Patent Application
20210207161
This invention relates to transgenic soybean plants, plant material and seeds, characterized by harboring a specific transformation event conferring nematode resistance and herbicide tolerance, at a specific location in the soybean genome, treated with compounds and/or biological control agents or mixtures as described herein. The invention also relates to seeds treated with compounds and/or biological control agents or mixtures as described herein, and to methods to improve yield in soybean comprising at least the elite event as described, wherein the soybean plants or seeds, or the soil in which soybean plants or seeds are grown or are intended to be grown, are treated with the compounds and/or biological control agents or mixtures as described herein. The soybean plants of the invention combine the nematode resistance and herbicide tolerance phenotype with an agronomic performance, genetic stability and functionality in different genetic backgrounds equivalent to the corresponding non-transformed soybean genetic background in the absence of HPPD inhibitor herbicide(s) or nematode infestation.
The phenotypic expression of a transgene in a plant is determined both by the structure of the gene or genes itself and by its or their location in the plant genome. At the same time the presence of the transgenes or “inserted T-DNA” at different locations in the genome will influence the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of a commercially interesting trait in a plant by genetic manipulation can be a lengthy procedure dependent on different factors. The actual transformation and regeneration of genetically transformed plants are only the first in a series of selection steps, which include extensive genetic characterization, introgression, and evaluation in field trials, eventually leading to the selection of an elite event.
The unequivocal identification of an elite event is becoming increasingly important in view of discussions on Novel Food/Feed, segregation of GMO and non-GMO products and the identification of proprietary material. Ideally, such identification method is both quick and simple, without the need for an extensive laboratory set-up. Furthermore, the method should provide results that allow unequivocal determination of the elite event without expert interpretation, but which hold up under expert scrutiny if necessary.
Planting nematode resistant and herbicide tolerant soybean EE-GM5 varieties provides growers with new options for nematode and weed control, using HPPD inhibitor herbicides such as isoxaflutole (IFT), topramezone or mesotrione (MST) herbicide. HPPD inhibitor herbicides offer an alternative weed control option for the soybean grower to help manage problem weed species and as an alternative mode of action tool to help slow the spread of herbicide resistant weeds.
Soybean cyst nematode (SCN) Heterodera glycines (Ichinohe), a worldwide problem for soybean production, is a continuing threat to producers. Since its first detection in the US in 1954 from a single county in North Carolina, SCN has spread to nearly every soybean-producing state in the United States and is estimated to cause more than $1.2 billion in annual yield losses in the US, making it the most damaging soybean pathogen there. SCN was first detected in Brazil in the early 1990s and has since spread throughout South America, and is one of the most important pathogens in Brazil causing losses in practically all Brazilian growing regions. Similarly, SCN continues to spread across soybean producing regions of China with detection in 15 provinces and yield loss estimates of more than $120 million. A multi-year study in the state of Iowa, USA (2001 to 2015) where almost all SCN-resistant soybean varieties contain SCN resistance from PI 88788, found that the virulence of SCN populations increased over the years, resulting in increased end-of-season SCN population densities and reduced yields of SCN-resistant soybean varieties with the PI88788 source of resistance (Mitchum (2016), Phytopathology 106(12):1444-1450, Allen et al. (2017) Plant Health Progr. 18:19-27, Arias et al. (2017) on the world wide web at researchgate.net/publication/266907703_RESISTANCE_TO_SOYBEAN_CYST_NEMATO DE_GENETICS_AND_BREEDING_IN_BRAZIL; McCarville et al. (2017) Plant Health Progress 18:146-155).
The root lesion nematode Pratylenchus brachyurus has become an increasingly important pathogen of soybean. It has a broad host range and is widely distributed in tropical and subtropical regions, especially in Brazil, Africa, and the Southern United States. Pratylenchus brachyurus has become a concern among cotton and soybean growers in the Brazilian Cerrado region and is considered the main nematode pathogen of soybean in the region. In soybean, this nematode can reduce yields 30 to 50%, with greater damage being observed on sandy soils.
The use of resistant soybean varieties would be the best way to control this nematode, however, P. brachyurus-resistant soybean varieties have not been identified to date. Although several soybean genotypes have been studied for Pratylenchus brachyurus resistance, and some cultivars identified with increased tolerance, breeding resistant cultivars against P. brachyurus is difficult due to the fact that this nematode is polyphagous and lacks a close interaction with its hosts (Machado (2014) Current Agricultural Science and Technology 20:26-35; Antonio et al. (2012) Soil productivity losses in area infested by the nematoid of the root lesions in Vera, MT. In: Brazilian Congress of Soy, 6, 2012, Cuiaba. Abstracts. Londrina: Embrapa Soja, 4pp; Rios et al. (2016) Ciencia Rural 46:580-584; Lima et al., 2017, Chapter 6 in the book: Soybean—The Basis of Yield, Biomass and Productivity; Edited by Minobu Kasai, ISBN 978-953-51-3118-2, Print ISBN 978-953-51-3117-5, InTech; Inomoto et al. (2011) Sucessão de culturas sob pivô central para controle de fitonematoides: variação populacional, patogenicidade e estimativa de perdas. Tropical Plant Pathology 36:178-185).
It is known that protecting plants against nematodes such as SCN can help plants to better cope with other stresses such as soil composition/content, weather conditions, pathogen stress, herbicide applications, etc. Particularly when such other stresses give a phenotype that is easily seen, such as chlorosis/yellowing of leaves, the effect of SCN control is more easy to see while otherwise often not “visible”. E.g., when soybean plants have Sudden Death Syndrome (SDS) or Iron Deficiency Chlorosis (IDC), protection from SCN will result in plants that are greener or have less severe SDS/IDC symptoms. Despite extensive research and variety screening efforts, iron deficiency remains a challenge in large soybean production areas in the North Central U.S. The importance of this problem has increased due to expanded soybean production on soils susceptible to iron deficiency and to possible interactions with cropping system changes. Iron deficiency occurs in soils with high pH and carbonates, but the expression of iron deficiency is highly variable in space due to interactions with spatially variable soil properties such as moisture content, salinity, availability of iron, and other micronutrient and metal concentrations. Further, iron deficiency expression interacts with biotic factors such as nitrogen fixation, pests, diseases and with management induced stresses such as herbicide application. Variety selection is the most important means to manage iron deficiency, but selecting varieties is complicated by a large genotype by environment interaction related to chlorosis tolerance (Hansen et al. (2004) Soil Sci. Plant Nutr. 50(7):983-987).
Sudden death syndrome (SDS) of soybean was first discovered in 1971 in Arkansas and since then has been confirmed throughout most soybean-growing areas of the USA. SDS is a fungal disease that also occurs in a disease complex with the soybean cyst nematode (SCN). SDS is among the most devastating soil-borne diseases of soybean in the USA. When this disease occurs in the presence of SCN, symptoms occur earlier and are more severe. SDS is caused by soil-borne fungi within a group of the Fusarium solani species complex. In North America, Fusarium virguliforme, formerly Fusarium solani f. sp. glycines, is the causal agent. In South America, F. brasiliense, F. cuneirostrum, F. tucumaniae, and F. virguliforme cause SDS symptoms. Although soybean cultivars that are less susceptible to SDS have been developed, no highly resistant cultivars are available. The fungus may infect roots of soybean seedlings soon after planting, but above ground symptoms of SDS rarely appear until soybean plants have reached reproductive stages. The fungus produces toxins in the roots that are translocated to the leaves. The first noticeable symptoms of SDS are yellowing and defoliation of upper leaves. If the disease develops early in the season, flowers and young pods will abort. When the disease develops later, the plant will produce fewer seeds per pod or smaller seeds. The earlier severe disease develops, the more the yield is reduced. Because the SDS fungus can persist in soil for long periods, larger areas of a field will show symptoms of the disease each growing season until most of the field is affected (Westphal et al. (2008). Sudden Death Syndrome of Soybean. The Plant Health Instructor. DOI:10.1094/PHI-I-2008-0102-01, on the world wide web at apsnet.org/edcenter/intropp/lessons/fungi/ascomycetes/Pages/SuddenDeath.aspx).
Currently, no soybean plants genetically engineered for nematode resistance are commercialized. Soybean plants comprising one or more herbicide tolerance genes have been disclosed in the art. WO2006/130436 describes a glyphosate tolerant soybean event comprising an epsps gene, and WO2011/034704 describes a dicamba-tolerant soybean event. WO2012/082548 describes soybean plants comprising both an hppd and pat gene. WO2011/063411 describes a soybean event with tolerance to HPPD inhibitors and glyphosate, while WO2011/063413 describes soybean plants with tolerance to HPPD inhibitors, glufosinate and glyphosate. WO2011/066384 describes a soybean event with tolerance to 2,4-D and glufosinate, while WO2012/075426 describes a soybean event with tolerance to 2,4-D, glufosinate and glyphosate and WO2017/059795 describes a soybean event with tolerance to glyphosate. WO2009/064652 describes a soybean event with resistance to lepidopteran insects, and WO2013/016527 describes a soybean event with resistance to lepidopteran insects and glufosinate tolerance.
HPPD genes and proteins that confer improved tolerance to HPPD inhibitor herbicides have been disclosed e.g., in WO2015138394, WO2015135881, WO2014043435, and nematicidal activity of Cry proteins has been described in, e.g., WO2010027805, WO2010027809, WO2010027804, WO2010027799, WO2010027808 and in WO2007147029.
None of the prior art disclosures teach or suggest an elite event in soybean comprising a nematode-active Cry gene, treated with the compounds and/or biological control agents or mixtures as described herein, and certainly not an elite event in soybean comprising a nematode-active Cry gene combined with a gene conferring tolerance to HPPD inhibitors, treated with the compounds and/or biological control agents or mixtures as described herein.
It is known in the art that getting a commercial elite transformation event in soybean plants with acceptable agronomic performance is by no means straightforward.
The present invention relates to a treated transgenic soybean plant, plant part, seed, cell or tissue thereof, or treated soil wherein a plant or seed is grown or is intended to be grown (followed by planting or sowing of said plant or seed in said soil), comprising, stably integrated into its genome, an expression cassette which comprises a nematode resistance gene comprising the coding sequence of the cry14Ab-1.b gene and a herbicide tolerance gene comprising the coding sequence of the hppdPf-4 Pa gene (both as described in Example 1.1 herein and as represented in SEQ ID No. 7 and 9, respectively), which provide resistance to plant parasitic nematodes such as soybean cyst nematode and tolerance to an HPPD inhibitor herbicides such as isoxaflutole, topramezone or mesotrione. In the absence of HPPD inhibitor herbicide and nematode pressure, such soybean plant has an agronomic performance which is substantially equivalent to the non-transgenic isogenic line. When encountering soybean cyst nematode (SCN) pressure affecting plant performance in the field, the plants of the invention will have a superior agronomic phenotype compared to a non-transgenic plant. Also, in the presence of weeds, after application of an HPPD inhibitor herbicide to which tolerance is provided, the plants of the invention will have a superior agronomic phenotype compared to plants that were not treated with herbicides. In one embodiment, the invention also relates to such plants or seeds treated with one or more of the compounds and/or biological control agents or mixtures as described herein, and to methods to improve yield in soybean comprising at least the elite event as described, wherein the soybean plants or seeds, or the soil in which soybean plants or seeds are grown or are intended to be grown, are treated with the compounds and/or biological control agents or mixtures thereof as described herein. In one embodiment, such treated plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
According to the present invention the soybean plant or seed, cells or tissues thereof comprise elite event EE-GM5 (also described herein as “the (elite) event of this invention”). In one embodiment, elite event EE-GM5 comprises the sequence of any one of SEQ ID No. 1, 3, 5, or 24, or the sequence of any one of SEQ ID No. 2, 4, 6, or 25, or any sequences essentially similar thereto. In one embodiment, EE-GM5 comprises the sequence of any one of SEQ ID No. 1, 3, 5 or 24 and the sequence of any one of SEQ ID No. 2, 4, 6, or 25, or any sequences essentially similar thereto, and the cry14Ab-1.b coding sequence of SEQ ID No. 7 and the hppdPf-4 Pa coding sequence of SEQ ID No. 9, or sequences essentially similar thereto. In one embodiment, elite event EE-GM5 is a T-DNA inserted at a specific position in the soybean genome, as is contained in reference seed deposited at the ATCC under deposit number PTA-123625. In one embodiment, such T-DNA in EE-GM5 comprises a chimeric Cry14Ab-1-encoding gene and an HPPD-4-encoding gene. In another embodiment, said event is characterized by the 5′ junction sequence of SEQ ID No. 1 or 3, or by the 3′ junction sequence of SEQ ID No. 2 or 4; or by the 5′ junction sequence of SEQ ID No. 1 or 3, and by the 3′ junction sequence of SEQ ID No. 2 or 4. In one embodiment, genomic DNA containing EE-GM5, when analyzed using a polymerase chain reaction (“PCR” herein) with two primers comprising the nucleotide sequence of SEQ ID No. 12 and SEQ ID No. 13 respectively, yields a DNA fragment of 85 bp. In one embodiment, genomic DNA containing EE-GM5, when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID No. 18 and SEQ ID No. 19 respectively, yields a DNA fragment of 84 bp. In one embodiment, the invention also relates to such plants or seeds treated with one or more of the compounds and/or biological control agents or mixtures thereof as described herein, particularly such plant or seeds also comprising one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
In one embodiment herein is provided a soybean plant, cell, plant part, seed or progeny thereof, each comprising elite event EE-GM5 in its genome, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-123625. In one embodiment, a plant or seed comprising EE-GM5 is obtainable by propagation of and/or breeding with a soybean plant grown from the seed deposited at the ATCC under deposit number PTA-123625. In one embodiment, the invention relates to such plants or seeds treated with one or more of the compounds and/or biological control agents or mixtures thereof as described herein, and in another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
More specifically, the present invention relates to a transgenic soybean plant, plant part, pollen, seed, cell or tissue thereof, the genomic DNA of which is characterized by the fact that, when analyzed in PCR as described herein, using at least two primers directed to the region formed by a part of the 5′ or 3′ T-DNA flanking region of EE-GM5 and part of the inserted T-DNA, a fragment is amplified that is specific for event EE-GM5. The primers may be directed against the 3′ T-DNA flanking region within SEQ ID NO: 6 or SEQ ID NO. 25 or soybean plant genomic DNA downstream thereof and contiguous therewith and the inserted T-DNA upstream thereof and contiguous therewith. The primers may also be directed against the 5′ T-DNA flanking region within SEQ ID NO: 5 or SEQ ID NO. 24 or soybean plant genomic DNA upstream thereof and contiguous therewith and the inserted T-DNA downstream of and contiguous therewith. In one embodiment, such primers comprise or consist (essentially) of the nucleotide sequence of SEQ ID NO: 12 and SEQ ID NO: 13, or of SEQ ID No. 18 and SEQ ID No. 19, or of SEQ ID NO. 26 and SEQ ID NO. 28, or of SEQ ID NO. 27 and SEQ ID NO. 29, respectively (e.g., a primer pair comprising a primer containing at its extreme 3′ end the nucleotide sequence of SEQ ID NO: 12 and a primer containing at its extreme 3′ end the nucleotide sequence of SEQ ID NO: 13, or a primer pair comprising a primer containing at its extreme 3′ end the nucleotide sequence of SEQ ID No. 18 and a primer containing at its extreme 3′ end the nucleotide sequence of SEQ ID No. 19, or a primer pair comprising a primer containing at its extreme 3′ end the nucleotide sequence of SEQ ID NO: 26 and a primer containing at its extreme 3′ end the nucleotide sequence of SEQ ID NO: 28, or a primer pair comprising a primer containing at its extreme 3′ end the nucleotide sequence of SEQ ID NO: 27 and a primer containing at its extreme 3′ end the nucleotide sequence of SEQ ID NO: 29), and yield a DNA fragment of between 50 and 1000 bp, such as a fragment of 85 bp or of 84 bp. Reference seed comprising the elite event of the invention (also referred to herein as EE-GM5) has been deposited at the ATCC under accession number PTA-123625. One embodiment of the invention is the elite event EE-GM5 as contained in seed deposited under accession number PTA-123625, which when introduced in a soybean plant will provide resistance to nematodes and tolerance to herbicides, particularly resistance to soybean cyst nematode (Heterodera glycines, “SCN” herein) and/or lesion nematode (lesion nematode as used herein refers to Pratylenchus spp. soybean pest nematodes, including but not limited to Pratylenchus brachyurus) and tolerance to HPPD inhibitors such as isoxaflutole, topramezone or mesotrione. The plants with EE-GM5 of this invention also control root knot nematode (root-knot nematode as used herein refers to Meloidogyne spp. soybean pest nematodes, including but not limited to Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne hapla, or Meloidogyne javanica, or any combination thereof), reniform nematode (Rotylenchulus reniformis) and Lance nematode (Hoplolaimus spp. such as H. columbus, H. galeatus, and H. magnistylus). Included in this invention are minor variants of this event such as a soybean event with HPPD inhibitor tolerance and SCN nematode resistance that has a nucleotide sequence with at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to the nucleotide sequence of EE-GM5 as contained in the seed deposited at the ATCC under deposit number PTA-123625, or a soybean event with HPPD inhibitor tolerance and SCN nematode resistance that has a nucleotide sequence differing in 1 to 200, 1 to 150, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 nucleotides from the nucleotide sequence of EE-GM5 as contained in the deposited seed of ATCC deposit PTA-123625, or that has a nucleotide sequence differing in 1 to 200, 1 to 150, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 nucleotides from the nucleotide sequence formed by the following consecutive nucleotide sequences (5′ to 3′): SEQ ID No. 5 or SEQ ID No. 24, SEQ ID No. 11 from nucleotide position 188 to nucleotide position 7101, and SEQ ID No. 6 or SEQ ID No. 25. In one embodiment, EE-GM5 comprises a nucleotide sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to the sequence formed by the following consecutive nucleotide sequences (5′ to 3′): SEQ ID No. 5 or 24, SEQ ID No. 11 from nucleotide position 188 to nucleotide position 7101, and SEQ ID No. 6 or 25. Due to natural genetic variation, single DNA base differences and small insertions and deletions in homologous DNA sequences (e.g., single-nucleotide polymorphisms (SNPs)) are commonly found in plants of the same species (Zhu et al. (2003) Genetics 163:1123-1134).
The seed of ATCC deposit number PTA-123625, is a pure seed lot of transgenic seeds homozygous for elite event EE-GM5 of the invention, which will grow into nematode resistant plants, whereby the plants are also tolerant to an HPPD inhibitor such as isoxaflutole, topramezone or mesotrione. The seed or progeny seed obtainable from the deposited seed (e.g., following crossing with other soybean plants with a different genetic background) can be sown and the growing plants can be treated with an HPPD inhibitor such as isoxaflutole, topramezone or mesotrione as described herein or can be tested for the presence of EE-GM5 as described herein to obtain plants comprising the elite event of the invention. The invention further relates to cells, seeds, tissues, progeny, and descendants from a plant comprising the elite event of the invention grown from the seed deposited at the ATCC having accession number PTA-123625. The invention further relates to plants obtainable from (such as by propagation of and/or breeding with) a soybean plant comprising the elite event of the invention (such as a plant grown from the seed deposited at the ATCC having accession number PTA-123625, or a plant comprising the HPPD-4 coding sequence of SEQ ID No. 9 and the cry14Ab-1.b coding sequence of SEQ ID No. 7 located between the sequence of SEQ ID No. 1, 3 or 5 and the sequence of SEQ ID No. 2, 4 or 6, or a plant comprising the hppdPf-4 Pa coding sequence of SEQ ID No. 9 and the cry14Ab-1.b coding sequence of SEQ ID No. 7 located between any one of the sequence of SEQ ID No. 1, 3, 5, or 24 and the sequence of any one of SEQ ID No. 2, 4, 6, or 25). The invention also relates to progeny plants and seeds obtained from the above plants or seed and that comprise the sequence of SEQ ID No. 1 and the sequence of SEQ ID No. 2, or the sequence of SEQ ID No. 3 and the sequence of SEQ ID No. 4, or the sequence of SEQ ID No. 5 and the sequence of SEQ ID No. 6, or the sequence of SEQ ID No. 24 and the sequence of SEQ ID No. 25. In one embodiment, the invention also relates to such plants or seeds treated with one or more of the compounds and/or biological control agents or mixtures thereof as described herein. In another embodiment, the invention relates to such plant or seeds also comprising one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
The transgenic plant, or cells or tissues thereof, comprising elite event EE-GM5, can be identified using methods described herein that are based on the presence of characterizing DNA sequences or amino acids encoded by such DNA sequences in the transgenic plant, cells or tissues. According to a preferred embodiment of the invention, such characterizing DNA sequences are sequences of 15 bp or at least 15 bp, preferably 20 bp or at least 20 bp, most preferably 30 bp or more which comprise the insertion site of the event, i.e., a sequence containing both a part of the inserted T-DNA containing an HPPD inhibitor and nematode resistance transgene and a part of the 5′ or 3′ T-DNA flanking region contiguous therewith that extends into the soybean plant genome, allowing specific identification of the elite event. Also described herein are methods for identifying elite event EE-GM5 in biological samples, which methods are based on primer pairs or probes which specifically recognize the 5′ and/or 3′ T-DNA flanking sequence and the inserted T-DNA sequence contiguous therewith in EE-GM5. Any other methods to identify EE-GM5, e.g., to identify its specific characterizing sequences, are also included herein, such as whole or partial (directed) genome sequencing.
Provided herein is a method for identifying elite event EE-GM5 in biological samples comprising amplifying a sequence of a nucleic acid present in said biological samples, using a polymerase chain reaction with at least two primers, or a polymerase chain reaction with at least two primers and a probe, wherein one of these primers recognizes the 5′ or 3′ T-DNA flanking region in EE-GM5, the other primer recognizes a sequence within the T-DNA comprising the herbicide tolerance and nematode resistance genes that is contiguous with said 5′ or 3′ T-DNA flanking region, preferably to obtain a DNA fragment of 50 to 1000 bp in size. In one embodiment, a first primer recognizes the 5′ T-DNA flanking region in EE-GM5, and a second primer recognizes a sequence within the T-DNA comprising the herbicide tolerance and nematode resistance genes that is contiguous with and downstream of said 5′ T-DNA flanking region, or a first primer recognizes the 3′ T-DNA flanking region in EE-GM5, and a second primer recognizes a sequence within the T-DNA comprising the herbicide tolerance and nematode resistance genes that is contiguous with and upstream of said 3′ T-DNA flanking region, to obtain a DNA fragment characteristic for elite event EE-GM5. In one embodiment, said polymerase chain reaction method further comprises the use of a probe that recognizes the DNA amplified by said primers, e.g., the junction DNA comprising part of the inserted T-DNA and part of the DNA flanking said T-DNA in EE-GM5 (at either the 5′ or 3′ side of the event, as applicable, such as a probe comprising the nucleotide sequence of SEQ ID No. 14 or 20 herein, or their complement), so as to detect the amplification product produced by said primers. The primers may recognize a sequence within the 5′ T-DNA flanking region of EE-GM5 (SEQ ID No. 5, from nucleotide position 1 to nucleotide position 166, or SEQ ID No. 24 from nucleotide position 1 to nucleotide position 1113) or within the 3′ T-DNA flanking region of EE-GM5 (complement of SEQ ID No. 6 from nucleotide position 359 to nucleotide position 691, or SEQ ID No. 25 from nucleotide position 359 to nucleotide position 1449) and a sequence within the inserted T-DNA (SEQ ID No. 5 from nucleotide position 167 to 353, or SEQ ID No. 6 from nucleotide position 1 to nucleotide position 358, or SEQ ID No. 23 from nucleotide position 1114 to 8572, or the complement thereof), respectively. The primer recognizing the 5′ or 3′ T-DNA flanking region may comprise the nucleotide sequence of SEQ ID No. 13, SEQ ID No. 19, SEQ ID No. 26 or SEQ ID No. 27, and the primer recognizing a sequence within the inserted T-DNA comprising nematode resistance and herbicide tolerance genes may comprise the nucleotide sequence of SEQ ID No. 12, SEQ ID No. 18, SEQ ID No. 28 or SEQ ID No. 29 described herein. Also described herein is an event-specific primer pair and the specific DNA amplified using such primer pair, as can be obtained by a person of ordinary skill in the art or as can be obtained from commercial sources from the EE-GM5 event sequences provided herein or contained in the seed deposited at the ATCC under accession number PTA-123625.
A method for identifying elite event EE-GM5 in biological samples, can comprise amplifying a sequence of a nucleic acid present in a biological sample, using a polymerase chain reaction with two primers comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 12 and SEQ ID No. 13 respectively, to obtain a DNA fragment of 85 bp or with two primers comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 18 and SEQ ID No. 19 respectively, to obtain a DNA fragment of 84 bp.
Described herein are the specific T-DNA flanking sequences of EE-GM5, which can be used to develop specific identification methods for EE-GM5 in biological samples. Such specific T-DNA flanking sequences may also be used as reference control material in identification assays. More particularly, the invention relates to the 5′ and/or 3′ T-DNA flanking regions of EE-GM5 which can be used for the development of specific primers and probes as further described herein. Also suitable as reference material are nucleic acid molecules, preferably of about 150-850 bp, comprising the sequence which can be amplified by primers comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 12 and SEQ ID No. 13 or of SEQ ID No. 18 and SEQ ID No. 19.
Identification methods for the presence of EE-GM5 in biological samples are described herein, based on the use of such specific primers or probes. Primers may comprise, consist or consist essentially of a nucleotide sequence of 17 to about 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 5 from nucleotide 1 to nucleotide 166 or SEQ ID No. 24 from nucleotide 1 to nucleotide 1113, or the complement of the nucleotide sequence of SEQ ID 6 from nucleotide 359 to nucleotide 691, or the complement of the nucleotide sequence of SEQ ID No. 25 from nucleotide 359 to nucleotide 1449, combined with primers comprising, consisting, or consisting essentially of a nucleotide sequence of 17 to about 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 11 from nucleotide 1 to nucleotide 7459 or SEQ ID No. 23 from nucleotide position 1114 to nucleotide position 8572, such as a nucleotide sequence of 17 to about 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 5 from nucleotide 167 to nucleotide 353 or the nucleotide sequence of SEQ ID No. 6 from nucleotide 1 to nucleotide 358, or the complement thereof. Primers may also comprise these nucleotide sequences located at their extreme 3′ end, and further comprise unrelated sequences or sequences derived from the mentioned nucleotide sequences, but comprising mismatches. In one embodiment, the primers as used herein, can also be identical to the target DNA or the complement thereof, wherein said target DNA is a hybrid containing nucleotide sequences from different origins, that do not occur in such combination in nature.
Kits for identifying elite event EE-GM5 in biological samples are also provided herein, said kits comprising at least one primer pair or probe which specifically recognizes the 5′ or 3′ T-DNA flanking region and the inserted T-DNA comprising a herbicide tolerance and a nematode resistance gene contiguous therewith in EE-GM5.
These kit may comprise, in addition to a primer which specifically recognizes the 5′ or 3′ T-DNA flanking region of EE-GM5, a second primer which specifically recognizes a sequence within the inserted T-DNA comprising an HPPD inhibitor herbicide tolerance and a nematode resistance gene of EE-GM5, for use in a PCR identification protocol. These kits may comprise at least two specific primers, one of which recognizes a sequence within the 5′ T-DNA flanking region of EE-GM5 or a sequence within the 3′ T-DNA flanking region of EE-GM5, and the other which recognizes a sequence within the inserted T-DNA comprising an HPPD inhibitor herbicide tolerance and a nematode resistance gene. The primer recognizing the 5′ T-DNA flanking region may comprise the nucleotide sequence of SEQ ID No. 19 and the primer recognizing the inserted T-DNA contiguous with said 5′ T-DNA flanking region may comprise the nucleotide sequence of SEQ ID No. 18, or the primer recognizing the 3′ T-DNA flanking region may comprise the nucleotide sequence of SEQ ID No. 13 and the primer recognizing the inserted T-DNA contiguous with said 3′ flanking region may comprise the nucleotide sequence of SEQ ID No. 12, or any other primer or primer combination as described herein or obtainable from the description or the seed deposit. The kit may further comprise a probe recognizing a sequence located between the primer recognizing the 5′ T-DNA flanking region and the primer recognizing the sequence within the inserted T-DNA, or recognizing a sequence between the primer recognizing the 3′ T-DNA flanking region and the primer recognizing the sequence within the inserted T-DNA, such as a probe comprising the sequence of SEQ ID No. 14 or a probe comprising the sequence of SEQ ID No. 20.
A kit for identifying elite event EE-GM5 in biological samples, can also comprise the PCR primers comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 12 and SEQ ID No. 13, or of the nucleotide sequence of SEQ ID No. 18 and SEQ ID No. 19 for use in the EE-GM5 PCR protocol described herein. Said kit comprising the primers comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 12 and SEQ ID No. 13 may further comprise a probe comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 14, and said kit comprising the primers comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 18 and SEQ ID No. 19 may further comprise a probe comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 20. Said kit can further comprise buffer and reagents such as anyone or each of the following compounds: dNTPs, (Taq) DNA polymerase, MgCl2, stabilizers, and optionally a dye.
A kit for identifying elite event EE-GM5 in biological samples, can also comprise a specific probe comprising or consisting (essentially) of a sequence which corresponds (or is complementary) to a sequence having 80% to 100% sequence identity with a specific region of EE-GM5, wherein such specific region comprises part of the 5′ or 3′ T-DNA flanking region of EE-GM5 and part of the inserted T-DNA contiguous therewith. In one embodiment, the sequence of the probe corresponds to a specific region comprising part of the 5′ or 3′ T-DNA flanking region of EE-GM5 and part of the inserted T-DNA contiguous therewith. Most preferably the specific probe comprises or consists (essentially) of (or is complementary to) a sequence having 80% to 100% sequence identity to the sequence of any one of SEQ ID No. 1, 3 or 5, or a sequence having 80% to 100% sequence identity to the sequence of any one of SEQ ID No. 2, 4 or 6, or the specific probe comprises or consists (essentially) of (or is complementary to) a sequence having 80% to 100% sequence identity to a part of at least 50 contiguous nucleotides of the sequence of SEQ ID No. 5, or a sequence having 80% to 100% sequence identity to a part of at least 50 contiguous nucleotides of the sequence of SEQ ID No. 6, wherein each of said part of SEQ ID No. 5 or 6 comprises sequences of inserted T-DNA and T-DNA flanking sequences of approximately equal length.
Also described herein are DNA molecules comprising sufficient length of polynucleotides of both the T-DNA flanking sequences and the inserted T-DNA of EE-GM5, so as to be useful as primer or probe for the detection of EE-GM5, or to characterize plants comprising event EE-GM5. Such sequences may comprise any one of at least 9, at least 10, at least 15, at least 20, or at least 30 nucleotides, or may comprise any one of 9, 10, 15, 20 or 30 nucleotides of the T-DNA flanking sequence and a similar number of nucleotides of the inserted T-DNA of EE-GM5, at each side of the junction site respectively, and this at either or both of the 5′ and 3′ junction site of the EE-GM5 event. Most preferably, such DNA molecules comprise the sequence of any one of SEQ ID No. 1, 3, or 5 or the sequence of any one of SEQ ID No. 2, 4, or 6. In one embodiment, such DNA molecules comprise the sequence of SEQ ID No. 23, 24 or 25. In one aspect of the invention, soybean plants and seeds are provided comprising such specific DNA molecules.
The methods and kits disclosed herein can be used for different purposes such as, but not limited to the following: to identify the presence or determine the (lower) threshold of EE-GM5 in plants, plant material or in products such as, but not limited to food or feed products (fresh or processed) comprising or derived from plant material; additionally or alternatively, the methods and kits of the present invention can be used to identify transgenic plant material for purposes of segregation between transgenic and non-transgenic material; additionally or alternatively, the methods and kits of the present invention can be used to determine the quality (i.e., percentage pure material) of plant material comprising EE-GM5.
Provided herein is also genomic DNA obtained from plants comprising elite event EE-GM5, particularly genomic DNA comprising EE-GM5 event-specific sequences, such as one or both of the EE-GM5 junction sequences (containing a part of T-DNA flanking DNA and inserted T-DNA contiguous therewith, characteristic for EE-GM5), e.g., any one of the sequences of SEQ ID No. 1, 3, 5, or 24 and/or any one of the sequences of SEQ ID No. 2, 4, 6, or 25. Such genomic DNA may be used as reference control material in the identification assays herein described.
Also provided herein is a transgenic nematode resistant and herbicide tolerant soybean plant, or cells, parts, seeds or progeny thereof, each comprising at least one elite event, said elite event comprises an inserted T-DNA comprising:
In one embodiment, elite event EE-GM5 comprises nucleotides 1 to 166 of SEQ ID No. 5 or 1 to 1113 of SEQ ID No. 24 immediately upstream of and contiguous with said inserted T-DNA and nucleotides 359 to 691 of SEQ ID No. 6 or nucleotides 359 to 1449 of SEQ ID No. 25 immediately downstream of and contiguous with said inserted T-DNA.
In a further embodiment, said elite event is obtainable by breeding with a soybean plant grown from reference seed comprising said event having been deposited at the ATCC under deposit number PTA-123625.
In another embodiment, the genomic DNA of said soybean plant, or cells, parts, seeds or progeny thereof when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID No. 12 and SEQ ID No. 13 respectively, yields a DNA fragment of 85 bp, or when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID No. 18 and SEQ ID No. 19 respectively, yields a DNA fragment of 84 bp.
Also provided herein is a method for identifying a transgenic soybean plant, or cells, parts, seed or progeny thereof with nematode resistance, such as SCN and/or Pratylenchus and/or root-knot and/or reniform nematode resistance, and tolerance to an HPPD inhibitor herbicide, such as isoxaflutole, topramezone or mesotrione, in biological samples, said method comprising amplifying a DNA fragment of between 50 and 150 bp from a nucleic acid present in biological samples using a polymerase chain reaction with at least two primers, one of said primers recognizing the 5′ T-DNA flanking region of the elite event EE-GM5, said 5′ T-DNA flanking region comprising the nucleotide sequence of SEQ ID No. 5 from nucleotide 1 to nucleotide 166 or of SEQ ID No. 24 from nucleotide 1 to nucleotide 1113, or recognizing the 3′ T-DNA flanking region of said elite event, said 3′ T-DNA flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID No. 25 from nucleotide 359 to nucleotide 1449, the other primer of said primers recognizing a sequence within the inserted T-DNA comprising the nucleotide sequence of the complement of SEQ ID No. 5 from nucleotide 167 to nucleotide 353 or the nucleotide sequence of SEQ ID No. 6 from nucleotide 1 to nucleotide 358, or wherein said inserted T-DNA comprises the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1 to nucleotide position 7459, or the complement thereof.
Also provided herein is a kit for identifying a transgenic soybean plant, or cells, parts, seed or progeny thereof with nematode resistance and tolerance to an HPPD inhibitor herbicide, in biological samples, said kit comprising one primer recognizing the 5′ T-DNA flanking region of elite event EE-GM5, said 5′ T-DNA flanking region comprising the nucleotide sequence of SEQ ID No. 5 from nucleotide 1 to nucleotide 166, or the nucleotide sequence of SEQ ID No. 24 from nucleotide 1 to nucleotide 1113, or one primer recognizing the 3′ T-DNA flanking region of said elite event, said 3′ T-DNA flanking region comprising the nucleotide sequence of the complement of SEQ ID No. 6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID No. 25 from nucleotide 359 to nucleotide 1449, and one primer recognizing a sequence within the inserted T-DNA, said inserted T-DNA comprising the nucleotide sequence of the complement of SEQ ID No. 5 from nucleotide 167 to nucleotide 353 or the nucleotide sequence of SEQ ID No. 6 from nucleotide 1 to nucleotide 358, or said inserted T-DNA comprising the nucleotide sequence of SEQ ID No. 11 from nucleotide position 1 to nucleotide position 7459, or the complement thereof.
In one embodiment, the inserted T-DNA of elite event EE-GM5, as used herein, comprises the nucleotide sequence of SEQ ID No. 5 from nucleotide 167 to nucleotide 353 or its complement, and the nucleotide sequence of SEQ ID No. 6 from nucleotide 359 to nucleotide 691 or its complement, or comprises a sequence with at least 95, 98, 99, 99.5, or 99.9% sequence identity to the nucleotide sequence of SEQ ID No. 11 from nucleotide position 7 to nucleotide position 7459, or its complement.
Also provided herein is a soybean plant, plant cell, tissue, or seed, comprising in their genome a nucleic acid molecule comprising the nucleotide sequence of any one of SEQ ID No. 1, 3, 5, or 24 or a nucleotide sequence of 80 to 100% sequence identity thereto and/or SEQ ID No. 2, 4, 6, or 25, or a nucleotide sequence of 80 to 100% sequence identity thereto, and a nucleotide sequence with at least 80, 85, 90, 95, 97, 98, 99, 99.5 or at least 99.9% sequence identity to the nucleotide sequence of SEQ ID No. 11 from nucleotide position 188 to nucleotide position 7101 or the complement thereof, wherein such plant, plant cell, tissue, or seed was treated with one or more of the compounds and/or biological control agents or mixtures as described herein. In one embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
Also provided herein is the use of an isolated nucleic acid molecule comprising a nucleotide sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID No. 7 or the complement thereof, or an isolated nucleic acid molecule comprising a nucleotide sequence hybridizing under standard stringency conditions to the nucleotide sequence of SEQ ID No. 7 or the complement thereof, wherein such nucleic acid molecule encodes a nematicidal toxin active to cyst nematodes and/or lesion nematodes and/or root-knot nematodes and/or reniform nematode, such as Heterodera glycines and/or Pratylenchus brachyurus and/or Meloidogyne incognita and/or Rotylenchulus reniformis, in combination with one or more of the compounds and/or biological control agents or mixtures as described herein, so as to ensure increased protection to such nematodes and/or delay or prevent the development of nematode resistance. In one embodiment, such nucleic acid molecule is operably-linked to a nucleic acid molecule comprising a (heterologous) plant-expressible promoter so as to form a chimeric gene. Also provided herein is the use of said nucleic acid molecule in transformed plants or seeds treated with said compounds and/or biological control agents or mixtures to control plant-pathogenic nematodes. Further provided herein is a method to control root-knot nematodes such as Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne hapla, or Meloidogyne javanica, particularly Meloidogyne incognita, comprising using a Cry14Ab protein or a DNA encoding a Cry14Ab protein or a plant or seed containing said DNA under the control of a plant-expressible promoter, wherein said Cry14Ab protein is the protein comprising the amino acid sequence of SEQ ID No. 8 or a protein with at least 96% or at least 98 or at least 99% sequence identity thereto, or a protein comprising the amino acid sequence of SEQ ID No. 8 from amino acid position 1 to amino acid position 706, or a protein with at least 96% or at least 98 or at least 99% sequence identity thereto, wherein said use also includes the use of the compounds and/or biological control agents or mixtures described herein on plants or seeds comprising said (DNA encoding) said Cry protein. Further provided herein is a method to control reniform nematodes (Rotylenchulus reniformis), comprising using a Cry14Ab protein or a DNA encoding a Cry14Ab protein, or a plant or seed containing said DNA, under the control of a plant-expressible promoter, wherein said Cry14Ab protein is the protein comprising the amino acid sequence of SEQ ID No. 8 or a protein with at least 96% or at least 98% or at least 99% sequence identity thereto, or a protein comprising the amino acid sequence of SEQ ID No. 8 from amino acid position 1 to amino acid position 706, or a protein with at least 96% or at least 98% or at least 99% sequence identity thereto, wherein said use also includes the use of the compounds and/or biological control agents or mixtures described herein on plants or seeds comprising said (DNA encoding) said Cry protein. Also provided herein is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID No. 11 from nucleotide position 131 to nucleotide position 7941, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein said nucleic acid molecule encodes a nematicidal Cry14Ab protein and an HPPD protein tolerant to HPPD inhibitors, wherein said use also includes the use of the compounds and/or biological control agents or mixtures described herein on plants or seeds comprising said nucleic acid molecule. In one embodiment, that nucleic acid molecule encodes the protein of SEQ ID No. 8 or a protein at least 99% identical thereto and the protein of SEQ ID No. 10, or a protein at least 99% identical thereto.
Also provided herein is a soybean plant, seed or cell comprising in its genome elite event EE-GM5 which is a foreign DNA or an inserted T-DNA at a defined locus, wherein the elite event EE-GM5 is as contained in reference seed deposited at the ATCC under deposit number PTA-123625, wherein said inserted T-DNA comprises a chimeric Cry14Ab-1-encoding gene and a chimeric HPPD-4-encoding gene, and wherein said elite event is characterized by the 5′ junction sequence of SEQ ID No. 1 or 3 and by the 3′ junction sequence of SEQ ID No. 2 or 4; or such cell which is a seed cell, or such cell, wherein the genomic DNA of said cell, when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID 12 and SEQ ID 13 respectively, yields a DNA fragment of 85 bp, wherein said plant, seed or cell are treated with the compounds and/or biological control agents or mixtures described herein.
In one embodiment, elite event EE-GM5 is as contained in reference seed deposited at the ATCC under deposit number PTA-123625, and is characterized by comprising a chimeric Cry14Ab-1-encoding gene and an HPPD-4-encoding gene, and comprising the sequence of SEQ ID No. 1 or 3 and the sequence of SEQ ID No. 2 or 4.
In one embodiment, elite event EE-GM5 contains a nucleic acid molecule comprising in order the following nucleotide sequences: a) the nucleotide sequence of SEQ ID NO. 5 from nucleotide 1 to 166 or a sequence at least 99% identical thereto, b) the nucleotide sequence of SEQ ID No. 11 from nucleotide 188 to nucleotide 7101 or a sequence at least 99% identical thereto, and c) the nucleotide sequence of SEQ ID NO. 6 from nucleotide 359 to nucleotide 691 or a sequence at least 99% identical thereto, such as such nucleic acid molecule comprising a sequence b) that is at least 99.5% or at least 99.9% identical to the nucleotide sequence of SEQ ID No. 11 from nucleotide 188 to nucleotide 7101.
In one embodiment, elite event EE-GM5 contains a nucleic acid molecule comprising in order the following nucleotide sequences: a) the nucleotide sequence of SEQ ID NO. 24 from nucleotide 1 to 1113 or a sequence at least 99% identical thereto, b) the nucleotide sequence of SEQ ID No. 23 from nucleotide 1114 to nucleotide 8572 or a sequence at least 99% identical thereto, and c) the nucleotide sequence of SEQ ID NO. 25 from nucleotide 359 to nucleotide 1449 or a sequence at least 99% identical thereto, such as such nucleic acid molecule comprising a sequence b) that is at least 99.5% or at least 99.9% identical to the nucleotide sequence of SEQ ID No. 23.
