Fungicidal compounds

Abstract

The present invention relates to a compound of formula (I) and a composition containing the compound, a method of producing the compound and a method of preventing or controlling fungi in plants using the compound and the composition. # imgabs0 #

Description

[0001] The present invention relates to novel compounds having pesticidal activity, compositions comprising the compounds, methods for preparing the compounds and the use of the compounds or the compositions in agriculture or horticulture for preventing or controlling phytopathogenic infestations of plants, harvested food crops, seeds or non-living materials. Background Art

[0002] Fungicides are widely used in agriculture to protect plants from damage caused by fungi. Fungicides can come from chemical or biological sources. Since chemical fungicides have some negative effects on the environment, there is a growing demand for fungicides of biological origin (e.g., microbial origin). Known microorganisms that produce antifungal antibiotics are actinomycetes, such as Streptomyces sp. A well-known species is Streptomyces natalensis, which produces the antifungal compound natamycin for food and crop protection. In US 5,356,624, a strain of Streptomyces rimosus is disclosed, which is found to be active against several wood-degrading fungi. In WO 2022 / 038180, a new species of Streptomyces is disclosed, which produces several known antifungal compounds, such as streptoglutarimide, natamycin (pimaricin) or white fungi. Extracts of these bacterial strains were found to be active against well-known plant pests such as Fusarium graminearum, Zymoseptoriatritici and Puccinia striiformis.

[0003] Another compound produced by Streptomyces sp. No. AC-69 is the lipopeptide antibiotic (Lipopeptin) A, which is known to be active against some plant pathogenic fungi (Tsuda, Suzuki (1980), The Journal of Antibiotics, Vol. 33, No. 2, pp. 247-248).

[0004] The development of fungal resistance to fungicides, coupled with government regulation and societal pressure, has led to a continued search for new compounds with fungicidal activity from biological sources. Summary of the invention

[0005] The present invention relates to a compound according to formula (I)

[0006]

[0007] or a salt thereof, wherein R1=CH3 or C2H5.

[0008] Surprisingly, it has been found that the novel compounds according to the present invention have a surprising level of biological activity against preventing or controlling plant pathogenic microorganisms such as fungi. As used herein, biological activity includes fungicidal activity.

[0009] In a second aspect, the present invention relates to a composition comprising a compound according to the invention and a microorganism capable of producing a compound according to the invention.

[0010] In a third aspect, the invention relates to a method for producing a compound or composition according to the invention, the method comprising culturing a microorganism in a suitable fermentation medium under conditions allowing the production of the compound.

[0011] In a fourth aspect, the present invention relates to a method for controlling or preventing infection of plants by phytopathogenic microorganisms, wherein an effective amount of a compound or salt thereof according to the present invention disclosed herein, or a composition according to the present invention is applied to the plant, its part or its locus.

[0012] According to a fifth aspect of the present invention there is provided the use of a compound or composition according to the present invention as a pesticidal agent, preferably a fungicide. According to this aspect of the present invention the use does not include a method of treating the human or animal body by surgery or therapy. DETAILED DESCRIPTION

[0013] The present invention relates to a compound according to formula (I)

[0014]

[0015] or a salt thereof, wherein R1 is CH3 or C2H5.

[0016] The compounds according to the invention comprise or are lipopeptides.

[0017] Therefore, the compounds according to the present invention comprise a compound according to formula I(a) and / or a compound according to formula I(b) or a salt thereof.

[0018] The compound according to formula I(a) comprises the structural formula

[0019]

[0020] or a salt thereof.

[0021] The compound according to formula I(a) comprises the molecular formula C 55 H 85 N11O 19, accurate mass is 1203.602 g. The solubility of the compound according to formula I(a) in DMSO is higher than 10,000 ppm. The compound according to formula I(a) comprises or is a lipopeptide.

[0022] The compound according to formula I(b) comprises the structural formula

[0023]

[0024] or a salt thereof.

[0025] The compound of formula I(b) comprises or has the molecular formula C 56 H 87 N 11 O 19 , with an exact mass of 1217.618 g. The solubility of the compound according to formula I(a) in DMSO is higher than 10,000 ppm. The compound according to formula I(b) comprises or is a lipopeptide.

[0026] In a preferred embodiment, the compound according to formula I is an isolated compound. The wording "isolated" with respect to a compound means that the compound has been separated from its natural environment.

[0027] In one aspect, the present invention relates to a composition comprising a compound according to the present invention and a microorganism capable of producing a compound disclosed herein. The compound or a composition comprising a compound according to the present invention and a microorganism capable of producing a compound according to the present invention is applied to a plant or part thereof to treat or protect the plant against diseases caused by plant pathogenic microorganisms (e.g., fungi, bacteria or viruses).

[0028] Surprisingly, it has now been found that the compounds and / or compositions according to the invention have a favourable level of biological activity for treating or protecting plants against diseases caused by infection with phytopathogenic microorganisms (e.g. fungi, bacteria or viruses). Surprisingly, the compounds and / or compositions according to the invention have a favourable fungicidal activity against various phytopathogenic fungi.

[0029] Preferably, the compounds and / or compositions according to the invention have fungicidal activity. Therefore, the compounds and / or compositions according to the invention are preferably fungicides.

[0030] As used herein, the term "compound having fungicidal activity" or "fungicide" means a compound that controls, modifies, or prevents the growth of fungi. The term "fungicidally effective amount" when used means an amount 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 deviations from natural development, such as killing, retardation, etc., and prevention includes the formation of a barrier or other defense in or on a plant to prevent fungal infection.

[0031] The compounds and / or compositions according to the invention may be produced in any suitable manner, preferably, the compounds and / or compositions of the invention are produced by culturing a microorganism in a suitable fermentation medium that allows the production of the compounds and / or compositions of the invention. The microorganism is preferably a Streptomyces species.

[0032] The microorganism capable of producing a compound or composition according to the invention comprises or is a microorganism containing at least one nucleotide sequence encoding a protein that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to the amino acid sequence according to SEQ ID NO: 47 to 91, preferably SEQ ID NO: 67 and / or SEQ ID NO: 68.

[0033] Preferably, the microorganism comprises a nucleotide sequence encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, at least twenty-six, at least twenty-seven, at least twenty-eight, at least twenty-nine, at least thirty, at least thirty-one, at least thirty-two, at least thirty-three, at least thirty-four, at least thirty-five, at least thirty-six, at least thirty-seven, at least thirty-eight, at least thirty-nine, at least forty, at least forty-one, at least forty-two, at least forty-three, at least forty-four, at least forty-five of the following amino acid sequences: SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 or SEQ ID NO:91, preferably SEQ ID NO:67 and / or SEQ ID NO:68, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto.

[0034] Microorganisms capable of producing compounds or compositions according to the present invention include microorganisms containing at least one nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity with the nucleotide sequence of SEQ ID NO: 2 to 46, preferably SEQ ID NO: 22 and / or SEQ ID NO: 23.

[0035] Preferably, the microorganism comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, at least twenty-five, at least twenty-six, at least twenty-seven, at least twenty-eight, at least twenty-nine, at least thirty, at least thirty-one, at least thirty-two, at least thirty-three, at least thirty-four, at least thirty-five, at least thirty-six, at least thirty-seven, at least thirty-eight, at least thirty-nine, at least forty, at least forty-one, at least forty-two, at least forty-three, at least forty-four, at least forty-five of the following nucleotide sequences: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 or SEQ ID NO:46 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0036] The microorganism that can produce the compound according to the present invention can be a naturally occurring microorganism or a recombinant microorganism. Recombinant microorganisms can be produced by methods known to those skilled in the art. Recombinant microorganisms can be produced by transforming microorganisms with the following nucleotide sequence: at least one of the nucleotide sequences encoding the protein of at least one of the amino acid sequences of SEQ ID NO:47-91, preferably SEQ ID NO:67 and SEQ ID NO:68, preferably at least one of the nucleotide sequences of SEQ ID NO:2 to 47, preferably at least one of the nucleotide sequences of SEQ ID NO:22 or 23, or a nucleotide sequence having at least 80% identity thereto, preferably at least 85%, preferably at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, preferably at least 99% identity thereto.

[0037] Preferably, the microorganism in the composition or method of the present invention disclosed herein is a bacterium of the genus Streptomyces, preferably the bacterium is Streptomyces chrestomyceticus, Streptomyces chrestomyceticus, Streptomyces paromomycinus or Streptomyces monomicini. Preferably, the composition comprises the Streptomyces species Saigon 413 deposited in the Fungal Culture Collection under the accession number CBS149411. Preferably, the microorganism is a Streptomyces species, for example, the Streptomyces species Saigon 413 deposited in the Fungal Culture Collection under the accession number CBS149411, wherein the Streptomyces species has a 16S RNA sequence that is 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 100% identical to SEQ ID NO:1.

[0038] Preferably, the microorganism (e.g., Streptomyces coronamicinus) in the composition or method according to the present invention comprises a genomic sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical or 100% identical to the whole genome of Streptomyces coronamicinus NRRL-3672 or the whole genome of Streptomyces species Saigon 413 deposited in the Fungal Culture Collection under the accession number CBS149411. In one embodiment, the composition or method according to the present invention comprises Streptomyces coronamicinus, which is Streptomyces species Saigon 413 deposited in the Fungal Culture Collection under the accession number CBS149411.

[0039] As used herein, the terms "percent identity" and "percent identical" refer to the relatedness of two or more nucleotide or amino acid sequences, which can be calculated by: (i) comparing two optimally aligned sequences over a comparison window, (ii) determining the number of positions at which the same nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) is present in the two sequences to produce the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the comparison window, and then (iv) multiplying the quotient by 100% to obtain the percent identity. If "percent identity" is calculated relative to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matched positions over the alignment region by the total length of the reference sequence. Therefore, for the purposes of the present invention, when two sequences (query sequence and subject sequence) are optimally aligned (allowing gaps in the alignment), the "percent identity" of 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 comparison window), and then multiplied by 100%.

[0040] The present invention also relates to a microorganism, which is the Streptomyces species Saigon 413 deposited in the Fungal Culture Collection Center with the deposit number CBS149411.

[0041] Surprisingly, it was found that the Streptomyces sp. Saigon 413 deposited with the Fungal Culture Collection under the accession number CBS 149411 has advantageous properties compared to the Streptomyces sp. known in the art.

[0042] Compound of the present invention or the composition comprising compound of the present invention can be used for agricultural sector and relevant use field, for example as the active ingredient of controlling plant pathogenic microorganisms.The feature of compound according to the present invention is that there is excellent activity under low application rate (for example 2 to 250ppm, for example 10 to 200ppm, for example 20 to 100ppm), plant tolerance is good and environmentally safe.It has very useful preventive characteristics and can be used for protecting many plants.Compound of the present invention can be used for suppressing or destroying plant pathogenic microorganisms occurring in different crops of plant or part (fruit, flower, leaf, stem, tuber, root) of plant.This compound can also protect those parts that plant grows later.

