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EP4598358A1 - Composé fongicide - Google Patents

Composé fongicide

Info

Publication number
EP4598358A1
EP4598358A1 EP23789246.8A EP23789246A EP4598358A1 EP 4598358 A1 EP4598358 A1 EP 4598358A1 EP 23789246 A EP23789246 A EP 23789246A EP 4598358 A1 EP4598358 A1 EP 4598358A1
Authority
EP
European Patent Office
Prior art keywords
compound
seq
formula
streptomyces
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23789246.8A
Other languages
German (de)
English (en)
Inventor
Stephane BIERI
Philip SIDEBOTTOM
Dimitrios PAPASOTIRIOU
Dianne IRWIN
Lauren RAY
Peter VAN DE VONDERVOORT
Leon Coulier
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syngenta Crop Protection AG Switzerland
Original Assignee
Syngenta Crop Protection AG Switzerland
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Syngenta Crop Protection AG Switzerland filed Critical Syngenta Crop Protection AG Switzerland
Publication of EP4598358A1 publication Critical patent/EP4598358A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/28—Streptomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K11/02—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof cyclic, e.g. valinomycins ; Derivatives thereof

Definitions

  • the present invention relates to a novel compound which has pesticidal activity.
  • the invention also relates to compositions comprising the compound, to a process of preparation of the compound and to the use of the compound or the compositions in agriculture or horticulture for preventing or controlling phytophatogenic infestation of plants, harvested food crops, seeds or non-living materials.
  • fungicidal activity or “fungicide” as used herein means a compound that controls, modifies, or prevents the growth of fungi.
  • fungicidally effective amount where used means the quantity of such a compound or combination of such compounds that is capable of producing an effect on the growth of fungi. Controlling or modifying effects include all deviation from natural development, such as killing, retardation and the like, and prevention includes barrier or other defensive formation in or on a plant to prevent fungal infection.
  • the microorganism in the composition or in the process of the present invention as disclosed herein is a bacterium of the genus Streptomyces, preferably the bacterium is a Streptomyces chrestomyceticus, S. rimosus, or S. paromomycinus, or S. monomicini.
  • the composition comprises Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411.
  • the microorganism is a Streptomyces sp., for instance Streptomyces sp.
  • Streptomyces sp. has a 16S RNA sequence which has at least 98%, preferably at least 98.2%, 98.4%, 98.6%, 98.8%, preferably at least 99%, 99.2%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% preferably at least 99.9%, or has 100% identity to SEQ ID NO: 1 .
  • the microorgansim in the composition or in the process according to tthe present invention for instance a Streptomyces chrestomyceticus, comprises a genome sequence which has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity or 100% identity to the whole genome of Streptomyces chrestomyceticus NRRL-3672 or to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411.
  • the composition or process according to the present invention comprises a Streptomyces chrestomyceticus which is Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411 .
  • percent identity refers to the relatedness of two or more nucleotide or amino acid sequences, which may be calculated by (i) comparing two optimally aligned sequences over a window of comparison, (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100 percent to yield the percent identity.
  • the percent identity is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present invention, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the "percent identity" for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100 percent.
  • the present invention also relates to a microorganims which is a Streptomyces sp. Saigon413 deposited with the Westerdijk Institute with accession number CBS149411 .
  • the compound of the present invention or a composition comprising the compound of the present invention can be used in the agricultural sector and related fields of use, e.g., as active ingredients for controlling phytopathogenic microorganisms.
  • the compound according to the present invention is distinguished by excellent activity at low rates of application such as from 2 to 250 ppm, for instance from 10 to 200 ppm, for instance from 20 to 100 ppm, by being well tolerated by plants and by being environmentally safe. It has very useful preventive properties and can be used for protecting numerous plants.
  • the compound of the present invention can be used to inhibit or destroy phytopathogenic microorganisms that occur on plants or parts of plants (fruit, blossoms, leaves, stems, tubers, roots) or different crops of plants. The compound may also protect those parts of the plants that grow later.
  • the compound according to the present invention and / or a composition comprising the compound according to the present invention can be used as such or formulated with an auxiliary, preferably an agricultural-acceptable auxiliary.
  • auxiliary preferably an agricultural-acceptable auxiliary.
  • Known formulations in the art are for instance emulsifiable concentratres, coatable pastes, sprayable or dilutable solutions or suspensions, powders, dusts, granulates and encapsulations.
  • auxiliaries include for example solvents, liquid carriers, solid carriers or fillers, surfactants, dispersants, emulsifiers, welters, adjuvants, solubilizers, penetration enhancers, protective colloids, adhesion agents, thickeners, humectants, repellents, attractants, feeding stimulants, compatibilizers, bactericides, anti-freezing agents, anti-foaming agents, colorants, tackifiers and binders.