Also provided herein is the use of soybean seed comprising elite event EE-GM5 to obtain a treated seed, wherein said elite event comprises the sequence of any one of SEQ ID NO. 1, 3, 5 or 24 and/or the sequence of any one of SEQ ID No. 2, 4, 6, or 25, and wherein said treatment is with one or more of the compounds and/or biological control agents or mixtures described herein.
Further provided herein is a method for producing a soybean plant or seed comprising elite event EE-GM5 combined with another SCN resistance locus/gene, such as by combining elite event EE-GM5 with another SCN resistance locus/gene occurring in soybean, and planting seed comprising EE-GM5 and said other SCN resistance locus/gene. In one embodiment, the plants, cells or seeds of the invention contain one or more other SCN resistance loci/genes that occur in soybean, to get a combination of different SCN resistance sources in the soybean plants, cells or seeds of the invention. Several soybean SCN resistance loci or genes are known and one or more of those can be combined with EE-GM5 in the same plant, cell or seed, such as any one of the SCN resistance genes/loci from the resistance sources PI 88788, PI 548402 (Peking), PI 437654 (Hartwig or CystX®), or any combination thereof, or one or more of the native SCN resistance loci/genes rhg1, rhg1-b, rhg2, rhg3, Rhg4, Rhg5, qSCN11, cqSCN-003, cqSCN-005, cqSCN-006, cqSCN-007, or any of the SCN resistance loci identified on any one of soybean chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of any combination thereof (Kim et al. 2016, Theor. Appl. Genet. 129(12):2295-2311; Kim and Diers 2013, Crop Science 53:775-785; Kazi et al. 2010, Theor. Appl. Gen. 120(3):633-644; Glover et al. 2004, Crop Science 44(3):936-941; on the world wide web at soybase.org; Concibido et al. 2004, Crop Science 44:1121-1131; Webb et al. 1995, Theor. Appl. Genet. 91:574-581). Also, in one embodiment the plants or seeds of the invention contain EE-GM5 when combined with one or more SCN resistance loci in soybean obtained from any one of SCN resistance sources PI 548316, PI 567305, PI 437654, PI 90763, PI 404198B, PI 88788, PI 468916, PI 567516C, PI 209332, PI 438489B, PI 89772, Peking, PI 548402, PI 404198A, PI 561389B, PI 629013, PI 507471, PI 633736, PI 507354, PI 404166, PI 437655, PI 467312, PI 567328, PI 22897, or PI 494182. Table 1 enclosed hereto provides a comprehensive list of soybean accessions reported as SCN resistant, of which the SCN resistance genes/loci (one or several) can be combined with EE-GM5 of the invention in the same soybean plant, cell or seed. In one embodiment, the invention also relates to such plants or seeds treated with one or more of the compounds and/or biological control agents or mixtures as described herein.
In one embodiment, the methods of the invention also comprise treating the soybean plants or seeds, or the soil in which the soybean plants or seeds are grown or are intended to be grown, with one or more of the compound(s) and/or biological control agent(s) or a mixture as described herein, such as wherein said compound or biological control agent is nematicidal, insecticidal, or fungicidal. In another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
Also provided herein is a method for protecting emerging soybean plants from competition by weeds, comprising treating a field in which seeds containing elite event EE-GM5 as described above were sown, with an HPPD inhibitor herbicide, wherein the plants are tolerant to the HPPD inhibitor herbicide. In one embodiment, in such method the HPPD inhibitor herbicide is isoxaflutole, topramezone or mesotrione. In one embodiment, such method comprises also treating the soybean plants or seeds, or the soil in which the soybean plants or seeds are grown or are intended to be grown, with one or more of the compound(s) and/or biological control agent(s) or a mixtures comprising them, as described herein, such as wherein said compound or biological control agent is nematicidal, insecticidal, or fungicidal, e.g. a compound or biological control agent or combination thereof selected from any one of group IAN1, IAN2, IAN3, IAN4, IAN5, IAN6, IAN7, IAN8, IAN9, IAN10, IAN11, IAN12, IAN13, IAN14, IAN15, IAN16, IAN17, IAN18, IAN19, IAN20, IAN21, IAN22, IAN23, IAN24, IAN25, IAN26, IAN27, IAN28, IAN29, IAN30, SIAN1, F1, F2, F3, F4, F5, F6, F7, F8, F9, F9, F10, F11, F12, F13, F14, F15, F16, SF1, P1, BCA1, BCA2, BCA3, BCA4, BCA5, BCA6, BCA7, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2, as described herein.
Also provided herein is method for protecting emerging soybean plants from competition by weeds and from damage caused by plant-pathogenic nematodes, comprising treating a field to be planted with soybean plants comprising elite event EE-GM5 as described above with an HPPD inhibitor herbicide and a nematicidal compound or biological control agent or combination thereof as described herein, before the soybean plants are planted or the seeds are sown, followed by planting or sowing of said soybean plants or seeds in said pre-treated field, wherein the plants are tolerant to the HPPD inhibitor herbicide. In one embodiment, provided herein is method for protecting emerging soybean plants from competition by weeds and from damage caused by plant-pathogenic nematodes, comprising treating a field to be planted with soybean plants comprising elite event EE-GM5 as described above with an HPPD inhibitor herbicide, before the soybean seeds are sown, followed by sowing of said soybean seeds in said pre-treated field, wherein the plants are tolerant to the HPPD inhibitor herbicide, and wherein said seeds are coated with a compound or biological control agent or combination thereof, as described herein, such as a compound or biological control agent or combination thereof selected from any one of group IAN1, IAN2, IAN3, IAN4, IAN5, IAN6, IAN7, IAN8, IAN9, IAN10, IAN11, IAN12, IAN13, IAN14, IAN15, IAN16, IAN17, IAN18, IAN19, IAN20, IAN21, IAN22, IAN23, IAN24, IAN25, IAN26, IAN27, IAN28, IAN29, IAN30, SIAN1, F1, F2, F3, F4, F5, F6, F7, F8, F9, F9, F10, F11, F12, F13, F14, F15, F16, SF1, P1, BCA1, BCA2, BCA3, BCA4, BCA5, BCA6, BCA7, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2, as described herein.
Also provided herein is a method for controlling weeds and for protection plants from damage caused by plant-pathogenic nematodes in a field of soybean plants comprising elite event EE-GM5 as described above, comprising treating said field with an effective amount of an HPPD inhibitor herbicide, and a nematicidal compound or biological control agent or combination thereof as described herein wherein the plants are tolerant to such herbicide, such as wherein said compound or biological control agent or combination is selected from any one of group IAN1, IAN2, IAN3, IAN4, IAN5, IAN6, IAN7, IAN8, IAN9, IAN10, IAN11, IAN12, IAN13, IAN14, IAN15, IAN16, IAN17, IAN18, IAN19, IAN20, IAN21, IAN22, IAN23, IAN24, IAN25, IAN26, IAN27, IAN28, IAN29, IAN30, SIAN1, F1, F2, F3, F4, F5, F6, F7, F8, F9, F9, F10, F11, F12, F13, F14, F15, F16, SF1, P1, BCA1, BCA2, BCA3, BCA4, BCA5, BCA6, BCA7, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2, as described herein.
In one embodiment, the methods of the invention, such as the methods for controlling weeds and for protecting plants from plant-pathogenic nematodes, comprise besides application of HPPD inhibitor herbicides, also comprise treating the soybean plants or seeds, or the soil in which the soybean plants or seeds are grown or are intended to be grown, with one or more of the compound(s) and/or biological control agent(s) or a mixture, as described herein, such as wherein said compound or biological control agent is nematicidal, insecticidal, or fungicidal, or a compound or biological control agent or combination thereof selected from any one of group IAN1, IAN2, IAN3, IAN4, IAN5, IAN6, IAN7, IAN8, IAN9, IAN10, IAN11, IAN12, IAN13, IAN14, IAN15, IAN16, IAN17, IAN18, IAN19, IAN20, IAN21, IAN22, IAN23, IAN24, IAN25, IAN26, IAN27, IAN28, IAN29, IAN30, SIAN1, F1, F2, F3, F4, F5, F6, F7, F8, F9, F9, F10, F11, F12, F13, F14, F15, F16, SF1, P1, BCA1, BCA2, BCA3, BCA4, BCA5, BCA6, BCA7, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2, as described herein. In another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
Even further provided herein is the use of a transgenic soybean plant, seed or progeny thereof, to control weeds in a soybean field, wherein each of said plant, seed or progeny comprises elite event EE-GM5 in its genome, wherein EE-GM5 which is a T-DNA at a defined locus, as contained in reference seed deposited at ATCC under deposit number PTA-123625, wherein said T-DNA comprises a chimeric Cry14Ab-1-encoding gene and a chimeric HPPD-4-encoding gene, and wherein said elite event is characterized by the 5′ junction sequence of SEQ ID No. 1 or 3 and by the 3′ junction sequence of SEQ ID No. 2 or 4. In one embodiment, in such use the transgenic soybean plant, seed or progeny thereof is resistant to nematodes and/or tolerant to an HPPD inhibitor herbicide. In one embodiment, said T-DNA comprises a chimeric Cry14Ab-1-encoding gene and a chimeric HPPD-4-encoding gene, and said elite event is characterized by the 5′ junction sequence of SEQ ID No. 5 or 24 and by the 3′ junction sequence of SEQ ID No. 6 or 25. In one embodiment, the invention also relates to such plants or seeds treated with one or more of the compounds and/or biological control agents or mixtures thereof as described herein. In another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
Also provided herein is the use of a soybean plant or seed comprising elite event EE-GM5 in its genome to grow a nematode-resistant and/or herbicide-tolerant plant, wherein said elite event EE-GM5 is an inserted T-DNA at a defined locus, as contained in reference seed deposited at ATCC under deposit number PTA-123625, wherein said inserted T-DNA comprises a chimeric Cry14Ab-1-encoding gene and a chimeric HPPD-4-encoding gene, and wherein said elite event is characterized by the 5′ junction sequence of SEQ ID No. 1 or 3 and by the 3′ junction sequence of SEQ ID No. 2 or 4, and said use include the use of one or more of the compound(s) and/or biological control agent(s) or mixture, as described herein, such as wherein said compound or biological control agent is nematicidal, insecticidal, or fungicidal, or a compound or biological control agent or combination thereof selected from any one of group IAN1, IAN2, IAN3, IAN4, IAN5, IAN6, IAN7, IAN8, IAN9, IAN10, IAN11, IAN12, IAN13, IAN14, IAN15, IAN16, IAN17, IAN18, IAN19, IAN20, IAN21, IAN22, IAN23, IAN24, IAN25, IAN26, IAN27, IAN28, IAN29, IAN30, SIAN1, F1, F2, F3, F4, F5, F6, F7, F8, F9, F9, F10, F11, F12, F13, F14, F15, F16, SF1, P1, BCA1, BCA2, BCA3, BCA4, BCA5, BCA6, BCA7, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2, as described herein. In one embodiment, in such use the soybean plant or seed is resistant to SCN nematodes and/or tolerant to an HPPD inhibitor herbicide. In one embodiment, said T-DNA comprises a chimeric Cry14Ab-1-encoding gene and a chimeric HPPD-4-encoding gene, and said elite event is characterized by the 5′ junction sequence of SEQ ID No. 5 or 24 and by the 3′ junction sequence of SEQ ID No. 6 or 25.
Also provided herein is the use of a soybean seed comprising elite event EE-GM5 as described herein, treated with at least one compound or biological control agent or combination as described herein, to produce a soybean crop. In one embodiment, said seed also comprises one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654. In another embodiment, said seed comprises another soybean transformation event such as a soybean transformation event providing tolerance to additional herbicides, a soybean transformation event providing tolerance to nematodes via another mode of action compared to Cry14Ab-1, or a soybean transformation event providing insect control, or any one of the following soybean transformation events: Event MON87751, Event pDAB8264.42.32.1, Event DAS-81419-2, Event FG-072, Event SYHT0H2, Event DAS-68416-4, Event DAS-81615-9, Event DAS-44406-6, Event MON87708, Event MON89788, Event DAS-14536-7, Event GTS 40-3-2, Event A2704-12, Event BPS-CV127-9, Event A5547-127, Event MON87754, Event DP-3Ø5423-1, Event MON87701, Event MON87705, Event MON87712, Event pDAB4472-1606, Event DP-356043-5, Event MON87769, Event IND-ØØ41Ø-5, Event DP305423, or any of the following soybean event combinations: MON89788×MON87708, HOS×GTS 40-3-2, FG-072×A5547-127, MON87701×MON 89788, DAS-81419-2×DAS-44406-6, DAS-81419-2×DAS-68416-4, DAS-68416-4×MON 89788, MON 87705×MON 89788, MON 87769×MON 89788, DP305423×GTS 40-3-2, DP305423×MON87708, Event DP305423×MON87708×Event MON89788, DP305423×MON89788, MON87705×MON87708, MON87705×MON87708×MON89788, MON89788×MON87708×A5547-127, MON87751×MON87701×MON87708×MON89788, SYHT0H2×MON89788, SYHT0H2×GTS 40-3-2, SYHT0H2×MON89788×MON87708.
In one embodiment, the uses of a soybean plant or seed of the invention as described herein also includes the use of one or more of the compound(s) and/or biological control agent(s) or a mixture, as described herein, for treating the soybean plants or seeds, or for treating the soil in which the soybean plants or seeds are grown or are intended to be grown, such as wherein said compound or biological control agent is nematicidal, insecticidal, or fungicidal. In another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654. In another embodiment, said plants or seeds (with or without a native soybean SCN resistance loci or genes) also comprise another soybean transformation event such as a soybean transformation event providing tolerance to additional herbicides, a soybean transformation event providing tolerance to nematodes (such as SCN, lesion nematodes, root-knot nematodes and/or reniform nematodes) via another mode of action compared to Cry14Ab-1, or a soybean transformation event providing insect control, or any one of the following soybean transformation events: Event MON87751, Event pDAB8264.42.32.1, Event DAS-81419-2, Event FG-072, Event SYHTOH2, Event DAS-68416-4, Event DAS-81615-9, Event DAS-44406-6, Event MON87708, Event MON89788, Event DAS-14536-7, Event GTS 40-3-2, Event A2704-12, Event BPS-CV127-9, Event A5547-127, Event MON87754, Event DP-3Ø5423-1, Event MON87701, Event MON87705, Event MON87712, Event pDAB4472-1606, Event DP-356043-5, Event MON87769, Event IND-ØØ41Ø-5, Event DP305423, or any of the following soybean event combinations: MON89788×MON87708, HOS×GTS 40-3-2, FG-072×A5547-127, MON87701×MON 89788, DAS-81419-2×DAS-44406-6, DAS-81419-2×DAS-68416-4, DAS-68416-4×MON 89788, MON 87705×MON 89788, MON 87769×MON 89788, DP305423×GTS 40-3-2, DP305423×MON87708, Event DP305423×MON87708×Event MON89788, DP305423×MON89788, MON87705×MON87708, MON87705×MON87708×MON89788, MON89788×MON87708×A5547-127, MON87751×MON87701×MON87708×MON89788, SYHTOH2×MON89788, SYHTOH2×GTS 40-3-2, SYHTOH2×MON89788×MON87708.
Also provided herein is a method for producing a soybean plant or seed comprising elite event EE-GM5, comprising crossing a plant comprising EE-GM5 with another soybean plant, and planting seed comprising EE-GM5 obtained from said cross. In one embodiment, such method includes a step of application of an HPPD inhibitor herbicide and a step of application of one or more of the compounds or biological control agents or combinations described herein, on said seed or plant, or to the soil wherein said seed or plant is grown or is intended to be grown.
In accordance with this invention, also provided is the use of a soybean seed comprising elite event EE-GM5 as described above, and an HPPD inhibitor herbicide, to control weeds in a soybean field, and the use of a soybean seed comprising elite event EE-GM5 in a method of growing soybeans tolerant to HPPD inhibitor herbicides, wherein said seed is a seed treated with the compounds or biological control agents or combinations described herein, such as the compounds or biological control agents or combinations described in group SIAN1, SF1, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2 as described herein.
Further provided herein is the use of elite event EE-GM5 as described above to confer resistance to nematodes and/or tolerance to an HPPD inhibitor herbicide to a soybean plant or seed, or the use of a soybean plant or seed comprising elite event EE-GM5, in combination with an HPPD inhibitor herbicide, for growing soybeans.
Also provided herein is a primer pair specific for EE-GM5, as well as kits or methods using such primer pair, wherein at least one primer of said pair is labeled (such as with a detectable or screenable moiety), or wherein the 5′ end of at least one of said primers comprises one or more mismatches or a nucleotide sequence unrelated to the 5′ or 3′ flanking sequences of EE-GM5 or unrelated to the T-DNA sequence of EE-GM5; or wherein at least one of said primers comprises a nucleotide sequence at their 3′ end spanning the joining region between the T-DNA flanking sequences and the T-DNA sequences, said joining region being at nucleotides 166-167 in SEQ ID No. 5, nucleotides 1113-1114 in SEQ ID No. 24, or at nucleotides 358-359 in SEQ ID No. 6 or 25, provided that the 17 consecutive nucleotides at the 3′ end are not derived exclusively from either the T-DNA or T-DNA flanking sequences in SEQ ID Nos. 5 or 24, or 6 or 25; or wherein at least one of said primers comprises a sequence which is between 80 and 100% identical to a sequence within the 5′ or 3′ flanking region of EE-GM5 or within the inserted T-DNA of EE-GM5, respectively, and said primer sequence comprises at least one mismatch with said 5′ or 3′ flanking region or said T-DNA, provided the at least one mismatch still allows specific identification of the elite event EE-GM5 with these primers under optimized detection conditions (e.g., optimized PCR conditions); or wherein the nucleotide sequence of at least one of said primers comprises the nucleotide sequence of a nucleic acid fused to a nucleic acid from another origin, or its complement.
The invention also relates to the above-described plants or seeds comprising elite event EE-GM5 treated with pesticidal (e.g., nematicidal, insecticidal and/or fungicidal) compounds and/or biological control agents or mixtures thereof, wherein said treatment can be achieved by treating the soil wherein said plants or seeds are to be grown, by treating a field sown with said seeds or planted with said plants, or by treatment of the seed to be planted. The treatment with compounds and/or biological control agents or mixtures as described herein, including the treatment with HPPD inhibitor herbicides can be sequentially or simultaneous. Application can be as a split application over time, or the application of the individual active agents or the mixtures comprising the active agents in a plurality of portions (sequential application), can be by pre-emergence application, by post-emergence application, by early post-emergence applications, or by medium or late post-emergence, or can be a combination thereof, particularly for different active ingredients. The skilled person knows the application timings and methods suited for each active ingredient/combination.
The invention further relates to methods to improve yield in soybean comprising elite event EE-GM5 as described above, wherein the soybean plants or seeds, or the soil in which soybean plants or seeds are grown or are intended to be grown, are treated with the compounds and/or biological control agents or mixtures thereof as described herein, as well as to use of the plants or seeds comprising EE-GM5 as described herein with compounds and/or biological control agents or mixtures thereof as described herein. One embodiment of the invention relates to seed comprising elite event EE-GM5 treated with pesticidal (e.g., nematicidal, insecticidal, acaricidal, or fungicidal) compounds and/or biological agents or mixtures comprising them, so as to ensure improved protection of the seed, the germinated plantlet and the plant grown from the seed from agricultural pests. In one embodiment of the invention, the seed comprising elite event EE-GM5 contains a coating of one or more nematicidal compounds and/or biological control agents, or mixtures comprising them, such as seed comprising elite event EE-GM5 coated with at least one nematicidal compound (such as tioxazafen, fluopyram, metam, oxamyl, or abamectin, or any of the nematicidal compounds described herein or known in the art), and at least one nematicidal biological control agent (such as Bacillus firmus, Pasteuria nishizawae, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, or Burkholderia rinojensis, or any of the nematicidal biological control agents described herein or known in the art). In one embodiment of the invention, that seed coating also contains (besides the nematicidal agents) one or more insecticidal compounds or biological control agents as described herein (such as clothianidin, tetraniliprole, spirotetramat, flupyradifurone, thiamethoxam, chlorpyrifos, gamma-cyhalothrin, lambda-cyhalothrin, chlorantraniliprole, bifethrin, imidacloprid, zetacypermethrin, cyfluthrin, Bacillus thuringiensis, diflubenzuron), and/or one or more fungicidal compounds or biological control agents, as described herein (such as any one or more of Sedaxane, Fludioxonil, Mefenoxam, flutriafol, fluxapyroxad, pyraclostrobin, tetraconazole, azoxystrobin, propiconazole, benzovindiflupyr, tebuconazole, azoxystrobin). In another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654. In another embodiment, said plants or seeds (with or without said native soybean SCN resistance loci or genes) also comprise another soybean transformation event such as a soybean transformation event providing tolerance to additional herbicides, a soybean transformation event providing tolerance to nematodes (such as SCN, lesion nematodes, root-knot nematodes and/or reniform nematodes) via another mode of action compared to Cry14Ab-1, or a soybean transformation event providing insect control, or any one of the following soybean transformation events: Event MON87751, Event pDAB8264.42.32.1, Event DAS-81419-2, Event FG-072, Event SYHTOH2, Event DAS-68416-4, Event DAS-81615-9, Event DAS-44406-6, Event MON87708, Event MON89788, Event DAS-14536-7, Event GTS 40-3-2, Event A2704-12, Event BPS-CV127-9, Event A5547-127, Event MON87754, Event DP-3Ø5423-1, Event MON87701, Event MON87705, Event MON87712, Event pDAB4472-1606, Event DP-356043-5, Event MON87769, Event IND-ØØ41Ø-5, Event DP305423, or any of the following soybean event combinations: MON89788×MON87708, HOS×GTS 40-3-2, FG-072×A5547-127, MON87701×MON 89788, DAS-81419-2×DAS-44406-6, DAS-81419-2×DAS-68416-4, DAS-68416-4×MON 89788, MON 87705×MON 89788, MON 87769×MON 89788, DP305423×GTS 40-3-2, DP305423×MON87708, Event DP305423×MON87708×Event MON89788, DP305423×MON89788, MON87705×MON87708, MON87705×MON87708×MON89788, MON89788×MON87708×A5547-127, MON87751×MON87701×MON87708×MON89788, SYHT0H2×MON89788, SYHT0H2×GTS 40-3-2, SYHT0H2×MON89788×MON87708. In one embodiment, said compound or biological control agent or combination is selected from any one of group IAN1, IAN2, IAN3, IAN4, IAN5, IAN6, IAN7, IAN8, IAN9, IAN10, IAN11, IAN12, IAN13, IAN14, IAN15, IAN16, IAN17, IAN18, IAN19, IAN20, IAN21, IAN22, IAN23, IAN24, IAN25, IAN26, IAN27, IAN28, IAN29, IAN30, SIAN1, F1, F2, F3, F4, F5, F6, F7, F8, F9, F9, F10, F11, F12, F13, F14, F15, F16, SF1, P1, BCA1, BCA2, BCA3, BCA4, BCA5, BCA6, BCA7, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2, as described herein.
The invention also relates to methods for controlling soybean pests, such as soybean nematodes, on plants or seed of the invention by treating said plants or seeds with compounds and/or biological control agents or mixtures thereof that act on soybean pests, such as soybean nematodes, and/or their habitat.
Also provided herein is a method to prevent or delay nematode resistance development to the Cry14Ab-1 protein or to elite event of the invention, comprising treating the plants or seeds of the invention with one or more nematicidal compound(s) and/or nematicidal biological control agent(s), or mixtures containing them, or treating the soil wherein the plants or seeds of the invention will be grown (which can be followed by planting or sowing said plants or seeds in said soil). In one embodiment, said use is of one or more nematicidal compound(s) and one or more nematicidal biological control agent(s). In one embodiment, said nematode is soybean cyst nematode, a Pratylenchus species nematode, a root-knot nematode, and/or a reniform nematode, such as any of said nematodes feeding on soybean. In one embodiment more than one nematicidal compound or more than one nematicidal biological control agent is used or more than one nematicidal compound and more than one nematicidal biological control agent is used, particularly when they each have a different mode of action. In one embodiment, seeds comprising EE-GM5 are treated with said compounds or biological control agents.
In one embodiment of the above methods for controlling soybean pests or to prevent or delay nematode resistance development, such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654. In another embodiment, said plants or seeds (with or without said native soybean SCN resistance loci or genes) also comprise another soybean transformation event such as a soybean transformation event providing tolerance to additional herbicides, a soybean transformation event providing tolerance to nematodes (such as SCN, lesion nematodes, root-knot nematodes and/or reniform nematodes) via another mode of action compared to Cry14Ab-1, or a soybean transformation event providing insect control, or any one of the following soybean transformation events: Event MON87751, Event pDAB8264.42.32.1, Event DAS-81419-2, Event FG-072, Event SYHT0H2, Event DAS-68416-4, Event DAS-81615-9, Event DAS-44406-6, Event MON87708, Event MON89788, Event DAS-14536-7, Event GTS 40-3-2, Event A2704-12, Event BPS-CV127-9, Event A5547-127, Event MON87754, Event DP-3Ø5423-1, Event MON87701, Event MON87705, Event MON87712, Event pDAB4472-1606, Event DP-356043-5, Event MON87769, Event IND-ØØ41Ø-5, Event DP305423, or any of the following soybean event combinations: MON89788×MON87708, HOS×GTS 40-3-2, FG-072×A5547-127, MON87701×MON 89788, DAS-81419-2×DAS-44406-6, DAS-81419-2×DAS-68416-4, DAS-68416-4×MON 89788, MON 87705×MON 89788, MON 87769×MON 89788, DP305423×GTS 40-3-2, DP305423×MON87708, Event DP305423×MON87708×Event MON89788, DP305423×MON89788, MON87705×MON87708, MON87705×MON87708×MON89788, MON89788×MON87708×A5547-127, MON87751×MON87701×MON87708×MON89788, SYHT0H2×MON89788, SYHT0H2×GTS 40-3-2, SYHT0H2×MON89788×MON87708. In one embodiment, said compound or biological control agent or combination is selected from any one of group IAN1, IAN2, IAN3, IAN4, IAN5, IAN6, IAN7, IAN8, IAN9, IAN10, IAN11, IAN12, IAN13, IAN14, IAN15, IAN16, IAN17, IAN18, IAN19, IAN20, IAN21, IAN22, IAN23, IAN24, IAN25, IAN26, IAN27, IAN28, IAN29, IAN30, SIAN1, F1, F2, F3, F4, F5, F6, F7, F8, F9, F9, F10, F11, F12, F13, F14, F15, F16, SF1, P1, BCA1, BCA2, BCA3, BCA4, BCA5, BCA6, BCA7, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2, as described herein. In one embodiment, said compound or biological control agent or combination is selected from any one of group IAN1, IAN2, IAN3, IAN4, IAN5, IAN6, IAN7, IAN8, IAN9, IAN10, IAN11, IAN12, IAN13, IAN14, IAN15, IAN16, IAN17, IAN18, IAN19, IAN20, IAN21, IAN22, IAN23, IAN24, IAN25, IAN26, IAN27, IAN28, IAN29, IAN30, SIAN1, F1, F2, F3, F4, F5, F6, F7, F8, F9, F9, F10, F11, F12, F13, F14, F15, F16, SF1, P1, BCA1, BCA2, BCA3, BCA4, BCA5, BCA6, BCA7, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2, as described herein.
In one embodiment, the plants and seeds that were treated or are to be treated in accordance with the invention contain (next to event EE-GM5), SCN resistance loci or genes from one or more of SCN resistance sources PI 548316, PI 567305, PI 437654, PI 90763, PI 404198B, PI 88788, PI 468916, PI 567516C, PI 209332, PI 438489B, PI 89772, Peking, PI 548402, PI 404198A, PI 561389B, PI 629013, PI 507471, PI 633736, PI 507354, PI 404166, PI 437655, PI 467312, PI 567328, PI 22897, or PI 494182.
In one embodiment, the compound(s) or the biological control agent(s), or mixtures, of the invention as described herein are selected from any one of the groups as described herein, such as H1, H2, H3, H4, H5, IAN1, IAN2, IAN3, IAN4, IAN5, IAN6, IAN7, IAN8, IAN9, IAN10, IAN11, IAN12, IAN13, IAN14, IAN15, IAN16, IAN17, IAN18, IAN19, IAN20, IAN21, IAN22, IAN23, IAN24, IAN25, IAN26, IAN27, IAN28, IAN29, IAN30, SIAN1, F1, F2, F3, F4, F5, F6, F7, F8, F9, F9, F10, F11, F12, F13, F14, F15, F16, SF1, P1, BCA1, BCA2, BCA3, BCA4, BCA5, BCA6, BCA7, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2, as well as the preferred combinations or mixtures described herein.
In one embodiment, a seed of the invention is treated with the compound(s) and/or the biological control agent(s), as described herein, or a combination described herein, wherein said compound or agent or combination is from group SIAN1, BCA8, BCA9, BCA10, NC1, NC2, NC3, SBCA1, SAI1, SC1 or SC2.
Other embodiments referred to in this invention are summarized in the following paragraphs:
The following Examples, not intended to limit the invention to the specific embodiments described, may be understood in conjunction with the accompanying Figures, incorporated herein by reference, in which:
EE-GM5 in original transformant background (Thorne) was tested in 9 different locations throughout Iowa, Illinois, Indiana, Missouri and Tennessee in 2015 and 2016, in SCN infested fields (ranging from low to high SCN infestation). The dot is the estimated yield of the homozygous event for each trial (as percent difference to the null), the horizontal lines represent the 95% confidence limits of the contrast between the homozygous event and the null segregate (if the line does not overlap the vertical line at 100 percent yield of null segregate, then the event was significantly different from the null segregate). “Across Locs” is the estimated yield of a combined analysis across all 9 locations.
EE-GM5 was introgressed (BC2F3) into an elite MG I (maturity group I) line that is susceptible to SCN and was tested at one location in Minnesota and one location in North Dakota in 2016 (each with high SCN infestation levels). The dot is the estimated yield of the homozygous event for each trial (as percent difference to the null), the horizontal line around the dot represents the 95% confidence limits of the contrast between the homozygous event and the null segregant (if the line does not overlap the vertical line at 100 percent yield of null segregant, then the event was significantly different from the null segregant). “Across Locs” is the estimated yield of a combined analysis across both locations.
EE-GM5 was crossed into an elite MG III (maturity group III) line that is resistant to SCN (due to the rhg1 locus from PI88788) and was tested at 3 locations in 2016 (trials starting with “16” such as 16-IN1) and at 7 locations in 2017 (trials starting with “17”, such as 17-IN1), ranging from low to high SCN infestation levels (see arrow, locations with low SCN pressure are at the bottom of the figure (e.g., 17-IL2) and locations with high SCN pressure at the top (e.g., 17-IN1)). SCN pressure was assigned by considering several factors including known field history, SCN populations in the soil, relative yields of resistant and susceptible control varieties, soil characteristics (pH and % sand) and a visual evaluation of root infestation in susceptible entries. The dot is the average yield difference (in tons per hectare) of the homozygous event in each trial compared to the null segregant, the horizontal line around the dot represents the 95% confidence limits of the contrast between the homozygous event and the null segregant (if the line does not overlap the vertical line at 0 difference with the null segregant, then the yield for the event was significantly different from the null segregant). “Avg” is the average yield across all locations in each year.
Elite soybean plants with EE-GM5 control Pratylenchus brachyurus in US greenhouse assays. Plants with EE-GM5 (“EE-GM5”) were compared to other elite soybean lines: one SCN susceptible Maturity Group (MG)3 line (“THORNE”), one MG3 SCN susceptible line, one MG 6.2 SCN susceptible line and one MG9 SCN susceptible line (“Susc WT” shows the average for these 3 lines), one MG3 SCN resistant line (with the rhg1 resistance allele from PI88788, “SCN Res (PI88788)”), and one MG 6.2 SCN resistant line with the rhg1 and Rhg4 SCN resistance from Peking (“SCN Res (Peking)”). Plotted are the average numbers of Pratylenchus in roots 30 days after infestation (5 plants per entry), also showing the variation observed across varieties (as typically seen in greenhouse assays). Results show ˜90% control of Pratylenchus across EE-GM5 lines. Soybean lines with native SCN resistance (from Peking or PI88788) do not control Pratylenchus brachyurus.
Soybean plants homozygous for EE-GM5 (“EE-GM5 HH”) significantly reduce Pratylenchus brachyurus in soybean roots. Pratylenchus brachyurus were isolated from local fields in Brazil. EE-GM5 plants (in two different US elite lines (both maturity group 6.2, one SCN-susceptible and one with Peking SCN-resistance (“EE-GM5”)) and five Brazilian soybean lines, with limited Pratylenchus control (“Brazil lines”), one Brazilian line labeled as low Rf (reproductive factor) for Pratylenchus (“BRS 7380 (low Rf)”), one US elite line (maturity group 6.2) that is SCN-susceptible (“SCN Susc”) and one US elite line (MG 6.2) with Peking SCN-resistance (“SCN Res (Peking)”) were evaluated for Pratylenchus control in a greenhouse assay in Brazil. Plotted are the averages of those entries, also showing the variation observed across varieties (as typically seen in greenhouse assays). One Brazilian soybean line (BRS 7380), showed 89% reduction of Pratylenchus. EE-GM5 lines gave ˜99% control of Pratylenchus. Soybean lines that carry Peking native resistance to SCN do not control Pratylenchus brachyurus.
Soybean plants homozygous for EE-GM5 (“HH”) showed significantly lower Pratylenchus brachyurus nematodes in soybean roots. The EE-GM5 event in the elite maturity group IX background was compared to segregating sister lines having lost the transgene (“Null”), as well as to the wild-type elite maturity group IX parent line (“WT”) in fields naturally infested with P. brachyurus. The data were compiled from two trial locations that were sampled at −98 days after planting. Plotted are the natural log of the averages of those entries, and the variation. The vertical lines indicate the standard error (“SEM” is the Standard Error of the Mean). Treatment means with different letters are significantly different at P<0.05.
In this invention, EE-GM5 has been identified as an elite event from a population of transgenic soybean plants in the development of nematode resistant soybean (Glycine max) comprising a gene coding for 4-hydroxy phenylpyruvate dioxygenase (HPPD) inhibitor tolerance combined with a gene conferring resistance to nematodes, each under control of a plant-expressible promoter. Specific tools for use in the identification of elite event EE-GM5 in biological samples are described herein.
The incorporation of a recombinant DNA molecule in the plant genome typically results from transformation of a cell or tissue. The particular site of incorporation is usually due to random integration.
The DNA introduced into the plant genome as a result of transformation of a plant cell or tissue with a recombinant DNA or “transforming DNA”, and originating from such transforming DNA is hereinafter referred to as “inserted T-DNA” comprising one or more “transgenes”. The transgenes of EE-GM5 are a nematode resistance and an HPPD inhibitor herbicide tolerance gene. “Plant DNA” in the context of the present invention will refer to DNA originating from the plant which is transformed. Plant DNA will usually be found in the same genetic locus in the corresponding wild-type plant. The inserted T-DNA can be characterized by the location and the configuration at the site of incorporation of the recombinant DNA molecule in the plant genome. The site in the plant genome where a recombinant DNA has been inserted is also referred to as the “insertion site” or “target site”. Insertion of the recombinant DNA into the region of the plant genome referred to as “pre-insertion plant DNA” (or “pre-insertion locus”) can be associated with a deletion of plant DNA, referred to as “target site deletion”. A “flanking region” or “flanking sequence” as used herein refers to a sequence of at least 10 bp, at least 20 bp, at least 50 bp, and up to 5000 bp of DNA different from the introduced T-DNA, preferably DNA from the plant genome which is located either immediately upstream of and contiguous with or immediately downstream of and contiguous with the inserted T-DNA. Transformation procedures leading to random integration of the inserted T-DNA will result in transformants with different flanking regions, which are characteristic and unique for each transformant. When the recombinant DNA is introduced into a plant through traditional crossing, its insertion site in the plant genome, or its flanking regions, will generally not be changed.
An “isolated nucleic acid (sequence/molecule)” or “isolated DNA (sequence/molecule)”, as used herein, refers to a nucleic acid or DNA (sequence/molecule) which is no longer in the natural environment it was isolated from, e.g., the nucleic acid sequence in another (bacterial) host or in a plant genome, or a nucleic acid or DNA (sequence/molecule) fused to DNA or nucleic acid (sequence/molecule) from another origin, such as when contained in a chimeric gene under the control of a (heterologous) plant-expressible promoter. Any nucleic acid or DNA of this invention, including any primer, can also be non-naturally-occurring, such as a nucleic acid or DNA with a sequence identical to a sequence occurring in nature, but having a label (missing from the naturally-occurring counterpart), or with a sequence having at least one nucleotide addition or replacement or at least one internal nucleotide deletion compared to a naturally-existing nucleotide, or with a sequence having a sequence identity below 100% (not identical) to a naturally-existing nucleic acid or DNA or a fragment thereof, or a nucleic acid or DNA with a sequence consisting of nucleotide sequences from different origins that do not occur together in nature (a chimeric or hybrid DNA), or a man-made synthetic nucleic acid or DNA with a sequence different from the natural nucleic acid or DNA or a fragment thereof.
An event is defined as a (artificial) genetic locus that, as a result of genetic engineering, carries an inserted T-DNA or transgene comprising at least one copy of a gene of interest or of the genes of interest. The typical allelic states of an event are the presence or absence of the inserted T-DNA. An event is characterized phenotypically by the expression of the transgene or transgenes. At the genetic level, an event is part of the genetic make-up of a plant. At the molecular level, an event can be characterized by the restriction map (e.g., as determined by Southern blotting), by the upstream and/or downstream flanking sequences of the transgene, the location of molecular markers and/or the molecular configuration of the transgene. Usually transformation of a plant with a transforming DNA comprising at least one gene of interest leads to a population of transformants comprising a multitude of separate events, each of which is unique. An event is characterized by the inserted T-DNA and at least one of the flanking sequences.
An elite event, as used herein, is an event which is selected from a group of events, obtained by transformation with the same transforming DNA, based on an optimal trait efficacy and superior expression, stability of the transgene(s) and its compatibility with optimal agronomic characteristics of the plant comprising it. Thus the criteria for elite event selection are one or more, preferably two or more, advantageously all of the following:
It is preferred that the inserted T-DNA is associated with a position in the plant genome that allows easy introgression into desired commercial genetic backgrounds.
The status of an event as an elite event is confirmed by introgression of the elite event in different relevant genetic backgrounds and observing compliance with one, two, three or all of the criteria e.g. a), b), c) and d) above.
An “elite event” thus refers to a genetic locus comprising an inserted T-DNA, which meets the above-described criteria. A plant, plant material or progeny such as seeds can comprise one or more different elite events in its genome.
The tools developed to identify an elite event or the plant or plant material comprising an elite event, or products which comprise plant material comprising the elite event, are based on the specific genomic characteristics of the elite event, such as, a specific restriction map of the genomic region comprising the inserted T-DNA, molecular markers or the sequence of the flanking region(s) of the inserted T-DNA.
Once one or both of the flanking regions of the inserted T-DNA have been sequenced, primers and/or probes can be developed which specifically recognize this (these) sequence(s) in the nucleic acid (DNA or RNA) of a sample by way of a molecular biological technique. For instance a PCR method can be developed to identify the elite event in biological samples (such as samples of plants, plant material or products comprising plant material). Such a PCR is based on at least two specific “primers”, one recognizing a sequence within the 5′ or 3′ T-DNA flanking region of the elite event and the other recognizing a sequence within the inserted T-DNA. The primers preferably have a sequence of between 15 and 35 nucleotides which under optimized PCR conditions “specifically recognize” a sequence within the 5′ or 3′ T-DNA flanking region of the elite event and the inserted T-DNA of the elite event respectively, so that a specific fragment (“integration fragment” or discriminating amplicon) is amplified from a nucleic acid sample comprising the elite event. This means that only the targeted integration fragment, and no other sequence in the plant genome or inserted T-DNA, is amplified under optimized PCR conditions.
PCR primers suitable for the invention may be the following:
It will be understood that primers recognizing the 5′ T-DNA flanking sequences can be used in a PCR reaction together with primers recognizing the inserted T-DNA which are selected from the complement of SEQ ID No. 5 from nucleotide 167 to nucleotide 353 or T-DNA sequences downstream thereof and contiguous therewith, whereas primers recognizing the 3′ T-DNA flanking sequences can be used in a PCR reaction together with primers recognizing the inserted T-DNA which are selected from the sequence of SEQ ID No. 6 from nucleotide 1 to nucleotide 358, or T-DNA upstream thereof and contiguous therewith. Primers recognizing inserted T-DNA can also be selected from the sequence of SEQ ID No. 11 from nucleotide 1 to nucleotide 7459, or the sequence of SEQ ID No. 23 from nucleotide 1114 to nucleotide 8572, or the complement thereof.
The primers may of course be longer than the mentioned 17 consecutive nucleotides, and may, e.g., be 20, 21, 30, 35, 50, 75, 100, 150, 200 nt long or even longer. The primers may entirely consist of nucleotide sequence selected from the mentioned nucleotide sequences of flanking sequences and inserted T-DNA sequences. However, the nucleotide sequence of the primers at their 5′ end (i.e., outside of the 17 consecutive nucleotides at the 3′ end) is less critical. Thus, the 5′ sequence of the primers may comprise or consist of a nucleotide sequence selected from the flanking sequences or inserted T-DNA, as appropriate, but may contain several (e.g., 1, 2, 5, or 10) mismatches in comparison with the T-DNA or T-DNA flanking DNA. The 5′ sequence of the primers may even entirely be a nucleotide sequence unrelated to the flanking sequences or inserted T-DNA, such as, e.g., a nucleotide sequence representing one or more restriction enzyme recognition sites, or such as nucleotide sequences capable of binding other oligonucleotides, such as labelled oligonucleotides, such as FRET cassettes (LGC genomics; see Semagn et al., 2014, Mol Breeding 33:1-14, and U.S. Pat. No. 7,615,620). Such unrelated sequences or flanking DNA sequences with mismatches should preferably not be longer than 100, more preferably not longer than 50 or even 25 nucleotides. The primers can also be modified with a label, such as a fluorescent label.
Moreover, suitable primers may comprise or consist (essentially) of a nucleotide sequence at their 3′ end spanning the joining region between the 5′ or 3′ T-DNA flanking region-derived sequences and the inserted T-DNA sequences (located at nucleotides 166 and 167 in SEQ ID No. 5 and nucleotides 358 and 359 in SEQ ID No. 6, or nucleotides 1113 and 1114 in SEQ ID No. 24 and nucleotides 358 and 359 in SEQ ID No. 25) provided the mentioned 3′-located 17 consecutive nucleotides are not derived exclusively from either the inserted T-DNA or the T-DNA flanking sequences in SEQ ID No. 5 or 6 or SEQ ID No. 24 or 25.
It will also be immediately clear to the skilled artisan that properly selected PCR primer pairs should also not comprise sequences complementary to each other.