[0043] The compound according to the invention and / or the composition comprising the compound according to the invention can be used as is or formulated with an adjuvant (preferably an agriculturally acceptable adjuvant) for use. Formulations known in the art are, for example, emulsifiable concentrates, coatable pastes, sprayable or dilutable solutions or suspensions, powders, dusts, granules and encapsulates.

[0044] Therefore, in one embodiment, the composition disclosed herein comprising the compound according to the present invention further comprises an adjuvant. Preferably, the adjuvant is an agriculturally acceptable adjuvant.

[0045] Suitable adjuvants are known in the art and include, for example, solvents, liquid carriers, solid carriers or fillers, surfactants, dispersants, emulsifiers, wetting agents, adjuvants, solubilizers, penetration enhancers, protective colloids, adhesives, thickeners, wetting agents, repellents, attractants, feeding stimulants, extenders, bactericides, antifreeze agents, defoamers, colorants, tackifiers and adhesives.

[0046] Suitable solvents and liquid vehicles include, for example, water, organic solvents, oils of plant or animal origin, cyclic and aromatic hydrocarbons, alcohols, esters, fatty acids, glycols or any other suitable liquid vehicle known in the art. The solvent or liquid vehicle may be water or DMSO (dimethyl sulfoxide).

[0047] Suitable solid carriers include, for example, talc, titanium dioxide, pyrophyllite clay, silica, attapulgite clay, kieselguhr, chalk, diatomaxeous earth, lime, calcium carbonate, bentonite, Fuller's earth, cottonseed hulls, wheat flour, soybean flour, pumice, wood flour, walnut shell flour and lignin.

[0048] Adjuvants can be surfactants, crystallization inhibitors, viscosity regulators, suspending agents, spray droplet regulators, pigments, antioxidants, foaming agents, defoamers, opacifiers, compatibilizers, masking agents, neutralizing and buffering agents, corrosion inhibitors, dyes, flavor enhancers, spreading agents, penetration aids, micronutrients, emollients, lubricants and fixatives.

[0049] The compositions disclosed herein are preferably agriculturally acceptable compositions.

[0050] The composition comprising the compound according to the present invention disclosed herein typically comprises 0.5w / w% to 95w / w% of active ingredients, such as 1% to 90w / w%, such as 2w / w% to 80w / w%, such as 5w / w% to 60w / w%. The compound according to the present invention can be the only active ingredient in the composition disclosed herein. In one embodiment, the composition comprising the compound of the present invention further comprises at least one other active ingredient. The active ingredient defined herein has fungicidal and / or insecticidal and / or herbicidal activity or has activity as a plant growth regulator. The compound or composition of the present invention can be mixed with one or more other ingredients (such as fungicides, insecticides, herbicides, bactericides, acaricides, nematicides) having pesticidal activity, and / or the other ingredients include plant growth regulators in appropriate cases. The pesticides mentioned herein using their common names are, for example, known from "The Pesticide Manual [Pesticide Manual]", 19th edition, British Crop Protection Council (British Crop Protection Council) 2021.

[0051] Additional ingredients with pesticidal activity (e.g., fungicidal activity) can promote unexpected synergistic activity. Therefore, compositions comprising lipopeptide compounds according to formula I and additional active ingredients (e.g., malonomicin) can show synergistic effects. As long as the effect of the active ingredient combination is greater than the sum of the effects of the individual components, there is a synergistic effect. For a given active ingredient combination, the expected effect E obeys the so-called COLBY formula and can be calculated as follows (COLBY, SR "Calculating synergistic and antagonistic responses of herbicide combination". Weeds [weeds], Vol. 15, pp. 20-22; 1967):

[0052] ppm = milligrams of active ingredient (=ai) per liter of spray mixture

[0053] X = effect of active ingredient A) in %, p ppm of active ingredient used

[0054] Y = action in % of active ingredient B), q ppm of active ingredient used.

[0055] According to COLBY, using p+q ppm of active ingredient, the expected (additive) effect of active ingredient A)+B) is

[0056]

[0057] If the effect actually observed (O) is greater than the expected effect (E), then the effect of the combination is superadditive, i.e. there is a synergistic effect. In mathematical terms, synergism corresponds to a positive value of the difference (OE). In the case of a superposition of completely complementary activities (expected activity), the difference (OE) is zero. A negative value of the difference (OE) indicates a loss of activity compared to the expected activity.

[0058] In addition to the actual synergistic effect with respect to the fungicidal activity, the compositions according to the invention may also have further unexpected advantageous properties. Examples of such advantageous properties that may be mentioned are: more favorable degradability; improved toxicological and / or ecotoxicological behavior; or improved characteristics of useful plants, including: emergence, crop yield, more developed root system, increased tillering, increased plant height, larger leaves, less basal leaf death, stronger tillering, greener leaf color, less fertilizer required, less seed required, more productive tillering, earlier flowering, earlier grain maturity, less plant lodging (lodging), enhanced shoot growth, improved plant vigor and early germination.

[0059] Other ingredients with pesticidal activity and / or as plant growth regulators can be combined with the composition of the present invention and used in the method of the present invention and applied simultaneously or sequentially with the composition of the present invention. When applied simultaneously, these other ingredients can be formulated or mixed in, for example, a spray tank together with the composition of the present invention. As an alternative to directly mixing these other ingredients with pesticidal activity, these components can be used in separate fungicidal, insecticidal or weeding applications as a part of a fungus, insect or grass control program carried out in part or throughout the growing season.

[0060] At least one other component having pesticidal activity and / or as a plant growth regulator can be any suitable known fungicide, insecticide, herbicide and / or plant growth regulator.At least one other component having pesticidal activity and / or as a plant growth regulator can be from a chemical source or a biological source, for example from a plant or microbial source.At least one other component having pesticidal activity in the composition disclosed herein can be produced by a microorganism according to a compound of formula (I) according to the present invention as disclosed above.

[0061] In addition, the compositions of the invention may also be administered with one or more inducers of systemic acquired resistance ("SAR" inducers). SAR inducers are known and described, for example, in U.S. Pat. No. 6,919,298, and include, for example, salicylates and the commercial SAR inducer acibenzolar-S-methyl.

[0062] The compounds and / or compositions according to the invention may induce resistance in plants by a priming mechanism. Priming is a mechanism leading to a physiological state that enables a plant to respond more quickly and / or more robustly after exposure to a biotic or abiotic stress, as described in the review article: P. Aranega-Bou et al. Priming of plant resistance by natural compounds. Hexanoic acid as a model. Front. Plant. Sci., October 1, 2014.

[0063] In one embodiment, the composition according to the present invention further comprises cyclothiazolin C, streptoglutarimide and / or malenomycin.

[0064] Cyclothiazolyl C is a known compound, the structure of which is disclosed on page 3 of WO 2015191789, and can be produced as disclosed in Example 4 of WO 2015 / 191789.

[0065] Malonomicin (sometimes spelled "malonomycin") is {[(2S)-2-amino-3-hydroxypropanoyl]amino}{2-[(5S)-5-(aminomethyl)-4-hydroxy-2-oxo-2,5-dihydro-1H-pyrrol-3-yl]-2-oxoethyl}malonic acid having formula II.

[0066]

[0067] Malenomycin can be produced as disclosed in Example I of WO 2006 / 078939. Malenomycin can also be prepared according to the methods disclosed in Examples IA and B of EP 1860939 or according to Law et al., 2018 (Nature Catalysis | Vol. 1 | December 2018 | 977-984).

[0068] Streptoglutarimide is a known compound having formula III

[0069]

[0070] Streptimidone can be synthesized according to the method disclosed in Kondo, H., Oritani, T., and Kiyota, H. Synthesis and antifungalactivity of the four stereoisomers of streptimidone, aglutarimide antibiotic from Streptomyces rimosus paromomycinus. Eur. J. Org. Chem. (20), 3459-3462 (2000).

[0071] The present invention also relates to a composition comprising a lipopeptide compound according to formula (I), preferably according to formula I (a), and malenomycin. Surprisingly, it was found that the composition comprising a lipopeptide compound according to formula (I) and malenomycin can exhibit unexpected synergistic fungicidal effects. For example, it was found that the composition comprising a lipopeptide compound according to formula (I), preferably according to formula I (a), and malenomycin had a surprising synergistic fungicidal effect on the following: wheat leaf blight fungus, Fusarium culmorum, Microdochium nivale, Botrytis cinerea, Puccinia recondita and Pyricularia oryzae.

[0072] In one embodiment, the active ingredients cyclothiazolin C, streptoglutarimide and / or malenomycin are produced by a microorganism capable of producing a compound according to formula (I) according to the invention as defined above.

[0073] The mixing ratio of the composition comprising a mixture of the compound according to the invention and at least one further active ingredient is preferably 100:1 to 1:6000, in particular 50:1 to 1:50, more particularly 20:1 to 1:20, even more particularly 10:1 to 1:10, very particularly 5:1 and 1:5, particularly preferably 2:1 to 1:2, and 4:1 to 2:1 ratios are likewise preferred, in particular 1:1, or 5:1, or 5:2, or 5:3, or 5:4, or 4:1, or 4:2, or 4:3, or or 1:300, or 1:150, or 1:35, or 2:35, or 4:35, or 1:75, or 2:75, or 4:75, or 1:6000, or 1:3000, or 1:1500, or 1:350, or 2:350, or 4:350, or 1:750, or 2:750, or 4:750. Those mixing ratios are by weight. The composition comprising a mixture of a lipopeptide compound according to formula (I) and malenomycin comprises the following ratio of the lipopeptide compound according to formula (I) and malenomycin: 2000: 1 to 1: 2000, preferably 1000: 1 to 1: 1000, such as 800: 1 to 1: 800, such as 600: 1 to 1: 600 or 500: 1 to 1: 500, 400: 1 to 1: 400, 300: 1 to 1: 300, or 300: 1 to 1: 200. The mixture as described above can be used in a method of controlling pests, which method comprises applying a composition comprising a mixture as described above to the pests or their environment, except for methods for treating the human or animal body by surgery or therapy and diagnostic methods performed on the human or animal body.

[0074] Compositions comprising a mixture of a compound of the invention and one or more active ingredients as described above (e.g., malenomycin) can be applied, for example, in a single "ready-to-use" form, as a combined spray mixture (which mixture consists of separate formulations of these single active ingredient components) (e.g., a "tank mix"), and when applied in a sequential manner (i.e., one after another within a suitably short period of time, such as a few hours or days) using a combination of these individual active ingredients.