  • solvents liquid carriers, solid carriers or fillers, surfactants, dispersants, emulsifiers, welters, adjuvants, solubilizers, penetration enhancers, protective colloids, adhesion agents, thickeners, humectants, repellents, attractants, feeding stimulants, compatibilizers, bactericides, anti-freezing agents, anti-foaming agents, colorants, tackifiers and binders.
  • Suitable solvents and liquid carriers include, for example water, organic solvents, oils of vegetable or animal origin, cyclic and aromatic hydrocarbons, alcohols, esters, fatty acids, a glycol or any other suitable liquid carrier known in the art.
  • the solvent or liquid carrier may be water or DMSO (dimethylsulphoxide).
  • Suitable solid carriers include, for example, talc, titanium dioxide, pyrophyllite clay, silica, attapulgite clay, kieselguhr, chalk, diatomaxeous earth, lime, calcium carbonate, bentonite clay, fuller’s earth, cotton seed hulls, wheat flour, soybean flour, pumice, wood flour, walnut shell flour and lignin.
  • An adjuvant may be a surface-active agent, crystallisation inhibitor, viscosity modifier, suspending agents, spray droplet modifiers, pigments, antioxidants, foaming agents, anti-foaming agents, light-blocking agents, compatibilizing agents, sequestering agents, neutralising agents and buffers, corrosion inhibitors, dyes, odorants, spreading agents, penetration aids, micronutrients, emollients, lubricants and sticking agents.
  • a composition as disclosed herein preferably is an agricultural-acceptable composition.
  • a compound or a composition of the present invention may be admixed with one or more additional ingredients having pesticidal activity such as fungicides, insecticides, herbicides, bactericides, acaricides, nematicides and I or the additional ingredient comprises plant growth regulators where appropriate.
  • pesticidal agents are referred to herein using their common name are known, for example, from "The Pesticide Manual", 19th Ed., British Crop Protection Council 2021 .
  • synergism corresponds to a positive value for the difference of (O-E).
  • expected activity said difference (O-E) is zero.
  • a negative value of said difference (O-E) signals a loss of activity compared to the expected activity.
  • Such advantageous properties are: more advantageous degradability; improved toxicological and/or ecotoxicological behaviour; or improved characteristics of the useful plants including: emergence, crop yields, more developed root system, tillering increase, increase in plant height, bigger leaf blade, less dead basal leaves, stronger tillers, greener leaf colour, less fertilizers needed, less seeds needed, more productive tillers, earlier flowering, early grain maturity, less plant verse (lodging), increased shoot growth, improved plant vigor, and early germination.
  • the additional ingredients having pesticidal activity and I or which is a plant growth regulator may be combined with a composition of the invention and used in a method of the invention and applied simultaneously or sequentially with a composition of the invention. When applied simultaneously, these further ingredients may be formulated together with the compositions of the invention or mixed in, for example, a spray tank. As an alternative to directly admixing these further ingredients having pesticidal activity, the components may be used in separate fungicidal, insecticidal or herbicidal applications as part of a programme of fungal, insect or herbal control spread over part or all of a growing season.
  • compositions of the invention may also be applied with one or more systemically acquired resistance inducers (“SAR” inducer).
  • SAR inducers are known and described in, for example, United States Patent No. US 6,919,298 and include, for example, salicylates and the commercial SAR inducer acibenzolar-S-methyl.
  • the compound and I or composition according to the present invention may induce resistance of a plant by a priming mechanism.
  • Priming is a mechanism which leads to a physiological state that enables plants to respond more rapidly and/or more robustly after exposure to biotic or abiotic stress as described for instance in review article: P. Aranega-Bou et. al. Priming of plant resistance by natural compounds. Hexanoic acid as a model. Front. Plant. Sci. 1 , October 2014.
  • Cyclothiazomycin C is a known compound and the structure of cyclothazomycin C is disclosed on p. 3 of WO2015191789 and can be produced as disclosed in Example 4 of WO2015/191789.
  • Malonomicin (sometimes spelt ‘malonomycin’) is ⁇ [(2S)-2-amino-3-hydroxypropanoyl]amino ⁇ ⁇ 2-
  • Malonomicin can be produced as disclosed in Example I of W02006/078939. Malonomicin may also be prepared according to the method disclosed in Example I A and B in EP 1860939. or according to Law et al, 2018 (Nature Catalys is
  • Streptimidone is a known compound of Formula III
  • Streptimidone can be synthesised following the method disclosed in Kondo, H., Oritani, T., and Kiyota, H. Synthesis and antifungal activity of the four stereoisomers of streptimidone, a glutarimide antibiotic from Streptomyces rimosus forma paromomycinus. Eur. J. Org. Chem. (20), 3459-3462 (2000).
  • the present invention also relates to a composition comprising a lipopeptide compound according to Formula (I), preferably according to Formula l(a), and malonomicin. It was surprisingly found that a composition comprising a lipopeptide compound according to Formula (I) and malonomicin can exhibit an unexpected synergistic fungicidal effect.