For the purpose of the invention, the “complement of a nucleotide sequence represented in SEQ ID No: X” is the nucleotide sequence which can be derived from the represented nucleotide sequence by replacing the nucleotides with their complementary nucleotide according to Chargaff's rules (A⇔T; G⇔C) and reading the sequence in the 5′ to 3′ direction, i.e., in opposite direction of the represented nucleotide sequence.
Examples of suitable primers are the oligonucleotide sequences of SEQ ID no. 13 or SEQ ID No. 19 or SEQ ID No. 26 or 27 (3′ or 5′ T-DNA flanking sequence recognizing primer), or SEQ ID No. 12 or SEQ ID No. 18 or SEQ ID No. 28 or 29 (inserted T-DNA recognizing primer for use with the 3′ or 5′ T-DNA flanking sequence recognizing primers).
Preferably, the amplified fragment has a length of between 50 and 500 nucleotides, such as a length between 50 and 150 nucleotides. The specific primers may have a sequence which is between 80 and 100% identical to a sequence within the 5′ or 3′ T-DNA flanking region of the elite event and the inserted T-DNA of the elite event, respectively, provided the mismatches still allow specific identification of the elite event with these primers under optimized PCR conditions. The range of allowable mismatches however, can easily be determined experimentally and are known to a person skilled in the art.
Detection of integration fragments can occur in various ways, e.g., via size estimation after gel analysis. The integration fragments may also be directly sequenced. Other sequence specific methods for detection of amplified DNA fragments are also known in the art. Amplified DNA fragments can also be detected using labelled sequences and detection of the label. For example, a labelled probe can be included in the reaction mixture which specifically binds to the amplified fragment. In one embodiment, the labelled probe (FRET hybridization probe) can comprise a fluorescent label and a quencher, such that the FRET cassette is no longer quenched and emits fluorescence when bound to the PCR product. Alternatively, a labelled FRET cassette, i.e., an oligonucleotide labeled with a fluorescent label and a quencher, can be included in the reaction mixture which specifically binds one of the primers in the reaction mixture, such as a FRET cassette directed to a 5′ extension of the primer used in the reaction mixture (see, e.g., Semagn et al., 2014, Mol Breeding 33:1-14, and U.S. Pat. No. 7,615,620). Fluorescence can be measured using methods known in the art. Fluorescence can be measured real-time, i.e., during each cycle of the PCR reaction. Fluorescence can also be measured at the end of the PCR reaction.
As the sequence of the primers and their relative location in the genome are unique for the elite event, amplification of the integration fragment will occur only in biological samples comprising (the nucleic acid of) the elite event. Preferably when performing a PCR to identify the presence of EE-GM5 in unknown samples, a control is included of a set of primers with which a fragment within a “housekeeping gene” of the plant species of the event can be amplified. Housekeeping genes are genes that are expressed in most cell types and which are concerned with basic metabolic activities common to all cells. Preferably, the fragment amplified from the housekeeping gene is a fragment which is larger than the amplified integration fragment. Depending on the samples to be analyzed, other controls can be included.
Standard PCR protocols are described in the art, such as in “PCR Applications Manual” (Roche Molecular Biochemicals, 2nd Edition, 1999, or 3rd Edition, 2006) and other references. The optimal conditions for the PCR, including the sequence of the specific primers, are specified in a “PCR (or Polymerase Chain Reaction) Identification Protocol” for each elite event. It is however understood that a number of parameters in the PCR Identification Protocol may need to be adjusted to specific laboratory conditions, and may be modified slightly to obtain similar results. For instance, use of a different method for preparation of DNA may require adjustment of, for instance, the amount of primers, polymerase and annealing conditions used. Similarly, the selection of other primers may dictate other optimal conditions for the PCR Identification Protocol. These adjustments will however be apparent to a person skilled in the art, and are furthermore detailed in current PCR application manuals such as the one cited above.
Alternatively, specific primers can be used to amplify an integration fragment that can be used as a “specific probe” for identifying EE-GM5 in biological samples. Contacting nucleic acid of a biological sample, with the probe, under conditions which allow hybridization of the probe with its corresponding fragment in the nucleic acid, results in the formation of a nucleic acid/probe hybrid. The formation of this hybrid can be detected (e.g., via labeling of the nucleic acid or probe), whereby the formation of this hybrid indicates the presence of EE-GM5. Such identification methods based on hybridization with a specific probe (either on a solid phase carrier or in solution) have been described in the art. The specific probe is preferably a sequence which, under optimized conditions, hybridizes specifically to a region comprising part of the 5′ or 3′ T-DNA flanking region of the elite event and part of the inserted T-DNA contiguous therewith (hereinafter referred to as “specific region”). Preferably, the specific probe comprises a sequence of between 50 and 500 bp, or of 100 to 350 bp which is at least 80%, or between 80 and 85%, or between 85 and 90%, or between 90 and 95%, or between 95% and 100% identical (or complementary), or is identical (or complementary) to the nucleotide sequence of a specific region of EE-GM5. Preferably, the specific probe will comprise a sequence of about 15 to about 100 contiguous nucleotides identical (or complementary) to a specific region of the elite event.
Oligonucleotides suitable as PCR primers for detection of the elite event EE-GM5 can also be used to develop a PCR-based protocol to determine the zygosity status of plants containing the elite event. To this end, two primers recognizing the wild-type locus before integration are designed in such a way that they are directed towards each other and have the insertion site located in between the primers. These primers may contain primers specifically recognizing the 5′ and/or 3′ T-DNA flanking sequences of EE-GM5. This set of primers recognizing the wild-type locus before integration, together with a third primer complementary to transforming DNA sequences (inserted T-DNA) allows simultaneous diagnostic PCR amplification of the EE-GM5 specific locus, as well as of the wild type locus. If the plant is homozygous for the transgenic locus or the corresponding wild type locus, the diagnostic PCR will give rise to a single PCR product typical, preferably typical in length, for either the transgenic or wild type locus. If the plant is hemizygous for the transgenic locus, two locus-specific PCR products will appear, reflecting both the amplification of the transgenic and wild type locus.
Alternatively, to determine the zygosity status of plants containing the elite event, two primers recognizing the wild-type locus before integration are designed in such a way that they are directed towards each other, and that one primer specifically recognizes the 5′ or the 3′ T-DNA flanking sequences contained in SEQ ID No. 5 or 6 or in SEQ ID No. 24 or 25, and that one primer specifically recognizes the 3′ or the 5′ T-DNA flanking sequences contained within SEQ ID No. 6 or 5 or SEQ ID No. 24 or 25, or specifically recognizes the pre-insertion locus. For the current invention, a suitable primer pair recognizing the wild type locus before integration is a primer pair containing one primer comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 21, and one primer comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 19. This set of primers, together with a third primer complementary to transforming DNA sequences (inserted T-DNA), or complementary to transforming DNA sequences and the 5′ or 3′ T-DNA flanking sequences contiguous therewith, and in a direction towards the primer which specifically recognizes the 5′ or the 3′ T-DNA flanking sequences (such as a primer comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 18, which is in a direction towards the primer comprising or consisting (essentially) of the nucleotide sequence of SEQ ID No. 19) allow simultaneous diagnostic PCR amplification of the EE-GM5 specific locus, as well as of the wild type locus. If the plant is homozygous for the transgenic locus or the corresponding wild type locus, the diagnostic PCR will give rise to a single PCR product typical for either the transgenic or wild type locus. If the plant is hemizygous for the transgenic locus, two locus-specific PCR products will appear, reflecting both the amplification of the transgenic and wild type locus.
Detection of the PCR products typical for the wild-type and transgenic locus can be based on determination of the length of the PCR products which can be typical for the wild-type and transgenic locus. Alternatively, detection of the PCR products typical for the wild-type and transgenic locus can be performed by modification of the primer specific for the pre-insertion locus and by modification of the primer specific for the inserted T-DNA, and detection of incorporation into a PCR product of the modified primers. For example, the primer specific for the pre-insertion locus and the primer specific for the inserted T-DNA can be labeled using a fluorescent label, wherein the labels are different for the two primers. Fluorescence can be detected when the primer is incorporated into a PCR product. If the plant is homozygous for the transgenic locus or the corresponding wild type locus, fluorescence can be detected of the label of the primer specific for the inserted T-DNA only or of the primer specific for the pre-insertion locus only. If the plant is hemizygous for the transgenic locus, fluorescence can be detected of both the label of the primer specific for the inserted T-DNA and of the primer specific for the pre-insertion locus, reflecting both the amplification of the transgenic and wild type locus. Alternatively, the primer specific for the pre-insertion locus and the primer specific for the inserted T-DNA can have a 5′ extension which specifically binds a labeled FRET cassette, i.e. an oligonucleotide labelled with a fluorescent label and a quencher, wherein the 5′ extension and the corresponding FRET cassettes are different for the two primers (see, e.g., Semagn et al., 2014, Mol Breeding 33:1-14, and U.S. Pat. No. 7,615,620). Fluorescence can be detected when the primer is incorporated into a PCR product and, subsequently, the FRET cassette is incorporated in the PCR product. If the plant is homozygous for the transgenic locus or the corresponding wild type locus, fluorescence can be detected of the FRET cassette specifically binding to the primer specific for the inserted T-DNA only or of the FRET cassette specifically binding to the primer specific for the pre-insertion locus only. If the plant is hemizygous for the transgenic locus, fluorescence can be detected of both of the FRET cassette specifically binding to the primer specific for the inserted T-DNA and of the FRET cassette specifically binding to the primer specific for the pre-insertion locus, reflecting both the amplification of the transgenic and wild type locus.
If the plant is homozygous for the transgenic locus or the corresponding wild type locus, the diagnostic PCR will give rise to a single PCR product typical, preferably typical in length, for either the transgenic or wild type locus. If the plant is hemizygous for the transgenic locus, two locus-specific PCR products will appear, reflecting both the amplification of the transgenic and wild type locus.
Alternatively, to determine the zygosity status of plants containing the elite event, presence of the event can be determined in a PCR reaction in a quantitative way as described in the Examples. To this end, two primers recognizing the event EE-GM5 are designed in such a way that they are directed towards each other, wherein one primer specifically recognizes the 5′ or 3′ T-DNA flanking sequence contained within SEQ ID No. 5 or 6 or within SEQ ID No. 24 or 25, and wherein one primer specifically recognizes the inserted T-DNA within SEQ ID no. 5 or 6 or within SEQ ID No. 24 or 25 or within SEQ ID No. 11 or 23. This set of primers allows PCR amplification of the EE-GM5 specific locus. The amplified DNA fragment can quantitatively be detected using a labeled probe which is included in the reaction mixture which specifically binds to the amplified fragment. The labeled probe can comprise a fluorescent label and a quencher, such that label is no longer quenched and emits fluorescence when bound to the PCR product. Fluorescence can be measured real-time, i.e. during each cycle of the PCR reaction, using methods known in the art. The PCR cycle at which the fluorescence exceeds a certain threshold level is a measure for the amount of EE-GM5 specific locus in the biological sample which is analyzed, and the zygosity status can be calculated based on reference homozygous and heterozygous samples.
Alternatively, zygosity status of plants comprising EE-GM5 can also be determined based on copy number analysis, using the Taqman chemistry and principles of Real-Time PCR. The alternative method will typically include a EE-GM5 specific reaction to quantify the EE-GM5 copy number, and a endogenous gene-specific reaction for normalization of the EE-GM5 copy number. Samples containing the EE-GM5 event in a homozygous state will have a relative copy number that is two-fold higher than hemizygous samples. Azygous samples will not amplify the EE-GM5 sequence in such a method.
Furthermore, detection methods specific for elite event EE-GM5 which differ from PCR based amplification methods can also be developed using the elite event specific sequence information provided herein. Such alternative detection methods include linear signal amplification detection methods based on invasive cleavage of particular nucleic acid structures, also known as Invader™ technology, (as described e.g. in U.S. Pat. No. 5,985,557 “Invasive Cleavage of Nucleic Acids”, U.S. Pat. No. 6,001,567 “Detection of Nucleic Acid sequences by Invader Directed Cleavage”, incorporated herein by reference). To this end, the target sequence is hybridized with a labeled first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 5 from nucleotide position 167 to nucleotide position 184 or its complement or comprising the nucleotide sequence of SEQ ID No. 6 from nucleotide position 341 to nucleotide position 358 or its complement, and is further hybridized with a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 5 from nucleotide 149 to nucleotide 166 or its complement or said nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 6 from nucleotide 359 to nucleotide 376 or its complement, wherein said first and second oligonucleotide overlap by at least one nucleotide.
The duplex or triplex structure which is produced by this hybridization allows selective probe cleavage with an enzyme (Cleavage®) leaving the target sequence intact. The cleaved labeled probe is subsequently detected, potentially via an intermediate step resulting in further signal amplification.
In one embodiment is provided a method of detecting the presence of elite event EE-GM5 in biological samples through hybridization with a substantially complementary labeled nucleic acid probe in which the probe:target nucleic acid ratio is amplified through recycling of the target nucleic acid sequence, said method comprising:
a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 5 from nucleotide position 167 to nucleotide position 184 or its complement or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 6 from nucleotide position 341 to nucleotide position 358 or its complement;
b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 5 from nucleotide 149 to nucleotide 166 or its complement or said second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No. 6 from nucleotide 359 to nucleotide 376 or its complement, wherein said first and second oligonucleotide overlap by at least one nucleotide and wherein either said first or said second oligonucleotide is labeled to be said labeled nucleic acid probe;
c) cleaving only the labeled probe within the probe:target nucleic acid sequence duplex with an enzyme which causes selective probe cleavage resulting in duplex disassociation, leaving the target sequence intact;
d) recycling of the target nucleic acid sequence by repeating steps (a) to (c); and
e) detecting cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence, and detecting the presence of elite event EE-GM5 in said biological samples.
Two nucleic acids are “substantially complementary” as used herein, when they are not the full complement of each other (as defined herein), such as when their sequences are at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary to each other.
A “kit” as used herein refers to a set of reagents for the purpose of performing the method of the invention, more particularly, the identification of the elite event EE-GM5 in biological samples or the determination of the zygosity status of EE-GM5 containing plant material. More particularly, a preferred embodiment of the kit of the invention comprises at least one or two specific primers, as described above for identification of the elite event, or three specific primers, or two specific primers and one specific probe, as described above for the determination of the zygosity status. Optionally, the kit can further comprise any other reagent described herein in the PCR Identification Protocol or any of the other protocols as described herein for EE-GM5 detection. Alternatively, according to another embodiment of this invention, the kit can comprise a specific probe, as described above, which specifically hybridizes with nucleic acid of biological samples to identify the presence of EE-GM5 therein. Optionally, the kit can further comprise any other reagent (such as but not limited to hybridizing buffer, label) for identification of EE-GM5 in biological samples, using the specific probe.
The kit of the invention can be used, and its components can be specifically adjusted, for purposes of quality control (e.g., purity of seed lots), detection of the presence or absence of the elite event in plant material or material comprising or derived from plant material, such as but not limited to food or feed or industrial products.
As used herein, “sequence identity” with regard to nucleotide sequences (DNA or RNA), refers to the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences. The alignment of the two nucleotide sequences is performed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983, Proc. Nat. Acad. Sci. USA 80:726) using a window-size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4. Computer-assisted analysis and interpretation of sequence data, including sequence alignment as described above, can, e.g., be conveniently performed using the sequence analysis software package of the Genetics Computer Group (GCG, University of Wisconsin Biotechnology Center). Sequences are indicated as “essentially similar” when such sequences have a sequence identity of at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9%. It is clear that when RNA sequences are said to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Also, it is clear that small differences or mutations may appear in DNA sequences over time and that some mismatches can be allowed for the event-specific primers or probes of the invention, so any DNA sequence indicated herein in any embodiment of this invention for any 3′ or 5′ T-DNA flanking DNA or for any insert or inserted T-DNA or any primer or probe of this invention, also includes sequences essentially similar to the sequences provided herein, such as sequences hybridizing to or with at least 90%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the sequence given for any 3′ or 5′ T-DNA flanking DNA, for any primer or probe or for any insert or inserted T-DNA of this invention, such as a nucleotide sequence differing in 1 to 200, 1 to 150, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 nucleotides from any given sequence.
The term “primer” as used herein encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR. Typically, primers are oligonucleotides from 10 to 30 nucleotides, but longer sequences can be employed. Primers may be provided in double-stranded form, though the single-stranded form is preferred. Probes can be used as primers, but are designed to bind to the target DNA or RNA and need not be used in an amplification process.
The term “recognizing” as used herein when referring to specific primers or probes, refers to the fact that the specific primers or probes specifically hybridize to a nucleic acid sequence in the elite event under the conditions set forth in the method (such as the conditions of the PCR Identification Protocol), whereby the specificity is determined by the presence of positive and negative controls.
The term “hybridizing” as used herein when referring to specific probes, refers to the fact that the probe binds to a specific region in the nucleic acid sequence of the elite event under standard stringency conditions. Standard stringency conditions as used herein refers to the conditions for hybridization described herein or to the conventional hybridizing conditions as described by Sambrook et al., 1989 (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY) which for instance can comprise the following steps: 1) immobilizing plant genomic DNA fragments on a filter, 2) prehybridizing the filter for 1 to 2 hours at 42° C. in 50% formamide, 5×SSPE, 2×Denhardt's reagent and 0.1% SDS, or for 1 to 2 hours at 68° C. in 6×SSC, 2×Denhardt's reagent and 0.1% SDS, 3) adding the hybridization probe which has been labeled, 4) incubating for 16 to 24 hours, 5) washing the filter for 20 min. at room temperature in 1×SSC, 0.1% SDS, 6) washing the filter three times for 20 min. each at 68° C. in 0.2×SSC, 0.1% SDS, and 7) exposing the filter for 24 to 48 hours to X-ray film at −70° C. with an intensifying screen.
As used in herein, a biological sample is a sample of a plant, plant material or products comprising plant material. The term “plant” is intended to encompass soybean (Glycine max) plant tissues, at any stage of maturity, as well as any cells, tissues, or organs taken from or derived from any such plant, including without limitation, any seeds, leaves, stems, flowers, roots, single cells, gametes, cell cultures, tissue cultures or protoplasts. “Plant material”, as used herein refers to material which is obtained or derived from a plant. Products comprising plant material relate to food, feed or other products which are produced using plant material or can be contaminated by plant material. It is understood that, in the context of the present invention, such biological samples are tested for the presence of nucleic acids specific for EE-GM5, implying the presence of nucleic acids in the samples. Thus the methods referred to herein for identifying elite event EE-GM5 in biological samples, relate to the identification in biological samples of nucleic acids which comprise the elite event.
As used herein “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, reagents or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA sequence which is functionally or structurally defined, may comprise additional DNA sequences, such as promoter, leader, trailer, and/or transcript termination sequences (possibly also including a DNA encoding a targeting or transit peptide).
The present invention also relates to the development of an elite event EE-GM5 in soybean plants comprising this event, the progeny plants and seeds comprising elite event EE-GM5 obtained from these plants and to the plant cells, or plant material derived from plants comprising this event, as well as to such plants or seeds treated with one or more of the compound(s) and/or biological control agent(s) of the invention, or a mixture as described herein. Plants comprising elite event EE-GM5 can be obtained as described in the Examples. This invention also relates to seed comprising elite event EE-GM5 deposited at the ATCC under deposit number PTA-123625 or derivatives therefrom comprising elite event EE-GM5. “Derivatives (of seed)” as used herein, refers to plants which can be grown from such seed, progeny resulting from selfing, crossing or backcrossing, as well as plant cells, organs, parts, tissue, cell cultures, protoplasts, and plant material of same. This includes such plants or seeds treated with one or more of the compound(s) and/or biological control agent(s) of the invention, or a mixture/combination as described herein.
Soybean plants or plant material comprising EE-GM5 can be identified according to any one of the identification protocols for EE-GM5 as described in the Examples, including the End-Point method for EE-GM5 identity analysis in Example 2.1, the End-Point method for EE-GM5 identity and zygosity analysis as described in Example 2.2, the Real-Time PCR method for EE-GM5 Low Level Presence analysis as described in Example 2.3, or the Real-Time PCR for EE-GM5 low level presence analysis as described in Example 2.4. Briefly, soybean genomic DNA present in the biological sample is amplified by PCR using a primer which specifically recognizes a sequence within the 5′ or 3′ T-DNA flanking sequence of EE-GM5 such as the primer with the sequence of SEQ ID NO: 13 or SEQ ID No. 19, and a primer which recognizes a sequence in the inserted T-DNA, such as the primer with the sequence of SEQ ID No. 12 or SEQ ID No. 18, or with a primer which recognizes the 5′ or 3′ T-DNA flanking sequence of EE-GM5 and the inserted T-DNA contiguous therewith. DNA primers which amplify part of an endogenous soybean sequence are used as positive control for the PCR amplification. If upon PCR amplification, the material yields a fragment of the expected size or gives rise to fluorescence of the expected fluorescent label, the material contains plant material from a soybean plant harboring elite event EE-GM5.
Plants harboring EE-GM5 are characterized by their nematode resistance, particularly SCN, lesion nematode and/or root-knot nematode (“RKN”) and/or reniform nematode resistance, as well as by their tolerance to HPPD inhibitors such as isoxaflutole, topramezone or mesotrione. Soybean plants in different commercially available varieties harboring EE-GM5 are also characterized by having agronomical characteristics that are comparable to the corresponding non-transgenic isogenic commercially available varieties, in the absence of HPPD inhibitor herbicide application and SCN infestation. It has been observed that the presence of an inserted T-DNA in the insertion region of the soybean plant genome described herein, confers particularly interesting phenotypic and molecular characteristics to the plants comprising this event.
Also provided herein is a method for producing a soybean plant resistant to SCN and tolerant to HPPD inhibitor herbicides, comprising introducing resistance to SCN and tolerance to HPPD inhibitor herbicides into the genome of a soybean plant by crossing a first soybean plant lacking a Cry14Ab-1-encoding gene and lacking an HPPD-4-encoding gene with an EE-GM5-containing soybean plant, and selecting a progeny plant resistant to SCN and tolerant to HPPD inhibitor herbicides. In one embodiment, this method includes the treatment of said plant with one or more of the compound(s) and/or biological control agent(s) of the invention, or a mixture as described herein, such as one or more of the nematicidal compound(s) and/or nematicidal biological control agent(s) as described herein. Resistance to SCN can be measured using a standard SCN greenhouse assay, e.g., on the world wide web at plantpath.iastate.edu/tylkalab/greenhouse-resistance-screening and on the world wide web at plantmanagementnetwork.org/pub/php/review/2009/sce08/.
One embodiment of this invention provides an elite event in soybean plants, obtainable by insertion of 2 transgenes at a specific location in the soybean genome, which elite event confers resistance to nematodes and tolerance to an HPPD inhibitor herbicide such as isoxaflutole, topramezone or mesotrione on such soybean plants, and wherein such elite event has an agronomic performance essentially similar to isogenic lines (as used herein, “isogenic lines” or “near-isogenic lines” are soybean lines of the same genetic background but lacking the transgenes, such as plants of the same genetic background as the plant used for transformation, or segregating sister lines (“nulls”) having lost the transgenes). Particularly, the current invention provides an elite event in soybean plants, wherein the insertion or presence of said elite event in the genome of such soybean plants does not appear to cause an increased susceptibility to disease, does not cause a yield penalty, or does not cause increased lodging, as compared to isogenic lines or to commercial soybean cultivars. Hence, the current invention provides an elite event in soybean plants, designated as EE-GM5, which results in soybean plants that have improved resistance to nematodes and can tolerate the application of an HPPD inhibitor herbicide such as isoxaflutole, topramezone or mesotrione without negatively affecting the yield of said soybean plants compared to isogenic lines, which soybean plants are not statistically significantly different in their disease susceptibility, or lodging, from isogenic soybean plants or from commercial soybean cultivars. These characteristics make the current elite event a valuable tool in a nematode control and weed resistance management program. In one embodiment, event EE-GM5 is combined with one or more soybean transformation events containing a herbicide tolerance gene, such as one or more soybean transformation events providing tolerance to any one or a combination of glyphosate-based, glufosinate-based, HPPD inhibitor-based, PPO inhibitor-based, sulfonylurea- or imidazolinone-based, AHAS- or ALS-inhibiting and/or auxin-type (e.g., dicamba, 2,4-D) herbicides, including but not limited to Event EE-GM3 (aka FG-072, MST-FG072-3, described in WO2011063411, USDA-APHIS Petition 09-328-01p), Event SYHTOH2 (aka OH2, SYN-ØØØH2-5, described in WO2012/082548 and 12-215-01p), Event DAS-68416-4 (aka Enlist Soybean, described in WO2011/066384 and WO2011/066360, USDA-APHIS Petition 09-349-01p), Event DAS-44406-6 (aka Enlist E3, DAS-444Ø6-6, described in WO2012/075426 and USDA-APHIS 11-234-01p), Event MON87708 (dicamba-tolerant event of Roundup Ready 2 Xtend Soybeans, described in WO2011/034704 and USDA-APHIS Petition 10-188-01p, MON-877Ø8-9), Event MON89788 (aka Genuity Roundup Ready 2 Yield, MON-89788-1, described in WO2006/130436 and USDA-APHIS Petition 06-178-01p), Event 40-3-2 (aka Roundup Ready, GTS 40-3-2, MON-Ø4Ø32-6, described in USDA-APHIS Petition 93-258-01), Event A2704-12 (aka LL27, ACS-GM005-3, described in WO2006108674 and USDA-APHIS Petition 96-068-01p), Event 127 (aka BPS-CV127-9, described in WO2010/080829), Event A5547-127 (aka LL55, EE-GM2, ACS-GMØØ6-4, described in WO2006108675 and in USDA-APHIS Petition 96-068-01p), event MON87705 (MON-877Ø5-6, Vistive Gold, published PCT patent application WO2010/037016, USDA-APHIS Petition 09-201-01p), Event IND-ØØ41Ø-5 (aka HB4 Soybean, US FDA BNF No. 000155, USDA-APHIS Docket No. APHIS-2017-0075, www.aphis.usda.gov/brs/aphisdocs/17_22301p.pdf), Event DP305423 (aka DP-3Ø5423-1, published PCT patent application WO2008/054747, USDA-APHIS Petition 06-354-01p), Event DAS-81419-2 (aka Conkesta™ Soybean, described in WO2013016527 and USDA-APHIS Petition 12-272-01p), Event 3560.4.3.5 (aka DP-356043-5, described in WO2008/002872), Event MON87712 (aka MON-87712-4, described in WO2012/051199, USDA-APHIS Petition 11-202-01p), Event MON87769 (aka MON-87769-7, described in WO2009102873 and in USDA-APHIS Petition 09-183-01p, or EE-GM5 is combined with a combination of the following events: Event MON89788×MON87708 (aka Roundup Ready 2 Xtend Soybeans, MON-877Ø8-9×MON-89788-1), Event HOS×Event 40-3-2 (aka Plenish High Oleic Soybeans×Roundup Ready Soybeans), Event EE-GM3×EE-GM2 (aka FG-072xLL55, described in WO2011063413), Event MON 87701×MON 89788 (aka Intacta RR2 Pro Soybean, MON-877Ø1-2×MON-89788-1), DAS-81419-2×DAS-44406-6 (aka Conkesta™ Enlist E3™ Soybean, DAS-81419-2×DAS-444Ø6-6), Event DAS-68416-4×Event MON 89788 (aka Enlist™ RoundUp Ready® 2 Soybean, DAS-68416-4×MON-89788-1), Event MON 87705×Event MON 89788 (aka Vistive Gold, MON-877Ø5-6×MON-89788-1), Event DP305423×Event 40-3-2 (DP-3Ø5423-1×MON-Ø4Ø32-6), Event DP305423×MON87708 (DP-3Ø5423-1×MON-877Ø8-9), Event DP305423×MON87708×Event MON89788 (DP-3Ø5423-1×MON-877Ø8-9×MON-89788-1), Event DP305423×Event MON89788 (DP-3Ø5423-1×MON-89788-1), Event MON87705×MON87708 (MON-877Ø5-6×MON-877Ø8-9), Event MON87705×MON87708×MON89788 (MON-877Ø5-6×MON-877Ø8-9×MON-89788-1), Event MON89788×MON87708×A5547-127 (MON-89788-1×MON-877Ø8-9×ACS-GM006-4), Event MON87751×MON87701×MON87708×MON89788 (MON-87751-7×MON-877Ø1-2×MON-877Ø8-9×MON-89788-1), or Event MON87769×Event MON89788 (aka Omega-3×Genuity Roundup Ready 2 Yield Soybeans, MON-87769-7×MON-89788-1). This includes such plants or seeds containing such combination of soybean GM events, treated with one or more of the compound(s) and/or biological control agent(s) of the invention, or a mixture, as described herein. In another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
In one embodiment of the invention, plants or seeds comprising event EE-GM5 also comprise another transformation event providing nematode control, such as a transformation event that produces double-stranded RNA interfering with one or more nematode genes critical for nematode feeding, development, or reproduction, or a transformation event that produces another nematicidal toxin (different from Cry14Ab-1). In another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
Provided herein is also a soybean plant or part thereof comprising event EE-GM5, wherein representative soybean seed comprising event EE-GM5 has been deposited under ATCC accession number PTA-123625. Further provided herein are seeds of such plants, comprising such event, as well as a soybean product produced from such seeds, wherein said soybean product comprises event EE-GM5. Such soybean product can be or can comprise soybean meal, ground soybean grain, soybean flakes, or a product comprising any of these processed soybean products. Particularly, such soybean product comprises a nucleic acid that produces an amplicon diagnostic of or specific for event EE-GM5, such amplicon comprising the sequence of any one of SEQ ID No. 1 or 3 or SEQ ID No. 2 or 4. Also provided herein is a method for producing a soybean product, comprising obtaining a soybean seed or grain comprising event EE-GM5, and producing such soybean product therefrom. Also provided herein is a method of obtaining processed food, feed or industrial products derived from soybean grain, such as soybean oil, soybean protein, lecithin, soybean milk, tofu, margarine, biodiesel, biocomposites, adhesives, solvents, lubricants, cleaners, foam, paint, ink, candles, soybean-oil or soybean protein-containing food or (animal) feed products, said method comprising obtaining grain comprising EE-GM5 and producing said processed food, feed or industrial product from said grain. In one embodiment, this process can also include the step of a obtaining a soybean seed or plant comprising event EE-GM5, growing said seed or plant in a field, and harvesting soybean grain. Optionally, this method includes application of an HPPD inhibitor herbicide such as IFT, topramezone or mesotrione before planting, before emergence, after emergence or over the top of plants comprising EE-GM5. In one embodiment, the above soybean-derived processed food, feed or industrial products are included in this invention, such as such processed products that produce an EE-GM5 event-specific amplicon using the methods described herein, or that comprise the nucleotide sequence of any one of SEQ ID No. 1, 3 or 5, or SEQ ID No. 2, 4, or 6.
Also provided herein is a soybean plant, which is progeny of any of the above soybean plants, and which comprises event EE-GM5, such as a progeny plant or seed of any one of the above soybean plants that comprises the sequence of SEQ ID No. 1 or 3 or the sequence of SEQ ID No. 2 or 4, or a progeny plant or seed of any one of the above soybean plants that comprises the sequence of SEQ ID No. 1 or 3 and the sequence of SEQ ID No. 2 or 4, or a progeny plant or seed of any one of the above soybean plants that comprises the sequence of SEQ ID No. 5 or SEQ ID No. 24 or the sequence of SEQ ID No. 6 or SEQ ID No. 25, or a progeny plant or seed of any one of the above soybean plants that comprises the sequence of SEQ ID No. 5 or SEQ ID No. 24 and the sequence of SEQ ID No. 6 or SEQ ID No. 25. In one embodiment, such plant or seed is treated with one or more of the compound(s) and/or biological control agent(s) of the invention, or a mixture as described herein. In another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
Further provided herein is a method for producing a soybean plant resistant to nematodes and tolerant to isoxaflutole and/or topramezone and/or mesotrione herbicide, comprising introducing into the genome of such plant event EE-GM5, particularly by crossing a first soybean plant lacking event EE-GM5 with a soybean plant comprising EE-GM5, and selecting a progeny plant resistant to nematodes and tolerant to isoxaflutole and/or topramezone and/or mesotrione herbicide. In one embodiment, such method comprises treating the plant or seed of the invention with one or more of the compound(s) and/or biological control agent(s) of the invention, or a mixture as described herein. In another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
Also provided herein is a soybean plant resistant to nematodes and tolerant to isoxaflutole, topramezone or mesotrione herbicide with acceptable agronomical characteristics, comprising a Cry14Ab-1-encoding gene and HPPD-4-encoding gene, and capable of producing an amplicon diagnostic for event EE-GM5. Also provided herein are the specific isolated amplicons (DNA sequence fragments) as such, that can be obtained using the specific detection tools described herein, particularly amplicons including in their sequence a DNA fragment originating from 5′ or 3′ T-DNA flanking DNA and the DNA inserted in the plant genome by transformation, as defined herein.
Further provided herein is a method for controlling weeds in a field of soybean plants comprising event EE-GM5, or a field to be planted with such soybean plants (wherein said plants are planted in said field after treatment), comprising treating the field with an effective amount of an HPPD inhibitor herbicide such as an isoxaflutole-based or topramezone-based or mesotrione-based herbicide, wherein such plants are tolerant to such herbicide. In one embodiment, such method comprises treating the soybean plants or seeds, or the soil in which the soybean plants or seeds are grown or are intended to be grown, with one or more of the compound(s) and/or biological control agent(s) or a mixture comprising them, as described herein, such as wherein said compound or biological control agent is nematicidal, insecticidal, or fungicidal.
Further provided herein is a DNA comprising the sequence of SEQ ID No. 5 or 6 or a sequence essentially similar thereto, and any plant, cell, tissue or seed, particularly of soybean, comprising such DNA sequence, such as a plant, cell, tissue, or seed comprising EE-GM5. Also included herein is any soybean plant, cell, tissue or seed, comprising the DNA sequence (heterologous or foreign to a conventional soybean plant, seed, tissue or cell) of SEQ ID No. 5 or 6, or comprising a DNA sequence with at least 99% or 99.5% sequence identity to the sequence of SEQ ID No. 5 or 24 or SEQ ID No. 6 or 25.
Also described is a chimeric DNA comprising an inserted T-DNA, wherein the sequence of said inserted T-DNA comprises the sequence of SEQ ID No. 11 from nucleotide 1 to nucleotide 7459, or SEQ ID No. 23 from nucleotide 1114 to nucleotide 8572, or a sequence with at least 97, 98, 99, 99.5 or at least 99.9% sequence identity thereto, flanked by a 5′ and a 3′ T-DNA flanking region, wherein the 5′ T-DNA flanking region immediately upstream of and contiguous with said inserted T-DNA is characterized by a sequence comprising the sequence of SEQ ID No. 5 from nucleotide 1 to nucleotide 166 or of SEQ ID No. 24 from nucleotide 1 to nucleotide 1113, and wherein the 3′ T-DNA flanking region immediately downstream of and contiguous with said inserted T-DNA is characterized by a sequence comprising the sequence of SEQ ID No. 6 from nucleotide 359 to nucleotide 691, or the nucleotide sequence of the complement of SEQ ID No. 25 from nucleotide 359 to nucleotide 1449. In one embodiment, the sequence of said inserted T-DNA consists of the sequence of SEQ ID No. 11 from nucleotide 1 to nucleotide 7459, or SEQ ID No. 23 from nucleotide 1114 to nucleotide 8572, or a sequence with at least 97, 98, 99, 99.5 or at least 99.9% sequence identity thereto, flanked by part of a 5′ and a 3′ T-DNA flanking region, wherein the part of said 5′ T-DNA flanking region immediately upstream of and contiguous with said inserted T-DNA is characterized by a sequence consisting of the sequence of SEQ ID No. 5 from nucleotide 1 to nucleotide 166 or of SEQ ID No. 24 from nucleotide 1 to nucleotide 1113, or a sequence with at least 97, 98, 99, 99.5 or at least 99.9% sequence identity thereto, and wherein the part of the 3′ T-DNA flanking region immediately downstream of and contiguous with said inserted T-DNA is characterized by a sequence consisting of the sequence of SEQ ID No. 6 from nucleotide 359 to nucleotide 691 or the nucleotide sequence of the complement of SEQ ID No. 25 from nucleotide 359 to nucleotide 1449, or a sequence with at least 97, 98, 99, 99.5 or at least 99.9% sequence identity thereto.
Chimeric DNA refers to DNA sequences, including regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric DNA may comprise DNA regions adjacent to each other that are derived from different sources, or which are arranged in a manner different from that found in nature. Examples of a chimeric DNA are the sequences of SEQ ID No. 5 or 6.
Also provided herein is a transgenic soybean plant, plant cell, tissue, or seed, comprising in their genome event EE-GM5 characterized by a nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID No. 1 or 3 and a nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID No. 2 or 4, or the complement of said sequences, as well as a soybean plant, plant cell, tissue, or seed, comprising in their genome event EE-GM5 characterized by a nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID No. 5 or 24 and SEQ ID No. 6 or 25, or the complement of said sequences.
Even further provided herein is a soybean plant, cell, tissue or seed, comprising EE-GM5, characterized by comprising in the genome of its cells a nucleic acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID No. 1, 3, 5 or 24 and a nucleic acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID No. 2, 4, 6, or 25, or the complement of said sequences.
In one embodiment, the above plant or seed comprising EE-GM5 is treated with one or more of the compound(s) and/or biological control agent(s) of the invention, or a mixture as described herein.
The term “isoxaflutole”, as used herein, refers to the herbicide isoxaflutole [i.e. (5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone], the active metabolite thereof, diketonitrile, and any mixtures or solutions comprising said compound. HPPD inhibiting herbicides useful for application on the event of this invention are the diketonitriles, e.g., 2-cyano-3-cyclopropyl-1-(2-methylsulphonyl-4-trifluoromethylphenyl)-propane-1,3-dione and 2-cyano-1-[4-(methylsulphonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-fione; other isoxazoles; and the pyrazolinates, e.g. topramezone [i.e. [3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methyl sulfonyl) phenyl](5-hydroxy-1-methyl-1H-pyrazol-4-yl)methanone], and pyrasulfotole [(5-hydroxy-1,3-dimethylpyrazol-4-yl(2-mesyl-4-trifluaromethylphenyl) methanone]; or mesotrione [2-[4-(Methylsulfonyl)-2-nitrobenzoyl]cyclohexane-1,3-dione]; or 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide]; or 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide; or pyrazofen [2-[4-(2,4-dichlorobenzoyl)-1,3-dimethylpyrazol-5-yloxy]acetophenone].
In one embodiment of this invention, a field to be planted with soybean plants containing the EE-GM5 event, can be treated with an HPPD inhibitor herbicide, such as isoxaflutole (‘IFT’), topramezone or mesotrione, or with both an HPPD inhibitor herbicide and glyphosate, before the soybean is sown, which cleans the field of weeds that are killed by the HPPD inhibitor and/or glyphosate, allowing for no-till practices, followed by planting or sowing of the soybeans in that same pre-treated field later on (burn-down application using an HPPD inhibitor herbicide). The residual activity of IFT will also protect the emerging and growing soybean plants from competition by weeds in the early growth stages. Once the soybean plants have a certain size, and weeds tend to re-appear, an HPPD inhibitor or a mixture of an HPPD inhibitor with a selective (conventional) soybean herbicide or a mixture of an HPPD inhibitor with a herbicide that is non-selective in soybean (e.g., glyphosate or glufosinate) but for which the plants contain a tolerance gene/locus so that said plants are tolerant to said herbicide, can be applied as post-emergent herbicide over the top of the plants.
In another embodiment of this invention, a field in which seeds containing the EE-GM5 event were sown, can be treated with an HPPD inhibitor herbicide, such as IFT, topramezone or mesotrione, before the soybean plants emerge but after the seeds are sown (the field can be made weed-free before sowing using other means, including conventional tillage practices such as ploughing, chisel ploughing, or seed bed preparation), where residual activity will keep the field free of weeds killed by the herbicide so that the emerging and growing soybean plants have no competition by weeds (pre-emergence application of an HPPD inhibitor herbicide). Once the soybean plants have a certain size, and weeds tend to re-appear, an HPPD inhibitor-or an HPPD inhibitor-soybean selective (conventional) herbicide mixture or a mixture of an HPPD inhibitor with a herbicide that is non-selective in soybean (e.g., glyphosate or glufosinate) but for which the plants contain a tolerance gene/locus so that said plants are tolerant to said herbicide—can be applied as post-emergent herbicide over the top of the plants.
In one embodiment of the invention is provided a process for weed control comprising sowing in a field EE-GM5-containing soybean seeds, and treating said field with an HPPD inhibitor herbicide before plants emerge from said seed, but after the seeds are sown.
In another embodiment of this invention, plants containing the EE-GM5 event can be treated with an HPPD inhibitor herbicide, such as IFT, topramezone or mesotrione, over the top of the soybean plants that have emerged from the seeds that were sown, which cleans the field of weeds killed by the HPPD inhibitor, which application can be together with (e.g., in a spray tank mix), followed by or preceded by a treatment with a selective soybean post-emergent herbicide, or a herbicide that is non-selective in soybean (e.g., glyphosate or glufosinate) for which the plants contain a tolerance gene/locus so that said plants are tolerant to said herbicide, over the top of the plants (post-emergence application of an HPPD inhibitor herbicide (with or without said soybean selective or non-selective herbicide)).
In one embodiment, such methods involving use of an HPPD inhibitor herbicide, also comprise treating the soybean plants or seeds, or the soil in which the soybean plants or seeds are grown or are intended to be grown, with one or more of the compound(s) and/or biological control agent(s) or a mixture comprising them, as described herein, such as wherein said compound or biological control agent is nematicidal, insecticidal, or fungicidal. In another embodiment such plants or seeds also comprise one or more native soybean SCN resistance loci or genes, such as one or more of the SCN resistance genes or loci from the resistance sources of Table 1, or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332, or PI 437654.
Also, in accordance with the current invention, soybean plants harboring EE-GM5 (which may also contain another herbicide tolerance soybean event/trait as described herein) may be treated with, or soybean seeds harboring EE-GM5 may be coated with, any soybean insectide, herbicide or fungicide. In one embodiment, such plants or seeds also contain one or more soybean SCN resistance genes from from the resistance sources of Table 1, or or one or more of the SCN resistance genes or loci from the resistance sources PI 88788, PI 548402, PI 209332 or PI 437654, or comprise one or more of the soybean SCN resistance loci or genes selected from the group consisting of: rhg1, rhg1-b, rhg2, rhg3, Rhg4, Rhg5, qSCN11, cqSCN-003, cqSCN-005, cqSCN-006, and cqSCN-007.