[0075] In one aspect, the present invention relates to a method for producing a compound or composition according to the present invention, the method comprising culturing a microorganism in a suitable fermentation medium under conditions that allow the production of the compound. The microorganism fermented in the method disclosed herein is a microorganism capable of producing a compound according to the present invention as defined above.

[0076] Microorganisms capable of producing the compounds according to the invention are, for example, bacteria of the genus Streptomyces disclosed above.

[0077] Cultivating the microorganism for producing the compound according to the present invention in a suitable fermentation medium is well known to those skilled in the art. Microorganism can ferment under aerobic or anaerobic conditions. Microorganisms belonging to Streptomyces species are typically cultivated under aerobic conditions. Suitable fermentation medium comprises nutrients, such as suitable carbon source (such as sugar cane or beet molasses, polysaccharides, flour, starch, sugar or glucose) and suitable nitrogen source (such as casein hydrolysate, tryptone, ammonium sulfate, ammonia, yeast extract, peptone or urea peptide or amino acid). The method for producing the compound according to the present invention can be carried out with batch culture, fed-batch culture or continuous culture.

[0078] In one embodiment, the method further comprises producing a composition comprising the compound according to the present invention as defined herein.Can produce the microorganism according to the compound of the present invention or composition as defined above.Can produce the microorganism according to the compound of the present invention or composition can produce other active ingredients as defined above, for example cyclothiazomycin C, streptoglutarimide and / or malenomycin.

[0079] The method according to the invention may further include the step of reclaiming the compound according to the invention or its salt. The compound according to the invention may be reclaimed by suitable method known in the art (e.g., via crystallization or chromatography, e.g., HPLC). Reclaiming the compound according to the invention may further include the step of purifying the compound.

[0080] The method for producing a compound according to the invention may further comprise the step of formulating the compound into a suitable formulation or composition as defined above.

[0081] In a further aspect, the present invention relates to a method for controlling or preventing infestation of plants, plant propagation materials and / or harvested food crops by phytopathogenic microorganisms by treating the plants, plant propagation materials and / or harvested food crops, wherein an effective amount of a compound or composition according to the invention is applied to the plant, its part or its locus, plant propagation material and / or harvested food crops.

[0082] Applying an effective amount of a compound or composition of the present invention in a method for controlling or preventing plant infestations comprises applying 0.01 g to 5 kg of a compound of the present invention (active ingredient (ai)) per hectare (ha), preferably 0.015 g to 500 g ai. / ha, preferably 0.020 g to 100 g ai. / ha, preferably 0.025 g to 50 g ai / ha, preferably 0.030 g to 5 g ai. / ha, preferably 0.035 g to 500 mg ai / ha.

[0083] When a compound of the invention or a composition of the invention is used to treat seed, a rate of 0.0001 to 10 g of compound of the invention per kg of seed, for example 0.0002 to 0.1 g per kg of seed, for example 0.0005 to 0.001 g per kg of seed is generally sufficient.

[0084] Suitably, the compounds or compositions of the invention are administered prophylactically (meaning before the disease develops) or therapeutically (meaning after the disease develops).

[0085] Phytopathogenic microorganisms affected by the compounds of the invention are fungi and fungal vectors of diseases as well as phytopathogenic bacteria and viruses. The phytopathogenic microorganisms in the method according to the invention include the following fungi and fungal vectors of diseases as well as phytopathogenic bacteria:

[0086] Absidia corymbifera, Albugo candida, Alternaria spp. (including A. solani), Aphanomyces spp., Ascochyta spp., Aspergillus spp. (including A. flavus, A. fumigatus, A. nidulans, A. niger, A. terrus), Aureobasidium spp. (including A. pullulans), Bacillus subtilis, Blastomyces dermatitidis, Blumeria graminis, Blumeriella jaapii), Botryosphaeria spp. (including B. dothidea, B. obtusa), Botrytis spp. (including Botrytis cinerea), Bremialactucae, Cadophora gregata, Candida spp. (including C. albicans, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, C. tropicalis), Cephaloascus fragrans, Ceratocystis spp., Cercospora spp.) include C.arachidicola, C.beticola, C.kikuchii, C.sojina), Cercosporidium personatum, Cladosporium spp., Clarireedia homoeocarpa, Clavibacter spp., Claviceps purpurea, Coccidioidesimmitis, Cochliobolus spp., Colletotrichum spp. including C. dematium, C. lindemuthianum, C. musae, C. orbiculare, C. truncatum, Corynespora cassicola, Cryptococcus neoformans, Diaporthe spp., Dickeya zeae), Didymella sp., Drechslera spp., Elsinoe spp., Epidermophyton spp., Eremothecium gossypiim, Erwinia spp. (including E. amylovora and E. carotovora), Erysiphe spp. (including E. cichoracearum and E. necator), Eutypa lata, Fusarium spp. spp.) (including Fusarium graminearum, Fusarium graminearum, Fusarium langsethiae, Fusarium moniliforme, Fusarium oxysporum, Fusarium poae, Fusarium proliferatum, Fusarium pseudograminearum, Fusarium sacchari, Fusarium sambucinum, Fusarium subglutinans, Fusarium solani, Fusarium sporotrichioides, Fusarium trilineata, Fusarium spp.tricinctum), F. virguliforme), Gaeumannomyces graminis, Gibberella spp. (including G. avenacea, G. fujikuroi, G. intricans, G. moniliformis, G. zeae), Gloeodes pomigena, Gloeosporium musarum, Glomerella cingulate, Golovinomyces cichoracearum, Gymnosporangium juniperi-virginianae, Guignardia bidwellii, Helminthosporium spp.), Hemileia spp., Histoplasma spp. (including H. capsulatum), Hyaloperonospora parasitica, Kabatiella zeae, Laetisaria fuciformis, Leptographium lundbergii, Leveillula taurica, Lophodermium seditiosum, Microdochium majus, Microdochium snow mold, Microsporum spp., Monilinia spp. (including M. fructicola), Monographella spp.) (including M. nivalis), Mucor spp., Mycosphaerella spp. (including M. arachidis, M. fijiensis, M. graminicola, M.pomi), Nakataea oryzae, Neopseudocercosporella spp., Oculimacula spp., Oncobasidium theobromaeon, Ophiostoma spp., Pantoea stewartia, Paracoccidioides spp., Parastagonospora nodorum, Pectobacterium spp., Penicillium spp. (including P. digitatum, P. italicum), Petriellidium spp., Peronosclerospora spp.) (including P. maydis, P. philippinensis and P. sorghi), Peronospora spp. (including P. destroyer), Phaeosphaeria nodorum, Phakopsora pachyrhizi, Phellinus igniarus, Phialophora spp., Phlyctema vagabunda, Phoma spp., Phomopsis viticola, Phyllactoderma pomigena, Phyllosticta spp., Physoderma maydis, Phytophthora spp. spp.) (including P. capsica, P. infestans), Plasmodiophora brassicae, Plasmopara spp. (including P. halstedii, P. viticola), Pleodorus spp., Pleospora spp., Podosphaera spp. (including P.leucotricha), Polymyxa graminis, Polymyxa betae, Pseudocercosporafijiensis, Pseudocercosporella herpotrichoides, Pseudomonas spp. (including P. syringae), Pseudoperonospora spp. (including P. cubensis, P. humuli), Pseudopeziza tracheiphila, Pseudopyrenochaeta lycopersici, Puccinia spp. spp.) (including P. hordei, P. cryptica, P. stripe, P. triticina), Pyrenopeziza spp., Pyrenophora spp., Pyricularia spp. (including Pyricularia oryzae), Pythium spp. (including P. ultimum), Ralstonia solanacearum, Ramularia spp., Rathayibacter spp., Remotididymella destructiva, Rhizoctonia spp., Rhizomucor pusillus, Rhizopus arrhizus, Rhynchosporium spp.), Robbsiaandropogonis, Sarocladium oryzae, Scedosporium spp. (including S. apiospermum and S. prolificans), Schizothyrium pomi, Sclerophthora macrospora, Sclerotinia spp. (including S.sclerotiorum), Sclerotium spp, Septoria spp (including S. nodorum, S. tritici), Setosphaeria turcica, Sphaerotheca macularis, Sphaerotheca fusca, Sphaerotheca fuliginea, Spiroplasma kunkelii, Sporothorix spp, Stagonospora nodorum, Stagonosporopsis cucurbitacearum, Stemphylium spp, Stenocarpella macrospora, Stereum hirsutum), Streptomyces species, Thanatephorus cucumeris, Thielaviopsis basicola, Tilletia spp., Tranzschelia discolor, Trichoderma spp. (including T. harzianum, T. pseudokoningii, T. viride), Trichophyton spp., Typhula spp., Uncinula necator, Urocystis spp., Uromyces spp., Ustilago spp., Venturia spp. (including V. inaequalis), Verticillium spp. spp), Wilson's sore throat (Wilsonomyces carpophilus) or Xanthomonas spp (including Xanthomonas oryzae and Xanthomonas campestris), Xylella spp, Psoralea corylifolia. .

[0087] Surprisingly, it has been found that the phytopathogenic microorganisms affected by the compounds and / or compositions according to the invention are fungi, for example belonging to the genus Blumeria, Botrytis sp., Cercospora sp., Fusarium sp., Glomerella, Microdochium, Glomerella, Zymoseptorias sp., Parastagonospora sp., Puccinia sp., Phaeosphaeria sp., Pyricularia sp., Pyricularia sp., Sclerotinia sp., Pyricular ... arachidis), Puccinia recondita, Puccinia recondita f.sp.tritici, Puccinia recondita, Puccinia spp.

[0088] Surprisingly, it has been found that the phytopathogenic microorganisms affected by the compounds and / or compositions according to the invention are fungi, for example fungi belonging to species of the genus Botrytis, species of the genus Aspergillus, species of Mycosphaeria, species of Puccinia, species of Puccinia, species of Pyrenophora, species of Pyrenophora or species of Psoralea, preferably fungi belonging to Botrytis cinerea, Aspergillus cucurbitae, Mycosphaeria arachidis, Puccinia cryptica, Puccinia graminearum, Pyrenophora teres or Psoralea graminearum.

[0089] Control or prevention means the reduction of infestation by phytopathogenic microorganisms, especially fungi, to a level that demonstrates improvement.