  • a surprising synergistic fungicidal effect of a composition comprising a lipopeptide compound according to Formula (I), preferably according to Formula l(a), and malonomicin was for instance found against Zymoseptoria tritici, Fusarium culmorum, Microdochium nivale, Botrytis cinerea, Puccinia recondita and Pyricularia oryzae.
  • the mixtures as described above can be used in a method for controlling pests, which comprises applying a composition comprising a mixture as described above to the pests or their environment, with the exception of a method for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body.
  • the process further comprises producing a composition comprising the compound according to the present invention as defined herein.
  • a microorganism able to produce the compound or composition according to the present invention is defined herein above.
  • a microorganism able to produce the compound or composition according to the present may be able to produce further active ingredients as defined herein above, for instance cyclothiazomycin C, streptimidone and / or malonomicin.
  • Applying an effective amount of the compound or composition of the invention in a method for controlling or preventing investation of a plant comprises applying from 0.01 g to 5 kg of the compound of the invention (active ingredient (a.i.) per hectare (ha), preferably from 0.015 g to 500 g a.i./ha, preferably from 0.020 g to 100g a.i./ha, preferably from 0.025 g to 50g a.i./ha, preferably from 0.030 g to 5 g a.i./ha, , preferably from 0.035 g to 500 mg a.i./ha.
  • active ingredient (a.i.) per hectare (ha) preferably from 0.015 g to 500 g a.i./ha, preferably from 0.020 g to 100g a.i./ha, preferably from 0.025 g to 50g a.i./ha, preferably from 0.030 g to 5 g a.i./ha, preferably
  • Zymoseptoria preferably the fungi belong to Blumeria graminis f.sp.tritici, Botrytis cinerea, Cercospora arachidicola, Fusarium culmorum, Glomerella lagenarium, Microdochium nivale Mycosphaerella arachidis, Parastagonospora nodorum, Puccinia recondite, Puccinia recondita f. sp. tritici, Phaeosphaeria nodorum, Pyrenophora
  • Phytopathogenic microorganisms that are found to be surpisingly affected by the compound or and I or composition according to the present invention, are fungi, for instance fungi belonging to Botrytis sp., Glomerella sp., Mycosphaerella sp., Puccinia sp., Phaeosphaeria sp. Pyrenophora sp, or Zymoseptoria sp. preferably fungi belonging to Botrytis cinerea, Glomerella lagenarium, Mycosphaerella arachidis, Puccinia recondita f. sp. tritici, Phaeosphaeria nodorum, Pyrenophora teres or Zymoseptoria tritici.
  • Controlling or preventing means reducing infestation by phytopathogenic microorganisms especially fungi, to such a level that an improvement is demonstrated.
  • a preferred method of controlling or preventing an infestation of crop plants by phytopathogenic microorganisms, especially fungi, or insects comprises the application of the compound or composition according to the present invention is foliar application.
  • the frequency of application and the rate of application will depend on the risk of infestation by the corresponding pathogen or insect.
  • the compound or composition according to the present invention can also penetrate the plant through the roots via the soil (systemic action) by drenching the locus of the plant with a liquid formulation, or by applying the compounds in solid form to the soil, e.g. in granular form (soil application). In crops of water rice such granulates can be applied to the flooded rice field.
  • the compound or composition according to the present invention may also be applied to seeds (coating) by impregnating the seeds or tubers either with a liquid formulation of the fungicide or coating them with a solid formulation.
  • the compound or composition according to the present invention as dressing agent for the treatment of plant propagation material, e.g., seed, such as fruits, tubers or grains, or plant cuttings, for the protection against fungal infections as well as against phytopathogenic fungi occurring in the soil.
  • plant propagation material e.g., seed, such as fruits, tubers or grains, or plant cuttings
  • the propagation material can be treated with a compound and / or a composition according to the present invention before planting: seed, for example, can be dressed before being sown.
  • the compound and I or composition according to the present invention can also be applied to grains (coating), either by impregnating the seeds in a liquid formulation or by coating them with a solid formulation.
  • the composition can also be applied to the planting site when the propagation material is being planted, for example, to the seed furrow during sowing. Disclosed herein are such methods of treating plant propagation material and the plant propagation material so treated.
  • locus means fields in or on which plants are growing, or where seeds of cultivated plants are sown, or where seed will be placed into the soil. It includes soil, seeds, and seedlings, as well as established vegetation.
  • plants refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage, and fruits. Germinated plants and young plants which are to be transplanted after germination or after emergence from the soil, may also be mentioned. These young plants can be protected before transplantation by a total or partial treatment by immersion.
  • plant propagation material is understood to denote generative parts of the plant, such as seeds, which can be used for the multiplication of the latter, and vegetative material, such as cuttings or tubers, (for example potatoes), roots, fruits, bulbs, rhizomes or parts of plants.
  • plants involve “useful plants” or “crops”.