The compounds (such as pesticides or herbicides), biological control agents, or plant growth regulators of the invention as referred to herein are described below, as well as formulations, mixtures and types of treatment. In one embodiment, the compound(s) and/or biological control agent(s) of the invention, or a mixture comprising them, is/are used as seed treatment on the seeds of the invention, as described below.
Insecticides/Acaricides/Nematicides
The active ingredients specified herein by their Common Name are known and described, for example, in The Pesticide Manual (17th Ed., British Crop Protection Council, updated version at on the world wide web at bcpc.org/product/bcpc-online-pesticide-manual-latest-version) or can be searched on the internet (e.g., on the world wide web at alanwood.net/pesticides). The classification is based on the current IRAC Mode of Action Classification Scheme at the time of filing of this patent application.
In one embodiment, an insecticide/acaricide/nematicide used in this invention is from the following groups IAN1 to IAN30:
IAN1: Acetylcholinesterase (AChE) inhibitors, preferably carbamates selected from alanycarb, aldicarb, bendiocarb, benfuracarb, butocarboxim, butoxycarboxim, carbaryl, carbofuran, carbosulfan, ethiofencarb, fenobucarb, formetanate, furathiocarb, isoprocarb, methiocarb, methomyl, metolcarb, oxamyl, pirimicarb, propoxur, thiodicarb, thiofanox, triazamate, trimethacarb, XMC and xylylcarb, or organophosphates selected from acephate, azamethiphos, azinphos-ethyl, azinphos-methyl, cadusafos, chlorethoxyfos, chlorfenvinphos, chlormephos, chlorpyrifos-methyl, coumaphos, cyanophos, demeton-S-methyl, diazinon, dichlorvos/DDVP, dicrotophos, dimethoate, dimethylvinphos, disulfoton, EPN, ethion, ethoprophos, famphur, fenamiphos, fenitrothion, fenthion, fosthiazate, heptenophos, imicyafos, isofenphos, isopropyl O-(methoxyaminothiophosphoryl) salicylate, isoxathion, malathion, mecarbam, methamidophos, methidathion, mevinphos, monocrotophos, naled, omethoate, oxydemeton-methyl, parathion-methyl, phenthoate, phorate, phosalone, phosmet, phosphamidon, phoxim, pirimiphos-methyl, profenofos, propetamphos, prothiofos, pyraclofos, pyridaphenthion, quinalphos, sulfotep, tebupirimfos, temephos, terbufos, tetrachlorvinphos, thiometon, triazophos, triclorfon and vamidothion.
IAN2: GABA-gated chloride channel blockers, preferably cyclodiene-organochlorines selected from chlordane and endosulfan or phenylpyrazoles (fiproles), for example ethiprole and fipronil.
IAN3: Sodium channel modulators, preferably pyrethroids selected from acrinathrin, allethrin, d-cis-trans allethrin, d-trans allethrin, bifenthrin, bioallethrin, bioallethrin s-cyclopentenyl isomer, bioresmethrin, cycloprothrin, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, gamma-cyhalothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, theta-cypermethrin, zeta-cypermethrin, cyphenothrin [(1R)-trans-isomer], deltamethrin, empenthrin [(EZ)-(1R)-isomer], esfenvalerate, etofenprox, fenpropathrin, fenvalerate, flucythrinate, flumethrin, tau-fluvalinate, halfenprox, imiprothrin, kadethrin, momfluorothrin, permethrin, phenothrin [(1R)-trans-isomer], prallethrin, pyrethrins (pyrethrum), resmethrin, silafluofen, tefluthrin, tetramethrin, tetramethrin [(1R)-isomer)], tralomethrin and transfluthrin or DDT or methoxychlor.
IAN4: Nicotinic acetylcholine receptor (nAChR) competitive modulators, preferably neonicotinoids selected from acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam or nicotine or sulfoxaflor or flupyradifurone.
IAN5: Nicotinic acetylcholine receptor (nAChR) allosteric modulators, preferably spinosyns selected from spinetoram and spinosad.
IAN6: Glutamate-gated chloride channel (GluCl) allosteric modulators, preferably avermectins/milbemycins selected from abamectin, emamectin benzoate, lepimectin and milbemectin.
IAN7: Juvenile hormone mimics, preferably juvenile hormone analogues selected from hydroprene, kinoprene and methoprene, or fenoxycarb or pyriproxyfen.
IAN8: Miscellaneous non-specific (multi-site) inhibitors, preferably alkyl halides selected from methyl bromide and other alkyl halides, or chloropicrine or sulphuryl fluoride or borax or tartar emetic or methyl isocyanate generators selected from diazomet and metam.
IAN9: Chordotonal organ TRPV channel modulators selected from pymetrozine, afidopyropen and pyrifluquinazone.
IAN10: Mite growth inhibitors selected from clofentezine, hexythiazox, diflovidazin and etoxazole.
IAN11: Microbial disruptors of the insect gut membrane selected from Bacillus thuringiensis subspecies israelensis, Bacillus sphaericus, Bacillus thuringiensis subspecies aizawai, Bacillus thuringiensis subspecies kurstaki, Bacillus thuringiensis subspecies tenebrionis, and B.t. plant proteins selected from Cry1Ab, Cry1Ac, Cry1Fa, Cry1A.105, Cry2Ab, Vip3A, mCry3A, Cry3Ab, Cry3Bb and Cry34Ab1/35Ab1.
IAN12: Inhibitors of mitochondrial ATP synthase, preferably ATP disruptors selected from diafenthiuron, or organotin compounds selected from azocyclotin, cyhexatin and fenbutatin oxide, or propargite or tetradifon.
IAN13: Uncouplers of oxidative phosphorylation via disruption of the proton gradient selected from chlorfenapyr, DNOC and sulfluramid.
IAN14: Nicotinic acetylcholine receptor channel blockers selected from bensultap, cartap hydrochloride, thiocylam and thiosultap-sodium.
IAN15: Inhibitors of chitin biosynthesis, type 0, selected from bistrifluron, chlorfluazuron, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, noviflumuron, teflubenzuron and triflumuron.
IAN16: Inhibitors of chitin biosynthesis, type 1 selected from buprofezin.
IAN17: Moulting disruptor (in particular for Diptera, i.e. dipterans) selected from cyromazine.
IAN18: Ecdysone receptor agonists selected from chromafenozide, halofenozide, methoxyfenozide and tebufenozide.
IAN19: Octopamine receptor agonists selected from amitraz.
IAN20: Mitochondrial complex III electron transport inhibitors selected from hydramethylnone, acequinocyl and fluacrypyrim.
IAN21: Mitochondrial complex I electron transport inhibitors, preferably METI acaricides selected from fenazaquin, fenpyroximate, pyrimidifen, pyridaben, tebufenpyrad and tolfenpyrad, or rotenone (Derris).
IAN22: Voltage-dependent sodium channel blockers selected from indoxacarb and metaflumizone.
IAN23: Inhibitors of acetyl CoA carboxylase, preferably tetronic and tetramic acid derivatives selected from spirodiclofen, spiromesifen and spirotetramat.
IAN24: Mitochondrial complex IV electron transport inhibitors, preferably phosphines selected from aluminium phosphide, calcium phosphide, phosphine and zinc phosphide, or cyanides selected from calcium cyanide, potassium cyanide and sodium cyanide.
IAN25: Mitochondrial complex II electron transport inhibitors, preferably beta-ketonitrile derivatives selected from cyenopyrafen and cyflumetofen, and carboxanilides selected from pyflubumide.
IAN28: Ryanodine receptor modulators, preferably diamides selected from chlorantraniliprole, cyantraniliprole, tetranaliiprole and flubendiamide.
IAN29: Chordotonal organ Modulators (with undefined target site) selected from flonicamid.
IAN30: further active compounds selected from the group consisting of: Afidopyropen, Afoxolaner, Azadirachtin, Benclothiaz, Benzoximate, Bifenazate, Broflanilide, Bromopropylate, Chinomethionat, Chloroprallethrin, Cryolite, Cyclaniliprole, Cycloxaprid, Cyhalodiamide, Dicloromezotiaz, Dicofol, epsilon-Metofluthrin, epsilon-Momfluthrin, Flometoquin, Fluazaindolizine, Fluensulfone, Flufenerim, Flufenoxystrobin, Flufiprole, Fluhexafon, Fluopyram (including the formulations described in WO2018046381, incorporated herein by reference), Fluralaner, Fluxametamide, Fufenozide, Guadipyr, Heptafluthrin, Imidaclothiz, Iprodione, kappa-Bifenthrin, kappa-Tefluthrin, Lotilaner, Meperfluthrin, Paichongding, Pyridalyl, Pyrifluquinazon, Pyriminostrobin, Spirobudiclofen, Tetramethylfluthrin, Tetraniliprole, Tetrachlorantraniliprole, Tigolaner, Tioxazafen (Nemastrike™), Thiofluoximate, Triflumezopyrim and iodomethane; furthermore preparations based on Bacillus firmus (1-1582, BioNeem, Votivo), and also the following compounds: 1-{2-fluoro-4-methyl-5-[(2,2,2-trifluoroethyl)sulphinyl]phenyl}-3-(trifluoromethyl)-1H-1,2,4-triazole-5-amine (known from WO2006/043635) (CAS 885026-50-6), {1′-[(2E)-3-(4-chlorophenyl)prop-2-en-1-yl]-5-fluorospiro[indol-3,4′-piperidin]-1(2H)-yl}(2-chloropyridin-4-yl)methanone (known from WO2003/106457) (CAS 637360-23-7), 2-chloro-N-[2-{1-[(2E)-3-(4-chlorophenyl)prop-2-en-1-yl]piperidin-4-yl}-4-(trifluoromethyl)phenyl]isonicotinamide (known from WO2006/003494) (CAS 872999-66-1), 3-(4-chloro-2,6-dimethylphenyl)-4-hydroxy-8-methoxy-1,8-diazaspiro[4.5]dec-3-en-2-one (known from WO 2010052161) (CAS 1225292-17-0), 3-(4-chloro-2,6-dimethylphenyl)-8-methoxy-2-oxo-1,8-diazaspiro[4.5]dec-3-en-4-yl ethyl carbonate (known from EP2647626) (CAS 1440516-42-6), 4-(but-2-yn-1-yloxy)-6-(3,5-dimethylpiperidin-1-yl)-5-fluoropyrimidine (known from WO2004/099160) (CAS 792914-58-0), PF1364 (known from JP2010/018586) (CAS 1204776-60-2), N-[(2E)-1-[(6-chloropyridin-3-yl)methyl]pyridin-2(1H)-ylidene]-2,2,2-trifluoroacetamide (known from WO2012/029672) (CAS 1363400-41-2), (3E)-3-[1-[(6-chloro-3-pyridyl)methyl]-2-pyridylidene]-1,1,1-trifluoro-propan-2-one (known from WO2013/144213) (CAS 1461743-15-6), N-[3-(benzylcarbamoyl)-4-chlorophenyl]-1-methyl-3-(pentafluoroethyl)-4-(trifluoromethyl)-1H-pyrazole-5-carboxamide (known from WO2010/051926) (CAS 1226889-14-0), 5-bromo-4-chloro-N-[4-chloro-2-methyl-6-(methylcarbamoyl)phenyl]-2-(3-chloro-2-pyridyl)pyrazole-3-carboxamide (known from CN103232431) (CAS 1449220-44-3), 4-[5-(3,5-dichlorophenyl)-4,5-dihydro-5-(trifluoromethyl)-3-isoxazolyl]-2-methyl-N-(cis-1-oxido-3-thietanyl)-benzamide, 4-[5-(3,5-dichlorophenyl)-4,5-dihydro-5-(trifluoromethyl)-3-isoxazolyl]-2-methyl-N-(trans-1-oxido-3-thietanyl)-benzamide and 4-[(5S)-5-(3,5-dichlorophenyl)-4,5-dihydro-5-(trifluoromethyl)-3-isoxazolyl]-2-methyl-N-(cis-1-oxido-3-thietanyl)benzamide (known from WO 2013/050317 A1) (CAS 1332628-83-7), N-[3-chloro-1-(3-pyridinyl)-1H-pyrazol-4-yl]-N-ethyl-3-[(3,3,3-trifluoropropyl)sulfinyl]-propanamide, (+)-N-[3-chloro-1-(3-pyridinyl)-1H-pyrazol-4-yl]-N-ethyl-3-[(3,3,3-trifluoropropyl)sulfinyl]-propanamide and (−)-N-[3-chloro-1-(3-pyridinyl)-1H-pyrazol-4-yl]-N-ethyl-3-[(3,3,3-trifluoropropyl)sulfinyl]-propanamide (known from WO 2013/162715 A2, WO 2013/162716 A2, US 2014/0213448 A1) (CAS 1477923-37-7), 5-[[(2E)-3-chloro-2-propen-1-yl]amino]-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile (known from CN 101337937 A) (CAS 1105672-77-2), 3-bromo-N-[4-chloro-2-methyl-6-[(methylamino)thioxomethyl]phenyl]-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxamide, (Liudaibenjiaxuanan, known from CN 103109816 A) (CAS 1232543-85-9); N-[4-chloro-2-[[(1,1-dimethylethyl)amino]carbonyl]-6-methylphenyl]-1-(3-chloro-2-pyridinyl)-3-(fluoromethoxy)-1H-Pyrazole-5-carboxamide (known from WO 2012/034403 A1) (CAS 1268277-22-0), N-[2-(5-amino-1,3,4-thiadiazol-2-yl)-4-chloro-6-methylphenyl]-3-bromo-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxamide (known from WO 2011/085575 A1) (CAS 1233882-22-8), 4-[3-[2,6-dichloro-4-[(3,3-dichloro-2-propen-1-yl)oxy]phenoxy]propoxy]-2-methoxy-6-(trifluoromethyl)-pyrimidine (known from CN 101337940 A) (CAS 1108184-52-6); (2E)- and 2(Z)-2-[2-(4-cyanophenyl)-1-[3-(trifluoromethyl)phenyl]ethylidene]-N-[4-(difluoromethoxy)phenyl]-hydrazinecarboxamide (known from CN 101715774 A) (CAS 1232543-85-9); 3-(2,2-dichloroethenyl)-2,2-dimethyl-4-(1H-b enzimidazol-2-yl)phenyl-cyclopropanecarboxylic acid ester (known from CN 103524422 A) (CAS 1542271-46-4); (4aS)-7-chloro-2,5-dihydro-2-[[(methoxycarbonyl)[4-[(trifluoromethyl)thio]phenyl]amino]carbonyl]-indeno[1,2-e][1,3,4]oxadiazine-4a(3H)-carboxylic acid methyl ester (known from CN 102391261 A) (CAS 1370358-69-2); 6-deoxy-3-O-ethyl-2,4-di-O-methyl-, 1-[N-[4-[1-[4-(1,1,2,2,2-pentafluoroethoxy)phenyl]-1H-1,2,4-triazol-3-yl]phenyl]carbamate]-α-L-mannopyranose (known from US 2014/0275503 A1) (CAS 1181213-14-8); 8-(2-cyclopropylmethoxy-4-trifluoromethyl-phenoxy)-3-(6-trifluoromethyl-pyridazin-3-yl)-3-aza-bicyclo[3.2.1]octane (CAS 1253850-56-4), (8-anti)-8-(2-cyclopropylmethoxy-4-trifluoromethyl-phenoxy)-3-(6-trifluoromethyl-pyridazin-3-yl)-3-aza-bicyclo[3.2.1]octane (CAS 933798-27-7), (8-syn)-8-(2-cyclopropylmethoxy-4-trifluoromethyl-phenoxy)-3-(6-trifluoromethyl-pyridazin-3-yl)-3-aza-bicyclo[3.2.1]octane (known from WO 2007040280 A1, WO 2007040282 A1) (CAS 934001-66-8), N-[3-chloro-1-(3-pyridinyl)-1H-pyrazol-4-yl]-N-ethyl-3-[(3,3,3-trifluoropropyl)thio]-propanamide (known from WO 2015/058021 A1, WO 2015/058028 A1) (CAS 1477919-27-9) and N-[4-(aminothioxomethyl)-2-methyl-6-[(methylamino)carbonyl]phenyl]-3-bromo-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxamide (known from CN 103265527 A) (CAS 1452877-50-7), 5-(1,3-dioxan-2-yl)-4-[[4-(trifluoromethyl)phenyl]methoxy]-pyrimidine (known from WO 2013/115391 A1) (CAS 1449021-97-9), 3-(4-chloro-2,6-dimethylphenyl)-4-hydroxy-8-methoxy-1-methyl-1,8-diazaspiro[4.5]dec-3-en-2-one (known from WO 2010/066780 A1, WO 2011/151146 A1) (CAS 1229023-34-0), 3-(4-chloro-2,6-dimethylphenyl)-8-methoxy-1-methyl-1,8-diazaspiro[4.5]decane-2,4-dione (known from WO 2014/187846 A1) (CAS 1638765-58-8), 3-(4-chloro-2,6-dimethylphenyl)-8-methoxy-1-methyl-2-oxo-1,8-diazaspiro[4.5]dec-3-en-4-yl-carbonic acid ethyl ester (known from WO 2010/066780 A1, WO 2011151146 A1) (CAS 1229023-00-0), N-[1-[(6-chloro-3-pyridinyl)methyl]-2(1H)-pyridinylidene]-2,2,2-trifluoro-acetamide (known from DE 3639877 A1, WO 2012029672 A1) (CAS 1363400-41-2), [N(E)]-N-[1-[(6-chloro-3-pyridinyl)methyl]-2(1H)-pyridinylidene]-2,2,2-trifluoro-acetamide, (known from WO 2016005276 A1) (CAS 1689566-03-7), [N(Z)]-N-[1-[(6-chloro-3-pyridinyl) methyl]-2(1H)-pyridinylidene]-2,2,2-trifluoro-acetamide, (CAS 1702305-40-5), 3-endo-3-[2-propoxy-4-(trifluoromethyl)phenoxy]-9-[[5-(trifluoromethyl)-2-pyridinyl]oxy]-9-azabicyclo[3.3.1]nonane (known from WO 2011/105506 A1, WO 2016/133011 A1) (CAS 1332838-17-1), the compounds described in WO2018087036 or WO2015169776, flupyrimin or mixtures thereof (see EP0268915, WO2012/029672, WO2013/129688), Spiropidion, Benzpyrimoxan, Pyrifluramide, the nematicides described in U.S. Pat. No. 9,433,218 or 9,701,673 (incorporated herein by reference), and N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide (described in WO2014/053450).
Also included as insecticide/acaricide/nematicides used in this invention are pyridazinamides including but not limited to 1-isopropyl-N,5-dimethyl-N-pyridazin-4-yl-pyrazole-4-carboxamide; 1-(1,2-dimethylpropyl)-N-ethyl-5-methyl-N-pyridazin-4-yl-pyrazole-4-carboxamide; N,5-dimethyl-N-pyridazin-4-yl-1-(2,2,2-trifluoro-1-methyl-ethyl)pyrazole-4-carboxamide; 1-[1-(1-cyanocyclopropyl)ethyl]-N-ethyl-5-methyl-N-pyridazin-4-yl-pyrazole-4-carboxamide; N-ethyl-1-(2-fluoro-1-methyl-propyl)-5-methyl-N-pyridazin-4-yl-pyrazole-4-carboxamide; 1-(1,2-dimethylpropyl)-N,5-dimethyl-N-pyridazin-4-yl-pyrazole-4-carboxamide; 1-[1-(1-cyanocyclopropyl)ethyl]-N,5-dimethyl-N-pyridazin-4-yl-pyrazole-4-carboxamide; N-methyl-1-(2-fluoro-1-methyl-propyl]-5-methyl-N-pyridazin-4-yl-pyrazole-4-carboxamide; 1-(4,4-difluorocyclohexyl)-N-ethyl-5-methyl-N-pyridazin-4-yl-pyrazole-4-carboxamide; or 1-(4,4-difluorocyclohexyl)-N,5-dimethyl-N-pyridazin-4-yl-pyrazole-4-carboxamide. In some embodiments, the pyridazinamide used in the invention is 1-(1,2-dimethylpropyl)-N-ethyl-5-methyl-N-pyridazin-4-yl-pyrazole-4-carboxamide.
Also included as insecticide/acaricide/nematicides used in this invention are mesoionics including but not limited to (3R)-3-(2-chlorothiazol-5-yl)-8-methyl-5-oxo-6-phenyl-2,3-dihydrothiazolo[3,2-a]pyrimidin-8-ium-7-olate; (3S)-3-(6-chloro-3-pyridyl)-8-methyl-5-oxo-6-phenyl-2,3-dihydrothiazolo[3,2-a]pyrimidin-8-ium-7-olate; (3S)-8-methyl-5-oxo-6-phenyl-3-pyrimidin-5-yl-2,3-dihydrothiazolo[3,2-a]pyrimidin-8-ium-7-olate; (3R)-3-(2-chlorothiazol-5-yl)-8-methyl-5-oxo-6-[3-(trifluoromethyl)phenyl]-2,3-dihydrothiazolo[3,2-a]pyrimidin-8-ium-7-olate; (3R)-3-(2-chlorothiazol-5-yl)-6-(3,5-dichlorophenyl)-8-methyl-5-oxo-2,3-dihydrothiazolo[3,2-a]pyrimidin-8-ium-7-olate; (3R)-3-(2-chlorothiazol-5-yl)-8-ethyl-5-oxo-6-phenyl-2,3-dihydrothiazolo[3,2-a]pyrimidin-8-ium-7-olate. In some embodiments, the mesoionic used in the invention is (3R)-3-(2-chlorothiazol-5-yl)-8-methyl-5-oxo-6-phenyl-2,3-dihydrothiazolo[3,2-a]pyrimidin-8-ium-7-olate.
Further nematicidal compound used in this invention include trifluoroethyl sulfoxide (known from Kumiai/Bayer); N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-(trifluoromethyl)benzamide (compound (1a or Ia; described in WO15144683); N-[1-(3,5-difluoropyridin-2-yl)-1H-pyrazol-3-yl]-2-(trifluoromethyl)benzamide (compound 1b or Ib); N-[2-(2,6-Difluorophenyl)-2H-1,2,3-triazol-4-yl]-2-(trifluoromethyl)benzamide (compound 1c or Ic).
An insecticide/insecticidal agent in crop protection, as described herein for use on or with the plants or seeds of the invention, is capable of controlling insects. The term “controlling insects”, or “insect-controlling” as used herein, means killing the insects (in any stage, preferably at least in the larval stage) or preventing or impeding their development, their reproduction, or their growth or preventing or impeding their penetration into or their sucking/feeding on plant tissue.
A nematicide/nematicidal agent in crop protection, as described herein for use on or with the plants or seeds of the invention, is capable of controlling nematodes. The term “controlling nematodes”, or “nematode-controlling”, as used herein, means killing the nematodes or preventing or impeding their development, their reproduction, or their growth, or preventing or impeding their penetration into or their sucking/feeding on plant tissue. Hence, a “nematicide” or “nematicidal agent”, as used herein, is an agent that can kill nematodes (such as plant-pathogenic nematodes) or can prevent or impede their development, their reproduction, or their growth, or can prevent or impede their penetration into or their sucking/feeding on plant tissue.
The efficacy of nematicidal compounds or biological control agents is determined by comparing mortalities, gall formation, cyst formation, nematode density per volume of soil, nematode density per root, number of nematode eggs per soil volume, mobility of the nematodes between a treated plant or plant part or the treated soil and an untreated plant or plant part or the untreated soil. In one embodiment the reduction achieved is 25-50% in comparison to an untreated plant, plant part or the untreated soil, in another embodiment 51-79% reduction in comparison to an untreated plant, plant part or the untreated soil and in yet another embodiment refers to the complete kill or the complete prevention of development and growth of the nematodes by a reduction of 80 to 100%. The control of nematodes as described herein also comprises the control of proliferation of the nematodes (development of cysts and/or eggs). Nematicidal compounds and/or or biological control agents can also be used to keep the soybean plants of the invention more healthy, and they can be employed curatively, preventatively or systemically for the control of nematodes.
The use of the nematode-controlling compounds or biological control agents can further increase the yield of the plants of the invention.
The nematode-controlling compounds or biological control agents as mentioned herein can be used to control plant nematodes such as Aglenchus agricola, Anguina tritici, Aphelenchoides arachidis, Aphelenchoides fragaria, and the stem and leaf endoparasites Aphelenchoides spp., Belonolaimus gracilis, Belonolaimus longicaudatus, Belonolaimus nortoni, Bursaphelenchus cocophilus, Bursaphelenchus eremus, Bursaphelenchus xylophilus and Bursaphelenchus spp., Cacopaurus pestis, Criconemella curvata, Criconemella onoensis, Criconemella ornata, Criconemella rusium, Criconemella xenoplax Mesocriconema xenoplax) and Criconemella spp., Criconemoides ferniae, Criconemoides onoense, Criconemoides ornatum and Criconemoides spp., Ditylenchus destructor, Ditylenchus dipsaci, Ditylenchus myceliophagus and also the stem and leaf endoparasites Ditylenchus spp., Dolichodorus heterocephalus, Globodera pallida (=Heterodera pallida), Globodera rostochiensis (yellow potato cyst nematode), Globodera solanacearum, Globodera tabacum, Globodera virginia and the non-migratory cyst-forming parasites Globodera spp., Helicotylenchus digonicus, Helicotylenchus dihystera, Helicotylenchus erythrine, Helicotylenchus multicinctus, Helicotylenchus nannus, Helicotylenchus pseudorobustus and Helicotylenchus spp., Hemicriconemoides, Hemicycliophora arenaria, Hemicycliophora nudata, Hemicycliophora parvana, Heterodera avenae, Heterodera cruciferae, Heterodera glycines, Heterodera oryzae, Heterodera schachtii, Heterodera zeae and the non-migratory cyst-forming parasites Heterodera spp., Hirschmaniella gracilis, Hirschmaniella oryzae, Hirschmaniella spinicaudata and the stem and leaf endoparasites Hirschmaniella spp., Hoplolaimus aegyptii, Hoplolaimus californicus, Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimus indicus, Hoplolaimus magnistylus, Hoplolaimus pararobustus, Longidorus africanus, Longidorus breviannulatus, Longidorus elongatus, Longidorus laevicapitatus, Longidorus vineacola and the ectoparasites Longidorus spp., Meloidogyne acronea, Meloidogyne africana, Meloidogyne arenaria, Meloidogyne arenaria thamesi, Meloidogyne artiella, Meloidogyne chitwoodi, Meloidogyne coffeicola, Meloidogyne ethiopica, Meloidogyne exigua, Meloidogyne fallax, Meloidogyne graminicola, Meloidogyne graminis, Meloidogyne hapla, Meloidogyne incognita, Meloidogyne incognita acrita, Meloidogyne javanica, Meloidogyne kikuyensis, Meloidogyne minor, Meloidogyne naasi, Meloidogyne paranaensis, Meloidogyne thamesi and the non-migratory parasites Meloidogyne spp., Meloinema spp., Nacobbus aberrans, Neotylenchus vigissi, Paraphelenchus pseudoparietinus, Paratrichodorus allius, Paratrichodorus lobatus, Paratrichodorus minor, Paratrichodorus nanus, Paratrichodorus porosus, Paratrichodorus teres and Paratrichodorus spp., Paratylenchus hamatus, Paratylenchus minutus, Paratylenchus projectus and Paratylenchus spp., Pratylenchus agilis, Pratylenchus alleni, Pratylenchus andinus, Pratylenchus brachyurus, Pratylenchus cerealis, Pratylenchus coffeae, Pratylenchus crenatus, Pratylenchus delattrei, Pratylenchus giibbicaudatus, Pratylenchus goodeyi, Pratylenchus hamatus, Pratylenchus hexincisus, Pratylenchus loosi, Pratylenchus neglectus, Pratylenchus penetrans, Pratylenchus pratensis, Pratylenchus scribneri, Pratylenchus teres, Pratylenchus thornei, Pratylenchus vulnus, Pratylenchus zeae and the migratory endoparasites Pratylenchus spp., Pseudohalenchus minutus, Psilenchus magnidens, Psilenchus tumidus, Punctodera chalcoensis, Quinisulcius acutus, Radopholus citrophilus, Radopholus similis, the migratory endoparasites Radopholus spp., Rotylenchulus borealis, Rotylenchulus parvus, Rotylenchulus reniformis and Rotylenchulus spp., Rotylenchus laurentinus, Rotylenchus macrodoratus, Rotylenchus robustus, Rotylenchus uniformis and Rotylenchus spp., Scutellonema brachyurum, Scutellonema bradys, Scutellonema clathricaudatum and the migratory endoparasites Scutellonema spp., Subanguina radiciola, Tetylenchus nicotianae, Trichodorus cylindricus, Trichodorus minor, Trichodorus primitivus, Trichodorus proximus, Trichodorus similis, Trichodorus sparsus and the ectoparasites Trichodorus spp., Tylenchorhynchus agri, Tylenchorhynchus brassicae, Tylenchorhynchus clarus, Tylenchorhynchus claytoni, Tylenchorhynchus digitatus, Tylenchorhynchus ebriensis, Tylenchorhynchus maximus, Tylenchorhynchus nudus, Tylenchorhynchus vulgaris and Tylenchorhynchus spp., Tylenchulus semipenetrans and the semiparasites Tylenchulus spp., Xiphinema americanum, Xiphinema brevicolle, Xiphinema dimorphicaudatum, Xiphinema index and the ectoparasites Xiphinema spp.
The above compounds with nematode-controlling activity are particularly suitable for controlling soybean nematodes, in particular nematodes selected from the group consisting of: Pratylenchus brachyurus, Pratylenchus pratensis, Pratylenchus penetrans, Pratylenchus scribneri, Belonolaimus longicaudatus, Heterodera glycines, Hoplolaimus columbus and also Pratylenchus coffeae, Pratylenchus hexincisus, Pratylenchus neglectus, Pratylenchus crenatus, Pratylenchus alleni, Pratylenchus agilis, Pratylenchus zeae, Pratylenchus vulnus, Belonolaimus gracilis, Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica, Meloidogyne hapla, Hoplolaimus columbus, Hoplolaimus galeatus and Rotylenchulus reniformis.
In one embodiment, nematicidal compounds to be used for treating seeds or plants of the invention, or the soil wherein said seeds or plants are growing or are intended to be grown, include these of group NC1: alanycarb, aldicarb, carbofuran, carbosulfan, fosthiazate, cadusafos, oxamyl, thiodicarb, dimethoate, ethoprophos, terbufos, abamectin, methyl bromide and other alkyl halides, methyl isocyanate generators selected from diazomet and metam, fluazaindolizine, fluensulfone, fluopyram, tioxazafen, N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide, cis-Jasmone, harpin, Azadirachta indica oil, or
Azadirachtin. In another embodiment, the plants or seeds of the invention, or the soil in which they are grown or are intended to be grown, are treated with any of the following nematicidal agents of group NC2: fosthiazate, cadusafos, thiodicarb, abamectin, fluazaindolizine, fluopyram, tioxazafen, N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide, cis-Jasmone, harpin, Azadirachta indica oil, or Azadirachtin, particularly the seeds of the invention are treated with said nematicidal compounds.
In one embodiment, the plant, cell, plant part or seed of the invention, comprising EE-GM5, is treated with a combination selected from the group NC3 consisting of: fosthiazate and cadusafos, fosthiazate and thiodicarb, fosthiazate and abamectin, fosthiazate and fluazaindolizine, fosthiazate and fluopyram, fosthiazate and tioxazafen, fosthiazate and N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide, fosthiazate and ci s-Jasmone, fosthiazate and harpin, fosthiazate and Azadirachta indica oil, fosthiazate and Azadirachtin, cadusafos and fosthiazate, cadusafos and thiodicarb, cadusafos and abamectin, cadusafos and fluazaindolizine, cadusafos and fluopyram, cadusafos and tioxazafen, cadusafos and N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide, cadusafos and cis-Jasmone, cadusafos and harpin, cadusafos and Azadirachta indica oil, cadusafos and Azadirachtin, thiodicarb and fosthiazate, thiodicarb and cadusafos, thiodicarb and abamectin, thiodicarb and fluazaindolizine, thiodicarb and fluopyram, thiodicarb and tioxazafen, thiodicarb and N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide, thiodicarb and cis-Jasmone, thiodicarb and harpin, thiodicarb and Azadirachta indica oil, abamectin and cadusafos, abamectin and thiodicarb, abamectin and fluazaindolizine, abamectin and fluopyram, abamectin and tioxazafen, abamectin and N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide, abamectin and cis-Jasmone, abamectin and harpin, abamectin and Azadirachta indica oil, abamectin and Azadirachtin, fluazaindolizine and abamectin, fluazaindolizine and fluopyram, fluazaindolizine and tioxazafen, fluazaindolizine and N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide, fluazaindolizine and cis-Jasmone, fluazaindolizine and harpin, fluazaindolizine and Azadirachta indica oil, fluazaindolizine and Azadirachtin, fluopyram and fluazaindolizine, fluopyram and tioxazafen, fluopyram and N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide, fluopyram and cis-Jasmone, fluopyram and harpin, fluopyram and Azadirachta indica oil, fluopyram and Azadirachtin, tioxazafen and N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide, tioxazafen and cis-Jasmone, tioxazafen and harpin, tioxazafen and Azadirachta indica oil, tioxazafen and Azadirachtin, N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide and cis-Jasmone, N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide and harpin, N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide and Azadirachta indica oil, N-[1-(2,6-difluorophenyl)-1H-pyrazol-3-yl]-2-trifluoromethylbenzamide and Azadirachtin, cis-Jasmone and harpin, cis-Jasmone and Azadirachta indica oil, cis-Jasmone and Azadirachtin, harpin and Azadirachta indica oil, and harpin and Azadirachtin.
Fungicides
The active ingredients specified herein by their Common Name are known and described, for example, in The Pesticide Manual (17th Ed. British Crop Protection Council, updated version at on the world wide web at bcpc.org/product/bcpc-online-pesticide-manual-latest-version) or can be searched on the internet (e.g. on the world wide web at alanwood.net/pesticides).
All named fungicidal mixing partners of the classes F1 to F15 can, if their functional groups enable this, optionally form salts with suitable bases or acids. All named mixing partners of the classes F1 to F15 can include tautomeric forms, where applicable.
F1: Inhibitors of the ergosterol biosynthesis, for example (1.001) cyproconazole, (1.002) difenoconazole, (1.003) epoxiconazole, (1.004) fenhexamid, (1.005) fenpropidin, (1.006) fenpropimorph, (1.007) fenpyrazamine, (1.008) fluquinconazole, (1.009) flutriafol, (1.010) imazalil, (1.011) imazalil sulfate, (1.012) ipconazole, (1.013) metconazole, (1.014) myclobutanil, (1.015) paclobutrazol, (1.016) prochloraz, (1.017) propiconazole, (1.018) prothioconazole, (1.019) Pyrisoxazole, (1.020) spiroxamine, (1.021) tebuconazole, (1.022) tetraconazole, (1.023) triadimenol, (1.024) tridemorph, (1.025) triticonazole, (1.026) (1R,2S,5S)-5-(4-chlorobenzyl)-2-(chloromethyl)-2-methyl-1-(1H-1,2,4-triazol-1-ylmethyl)cyclopentanol, (1.027) (1S,2R,5R)-5-(4-chlorobenzyl)-2-(chloromethyl)-2-methyl-1-(1H-1,2,4-triazol-1-ylmethyl)cyclopentanol, (1.028) (2R)-2-(1-chlorocyclopropyl)-4-[(1R)-2,2-dichlorocyclopropyl]-1-(1H-1,2,4-triazol-1-yl)butan-2-ol, (1.029) (2R)-2-(1-chlorocyclopropyl)-4-[(1S)-2,2-dichlorocyclopropyl]-1-(1H-1,2,4-triazol-1-yl)butan-2-ol, (1.030) (2R)-2-[4-(4-chlorophenoxy)-2-(trifluoromethyl)phenyl]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol, (1.031) (2S)-2-(1-chlorocyclopropyl)-4-[(1R)-2,2-dichlorocyclopropyl]-1-(1H-1,2,4-triazol-1-yl)butan-2-ol, (1.032) (2S)-2-(1-chlorocyclopropyl)-4-[(1S)-2,2-dichlorocyclopropyl]-1-(1H-1,2,4-triazol-1-yl)butan-2-ol, (1.033) (2S)-2-[4-(4-chlorophenoxy)-2-(trifluoromethyl)phenyl]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol, (1.034) (R)-[3-(4-chloro-2-fluorophenyl)-5-(2,4-difluorophenyl)-1,2-oxazol-4-yl](pyridin-3-yl)methanol, (1.035) (S)-[3-(4-chloro-2-fluorophenyl)-5-(2,4-difluorophenyl)-1,2-oxazol-4-yl](pyridin-3-yl)methanol, (1.036) [3-(4-chloro-2-fluorophenyl)-5-(2,4-difluorophenyl)-1,2-oxazol-4-yl](pyridin-3-yl)methanol, (1.037) 1-({(2R,4S)-2-[2-chloro-4-(4-chlorophenoxy)phenyl]-4-methyl-1,3-dioxolan-2-yl}methyl)-1H-1,2,4-triazole, (1.038) 1-({(2S,4S)-2-[2-chloro-4-(4-chlorophenoxy)phenyl]-4-methyl-1,3-dioxolan-2-yl}methyl)-1H-1,2,4-triazole, (1.039) 1-{[3-(2-chlorophenyl)-2-(2,4-difluorophenyl)oxiran-2-yl]methyl}-1H-1,2,4-triazol-5-yl thiocyanate, (1.040) 1-{[rel(2R,3R)-3-(2-chlorophenyl)-2-(2,4-difluorophenyl)oxiran-2-yl]methyl}-1H-1,2,4-triazol-5-yl thiocyanate, (1.041) 1-{[rel(2R,3S)-3-(2-chlorophenyl)-2-(2,4-difluorophenyl)oxiran-2-yl]methyl}-1H-1,2,4-triazol-5-yl thiocyanate, (1.042) 2-[(2R,4R,5R)-1-(2,4-dichlorophenyl)-5-hydroxy-2,6,6-trimethylheptan-4-yl]-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.043) 2-[(2R,4R,5S)-1-(2,4-dichlorophenyl)-5-hydroxy-2,6,6-trimethylheptan-4-yl]-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.044) 2-[(2R,4S,5R)-1-(2,4-dichlorophenyl)-5-hydroxy-2,6,6-trimethylheptan-4-yl]-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.045) 2-[(2R,4S,5S)-1-(2,4-dichlorophenyl)-5-hydroxy-2,6,6-trimethylheptan-4-yl]-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.046) 2-[(2S,4R,5R)-1-(2,4-dichlorophenyl)-5-hydroxy-2,6,6-trimethylheptan-4-yl]-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.047) 2-[(2S,4R,5S)-1-(2,4-dichlorophenyl)-5-hydroxy-2,6,6-trimethylheptan-4-yl]-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.048) 2-[(2S,4S,5R)-1-(2,4-dichlorophenyl)-5-hydroxy-2,6,6-trimethylheptan-4-yl]-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.049) 2-[(2S,4S,5S)-1-(2,4-dichlorophenyl)-5-hydroxy-2,6,6-trimethylheptan-4-yl]-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.050) 2-[1-(2,4-dichlorophenyl)-5-hydroxy-2,6,6-trimethylheptan-4-yl]-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.051) 2-[2-chloro-4-(2,4-dichlorophenoxy)phenyl]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol, (1.052) 2-[2-chloro-4-(4-chlorophenoxy)phenyl]-1-(1H-1,2,4-triazol-1-yl)butan-2-ol, (1.053) 2-[4-(4-chlorophenoxy)-2-(trifluoromethyl)phenyl]-1-(1H-1,2,4-triazol-1-yl)butan-2-ol, (1.054) 2-[4-(4-chlorophenoxy)-2-(trifluoromethyl)phenyl]-1-(1H-1,2,4-triazol-1-yl)pentan-2-ol, (1.055) 2-[4-(4-chlorophenoxy)-2-(trifluoromethyl)phenyl]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol, (1.056) 2-{[3-(2-chlorophenyl)-2-(2,4-difluorophenyl)oxiran-2-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.057) 2-{[rel(2R,3R)-3-(2-chlorophenyl)-2-(2,4-difluorophenyl)oxiran-2-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.058) 2-{[rel(2R,3S)-3-(2-chlorophenyl)-2-(2,4-difluorophenyl)oxiran-2-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione, (1.059) 5-(4-chlorobenzyl)-2-(chloromethyl)-2-methyl-1-(1H-1,2,4-triazol-1-ylmethyl)cyclopentanol, (1.060) 5-(allylsulfanyl)-1-{[3-(2-chlorophenyl)-2-(2,4-difluorophenyl)oxiran-2-yl]methyl}-1H-1,2,4-triazole, (1.061) 5-(allylsulfanyl)-1-{[rel(2R,3R)-3-(2-chlorophenyl)-2-(2,4-difluorophenyl)oxiran-2-yl]methyl}-1H-1,2,4-triazole, (1.062) 5-(allylsulfanyl)-1-{[rel(2R,3S)-3-(2-chlorophenyl)-2-(2,4-difluorophenyl)oxiran-2-yl]methyl}-1H-1,2,4-triazole, (1.063) N′-(2,5-dimethyl-4-{[3-(1,1,2,2-tetrafluoroethoxy)phenyl]sulfanyl}phenyl)-N-ethyl-N-methylimidoformamide, (1.064) N′-(2,5-dimethyl-4-{[3-(2,2,2-trifluoroethoxy)phenyl]sulfanyl}phenyl)-N-ethyl-N-methylimidoformamide, (1.065) N′-(2,5-dimethyl-4-{[3-(2,2,3,3-tetrafluoropropoxy)phenyl]sulfanyl}phenyl)-N-ethyl-N-methylimidoformamide, (1.066) N′-(2,5-dimethyl-4-{[3-(pentafluoroethoxy)phenyl]sulfanyl}phenyl)-N-ethyl-N-methylimidoformamide, (1.067) N′-(2,5-dimethyl-4-{3-[(1,1,2,2-tetrafluoroethyl)sulfanyl]phenoxy}phenyl)-N-ethyl-N-methylimidoformamide, (1.068) N′-(2,5-dimethyl-4-{3-[(2,2,2-trifluoroethyl)sulfanyl]phenoxy}phenyl)-N-ethyl-N-methylimidoformamide, (1.069) N′-(2,5-dimethyl-4-{3-[(2,2,3,3-tetrafluoropropyl)sulfanyl]phenoxy}phenyl)-N-ethyl-N-methylimidoformamide, (1.070) N′-(2,5-dimethyl-4-{3-[(pentafluoroethyl)sulfanyl]phenoxy}phenyl)-N-ethyl-N-methylimidoformamide, (1.071) N′-(2,5-dimethyl-4-phenoxyphenyl)-N-ethyl-N-methylimidoformamide, (1.072) N′-(4-{[3-(difluoromethoxy)phenyl]sulfanyl}-2,5-dimethylphenyl)-N-ethyl-N-methylimidoformamide, (1.073) N′-(4-{3-[(difluoromethyl)sulfanyl]phenoxy}-2,5-dimethylphenyl)-N-ethyl-N-methylimidoformamide, (1.074) N′-[5-bromo-6-(2,3-dihydro-1H-inden-2-yloxy)-2-methylpyridin-3-yl]-N-ethyl-N-methylimidoformamide, (1.075) N′-{4-[(4,5-dichloro-1,3-thiazol-2-yl)oxy]-2,5-dimethylphenyl}-N-ethyl-N-methylimidoformamide, (1.076) N′-{5-bromo-6-[(1R)-1-(3,5-difluorophenyl)ethoxy]-2-methylpyridin-3-yl}-N-ethyl-N-methylimidoformamide, (1.077) N′-{5-bromo-6-[(1S)-1-(3,5-difluorophenyl)ethoxy]-2-methylpyridin-3-yl}-N-ethyl-N-methylimidoformamide, (1.078) N′-{5-bromo-6-[(cis-4-isopropylcyclohexyl)oxy]-2-methylpyridin-3-yl}-N-ethyl-N-methylimidoformamide, (1.079) N′-{5-bromo-6-[(trans-4-isopropylcyclohexyl)oxy]-2-methylpyridin-3-yl}-N-ethyl-N-methylimidoformamide, (1.080) N′-{5-bromo-6-[1-(3,5-difluorophenyl)ethoxy]-2-methylpyridin-3-yl}-N-ethyl-N-methylimidoformamide, (1.081) Mefentrifluconazole, (1.082) Ipfentrifluconazole, (1.083) Azaconazole, (1.084) Hexaconazole, (1.085) Triadimefon, (1.086) Oxpoconazole, (1.087) Penconazole, (1.088) Prochloraz, (1.089) Triflumizole, (1.090) Triforine, (1.091) Pyrifenox, (1.092) Fenarimol, (1.093) Nuarimol, (1.094) Bitertanol, (1.095) Bromuconazole, (1.096) Diniconazole, (1.097) Epoxiconazole, (1.098) Fenfuconazole, (1.099) to Imibenconazole.