[0090] The preferred method (which includes applying the compound or composition according to the present invention) for controlling or preventing crop plants from being infected by plant pathogenic microorganisms (especially fungi) or insects is foliar application. The frequency of application and the rate of application will depend on the risk of infection by the corresponding pathogen or insect. However, the compound or composition according to the present invention can also be applied to the plant by soaking the plant in a liquid formulation or by applying the compound in a solid form, for example, in a granular form to the soil (soil application) and infiltrating the plant through the root (systemic effect) via the soil. In rice crops, such granules can be applied to the paddy field of irrigation. The compound or composition according to the present invention can also be applied to seeds (coating) by impregnating seeds or tubers with a liquid formulation of a fungicide or coating it with a solid formulation.

[0091] It is also possible to use the compound or composition according to the present invention as a seed dressing for treating plant propagation materials (e.g., seeds, such as fruits, tubers or cereals, or plant cuttings) to prevent fungal infections and prevent plant pathogenic fungi that occur in the soil. Propagation materials can be treated with the compound and / or composition according to the present invention before planting: for example, seeds can be dressed before sowing. The compound and / or composition according to the present invention can also be applied to cereals (coating) by dipping seeds in a liquid formulation or coating them with a solid formulation. It is also possible to apply the composition to the planting site when planting propagation materials, such as being applied to the furrows of seeds during sowing. Such a method for treating plant propagation materials, and the plant propagation materials so treated are disclosed herein.

[0092] As used herein, the term "locus" means a place where or on which plants grow, or where seeds of cultivated plants are sown, or where seeds are to be placed in the soil. It includes soil, seeds, and seedlings, together with established vegetation.

[0093] The term "plant" refers to all tangible parts of a plant, including seeds, seedlings, young trees, roots, tubers, stems, stalks, leaves, and fruits. Germinated plants and young plants that will be transplanted after germination or after emergence can also be mentioned. These young plants can be protected before transplanting by fully or partially processing through dipping.

[0094] The term "plant propagation material" is understood to mean reproductive parts of plants, such as seeds, which can be used for the multiplication of plants, and vegetative material, such as cuttings or tubers (such as potatoes), roots, fruits, bulbs, rhizomes or parts of plants.

[0095] The term plant relates to "useful plants" or "crops". The expressions "useful plants" and "crops" are used interchangeably herein. "Useful plants" and "crops" include 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; fiber plants, for example cotton, flax, hemp, jute and sisal; field crops, for example sugar beets and fodder beets, coffee beans, hops, mustard, rapeseed (canola), poppies, sugar cane, sunflower, tea and tobacco; fruit trees, for example apples, apricots, avocados, bananas, cherries, citrus, nectarines, peaches, pears and plums; grasses, for example Bermuda grass, bluegrass, bentgrass, centipede grass, fescue, ryegrass, St. Augustine's The term "plant" also includes woody crops, such as pine or pine, and woody plants.

[0096] The term "useful plants" is to be understood as also including useful plants that have been rendered tolerant to herbicides such as bromoxynil or classes of herbicides such as, for example, HPPD inhibitors, ALS inhibitors, for example primisulfuron, prosulfuron and trifloxysulfuron, EPSPS (5-enol-pyruvate-shikimate-3-phosphate-synthetase) inhibitors, GS (glutamine synthetase) inhibitors or PPO (protoporphyrinogen oxidase) inhibitors as a result of conventional methods of breeding or genetic engineering.

[0097] The term "useful plants" is to be understood as also including useful plants which have been transformed by the use of recombinant DNA techniques in such a way that they are able to synthesize one or more selectively acting toxins, as are known, for example, from toxigenic bacteria, in particular those of the genus Bacillus.

[0098] Any suitable plant, plant propagation material or food crop can be processed according to the method of the present invention as defined herein. Preferably, plant, plant propagation material or food crop comprise or are potato, tomato, grape, canola / oilseed rape / cabbage type rape, melon, peanut, wheat, barley, corn, rice, banana, soybean, preferably this plant is wheat or barley.

[0099] In another aspect, the present invention relates to the use of a compound or composition according to the invention as a pesticidal agent, preferably as a fungicide and / or as an initiator. The features associated with the compounds and compositions according to the invention are as disclosed above. Therefore, the present invention relates to a method for using a compound and / or composition according to the invention as a fungicide. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 . Light absorption (UV-VIS) spectrum (200-400nm) of the compound of formula I (a), formula I (b) or lipopeptide antibiotics

[0101] Figure 2 . Precursor of formula I (a) 1204.6m / z (M+H) + The LC-ESI-MS / MS spectrum of the amino acid β-glutamine shows fragmentation peaks consistent with the following amino acids: aspartic acid, hydroxyglutamine, serine, methylasparagine, methylphenylalanine

[0102] Figure 3 LC-ESI-MS / MS / MS spectrum of the precursor of Formula I(a) at 294.2 m / z depicting a peak consistent with the molecule C14H25-OH2-C4H5ON

[0103] Figure 4 . 1D of compounds according to formula I(a) in CD3OD at 600 MHz 1 High field region of the HNMR spectrum

[0104] Figure 5 . 1D of compounds according to formula I(a) in CD3OD at 600 MHz 1 Low-field region of the HNMR spectrum

[0105] Figure 6 . Schematic representation of the lipopeptide gene cluster

[0106] Examples

[0107] Example 1. Source and extraction of compounds of the present invention

[0108] 1.1. Fermentation of Streptomyces species

[0109] Streptomyces species were ordered from the culture collections disclosed in Table 1. Streptomyces species Saigon 413 was isolated in Vietnam before 1961. Streptomyces species Saigon 413 was deposited with the Fungal Culture Collection under the accession number CBS 149411. The deposit was made by Syngenta Ltd. at Jealott's Hill Research International Centre, Bracknell, Berkshire RG42 6EY, United Kingdom under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0110] Streptomyces species were cultured in Erlenmeyer flasks containing a liquid medium consisting of (g / l) casein hydrolysate 10, glucose 40, K2HPO4 1.25, soytone 2, tryptone 8, and incubated at 28°C in an incubator with shaking at 150 rpm and an amplitude of 25 mm for 4 days.

[0111] The presence of compounds according to formula I (eg compounds according to formula I(a) and formula I(b)) in the fermentation broth is determined by separation and purification according to the methods described in Section 1.5 and identified as disclosed in Section 2.

[0112] The results in Table 1 show that compounds according to Formula I(a) are present in the fermentation broths of various Streptomyces species.

[0113] 1.2.16S rDNA Isolation, Whole Genome Sequencing and Species Identification

[0114] Genomic DNA was isolated from Streptomyces species Saigon 413 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 species Saigon 413 is shown in SEQ ID NO: 1.

[0115] The species of strain Streptomyces species Saigon413 was identified by comparing the 16S rRNA sequence according to SEQ ID NO: 1 with the publicly available 16S rRNA sequence extracted using the whole genome sequence assembly of the Streptomyces species genome (based on the genome classification database GTDB (Parks, DH, et al. (2021). GTDB: Nucleic Acids Research [Nucleic Acids Research], 50: D785-D794)), using barrnap v0.9. Based on this comparative analysis, Streptomyces species Saigon413 was identified as a Streptomyces species. The sequence identity between the 16S rRNA sequence of Streptomyces species Saigon413 and the publicly available Streptomyces crown mycelium NRRL-3672 determined using Muscle v3.8.31 and the R software package Seqinrv4.2-16 was 99.87%.

[0116] In addition, whole genome sequencing was completed using genomic DNA from Streptomyces species Saigon 413 using Pacific Biosciences and Illumina sequencing technologies. The genome was assembled using HFAP4 and perfected using Pilon using Illumina reads. Genomic DNA was also extracted from Streptomyces rimosus CBS 492.64, Streptomyces rimosus CBS 570.66, Streptomyces rimosus CBS 569.66, Streptomyces rimosus DSM 41224, Streptomyces rimosus subsp. rimosus DSM 40673, and Streptomyces rimosus subsp. rimosus DSM 41057 using the method described in Kieser et al., (2000) Practical Streptomyces Genetics. Whole genome sequencing of these strains was completed using nanopore sequencing technology, and the genomes were assembled using Flye (Kolmogorov, M., et al., (2019), Nature Biotechnology, 37, 540).

[0117] After assembling the genome of Streptomyces species Saigon 413 and publicly available genomes, the average nucleotide identity (ANI) between Streptomyces species Saigon 413 and closely related Streptomyces strains was calculated using fastANI (Jain, C., et al. (2018), Nature Communications, 9, 5114) (Table 1). The highest percentage identity (ANI) of the genome of Streptomyces species Saigon 413 with the genome of the publicly available Streptomyces coronatus NRRL B-3672 was 96.9%.

[0118] Using 16S RNA sequence identity and ANI scores (%), strains CBS 596.66, CBS 570.66, and DSM 41429 were also found to be Streptomyces coronatus strains, rather than Streptomyces rimosus or Streptomyces paromomycinus strains as indicated by the depository institutes.

[0119] Table 1 shows the percentage identities of the whole genomes and 16S RNA sequences of several Streptomyces species with the whole genomes and 16S RNA sequences of Streptomyces species Saigon 413.

[0120] Table 1: Identification of lipopeptide compounds having formula I(a) in different Streptomyces species

[0121]

[0122]

[0123] CBS Fungal Biodiversity Centre or Westerdijk Fungal Diversity Institute: Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

[0124] DSMZ - German Collection of Microorganisms and Cell Cultures: Inhoffenstrasse 7B, 38124 Braunschweig, Germany.

[0125] ARS - ARS Culture Collection Centre (NRRL), 1815 North University Street, Peoria, Illinois 61604, USA

[0126] 1.3. Identification of the biosynthetic gene cluster in Streptomyces species Saigon 413 that produces the lipopeptide according to formula I

[0127] To identify genes involved in the production of lipopeptides according to Formula I, the assembled genome (see Example 1.2) was run with AntiSMASH (version 5.1.1, Blin et al., Nucleic Acids Res [Nucleic Acids Research] (2019) doi: 10.1093 / nar / gkz310), which is a commonly used tool to assist in the identification of biosynthetic gene clusters responsible for the production of secondary metabolites.

[0128] By identifying that the lipopeptide compound belongs to the lipopeptide family (see 1 above, and the AntiSMASH output, we were able to infer that the lipopeptide compound is produced by a non-ribosomal peptide synthetase (NRPS gene cluster). The identification of the NRPS gene cluster responsible for the biosynthesis of the lipopeptide compound is based on the structural analysis of the lipopeptide compound and the amino acids incorporated into the depsipeptide core of the lipopeptide compound. In Streptomyces species Saigon 413, only one NRPS biosynthetic gene cluster was identified (Figure 11), which enables the incorporation of amino acid precursors including asparagine, aspartic acid, glutamic acid, phenylalanine, serine and threonine, and is therefore associated with the production of lipopeptide compounds of Formula I(a) and I(b).