  • “Useful plants” and “crops” comprise perennial and annual crops, such as berry plants for example blackberries, blueberries, cranberries, raspberries and strawberries; cereals for example barley, maize (corn), millet, oats, rice, rye, sorghum triticale and wheat; fibre plants for example cotton, flax, hemp, jute and sisal; field crops for example sugar and fodder beet, coffee, hops, mustard, oilseed rape (canola), poppy, sugar cane, sunflower, tea and tobacco; fruit trees for example apple, apricot, avocado, banana, cherry, citrus, nectarine, peach, pear and plum; grasses for example Bermuda grass, bluegrass, bentgrass, centipede grass, fescue, ryegrass, St.
  • plants also includes wood crops, such as pine trees, or woody plants.
  • useful plants is to be understood as also including useful plants that have been rendered tolerant to herbicides like bromoxynil or classes of herbicides (such as, for example, HPPD inhibitors, ALS inhibitors, for example primisulfuron, prosulfuron and trifloxysulfuron, EPSPS (5-enol- pyrovyl-shikimate-3-phosphate-synthase) inhibitors, GS (glutamine synthetase) inhibitors or PPO (protoporphyrinogen-oxidase) inhibitors) as a result of conventional methods of breeding or genetic engineering.
  • herbicides like bromoxynil or classes of herbicides
  • ALS inhibitors for example primisulfuron, prosulfuron and trifloxysulfuron
  • EPSPS (5-enol- pyrovyl-shikimate-3-phosphate-synthase) inhibitors
  • GS glutamine synthetase
  • PPO protop
  • useful plants is to be understood as also including useful plants which have been so transformed by the use of recombinant DNA techniques that they are capable of synthesising one or more selectively acting toxins, such as are known, for example, from toxin-producing bacteria, especially those of the genus Bacillus.
  • any suitable plant, plant propagation material orfood crop may be treated in a method according according to the present invention as defined herein.
  • the plant, plant propagation material or food crop comprises or is potato, tomato, grape, canola/oilseed rape/colza, cucurbits, groundnut, wheat, barley, corn, rice banana soya, preferably the plant is wheat or barley.
  • the invention relates to the use of a compound or a composition according to the present invention as a presticide, preferably as a fungicide, and / or as a priming agent.
  • a compound or a composition according to the present invention as a presticide, preferably as a fungicide, and / or as a priming agent.
  • the features related to the compound and composition according to the present invention are as disclosed herein above. Accordingly, the present invention relates to a method for using a compound and I or composition according to the present invention as a fungicide.
  • FIG. 1 Spectrum of light absorption (UV-VIS) 200-400nm of a compound according to Formula I (a), Formula I (b) or Lipopetin
  • Streptomyces species were ordered from culture collections disclosed in Table 1. Streptomyces sp. Saigon413 was isolated in Vietnam before 1961. Streptomyces sp. Saigon413 was deposited at the Westerdijk institute under accession number CBS149411. The deposit was made by Syngenta Ltd., Jealott’s Hill Research International Centre, Bracknell, Berkshire, RG42 6EY, UK under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
  • Genomic DNA was isolated from Streptomyces sp. Saigon413 using the method described in Kutchma et al. (1998) Biotechniques 24(3):452-457.
  • the 16S rRNA gene was amplified using universal 16S primers and sequenced using Sanger sequencing.
  • the 16S rRNA of Streptomyces sp. Saigon413 is shown in SEQ ID NO: 1 .
  • the species of strain Streptomyces sp. Saigon413 was identified by comparing the 16 S rRNA sequence according to SEQ ID NO:1 with publicly available 16S rRNA sequences that were extracted using whole genome sequence assembly of genomes from Streptomyces species (based on The Genome Taxonomy Database GTDB (Parks, D.H., et al. (2021). GTDB: Nucleic Acids Research, 50: D785-D794) using barrnap v0.9. Based on this comparative analysis Streptomyces sp. Saigon413 was identified as a Streptomyces chrestomyceticus species. The sequence identity between the 16S rRNA sequence of Streptomyces sp. Saigon413 and the publicly available S. chrestomyceticus NRRL-3672 was 99.87%, which was determined using Muscle v3.8.31 and R package Seqinr v4.2-16.
  • Genomic DNA was also extracted from Streptomyces rimosus CBS 492.64, Streptomyces rimosus CBS 570.66, Streptomyces rimosus CBS 569.66, Streptomyces chrestomyceticus DSM 41224, Streptomyces rimosus subsp. rimosus DSM 40673, and Streptomyces rimosus subsp.
  • rimosus DSM 41057 using a method described in Kieser et. al., (2000) Practical Streptomyces Genetics. Whole genome sequencing for these strains was completed using Nanopore Sequencing technology and the genomes were assembled with Flye (Kolmogorov, M., et. al., (2019), Nature Biotechnology, 37, 540). Following assembly of the genome from Streptomyces sp. Saigon 413 and publicly available genomes, the average nucleotide identity (ANI) was calculated between Streptomyces sp. Saigon413 and closely related Streptomyces strains using fastANI (Jain, C., et. al. (2016), Nature Communications, 9, 5114) (Table 1).