F2: Inhibitors of the respiratory chain at complex I or II, for example (2.001) benzovindiflupyr, (2.002) bixafen, (2.003) boscalid, (2.004) carboxin, (2.005) fluopyram, (2.006) flutolanil, (2.007) fluxapyroxad, (2.008) furametpyr, (2.009) Isofetamid, (2.010) isopyrazam (anti-epimeric enantiomer 1R,4S,9S), (2.011) isopyrazam (anti-epimeric enantiomer 1S,4R,9R), (2.012) isopyrazam (anti-epimeric racemate 1RS,4SR,9SR), (2.013) isopyrazam (mixture of syn-epimeric racemate 1RS,4SR,9RS and anti-epimeric racemate 1RS,4SR,9SR), (2.014) isopyrazam (syn-epimeric enantiomer 1R,4S,9R), (2.015) isopyrazam (syn-epimeric enantiomer 1S,4R,9S), (2.016) isopyrazam (syn-epimeric racemate 1RS,4SR,9RS), (2.017) penflufen, (2.018) penthiopyrad, (2.019) pydiflumetofen, (2.020) Pyraziflumid, (2.021) sedaxane, (2.022) 1,3-dimethyl-N-(1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl)-1H-pyrazole-4-carboxamide, (2.023) 1,3-dimethyl-N-[(3R)-1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl]-1H-pyrazole-4-carboxamide, (2.024) 1,3-dimethyl-N-[(3S)-1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl]-1H-pyrazole-4-carboxamide, (2.025) 1-methyl-3-(trifluoromethyl)-N-[2′-(trifluoromethyl)biphenyl-2-yl]-1H-pyrazole-4-carboxamide, (2.026) 2-fluoro-6-(trifluoromethyl)-N-(1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl)benzamide, (2.027) 3-(difluoromethyl)-1-methyl-N-(1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl)-1H-pyrazole-4-carboxamide, (2.028) 3-(difluoromethyl)-1-methyl-N-[(3R)-1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl]-1H-pyrazole-4-carboxamide, (2.029) 3-(difluoromethyl)-1-methyl-N-[(3S)-1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl]-1H-pyrazole-4-carboxamide, (2.030) 3-(difluoromethyl)-N-(7-fluoro-1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl)-1-methyl-1H-pyrazole-4-carboxamide, (2.031) 3-(difluoromethyl)-N-[(3R)-7-fluoro-1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl]-1-methyl-1H-pyrazole-4-carboxamide, (2.032) 3-(difluoromethyl)-N-[(3S)-7-fluoro-1,1,3-trimethyl-2,3-dihydro-1H-inden-4-yl]-1-methyl-1H-pyrazole-4-carboxamide, (2.033) 5,8-difluoro-N-[2-(2-fluoro-4-{[4-(trifluoromethyl)pyridin-2-yl]oxy}phenyl)ethyl]quinazolin-4-amine, (2.034) N-(2-cyclopentyl-5-fluorobenzyl)-N-cyclopropyl-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.035) N-(2-tert-butyl-5-methylbenzyl)-N-cyclopropyl-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.036) N-(2-tert-butylbenzyl)-N-cyclopropyl-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.037) N-(5-chloro-2-ethylbenzyl)-N-cyclopropyl-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.038) N-(5-chloro-2-isopropylbenzyl)-N-cyclopropyl-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.039) N-[(1R,4S)-9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, (2.040) N-[(1S,4R)-9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, (2.041) N-[1-(2,4-dichlorophenyl)-1-methoxypropan-2-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, (2.042) N-[2-chloro-6-(trifluoromethyl)benzyl]-N-cyclopropyl-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.043) N-[3-chloro-2-fluoro-6-(trifluoromethyl)benzyl]-N-cyclopropyl-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.044) N-[5-chloro-2-(trifluoromethyl)benzyl]-N-cyclopropyl-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.045) N-cyclopropyl-3-(difluoromethyl)-5-fluoro-1-methyl-N-[5-methyl-2-(trifluoromethyl)benzyl]-1H-pyrazole-4-carboxamide, (2.046) N-cyclopropyl-3-(difluoromethyl)-5-fluoro-N-(2-fluoro-6-isopropylbenzyl)-1-methyl-1H-pyrazole-4-carboxamide, (2.047) N-cyclopropyl-3-(difluoromethyl)-5-fluoro-N-(2-isopropyl-5-methylbenzyl)-1-methyl-1H-pyrazole-4-carboxamide, (2.048) N-cyclopropyl-3-(difluoromethyl)-5-fluoro-N-(2-isopropylbenzyl)-1-methyl-1H-pyrazole-4-carbothioamide, (2.049) N-cyclopropyl-3-(difluoromethyl)-5-fluoro-N-(2-isopropylbenzyl)-1-methyl-1H-pyrazole-4-carboxamide, (2.050) N-cyclopropyl-3-(difluoromethyl)-5-fluoro-N-(5-fluoro-2-isopropylbenzyl)-1-methyl-1H-pyrazole-4-carboxamide, (2.051) N-cyclopropyl-3-(difluoromethyl)-N-(2-ethyl-4,5-dimethylbenzyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.052) N-cyclopropyl-3-(difluoromethyl)-N-(2-ethyl-5-fluorobenzyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.053) N-cyclopropyl-3-(difluoromethyl)-N-(2-ethyl-5-methylbenzyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.054) N-cyclopropyl-N-(2-cyclopropyl-5-fluorobenzyl)-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.055) N-cyclopropyl-N-(2-cyclopropyl-5-methylbenzyl)-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.056) N-cyclopropyl-N-(2-cyclopropylbenzyl)-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide, (2.057) Benzovindiflupyr, (2.058) Isoflucypram.
F3: Inhibitors of the respiratory chain at complex III, for example (3.001) ametoctradin, (3.002) amisulbrom, (3.003) azoxystrobin, (3.004) coumethoxystrobin, (3.005) coumoxystrobin, (3.006) cyazofamid, (3.007) dimoxystrobin, (3.008) enoxastrobin, (3.009) famoxadone, (3.010) fenamidone, (3.011) flufenoxystrobin, (3.012) fluoxastrobin, (3.013) kresoxim-methyl, (3.014) metominostrobin, (3.015) orysastrobin, (3.016) picoxystrobin, (3.017) pyraclostrobin, (3.018) pyrametostrobin, (3.019) pyraoxystrobin, (3.020) trifloxystrobin, (3.021) (2E)-2-{2-[({[(1E)-1-(3-{[(E)-1-fluoro-2-phenylvinyl]oxy}phenyl)ethylidene]amino}oxy)methyl]phenyl}-2-(methoxyimino)-N-methylacetamide, (3.022) (2E,3Z)-5-{[1-(4-chlorophenyl)-1H-pyrazol-3-yl]oxy}-2-(methoxyimino)-N,3-dimethylpent-3-enamide, (3.023) (2R)-2-{2-[(2,5-dimethylphenoxy)methyl]phenyl}-2-methoxy-N-methylacetamide, (3.024) (2S)-2-{2-[(2,5-dimethylphenoxy)methyl]phenyl}-2-methoxy-N-methylacetamide, (3.025) (3S,6S,7R,8R)-8-benzyl-3-[({3-[(isobutyryloxy)methoxy]-4-methoxypyridin-2-yl}carbonyl)amino]-6-methyl-4,9-dioxo-1,5-dioxonan-7-yl 2-methylpropanoate, (3.026) 2-{2-[(2,5-dimethylphenoxy)methyl]phenyl}-2-methoxy-N-methylacetamide, (3.027) N-(3-ethyl-3,5,5-trimethylcyclohexyl)-3-formamido-2-hydroxybenzamide, (3.028) (2E,3Z)-5-{[1-(4-chloro-2-fluorophenyl)-1H-pyrazol-3-yl]oxy}-2-(methoxyimino)-N,3-dimethylpent-3-enamide, (3.029) methyl {5-[3-(2,4-dimethylphenyl)-1H-pyrazol-1-yl]-2-methylbenzyl}carbamate, (3.030) mandestrobin.
F4: Inhibitors of the mitosis and cell division, for example (4.001) carbendazim, (4.002) diethofencarb, (4.003) ethaboxam, (4.004) fluopicolide, (4.005) pencycuron, (4.006) thiabendazole, (4.007) thiophanate-methyl, (4.008) zoxamide, (4.009) 3-chloro-4-(2,6-difluorophenyl)-6-methyl-5-phenylpyridazine, (4.010) 3-chloro-5-(4-chlorophenyl)-4-(2,6-difluorophenyl)-6-methylpyridazine, (4.011) 3-chloro-5-(6-chloropyridin-3-yl)-6-methyl-4-(2,4,6-trifluorophenyl)pyridazine, (4.012) 4-(2-bromo-4-fluorophenyl)-N-(2,6-difluorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.013) 4-(2-bromo-4-fluorophenyl)-N-(2-bromo-6-fluorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.014) 4-(2-bromo-4-fluorophenyl)-N-(2-bromophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.015) 4-(2-bromo-4-fluorophenyl)-N-(2-chloro-6-fluorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.016) 4-(2-bromo-4-fluorophenyl)-N-(2-chlorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.017) 4-(2-bromo-4-fluorophenyl)-N-(2-fluorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.018) 4-(2-chloro-4-fluorophenyl)-N-(2,6-difluorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.019) 4-(2-chloro-4-fluorophenyl)-N-(2-chloro-6-fluorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.020) 4-(2-chloro-4-fluorophenyl)-N-(2-chlorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.021) 4-(2-chloro-4-fluorophenyl)-N-(2-fluorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.022) 4-(4-chlorophenyl)-5-(2,6-difluorophenyl)-3,6-dimethylpyridazine, (4.023) N-(2-bromo-6-fluorophenyl)-4-(2-chloro-4-fluorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.024) N-(2-bromophenyl)-4-(2-chloro-4-fluorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine, (4.025) N-(4-chloro-2,6-difluorophenyl)-4-(2-chloro-4-fluorophenyl)-1,3-dimethyl-1H-pyrazol-5-amine.
F5: Compounds capable to have a multisite action, for example (5.001) bordeaux mixture, (5.002) captafol, (5.003) captan, (5.004) chlorothalonil, (5.005) copper hydroxide, (5.006) copper naphthenate, (5.007) copper oxide, (5.008) copper oxychloride, (5.009) copper(2+) sulfate, (5.010) dithianon, (5.011) dodine, (5.012) folpet, (5.013) mancozeb, (5.014) maneb, (5.015) metiram, (5.016) metiram zinc, (5.017) oxine-copper, (5.018) propineb, (5.019) sulfur and sulfur preparations including calcium polysulfide, (5.020) thiram, (5.021) zineb, (5.022) ziram, (5.023) 6-ethyl-5,7-dioxo-6,7-dihydro-5H-pyrrolo[3′,4′: 5,6][1,4]dithiino[2,3-c][1,2]thiazole-3-carbonitrile.
F6: Compounds capable to induce a host defence, for example (6.001) acibenzolar-S-methyl, (6.002) isotianil, (6.003) probenazole, (6.004) tiadinil.
F7: Inhibitors of the amino acid and/or protein biosynthesis, for example (7.001) cyprodinil, (7.002) kasugamycin, (7.003) kasugamycin hydrochloride hydrate, (7.004) oxytetracycline, (7.005) pyrimethanil, (7.006) 3-(5-fluoro-3,3,4,4-tetramethyl-3,4-dihydroisoquinolin-1-yl)quinoline.
F8: Inhibitors of the ATP production, for example (8.001) silthiofam.
F9: Inhibitors of the cell wall synthesis, for example (9.001) benthiavalicarb, (9.002) dimethomorph, (9.003) flumorph, (9.004) iprovalicarb, (9.005) mandipropamid, (9.006) pyrimorph, (9.007) valifenalate, (9.008) (2E)-3-(4-tert-butylphenyl)-3-(2-chloropyridin-4-yl)-1-(morpholin-4-yl)prop-2-en-1-one, (9.009) (2Z)-3-(4-tert-butylphenyl)-3-(2-chloropyridin-4-yl)-1-(morpholin-4-yl)prop-2-en-1-one.
F10: Inhibitors of the lipid and membrane synthesis, for example (10.001) propamocarb, (10.002) propamocarb hydrochloride, (10.003) tolclofos-methyl.
F11: Inhibitors of the melanin biosynthesis, for example (11.001) tricyclazole, (11.002) 2,2,2-trifluoroethyl {3-methyl-1-[(4-methylbenzoyl)amino]butan-2-yl}carbamate.
F12: Inhibitors of the nucleic acid synthesis, for example (12.001) benalaxyl, (12.002) benalaxyl-M (kiralaxyl), (12.003) metalaxyl, (12.004) metalaxyl-M (mefenoxam), (12.005) furalaxyl.
F13: Inhibitors of the signal transduction, for example (13.001) fludioxonil, (13.002) iprodione, (13.003) procymidone, (13.004) proquinazid, (13.005) quinoxyfen, (13.006) vinclozolin.
F14: Compounds capable to act as an uncoupler, for example (14.001) fluazinam, (14.002) meptyldinocap.
F15: Further compounds, for example (15.001) Abscisic acid, (15.002) benthiazole, (15.003) bethoxazin, (15.004) capsimycin, (15.005) carvone, (15.006) chinomethionat, (15.007) cufraneb, (15.008) cyflufenamid, (15.009) cymoxanil, (15.010) cyprosulfamide, (15.011) flutianil, (15.012) fosetyl-aluminium, (15.013) fosetyl-calcium, (15.014) fosetyl-sodium, (15.015) methyl isothiocyanate, (15.016) metrafenone, (15.017) mildiomycin, (15.018) natamycin, (15.019) nickel dimethyldithiocarbamate, (15.020) nitrothal-isopropyl, (15.021) oxamocarb, (15.022) Oxathiapiprolin, (15.023) oxyfenthiin, (15.024) pentachlorophenol and salts, (15.025) phosphorous acid and its salts, (15.026) propamocarb-fosetylate, (15.027) pyriofenone (chlazafenone), (15.028) tebufloquin, (15.029) tecloftalam, (15.030) tolnifanide, (15.031) 1-(4-{4-[(5R)-5-(2,6-difluorophenyl)-4,5-dihydro-1,2-oxazol-3-yl]-1,3-thiazol-2-yl}piperidin-1-yl)-2-[5-methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]ethanone, (15.032) 1-(4-{4-[(5S)-5-(2,6-difluorophenyl)-4,5-dihydro-1,2-oxazol-3-yl]-1,3-thiazol-2-yl}piperidin-1-yl)-2-[5-methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]ethanone, (15.033) 2-(6-benzylpyridin-2-yl)quinazoline, (15.034) 2,6-dimethyl-1H,5H-[1,4]dithiino[2,3-c:5,6-c′]dipyrrole-1,3,5,7(2H,6H)-tetrone, (15.035) 2-[3,5-bis(difluoromethyl)-1H-pyrazol-1-yl]-1-[4-(4-{5-[2-(prop-2-yn-1-yloxy)phenyl]-4,5-dihydro-1,2-oxazol-3-yl}-1,3-thiazol-2-yl)piperidin-1-yl]ethanone, (15.036) 2-[3,5-bis(difluoromethyl)-1H-pyrazol-1-yl]-1-[4-(4-{5-[2-chloro-6-(prop-2-yn-1-yloxy)phenyl]-4,5-dihydro-1,2-oxazol-3-yl}-1,3-thiazol-2-yl)piperidin-1-yl]ethanone, (15.037) 2-[3,5-bis(difluoromethyl)-1H-pyrazol-1-yl]-1-[4-(4-{5-[2-fluoro-6-(prop-2-yn-1-yloxy)phenyl]-4,5-dihydro-1,2-oxazol-3-yl}-1,3-thiazol-2-yl)piperidin-1-yl]ethanone, (15.038) 2-[6-(3-fluoro-4-methoxyphenyl)-5-methylpyridin-2-yl]quinazoline, (15.039) 2-{(5R)-3-[2-(1-{[3,5-bis(difluoromethyl)-1H-pyrazol-1-yl]acetyl}piperidin-4-yl)-1,3-thiazol-4-yl]-4,5-dihydro-1,2-oxazol-5-yl}-3-chlorophenyl methanesulfonate, (15.040) 2-{(5S)-3-[2-(1-{[3,5-bis(difluoromethyl)-1H-pyrazol-1-yl]acetyl}piperidin-4-yl)-1,3-thiazol-4-yl]-4,5-dihydro-1,2-oxazol-5-yl}-3-chlorophenyl methanesulfonate, (15.041) 2-{2-[(7,8-difluoro-2-methylquinolin-3-yl)oxy]-6-fluorophenyl}propan-2-ol, (15.042) 2-{2-fluoro-6-[(8-fluoro-2-methylquinolin-3-yl)oxy]phenyl}propan-2-ol, (15.043) 2-{3-[2-(1-{[3,5-bis(difluoromethyl)-1H-pyrazol-1-yl]acetyl}piperidin-4-yl)-1,3-thiazol-4-yl]-4,5-dihydro-1,2-oxazol-5-yl}-3-chlorophenyl methanesulfonate, (15.044) 2-{3-[2-(1-{[3,5-bis(difluoromethyl)-1H-pyrazol-1-yl]acetyl}piperidin-4-yl)-1,3-thiazol-4-yl]-4,5-dihydro-1,2-oxazol-5-yl}phenyl methanesulfonate, (15.045) 2-phenylphenol and salts, (15.046) 3-(4,4,5-trifluoro-3,3-dimethyl-3,4-dihydroisoquinolin-1-yl)quinoline, (15.047) 3-(4,4-difluoro-3,3-dimethyl-3,4-dihydroisoquinolin-1-yl)quinoline, (15.048) 4-amino-5-fluoropyrimidin-2-ol (tautomeric form: 4-amino-5-fluoropyrimidin-2(1H)-one), (15.049) 4-oxo-4-[(2-phenylethyl)amino]butanoic acid, (15.050) 5-amino-1,3,4-thiadiazole-2-thiol, (15.051) 5-chloro-N′-phenyl-N′-(prop-2-yn-1-yl)thiophene-2-sulfonohydrazide, (15.052) 5-fluoro-2-[(4-fluorobenzyl)oxy]pyrimidin-4-amine, (15.053) 5-fluoro-2-[(4-methylbenzyl)oxy]pyrimidin-4-amine, (15.054) 9-fluoro-2,2-dimethyl-5-(quinolin-3-yl)-2,3-dihydro-1,4-benzoxazepine, (15.055) but-3-yn-1-yl {6-[({[(Z)-(1-methyl-1H-tetrazol-5-yl)(phenyl)methylene]amino}oxy)methyl]pyridin-2-yl}carbamate, (15.056) ethyl (2Z)-3-amino-2-cyano-3-phenylacrylate, (15.057) phenazine-1-carboxylic acid, (15.058) propyl 3,4,5-trihydroxybenzoate, (15.059) quinolin-8-ol, (15.060) quinolin-8-ol sulfate (2:1), (15.061) tert-butyl {6-[({[(1-methyl-1H-tetrazol-5-yl)(phenyl)methylene]amino}oxy)methyl]pyridin-2-yl}carbamate, (15.062) 5-fluoro-4-imino-3-methyl-1-[(4-methylphenyl)sulfonyl]-3,4-dihydropyrimidin-2(1H)-one, (15.063) any of the pesticides described in US20140221362 (incorporated herein by reference), such as any of the compounds of claim 1 therein, (15.064) fenpicoxamid, (15.065) tolprocarb, (15.066) picarbutrazox, (15.067) pyraziflumid, (15.068) Inpyrfluxam (WO2011162397), (15.069) quinofumelin, (15.070) fluindapyr, (15.071) pyrapropoyne, (15.072) 5-fluoropyrimidone, (15.073) any fungicidal compounds from published patent applications WO2014/201326, WO2014/201327, WO2013/071169, WO2014/182951, WO2014/182945, WO2014/182950, incorporated herein by reference.
Particularly preferred mixtures of fungicides used in the context of the present invention are selected from the group F16 consisting of: mixtures of Prothioconazole and Fluopyram, mixtures of Prothioconazole and Tebuconazole, mixtures of Prothioconazole, Bixafen, and Tebuconazole, mixtures of Prothioconazole, Bixafen, and Trifloxystrobin, mixtures of Prothioconazole, Bixafen, and Spiroxamine, mixtures of Prothioconazole, Bixafen, and Chlorothalonil, mixtures of Prothioconazole and Tebuconazole and Metalaxyl, mixtures of Prothioconazole, Bixafen, and Fluopyram, mixtures of Prothioconazole and Fluoxastrobin, mixtures of Prothioconazole and Trifloxystrobin, mixtures of Prothioconazole and Pencycuron, mixtures of Prothioconazole and Spiroxamine, mixtures of Tebuconazole and Bixafen, mixtures of Tebuconazole and Metalaxyl, mixtures of Tebuconazole and Fluoxastrobin, mixtures of Tebuconazole and Trifloxystrobin, mixtures of Tebuconazole and Fenhexamid, mixtures of Tebuconazole and Fluopicolide, mixtures of Tebuconazole and Spiroxamine, mixtures of Tebuconazole and Pencycuron, mixtures of Fluopyram and Tebuconazole, mixtures of Fluopyram and Spiroxamine, mixtures of Fluopyram and Pyrimethanil, mixtures of Fluopyram and Trifloxystrobin, mixtures of Fluopyram and Triadimenol, mixtures of Fluopyram and Fosetyl or salts and esters thereof, in particular Fosetyl-aluminium, mixtures of Fluopyram and Imidacloprid, mixtures of Fluopyram and Spiroxamine, mixtures of Fluopyram and Fluoxastrobin, mixtures of Bixafen and Spiroxamine, mixtures of Bixafen and Trifloxystrobin, mixtures of Bixafen and Chlorothalonil, mixtures of Bixafen and Fluoxastrobin, mixtures of Trifloxystrobin and Isotianil, mixtures of Trifloxystrobin and Metalaxyl, mixtures of Trifloxystrobin and Propineb, mixtures of Trifloxystrobin and Pyrimethanil, mixtures of Trifloxystrobin and Fosetyl or salts and esters thereof, in particular Fosetyl-aluminium, mixtures of Trifloxystrobin and Fluopicolide, mixtures of Propineb and Fluopicolide, mixtures of Fosetyl or salts and esters thereof, in particular Fosetyl-aluminium, and Fluopicolide, mixtures of Bixafen and Ipfentrifluconazole, mixtures of Bixafen and Mefentrifluconazole, mixtures of Metalaxyl and Ipfentrifluconazole, mixtures of Metalaxyl and Mefentrifluconazole, mixtures of Prothioconazole and Ipfentrifluconazole, mixtures of Prothioconazole and Mefentrifluconazole, mixtures of Tebuconazole and Ipfentrifluconazole, mixtures of Tebuconazole and Mefentrifluconazole, mixtures of Trifloxystrobin and Ipfentrifluconazole, mixtures of Trifloxystrobin and Mefentrifluconazole, mixtures of Fluoxastrobin and Ipfentrifluconazole, mixtures of Fluoxastrobin and Mefentrifluconazole, mixtures of Fluopyram and Ipfentrifluconazole, mixtures of Fluopyram and Mefentrifluconazole, mixtures of Spiroxamine and Ipfentrifluconazole, mixtures of Spiroxamine and Mefentrifluconazole, mixtures of Pydiflumetofen and Ipfentrifluconazole, mixtures of Pydiflumetofen and Mefentrifluconazole, mixtures of Pyraclostrobin and Ipfentrifluconazole, mixtures of Pyraclostrobin and Mefentrifluconazole, mixtures of Fluxapyroxad and Ipfentrifluconazole, mixtures of Fluxapyroxad and Mefentrifluconazole.
A fungicide/fungicidal agent in crop protection, as described herein for use on or with the plants or seeds of the invention, is capable of controlling fungi or oomycetes. The term “controlling fungi/oomycetes”, as used herein, means killing the fungi/oomycetes or preventing or impeding their development or their growth or preventing or impeding their penetration into or their feeding on plant tissue.
Herbicides
Examples for herbicides useful in the context of the present invention are disclosed in group H1:
Acetochlor, acifluorfen, acifluorfen-sodium, aclonifen, alachlor, allidochlor, alloxydim, alloxydim-sodium, ametryn, amicarbazone, amidochlor, amidosulfuron, 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methylphenyl)-5-fluoropyridine-2-carboxylic acid, aminocyclopyrachlor, aminocyclopyrachlor-potassium, aminocyclopyrachlor-methyl, aminopyralid, amitrole, ammoniumsulfamate, anilofos, asulam, atrazine, azafenidin, azimsulfuron, beflubutamid, benazolin, benazolin-ethyl, benfluralin, benfuresate, bensulfuron, bensulfuron-methyl, bensulide, bentazone, benzobicyclon, benzofenap, bicyclopyron, bifenox, bilanafos, bilanafos-sodium, bispyribac, bispyribac-sodium, bromacil, bromobutide, bromofenoxim, bromoxynil, bromoxynil-butyrate, -potassium, -heptanoate, and -octanoate, busoxinone, butachlor, butafenacil, butamifos, butenachlor, butralin, butroxydim, butylate, cafenstrole, carbetamide, carfentrazone, carfentrazone-ethyl, chloramben, chlorbromuron, chlorfenac, chlorfenac-sodium, chlorfenprop, chlorflurenol, chlorflurenol-methyl, chloridazon, chlorimuron, chlorimuron-ethyl, chlorophthalim, chlorotoluron, chlorthal-dimethyl, chlorsulfuron, cinidon, cinidon-ethyl, cinmethylin, cinosulfuron, clacyfos, clethodim, clodinafop, clodinafop-propargyl, clomazone, clomeprop, clopyralid, cloransulam, cloransulam-methyl, cumyluron, cyanamide, cyanazine, cycloate, cyclopyrimorate, cyclosulfamuron, cycloxydim, cyhalofop, cyhalofop-butyl, cyprazine, 2,4-D, 2,4-D-butotyl, -butyl, -dimethylammonium, -diolamin, -ethyl, -2-ethylhexyl, -isobutyl, -isooctyl, -isopropyl ammonium, -potassium, -triisopropanolammonium, and -trolamine, 2,4-DB, 2,4-DB-butyl, -dimethylammonium, -isooctyl, -potassium, and -sodium, daimuron (dymron), dalapon, dazomet, n-decanol, desmedipham, detosyl-pyrazolate (DTP), dicamba, dichlobenil, 2-(2,4-dichlorobenzyl)-4,4-dimethyl-1,2-oxazolidin-3-one, 2-(2,5-dichlorobenzyl)-4,4-dimethyl-1,2-oxazolidin-3-one, dichlorprop, dichlorprop-P, diclofop, diclofop-methyl, diclofop-P-methyl, diclosulam, difenzoquat, diflufenican, diflufenzopyr, diflufenzopyr-sodium, dimefuron, dimepiperate, dimethachlor, dimethametryn, dimethenamid, dimethenamid-P, dimetrasulfuron, dinitramine, dinoterb, diphenamid, diquat, diquat-dibromid, dithiopyr, diuron, DNOC, endothal, EPTC, esprocarb, ethalfluralin, ethametsulfuron, ethametsulfuron-methyl, ethiozin, ethofumesate, ethoxyfen, ethoxyfen-ethyl, ethoxysulfuron, etobenzanid, F-9600, F-5231, i.e. N-{2-chloro-4-fluoro-5-[4-(3-fluoropropyl)-5-oxo-4,5-dihydro-1H-tetrazol-1-yl]phenyl}ethanesulfonamide, F-7967, i. e. 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)pyrimidine-2,4(1H,3H)-dione, fenoxaprop, fenoxaprop-P, fenoxaprop-ethyl, fenoxaprop-P-ethyl, fenoxasulfone, fenquinotrione, fentrazamide, flamprop, flamprop-M-isopropyl, flamprop-M-methyl, flazasulfuron, florasulam, fluazifop, fluazifop-P, fluazifop-butyl, fluazifop-P-butyl, flucarbazone, flucarbazone-sodium, flucetosulfuron, fluchloralin, flufenacet, flufenpyr, flufenpyr-ethyl, flumetsulam, flumiclorac, flumiclorac-pentyl, flumioxazin, fluometuron, flurenol, flurenol-butyl, -dimethylammonium and -methyl, fluoroglycofen, fluoroglycofen-ethyl, flupropanate, flupyrsulfuron, flupyrsulfuron-methyl-sodium, fluridone, flurochloridone, fluroxypyr, fluroxypyr-meptyl, flurtamone, fluthiacet, fluthiacet-methyl, fomesafen, fomesafen-sodium, foramsulfuron, fosamine, glufosinate, glufosinate-ammonium, glufosinate-P-sodium, glufosinate-P-ammonium, glufosinate-P-sodium, glyphosate, glyphosate-ammonium, -isopropyl-ammonium, -diammonium, -dimethylammonium, -potassium, -sodium, and -trimesium, H-9201, i.e. O-(2,4-dimethyl-6-nitrophenyl) O-ethyl isopropylphosphoramidothioate, halauxifen, halauxifen-methyl, halosafen, halosulfuron, halosulfuron-methyl, haloxyfop, haloxyfop-P, haloxyfop-ethoxyethyl, haloxyfop-P-ethoxyethyl, haloxyfop-methyl, haloxyfop-P-methyl, hexazinone, HW-02, i.e. 1-(dimethoxyphosphoryl)ethyl-(2,4-dichlorophenoxy)acetate, imazamethabenz, imazamethabenz-methyl, imazamox, imazamox-ammonium, imazapic, imazapic-ammonium, imazapyr, imazapyr-isopropylammonium, imazaquin, imazaquin-ammonium, imazethapyr, imazethapyr-immonium, imazosulfuron, indanofan, indaziflam, iodosulfuron, iodosulfuron-methyl-sodium, ioxynil, ioxynil-octanoate, -potassium and -sodium, ipfencarbazone, isoproturon, isouron, isoxaben, isoxaflutole, karbutilate, KUH-043, i.e. 3-({[5-(difluoromethyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]methyl}sulfonyl)-5,5-dimethyl-4,5-dihydro-1,2-oxazole, ketospiradox, lactofen, lenacil, linuron, MCPA, MCPA-butotyl, -dimethylammonium, -2-ethylhexyl, -isopropylammonium, -potassium, and -sodium, MCPB, MCPB-methyl, -ethyl, and -sodium, mecoprop, mecoprop-sodium, and -butotyl, mecoprop-P, mecoprop-P-butotyl, -dimethylammonium, -2-ethylhexyl, and -potassium, mefenacet, mefluidide, mesosulfuron, mesosulfuron-methyl, mesotrione, methabenzthiazuron, metam, metamifop, metamitron, metazachlor, metazosulfuron, methabenzthiazuron, methiopyrsulfuron, methiozolin, methyl isothiocyanate, metobromuron, metolachlor, S-metolachlor, metosulam, metoxuron, metribuzin, metsulfuron, metsulfuron-methyl, molinat, monolinuron, monosulfuron, monosulfuron-ester, MT-5950, i.e. N-(3-chloro-4-isopropylphenyl)-2-methylpentan amide, NGGC-011, napropamide, NC-310, i.e. [5-(benzyloxy)-1-methyl-1H-pyrazol-4-yl](2,4-dichlorophenyl)methanone, neburon, nicosulfuron, nonanoic acid (pelargonic acid), norflurazon, oleic acid (fatty acids), orbencarb, orthosulfamuron, oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxaziclomefon, oxyfluorfen, paraquat, paraquat dichloride, pebulate, pendimethalin, penoxsulam, pentachlorphenol, pentoxazone, pethoxamid, petroleum oils, phenmedipham, picloram, picolinafen, pinoxaden, piperophos, pretilachlor, primisulfuron, primisulfuron-methyl, prodiamine, profoxydim, prometon, prometryn, propachlor, propanil, propaquizafop, propazine, propham, propisochlor, propoxycarbazone, propoxycarbazone-sodium, propyrisulfuron, propyzamide, prosulfocarb, prosulfuron, pyraclonil, pyraflufen, pyraflufen-ethyl, pyrasulfotole, pyrazolynate (pyrazolate), pyrazosulfuron, pyrazosulfuron-ethyl, pyrazoxyfen, pyribambenz, pyribambenz-isopropyl, pyribambenz-propyl, pyribenzoxim, pyributicarb, pyridafol, pyridate, pyriftalid, pyriminobac, pyriminobac-methyl, pyrimisulfan, pyrithiobac, pyrithiobac-sodium, pyroxasulfone, pyroxsulam, quinclorac, quinmerac, quinoclamine, quizalofop, quizalofop-ethyl, quizalofop-P, quizalofop-P-ethyl, quizalofop-P-tefuryl, rimsulfuron, saflufenacil, sethoxydim, siduron, simazine, simetryn, SL-261, sulcotrione, sulfentrazone, sulfometuron, sulfometuron-methyl, sulfosulfuron, SYN-523, SYP-249, i.e. 1-ethoxy-3-methyl-1-oxobut-3-en-2-yl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate, SYP-300, i.e. 1-[7-fluoro-3-oxo-4-(prop-2-yn-1-yl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-3-propyl-2-thioxoimidazolidine-4,5-dione, 2,3,6-TBA, TCA (trichloroacetic acid), TCA-sodium, tebuthiuron, tefuryltrione, tembotrione, tepraloxydim, terbacil, terbucarb, terbumeton, terbuthylazin, terbutryn, thenylchlor, thiazopyr, thiencarb azone, thiencarbazone-methyl, thifensulfuron, thifensulfuron-methyl, thiobencarb, tiafenacil, tolpyralate, topramezone, tralkoxydim, triafamone, tri-allate, triasulfuron, triaziflam, tribenuron, tribenuron-methyl, triclopyr, trietazine, trifloxysulfuron, trifloxysulfuron-sodium, trifludimoxazin, trifluralin, triflusulfuron, triflusulfuron-methyl, tritosulfuron, urea sulfate, vernolate, XDE-848, ZJ-0862, i.e. 3,4-dichloro-N-{2-[(4,6-dimethoxypyrimidin-2-yl)oxy]benzyl}aniline, and the following compounds:
Group H2 discloses herbicides especially used in the context of the present invention are selected from the group consisting of:
Acetochlor, Alachlor, Dicamba, Diflufenican, Flufenacet, Fluroxypyr, Foramsulfuron, Glufosinate, L-Glufosinate, Glyphosate, Hexazinone, Imazamox, Imazethapyr, Indaziflam, Iodosulfuron, Isoxaflutole, Mesosulfuron, Mesotrione, Metolachlor, Metribuzin, Metsulfuron, Pendimethalin, Penoxsulam, Picloram, Pinoxaden, Pyroxsulam, Quinmerac, Rimsulfuron, Thiencarbazone, Tembotrione, Thifensulfuron, and/or Tribenuron, and the esters and/or agronomically acceptable salts of these herbicides.
Group H3 discloses preferred herbicides are selected from the group consisting of:
Acetochlor, Dicamba, Diflufenican, Flufenacet, Foramsulfuron, Glufosinate, L-Glufosinate, Glyphosate, Iodosulfuron, Isoxaflutole, Mesosulfuron, Mesotrione, Metribuzin, Pinoxaden, Tembotrione, and/or Thiencarbazone, and the esters and/or agronomically acceptable salts of these herbicides.
If Glyphosate salts are used as herbicide, preference is given to Glyphosate isopropylamine salt, Glyphosate potassium salt, Glyphosate sodium salt, Glyphosate trimethylsulfonium salt.
If (L-)Glufosinate salts are used as herbicide, preference is given to (L-)Glufosinate-ammonium salt, (L-) Glufosinate potassium salt and (L-)Glufosinate sodium salt.
If Foramsulfuron salts are used as herbicide, preference is given to Foramsulfuron-sodium.
If Iodosulfuron esters and/or salts thereof are used as herbicide, preference is given to Iodosulfuron-methyl and Iodosulfuron-methyl-sodium.
If Mesosulfuron esters and/or salts thereof are used as herbicide, preference is given to Mesosulfuron-methyl and Mesosulfuron-methyl-sodium.
If Thiencarbazone esters and/or salts thereof are used as herbicide, preference is given to Thiencarbazone-methyl and Thiencarbazone-methyl-sodium.
Preferred mixtures of herbicides used in the context of the present invention are disclosed in group H4: mixtures of Acetochlor and other herbicides mentioned herein, mixtures of Dicamba, esters and/or salts thereof and other herbicides mentioned herein, mixtures of Diflufenican and other herbicides mentioned herein, mixtures of Flufenacet and other herbicides mentioned herein, mixtures of Glufosinate and/or salts thereof (preferably Glufosinate-ammonium salt, Glufosinate potassium salt and Glufosinate sodium salt) and other herbicides mentioned herein, mixtures of L-Glufosinate and/or salts thereof (preferably L-Glufosinate-ammonium salt, L-Glufosinate potassium salt and L-Glufosinate sodium salt) and other herbicides mentioned herein, mixtures of Indaziflam and other herbicides mentioned herein, mixtures of Isoxaflutole and other herbicides mentioned herein, mixtures of Mesosulfuron esters and/or salts thereof (preferably Mesosulfuron-methyl(-sodium)) and other herbicides mentioned herein, mixtures of Metribuzin and other herbicides mentioned herein, mixtures of Thiencarbazone esters and/or salts thereof (preferably Thiencarbazone-methyl(-sodium)) and other herbicides mentioned herein, mixtures of Tembotrione and other herbicides mentioned herein.
Particularly preferred mixtures of herbicides used in the context of the present invention are disclosed in group H5:
mixtures of Acetochlor and Isoxaflutole, mixtures of Acetochlor and Tembotrione mixtures of Diflufenican and Flufenacet, mixtures of Diflufenican and Metribuzin, mixtures of Glufosinate and/or salts thereof and Indaziflam, in particular mixtures of Glufosinate-ammonium and Indaziflam, mixtures of L-Glufosinate and/or salts thereof and Indaziflam, in particular mixtures of L-Glufosinate-ammonium and Indaziflam, mixtures of Mesosulfuron-methyl(-sodium) and Iodosulfuron-methyl(-sodium), mixtures of Mesosulfuron-methyl(-sodium), Iodosulfuron-methyl(-sodium) and Flufenacet, mixtures of Tembotrione and Isoxaflutole, mixtures of Tembotrione, Isoxaflutole, and Acetochlor.
The herbicides and the mixtures of herbicides mentioned herein may be used in pre-emergence applications and/or in post-emergence applications.
In accordance with the present invention, the herbicides and the mixtures of herbicides mentioned herein may be applied as a split application over time. Another possibility is the application of the individual active ingredients or the mixtures comprising the active ingredients in a plurality of portions (sequential application), for example pre-emergence applications, followed by post-emergence applications or early post-emergence applications, followed by applications at medium or late post-emergence.
In one embodiment, an herbicide as listed in any one of groups H1 to H5 is used on seeds or plants comprising elite event EE-GM5 of the invention, or on soil wherein said seeds or plants are to be planted/sown, and said herbicide is not isoxaflutole, topramezone or mesotrione. In another embodiment, a herbicide as listed in any one of groups H1 to H5 is used on seeds or plants comprising elite event EE-GM5 of the invention, or on soil wherein said seeds or plants are to be planted/sown, wherein said plant or seed comprising EE-GM5 also contains one or more soybean SCN resistance genes from PI 88788, PI 548402, PI 209332 or PI 437654, or comprises one or more of the soybean SCN resistance loci or genes selected from the group consisting of: rhg1, rhg1-b, rhg2, rhg3, Rhg4, Rhg5, qSCN11, cqSCN-003, cqSCN-005, cqSCN-006, and cqSCN-007.
Examples for plant growth regulators are useful in the context of the present invention are disclosed in group P1:
Acibenzolar, acibenzolar-S-methyl, 5-aminolevulinic acid, ancymidol, 6-benzylaminopurine, Brassinolid, catechine, chlormequat chloride, cloprop, cyclanilide, 3-(cycloprop-1-enyl) propionic acid, daminozide, dazomet, n-decanol, dikegulac, dikegulac-sodium, endothal, endothal-dipotassium, -disodium, and -mono(N,N-dimethylalkylammonium), ethephon, flumetralin, flurenol, flurenol-butyl, flurprimidol, forchlorfenuron, gibberellic acid, inabenfide, indol-3-acetic acid (IAA), 4-indol-3-ylbutyric acid, isoprothiolane, probenazole, jasmonic acid, maleic hydrazide, mepiquat chloride, 1-methylcyclopropene, methyl jasmonate, 2-(1-naphthyl)acetamide, 1-naphthylacetic acid, 2-naphthyloxyacetic acid, nitrophenolate-mixture, paclobutrazol, N-(2-phenylethyl)-beta-alanine, N-phenylphthalamic acid, prohexadione, prohexadione-calcium, prohydrojasmone, salicylic acid, strigolactone, tecnazene, thidiazuron, triacontanol, trinexapac, trinexapac-ethyl, tsitodef, uniconazole, uniconazole-P.