[0129] The NRPS biosynthetic gene cluster contains 45 coding sequences, including the coding sequences of two NRPS genes, the coding sequence of the regulator, and the coding sequence responsible for the biosynthesis of the precursor incorporated into the lipopeptide having Formula I ( Figure 6 and Table 4).

[0130] Table 4. Coding sequences present in the NRPS biosynthetic gene cluster responsible for the production of lipopeptides according to Formula I. The annotations provided are based on a pBLAST search using non-redundant protein sequences on the National Centre for Biotechnology Information database.

[0131]

[0132]

[0133]

[0134] 1.4. Deletion of the genomic region including CDS_21 (SEQ ID NO: 22) and CDS_22 (SEQ ID NO: 23) from Streptomyces sp. Saigon 413 and phenotypic analysis

[0135] To confirm that the identified biosynthetic gene cluster was relevant to the production of [insert compound ID here], a region containing two non-ribosomal peptide synthetase genes encoded by ctg_7318 and ctg_7319 (SEQ ID NOs: 22 and 23) was deleted from Streptomyces sp. Saigon 413. To generate Streptomyces sp. Saigon 413 Δ7318-7319, plasmid pBCon2192 was used. Plasmid pBCCon2192 was prepared from pRAR017 and contains regions of homology to either side of the region to be deleted from the strain (facilitating primary and secondary crossover).

[0136] Plasmid pBCon2192 was used to transform E. coli ET12567 / pUZ8002 using standard electroporation methods and then introduced into Streptomyces species Saigon413 by mycelial conjugation (T. Kieser et al., Practical Streptomyces Genetics, 2000, John Innes Foundation, Norwich). Thiostrepton resistant colonies were spotted on ISP-4 agar medium supplemented with 40 μg / ml thiostrepton and 25 μg / ml nalidixic acid. These spotted colonies were initially incubated at 28°C for 6 days to allow plasmid replication. After 6 days at 28°C, the strains were re-spotted on ISP-4 agar medium supplemented with 40 μg / ml thiostrepton and incubated at 37°C for another 6 days to force primary integration. After 6 days at 37°C, the obtained strains were transferred to ISP-4 solid agar medium without selection and incubated at 28°C for 15 days to allow secondary crossing.

[0137] After 15 days of growth, strains were collected in 20% glycerol. 100 μl of the cell suspension was used to inoculate fresh plates and plated for up to 10 -10 Then 100 μl of 10 -8 Up to 10 -10 Plate onto ISP-4 agar plates. Incubate plates at 28°C until single colonies are observed.

[0138] Single colonies were double-plated on non-selective and thiostrepton selective ISP-4 agar media. Sensitive colonies (representing secondary recombinants) were then screened via PCR using gDNA isolated using the Soil FastSpin kit (MP Biomedicals) to identify the correct colonies.

[0139] To confirm that the region containing SEQ ID NO:22 and SEQ ID NO:23 had been removed from the strain, a primer pair that binds to the outside of the deleted region was used. Sanger sequencing of the PCR product and alignment with the genome of Streptomyces sp. Saigon 413 confirmed the deletion of the genomic region containing SEQ ID NO:22 and SEQ ID NO:23. In addition, whole genome analysis using PCR-free sequencing by Illumina confirmed that no other changes had occurred in the genome. Cultivation of Streptomyces sp. Saigon 413Δ7318-7319 and analysis of extracts from the strain confirmed that the strain no longer produced the lipopeptide compound according to formula I(a), thereby confirming that SEQ ID NO:22 and SEQ ID NO:23 are essential for the production of the lipopeptide compound according to formula I(a).

[0140] 1.5. Purification of the compounds according to the invention

[0141] Mycelia from the fermentation broth of the Streptomyces strains disclosed in Table 1 were separated via centrifugation and the supernatant was treated with butanol. The butanol was removed and the extract was partitioned between water and ethyl acetate. The lipopeptides were purified from the ethyl acetate fraction by preparative reverse phase (C18) HPLC. The lipopeptides are relatively non-polar and eluted in the higher organic fractions in a gradient system containing 0.1% formic acid and acetonitrile (0.1% formic acid). The compounds according to Formula I (a) and Formula I (b) were separated using a gradient of the above solvents from 60% aqueous phase to 40% aqueous phase.

[0142] Similarly, the fermentation broth of Streptomyces sp. Saigon 413 was treated.

[0143] Stock solutions of compounds of Formula I(a) and Formula I(b) were generated in DMSO (up to 10 mg / ml) and further diluted with water plus 0.025% Tween 20 to produce appropriate working concentrations for bioefficacy assays. Figure 1 )、Mass Spectrometry( Figure 2 and Figure 3 ) and NMR spectra ( Figure 4 and Figure 5 ) test compounds.

[0144] 1.6. Lipopeptide antibiotic A

[0145] Lipopeptide antibiotic A was purchased from Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores enAndalucía, Avda. del Conocimiento 34, Edificio Centro de Desarrollo Farmacéutico y Alimentario, Parque Tecnológico de Ciencias de la Salud, 18016 Granada (Spain). ).

[0146] Example 2. Characterization of compounds having formula I

[0147] 2.1 Liquid chromatography and high-resolution mass spectrometry

[0148] Spectra were recorded on an Orbitrap ID-XTribrid mass spectrometer from Thermo Scientific equipped with an OptaMax NG heated electrospray source (spray voltage: static, polar ion (V): 3400 (positive ion mode) and 2400 (negative ion mode), sheath gas (Arb): 40, auxiliary gas (Arb): 5, sweep gas (Arb): 1, ion transfer tube temperature: 350°C, vaporizer temperature: 350°C). Scan parameters were as follows;

[0149] Experiment 1: MS OT (Orbitrap resolution: 50,000, Scan range (m / z): 200 to 2000, RF lens (%): 60, AGC target: standard, Max injection time mode: automatic, Micro scan: 1, Data type: profile, Polarity: both),

[0150] Experiment 2: tMS2 OT CID (MSn level (n): 2, separation window (m / z): 1.0, activation type: CID, CID collision energy (%): 30, detector type: Orbitrap, Orbitrap resolution: 30,000, RF lens (%): 60, polarity: positive),

[0151] Experiment 3: tMS2 OT HCD (MSn level (n): 2, separation 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, separation window (m / z): 1.6, activation type: HCD, HCD collision energy (%): 30, MS2 separation 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 from Thermo Fisher Scientific. UHPLC using a Vanquish split sampler FT, Vanquish binary pump F, Vanquish column chamber H, Vanquish diode array detector FG and Vanquish charged aerosol detector.

[0152] Liquid chromatography conditions included: Waters ACQUITY UPLC C18 column 1.7 μm 3.0x50 mm, PN186004660. Temperature: 40°C, DAD wavelength range: 250 to 260 nm, solvent gradient: solvent A: H2O containing 0.1% formic acid, solvent B: CH3CN containing 0.1% formic acid, gradient: 0 min 10% B, 90% A; 4.00 min 90% B, 10% A; 4.25 min 90% B, 10% A; 4.50 min 10% B, 90% A; 5.00 min 10% B, 90% A, flow rate: 1.0 ml / min, injection volume: 2 uL, total run time: 5.0 min. Inject the purified fermentation broth as described above.

[0153] Figure 2 and Figure 3 The LC-ESI-MS / MS / MS spectrum of the compound having formula I(a) is shown.

[0154] Compounds according to formula I(a) and formula I(b) were identified in the fermentation broth of Streptomyces sp. Saigon 413 deposited under accession number CBS149411.

[0155] NMR spectroscopy

[0156] NMR spectra were recorded on a Bruker AVIII 600 NMR spectrometer equipped with a 5 mm Bruker (1 H / 19 F) / 13 C / 15 N TCI cryoprobe. The samples were dissolved in CD3OD, spectra were recorded at 300°K, and for 1 H, referenced to the residual solvent signal at 3.31 ppm. Figure 4 and Figure 5 In each case half of the 1H NMR spectrum of the compound according to formula 1(a) is covered.

[0157] 2.3. Molecular composition and mass

[0158] The molecular composition and mass of the compounds according to formula I(a) and formula I(b) were determined using liquid chromatography and high resolution mass spectrometry results as disclosed in 2.1. The compounds having formula I(a) and formula I(b) have the following composition.

[0159] Formula I(a) lipopeptide 1204: molecular composition is C55H85N11O19, and the exact mass is 1203.602.

[0160] Formula I(b) lipopeptide 1218: molecular composition is C56H87N11O19, and the exact mass is 1217.618.

[0161] The reference lipopeptide antibiotic A has the following composition: C54H84N10O19 and an exact mass of 1176.591421.

[0162] 2.4. Solubility

[0163] The solubility of the compounds of Formula I(a), Formula I(b) and lipopeptide antibiotic A in water and DMSO was determined:

[0164]

[0165] Example 3. Activity of the compounds according to the invention on plants

[0166] 3.1 Fungicidal activity in liquid culture assay

[0167] Mycelial fragments or conidia suspensions freshly prepared from liquid cultures of the fungus or from frozen stocks of the fungus are mixed directly into the nutrient broth.

[0168] Stock solutions of compounds of Formula I(a) and Formula I(b) (up to 10 mg / ml) were produced in DMSO and diluted with water to 0.025% 20 dilutions were made to produce 10-fold concentrated samples, and 10 μl of the solution was pipetted into a microtiter plate (96-well format). Nutrient broth containing fungal spores / mycelial fragments was then added to obtain the final concentration of the test compound. The test plates were incubated in the dark at 24° C. and 96% rh. Depending on the disease system, inhibition of fungal growth was determined photometrically after 2 to 7 days, and the percentage of antifungal activity relative to the untreated control was calculated.

[0169] Under the conditions specifically outlined above and below, the test compounds (Formula I(a), I(b) and lipopeptide antibiotic A) were tested against the following fungi:

[0170] Botryotiniafuckeliana (Botrytis cinerea) / liquid culture (gray mold)

[0171] Conidia of the fungus from frozen storage were mixed directly into nutrient broth (Vogels broth). Growth inhibition was determined photometrically 3-4 days after application.

[0172] Colletotrichum cucurbitae (Cucurbit anthracnose) / liquid culture (anthracnose)

[0173] Conidia of the fungus from frozen stocks were mixed directly into nutrient broth (PDB: Potato Dextrose Broth). Growth inhibition was measured photometrically 3-4 days after application.

[0174] Arachis coccidioides (Cercospora arachidis) / liquid culture (early leaf spot)

[0175] Conidia of the fungus from frozen stocks were mixed directly into nutrient broth (PDB: Potato Dextrose Broth). Growth inhibition was determined photometrically 4-5 days after application.