  • ANI average nucleotide identity
  • lipopeptide compound being a lipopeptide family (see 1 above, and the AntiSMASH output we were able to deduce that the lipopeptide compound is produced by a non- ribosomal peptide synthetase (NRPS gene cluster).
  • NRPS gene cluster responsible for the biosynthesis of the lipoeptide compound was based upon the structural analysis of the compound and the amino acids that are incorporated into the depsipeptide core of the lipopeptide compound.
  • the NRPS biosynthetic gene cluster contains 45 coding sequences including two coding sequences for NRPS genes, a coding sequence for a regulator, and coding sequences responsible for the biosynthesis of a precursor incorporated into lipopeptide of Formula I ( Figure 6 and Table 4).
  • Table 4 Coding sequences present in NRPS biosynthetic gene cluster responsible for the production of the lipopeptide according to Formula I. Annotations provided are based upon pBLAST search using the non-redundant protein sequences on the National Centre for Biotechnology Information database. .4. Deletion of genomic region including CDS_21 (SEQ ID NO: 22) and CDS_22 (SEQ ID NO:3) from Streptomyces sp.
  • Plasmid pBCon2192 was used to transform E. coli ET12567/pUZ8002 using a standard electroporation method, and then introduced into Streptomyces sp. Saigon413 by mycelial conjugation (T. Kieser et. al., Practical Streptomyces Genetics, 2000, John Innes Foundation, Norwich). Thiostrepton resistant colonies were patched on ISP-4 agar media supplemented with 40 pg/ml thiostrepton and 25 pg/ml nalidixic acid. These patches were initially incubated for 6 days at 28 °C allowing plasmid replication.
  • strains were re-patched on ISP-4 agar media supplemented with 40 pg/ml thiostrepton and incubated at 37°C for further 6 days to force primary integration. After 6 days at 37°C, the obtained strains were transferred onto ISP-4 solid agar media without selection and incubated for 15 days at 28°C to allow a second crossover.
  • strains were collected in 20 % glycerol. 100 pl of the cell suspension was used to inoculate fresh plates, as well as to make serial dilutions up to 10 -10 . 100 pl of 10 -8 to 10 -10 were then plated onto ISP-4 agar plate. Plates were incubated at 28°C until single colonies were observed.
  • the mycelia from fermentation broth from Streptomyces strains disclosed in Table 1 were separated via centrifugation and the supernatant was treated with butanol.
  • the butanol was removed and the extract partitioned between water and ethyl acetate.
  • the lipopeptides were purified from the ethyl acetate fraction by preparative reverse phase (C18) HPLC.
  • the lipopeptides were relatively aploar and elute in the higher organic fraction in a gradient system with 0.1 % formic acid and acetonirile (0.1 % formic acid).
  • a gradient of 60% Aqueous to 40% Aqueous with the above solvents allowed separation of a compound according to Formula l(a) and Formula l(b).
  • the fermentation broth of Streptomyces sp. Saigon413 was processed.
  • a stock solution for the compound of Formula l(a) and Formula l(b) was produced in DMSO (max. 10 mg/ml), which was further diluted with water plus 0.025% Tween20 to produce suitable working concentrations for biological efficacy assays.
  • Compounds were detected by UV-VIS ( Figure 1), mass spectrometry ( Figures 2 and 3) and NMR spectrometry ( Figures 4 ad 5).
  • Lipopeptin A was purchased from Fundacion MEDINA, Centro de Excelencia en Investigation de Medicamentos Innovadores en Andalucia, Avda. del Conocimiento 34, Edificio Centro de Desarrollo Farmaceutico y Alimentario, Parque Tecnologico de Ciencias de la Salud, 18016 Granada (ESPANA).
  • MS OT Orbitrap Resolution: 50,000, Scan Range (m/z): 200 to 2000, RF Lens (%): 60, AGC Target: Standard, Maximum Injection Time Mode: Auto, Microscans: 1 , Data Type: Profile, Polarity: Both),
  • Experiment 3 tMS2 OT HCD (MSn Level (n): 2, Isolation Window (m/z): 1.0, Activation Type: HCD, HCD Collision Energy (%): 30, Detector Type: Orbitrap, Orbitrap Resolution: 30,000, RF Lens (%): 60, Polarity: Positive),
  • Experiment 4 tMS3 OT HCD (MSn Level (n): 3, Isolation Window (m/z): 1.6, Activation Type: HCD, HCD Collision Energy (%): 30, MS2 Isolation Window (m/z): 2, MS2 Activation Type: HCD, MS2 HCD Collision Energy (%): 30, Detector Type: Orbitrap, Orbitrap Resolution: 30,000, RF Lens (%): 60, Polarity: Positive).
  • the mass spectrometer was connected to a Vanquish Flex UHPLC from Thermo Scientific using a Vanquish Split Sampler FT, Vanquish Binary Pump F, Vanquish Column Compartment H, Vanquish Diode Array Detector FG and Vanquish Charged Aerosol Detector.