Biological Control Agent (BCA) Groups:
Biological control agents are, in particular, bacteria, fungi or yeasts, protozoa, viruses, entomopathogenic nematodes, products produced by microorganisms including proteins or secondary metabolites and botanicals, especially botanical extracts, that support or enhance plant or seed growth or development so as to protect or increase plant yield, particularly when plants or seeds experience stresses such as drought or attack by plant pathogens/pests (e.g., by killing plant pathogens or plant pests or preventing or impeding their development or their growth or preventing or impeding their penetration into or their sucking/feeding on plant tissue). Therefore, the Biological Control Agent (BCA) Groups (1) to (7) according to the invention are:
BCA Group (1): bacteria
BCA Group (2): fungi or yeasts
BCA Group (3): protozoa
BCA Group (4): viruses
BCA Group (5): entomopathogenic nematodes
BCA Group (6): products produced by microorganisms including proteins or secondary metabolites
BCA Group (7): botanicals, especially botanical extracts.
These Biological Control Agent (“BCA”) Groups BCA1 to BCA7 are further characterized as follows:
BCA1: According to the invention biological control agents which are summarized under the term “bacteria” include but are not limited to spore-forming, root-colonizing bacteria, or bacteria useful as bioinsecticide, biofungicide or bionematicide. Such bacteria to be used or employed according to the invention include but are not limited to:
(1.1) Agrobacterium radiobacter, in particular strain K84 (product known as Galltrol-A from AgBioChem, CA) or strain K1026 (product known as Nogall from Becker Underwood, US), (1.2)Agrobacterium vitis, in particular the non-pathogenic strain VAR03-1, (1.3) Azorhizobium caulinodans, preferably strain ZB-SK-5, (1.4) Azospirillum amazonense, (1.5) Azospirillum brasilense, (1.6) Azospirillum halopraeference, (1.7) Azospirillum irakense, (1.8) Azospirillum lipoferum, (1.9) Azotobacter chroococcum, preferably strain H 23 (CECT 4435) (cf. Applied Soil Ecology 12 (1999) 51±59), (1.10) Azotobacter vinelandii, preferably strain ATCC 12837 (cf. Applied Soil Ecology 12 (1999) 51±59), (1.11) Bacillus sp. strain AQ175 (ATCC Accession No. 55608), (1.12) Bacillus sp. strain AQ177 (ATCC Accession No. 55609), (1.13) Bacillus sp. strain AQ178 (ATCC Accession No. 53522), (1.14) Bacillus acidocaldarius, (1.15) Bacillus acidoterrestris, (1.16) Bacillus agri (cf. WO 2012/140207), (1.17) Bacillus aizawai (cf. WO 2012/140207), (1.18) Bacillus albolactis (cf. WO 2012/140207), (1.19) Bacillus alcalophilus, (1.20) Bacillus alvei, (1.21) Bacillus aminoglucosidicus, (1.22) Bacillus aminovorans, (1.23) Bacillus amylolyticus (also known as Paenibacillus amylolyticus), (1.24) Bacillus amyloliquefaciens, in particular B. amyloliquefaciens strain IN937a (cf. WO 2012/140207), or B. amyloliquefaciens strain FZB42 (DSM 231179) (product known as RhizoVital® from ABiTEP, DE), or B. amyloliquefaciens strain B3, or B. amyloliquefaciens strain D747, (products known as Bacstar® from Etec Crop Solutions, NZ, or Double Nickel™ from Certis, US), B. amyloliquefaciens strain APM-1 (ATCC accession number PTA-4838, known as Aveo EZ from Valent), Bacillus amyloliquefaciens TJ1000 (also known as Bacillus amyloliquefaciens (Fukumoto) Priest et al. (ATCC BAA-390), or the B. amyloliquefaciens strains from US2012/0149571, such as B. amyloliquefaciens AP-136 (NRRL B-50330; NRRL B-50614), B. amyloliquefaciens AP-188 (NRRL B-50331; NRRL B-50615), B. amyloliquefaciens AP-218 (NRRL B-50618), B. amyloliquefaciens AP-219 (NRRL B-50332, NRRL B-50619), and B. amyloliquefaciens AP-295 (NRRL B-50333, NRRL B-50620), or Bacillus amyloliquefaciens strain MBI 600 (previously Bacillus subtilis strain MBI 600), or Bacillus amyloliquefaciens strain MBI 600 in combination with cis-Jasmone, or Bacillus amyloliquefaciens strain F727 (1.25) Bacillus aneurinolyticus, (1.26) Bacillus atrophaeus, (1.27) Bacillus azotoformans, (1.28) Bacillus badius, (1.29) Bacillus cereus (synonyms: Bacillus endorhythmos, Bacillus medusa), in particular spores of B. cereus strain CNCM I-1562 (cf. U.S. Pat. No. 6,406,690), or strain BP01 (ATCC 55675), (products known as Mepichlor from Arysta, US or Mepplus, Micro-Flo Company LLC, US), (1.30) Bacillus chitinosporus, in particular strain AQ746 (Accession No. NRRL B-21618), (1.31) Bacillus circulans (1.32) Bacillus coagulans, in particular strain TQ33, (1.33) Bacillus fastidiosus, (1.34) Bacillus firmus, in particular strain I-1582 (products known as Bionem, Flocter or VOTIVO from Bayer CropScience, CNCM I-1582), Bacillus firmus strain NRRL B-67003, or Bacillus firmus strain NRRL B-67518, (1.35) Bacillus kurstaki, (1.36) Bacillus lacticola, (1.37) Bacillus lactimorbus, (1.38) Bacillus lactis, (1.39) Bacillus laterosporus (also known as Brevibacillus laterosporus), (product known as Bio-Tode from Agro-Organics, SA), Bacillus laterosporus ATCC PTA-3952, Bacillus laterosporus ATCC PTA-3593, (1.40) Bacillus lautus, (1.41) Bacillus lentimorbus, (1.42) Bacillus lentus, (1.43) Bacillus licheniformis, in particular strain SB3086 (product known as EcoGuard™ Biofungicide or Green Releaf from Novozymes Biologicals, US), Bacillus licheniformis CH200, or Bacillus licheniformis RTI 184, or a combination of Bacillus licheniformis CH200 and Bacillus licheniformis RTI 184, or Bacillus licheniformis ATCC PTA-6175, (1.44) Bacillus maroccanus, (1.45) Bacillus medusa, (1.46) Bacillus megaterium, (products known as Bioarc®, from BioArc), or B. megaterium strain YFM3.25, (1.47) Bacillus metiens, (1.48) Bacillus mojavensis, in particular strain SR11 (CECT-7666), or Bacillus mojavensis AP-209 (US 2012/0149, NRRL B-50616), (1.49) Bacillus mycoides, in particular strain AQ726 (Accession No. NRRL B21664) or isolate J, (product known as BmJ from Certis USA), (1.50) Bacillus nematocida, (1.51) Bacillus nigrificans, (1.52) Bacillus popilliae, (product known as Cronox from Bio-Crop, CO), (1.53) Bacillus psychrosaccharolyticus, (1.54) Bacillus pumilus, in particular strain GB34 (Accession No. ATCC 700814), (products known as Yield Shield from Bayer Crop Science, DE), and strain QST2808 (Accession No. NRRL B-30087), (products known as Sonata QST 2808 from Bayer Crop Science), or strain BU F-33, (product known as Integral F-33 from Becker Underwood, US), or strain AQ717 (Accession No. NRRL B21662), (1.55) Bacillus siamensis, in particular strain KCTC 13613T, (1.56) Bacillus smithii, (1.57) Bacillus sphaericus, in particular Serotype H5a5b strain 2362, (product known as VectoLex® from Valent BioSciences, US), (1.58) Bacillus subtilis, in particular strain GB03 (Accession No. ATCC SD-1397), (product known as Kodiak® from Bayer Crop Science, DE), and strain QST713/AQ713 (Accession No. NRRL B-21661), (products known as Serenade QST 713®, Serenade Soil and Serenade Max from Bayer Crop Science) and strain AQ 153 (ATCC accession No. 55614), and strain AQ743 (Accession No. NRRL B-21665), and strain DB 101, (products known as Shelter from Dagutat Bio lab, ZA), and strain DB 102, (product known as Artemis from Dagutat Bio lab, ZA), and Bacillus subtilis strain MBI 600 (now Bacillus amyloliquefaciens strain MBI 600), (e.g., products known as Subtilex from Becker Underwood, US), or Bacillus subtilis Y1336 (available as BIOBAC® WP from Bion-Tech, Taiwan, registered as a biological fungicide in Taiwan under Registration Nos. 4764, 5454, 5096 and 5277), or B. subtilis var. amyloliquefaciens strain FZB24, (product known as Taegro® from Novozymes, US), a mutant of FZB24 that was assigned Accession No. NRRL B-50349 by the Agricultural Research Service Culture Collection and is also described in U.S. Patent Publication No. 20110230345, Bacillus amyloliquefaciens FZB42, available from ABiTEP GMBH, Germany, as the plant strengthening product RHIZOVITAL and described in European Patent Publication No. EP2179652, mutants of FZB42 described in International Application Publication No. WO 2012/130221, including Bacillus amyloliquefaciens ABI01, which was assigned Accession No. DSM 10-1092 by the DSMZ—German Collection of Microorganisms and Cell Cultures, or B. subtilis subspecies natto (formerly B. natto), or B. subtilis isolate B246, (product known as Avogreen from RE at UP) or strain MBI600 (products known as Subtilex or Hi Stick N/T from Becker Underwood), or strain QST30002/AQ30002 (Accession No. NRRL B-50421, cf. WO 2012/087980), or strain QST30004/AQ30004 (Accession No. NRRL B-50455, cf. WO 2012/087980), or Bacillus subtilis (including Bacillus subtilis var. amyloliquefaciens) strains and Bacillus amyloliquefaciens strains that produce a fungicidal combination of lipopeptides, including (a) fengycin or plipastatin-type compound(s), (an) iturin-type compound(s), and/or (a) surfactin-type compound(s) (Ongena et al., Trends in Microbiology, Vol 16, No. 3, March 2008, pp. 115-125), (1.59) Bacillus tequilensis, in particular strain NII-0943, (1.60) Bacillus thuringiensis, in particular B. thuringiensis subspecies israelensis (serotype H-14), strain AM65-52 (Accession No. ATCC 1276), (product known as VectoBac® from Valent BioSciences, US), or B. th. israelensis strain BMP 144, (product known as Aquabac from Becker Microbial Products IL), or B. thuringiensis subsp. aizawai, in particular strain ABTS-1857 (SD-1372), (products known as XenTari from Bayer Crop Science, DE) or strain GC-91 (Accession No. NCTC 11821), or serotype H-7, (product known as Florbac WG from Valent BioSciences, US), or B. thuringiensis subsp. kurstaki strain HD-1, (product known as Diper ES from Valent BioSciences, US), or strain BMP 123 from Becker Microbial Products, IL, or strain ABTS 351 (Accession No. ATCC SD-1275), or strain PB 54 (Accession No. CECT 7209), or strain SA 11 (Accession No. NRRL B-30790), or strain SA 12 (Accession No. NRRL B-30791), or strain EG 2348 (Accession No. NRRL B-18208), or strain EG-7841 (product known as Crymax from Certis USA), or B. thuringiensis subsp. tenebrionis strain NB 176 (SD-5428), (product known as Novodor® FC from BioFa DE), or B. thuringiensis subspecies. aegypti, (product known as Agerin), or B. thuringiensis var. colmeri (product known as TianBaoBTc from Changzhou Jianghai Chemical Factory), or B. thuringiensis var. darmstadiensis strains 24-91 (product known as Baciturin), or B. thuringiensis var. dendrolimus (products known as Dendrobacillin), or B. thuringiensis subsp. galleriae (product known as GrubGone or BeetleGone from Phyllom BioProducts), or B. thuringiensis var. japonensis strain Buibui, or B. thuringiensis subsp. morrisoni, or B. thuringiensis var. san diego (product known as M-One® from Mycogen Corporation, US), or B. thuringiensis subsp. thuringiensis serotype 1, strain MPPL002, or B. thuringiensis var. thuringiensis, or B. thuringiensis var 7216 (product known as Amactic, Pethian), or B. thuringiensis var T36 (product known as Cahat) or B. thuringiensis strain BD #32 (Accession No. NRRL B-21530) from Bayer Crop Science, DE, or B. thuringiensis strain AQ52 (Accession No. NRRL B-21619) from Bayer Crop Science, DE, or B. thuringiensis strain CR-371 (Accession No. ATCC 55273), (1.61) Bacillus uniflagellatus, (1.62) Bradyrhizobium japonicum (product known as Optimize from Novozymes), (1.63) Brevibacillus brevis (formerly Bacillus brevis), (product known as Brevisin), in particular strains SS86-3, SS86-4, SS86-5, 2904, (1.64) Brevibacillus laterosporus (formerly Bacillus laterosporus), in particular strains ATCC 64, NRS 1111, NRS 1645, NRS 1647, BPM3, G4, NCIMB 41419, (1.65) Burkholderia spp., in particular Burkholderia rinojensis strain A396 (Accession No. NRRL B-50319), (product known as MBI-206 TGAI from Marrone Bio Innovations), or B. cepacia (product known as Deny from Stine Microbial Products), (1.66) Chromobacterium subtsugae, in particular strain PRAA4-1T (MBI-203), (product known as Grandevo from Marrone Bio Innovations), (1.67) Corynebacterium paurometabolum, (1.68) Delftia acidovorans, in particular strain RAY209 (product known as BioBoost® from Brett Young Seeds), (1.69) Gluconacetobacter diazotrophicus, (1.70) Herbaspirilum rubrisubalbicans, (1.71) Herbaspirilum seropedicae, (1.72) Lactobacillus sp. (product known as Lactoplant from LactoPAFI), (1.73) Lactobacillus acidophilus (product known as Fruitsan from Inagrosa-Industrias Agrobiológicas, S.A), (1.74) Lysobacter antibioticus, in particular strain 13-1 (cf. Biological Control 2008, 45, 288-296), (1.75) Lysobacter enzymogenes, in particular strain C3 (cf. J Nematol. 2006 June; 38(2): 233-239), (1.76) Paenibacillus alvei, in particular strains III3DT-1A, 1112E, 46C3, 2771 (Bacillus genetic stock center, November 2001), (1.77) Paenibacillus macerans, (1.78) Paenibacillus polymyxa, in particular strain AC-1 (product known as Topseed from Green Biotech Company Ltd.), (1.79) Paenibacillus popilliae (formerly Bacillus popilliae) product known as Milky spore disease from St. Gabriel Laboratories), (1.80) P antoea agglomerans, in particular strain E325 (Accession No. NRRL B-21856), (product known as Bloomtime Biological FD Biopesticide from Northwest Agricultural Products), (1.81) Pasteuria nishizawae, such as the product known as oyacyst LF/ST from Pasteuria Bioscience, or Pasteuria nishizawae Pn1 (Clariva from Syngenta), (1.82) Pasteuria penetrans (formerly Bacillus penetrans), (product known as Pasteuria wettable powder from Pasteuria Bioscience), (1.83) Pasteuria ramosa, (1.84) Pasteuria reniformis, (1.85) Pasteuria thornei, (1.86) Pasteuria usgae (products known as Econem™ from Pasteuria Bioscience), (1.87) Pectobacterium carotovorum (formerly Erwinia carotovora), (product known as BioKeeper from Nissan, JP), (1.88) Pseudomonas aeruginosa, in particular strains WS-1 or PN1, (1.89) Pseudomonas aureofaciens, in particular strain TX-1 (product known as Spot-Less Biofungicide from Eco Soils Systems, CA), (1.90) Pseudomonas cepacia (formerly known as Burkholderia cepacia), in particular type Wisconsin, strains M54 or J82, (1.91) Pseudomonas chlororaphis, in particular strain MA 342 (products known as Cedomon from Bioagri, S), or strain 63-28 (product known as ATEze from EcoSoil Systems, US), (1.92) Pseudomonas fluorescens, in particular strain A506 (products known as Blightban from NuFarm or Frostban B from Frost Technology Corp), or strain 1629RS (product known as Frostban D from Frost Technology Corp), (1.93) Pseudomonas proradix (product known as Proradix from Sourcon Padena), (1.94) Pseudomonas putida, (1.95) Pseudomonas resinovorans (product known as Solanacure from Agricultural Resaerch Council, SA), (1.96) Pseudomonas syringae, in particular strain MA-4 (product known as Biosave from EcoScience, US), or strain 742RS (product known as Frostban C from Frost Technology Corp, (1.97) Rhizobium fredii, (1.98) Rhizobium leguminosarum, in particular by. viceae strain Z25 (Accession No. CECT 4585), (1.99) Rhizobium loti, (1.100) Rhizobium meliloti, (1.101) Rhizobium trifolii, (1.102) Rhizobium tropici, (1.103) Rhodococcus globerulus strain AQ719 (Accession No. NRRL B21663) from Bayer Crop Science, DE, (1.104) Serratia entomophila (product known as Invade® from Wrightson Seeds), (1.105) Serratia marcescens, in particular strain SRM (Accession No. MTCC 8708) or strain R35, (1.106) Streptomyces sp. strain NRRL B-30145 from Bayer Crop Science, DE, or strains WYE 20 (KCTC 0341BP) and WYE 324 (KCTC0342BP), (1.107) Streptomyces acidiscabies, in particular strain RL-110T, (product known as MBI-005EP from Marrone Bioinnovations, CA), (1.108) Streptomyces candidus, in particular strain Y21007-2, (products known as BioBac or BioAid from Biontech, TW), (1.109) Streptomyces colombiensis (1.110) Streptomyces galbus (=Streptomyces griseoviridis), in particular strain K61 (Accession No. DSM 7206) (product known as Mycostop® from Verdera, cf. Crop Protection 2006, 25, 468-475) or strain QST 6047 (=strain NRRL B-30232) (product known as Virtuoso from Bayer Crop Science, DE), (1.111) Streptomyces goshikiensis, (1.112) Streptomyces lavendulae, (1.113) Streptomyces lydicus, in particular strain WYCD108US) or strain WYEC108 (product known as Actinovate from Natural Industries, US), (1.114) Streptomyces microflavus, in particular strain AQ6121 (=QRD 31.013, NRRL B-50550) from Bayer Crop Science, or strain M (=AQ6121.002) (091013-02 deposited with the Canadian International Depository Authority) from Bayer Crop Science, (1.115) Streptomyces prasinus (cf. “Prasinons A and B: potent insecticides from Streptomyces prasinus”, Applied microbiology 1973 November), (1.116) Streptomyces rimosus, (1.117) Streptomyces saraceticus (product known as Clanda from A & A Group (Agro Chemical Corp.)), (1.118) Streptomyces venezuelae, (1.119) Thiobacillus sp. (product known as Cropaid from Cropaid Ltd UK), (1.120) Virgibacillus pantothenticus (formerly Bacillus pantothenticus), in particular strain ATCC 14576/DSM 491, (1.121) Xanthomonas campestris (herbicidal activity), in particular pv poae (product known as Camperico), (1.122) Xenorhabdus (=Photorhabdus) luminescens, (1.123) Xenorhabdus (=Photorhabdus) nematophila, and (1.124) Bacillus solisalsi, such as Bacillus solisalsi AP-217 (US 2012/0149571, NRRL B-50617), Bacillus thuringiensis strain EX297512, Bacillus licheniformis FMCH001 (contained in product known as “PRESENCE” by Chr. Hansen), Bacillus subtilis FMCH002 (contained in the product known as “PRESENCE” by Chr. Hansen), Bacillus amyloliquefaciens MBI-600 (contained in TRUNEMCO), wherein said mentioned bacteria are preferred.
BCA2: According to the invention biological control agents that are summarized under the term “fungi” or “yeasts” include but are not limited to:
(2.1) Ampelomyces quisqualis, in particular strain AQ 10 (Accession No. CNCM I-807) (product known as AQ 10® from IntrachemBio Italia), (2.2) Arkansas Fungus 18 (ARF18, cf. WO2012/140207), (2.3) Arthrobotrys dactyloides (cf. WO 2012/140207), (2.4) Arthrobotrys oligospora (cf. WO 2012/140207), (2.5) Arthrobotrys superba, (cf. WO 2012/140207), (2.6) Aschersonia aleyrodis (cf. Berger, 1921. Bull. State Pl. Bd. 5:141), (2.7) Aspergillus flavus, in particular strain NRRL 21882 (product known as Afla-Guard® from Syngenta) or strain AF36 (product known as AF36 from Arizona Cotton Research and Protection Council, US), (2.8) Aureobasidium pullulans, in particular blastospores of strain DSM14940 or blastospores of strain DSM 14941 or mixtures thereof (products known as Botector® or Blossom Protect from bio-ferm, CH), (2.9) Beauveria bassiana, in particular strain ATCC 74040 (product known as Naturalis® from Intrachem Bio Italia) and strain GHA (Accession No. ATCC74250) (products known as BotaniGuard Es or Mycontrol-O from Laverlam International Corporation), or strain ATP02 (Accession No. DSM 24665, cf. WO/2011/117351), or strain CG 716 (product known as BoveMax® from Novozymes), or strain ANT-03 (from Anatis Bioprotection, CA), (2.10) Beauveria brongniartii (product known as Beaupro from Andermatt Biocontrol AG), (2.11) Candida oleophila, in particular strain 0 (product known as Nexy® from BioNext) or isolate I-182 (product known as Aspire® from Ecogen, US), (2.12) Candida saitoana, in particular strain NRRL Y-21022 (cf. U.S. Pat. No. 5,591,429), (2.13) Chaetomium cupreum, (2.14) Chaetomium globosum, (2.15) Cladosporium cladosporioides, in particular strain H39, (2.16) Colletotrichum gloeosporioides, in particular strain ATCC 20358, (2.17) Conidiobolus obscurus, (2.18) Coniothyrium minitans, in particular strain CON/M/91-8 (Accession No. DSM-9660), (product known as Contans® from Bayer Crop Science, DE), (2.19) Cryptococcus albidus (product known as YieldPlus® from Anchor Bio-Technologies, ZA), (2.20) Cryptococcus flavescens, in particular strain 3C (NRRL Y-50378) and strain 4C (NRRL Y-50379) (described in U.S. Pat. No. 8,241,889), (2.21) Cylindrocarpon heteronema, (2.22) Dactylaria candida, (2.23) Dilophosphora alopecuri (product known as Twist Fungus®), (2.24) Entomophthora virulenta (product known as Vektor), (2.25) Exophiala jeanselmei, (2.26) Exophilia pisciphila, (2.27) Fusarium oxysporum, in particular strain Fo47 (non-pathogenic) (product known as Fusaclean from Natural Plant Protection, FR), (2.28) Fusarium solani, for example strain Fs5 (non-pathogenic), (2.29) Gigaspora margarita, (2.30) Gigaspora monosporum, (2.31) Gliocladium catenulatum (Synonym: Clonostachys rosea f. catenulate) in particular strain J1446 (products known as Prestop® from AgBio Inc. or Primastop® from Kemira Agro Oy), (2.32) Gliocladium roseum, in particular strain 321U, (2.33) Glomus aggregatum, (2.34) Glomus brasilianum, (2.35) Glomus clarum, (2.36) Glomus deserticola, (2.37) Glomus etunicatum, (2.38) Glomus intraradices, (2.39) Glomus iranicum, (2.40) Glomus monosporum, (2.41) Glomus mosseae, (2.42) Harposporium anguillullae, (2.43) Hirsutella citriformis, (2.44) Hirsutella minnesotensis, (2.45) Hirsutella rhossiliensis, (2.46) Hirsutella thompsonii (products known as Mycohit or ABTEC from Agro Bio-tech Research Centre, IN), (2.47) Laccaria bicolor, (2.48) Laccaria laccata, (2.49) Lagenidium giganteum (product known as Laginex®, Bayer Crop Science, DE), (2.50) Lecanicillium spp., in particular strain HRO LEC 12 from Bayer Crop Science, DE, (2.51) Lecanicillium lecanii (formerly known as Verticillium lecanii) in particular conidia of strain KV01 (products known as Mycotal® or Vertalec®, Koppert/Arysta), or strain DAOM198499, or strain DAOM216596, (2.52) Lecanicillium muscarium (formerly Verticillium lecanii), in particular strain 1/1 from Bayer Crop Science, DE, or strain VE 6/CABI(=IMI) 268317/CBS102071/ARSEF5128, (2.53) Meristacrum asterospermum (2.54) Metarhizium anisopliae, in particular strain F52 (DSM3884/ATCC 90448) (products known as BIO 1020, Bayer Crop Science, DE, or Met52, Novozymes), or M. anisopliae var acridum (product known as GreenGuard, Becker Underwood, US), or M. anisopliae var acridum isolate IMI 330189 (ARSEF7486), (product known as Green Muscle from Biological Control Products), (2.55) Metarhizium flavoviride, (2.56) Metschnikowia fructicola, in particular the strain NRRL Y-30752 (product known as Shemer® from Bayer Crop Science, DE), (2.57) Microdochium dimerum, in particular strain L13 (products known as ANTIBOT® from Agrauxine), (2.58) Microsphaeropsis ochracea (product known as Microx® from Bayer Crop Science, DE), (2.59) Monacrosporium cionopagum, (2.60) Monacrosporium psychrophilum, (2.61) Monacrosporium drechsleri, (2.62) Monacrosporium gephyropagum (2.63) Mucor haemelis (product known as BioAvard from Indore Biotech Inputs & Research), (2.64) Muscodor albus, in particular strain QST 20799 (Accession No. NRRL 30547) (products known as Arabesque™, Glissade™, or Andante™ from Bayer Crop Science, DE), (2.65) Muscodor roseus strains A3-5 (Accession No. NRRL 30548), (2.66) Myrothecium verrucaria, in particular strain AARC-0255 (product known as DiTera™ from Valent Biosciences), (2.67) Nematoctonus geogenius, (2.68) Nematoctonus leiosporus, (2.69) Neocosmospora vasinfecta, (2.70) Nomuraea rileyi, in particular strains SA86101, GU87401, SR86151, CG128 and VA9101, (2.71) Ophiostoma piliferum, in particular strain D97 (product known as Sylvanex), (2.72) Paecilomyces fumosoroseus (new: Isaria fumosorosea), in particular strain IFPC 200613, or strain apopka 97 (product known as PreFeRal® WG from Biobest) or strain FE 9901 (products known as NoFly® from Natural Industries Inc., US), (2.73) Paecilomyces lilacinus, in particular spores of P. lilacinus strain 251 (AGAL 89/030550), (product known as BioAct® from Bayer Crop Science, DE; cf. Crop Protection 2008, 27, 352-361), (2.74) Paecilomyces variotii, in particular strain Q-09 (product known as Nemaquim® from Quimia, MX), (2.75) Pandora delphacis, (2.76) Paraglomus sp, in particular P. brasilianum, (2.77) Penicillium bilaii, in particular strain ATCC 22348 (products known as JumpStart® from Novozymes, PB-50, Provide), (2.78) Penicillium vermiculatum, (2.79) Phlebiopsis (or Phlebia or Peniophora) gigantea, in particular the strains VRA 1835 (ATCC 90304), VRA 1984 (DSM16201), VRA 1985 (DSM16202), VRA 1986 (DSM16203), FOC PG B20/5 (IMI390096), FOC PG SP log 6 (IMI390097), FOC PG SP log 5 (IMI390098), FOC PG BU3 (IMI390099), FOC PG BU4 (IMI390100), FOC PG 410.3 (IMI390101), FOC PG 97/1062/116/1.1 (IMI390102), FOC PG B22/SP1287/3.1 (IMI390103), FOC PG SH1 (IMI390104), FOC PG B22/SP1190/3.2 (IMI390105), (products known as Rotstop® from Verdera, FIN, PG-Agromaster®, PG-Fungler®, PG-IBL®, PG-Poszwald®, Rotex® from e-nema, DE), (2.80) Phoma macrostroma, in particular strain 94-44B (products known as Phoma H or Phoma P from Scotts, US), (2.81) Pichia anomala, in particular strain WRL-076 (NRRL Y-30842), (2.82) Pisolithus tinctorius, (2.83) Pochonia chlamydosporia (for example the product known as Rizotec; the bacterial strain is also known as Vercillium chlamydosporium), in particular var catenulata (IMI SD 187) (product known as KlamiC from The National Center of Animal and Plant Health (CENSA), CU), or P. chlamydosporia var chlamydosporia (resp. V. chlamydosporium var chlamydosporium), (2.84) Pseudozyma aphidis (2.85), Pseudozyma flocculosa, in particular strain PF-A22 UL (product known as Sporodex® L from Plant Products Co., CA), (2.86) Pythium oligandrum, in particular strain DV74 or M1 (ATCC 38472), (product known as Polyversum from Bioprepraty, CZ), (2.87) Rhizopogon amylopogon, (2.88) Rhizopogon fulvigleba, (2.89) Rhizopogon luteolus, (2.90) Rhizopogon tinctorus, (2.91) Rhizopogon villosullus, (2.92) Saccharomyces cerevisae, in particular strain CNCM No. I-3936, strain CNCM No. 1-3937, strain CNCM No. 1-3938, strain CNCM No. 1-3939 (patent application US 2011/0301030), (2.93) Scleroderma citrinum, (2.94) Sclerotinia minor, in particular strain IMI 344141 (product known as Sarritor), (2.95) Sporothrix insectorum (product known as Sporothrix Es from Biocerto, BR), (2.96) Stagonospora atriplicis, (2.97) Stagonospora heteroderae, (2.98) Stagonospora phaseoli, (2.99) Suillus granulatus, (2.100) Suillus punctatapies, (2.101) Talaromyces flavus, in particular strain V117b (product known as PROTUS® WG from Bayer Crop Science, DE), (2.102) Trichoderma album (product known as Bio Zeid® from Organic Biotechnology, EG), (2.103) Trichoderma asperellum, in particular strain ICC 012 (CABI CC IMI 392716) (also known as Trichoderma harzianum ICC012), or strain SKT-1 (product known as ECO-HOPE® from Kumiai Chemical Industry) or strain T34 (product known as T34 Biocontrol from Bioncontrol Technologies, ES) or isolate SF04 (URM-5911) or strain TV1 (MUCL 43093) (also known as Trichoderma viride TV1) or strain T11 (CECT 20178) (also known as Trichoderma viride T25), (2.104) Trichoderma atroviride, in particular strain CNCM I-1237 (product known as Esquive® WP from Agrauxine, FR,) or the strains NMI No. V08/002387, NMI No. V08/002388, NMI No. V08/002389, NMI No. V08/002390 (patent application US 2011/0009260) or strain ATCC 20476 (IMI 206040) or strain T11 (IMI352941/CECT20498) or strain LC52 (products known as Tenet® or Sentinel® from Agrimm Technologies, NZ), or strain SC1 from Bayer Crop Science, DE, or the strains SKT-1 (FERM P-16510), SKT-2 (FERM P-16511) and SKT-3 (FERM P-17021), (2.105) Trichoderma gamsii (formerly T. viride), in particular strain ICC080 (IMI CC 392151 CABI) (product known as Bioderma), (2.106) Trichoderma harmatum, in particular strain TH382 (product known as Incept from Syngenta), (2.107) Trichoderma harzianum, in particular T. harzianum rifai T39 (product known as Trichodex® from Makhteshim, US), or T. harzianum rifai strain KRL-AG2 (strain T-22, /ATCC 208479) (products known as PLANTSHIELD T-22G, Rootshield® and TurfShield from BioWorks, US), or strain KD (products known as Trichoplus from Biological Control Products, SA, or Eco-T from Plant Health Products, SZ), or strain ITEM 908 (CBS 118749), or strain TH 35 (formerly known as Trichoderma lignorum), (product known as Root Pro from Mycontrol), or strain DB 103 (product known as T-Gro from Dagutat Biolab), or strain TSTh20 (Patent Deposit Designation number PTA-0317), or strain 1295-22, (2.108) Trichoderma koningii, (2.109) Trichoderma lignorum, in particular strain TL-0601 (product known as Mycotric from Futureco Bioscience, ES), (2.110) Trichoderma polysporum, in particular strain IMI 206039/ATCC 20475, (2.111) Trichoderma saturnisporium, in particular strain PBP-TH-001 from Bayer Crop Science, DE, (2.112) Trichoderma stromaticum (product known as TRICOVAB® from Ceclap, BR), (2.113) Trichoderma virens (also known as Gliocladium virens), in particular strain GL-21 (product known as SoilGard from Certis, US) or strain G41 or Trichoderma (Gliocladium) virens strain GI-3 (or G1-3, ATCC 58678), (2.114) Trichoderma viride, in particular strain TV1, (2.115) Tsukamurella paurometabola, in particular strain C-924 (product known as HeberNem®), (2.116) Ulocladium oudemansii, in particular strain HRU3 (product known as Botry-Zen® from Botry-Zen Ltd, NZ), (2.117) Verticillium albo-atrum (formerly V. dahliae), in particular strain WCS850 (CBS 276.92), (2.118) Verticillium chlamydosporium, (2.119) Verticillium dahlia, (2.120) Zoophtora radicans, and (2.121) a Penicillium strain, such as Penicillium steckii strain IBWF104-06 (accession number DSM 27859).
wherein said mentioned fungi or yeasts are preferred.
BCA3: According to the invention biological control agents that are summarized under the term “protozoa” include but are not limited to:
(3.1) Nosema locustae (product known as NoloBait), (3.2) Thelohania solenopsis and (3.3) Vairimorpha spp,
wherein said mentioned protozoa are preferred.
BCA4: According to the invention biological control agents that are summarized under the term “viruses” include but are not limited to:
(4.1) Adoxophyes orana (summer fruit tortrix) granulosis virus (GV), (product known as Capex® from BIOFA), (4.2) Agrotis segetum (turnip moth) nuclear polyhedrosis virus (NPV), (4.3) Anagrapha falcifera (celery looper) NPV, (4.4) Anticarsia gemmatalis (woolly pyrol moth) multiple NPV (product known as Coopervirus PM by Coodetec), (4.5) Autographa californica (alfalfa looper) mNPV (product known as VPN80 from Agricola El Sol; GT), (4.6) Biston suppressaria (tea looper) NPV, (4.7) Bombyx mori (silkworm) NPV, (4.8) Cryptophlebia leucotreta (false codling moth) GV (products known as Cryptex from Andermatt Biocontrol, CH), (4.9) Cydia pomonella (codling moth) granulosis virus (GV) (product known as Madex Plus from Andermatt Biocontrol, CH), (4.10)Dendrolimus punctatus (masson pine moth) CPV, (4.11) Hehcoverpa armigera (cotton bollworm) NPV (product known as Helicovex from Andermatt Biocontrol, CH), (4.12) Hehcoverpa (previously Heliothis) zea (corn earworm) NPV (product known as Elcar), (4.13) Leucoma salicis (satin moth) NPV, (4.14) Lymantria dispar (gypsy moth) NPV (product known as Gypcheck, US Forest Service), (4.15) Neodiprion abietis (balsam-fir sawfly) NPV (product known as Abietiv™), (4.16) Neodiprion lecontei (red-headed pine sawfly) NPV (product known as Lecontvirus from the Canadian Forestry Service), (4.17) Neodiprion sertifer (pine sawfly) NPV (product known as Neocheck-S from the US Forest service), (4.18) Orgyia pseudotsugata (douglas-fir tussock moth) NPV (product known as TM-BioControl-1™), (4.19) Phthorimaea operculella (tobacco leaf miner) GV (product known as Matapol Plus), (4.20) Pieris rapae (small white butterfly) GV, (4.21) Plutella xylostella (diamondback moth) GV, (4.22) Spodoptera albula (gray-streaked armywom moth) mNPV (product known as VPN 82, Agricola El Sol, GT), (4.23) Spodoptera exempta (true armyworm) mNPV, (4.24) Spodoptera exigua (sugarbeet armyworm) mNPV (product known as Spexit from Andermatt Biocontrol), (4.25) Spodoptera frupperda (fall armyworm) mNPV), (4.26) Spodoptera littoralis (tobacco cutworm) NPV (procucts known as Littovir from Andermatt Biocontrol, CH or Spodoptrin from NPP Calliope France), and (4.27) Spodoptera litura (oriental leafworm moth) NPV (products known as Littovir), wherein said mentioned viruses are preferred.
BCA5: According to the invention biological control agents that are summarized under the term “entomopathogenic nematodes” include but are not limited to:
(5.1) Abbreviata caucasica, (5.2) Acuaria spp., (5.3) Agamermis decaudata, (5.4) Allantonema spp., (5.5) Amphimermis spp., (5.6) Beddingia (=Deladenus) siridicola, (5.7) Bovienema spp., (5.8) Cameronia spp., (5.9) Chitwoodiella ovofilamenta, (5.10) Contortylenchus spp., (5.11) Culicimermis spp., (5.12) Diplotriaena spp., (5.13) Empidomermis spp., (5.14) Filipjevimermis leipsandra, (5.15) Gastromermis spp., (5.16) Gongylonema spp., (5.17) Gynopoecilia pseudovipara, (5.18) Heterorhabditis spp., in particular (5.19) Heterorhabditis bacteriophora (products known as B-Green® or Larvanem®, Koppert or Nemasys® G, Becker Underwood), or (5.20) Heterorhabditis baujardi, or (5.21) Heterorhabditis heliothidis (products known as Nematon®, biohelp GmbH), or (5.22) Heterorhabditis indica, (5.23) Heterorhabditis marelatus, (5.24) Heterorhabditis megidis (products known as Larvanem® M, Koppert or Meginem®, Andermatt Biocontrol AG or Nemasys-H®), (5.25) Heterorhabditis zealandica, (5.26) Hexamermis spp., (5.27) Hydromermis spp., (5.28) Isomermis spp., (5.29) Limnomermis spp., (5.30) Maupasina weissi, (5.31) Mermis nigrescens, (5.32) Mesomermis spp., (5.33) Neomesomermis spp., (5.34) Neoparasitylenchus rugulosi, (5.35) Octomyomermis spp., (5.36) Parasitaphelenchus spp., (5.37) Parasitorhabditis spp., (5.38) Parasitylenchus spp., (5.39) Perutilimermis culicis, (5.40) Phasmarhabditis hermaphrodita (product known as Nemaslug from BASF AG), (5.41) Physaloptera spp., (5.42) Protrellatus spp., (5.43) Pterygodermatites spp., (5.44) Romanomermis spp., (5.45) Seuratum cadarachense, (5.46) Sphaerulariopsis spp., (5.47) Spirura guianensis, (5.48) Steinernema spp. (=Neoaplectana spp.), in particular (5.49) Steinernema bibionis (product known as Nematoden gegen Trauermücken®), or (5.50) Steinernema carpocapsae (products known as Biocontrol, Nemasys-C®, NemAttack®), or (5.51) Steinernema feltiae (=Neoaplectana carpocapsae), (products known as Nemasys®, Nemaflor®, Nemaplus®, NemaShield®), or (5.52) Steinernema glaseri (products known as Biotopia®), or (5.53) Steinernema kraussei (products known as Exhibitline®, Grubsure®, Kraussei System®, Larvesure®), or (5.54) Steinernema riobrave (products known as Biovector®), or (5.55) Steinernema scapterisci (products known as Nematac® S), or (5.56) Steinernema scarabaei, or (5.57) Steinernema siamkayai, (5.58) Steinernema thailandse (products known as Nemanox®), (5.59) Strelkovimermis peterseni, (5.60) Subulura spp., (5.61) Sulphuretylenchus elongatus, and (5.62) Tetrameres spp., wherein said mentioned entomopathogenic nematodes are preferred.
BCA6: Biological control agents which are summarized under the term “proteins or secondary metabolites” include but are not limited to:
(6.1) Bacillus thuringiensis toxins (isolated from different subspecies of B. thuringiensis), (6.2) Gougerotin (isolated from Streptomyces microflavus strain AQ 6121, from Bayer Crop Science), (6.3) Harpin (isolated from Erwinia amylovora, products known as Harp-N-Tek™, Messenger®, Employ™, ProAct™), (6.4) the spider toxin GS-omega/kappa-Hxtx-Hv1a, product known as Versitude from Vestaron,
wherein said mentioned proteins or secondary metabolites are preferred.
BCA7: Biological control agents which are summarized under the term “botanical extracts” include but are not limited to:
(7.1) Thymol, extracted e. g. from thyme (Thymus vulgaris), (7.2) Neem tree (Azadirachta indica) oil, and therein Azadirachtin, (7.3) Pyrethrum, an extract made from the dried flower heads of different species of the genus Tanacetum, and therein Pyrethrins (the active components of the extract), (7.4) extract of Cassia nigricans, (7.5) wood extract of Quassia amara (bitterwood), (product known as Quassan from Andermatt Biocontrol AG), (7.6) Rotenon, an extract from the roots and stems of several tropical and subtropical plant species, especially those belonging to the genera Lonchocarpus and Derris, (7.7) extract of Allium sativum (garlic), (7.8) Quillaja extract, made from the concentrated purified extract of the outer cambium layer of the Quillaja Saponaria Molina tree, (7.9) Sabadilla (Sabadilla=Schoenocaulon officinale) seeds, in particular Veratrin (extracted from the seeds), (7.10) Ryania, an extract made from the ground stems of Ryania speciosa, in particular Ryanodine (the active component of the extract), (7.11) extract of Viscum album (mistletoe), (7.12) extract of Tanacetum vulgare (tansy), (7.13) extract of Artemisia absinthium (wormwood), (7.14) extract of Urtica dioica (stinging nettle), (7.15) extract of Symphytum officinale (common comfrey), (7.16) extract of Tropaeulum majus (monks cress), (7.17) leaves and bark of Quercus (oak tree) (7.18) Yellow mustard powder, (7.19) oil of the seeds of Chenopodium anthelminticum (wormseed goosefoot), (7.20) dried leaves of Dryopteris filix-mas (male fern), (7.21) bark of Celastrus angulatus (chinese bittersweet), (7.22) extract of Equisetum arvense (field horsetail), (7.23) Chitin (7.24) natural extracts or simulated blend of Chenopodium ambrosioides (wormseed), (product known as Requiem® from Bayer Crop Science) which contains a mixture of three terpenes, i.e. α-terpinene (around 10%), p-cymene (around 3.75%) and limonene (around 3%) as pesticidally active ingredients; it is disclosed in US 2010/0316738 corresponding to WO 2010/144919), (7.25) Saponins of Chenopodium quinoa (quinoa goosefoot), (product known as Heads Up), (7.26) Maltodextrin (product known as Majestik from Certis Europe), (7.27) orange oil (product known as PREV-AM from Oro Agri B.V.), sesame oil (product known as Dragon-fire-CCP, U.S. Pat. No. 6,599,539), wherein said mentioned botanical extracts are preferred.