[0176] Wheat leaf blight pathogen (Septorial eaf) / liquid culture blotch))

[0177] Conidia of the fungus from frozen stocks were mixed directly into nutrient broth (PDB: Potato Dextrose Broth). Growth inhibition was determined photometrically 4-5 days after application.

[0178] The results are shown in Tables 2 and 3.

[0179] Table 2. Control of fungal development by compounds according to formula I(a) and I(b) in liquid culture.

[0180]

[0181] Table 3: Control of fungal development by compounds according to formula I(a) and I(b) in liquid culture.

[0182]

[0183] The results in Tables 2 and 3 show that the lipopeptide compounds of Formula I(a) and Formula I(b) are active against several plant pathogenic fungi, providing control rates up to 100%. The compounds of Formula I(a) and Formula I(b) have similar efficacy, while the activity of lipopeptide antibiotic A is lower.

[0184] 3.2. Leaf disc or leaf segment testing in orifice plates

[0185] Leaf discs or leaf segments of different plant species were cut from plants grown in a greenhouse. The cut leaf discs or leaf segments were placed on water agar in multiwell plates (24-well format). The leaf discs were sprayed with the test solutions before inoculation with fungal spores. The compounds to be tested were prepared as DMSO solutions (up to 10 mg / ml) which were diluted with water at 0.025% 20 to the appropriate concentration. The inoculated leaf discs or leaf segments are incubated under defined conditions (temperature, relative humidity, light, etc.) according to the corresponding test system. A single assessment of the disease level is made by visual assessment 3-9 days after inoculation, depending on the disease system as shown below. The percentage of disease control relative to the untreated control leaf discs or leaf segments is then calculated.

[0186] Puccinia recursa wheat specific type / wheat / leaf discs (brown rust)

[0187] Wheat leaf segments cv. Kanzler were placed on agar in multiwell plates (24-well format) and sprayed with formulated test compounds diluted in water. One day after application, the leaf disks were inoculated with a spore suspension of the fungus. The inoculated leaf segments were incubated in a climate chamber at 19° C. and 75% rh under a lighting regime of 12 h light / 12 h dark, and the activity of the compound was assessed as the percentage of disease control compared to the untreated when appropriate levels of disease damage appeared on the untreated control leaf segments (7-9 days after application).

[0188] Septoria glumae (Septoria glumae) / wheat / leaf discs (leaf blight)

[0189] Wheat leaf segments cv. Kanzler were placed on agar in multiwell plates (24-well format) and sprayed with the formulated test compounds diluted in water. Two days after application, the leaf discs were inoculated with a spore suspension of the fungus. The inoculated test leaf discs were incubated in a climate chamber under a lighting regime of 12 h light / 12 h dark at 20° C. and 75% rh, and the activity of the compounds was assessed as the percentage of disease control compared to the untreated when the appropriate level of disease damage appeared on the untreated control leaf discs (5-7 days after application).

[0190] Pyricularia terrestris / barley / leaf disc preventive (net spot)

[0191] Barley leaf segments cv. Hasso were placed on agar in multiwell plates (24-well format) and sprayed with formulated test compounds diluted in water. 2 days after application, the segments were inoculated with a spore suspension of the fungus. The inoculated segments were incubated in a climate chamber at 20°C and 65% rh under a lighting regime of 12h light / 12h dark, and the activity of the compounds was assessed as disease control compared to the untreated when appropriate levels of disease damage appeared on untreated control leaf segments (5-7 days after application).

[0192] Table 4: Control of fungal development on leaf disks or segments by compounds according to formula I (a) or (b).

[0193]

[0194]

[0195] Table 5: Control of fungal development on leaf disks or segments by compounds according to formula I (a) or (b) or lipopeptide antibiotic A.

[0196]

[0197] The results in Tables 4 and 5 show that compounds according to formula I(a) or I(b) are active in the assay, providing growth reduction percentages of up to 95% against several fungal species. Compounds of formula I(a) and formula I(b) have similar efficacy, while lipopeptide antibiotic A is less active.

[0198] 3.3. Wheat seedling leaf smear assay

[0199] Wheat variety Riband (for wheat leaf blight test) or variety Arina (for reclusive rust test) seedlings are grown in a greenhouse until 14 days after sowing. At this time, such seedlings usually have a fully emerged first leaf (designated L1), a fully emerged second leaf (L2) and a partially emerged and developing third leaf (L3). Use a permanent pen to apply two dots on the second leaf to form three roughly equal-sized segments: segment A (leaf base), segment B (leaf middle) and segment C (leaf top). A stock solution of the test compound at a concentration of 10'000 ppm is produced in DMSO. The stock solution is then added to a 5% DMSO solution supplemented with 1% DMSO. The test compound (containing 0.05% 20 and 2% DMSO) to a final concentration of 200 ppm or higher. The diluted compound was applied to the middle section of L2 using a regular cotton swab; the cotton swab was soaked in the diluted compound and rubbed several times on the adaxial surface of the leaf between the two marks. One day later, the fungal spore suspension was inoculated onto the entire plant using a paintbrush and the spore suspension was applied until it was almost running down.

[0200] For infection with Psoralea corylifolia: 1 day after application, the test plants were sprayed with a spore suspension (1.5 million spores / ml in a solution supplemented with 0.01% 20% water). After a 4-day incubation period at 22°C / 21°C (day / night) and 95% rh, the inoculated test plants were maintained in a greenhouse at 22°C / 21°C (day / night) and 70% rh. Efficacy was assessed directly by visual assessment when appropriate levels of disease appeared on untreated control plants (usually >80% disease coverage 16-19 days after inoculation).

[0201] For infection with Puccinia recondita: 1 day after application, the test plants were sprayed with a spore suspension (80'000 spores / ml of spore suspension in a humidified soil supplemented with 20 (0.1%) in water). After a 1-day incubation period at 20°C and 95% rh, the inoculated test plants were kept in a greenhouse at 20°C and 60% rh. When appropriate levels of disease appeared on untreated control plants (usually 50%-80% disease coverage 9-12 days after infection), the percentage of leaf area covered by disease was assessed by visual assessment.

[0202] The three segments of the leaf were evaluated separately.

[0203] Table 6: Disease control efficacy of compounds according to formula I(a) and I(b) in leaf application assay in greenhouse

[0204]

[0205] The results in Table 6 show that compounds of Formula I(a) and Formula I(b) were active in the assay, providing up to 70% control of Puccinia recondita and Puccinia tritici on the treated area (mid-section). Compounds of Formula I(a) and Formula I(b) had similar efficacy.

[0206] 3.4. Reactive oxygen species (ROS) burst assay in wheat leaf discs

[0207] 200 μL of the test solution (analyte) or the corresponding control (water) of the appropriate concentration are pipetted into a white 96-well plate (Nunc, Langenselbold, Germany). A 5 mm leaf disk of a 2-week-old wheat plant is obtained using a tissue punch and then floated on the test solution. The plate is stored for 24 h at room temperature (RT). The next day, 50 μL ddH2O replaces the solution, and the leaf disk is placed in the dark at RT for at least one hour for regeneration. At the same time, a freshly prepared appropriate master mixture with or without the excitation flg22 (see below) is prepared in a black 5 mL reaction tube. After regeneration, 50 μL of the corresponding master mixture is added to the hole containing the leaf disk. Subsequently, luminescence is recorded with a plate reader (BMG Labtech; Ortenberg, Germany) for 40 minutes. The compound INA (2,6-dichloro-isonicotinic acid, CAS: 5398-44-7) is a synthetic salicylic acid analogue and was included as a reference for elicitor activity (Kauss et al., 1992).

[0208] Master mix - flg22: 4.98 mL ddH2O, 10 μL HRP (10 mg / mL), 10 μL L-012 (20 mM)

[0209] Master mix + flg22: 4.979 mL ddH2O, 10 μL HRP (10 mg / mL), 10 μL L-012 (20 mM), 1 μL flg22 (10 μM)

[0210] Abbreviations: flg22 (22 amino acid flagellin peptide, Eurogentec catalog number AS-62633); HRP (horseradish peroxidase), L-012 sodium salt (CAS number: 143556-24-5) was used.

[0211] Table 7. Fold changes in peak ROS production. Values ​​represent the ratio of peak values ​​observed in the treated groups to the control group, using the maximum value measured in each condition over a 40-minute time course. Values ​​indicated are the average of two replicates.

[0212]

[0213] The results in Table 7 show that wheat pre-treated with compounds of Formula I (a) or Formula I (b) at a concentration of 100 ppm increased ROS production induced by peptide flg22 by 2.7-fold or more. This response is similar to or stronger than that observed with treatment with INA (2.6-dichloro-isonicotinic acid), a well-known elicitor (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).

[0214] Example 4. Fungicidal activity of a mixture of a lipopeptide according to formula I(a) and malenomycin in a liquid culture assay

[0215] method

[0216] A stock solution of the compound of formula I(a) was produced in DMSO (up to 10 mg / ml). Malenomycin was produced according to Law et al., 2018 (Nature Catalysis | Vol. 1 | December 2018 | 977-984). Add 0.025% A stock solution of malenomycin was generated in 20.

[0217] To test the efficacy of a mixture of a lipopeptide compound of Formula I(a) in combination with malenomycin in controlling fungal pathogens in a liquid culture assay, an assay for 96-well plates was designed as outlined in Table 1:

[0218] Table 8. Overview of the 96-well assay plate (including the concentration of the compound in each well). The upper numbers in the cells indicate the concentration of malenomycin (ppm), and the lower numbers indicate the concentration of the compound of Formula I(a) (ppm). The cells (1) are the dilution series of the compound of Formula I(a), (2) are the dilution series of malenomycin, and (12) are the untreated controls.

[0219] ppm (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) A 0 1000 500 250 125 62.5 31.25 15.63 7.81 3.00 1.00 0 100 100 100 100 100 100 100 100 100 100 100 0 B 0 1000 500 250 125 62.5 31.25 15.63 7.81 3.00 1.00 0 50 50 50 50 50 50 50 50 50 50 50 0 C 0 1000 500 250 125 62.5 31.25 15.63 7.81 3.00 1.00 0 25 25 25 25 25 25 25 25 25 25 25 0 D 0 1000 500 250 125 62.5 31.25 15.63 7.81 3.00 1.00 0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0 E 0 1000 500 250 125 62.5 31.25 15.63 7.81 3.00 1.00 0 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 6.25 0 F 0 1000 500 250 125 62.5 31.25 15.63 7.81 3.00 1.00 0 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 3.125 0 G 0 1000 500 250 125 62.5 31.25 15.63 7.81 3.00 1.00 0 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 0 H 0 1000 500 250 125 62.5 31.25 15.63 7.81 3.00 1.00 0 0 0 0 0 0 0 0 0 0 0 0 0

[0220] The 96-well plate design allows for comparison of disease control of mixtures with disease control of the corresponding single compounds at the same rate. Comparison of the estimated efficacy of a mixture with the efficacy of the same mixture according to the Colby calculation allows for determination of whether the mixture is additive (efficacy similar to the Colby calculation), synergistic (efficacy better than the Colby calculation), or antagonistic (efficacy less than the Colby calculation).