  • Figures 2 and 3 show LC-ESI-MS/MS/MS spectra of a compound of Formula I (a).
  • a compound according to Formula 1(a) and Formula 1(b) was identified in the fermentation broth of Streptomyces sp. Saigon413 deposited under accession number CBS 149411 .
  • NMR spectra were recorded on a Bruker AVIII 600 NMR spectrometer, equipped with a 5 mm Bruker (1H/ 19 F)/ 13 C/ 15 N TCI cryoprobe fitted with Z gradients, using standard Bruker pulse sequences. Samples were dissolved in CD3OD, and the spectra were recorded at 300° K and referenced to the residual solvent signal at 3.31 ppm for 1 H. Figures 4 and 5 each cover half of the 1 H NMR spectrum of a compound according to Formula 1 (a).
  • the molecular composition and mass of a compound according to Formula l(a) and Formula l(b) was determined using the results of liquid chromatography and high-resolution mass spectrometry as disclosed in 2.1.
  • the compounds of Formula l(a) and l(b) have the following composition.
  • Mycelia fragments or conidia suspensions of a fungus prepared either freshly from liquid cultures of the fungus or from cryogenic storage, were directly mixed into nutrient broth.
  • a stock solution for compound of Formula l(a) and Formula l(b) was produced in DMSO (max. 10 mg/ml), which was diluted with water plus 0.025% Tween®20 to produce a 10x concentrated sample and 10 pl of this solution was pipetted into a microtiter plate (96-well format). The nutrient broth containing the fungal spores/mycelia fragments was then added to give an end concentration of the tested compound. The test plates were incubated in the dark at 24°C and 96% rh. The inhibition of fungal growth was determined photometrically after 2 - 7 days, depending on the pathosystem, and percent antifungal activity relative to the untreated check was calculated. The effect of the test compound (Formula 1(a), 1(b) and Lipopeptin A) was tested against the following fungi under the conditions as outlined above and specifically herein below:
  • Botryotinia fuckeliana Botrytis cinerea
  • liquid culture Gram mould
  • Conidia of the fungus from cryogenic storage were directly mixed into nutrient broth (Vogels broth). The inhibition of growth is determined photometrically 3-4 days after application.
  • Glomerella laqenarium (Colletotrichum laqenarium) / liquid culture (Anthracnose)
  • Conidia of the fungus from cryogenic storage were directly mixed into nutrient broth (PDB potato dextrose broth). The inhibition of growth was measured photometrically 3-4 days after application.
  • Conidia of the fungus from cryogenic storage were directly mixed into nutrient broth (PDB potato dextrose broth). The inhibition of growth was determined photometrically 4-5 days after application.
  • Table 2 Control of fungal development by a compound according to Formula 1(a) and 1(b) in liquid culture.
  • Table 3 Control of fungal development by a compound according to Formula 1(a) and 1(b) in a liquid culture.
  • Formula l(a) and Formula l(b) have similar efficacy, while Lipopeptin A was less active.
  • Wheat leaf segments cv. Kanzler were placed on agar in multiwell plates (24-well format) and sprayed with the formulated test compound diluted in water.
  • the leaf disks were inoculated with a spore suspension of the fungus 1 day after application.
  • the inoculated leaf segments were incubated at 19°C and 75% rh under a light regime of 12 h light / 12 h darkness in a climate cabinet and the activity of a compound was assessed as percent disease control compared to untreated when an appropriate level of disease damage appears in untreated check leaf segments (7 - 9 days after application).
  • Phaeosphaeria nodorum (Septoria nodorum) /wheat / leaf disc (Glume blotch)
  • Barley leaf segments cv. Hasso were placed on agar in a multiwell plate (24-well format) and sprayed with the formulated test compound diluted in water.
  • the leaf segmens were inoculated with a spore suspension of the fungus 2 days after application.
  • the inoculated leaf segments were incubated at 20°C and 65% rh under a light regime of 12 h light / 12 h darkness in a climate cabinet and the activity of a compound was assessed as disease control compared to untreated when an appropriate level of disease damage appears in untreated check leaf segments (5 - 7 days after application).
  • the stock solution was then further diluted in water supplemented with Tween®20 to a final concentration of 200ppm or higher of test compound, 0.05% Tween®20 and 2% DMSO.
  • the diluted compound was applied to the middle segment of L2 using a conventional cotton stick; the cotton stick was soaked in the diluted compound and rubbed several times on the adaxial leaf surface between the two marks. 1 day later, the complete plant was inoculated with a fungal spore suspension using a paint brush and application of the spore suspension until before run-off.