Also included are bacteria and fungi which are added as ‘inoculant’ to plants or plant parts or plant organs and which, by virtue of their particular properties, promote plant growth and plant health. Examples which may be mentioned are:
Agrobacterium spp., Azorhizobium caulinodans, Azospirillum spp., Azotobacter spp., Bradyrhizobium spp., Burkholderia spp., in particular Burkholderia cepacia (formerly known as Pseudomonas cepacia), Gigaspora spp., or Gigaspora monosporum, Glomus spp., Laccaria spp., Lactobacillus buchneri, Paraglomus spp., Pisolithus tinctorus, Pseudomonas spp., Rhizobium spp., in particular Rhizobium trifolii, Rhizopogon spp., Scleroderma spp., Suillus spp., Streptomyces spp., Rhizophagus irregularis (previously known as Glomus intraradices).
Also included herein are any of the biological or chemical control agents ((bio)pesticides) described in US2017/0188584 (incorporated by reference herein, such as any of the biological or chemical control agents from the groups A) to O), or the biopesticides listed in paragraph 127 in US2017/0188584). Biological control agents as used herein can be obtained from culture collections and deposition centers (often referred to by their acronym (on the world wide web at wfcc.info/ccinfo/collection/by_acronym/)) or strain prefix herein, such as strains with the following prefixes from the following collections: AGAL or NMI from: National Measurement Institute, 1/153 Bertie Street, Port Melbourne, Victoria, Australia 3207; ATCC from American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, USA; BR from Embrapa Agrobiology Diazothrophic Microbial Culture Collection, P.O. Box 74.505, Seropedica, Rio de Janeiro, 23.851-970, Brazil; CABI or IMI from CABI Europe—International Mycological Institute, Bakeham Lane, Egham, Surrey, TW20 9TYNRRL, UK; CB from The CB Rhizobium Collection, School of Environment and Agriculture, University of Western Sydney, Hawkesbury, Locked Bag 1797, South Penrith Distribution Centre, NSW 1797, Australia; CBS from Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Uppsalaan 8, PO Box 85167, 3508 AD Utrecht, Netherlands; CC from the Division of Plant Industry, CSIRO, Canberra, Australia; CNCM from Collection Nationale de Cultures de Microorganismes, Institute Pasteur, 25 rue du Docteur Roux, F-75724 PARIS Cedex 15; CPAC from Embrapa-Cerrados, CX. Postal 08223, Planaltina, DF, 73301-970, Brazil; DSM from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstra.beta.e 7 B, 38124 Braunschweig, Germany; IDAC from International Depositary Authority of Canada Collection, Canada; IC1VIP from Interntional Collection of Micro-organisms from Plants, Landcare Research, Private Bag 92170, Auckland Mail Centre, Auckland 1142, New Zealand; IITA from IITA, PMB 5320, Ibadan, Nigeria; INTA from Agriculture Collection Laboratory of the Instituto de Microbiologia y Zoologia Agricola (IMYZA), Instituto Nacional de Tecnologi'a Agropecuaria (INTA), Castelar, Argentina; MSDJ from Laboratoire de Microbiologie des Sols, INRA, Dijon, France; MUCL from Mycotheque de l'Universite catholique de Louvain, Croix du Sud 2, box L7.05.06, 1348 Louvain-la-Neuve, Belgium; NCIMB or NICB from The National Collections of Industrial and Marine Bacteria Ltd., Torry Research Station, P.O. Box 31, 135 Abbey Road, Aberdeen, AB9 8DG, Scotland; Nitragin from Nitragin strain collection, The Nitragin Company, Milwaukee, Wis., USA, NRRL or ARSEF (collection of entomopathogenic fungi) from ARS Culture Collection of the National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Ill. 61604, USA; NZP from the Department of Scientific and Industrial Research Culture Collection, Applied Biochemistry Division, Palmerston North, New Zealand; PPM from ARC-Plant Protection Research Institute, Private Bag X134, Queenswood Pretoria, Gauteng, 0121, South Africa; SEMIA from FEPAGRO-Fundacao Estadual de Pesquisa Agropecuaria, Rua Gonsalves Dias, 570, Bairro Menino Deus, Porto Alegre/RS, Brazil; SRDI from SARDI, Adelaide, South Australia; USDA from U.S. Department of Agriculture, Agricultural Research Service, Soybean and Alfalfa Research Laboratory, BARC-West, 10300 Baltimore Boulevard, Building 011, Beltsville, Md. 20705, USA (Beltsville Rhiz. Cult. Catalog: on the world wide web at pdf.usaid.gov/pdf docs/PNAAW891.pdf); and WSM from Murdoch University, Perth, Western Australia.
Also included herein as biological control agent are any of the following biochemical pesticides: citral, (E,Z)-7,9-dodecadien-1-yl acetate, ethyl formate, (E,Z)-2,4-ethyl decadienoate (pear ester), (Z,Z,E)-7,11,13-hexadecatrienal, heptyl butyrate, isopropyl myristate, lavanulyl senecioate, 2-methyl 1-butanol, methyl eugenol, (E,Z)-2,13-octadecadien-1-ol, (E,Z)-2,13-octadecadien-1-ol acetate, (E,Z)-3,13-octadecadien-1-ol, R-1-octen-3-ol, pentatermanone, potassium silicate, sorbitol actanoate, (E,Z,Z)-3,8,11-tetradecatrienyl acetate, (Z,E)-9,12-tetradecadien-1-yl acetate, Z-7-tetradecen-2-one, Z-9-tetradecen-1-yl acetate, Z-11-tetradecenal, Z-11-tetradecen-1-ol, Acacia negra extract, and extract of grapefruit seeds and pulp.
Also included herein as biological control agent is any of the biopesticides mentioned at: (on the world wide web at sitem.herts.ac.uk/aeru/bpdb/atoz.htm, gcm.wfcc.info/, landcareresearch.co.nz/resources.collections/icmp, epa.gov/opp00001/biopesticides/, omri.org/omri-lists, and included as compound herein is any of the pesticides mentioned at sitem.herts.ac.uk/aeru/ppdb/en/atoz.htm.
In one embodiment, a biological control agent for use in the current invention includes one or more biological control agents selected from group BCA8: a Bacillus species strain, a Brevibacillus species strain, a Burkholderia species strain, a Lysobacter species strain, a Pasteuria species strain, an Arthrobotrys species strain, a Nematoctonus species strain, a Myrothecium species strain, a Paecilomyces species strain, a Trichoderma species strain, and a Tsukamurella species strain.
In another embodiment, the plant, cell, plant part or seed of the invention, or the soil in which they are grown or are intended to be grown, are treated with a biological control agent selected from group BCA9 consisting of: Bacillus amyloliquefaciens, Bacillus firmus, Bacillus laterosporus, Bacillus lentus, Bacillus licheniformis, Bacillus nematocida, Bacillus pumilus, Bacillus subtilis, Bacillus penetrans, Bacillus thuringiensis, Brevibacillus laterosporus, Burkholderia rinojensis, Lysobacter antibioticus, Lysobacter enzymogenes, Pasteuria nishizawae, Pasteuria penetrans, Pasteuria ramosa, Pasteuria reniformis, Pasteuria thornei, Pasteuria usage, Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys superba, Nematoctonus geogenius, Nematoctonus leiosporus, Myrothecium verrucaria, Paecilomyces lilacinus, Paecilomyces variotii, Trichoderma asperellum, Trichoderma harzianum, Trichoderma viride, Trichoderma harzianum rifai, and Tsukamurella paurometabola.
In one embodiment, the plant, cell, plant part or seed of the invention, or the soil in which they are grown or are intended to be grown, are treated with a biological control agent selected from group BCA10 consisting of: Bacillus amyloliquefaciens strain IN937a, Bacillus amyloliquefaciens strain FZB42, Bacillus amyloliquefaciens strain FZB24, Bacillus amyloliquefaciens strain ABI01, Bacillus amyloliquefaciens strain B3, Bacillus amyloliquefaciens strain D747, Bacillus amyloliquefaciens strain APM-1, Bacillus amyloliquefaciens strain TJ1000, Bacillus amyloliquefaciens strain AP-136, Bacillus amyloliquefaciens strain AP-188, Bacillus amyloliquefaciens strain AP-218, Bacillus amyloliquefaciens strain AP-219, Bacillus amyloliquefaciens strain AP-295, Bacillus amyloliquefaciens strain MBI 600, Bacillus amyloliquefaciens strain PTA-4838, Bacillus amyloliquefaciens strain F727, Bacillus firmus strain I-1582, Bacillus firmus strain NRRL B-67003, Bacillus firmus strain NRRL B-67518, Bacillus firmus strain GB126, Bacillus laterosporus strain ATCC PTA-3952, Bacillus laterosporus strain ATCC PTA-3593, Bacillus licheniformis strain ATCC PTA-6175, Bacillus thuringiensis strain EX297512, Brevibacillus laterosporus strain ATCC 64, Brevibacillus laterosporus strain NRS 1111, Brevibacillus laterosporus strain NRS 1645, Brevibacillus laterosporus strain NRS 1647, Brevibacillus laterosporus strain BPM3, G4, Brevibacillus laterosporus strain NCIMB 41419, Burkholderia rinojensis strain A396, Lysobacter antibioticus strain 13-1, Lysobacter enzymogenes strain C3, Myrothecium verrucaria strain AARC-0255, Paecilomyces lilacinus strain 251, Paecilomyces variotii strain Q-09, Trichoderma asperellum strain ICC 012, Trichoderma asperellum strain SKT-1, Trichoderma asperellum strain T34, Trichoderma asperellum strain T25, Trichoderma asperellum strain SF04, Trichoderma asperellum strain TV1, Trichoderma asperellum strain T11, Trichoderma harzianum strain ICC012, Trichoderma harzianum rifai T39, Trichoderma harzianum rifai strain KRL-AG2, Trichoderma viride strain TV1 or strain TV25, Trichoderma atroviride strain CNCM I-1237, and Tsukamurella paurometabola strain C-924.
All plants and plant parts can be treated with the compounds and/or biological control agents and/or mixtures in accordance with the invention. Plants of the invention can be soybean plants containing traits obtained by conventional breeding and optimization methods or by biotechnological methods or combinations of these methods. Plant parts should be understood to mean all parts and organs of the plants above and below ground, such as shoot, leaf, flower and root, examples given being leaves, stems, flowers, pods and seeds, and also roots. Parts of plants also include harvested plants or harvested plant parts and vegetative and generative propagation material, for example seedlings, cuttings or seeds.
Treatment according to the invention of the plants and plant parts of the invention with the compounds and/or biological control agents and/or mixtures in accordance with the invention is carried out directly or by allowing the compounds and/or biological control agents and/or mixtures to act on the surroundings, environment or storage space by the customary treatment methods, for example by immersion, spraying, evaporation, fogging, scattering, painting on, injection and, in the case of propagation material, in particular in the case of seeds, also by applying one or more coats.
The plants comprising the elite event of the invention which can be treated in accordance with the invention include also plants which, through genetic modification or breeding, received genetic material which imparts particular advantageous useful properties (“traits”) to these plants, besides the (soybean or engineered) traits contained in the event of the invention. Examples of such properties are better plant growth, increased tolerance to high or low temperatures, increased tolerance to drought or to levels of water or soil salinity, enhanced flowering performance, easier harvesting, accelerated ripening, higher yields, higher quality and/or a higher nutritional value of the harvested products, better storage life and/or processability of the harvested products. Further and particularly emphasized examples of such properties are increased resistance of the plants against pests, such as animal or microbial pests, such as against insects, arachnids, nematodes, mites, slugs and snails owing, for example, to toxins formed in the plants, in particular those toxins derived from Bacillus thuringiensis (for example the toxins known as Cry1 Aa, Cry1 Ab, Cry1 Ac, Cry2Ab, Cry2Ae, Cry3Aa, Cry9c, Cry3Bb and Cry1Fa and also any mutants thereof such as Cry1A.105, or combinations of such toxins, such as Cry1Ac and Cry1F, or Cry1Ac, Cry1A.105, and Cry2Ab), furthermore increased resistance of the plants against phytopathogenic fungi, bacteria and/or viruses owing, for example, to systemic acquired resistance (SAR), systemin, phytoalexins, elicitors and also resistance genes and correspondingly expressed proteins and toxins, and also increased tolerance of the plants to certain herbicidally active compounds, for example imidazolinones, sulphonylureas, glyphosate, PPO inhibitors, metribuzin, or phosphinothricin (for example the “PAT” gene). The genes which impart the desired traits in question may also be present in combinations with one another in the transgenic plants.
Crop Protection—Types of Treatment
The treatment of the plants and plant parts with the compounds and/or biological control agents and/or mixtures is carried out directly or by action on their surroundings, habitat (such as the soil or the field in which the plants of the invention were planted or sown or will be planted or sown) or storage space using customary treatment methods, for example by dipping, spraying, atomizing, irrigating, evaporating, dusting, fogging, broadcasting, foaming, painting, spreading-on, injecting, watering (drenching), drip irrigating and, in the case of propagation material, in particular in the case of seed, furthermore as a powder for dry seed treatment, a solution for liquid seed treatment, a water-soluble powder for slurry treatment, by incrusting, by coating with one or more coats, etc. It is furthermore possible to apply the compounds and/or biological control agents and/or mixtures by the ultra-low volume method or to inject the application form or the compounds and/or biological control agents and/or mixtures itself into the soil (e.g., in furrow or pre-plant application).
One treatment of the plants of the invention is foliar application, i.e. the compounds and/or biological control agents and/or mixtures are applied to the foliage, where treatment frequency and the application rate should be adjusted according to the level of infestation with the pest in question.
In the case of systemically active compounds and/or biological agents, the compounds and/or biological control agents and/or mixtures also access the plants via the root system. The plants are then treated by the action of the compounds and/or biological control agents and/or mixtures on the habitat of the plant. This may be done, for example, by drenching, or by mixing into the soil or the nutrient solution, i.e. the locus of the plant (e.g., soil) is impregnated with a liquid form of the compounds and/or biological control agents and/or mixtures thereof, or by soil application, i.e. the compounds and/or biological control agents and/or mixtures according to the invention are introduced in solid form (e.g., in the form of granules) into the locus of the plants, or by drip application (often also referred to as “chemigation”), i.e. the liquid application of the compounds and/or biological control agents and/or mixtures according to the invention from surface or sub-surface driplines over a certain period of time together with varying amounts of water at defined locations in the vicinity of the plants.
Seed Treatment
The control of pests by treating the seed of plants has been known for a long time and is the subject of continuous improvements. Methods for the treatment of seed can also take into consideration the intrinsic insecticidal or nematicidal properties of pest-resistant or -tolerant plants in order to achieve optimum protection of the seed and also the germinating plant with a minimum of pesticides being employed.
The present invention therefore in particular also relates to a method for the protection of seed and germinating plants containing the event of the invention, from attack by pests, by treating the seed with one or more of the compound(s) and/or biological control agent(s) and/or mixtures described herein. The method according to the invention for protecting seed and germinating plants against attack by pests furthermore comprises a method where the seed is treated simultaneously in one operation or sequentially with a compounds and/or biological control agents and one or more mixing components. It also comprises a method where the seed is treated at different times with a compound, a biological control agent and a mixing component.
The invention likewise relates to the use of the compounds and/or biological control agents and/or mixtures as described herein for the treatment of seed containing the elite event of the invention for protecting that seed and the resulting plant from pests.
Furthermore, the invention relates to seed containing the elite event of the invention which has been treated with a compound and/or biological control agent and/or mixture or combination according to the invention so as to afford protection from pests. The invention also relates to seed which has been treated simultaneously with a compound and/or biological control agent and a mixing component. The invention furthermore relates to seed which has been treated at different times with a compound and/or biological control agent and a mixing component. In the case of seed which has been treated at different points in time with compounds and/or biological control agents and/or mixtures as described herein, the individual substances may be present on the soybean seed of the invention in different layers. Here, the layers comprising a compound and/or biological control agent and/or mixture may optionally be separated by an intermediate layer. The invention also relates to seed of the invention where a compound and/or biological control agent and/or mixture have been applied as component of a coating or as a further layer or further layers in addition to a coating.
Furthermore, the invention relates to seed which, after the treatment with a compound and/or biological control agent and/or mixture as described herein, is subjected to a film-coating process to prevent dust abrasion on the seed.
One of the advantages encountered with a systemically acting compound is the fact that, by treating the seed, not only the seed itself but also the plants resulting therefrom are, after emergence, protected against pests. In this manner, the immediate treatment of the crop at the time of sowing or shortly thereafter can be dispensed with.
Also, treatment of the seed with a compound and/or biological control agent and/or mixture germination as described herein, and emergence of the treated seed may be enhanced.
Furthermore, compound or biological control agents or mixtures thereof can be employed in combination with compositions or compounds of signalling technology, leading to better colonization by symbionts such as, for example, rhizobia, mycorrhizae, such as Rooteila® mycorrhiza, and/or endophytic bacteria or fungi, and/or to optimized nitrogen fixation.
In the context of the present invention, the seed is treated in a state in which it is stable enough to avoid damage during treatment. In general, the seed may be treated at any point in time between harvest and sowing. The seed usually used has been separated from the plant and freed from the pods or other plant parts. For example, it is possible to use seed which has been harvested, cleaned and dried down to a moisture content which allows storage. Alternatively, it is also possible to use seed which, after drying, has been treated with, for example, water and then dried again, as in the case of primed seed.
When treating the seed, the amount of the compound or biological agent or mixture described herein applied to the seed and/or the amount of further additives is chosen in such a way that the germination of the seed is not adversely affected, or that the resulting plant is not damaged so that yield is negatively affected.
In general, the compounds or biological agents are applied to the seed in a suitable formulation. Suitable formulations and processes for seed treatment are known to the person skilled in the art.
The compounds and/or biological agents and/or mixtures thereof described herein can be converted to the customary seed dressing formulations, such as solutions, emulsions, suspensions, powders, foams, slurries or other coating compositions for seed, and also ULV formulations.
These formulations are prepared in a known manner, by mixing the compounds and/or biological agents and/or mixtures thereof with customary additives such as, for example, customary extenders and also solvents or diluents, colorants, wetting agents, dispersants, emulsifiers, antifoams, preservatives, secondary thickeners, adhesives, gibberellins and also water.
Colorants which may be present in the seed-dressing formulations which can be used in accordance with the invention are all colorants which are customary for such purposes. It is possible to use either pigments, which are sparingly soluble in water, or dyes, which are soluble in water. Examples include the dyes known by the names Rhodamine B, C.I. Pigment Red 112 and C.I. Solvent Red 1.
Useful wetting agents which may be present in the seed dressing formulations usable in accordance with the invention are all substances which promote wetting and which are conventionally used for the formulation of agrochemically active compounds. Preference is given to using alkylnaphthalenesulphonates, such as diisopropyl- or diisobutylnaphthalenesulphonates.
Useful dispersants and/or emulsifiers which may be present in the seed dressing formulations usable in accordance with the invention are all nonionic, anionic and cationic dispersants conventionally used for the formulation of active agrochemical ingredients. Preference is given to using nonionic or anionic dispersants or mixtures of nonionic or anionic dispersants. Suitable nonionic dispersants include in particular ethylene oxide/propylene oxide block polymers, alkylphenol polyglycol ethers and tristryrylphenol polyglycol ethers, and the phosphated or sulphated derivatives thereof. Suitable anionic dispersants are in particular lignosulphonates, polyacrylic acid salts and arylsulphonate/formaldehyde condensates.
Antifoams which may be present in the seed dressing formulations usable in accordance with the invention are all foam-inhibiting substances conventionally used for the formulation of active agrochemical ingredients. Preference is given to using silicone antifoams and magnesium stearate.
Preservatives which may be present in the seed dressing formulations usable in accordance with the invention are all substances usable for such purposes in agrochemical compositions. Examples include dichlorophene and benzyl alcohol hemiformal.
Secondary thickeners which may be present in the seed dressing formulations usable in accordance with the invention are all substances which can be used for such purposes in agrochemical compositions. Cellulose derivatives, acrylic acid derivatives, xanthan, modified clays and finely divided silica are preferred.
Adhesives which may be present in the seed dressing formulations usable in accordance with the invention are all customary binders usable in seed dressing products. Polyvinylpyrrolidone, polyvinyl acetate, polyvinyl alcohol and tylose may be mentioned as being preferred.
Gibberellins which can be present in the seed-dressing formulations which can be used in accordance with the invention are preferably the gibberellins A1, A3 (=gibberellic acid), A4 and A7; gibberellic acid is especially preferably used.
For treatment of seed with the seed dressing formulations usable in accordance with the invention, or the use forms prepared therefrom by adding water, all mixing units usable customarily for the seed dressing are useful. Specifically, the procedure in the seed dressing is to place the seed into a mixer, operated batch-wise or continously, to add the particular desired amount of seed dressing formulations, either as such or after prior dilution with water, and to mix everything until the formulation is distributed homogeneously on the seed. If appropriate, this is followed by a drying operation.
The application rate of the seed dressing formulations usable in accordance with the invention can be varied within a relatively wide range. It is guided by the particular content of the compounds and/or biological agents and/or mixtures thereof in the formulations. The application rates of a chemical compound are generally between 0.001 and 50 g per kilogram of seed, preferably between 0.01 and 15 g per kilogram of seed.
Preferred fungicides for seed treatment of seeds containing the event of the invention are selected from the group named SF1 consisting of:
Benzovindiflupyr, Carbendazim, Carboxin, Difenoconazole, Ethaboxam, Fludioxonil, Fluquinconazole, Fluxapyroxad, Ipconazole, Ipfentrifluconazole, Isotianil, Mefenoxam, Mefentrifluconazole, Metalaxyl, Pencycuron, Penflufen, Penthiopyrad, Prothioconazole, Prochloraz, Pyraclostrobin, Sedaxane, Silthiofam, Tebuconazole, Trifloxystrobin, Triticonazole, Ethaboxam (SCC), Penthiopyrad (DPX pipeline), Benzovindiflupyr (SYN pipeline), Bixafen, (see biologicals), Dimethomorph, Fenamidone, Fluopicolide, Fluoxastrobin, Flutolanil, Tolclophos-methyl, Azoxystrobin, Chlorothalonil, Cyproconazole, Cyprodinil, Diniconazole, Fluopyram, Flutriafol, Fluxapyroxad, Imazalil, Isopyrazam, Isotianil, Iprodione, Metconazole, Myclobutanil, Picoxystrobin, Pyrimethanil, Tetraconazole, Thiabendazole, Thiophanate-methyl, Triadimenol, Thiram, Triflumizole, Ziram, 2,6-dimethyl-1H,5H-[1,4]dithiino[2,3-c:5,6-c′]dipyrrole-1,3,5,7(2H,6H)-tetrone, 2-{3-[2-{[3,5-bis(difluoromethyl)-1H-pyrazol-1-yl]acetyl}piperidin-4-yl)-1,3-thiazol-4-yl]-4,5-dihydro-1,2-oxazol-5-yl}-3-chlorophenyl methanesulfonate (Thiazolylpiperidin, N-(5-chloro-2-isopropylbenzyl)-N-cyclopropyl-3-(difluoromethyl)-5-fluoro-1-methyl-1H-pyrazole-4-carboxamide Cypropamide, Picarbutrazox Oxathiapiprolin, Picoxystrobin, Isoflucypram, and Pydiflumetofen.
Preferred insecticides/acaricides/nematicides for seed treatment of seeds are selected from the group named SIAN1 consisting of:
Abamectin, Afidopyropen, Bifenthrin, Carbofuran, Carbendazim, Clothianidin, Cyazypyr, Cypermethrin, Deltamethrin, Difenoconazole, Ethoxysulfuron, Fenamidon, Fenoxaprop-P-Ethyl, Ethiprole, Fipronil, Fluazaindolizine, Flupyradifurone (Sivanto™), Flubendiamide, Fluopicolide, Fluopyram, Fluquinconazole, Fosetyl-A1, Imidacloprid, Prochloraz, Propineb, Lambda-Cyhalothrin, Methiocarb, Chlorantraniliprole, Spirotetramat, Spinosad, Tebuconazole, Thiacloprid, Thiametoxam, Thiodicarb, Tioxazafen, (Nemastrike™), Fluazaindolizine (nematicide), Lambda-Cyhalothrin, Bifenthrin, Cypermethrin, Acetamiprid, β-Cyfluthrin, Flubendiamide, Thiacloprid, Chlorphyriphos, Metamidophos, Phorate, Sulfoxaflor, Tefluthrin, Triflumuron, Tetraniliprole, Triflumuron, Broflanilide (MCI 8007—Mitsui/BASF), Cycloxaprid, Fosthiazate, Fluensulfone and Spinetoram.
Preferred Biologicals and biological active ingredients for seed treatment are selected from the group named SBCA1, consisting of:
Pasteuria nishizawae, such as Pasteuria nishizawae Pn1 (product known as Clariva form Syngenta), a Burkholderia strain, in particular strain A396, Bacillus amyloliquefaciens, such as Bacillus amyloliquefaciens strain PTA-4838 (known as Aveo EZ from Valent), or Bacillus amyloliquefaciens TJ1000, Bacilus firmus, such as Bacillus firmus GB126, Bacillus subtilis, Bacillus pumilus such as Bacillus pumilus QST 2808 or Bacillus pumilus GB34, Rhizobium spp strains, especially Rhizobium tropici SP25, Pseudomonas fluorescens, Pseudomonas chloropsis, Penicilium bilaii, Rhizobium japonicum, Purnate Varroacide, Chenopodioum quinoa saponins, Agrobacterium radiobacter, Bacillus cereus, Bacillus thuringiensis, Beauveria bassiana, Beauveria brongniartii, Burkholderia spp., Chromobacterium subtsugae, Flavobacterium spp., Heterorhabditis bacteriophora, Isaria fumosorosea, Lecanicillium longisporum, Lecanicillium muscarium, Metarhizium anisopliaze, Metarhizium anisopliae var. anisopliae, Metarhizium anisopliae var. acridum, Nomuraea rileyi; Paecilomyces lilacinus, Paenibacillus popilliae, Pasteuria spp., Pasteuria nishizawae, Pasteuria penetrans, Pasteuria ramosa, Pasteuria thornea, Pasteuria usage, Steinernema carpocapsae, Steinernema feltiae, Steinernema kraussei, Streptomyces galbus, Streptomyces microflavus, a recombinant exosporium-producing Bacillus cell, such as a Bacillus species cell, including a Bacillus thuringienses cell (such as B. thuringiensis BT013A) that expresses a fusion protein comprising:
In one embodiment, biologicals and biologically active ingredients for seed treatment of the seeds comprising the elite event of the invention are selected from the group named SBCA2 consisting of:
Bacilus firmus, especially Bacillus firmus GB126, Bacillus subtilis, Bacillus pumilus such as Bacillus pumilus QST 2808 or Bacillus pumilus GB34, the above-described recombinant exosporium-producing Bacillus, such as a Bacillus thuringienses cell expressing the above-described fusion protein.
In another more preferred embodiment, preferred active ingredients for seed treatment of seeds comprising the elite event of the invention are selected from the group named SAI1 consisting of:
β-Cyfluthrin, Bradyrhizobium japonicum, Carbendazim, Carboxin Clothianidin, Cypermethrin, Deltamethrin, Difenoconazole, Ethoxysulfuron, Fenamidon, Fenoxaprop-P-Ethyl, Flubendiamide, Fluopicolide, Fluopyram, Fluoxastrobin, Fluquinconazole, Fosetyl-A1, Fipronil, Prochloraz, Propineb, Prothioconazole, Pseudomonas Fluorescens, Spirotetramat, Tebuconazole, Tembotrione, Thiacloprid, Thiodicarb, Thiram, Triadimenol, Trifloxystrobin, Triflumuron, Penflufen, Imidacloprid, Metalaxyl, Mefonoxam, Fludioxinil, Pryaclostrobin, Azoxystrobin, Sedaxane, Ipconazole, Picoxystrobin, Cyantraniliprole, Chlorantraniliprole, Tetraniliprole, Penthiopyrad, Pasteuria nishizawae, such as Pasteuria nishizawae Pn1, Burkholderia spp., such as strain A396, Bacillus amyloliquefaciens, such as Bacillus amyloliquefaciens strain PTA-4838, Bacillus amyloliquefaciens strain D747 or Bacillus amyloliquefaciens TJ1000, Harpin protein, Thiamethoxam, Flupyradifurone, Bacillus firmus, such as Bacillus firmus GB126, Fluoxapyrad, Ethoboxam, Thiophanate-methyl, Pydiflumetofen, and Thiabendazole.
Particularly preferred combinations of compounds and/or biological agents for seed treatment of seeds comprising the elite event of the invention used in the context of the present invention are selected from the group named SC1 consisting of:
combination of Clothianidin and Bacillus firmus (such as B. firmus GB126), combination of Clothianidin, Bacillus thuringiensis (such as B. thuringiensis strain EX297512) and Bacillus firmus (such as B. firmus GB126), combination of Imidacloprid and Thiodicarb, combination of Imidacloprid and Prothioconazole, combination of Clothianidin and Carboxin and Metalaxyl and Trifloxystrobin, combination of Metalaxyl and Prothioconazole and Tebuconazole, combination of Clothianidin and beta-Cyfluthrin, combination of Prothioconazole and Tebuconazole, combination of Clothianidin and Imidacloprid and Prothioconazole and Tebuconazole, combination of Carbendazim and Thiram, combination of Imidacloprid and Methiocarb, combination of Metalaxyl, Penflufen and Prothioconazole, combination of Metalaxyl, Penflufen, Prothioconazole and imidacloprid, combination of Metalaxyl, Penflufen, Prothioconazole, imidacloprid and fluopyram, combination of Metalaxyl, Penflufen, Prothioconazole, imidacloprid, fluopyram and Bacillus firmus (such as B. firmus GB126), combination of Metalaxyl, Penflufen, Prothioconazole, imidacloprid, fluopyram, Bacillus firmus (such as B. firmus GB126) and Bacillus thuringiensis (such as B. thuringiensis strain EX297512), combination of Metalaxyl, Penflufen, Prothioconazole and Clothianidin, combination of Metalaxyl, Penflufen, Prothioconazole, Clothianidin and Bacillus firmus (such as B. firmus GB126), combination of Metalaxyl, Penflufen, Prothioconazole, Clothianidin, Bacillus firmus (such as B. firmus GB126) and Bacillus thuringiensis (such as B. thuringiensis strain EX297512), combination of Metalaxyl, Penflufen, Prothioconazole, Clothianidin, Bacillus firmus (such as B. firmus GB126) and fluopyram, combination of Metalaxyl, Penflufen, Prothioconazole, Clothianidin, Bacillus firmus (such as B. firmus GB126), fluopyram and Bacillus thuringiensis (such as B. thuringiensis strain EX297512), combination of Imidacloprid and Prothioconazole, combination of Imidacloprid and Tefluthrin, combination of Imidacloprid and Pencycuron, combination of Imidacloprid and Penflufen, combination of Fluoxastrobin, Prothioconazole and Tebuconazole, combination of Fluoxastrobin, Prothioconazole and metalaxyl, combination of Fluoxastrobin, Prothioconazole, metalaxyl and imdiacloprid, combination of Fluoxastrobin, Prothioconazole, metalaxyl, imdiacloprid and fluopyram, combination of Fluoxastrobin, Prothioconazole, metalaxyl, imdiacloprid, fluopyram and Bacillus firmus (such as B. firmus GB126), and combination of Fluoxastrobin, Prothioconazole, metalaxyl, imdiacloprid, fluopyram, Bacillus firmus (such as B. firmus GB126), and Bacillus thuringiensis (such as B. thuringiensis strain EX297512), combination of Fluoxastrobin, Prothioconazole, metalaxyl, clothianidin and fluopyram, combination of Fluoxastrobin, Prothioconazole, metalaxyl, clothianidin and Bacillus firmus (such as B. firmus GB126), combination of Fluoxastrobin, Prothioconazole, metalaxyl, clothianidin, Bacillus firmus (such as B. firmus GB126), and Bacillus thuringiensis (such as B. thuringiensis strain EX297512), combination of Fluoxastrobin, Prothioconazole, metalaxyl, clothianidin and Bacillus firmus (such as B. firmus GB126), combination of Fluoxastrobin, Prothioconazole, metalaxyl, clothianidin, Bacillus firmus (such as B. firmus GB126), and Bacillus thuringiensis (such as B. thuringiensis strain EX297512), combination of Metalaxyl and Trifloxystrobin, combination of Penflufen and Trifloxystrobin, combination of Prothioconazole and Tebuconazole, combination of Fluoxastrobin and Prothioconazole and Tebuconazole and Triazoxide, combination of Imidacloprid and Methiocarb and Thiram, combination of Clothianidin and beta-Cyfluthrin, combination of Clothianidin and Fluoxastrobin and Prothioconazole and Tebuconazole, combination of Fluopyram and Fluoxastrobin and Triadimenol, combination of Metalaxyl and Trifloxystrobin, combination of Imidacloprid and Ipconacole, combination of Difenoconazol and Fludioxonil and Tebuconazole, combination of Imidacloprid and Tebuconazole, combination of Imidacloprid, Prothioconazole and Tebuconazole, combination of Metalaxyl, Prothioconazole and Tebuconazole, combination of fluopyram and Bacillus firmus (such as B. firmus GB126), combination of fluopyram, Bacillus firmus (such as B. firmus GB126), and Bacillus thuringiensis (such as B. thuringiensis strain EX297512), Combination of Pasteria nishazawae (such as P. nishizawae Pn1), thiamethoxam, sedexane, fludioxinil and mefonaxam, Combination of thiamethoxam, sedexane, fludioninil and mefonaxam, Combination of thiamethoxam, fludioxinil and mefonaxam, Combination of fludioxinil and mefonaxam, Combination of pyraclostrobin and fluoxayprad, Combination of abamectin and thiamethoxam, Combination of Burkholderia spp. strain (such as strain A396) and imidacloprid, Combination of Bacillus amyloliquefaciens (such as B. amyloliquefaciens strain PTA-4838) and clothianidin, Combination of tioxazafen, imidacloprid, prothioconazole, fluoxastrobin and metalaxyl, Combination of tioxazafen, clothianidin, prothioconazole, fluoxastroin and metalaxyl, Combination of tioxazafen, clothianidin, Bacillus firmus (such as B. firmus GB126), prothioconazole, fluoxastrobin and metalaxyl, Combination of tioxazafen, clothianidin, Bacillus firmus (such as B. firmus GB126), Bacillus thuringiensis (such as B. thuringiensis strain EX297512), prothioconazole, fluoxastrobin and metalaxyl, combination of tioxazafen, prothioconazole, fluoxastrobin and metalaxyl, combination of tioxazafen, pyraclostrobin, fluoxyprad, metalaxyl and imidacloprid, combination of clothianidin, fluopyram and Bacillus firmus (such as B. firmus GB126), combination of clothianidin, fluopyram, tioxazafen and Bacillus firmus (such as B. firmus GB126); combination of clothianidin, fluopyram, Bacillus firmus (such as B. firmus GB126) and Bacillus thuringiensis (such as B. thuringiensis strain EX297512), combination of clothianidin, fluopyram, tioxazafen, Bacillus firmus (such as B. firmus GB126) and Bacillus thuringiensis (such as B. thuringiensis strain EX297512).
In one embodiment of the invention, seeds comprising EE-GM5 of the invention are treated with a combination of prothioconazole, penfluten and metalaxyl, or with a combination of prothioconazole, penfluten, metalaxyl and clothianidine, wherein said seeds or plants also contain one or more soybean SCN resistance genes from PI 548402, PI 209332 or PI 437654, or one or more of the soybean SCN resistance loci or genes selected from the group consisting of: rhg1, rhg1-b, rhg2, rhg3, Rhg4, Rhg5, qSCN11, cqSCN-003, cqSCN-005, cqSCN-006, and cqSCN-007.
In one embodiment of the invention, a combination of active ingredients for seed treatment is selected from the group named SC2 consisting of:
combination of Clothianidin and Bacillus firmus (such as B. firmus GB126), combination of Imidacloprid and Thiodicarb, combination of Imidacloprid and Prothioconazole, combination of Clothianidin Carboxin, Metalaxyl, Trifloxystrobin, combination of Metalaxyl, Prothioconazole and Tebuconazole, combination of Clothianidin and beta-Cyfluthrin, combination of Prothioconazole and Tebuconazole, combination of fluopyram, Bacillus firmus (such as Bacillus firmus GB126), combination of Pasteria nishazawe (such as P. nishizawae Pn1), thiamethoxam, sedexane, fludioxinil and mefonaxam, combination of abamectin and thiamethoxam, combination of penflufen, prothioconazole and metalaxyl, combination of penflufen and trifloxystrobin.
Preferred agents for use in seed treatment in accordance with this invention, are one or more of the nematicidal agents of group NC1, NC2 or N3, or one or more of the biological control agents of group BCA8, BCA9 or BCA10, or a combination of one of more of such nematicidal agents and biological control agents.
Formulations
The present invention further relates to formulations and use forms for the above-mentioned compounds and/or biological control agents and/or mixtures, for example drench, drip and spray liquors, for application to the plants or seeds of the invention, or for application to the soil wherein the plants or seeds of the invention were planted, or for application to the soil wherein the plants or seeds of the invention are to be planted (followed by planting of the plants or sowing of the seeds of the invention). In some cases, the use forms comprise further pesticides and/or adjuvants which improve action, such as penetrants, e.g. vegetable oils, for example rapeseed oil, sunflower oil, mineral oils, for example paraffin oils, alkyl esters of vegetable fatty acids, for example rapeseed oil methyl ester or soya oil methyl ester, or alkanol alkoxylates and/or spreaders, for example alkylsiloxanes and/or salts, for example organic or inorganic ammonium or phosphonium salts, for example ammonium sulphate or diammonium hydrogenphosphate and/or retention promoters, for example dioctyl sulphosuccinate or hydroxypropyl guar polymers and/or humectants, for example glycerol and/or fertilizers, for example ammonium-, potassium- or phosphorus-containing fertilizers.
Customary formulations are, for example, water-soluble liquids (SL), emulsion concentrates (EC), emulsions in water (EW), suspension concentrates (SC, SE, FS, OD), water-dispersible granules (WG), granules (GR) and capsule concentrates (CS); these and further possible formulation types are described, for example, by Crop Life International and in Pesticide Specifications, Manual on development and use of FAO and WHO specifications for pesticides, FAO Plant Production and Protection Papers—173, prepared by the FAO/WHO Joint Meeting on Pesticide Specifications, 2004, ISBN: 9251048576. The formulations, in addition to one or more of the above compounds, optionally comprise further agrochemically active compounds.
These are preferably formulations or use forms which comprise auxiliaries, for example extenders, solvents, spontaneity promoters, carriers, emulsifiers, dispersants, frost protectants, biocides, thickeners and/or further auxiliaries, for example adjuvants. An adjuvant in this context is a component which enhances the biological effect of the formulation, without the component itself having any biological effect. Examples of adjuvants are agents which promote retention, spreading, attachment to the leaf surface or penetration.
These formulations are prepared in a known way, for example by mixing the compounds with auxiliaries such as, for example, extenders, solvents and/or solid carriers and/or other auxiliaries such as, for example, surfactants. The formulations are prepared either in suitable facilities or else before or during application.
The auxiliaries used may be substances suitable for imparting special properties, such as certain physical, technical and/or biological properties, to the formulation of the compounds, or to the use forms prepared from these formulations (for example ready-to-use pesticides such as spray liquors or seed dressing products).
Suitable extenders are, for example, water, polar and nonpolar organic chemical liquids, for example from the classes of the aromatic and non-aromatic hydrocarbons (such as paraffins, alkylbenzenes, alkylnaphthalenes, chlorobenzenes), the alcohols and polyols (which, if appropriate, may also be substituted, etherified and/or esterified), the ketones (such as acetone, cyclohexanone), the esters (including fats and oils) and (poly)ethers, the unsubstituted and substituted amines, amides, lactams (such as N-alkylpyrrolidones) and lactones, the sulphones and sulphoxides (such as dimethyl sulphoxide), the carbonates and the nitriles.
If the extender used is water, it is also possible to employ, for example, organic solvents as auxiliary solvents. Essentially, suitable liquid solvents are: aromatics such as xylene, toluene or alkylnaphthalenes, chlorinated aromatics or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chloroethylenes or methylene chloride, aliphatic hydrocarbons such as cyclohexane or paraffins, for example mineral oil fractions, mineral and vegetable oils, alcohols such as butanol or glycol and their ethers and esters, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, strongly polar solvents such as dimethylformamide or dimethyl sulphoxide, carbonates such as propylene carbonate, butylene carbonate, diethyl carbonate or dibutyl carbonate, or nitriles such as acetonitrile or propanenitrile.
In principle, it is possible to use all suitable solvents. Examples of suitable solvents are aromatic hydrocarbons, such as xylene, toluene or alkylnaphthalenes, chlorinated aromatic or chlorinated aliphatic hydrocarbons, such as chlorobenzene, chloroethylene or methylene chloride, aliphatic hydrocarbons, such as cyclohexane, paraffins, petroleum fractions, mineral and vegetable oils, alcohols, such as methanol, ethanol, isopropanol, butanol or glycol and their ethers and esters, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, strongly polar solvents, such as dimethyl sulphoxide, carbonates such as propylene carbonate, butylene carbonate, diethyl carbonate or dibutyl carbonate, nitriles such as acetonitrile or propanenitrile, and also water.
In principle, it is possible to use all suitable carriers. Useful carriers include especially: for example ammonium salts and ground natural minerals such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth, and ground synthetic materials such as finely divided silica, alumina and natural or synthetic silicates, resins, waxes and/or solid fertilizers. Mixtures of such carriers can likewise be used. Useful carriers for granules include: for example crushed and fractionated natural rocks such as calcite, marble, pumice, sepiolite, dolomite, and synthetic granules of inorganic and organic meals, and also granules of organic material such as sawdust, paper, coconut shells, corn cobs and tobacco stalks.
Liquefied gaseous extenders or solvents can also be used. Particularly suitable extenders or carriers are those which are gaseous at ambient temperature and under atmospheric pressure, for example aerosol propellant gases, such as halohydrocarbons, and also butane, propane, nitrogen and carbon dioxide.
Examples of emulsifiers and/or foam-formers, dispersants or wetting agents with ionic or nonionic properties, or mixtures of these surfactants, are salts of polyacrylic acid, salts of lignosulphonic acid, salts of phenolsulphonic acid or naphthalenesulphonic acid, polycondensates of ethylene oxide with fatty alcohols or with fatty acids or with fatty amines, with substituted phenols (preferably alkylphenols or arylphenols), salts of sulphosuccinic esters, taurine derivatives (preferably alkyl taurates), isethionate derivatives, phosphoric esters of polyethoxylated alcohols or phenols, fatty esters of polyols, and derivatives of the compounds containing sulphates, sulphonates and phosphates, for example alkylaryl polyglycol ethers, alkyl sulphonates, alkyl sulphates, aryl sulphonates, protein hydrolysates, lignosulphite waste liquors and methylcellulose. The presence of a surfactant is advantageous if one of the pesticides and/or one of the inert carriers is insoluble in water and when the application takes place in water.
