, reduction in anti-nutritional compounds, improved processability and better storage stability. Plants and plant cultivars which may also be treated according to the invention, are those plants characterized- besides nematode resistance / tolerance - by enhanced yield characteristics.
• Increased yield in said plants can be the result of, for example, improved plant physiology, growth and development, such as water use efficiency, water retention efficiency, improved nitrogen use, enhanced carbon assimilation, improved photosynthesis, increased germination efficiency and accelerated maturation.
• Yield can furthermore be affected by improved plant architecture (under stress and non-stress conditions), including but not limited to, early flowering, flowering control for hybrid seed production, seedling vigor, plant size, internode number and distance, root growth, seed size, fruit size, pod size, pod or ear number, seed number per pod or ear, seed mass, enhanced seed filling, reduced seed dispersal, reduced pod dehiscence and lodging resistance.
• Further yield traits include seed composition, such as carbohydrate content, protein content, oil content and composition, nutritional value, reduction in anti-nutritional compounds, improved processability and better storage stability.
• Table A Non-exclusive list of transgenic plants and events for the design of experiments with the compound of formula (I) related to the invention (source: ( AG BIOS. P.O. Box 475, 106 St. John St. Merrickville, Ontario 0G1N0, CANADA) accessible under: http://www.agbios.com/dbase.php.
• A-l ASR-368 Scotts Seeds Glyphosate tolerance derived Agrostis US 2006- by inserting a modified 5- stolonifera 162007 enolpyruvylshikimate-3 - Creeping
• EPSPS phosphate synthase
• EPSPS 5- enolypyruvyl shikimate -3 - phosphate synthase
• Acetylated PPT is
• Acetylated PPT is
• Acetylated PPT is
• MS lines contained
• NS738 contains the P2
• HN28 CropScience acetyltransferase (PAT) napus (Argen
• Acetylated PPT is
• EPSPS phosphate synthase
• A-31 A, B Agritope inc.
• SAM S- Cucumis adenosylmethionine
• Melon melo
• EPSPS modified 5- enolpyruvylshikimate-3 - phosphate synthase
• Virus leader > ribulose 1 ,5- biphosphate carboxylase
• virus TPotp Y coding sequence of an optimized
• h3At first intron of gene 11 of
• the aad-12 gene (originally
• AAD-12 dioxygenase
• cry2Ab gene from B. thuringiensis subsp.
• Seeds, Inc. produced by inserting the hirsutum 130175, vip3A(a) gene from Bacillus L. (Cotton) WO2004039 thuringiensis AB 88.
• the 986, US APH4 encoding gene from E. 2010298553 coii was introduced as a
• VIP3A Syngenta Insect resistance
• VIP3 Cot202 Syngenta insect resistance
• Seeds, Inc. produced by inserting a full- hirsutum
• insects from MON15985 are insects from MON15985.
• EPSPS 5- enolypyruwl shikimate -3 - phosphate synthase
• Agrobacterium tumefaciens ;
• A- 104 B Da. F Zeneca Seeds Delayed softening tomatoes Lycopersicon produced by inserting a esculentum ( truncated version of the Tomato) polygalacturonase (PG)
• A-108 Vector Vector Reduced nicotine content Nicotiana A-108 Vector Vector Reduced nicotine content Nicotiana
• A- 120 Kefeng CHINA NAT Transgenic rice Kefeng 6 is a Oryza CN
• ATBT04 subsp. Tenebrionis.
• SEMT15 from Bacillus thuringiensis
• EPSPS enolpvruvylshikimate-3 - phosphate synthase
• A- 143 B16 Dekalb Glufosinate ammonium Zea mays

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