  • test plants were inoculated by spraying a spore suspension on them 1 day after application (1 ,5Mio spores per ml in water supplemented with 0.01 % Tween®20). After an incubation period of 4 days at 22°C/21 °C (day/night) and 95% rh, the inoculated test plants were kept at 22°C/21°C (day/night) and 70% rh in a greenhouse. Efficacy was assessed by visual evaluation directly when an appropriate level of disease appears on untreated check plants (usually >80% disease cover at 16 - 19 days after inoculation).
  • test plants were inoculated by spraying them with a spore suspension 1 day after application (spore suspension at 80’000 spores per ml in water supplemented with Tween®20 at 0.1 %). After an incubation period of 1 day at 20° C and 95% rh, the inoculated test plants were kept at 20° C and 60% rh in a greenhouse. The percentage leaf area covered by disease was assessed by visual evaluation when an appropriate level of disease appeared on untreated check plants (usually 50-80% disease cover at 9 - 12 days after infection).
  • test solution 200 pL of the test solution (analyte) or the respective control (water) in the appropriate concentration was pipetted into a white 96-well plate (Nunc, Langenselbold, Germany). 5 mm leaf discs from 2-week- old wheat plants were obtained using a tissue punch and subsequently floated on the test solution. The plates were stored for 24 h at Room Temperature (RT). The next day, the solution was replaced with 50 pL ddbW and leaf discs were left for regeneration for at least one hour at RT in the dark. Meanwhile appropriate master mixes, either with or without the elicitor flg22 (see below), were freshly prepared in black 5 mL reaction tubes.
  • INA 2.6-dichloro-isonicotinic acid, CAS: 5398- 44-7) is a synthetic salicylic acid analog and was included as a reference for priming activity (Kauss et al., 1992).
  • Table 7 demonstrate that wheat pretreated with a compound of Formula l(a) or Formula l(b) in a concentration of 100 ppm increased ROS production induced by the peptide flg22 by a factor of 2.7 or higher. This is a response that is similar to or stronger than the response observed from a treatment with INA (2.6-dichloro-isonicotinic acid), a well-known priming agent (Krauss et al., 1992: Dichloroisonicotinic and salicylic acid, inducers of systemic acquired resistance, enhance fungal elicitor responses in parsley cells, Plant Journal 2:655-60).
  • INA 2.6-dichloro-isonicotinic acid
  • Example 4 Fungicidal activity of a mixture of a lipopeptide according to Formula l(a) and malonomicin in liquid culture assays
  • Table 9 Mixture ratio of two compounds in the 96well plate assay design as outlined in Table 8. The number represents the ratio of compound 1 : compound 2 .
  • Compound 1 is malonomicin
  • compound 2 is the lipopeptyide compound of Formula 1 (a).
  • the plate design spans a wide range from 641 :1 to 1 :100.
  • Conidia of the fungus from cryogenic storage were directly mixed into nutrient broth (PDB: potato dextrose broth). The test plates were incubated at 24°C and the inhibition of growth was determined photometrically after 72 hrs.
  • PDB potato dextrose broth
  • Cercospora arachidicola (EPPO code: MYCOAR) Conidia of the fungus from cryogenic storage were directly mixed into nutrient broth (PDB: potato dextrose broth). The test plates were incubated at 24°C and the inhibition of growth was determined photometrically after 5-6 days.
  • Mycelial fragments of the fungus prepared from a fresh liquid culture were directly mixed into nutrient broth (PDB: potato dextrose broth). The test plates were incubated at 24°C and the inhibition of growth was determined photometrically after 72 hrs.
  • PDB potato dextrose broth
  • Table 10 Control of Zymoseptoria tritici for solo compounds and mixtures.
  • the plate design including the concentration of compound of Formula l(a) and malonomicin are indicated in Table 8. Values indicate control of fungal growth (% reduction of growth in the test well as compared to untreated check).
  • Table 11 Comparison of measured values for disease control of Zymoseptoria tritici (as reported in Table 10) with calculated values using the formula from Colby for the same mixture. Numbers reported in the table represent the difference from measured efficacy (in %) minus calculated efficacy (in %). Values around 0 (zero) indicate additive activity, while positive values suggest synergistic activity.
  • Table 13 Comparison of measured values for disease control of Fusarium culmorum (as reported in Table 12) with calculated values using the formula from Colby for the same mixture. Numbers reported in the table represent the difference from measured efficacy (in %) minus calculated efficacy (in %). Values around 0 (zero) indicate additive activity, while positive values suggest synergistic activity.
  • Table 14 Control of Microdochium nivale for solo compounds and mixtures.
  • the plate design including the concentration of compound of Formula l(a) and malonomicin are indicated in Table 8. Values indicate control of fungal growth (% reduction of growth in the test well as compared to untreated check)
  • the respective mixture ratio can be assigned from Table 9. Effective mixtures for the control of Microdochium nivale were found from 641 :1 to 1 :3.
  • Table 15 Comparison of measured values for disease control of Microdochium nivale (as reported in Table 14) with calculated values using the formula from Colby for the same mixture. Numbers reported in the table represent the difference from measured efficacy (in %) minus calculated efficacy (in %). Values around 0 (zero) indicate additive activity, while positive values suggest synergistic activity.