It is possible to use colorants such as inorganic pigments, for example iron oxide, titanium oxide and Prussian Blue, and organic dyes such as alizarin dyes, azo dyes and metal phthalocyanine dyes, and nutrients and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc as further auxiliaries in the formulations and the use forms derived therefrom.
Additional components may be stabilizers, such as low-temperature stabilizers, preservatives, antioxidants, light stabilizers or other agents which improve chemical and/or physical stability. Foam formers or antifoams may also be present.
Tackifiers such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or latices, such as gum arabic, polyvinyl alcohol and polyvinyl acetate, or else natural phospholipids such as cephalins and lecithins and synthetic phospholipids may also be present as additional auxiliaries in the formulations and the use forms derived therefrom. Further possible auxiliaries are mineral and vegetable oils.
Optionally, further auxiliaries may be present in the formulations and the use forms derived therefrom. Examples of such additives include fragrances, protective colloids, binders, adhesives, thickeners, thixotropic agents, penetrants, retention promoters, stabilizers, sequestrants, complexing agents, humectants, spreaders. In general, the compounds can be combined with any solid or liquid additive commonly used for formulation purposes.
Useful retention promoters include all those substances which reduce the dynamic surface tension, for example dioctyl sulphosuccinate, or increase the viscoelasticity, for example hydroxypropylguar polymers.
Suitable penetrants in the present context are all those substances which are usually used for improving the penetration of agrochemical active compounds into plants. Penetrants are defined in this context by their ability to penetrate from the (generally aqueous) application liquor and/or from the spray coating into the cuticle of the plant and thereby increase the mobility of active compounds in the cuticle. The method described in the literature (Baur et al., 1997, Pesticide Science 51, 131-152) can be used to determine this property. Examples include alcohol alkoxylates such as coconut fatty ethoxylate (10) or isotridecyl ethoxylate (12), fatty acid esters, for example rapeseed oil methyl ester or soya oil methyl ester, fatty amine alkoxylates, for example tallowamine ethoxylate (15), or ammonium and/or phosphonium salts, for example ammonium sulphate or diammonium hydrogenphosphate.
The formulations preferably comprise between 0.00000001 and 98% by weight of the compound or, with particular preference, between 0.01% and 95% by weight of the compound, more preferably between 0.5% and 90% by weight of the compound, based on the weight of the formulation.
The content of the compound in the use forms prepared from the formulations (in particular pesticides) may vary within wide ranges. The concentration of the compound in the use forms is usually between 0.00000001 and 95% by weight of the compound, preferably between 0.00001 and 1% by weight, based on the weight of the use form. The compounds are employed in a customary manner appropriate for the use forms.
Mixtures
The compounds mentioned herein may also be employed as a mixture with one or more suitable fungicides, bactericides, acaricides, molluscicides, nematicides, insecticides, microbiologicals, beneficial species, herbicides, fertilizers, bird repellents, phytotonics, sterilants, safeners, semiochemicals and/or plant growth regulators, e.g., to broaden the spectrum of action, to prolong the duration of action, to increase the rate of action, to prevent repulsion or prevent evolution of resistance. In addition, such active compound combinations may improve plant growth and/or tolerance to abiotic factors, for example high or low temperatures, to drought or to elevated water content or soil salinity. It is also possible to improve flowering and fruiting performance, optimize germination capacity and root development, facilitate harvesting and improve yields, influence maturation, improve the quality and/or the nutritional value of the harvested products, prolong storage life and/or improve the processability of the harvested products.
Furthermore, the compounds can be present in a mixture with other active compounds or semiochemicals such as attractants and/or bird repellants and/or plant activators and/or growth regulators and/or fertilizers. Likewise, the compounds can be used to improve plant properties such as, for example, growth, yield and quality of the harvested material.
In a particular embodiment according to the invention, the compounds are present in formulations or the use forms prepared from these formulations in a mixture with further compounds, preferably those as described herein.
If one of the compounds mentioned herein can occur in different tautomeric forms, these forms are also included even if not explicitly mentioned in each case. Further, all named mixing partners can, if their functional groups enable this, optionally form salts with suitable bases or acids.
In one embodiment at least one active ingredient selected from group H1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group H2 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group H3 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient mixture selected from group H4 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient mixture selected from group H5 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN2 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN3 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN4 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN5 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN6 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN7 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN8 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN9 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN9 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN10 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN11 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN12 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN13 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN14 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN15 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN16 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN17 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN18 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN19 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN20 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN21 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN22 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN23 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN24 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN25 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN26 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN27 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN28 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN29 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group IAN30 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group SIAN1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F2 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F3 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F4 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F5 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F6 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F7 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F8 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F9 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F10 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F11 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F12 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F13 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F14 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group F15 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group SF1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient mixture selected from group F16 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group P1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group BCA1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group BCA2 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group BCA3 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group BCA4 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group BCA5 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group BCA6 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group BCA7 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group BCA8 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group BCA9 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group BCA10 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group SBCA1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient combination selected from group SAI1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient combination selected from group SC1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient combination selected from group SC2 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group NC1 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient selected from group NC2 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In one embodiment at least one active ingredient combination selected from group NC3 is used on a plant comprising EE-GM5 or plant parts thereof (such as a seed), preferably for increasing yield.
In the context of the present invention, “increasing yield” means a significant increase in yield by compared with the untreated plant, preferably a significant increase by at least 1% such as by 1% to 3%, compared with the untreated plant (100% yield), i.e. the yield of the treated plants is at least 101% compared to the yield (100%) of the untreated plant; more preferably, the yield is even more increased by at least 2%, more preferably at least 5%, even more preferably by at least 10% such as by 2% to 5% (yield from 102% to 105%), by 2 to 10% (yield from 102% to 110%), by 5% to 20% (yield of from 105% to 120%), or by 10 to 30% (yield from 110% to 130%). The yield increase may be achieved by curative treatment, i.e. for treatment of already infected plants, or by protective treatment, for protection of plants which have not yet been infected.
In the context of the present invention, “to treat with” means to contact a plant or part of a plant with an effective amount of an active ingredient or a combination thereof or to coat a seed with an active ingredient or a combination thereof.
“Active ingredient” refers to compounds or biological control agents used in agriculture. Active ingredients according to the invention are not applied to humans or animals as a medical or therapeutic treatment.
In the context of the present invention the active ingredients according to the invention or to be used according to the invention may be a composition (i. e. a physical mixture) comprising at least one active ingredient. It may also be a combination of active ingredients composed from separate formulations of a single active ingredient component being active ingredient (tank-mix). Another example of a combination of active ingredients according to the invention is that the active ingredients are not present together in the same formulation, but packaged separately (combipack), i.e., not jointly preformulated. As such, combipacks include one or more separate containers such as vials, cans, bottles, pouches, bags or canisters, each container containing a separate component for an agrochemical composition, here at least one active ingredient. One example is a two-component combipack. Accordingly the present invention also relates to a two-component combipack, comprising a first component which in turn comprises an active ingredient, a liquid or solid carrier and, if appropriate, at least one surfactant and/or at least one customary auxiliary, and a second component which in turn comprises another active ingredient, a liquid or solid carrier and, if appropriate, at least one surfactant and/or at least one customary auxiliary. More details, e.g. as to suitable liquid and solid carriers, surfactants and customary auxiliaries are described below. A mixture or combination according to the invention shall mean/encompass a tank mix or a combipack.
In one embodiment, besides protection against nematodes, the compound(s) and/or biological control agent(s) as described herein are used to also protect soybean plants, parts or seeds from the following pests, bacterial diseases or fungi: hairy caterpillar, Spilarctia obliqua (Walker); leaf roller, Lamprosema indicata F; common cutworm, Spodoptera litura F; pod borer, Armyworms, especially Spodoptera exigua and S. praefica, Helicoverpa armigera (Hubner); stem fly, Ophiomyia phaseoli (Tryon) and white fly; Bemisia tabaci Genn., Pseudomonas syringae, Xanthomonas campestris, Septoria glycines, Macrophomina phaseolina, Cucumber beetles, especially Acalymma vittata or Diabrotica undecimpunctata, Downy mildew (Peronospora manshurica), Frogeye leaf spot (Cercospora sojina), Mexican Bean beetle (Epilachna varivestis), Phytophthora megasperma, Rhizoctonia solani, Rust (Phakopsora pachyrhizi), Sclerotinia sclerotiorum.
The following examples describe the development and identification of elite event EE-GM5, the development of different soybean lines comprising this event, and the development of tools for the specific identification of elite event EE-GM5 in biological samples.
Unless stated otherwise in the Examples, all recombinant techniques are carried out according to standard protocols as described in “Sambrook J and Russell D W (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, New York” and in “Ausubel F A, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A and Struhl K (eds.) (2006) Current Protocols in Molecular Biology. John Wiley & Sons, New York”.
Standard materials and references are described in “Croy R D D (ed.) (1993) Plant Molecular Biology LabFax, BIOS Scientific Publishers Ltd., Oxford and Blackwell Scientific Publications, Oxford” and in “Brown T A, (1998) Molecular Biology LabFax, 2nd Edition, Academic Press, San Diego”. Standard materials and methods for polymerase chain reactions (PCR) can be found in “McPherson M J and Møller S G (2000) PCR (The Basics), BIOS Scientific Publishers Ltd., Oxford” and in “PCR Applications Manual, 3rd Edition (2006), Roche Diagnostics GmbH, Mannheim or on the world wide web at roche-applied-science.com”.
It should be understood that a number of parameters in any lab protocol such as the PCR protocols in the below Examples may need to be adjusted to specific laboratory conditions, and may be modified slightly to obtain similar results. For instance, use of a different method for preparation of DNA or the selection of other primers in a PCR method may dictate other optimal conditions for the PCR protocol. These adjustments will however be apparent to a person skilled in the art, and are furthermore detailed in current PCR application manuals.
In the description and examples, reference is made to the following sequences in the enclosed
1. Transformation of Glycine max with a Nematode Resistance and an Herbicide Tolerance Gene
1.1. Description of the Inserted T-DNA Comprising the cry14Ab-1.b and hppdPf-4 Pa Chimeric Genes
EE-GM5 soybean was developed through Agrobacterium-mediated transformation using the vector pSZ8832 containing hppdPf-4 Pa and cry14Ab-1.b expression cassettes:
(i) The mutant hppdPf-4 Pa gene that encodes for the HPPD-4 protein (the amino acid sequence of which is shown in SEQ ID No. 10). The hppdPf-4 Pa coding sequence was developed by introducing point mutations at position 335 (substitution of Glu by Pro), at position 336 (substitution of Gly by Trp), at position 339 (substitution of Lys by Ala) and at position 340 (substitution of Ala by Gln) in a DNA encoding the HPPD protein derived from Pseudomonas fluorescens strain A32. Expression of the HPPD-4 protein confers tolerance to HPPD inhibitor herbicides, such as isoxaflutole, topramezone or mesotrione.
(ii) The cry14Ab-1.b gene encodes for the Cry14Ab-1 protein (the amino acid sequence of which is shown in SEQ ID No. 8). Expression of the Cry14Ab-1 protein confers resistance to nematodes such as the soybean cyst nematode Heterodera glycines.
Plasmid pSZ8832 is a plant transformation vector which contains a chimeric cry14Ab-1.b gene and a chimeric hppdPf-4 Pa gene located between the right T-DNA border (RB) and the left T-DNA border (LB). A description of the genetic elements comprised in the T-DNA between the right and the left T-DNA border is given in Table 2 below. Confirmatory sequencing of the T-DNA (between the T-DNA borders) of this plasmid resulted in the sequence of SEQ ID No. 11. The nucleotide sequence of the cry14Ab-1.b and hppdPf-4 Pa coding sequences (showing the coding strand) is represented in SEQ ID No. 7 and 9, respectively.
Pseudomonas fluorescens
Zea mays and Helianthus annuus (U.S. Pat. No. 5,510,471)
1.2. Event EE-GM5
The T-DNA vector pSZ8832 was introduced into Agrobacterium tumefaciens and transformed soybean plants (var. Thorne) were selected using HPPD inhibitor tolerance according to methods known in the art. The surviving plants were then self-pollinated to generate T1 seed. Subsequent generations were produced through self-pollination, or through crossing into other soybean germplasm.
1.2.1 Identification of Elite Event EE-GM5
Elite event EE-GM5 was selected based on an extensive selection procedure (based on parameters including but not limited to trait efficacy in the greenhouse and the field, molecular characteristics, and agronomic characteristics) from a wide range of different transformation events obtained using the same chimeric genes. Soybean plants containing EE-GM5 were found to have an insertion of the transgenes at a single locus in the soybean plant genome, to have overall agronomy similar to the parent plants used for transformation, to cause no yield penalty by the insertion of the transforming DNA (as compared to a corresponding isogenic line without the event, such as a “null” plant line obtained from a transformed plant in which the transgenes segregated out), to result in a significant reduction of adult females infesting the roots in a standard SCN greenhouse assay, and to have improved yield under SCN nematode pressure in the field compared to the isogenic null line not containing EE-GM5. Additionally, tolerance to HPPD inhibitor herbicide application was measured in field trials, but herbicide tolerance was not a selection criterion for elite event selection.
1.2.1.1 Molecular Analysis of the Event
Southern blot results showed that EE-GM5 contains a single transgenic locus which contains a single copy of the cry14Ab-1.b chimeric gene and a single copy of the hppdPf-4 Pa chimeric gene. EE-GM5 is missing a part of the 35S promoter of the hppdPf-4 Pa chimeric gene (indicating that not the entire T-DNA of SEQ ID No. 11 was inserted in the soybean genome during transformation). No PCR fragments were obtained upon PCR analysis using primers targeting vector backbone sequences that are flanking the left and right border of the T-DNA as well as the aadA sequence. Also, the presence of identical EE-GM5 integration fragments in multiple generations of EE-GM5 demonstrates the structural stability of the event.
1.2.1.2 Inheritance of the Event
Inheritance of the inserted T-DNA insert in subsequent generations by testing the genotype of hppdPf-4 Pa and cry14Ab-1.b genes by PCR analysis shows that the hppdPf-4 Pa and cry14Ab-1.b genes contained within the EE-GM5 insert are inherited in a predictable manner and as expected for a single insertion. These data are consistent with Mendelian principles and support the conclusion that the EE-GM5 event consists of a single insert integrated into a single chromosomal locus within the soybean nuclear genome.
Also, analysis of the segregation patterns of EE-GM5 in subsequent generations upon introgression of EE-GM5 into 5 elite soybean lines confirmed normal Mendelian segregation. Table 3 shows the observed segregation of EE-GM5 in different segregating populations.
In Table 3, “HH” stands for homozogous plants, “Hemi” for hemizygous plants, and “null” for null-segregants having lost EE-GM5, and “ns” means not statistically significant (as to any variation from normal/expected segregation). In these trials, Parent 1 was a MG VI line with Rhg1 and Rhg4 native SCN resistance, Parent 2 was a MG VI line susceptible to SCN, Parent 3 was a MG IX line susceptible to SCN, Parent 4 was a MG III line with Rhg1 native SCN resistance, and Parent 5 was a MG I line susceptible to SCN.
1.2.1.3 Stability of Protein Expression
Protein expression levels of HPPD-4 and Cry14Ab-1 proteins in greenhouse-grown plants were determined by sandwich enzyme-linked immunosorbent assay (ELISA) in leaf, root and seed samples collected from different generations (e.g., T4, T6 and BC2F3) of EE-GM5 soybean. HPPD-4 and Cry14Ab-1 exhibit similar mean expression levels in leaf, root and seed across all generations tested. Any differences observed in Cry14Ab-1 and HPPD-4 concentrations were attributed to natural plant-to-plant variability.
1.2.1.4 Agronomic Performance and Tolerance to HPPD Inhibitor Herbicides
In agronomic equivalency trials, plants comprising EE-GM5 in the original transformation background (Thorne) were compared to segregating nulls (lacking EE-GM5) and to wild-type Thorne plants when grown in the absence of SCN. Plots were not treated with HPPD herbicides but were maintained as weed free through the use of conventional herbicides and hand weeding where necessary. No differences impacting agronomic performance in a biologically significant way were observed between the plants containing the event and the segregating nulls (lacking EE-GM5) when grown in comparable trials at different locations when checking for qualitative plant characteristics such as flower color, pod color, seed color and pubescence and for quantitative characteristics like yield, height, lodging, stand, and days to maturity. Hence, plants comprising EE-GM5 showed normal agronomic characteristics comparable to the corresponding non-transgenic plants.
Additional trials with EE-GM5 in the original Thorne transformation background were conducted in 2017. Preliminary trials wherein EE-GM5 was in elite MG1 and MG3 genetic backgrounds were also established at a limited number of locations in 2017. When checking for qualitative plant characteristics such as flower color, pod color, seed color and pubescence and for quantitative characteristics like yield, height, lodging, stand, test weight, and days to maturity, no consistent and meaningful differences between the EE-GM5 event and the segregating nulls (lacking EE-GM5) were detected in any of the three genetic backgrounds, confirming that plants comprising EE-GM5 showed normal agronomic characteristics.
Tolerance of plants comprising EE-GM5 to HPPD inhibitor herbicides was tested at different locations in the field over 2 years. In these trials, it was found that plants with EE-GM5 had commercially relevant tolerance to isoxaflutole (IFT) when applied pre-emergence as well as when applied post-emergence, but crop damage was a bit higher for the IFT pre-emergence application. These trials also showed that plants containing event EE-GM5 had commercially relevant tolerance to mesotrione (MST) when applied pre-emergence or when applied post-emergence. All post-emergence treatments were at the V2-V3 stage, with adjuvants crop oil concentrate and ammonium sulfate added to increase herbicide activity.
In a 3rd year, plants with EE-GM5 (in Thorne background) when treated with isoxaflutole (IFT, at 410 g/ha) pre-emergence at one field trial location, had 9% maximum phytotoxicity, and when treated with isoxaflutole (IFT) post-emergence (V2-V3 stage, at 210 h/ha) at 4 locations, had an average of 10.9% maximum phytotoxicity, confirming the tolerance observed before.
Also, in several field trials across 2 years, soybean plants with event EE-GM5 had good tolerance towards experimental HPPD inhibitor compound 2-methyl-N-(5-methyl-1,3,4-oxadiazol-2-yl)-3-(methylsulfonyl)-4-(trifluoromethyl)benzamide (U.S. Pat. No. 9,101,141) when applied pre-emergence at 400 gr ai/ha or post-emergence at 200 gr ai/ha, respectively (the average maximum phytotoxicity value for each treatment was below 20%). In these trials, soybean plants with event EE-GM5 also showed good tolerance (average maximum phytotoxicity of 20%) to experimental HPPD inhibitor compound 2-chloro-3-(methylsulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide (U.S. Pat. No. 8,481,749) when applied post-emergence at 100-150 gr ai/ha. All post-emergence treatments were at the V2-V3 stage, with adjuvants crop oil concentrate and ammonium sulfate added to increase herbicide activity. In a 3rd year, plants with EE-GM5 (in Thorne background) when treated with 2-chloro-3-(methyl sulfanyl)-N-(1-methyl-1H-tetrazol-5-yl)-4-(trifluoromethyl)benzamide at 150 g/ha post-emergence at 3 field trial locations, had an average maximum phytotoxicity of 13.3%.
The same or very similar average maximum phytotoxicity ratings as those described in
Also, plants with EE-GM5 when treated post-emergent (V2-V3) with topramezone at 36 g ai/ha (+COC and AMS) in 2 field trials in the US gave an average maximum phytotoxicity of 11%, showing EE-GM5 also confers good tolerance to this HPPD inhibitor.
1.2.1.5 Nematode Resistance
Standard SCN assays measuring female index in the greenhouse showed a significant reduction of SCN cysts on roots of plants containing EE-GM5 when compared to Thorne wild-type soybean plants. In addition, standard SCN assays measuring female index in the greenhouse also showed that soybean plants containing event EE-GM5 and native SCN resistance showed a significant reduction of SCN cysts on roots compared to SCN resistant elite soybean lines without EE-GM5. When EE-GM5 was introgressed into an elite soybean line with PI88788 soybean resistance (maturity group 3), or into an elite soybean line with Peking soybean resistance (maturity group 6.2), consistently a reduced number of SCN cysts was seen on the roots compared to roots with native resistance alone.
In field trials across 2 years at several locations, soybean plants containing EE-GM5 gave a significant yield increase compared to the isogenic null segregants in SCN-infested fields.
In a study to evaluate the effect of event EE-GM5 on yield when combined with native SCN resistance, a series of F3 populations were developed from the single cross of EE-GM5 with an elite MG III conventional line carrying the rhg1 resistance gene from PI88788. In the F3 populations one ‘stacked’ population that is homozygous for both event EE-GM5 and the rhg1 allele, was compared to a population homozygous for just the rhg1 allele (lacking EE-GM5). Yield trials were established with these populations in 2016 at three locations with moderate to high infestation of SCN and in 2017 at seven locations ranging from low to high SCN pressure. The results are shown in
Conducting yield trials under moderate to high SCN infestation are challenging due many factors that have an impact on the results. SCN population densities within fields can vary substantially and so the overall impact of SCN on yield can also vary from one plot to the next (see, e.g., on the world wide web at plantmanagementnetwork.org/pub/php/review/2009/sce08/). Favorable soil types, good fertility and adequate rainfall can mitigate the impact of SCN infestation on the soybean plant and can minimize yield impacts even under high SCN populations. Many fields with very high SCN populations tend to have poor soils and thus lower yield potential, making it difficult to discern statistically significant impacts on yield. Thus, yield data from SCN field trials can be quite variable and one would not expect to see significant improvements in yield in every trial with high SCN populations. The overall trends across trials are the most relevant criteria for judging performance of an event.
SCN field trials that were done with plants containing EE-GM5 were established in field with natural SCN infestation. Experimental units consisted of a field plot containing 2 to 4 rows spaced 0.76 m apart and ranging from 3.8 to 9.1 m long. The number of rows per plot and plot length varied from location to location based on field size and equipment configurations. Plots were seeded at 26 seeds per meter and so each experimental unit contained between 200 and 960 seeds. Plots were randomized in the field using a split-plot or split-split plot design. Split plot designs are well suited to help minimize the effect of high variability in soil type or SCN populations which is common in SCN infested fields. In SCN field trials plants comprising EE-GM5 were planted in a sub plot next to, or very close to, a companion sub plot containing segregating null plants (without EE-GM5). The close proximity of the two plots helps minimize the effect of (SCN) field variability on the estimate of the difference between the plants with and without event EE-GM5. Most trials were replicated four times, but a few were replicated three times and a few were replicated five or six times.
Moderate to severe infestations of Sudden Death Syndrome were observed at two locations (Indiana and Iowa) in 2016. Plots at these two locations were rated for incidence and severity of SDS symptoms and the SDS Disease Index (DX) was calculated using the “SIUC Method of SDS Scoring” (on the world wide web at scnresearch.info/462.pdf). DX ratings on plants homozygous for EE-GM5 were 61% lower in Indiana and 55% lower in Iowa than on the susceptible null segregate (lacking EE-GM5), indicating that the event was providing protection against SDS infection. SDS and SCN are often closely associated in the field and will show some interactions in the plant (see, e.g., on the world wide web at soybeanresearchinfo.com/pdf docs/sdsupdate.pdf, and on the world wide web at apsnet.org/edcenter/intropp/lessons/fungi/ascomycetes/pages/suddendeath.aspx).
In 2017, Iron Deficiency Chlorosis (IDC) scores were gathered on plants with EE-GM5 (and their null segregants) at one trial location in the US (with high SCN infestation) where IDC symptoms were observed. The trial was a split-plot design looking at the effect of event in three different backgrounds. IDC ratings were taken as described by Cianzio et al. (1979) Crop Science 19: 644-646.
Also, non-transformed Thorne and EE-GM5 seeds were geminated and planted in the greenhouse to check for control of the lesion nematode, Pratylenchus brachyurus. Pratylenchus brachyurus nematodes (#1500/plant, different developmental stages) were applied to the plants when 2 weeks old. 30 days after application, Pratylenchus nematodes were extracted from the roots and counted. The average number of nematodes found in the roots of plants containing EE-GM5 were compared with the average number of Pratylenchus nematodes found in the wild-type Thorne plant roots. On average about 80 to 90% fewer Pratylenchus nematodes were found in roots of plants containing EE-GM5 when compared with the Thorne control roots, indicating significant control of lesion nematodes by soybean event EE-GM5.
In these field trials, soybean plants containing EE-GM5 showed a significant reduction in the total number of Pratylenchus brachyurus (adult and juvenile) nematodes in soybean roots, compared to the null segregants lacking the event (see
Also, plants containing EE-GM5 can be used to control root-knot nematodes (RKN) such as Meloidogyne incognita. Even though the population of Meloidogyne incognita does not infest Thorne wild-type soybean very well, Thorne plants with EE-GM5 show a further reduction in the number of RKN eggs/root mass on average, as compared to untransformed Thorne plants.
1.2.2 Identification of the Flanking Regions and Inserted T-DNA of Elite Event EE-GM5
The sequence of the regions flanking the inserted T-DNA and the T-DNA contiguous therewith as contained in the EE-GM5 elite event are shown in the enclosed Sequence Listing.
1.2.2.1 5′ T-DNA Flanking Region
A fragment identified as comprising the 5′ T-DNA flanking region of EE-GM5 was sequenced and its nucleotide sequence is represented in SEQ ID No. 5, nucleotides 1-166. This 5′ T-DNA flanking region is made up of soybean genomic sequences corresponding to the pre-insertion locus sequence (SEQ ID No. 5, nucleotides 1-166). The 5′ junction region comprising part of the inserted T-DNA sequence and part of the T-DNA 5′ flanking sequence contiguous therewith is represented in SEQ ID No. 1 and 3.
1.2.2.2 3′ T-DNA Flanking Region
A fragment identified as comprising the 3′ T-DNA flanking region of EE-GM5 was sequenced and its nucleotide sequence is represented in SEQ ID No. 6, nucleotides 359-691. This 3′ T-DNA flanking region is made up of a 39 nucleotide filler DNA sequence (from position 359 to position 397 in SEQ ID No. 6), followed by soybean genomic sequences corresponding to the pre-insertion locus sequence (from position 398 to position 691 in SEQ ID No. 6). The 3′ junction region comprising part of the inserted T-DNA sequence and part of the T-DNA 3′ flanking sequence contiguous therewith is represented in SEQ ID No. 2 and 4.
1.2.2.3 Inserted T-DNA of EE-GM5
The inserted T-DNA contiguous with the above 5′ T-DNA flanking sequence was sequenced and its nucleotide sequence is represented in SEQ ID No. 5, nucleotides 167-353. Also, the inserted T-DNA contiguous with the above 3′ T-DNA flanking sequence was sequenced and its nucleotide sequence is represented in SEQ ID No. 6, nucleotides 1-358. During transformation, 63 bp of genomic DNA were deleted at the pre-insertion locus sequence, and these were replaced by the inserted DNA (made up of T-DNA and a small part of filler DNA).
Sequencing of the T-DNA region in transformation plasmid pSZ8832 (the part between the T-DNA borders) resulted in the sequence reported in SEQ ID No. 11. The chimeric cry14Ab-1.b gene sequence (comprising the Ubi10 promoter and the 35S 3′ untranslated region) is represented in SEQ ID No. 11 from nucleotides 131-5276 (counterclockwise). The inserted T-DNA sequence at the 5′ flanking region in SEQ ID No. 5 (nucleotide 167-353) is identical to the nucleotide sequence in SEQ ID No. 11 from nucleotide 1 to nucleotide 187, and the inserted T-DNA sequence at the 3′ flanking region in SEQ ID No. 6 (nucleotide 1-358) is identical to the nucleotide sequence in SEQ ID No. 11 from nucleotide 7102 to nucleotide 7459. Hence, the 5′ end of the T-DNA inserted in EE-GM5 corresponds to nucleotide 1 in the transformation plasmid sequence of SEQ ID No. 11 and the 3′ end of the T-DNA inserted in EE-GM5 corresponds to nucleotide 7459 in the transformation plasmid sequence of SEQ ID No. 11. The T-DNA inserted in EE-GM5 between the sequence of SEQ ID No. 5 and the sequence of SEQ ID No. 6 is contained in the seed deposited at the ATCC under accession number PTA-123625, and has a sequence essentially similar or identical to the sequence of SEQ ID No. 11 from nucleotide 188 to nucleotide 7101.
The insertion locus for event EE-GM5 can be determined from wild-type soybean var. Thorne based on the 5′ and 3′ T-DNA flanking sequences provided herein (SEQ ID No. 5 from nt 1 to nt 166 and SEQ ID No. 6 from nt 359 to nt 691) by methods known in the art. The pre-insertion locus sequence in the soybean genome corresponds to the following sequences in order: nucleotide position 1 to nucleotide position 166 in SEQ ID No. 5, a 63 nt deletion, and nucleotide position 398 to nucleotide position 691 in SEQ ID No. 6. The complete pre-insertion locus sequence is given in SEQ ID No. 33, wherein nt 1-1000 are 5′ flanking genomic sequences, nt 1001-1063 are the target site deletion, and 1064-2063 are 3′ flanking genomic sequences.
1.2.3 Confirmation of the Flanking Regions and Inserted T-DNA of Elite Event EE-GM5
PCR amplification using primers targeted to the plant DNA upstream and downstream of the inserted T-DNA and to the inserted T-DNA in EE-GM5 confirmed and extended the 5′ and 3′ flanking sequences of EE-GM5.
1.2.3.1. 5′ Junction Sequence EE-GM5-Specific Reaction
Composition of the reaction mixture for the 5′ junction sequence reaction:
Thermocycling conditions for the 5′ junction sequence reaction:
1.2.3.2. 3′ Junction Sequence EE-GM5-Specific Reaction
Composition of the reaction mixture for the 3′ junction sequence reaction:
Thermocycling conditions for the 3′ junction sequence reaction:
Since the resulting amplicons in the above 2 reactions overlapped, this allowed a reconstruction of the sequence of the EE-GM5 inserted T-DNA and the extended 5′ and 3′ flanking sequences, which is shown in SEQ ID No. 23. The 5′ T-DNA flanking sequence in SEQ ID No. 23 is from nucleotide position 1 to nucleotide position 1113 (corresponding to pre-insertion locus genomic sequences), the inserted T-DNA sequence is from nucleotide position 1114 to nucleotide position 8572 and the 3′ T-DNA flanking sequence in SEQ ID No. 23 is from nucleotide position 8573 to nucleotide position 9663 (corresponding to 39 nt filler DNA (nt 8573-8611 in SEQ ID No. 23) and pre-insertion locus genomic sequences (nt 8612-9663 in SEQ ID No. 23)).
2. Development of Identification Protocols for EE-GM5
2.1. End-Point Method for EE-GM5 Identity Analysis
This method describes a polymerase chain reaction detection method to analyze the presence of event EE-GM5-specific DNA sequences in DNA samples obtained from biological samples, such as plant materials (e.g., leaf or seed) using standard DNA extraction procedures.
The method description outlines the method design, including the oligonucleotide primer and probe sequences, the composition of the reaction mixture, the thermocycling conditions required to perform the reaction, and the fluorescent reader settings found appropriate for amplicon detection. It also provides general recommendations on the nature and use of control samples. In addition, guidance is provided for data analysis and interpretation, including an example of a method result taking into account the recommendations on the use of control materials and the guidance for data analysis.
2.1.1. Method Design
The method uses the Taqman chemistry to amplify and detect two target sequences: a EE-GM5 specific reaction determines the presence of the event, a taxon-specific reaction validates negative results for the event-specific reaction.
2.1.1.1. EE-GM5-Specific Reaction
2.1.1.2. Taxon-Specific Specific Reaction
2.1.2. Composition of the Reaction Mixture
2.1.3. Thermocycling Conditions
2.1.4. Wavelength and Bandwidth Settings
2.1.5. Control Samples
Following control samples should be included in the experiment to validate the results of test samples:
2.2. End-Point Method for EE-GM5 Identity and Zygosity Analysis
2.2.1. Method Design
2.2.1.1. EE-GM5-Specific Reaction
A. Option 1
B. Option 2
2.2.1.1. Pre-Insertion Locus Specific Reaction
A. Option 1
B. Option 2
2.2.2. Composition of the Reaction Mixture
2.2.3. Thermocycling Conditions (the Same for Option 1 and 2)
2.2.4. Wavelength and Bandwidth Settings (the Same for Option 1 and 2)
2.2.5. Control Samples (the Same for Option 1 and 2)
2.2.6. Data Analysis (the Same for Option 1 and 2)
2.3. Real-Time PCR Method for EE-GM5 Identity and Zygosity Analysis
The method describes a quantitative Real-Time polymerase chain reaction detection method to analyze the zygosity status of event EE-GM5 DNA sequences in DNA samples obtained from biological samples, such as plant materials (e.g., leaf or seed) using standard DNA extraction procedures.
The method description outlines the reaction reagents, the oligonucleotide primer and probe sequences, the thermocycling conditions required to perform the reaction, incl the fluorescent reader settings found appropriate for amplicon detection. It also provides general recommendations on the nature and use of control samples. In addition, guidance is provided for data analysis and interpretation.
This method is not variety dependent and can be used as alternative zygosity analysis method to the method described in section 2.2 above.
2.3.1. Method Design
The method uses the Taqman chemistry and principles of Real-Time PCR to quantify the relative copy number of a EE-GM5 specific sequence.
The method includes a EE-GM5 specific reaction to quantify the EE-GM5 copy number, and a taxon-specific reaction for normalization of the EE-GM5 copy number.
Samples containing the EE-GM5 insertion sequence in a homozygous state have a relative copy number that is two-fold higher than hemizygous samples. Azygous samples do not amplify the EE-GM5 sequence.
2.3.2. EE-GM5-Specific Reaction
2.3.3. Taxon-Specific Reaction
2.3.4. Composition of the Reaction Mixture
2.3.5. Thermocycling Conditions
2.3.6. Control Samples
Following control samples should be included in the experiment to validate the results of test samples
2.3.7. Data Analysis
2.4. Real-Time PCR Method for EE-GM5 Low Level Presence Analysis
This method describes a detection method to analyze the Low Level Presence of event EE-GM5 DNA sequences obtained from bulked plant materials (e.g., leaf or seed) or processed materials (e.g., food or feed containing processed soybean grain) using standard DNA extraction procedures.
The method description outlines the reaction reagents, the oligonucleotide primer and probe sequences, and the thermocycling conditions required to perform the reaction. It also provides general recommendations on the concurrent use of a taxon-specific method to support data analysis and result interpretation. In addition, recommendations are provided on the nature and use of control samples.
It is noted that alternative methods may be available for the intended purpose, including but not limited to digital droplet PCR methods. Digital droplet PCR methods use End-Point methods for event identity analysis, as described in section 1.1, in combination with principles of subsampling on the extracted DNA sample. In this method the low level presence of the event is determined based on the ratio of DNA subsamples found positive and negative for the event sequence.
1.1.1 Method Design
1.1.2 Composition of the Reaction Mixture
1.1.3 Thermocycling Conditions
1.1.4 Taxon Specific Method
1.1.5 Test Samples, Calibration Samples and Control Samples
1.1.6 Data Analysis
3. Introgression of EE-GM5 into Preferred Cultivars
Elite event EE-GM5 was introduced by repeated back-crossing into six different elite soybean lines. The lines were selected to represent a range of maturities: two lines from MG I, one line from MG III, two lines from MG VI and one line from MG IX. One of the MG I lines and the MG III line contained the Rhg1 native resistance allele from PI88788, and one of the MG VI lines carried the Rhg1 and Rhg4 native resistance alleles from PI437654. The other three lines were susceptible to SCN.
Also, in initial testing, in several experiments, no biologically significant differences were observed for Cry14Ab-1 of HPPD-4 protein expression levels measured in leaves of greenhouse-grown plants (as measured with ELISA or Western blot (only normal assay variation was seen)), and no significant differences were seen in the standard greenhouse SCN assay results (measuring % reduction in SCN cysts vs. the Thorne control), when event EE-GM5 was introgressed from Thorne background into other soybean germplasm backgrounds (of different maturity, at different stages of introgression), compared to what was found for EE-GM5 in the Thorne background.
Introgression of the elite event EE-GM5 into other soybean cultivars does not significantly influence any of the desirable phenotypic or agronomic characteristics of these cultivars (no linkage drag) while expression of the transgenes meets commercially acceptable levels. This confirms the status of event EE-GM5 as an elite event.
Furthermore, elite event EE-GM5 is advantageously combined with other soybean elite transformation events. Particularly useful plants according to the invention are plants containing EE-GM5 combined with another soybean transformation event, or a combination of more than one other soybean transformation event, such as those listed in the databases of various national or regional regulatory agencies, including but not limited to Event MON87751 (aka MON-87751-7, described in WO2014201235 and USDA-APHIS Petition 13-337-01p), Event pDAB8264.42.32.1 (described in WO2013010094), Event DAS-81419-2 (aka Conkesta™ Soybean, described in WO2013016527 and USDA-APHIS Petition 12-272-01p), Event EE-GM3 (aka FG-072, MST-FG072-3, described in WO2011063411, USDA-APHIS Petition 09-328-01p), Event SYHTOH2 (aka OH2, SYN-ØØØH2-5, described in WO2012/082548 and 12-215-01p), Event DAS-68416-4 (aka Enlist Soybean, described in WO2011/066384 and WO2011/066360, USDA-APHIS Petition 09-349-01p), Event DAS-81615-9 (described in WO2014004458), Event DAS-44406-6 (aka Enlist E3, DAS-444Ø6-6, described in WO2012/075426 and USDA-APHIS 11-234-01p), Event MON87708 (Xtend Soybeans, described in WO2011/034704 and USDA-APHIS Petition 10-188-01p), Event MON89788 (aka Genuity Roundup Ready 2 Yield, described in WO2006/130436 and USDA-APHIS Petition 06-178-01p), Event DAS-14536-7 (described in WO2012/075429), Event 40-3-2 (aka RoundUp Ready, MON-Ø4Ø32-6, described in USDA-APHIS Petition 93-258-01), Event A2704-12 (aka LL27, ACS-GMØØ5-3, described in WO2006108674 and USDA-APHIS Petition 96-068-01p), Event 127 (aka BPS-CV127-9, described in WO2010/080829), Event A5547-127 (aka LL55, ACS-GMØØ6-4, described in WO2006108675 and in USDA-APHIS Petition 96-068-01p), Event MON87754 (aka Vistive III, MON-87754-1, described in WO2010/024976), Event HOS (aka DP-3Ø5423-1, Plenish High Oleic Soybean, described in WO2008054747), Event MON87701 (aka MON-877Ø1-2, described in WO2009064652 and USDA-APHIS Petition 09-082-01p), Event MON 87705 (aka MON-877Ø5-6, described in WO2010/037016 and USDA-APHIS Petition 09-201-01p), Event MON87712 (aka MON-87712-4, described in WO2012/051199, USDA-APHIS Petition 11-202-01p), Event pDAB4472-1606 (aka Event 1606, described in WO2012/033794), Event 3560.4.3.5 (aka DP-356043-5, described in WO2008/002872), Event MON87769 (aka MON-87769-7, described in WO2009102873 and in USDA-APHIS Petition 09-183-01p), event IND-ØØ41Ø-5 (aka HB4 Soybean, US FDA BNF No. 000155, USDA-APHIS Docket No. APHIS-2017-0075, www.aphis.usda.gov/brs/aphisdocs/17_22301p.pdf), event DP305423 (aka DP-3Ø5423-1, published PCT patent application WO2008/054747, USDA-APHIS Petition 06-354-01p), or any combination of EE-GM5 with several of these other transgenic soybean events, such as a combination of EE-GM5 with any one of the following combinations: Event MON89788×MON87708 (aka Roundup Ready 2 Xtend Soybeans, MON-877Ø8-9×MON-89788-1), Event HOS×Event 40-3-2 (aka Plenish High Oleic Soybeans×Roundup Ready Soybeans), Event EE-GM3×EE-GM2 (aka FG-072xLL55, described in WO2011063413), Event MON 87701×MON 89788 (aka Intacta RR2 Pro Soybean, MON-877Ø1-2×MON-89788-1), DAS-81419-2×DAS-44406-6 (aka Conkesta™ Enlist E3™ Soybean, DAS-81419-2×DAS-444Ø6-6), Event DAS-81419-2×Event DAS-68416-4 (described in WO2013016516), Event DAS-68416-4×Event MON 89788 (aka Enlist™ RoundUp Ready® 2 Soybean, DAS-68416-4×MON-89788-1), MON 87705×MON 89788 (aka Vistive Gold, MON-877Ø5-6×MON-89788-1), MON 87769×MON 89788 (aka Omega-3×Genuity Roundup Ready 2 Yield Soybeans, MON-87769-7×MON-89788-1), Event DP305423×Event 40-3-2 (DP-3Ø5423-1×MON-Ø4Ø32-6), Event DP305423×MON87708 (DP-3Ø5423-1×MON-877Ø8-9), Event DP305423×MON87708×Event MON89788 (DP-3Ø5423-1×MON-877Ø8-9×MON-89788-1), Event DP305423×Event MON89788 (DP-3Ø5423-1×MON-89788-1), Event MON87705×MON87708 (MON-877Ø5-6×MON-877Ø8-9), Event MON87705×MON87708×MON89788 (MON-877Ø5-6×MON-877Ø8-9×MON-89788-1), Event MON89788×MON87708×A5547-127 (MON-89788-1×MON-877Ø8-9×ACS-GMØØ6-4), Event MON87751×MON87701×MON87708×MON89788 (MON-87751-7×MON-877Ø1-2×MON-877Ø8-9×MON-89788-1).
As used in the claims below, unless otherwise clearly indicated, the term “plant” is intended to encompass plant tissues, at any stage of maturity, as well as any cells, tissues, or organs taken from or derived from any such plant, including without limitation, any seeds, leaves, stems, flowers, roots, single cells, gametes, cell cultures, tissue cultures or protoplasts.
Reference seed comprising elite event EE-GM5 was deposited at the ATCC (10801 University Blvd., Manassas, Va. 20110-2209) on Nov. 9, 2016, under ATCC accession number PTA-123625, and the viability thereof was confirmed. Alternative names for EE-GM5 are event GMB151 or BCS-GM151-6.
The above description of the invention is intended to be illustrative and not limiting.
Various changes or modifications in the embodiments described may occur to those skilled in the art. These can be made without departing from the spirit or scope of the invention.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/676,445, filed May 25, 2018, U.S. Provisional Application Ser. No. 62/685,524 filed Jun. 15, 2018, and U.S. Provisional Application Ser. No. 62/686,666, filed Jun. 18, 2018, the contents of which are herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/033992 | 5/24/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62676445 | May 2018 | US | |
| 62685524 | Jun 2018 | US | |
| 62686666 | Jun 2018 | US |