[0221] The same 96-well plate design also allows for the evaluation of whether two compounds can be mixed at different usage rates and mixing ratios. The plate design outlined in Table 1 will provide the following mixing ratios, as shown in Table 2.

[0222] Table 9. Mixing ratios of two compounds in the 96-well plate assay design as outlined in Table 8. Numbers represent the ratio of Compound 1: Compound 2. Compound 1 is malenomycin and Compound 2 is a lipopeptide compound of Formula 1(a). The plate design spans a wide range from 641:1 to 1:100.

[0223]

[0224] Prepared by adding 0.025% DMSO (from the stock solution of the compound having Formula I(a)) and The concentration of 20 was kept constant in all wells of the mother plate. 10 μl of the solution was transferred from the mother plate to a 96-well test plate. Nutrient broth containing fungal spores / mycelial fragments was then added to the test plate to give a final concentration of 1 times the test compound (as summarized in Table 8). The test plate was incubated in the dark at 24° C. and 96% rh. The inhibition of fungal growth was determined photometrically after approximately 3 days, and the percentage reduction in fungal growth relative to the untreated control was calculated.

[0225] The efficacy of the mixture was tested against different fungal species:

[0226] Triticum truncatum (EPPO code: SEPTTR)

[0227] Fungal conidia from frozen stocks were mixed directly 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 h.

[0228] Fusarium scabra (EPPO code: FUSACU)

[0229] Fungal conidia from frozen stocks were mixed directly 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 h.

[0230] Microspores of snow mold (EPPO code: MONGNI)

[0231] Fungal conidia from frozen stocks were mixed directly 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 h.

[0232] Botrytis cinerea (EPPO code: BOTRCI)

[0233] Fungal conidia from frozen stocks were mixed directly into nutrient broth (Vogel's minimal medium).The test plates were incubated at 24°C and the inhibition of growth was determined photometrically after 72 h.

[0234] Pyriorhizium oryzae (EPPO code: PYRIOR)

[0235] Fungal conidia from frozen stocks were mixed directly 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 h.

[0236] Cercosporium arachidis (EPPO code: MYCOAR)

[0237] Conidia of the fungus from frozen stocks were mixed directly 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.

[0238] Sclerotinia sclerotiorum (EPPO code: SCLESC)

[0239] Mycelial fragments of the fungus prepared from fresh liquid cultures were mixed directly into the nutrient broth (PDB: Potato Dextrose Broth). The test plates were incubated at 24° C. and the inhibition of growth was determined photometrically after 72 h.

[0240] result

[0241] Table 10. Control of Pseudomonas aeruginosa by single compounds and mixtures. The plate design including the compound of formula I(a) and malenomycin concentrations is shown in Table 8. The values ​​indicate control of fungal growth (% reduction in growth in the test wells compared to untreated controls).

[0242]

[0243] For each test condition in Table 10 with an efficacy of 50% or more (hereinafter referred to as: effective mixture), the corresponding mixing ratio can be specified according to Table 9. The effective mixing ratio for controlling wheat leaf blight pathogen was found to be 641:1 to 1:100.

[0244] Table 11. Comparison of measured values ​​for disease control of Psoralea corylifolia (as reported in Table 10) with values ​​calculated for the same mixture using Colby's formula. The numbers reported in the table represent the difference between the measured efficacy (in %) minus the calculated efficacy (in %). Values ​​near 0 (zero) indicate additive activity, while positive values ​​indicate synergistic activity.

[0245]

[0246] Table 12. Control of Fusarium culmorum by single compounds and mixtures. The plate design including the compound having Formula I(a) and malenomycin concentrations is shown in Table 8. The values ​​indicate control of fungal growth (% reduction in growth in the test wells compared to untreated controls).

[0247]

[0248] For each test condition (effective mix) in Table 12 with an efficacy of 50% or greater, the corresponding mix ratio can be assigned according to Table 9. The effective mix ratios for controlling Fusarium culmorum were found to be 641:1 to 20:1.

[0249] Table 13. Comparison of measured values ​​for disease control of Fusarium culmorum (as reported in Table 12) with values ​​calculated for the same mixture using Colby's formula. The numbers reported in the table represent the difference between the measured efficacy (in %) minus the calculated efficacy (in %). Values ​​near 0 (zero) indicate additive activity, while positive values ​​indicate synergistic activity.

[0250]

[0251] Table 14. Control of Microdochium nivale by single compounds and mixtures. The plate design including the compound having Formula I(a) and malenomycin concentrations is shown in Table 8. The values ​​indicate control of fungal growth (% reduction in growth in the test wells compared to untreated controls).

[0252]

[0253] For each test condition (effective mix) in Table 14 with an efficacy of 50% or greater, the corresponding mix ratio can be assigned according to Table 9. The effective mix ratios found to control Microdochium nivale ranged from 641:1 to 1:3.

[0254] Table 15. Comparison of measured values ​​for disease control of Microdochium nivale (as reported in Table 14) with values ​​calculated for the same mixture using Colby's formula. The numbers reported in the table represent the difference between the measured efficacy (in %) minus the calculated efficacy (in %). Values ​​near 0 (zero) indicate additive activity, while positive values ​​indicate synergistic activity.

[0255]

[0256] Table 16. Control of Botrytis cinerea by single compounds and mixtures. The plate design including the lipopeptide compounds of Formula I(a) and malenomycin concentrations is shown in Table 8. The values ​​indicate control of fungal growth (% reduction in growth in the test wells compared to untreated controls).

[0257]

[0258] For each test condition (effective mix) in Table 16 with an efficacy of 50% or more, the corresponding mix ratio can be assigned according to Table 9. The effective mix ratios found to control Botrytis cinerea were 641:1 to 1:6.

[0259] Table 17. Comparison of measured values ​​for disease control of Botrytis cinerea (as reported in Table 9) with values ​​calculated for the same mixture using Colby's formula. The numbers reported in the table represent the difference between the measured efficacy (in %) minus the calculated efficacy (in %). Values ​​near 0 (zero) indicate additive activity, while positive values ​​indicate synergistic activity.

[0260]

[0261] Table 18. Control of Pyricularia oryzae by single compounds and mixtures. The plate design including the lipopeptide compound having Formula I(a) and malenomycin concentrations is shown in Table 8. The values ​​indicate control of fungal growth (% reduction in growth in the test wells compared to untreated controls).

[0262]

[0263] For each test condition (effective mix) with an efficacy of 50% or more in Table 18, the corresponding mix ratio can be assigned according to Table 9. The effective mix ratio for controlling Pyricularia oryzae was found to be 641:1 to 1:3.

[0264] Table 19. Comparison of measured values ​​for disease control of Pyricularia oryzae (as reported in Table 18) with values ​​calculated for the same mixture using Colby's formula. The numbers reported in the table represent the difference between the measured efficacy (in %) minus the calculated efficacy (in %). Values ​​near 0 (zero) indicate additive activity, while positive values ​​indicate synergistic activity.

[0265]

[0266] Table 20. Control of Cercospora arachidis by single compounds and mixtures. The plate design including the compound having Formula I(a) and malenomycin concentrations is shown in Table 8. The values ​​indicate control of fungal growth (% reduction in growth in the test wells compared to untreated controls).

[0267]

[0268] For each test condition (effective mix) in Table 20 with an efficacy of 50% or more, the corresponding mix ratio can be assigned according to Table 9. The effective mix ratios found to control Cercospora arachidis were 641:1 to 1:100.

[0269] Table 21. Control of Sclerotinia sclerotiorum by single compounds and mixtures. The plate design including the compound having Formula I(a) and malenomycin concentrations is shown in Table 8. The values ​​indicate control of fungal growth (% reduction in growth in the test wells compared to untreated controls).

[0270]

[0271] For each assay condition (effective mix) in Table 21 with an efficacy of 50% or more, the corresponding mix ratio can be assigned according to Table 9. The effective mix ratios for controlling Sclerotinia sclerotiorum were found to be 641:1 to 1:2.

[0272] in conclusion

[0273] Mixtures of lipopeptide compounds of formula I(a) and malenomycin control a variety of fungal pathogens. Various mixing ratios of the two compounds (ranging from 641:1 to 1:100, including examples having ratios between the ratios (ratio of compound malenomycin: compound of formula I(a))) produced 50% or greater fungal growth control against several fungal species. Surprisingly, the efficacy of many mixtures was better than the efficacy predicted based on Colby's calculations, indicating that mixtures of lipopeptide compounds of formula I(a) and malenomycin have a synergistic effect on the control of fungal pathogens. This surprising synergistic effect was observed in tests to control Pseudomonas aeruginosa, Fusarium solani, Microdochium nivalis, Botrytis cinerea, and Pyricularia oryzae. Example 5. Fungicidal activity of mixtures of lipopeptide compounds according to formula I(a) and malenomycin in leaf disc assays

[0274] A stock solution of the lipopeptide compound of Formula I(a) was produced in DMSO (up to 10 mg / ml). Malenomycin was produced according to Law et al., 2018 (Nature Catalysis | Vol. 1 | December 2018 | 977-984). Add 0.025% A stock solution of malenomycin was generated in 20.

[0275] To test the efficacy of a mixture of a compound of Formula I(a) in combination with malenomycin in controlling fungal pathogens in a leaf disc assay, an assay for two 24-well plates was designed.

[0276] Table 22 and Table 23. Overview of 24-well test plates (1) and (2) (including the concentration of the compound sprayed in each well). The upper number in the cell indicates the concentration of the compound of Formula I (a) (ppm), and the lower number indicates the concentration of malenomycin (ppm). (1) is a dilution series of the compound of Formula I (a), and (2-D) is a dilution series of malenomycin. Well 2-D-(1) represents an untreated control. This design was applied to a test involving the use of preventive and curative spray times for Puccinia recursa (EPPO code: PUCCRE).

[0277] 24-well plate (1)

[0278]

[0279]

[0280] 24-well plates (2)

[0281]

[0282] Table 24. Mixing ratios of compounds for nebulization in 24-well plate assays are summarized in Tables 22 and 23. Numbers represent the ratio of Compound 1: Compound 2. Compound 1 is malenomycin and Compound 2 is a compound of Formula I(a). The plate designs for both 24-well plates span a wide range from 64:1 to 1:270.