  • Table 17 Comparison of measured values for disease control of Botrytis cinerea (as reported in Table 9) with calculated values using the formula from Colby for the same mixture. Numbers reported in the table represent the difference from measured efficacy (in %) minus calculated efficacy (in %). Values around 0 (zero) indicate additive activity, while positive values suggest synergistic activity.
  • Table 18 Control of Pyricularia oryzae for solo compounds and mixtures.
  • the plate design including the concentration of the lipopeptide compound of Formula l(a) and malonomicin are indicated in Table 8. Values indicate control of fungal growth (% reduction of growth in the test well as compared to untreated check)
  • Table 19 Comparison of measured values for disease control of Pyricularia oryzae (as reported in Table 18) with calculated values using the formula from Colby for the same mixture. Numbers reported in the table represent the difference from measured efficacy (in %) minus calculated efficacy (in %). Values around 0 (zero) indicate additive activity, while positive values suggest synergistic activity.
  • Table 20 Control of Cercospora arachidicola for solo compounds and mixtures.
  • the plate design including the concentration of compound of Formula l(a) and malonomicin are indicated in Table 8. Values indicate control of fungal growth (% reduction of growth in the test well as compared to untreated check)
  • Table 21 Control of Sclerotinia sclerotiorum for solo compounds and mixtures.
  • the plate design including the concentration of compound of Formula l(a) and malonomicin are indicated in Table 8. Values indicate control of fungal growth (% reduction of growth in the test well as compared to untreated check)
  • a mixture of the lipopeptide compound of Formula l(a) and malonomicin controls various fungal pathogens.
  • a large variety of mixture ratios of the two compounds produces 50% or more control of the fungal growth, ranging from 641 :1 to 1 :100, for several fungal species, including examples with a ratio in between the mentioned ratios (ratio of compounds malonomicin: compound of Formulal(a)) .
  • the efficacy of many mixtures is better than the efficacy predicted based on the calculation from Colby, indicating that the mixture of the lipopeptide compound of Formulal(a) with malonomicin has a synergistic effect on the control of fungal pathogens.
  • This surprising synergistic effect was observed in tests to control Zymoseptoria tritici, Fusarium culmorum, Microdochium nivale, Botrytis cinerea and Pyricularia oryzae.
  • Example 5 Fungicidal activity of a mixture of the lipopeptide compounds according to Formula l(a) and malonomicin in leaf disc assays
  • a stock solution for the lipopeptide compound of Formula l(a) was produced in DMSO (max. 10 mg/ml).
  • Malonomicin was produced according to Law et al, 2018 (Nature Catalys is
  • a stock solution of malonomicin was produced in water plus 0.025% Tween®20.
  • Assays to test efficacy of mixtures of compound of Formula l(a) in combination with malonomicin on the control of fungal pathogens in a leaf disc assay were designed for two 24well plates. Table 22 and Table 23. Outline of 24well testplate(l) and testplate(2) including concentration of compounds sprayed per well. The upper number in the cell indicates compound of Formula 1(a) concentration (ppm), the lower number malonomicin concentration (ppm). Column (1) holds a dilution series of compound of Formula l(a), row (2-D) hold a dilution series of malonomicin. Well 2-D-(1) represents an untreated check. This design was applied for tests including Puccinia recondita (EPPO code: PUCCRE) with preventive and curative spray timing.
  • EPPO code Puccinia recondita
  • leaf segments were infected with fungal spores to obtain a preventive application timing.
  • Alternativeley leaf segments infected one day before spray of the compounds were used, resulting in a curative spray timing.
  • several plates were produced where leaf segments were sprayed 2x in absence of test compound (with only DMSO and Tween20), representing the untreated check samples. For each leaf segment percent leaf coverage of disease symptoms was assessed. Percent leaf coverage reduction relative to the untreated check was calculated. Efficacy of the mixtures was tested in duplicate and on different fungal species. Reported efficacy values are the average of two replicate results.
  • Puccinia recondita EPPO code: PUCCRE

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Abstract

La présente invention concerne un composé de formule (I) et une composition le contenant, un procédé de production du composé et un procédé d'utilisation du composé et des compositions pour la prévention ou la lutte contre des champignons dans des plantes.
EP23789246.8A 2022-10-07 2023-10-05 Composé fongicide Pending EP4598358A1 (fr)

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US5356624A (en) 1993-04-23 1994-10-18 The United States Of America As Represented By The Secretary Of Agriculture Biological treatment for controlling wood deteriorating fungi
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US7887827B2 (en) 2005-01-21 2011-02-15 Dow Agrosciences Llc Use of malonomicin and analogs in fungicidal applications
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CA2951565A1 (fr) 2014-06-10 2015-12-17 The Board Of Trustees Of The University Of Illinois Criblage fonde sur la reactivite utilisable en vue de la decouverte de produits naturels
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