[0283]

[0284] Table 25 and Table 26. Overview of 24-well test plates (3) and test plates (4) (including the concentration of compounds in each well). The upper numbers in the cells indicate the concentration of the lipopeptide compound having Formula I (a) (ppm), and the lower numbers indicate the concentration of malenomycin (ppm). (1) is listed as a dilution series of the compound having Formula I (a), and (4-D) is a dilution series of malenomycin. Well 4-D-(1) represents an untreated control. This design was applied to tests including Blepharitis graminis wheat-specific type (EPPO code: ERYSGT) with a preventive spraying time and Ascopolysaccharide graminearum (EPPO code: LEPTNO) with a preventive spraying time.

[0285] 24-well plates (3)

[0286]

[0287] 24-well plates (4)

[0288]

[0289] Table 27. Mixing ratios of compounds nebulized in 24-well plate assays are summarized in Table 25 and Table 26. Numbers represent the ratio of Compound 1: Compound 2. Compound 1 is malenomycin and Compound 2 is a lipopeptide compound of Formula I(a). The plate designs for both 24-well plates span a wide range from 192:1 to 1:27.

[0290]

[0291] A first set of mother plates with a 1-fold concentrated spray solution of a lipopeptide compound stock solution of Formula I (a) diluted in water according to the concentrations of Table 22, Table 23, Table 25 or Table 26, respectively, was prepared. Each well contained 2% DMSO and 0.025% Tween20. Accordingly, a second set of mother plates with a 1-fold concentrated malenomycin stock solution diluted in water was prepared. Each well of the second set contained 0.025% Tween20. Leaf segments placed on agar in a 24-well plate were sprayed with 8 μl of a solution containing a lipopeptide compound of Formula I (a) from the mother plate, the leaf segments were allowed to dry, and 2 hours later, 8 μl of a solution containing malenomycin from the mother plate was sprayed. After the second spray was dried, the leaf segments were infected with fungal spores to obtain a preventive application time. Alternatively, leaf segments that had been infected one day before spraying the compound were used to obtain a therapeutic spray time. In addition, several plates were made, which were sprayed twice in the absence of the test compound (containing only DMSO and Tween20) as untreated control samples. The leaf coverage percentage of disease symptoms for each leaf segment was evaluated. The leaf coverage reduction percentage relative to the untreated control was calculated. The efficacy of the mixture was tested in duplicate for different fungal species. The reported efficacy value is the average of the two replicate results.

[0292] For Puccinia recondita (EPPO code: PUCCRE), use preventive spraying schedule.

[0293] Wheat (cultivar Kanzler) leaf segments were placed on agar in a multiwell plate (24-well format) and sprayed with the test solution (8 ul / well). After drying, the leaf disks were inoculated with a spore suspension of the fungus. After appropriate incubation, the activity of the compounds 8 days after inoculation (8 dpi) was evaluated as preventive fungicidal activity.

[0294] For Puccinia recondita (EPPO code: PUCCRE), use curative spraying schedules.

[0295] Wheat (cultivar Kanzler) leaf segments were placed on agar in a multiwell plate (24-well format). These leaf disks were then inoculated with a spore suspension of the fungus. One day after inoculation, the test solution (8ul / well) was sprayed. After appropriate incubation, the activity of the compound after 8 days of inoculation (8dpi) was evaluated as a therapeutic fungicidal activity.

[0296] Gramineae powdery mildew wheat-specific type (EPPO code: ERYSGT), use preventive spraying time

[0297] Wheat (cultivar Kanzler) leaf segments were placed on agar in a multiwell plate (24-well format) and sprayed with the test solution (8 ul / well). After drying, the leaf disks were inoculated with fungal spores. After appropriate incubation, the compound activity was evaluated 7 days after inoculation (7 dpi) as preventive fungicidal activity.

[0298] Ascopolysporus lemborius (EPPO code: LEPTNO)

[0299] Wheat (cultivar Kanzler) leaf segments were placed on agar in a multiwell plate (24-well format) and sprayed with the test solution (8 ul / well). After drying, the leaf disks were inoculated with a spore suspension of the fungus. After appropriate incubation, the activity of the compounds 4 days after inoculation (4 dpi) was evaluated as preventive fungicidal activity.

[0300] result

[0301] Table 28. Control of Puccinia recondita (preventive) by single compounds and mixtures. Plate designs including compounds of Formula I(a) and malenomycin concentrations are shown in Tables 22 and 23. Values ​​indicate control of fungal growth (% reduction in symptoms on leaf segments compared to untreated controls).

[0302]

[0303] For each test condition in Table 28 with an efficacy of 50% or greater (effective mix), the corresponding mixing ratio can be assigned according to Table 24. Effective mixing ratios for controlling Puccinia reconnaissance (preventative) were found to be 64:1 to 1:270.

[0304] Table 29. Control of Puccinia recondita (curative) by single compounds and mixtures. Plate designs including compounds of Formula I(a) and malenomycin concentrations are shown in Tables 22 and 23. Values ​​indicate control of fungal growth (% reduction in symptoms on leaf segments compared to untreated controls).

[0305]

[0306] For each test condition in Table 29 where the efficacy was 50% or greater (effective mix), the corresponding mix ratio can be assigned according to Table 24. Effective mix ratios for controlling Puccinia recondita (curative) were found to be 64:1 to 1:270.

[0307] Table 30. Comparison of measured values ​​for disease control of Puccinia recondita (curative) (as reported in Table 29) with values ​​calculated for the same mixture using Colby's formula. The numbers reported in the table represent the difference between the measured efficacy (in %) minus the calculated efficacy (in %). Values ​​near 0 (zero) indicate additive activity, while positive values ​​indicate synergistic activity.

[0308]

[0309]

[0310] Table 31: Control of Blumeria graminearum (preventive) by single compounds and mixtures. The plate design including the compound of formula I(a) and the concentration of malenomycin is shown in Table 25 and Table 27. The values ​​indicate the control of fungal growth (% reduction of symptoms on leaf segments compared to untreated controls).

[0311]

[0312] in conclusion

[0313] Compounds of formula I (a) can be mixed with malenomycin to obtain complete or partial control of a variety of fungal pathogens. In the mixture, the mixing ratio of the two compounds can vary greatly, but when sprayed on the leaves, 50% or more control of fungal growth is still produced. For several fungal species, examples with ratios ranging from 192: 1 to 1: 270 are shown, including many examples with ratios between the ratios (compound malenomycin: ratio of lipopeptide compounds with formula I (a)). Surprisingly, the efficacy of many mixtures is better than the efficacy predicted by Colby's calculations, indicating that the mixture of compounds of formula I (a) and malenomycin has a synergistic effect on the control of fungal pathogens when sprayed on the leaves. This surprising synergistic effect was observed in tests to control cryptic rust.

[0314]

Claims

1. A compound according to formula (I), or a salt thereof, wherein R1 is CH3 or C2H5.

2. A composition comprising the compound according to claim 1 and a microorganism capable of producing the compound according to claim 1.

3. A composition comprising the compound according to claim 1 or the composition according to claim 2, wherein the composition further comprises an auxiliary agent.

4. A compound according to claim 1 or a composition according to claim 2 or 3, wherein the compound has fungicidal activity.

5. The composition according to any one of claims 2 to 4, further comprising at least one additional ingredient having pesticidal activity and / or at least one plant growth regulator.

6. The composition according to claim 5, wherein the additional ingredients comprise cyclothiazolin C, streptoglutarimide and / or malenomycin, preferably malenomycin.

7. A method for producing a compound according to claim 1 or a composition according to any one of claims 2 to 6, the method comprising culturing a microorganism in a suitable fermentation medium under conditions that allow the production of the compound or the composition.

8. A composition according to any one of claims 2 to 6, or a method according to claim 7, wherein the microorganism comprises at least one nucleotide sequence encoding a protein having at least 80% identity with at least one amino acid sequence according to SEQ ID NOs: 47 to 91, preferably SEQ ID NO: 67 or SEQ ID NO: 68, preferably wherein the at least one nucleotide sequence has at least 80% identity with at least one of the nucleotide sequences according to SEQ ID NOs: 2 to 46, preferably SEQ ID NO: 22 or SEQ ID NO:

23.

9. A composition according to any one of claims 2 to 6 or 8, or a method according to any one of claims 7 or 8, wherein the microorganism is a Streptomyces species, preferably Streptomyces coronavirinus, preferably a Streptomyces species comprising a 16S RNA sequence having at least 98% identity to SEQ ID NO: 1, preferably a Streptomyces species deposited at the Fungal Culture Collection Saigon 413 under the accession number CBS149411.

10. A microorganism, which is Streptomyces species Saigon 413 deposited in the Fungal Culture Collection Center under the accession number CBS149411.

11. A method for controlling or preventing infection of plants, plant propagation materials and / or food crops harvested by treating plants, plant propagation materials and / or food crops harvested, wherein an effective amount of a compound according to claim 1 or a composition according to any one of claims 2 to 6, 8 or 9 is applied to the plants, parts thereof or their locations, the plant propagation materials and / or food crops harvested.

12. The method of claim 11, wherein the effective amount comprises 0.001 g to 5 kg of the compound of formula (I) per hectare.

13. The method of claim 11, wherein the plant propagation material is seeds and the effective amount comprises 0.0001 to 50 g of the compound of formula (I) per kg of seeds.

14. The method according to any one of claims 11 to 13, wherein the plant pathogenic microorganism is a fungus, preferably a fungus belonging to the genus Bulrushia, species of Botrytis, species of Cercospora, species of Fusarium, species of Aspergillus, species of Microspore, Glomerella, species of Psoralea, species of Puccinia, species of Dark Coelenteroides, species of Pyricularia, species of Pyricularia, species of Sclerotinia, species of Sclerotinia, and species of Psoralea. Preferably, the fungus belongs to the Poaceae family Bulrushia, specialized type of wheat, Botrytis cinerea, Cercospora arachidis, Fusarium scabra, Aspergillus cucurbitae, Microspore, Glomerella arachidis, Psoralea glumae, Psoralea cryptica, specialized type of Puccinia cryptica, Dark Coelenteroides, Pyricularia tetrasia, Pyricularia oryzae, Sclerotinia sclerotiorum, or Psoralea tritici.

15. The method according to any one of claims 11 to 14, wherein the plant comprises potato, tomato, grape, canola, melon, peanut, wheat and / or barley, corn, rice, soybean, banana.

16. Use of a compound according to claim 1, or a composition according to any one of claims 2 to 6, 8 or 9 as a pesticide, preferably as a fungicide and / or as an initiator.

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