Means and methods for controlling plant pathogens and pests

Abstract

The present invention relates to microbial strains and compositions comprising Streptomyces strains, methods of producing the microbial strains or compositions, and methods of preventing or controlling pests in plants using the microbial strains and compositions.

Description

[0001] The present invention relates to novel microbial strains having pesticidal activity. The present invention also relates to compositions comprising Streptomyces, methods for preparing microbial strains or compositions, and methods for preventing or controlling phytopathogenic infections of plants, harvested food crops, seeds or non-living materials using Streptomyces chrestomyceticus or these compositions in agriculture or horticulture. Background Art

[0002] Pesticides are widely used in agriculture to protect plants from damage caused by plant pathogenic microorganisms. Pesticides can come from chemical sources or biological sources. Due to some negative effects of chemical pesticides on the environment, there is a growing demand for pesticides from biological sources (e.g., microbial sources). Known microorganisms that produce antifungal antibiotics are actinomycetes, such as Streptomyces species (Streptomyces sp.). A very 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 novel Streptomyces species is disclosed, which produces several known antifungal compounds or metabolites, 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, Zymoseptoria tritici and Puccinia striiformis.

[0003] Due to the development of resistance of phytopathogenic microorganisms to pesticides, government regulations and societal pressure, there continues to be a need for improved products (eg, microbial strains or compounds from biological sources) having activity against phytopathogenic microorganisms. Summary of the invention

[0004] The present invention relates to an isolated microbial strain comprising a genome sequence having at least 99.8%, 99.9% or 100% identity with the whole genome of the Streptomyces species Saigon 413 deposited in the Fungal Culture Collection Center under the deposit number CBS149411.

[0005] In a second aspect, the present invention relates to an isolated microbial strain, wherein the strain comprises a nucleotide sequence having at least 99.8% identity to SEQ ID NO:1.

[0006] In a third aspect, the present invention relates to a composition comprising a strain of Streptomyces, malonomicin, and at least one compound selected from the group consisting of cyclothiazolylmycin C, and streptoglutarimide, a compound according to compound I, the compound I comprising a compound according to C 53 H 90 N2O 44 The molecular formula is further characterized by the NMR spectra listed in Table 2 and Table 3, preferably characterized by structural formula I,

[0007]

[0008] A lipopeptide according to formula II, or a salt thereof, wherein R1 = CH3 or C2H5

[0009]

[0010] and a polyene compound, the polyene compound being obtained by 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0011] In a fourth aspect, the present invention relates to a method for producing a microbial strain as disclosed herein or a composition as disclosed herein, the method comprising culturing the microbial strain or a microbial strain of the genus Streptomyces in a suitable fermentation medium under suitable fermentation conditions, and optionally comprising a step of recovering the microbial strain or the composition.

[0012] In a fifth aspect, the present invention relates to a method for controlling or preventing infection of plants, plant propagation materials and / or harvested food crops by plant pathogenic microorganisms, the method comprising treating the plant, plant propagation materials and / or harvested food crops by applying an effective amount of Streptomyces coronamicin, an isolated microbial strain as disclosed herein or a composition as disclosed herein to the plant, part thereof or its locus, the plant propagation material and / or harvested food crops.

[0013] In a sixth aspect, the present invention relates to a plant or plant propagation material treated with a microbial strain or a composition according to the invention.

[0014] In a seventh aspect, the present invention relates to the use of a microbial strain according to the present invention, or Streptomyces coronamicinus as disclosed herein, or a composition according to the present invention as a pesticide, preferably as a fungicide.

[0015] In an eighth aspect, the present invention relates to the use of a microbial strain according to the present invention, or a Streptomyces genus having at least 95% identity with the whole genome of Streptomyces coronomicinus NRRL B-3672, or having at least 95% identity with the whole genome of Streptomyces species Saigon 413 deposited in the Fungal Culture Collection under the accession number CBS149411, for producing at least one of malenomycin and a compound selected from the group consisting of cyclothiazolin C, streptoglutarimide, an oligosaccharide compound according to compound I (the compound I comprises a compound according to C 53 H 90 N2O 44 , further characterized by the NMR spectra listed in Tables 2 and 3, preferably characterized by structural formula I), lipopeptides according to formula II or salts thereof (wherein R1 = CH3 or C2H5) and polyene compounds (which are characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0016] Surprisingly, it has been found that a Streptomyces strain, such as an isolated microbial strain as disclosed herein, such as Streptomyces sp. Saigon 413, and / or metabolites thereof can effectively treat phytopathogenic microbial diseases of crops. DETAILED DESCRIPTION

[0017] The present invention relates to an isolated microbial strain comprising a genome sequence having at least 99.8%, 99.9% or 100% identity with the whole genome of the Streptomyces species Saigon 413 deposited in the Fungal Culture Collection Center under the deposit number CBS149411.

[0018] The present invention also relates to an isolated microbial strain, wherein the strain comprises a nucleotide sequence that is at least 99.9%, 99.91%, 99,92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99% or 100% identical to SEQ ID NO:1.

[0019] The isolated microbial strain according to the present invention is preferably a microbial strain comprising a nucleotide sequence having at least 99.9%, 99.91%, 99,92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99% or 100% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genome sequence having at least 99.8%, 99.9% or 100% identity to the whole genome of Streptomyces sp. Saigon 413 deposited in the Fungal Culture Collection under the deposit number CBS149411.

[0020] For example, the microbial strain may comprise a nucleotide sequence having at least 99.9% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity, or 100% identity to the whole genome of Streptomyces species Saigon 413 deposited at the Fungal Culture Collection under the deposit number CBS 149411. By way of another example, the microbial strain may comprise a nucleotide sequence having at least 99.91% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity, or 100% identity to the whole genome of Streptomyces species Saigon 413 deposited at the Fungal Culture Collection under the deposit number CBS 149411. By another example, the microbial strain may comprise a nucleotide sequence having at least 99.92% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity, or 100% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under the deposit number CBS 149411. By another example, the microbial strain may comprise a nucleotide sequence having at least 99.93% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity, or 100% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under the deposit number CBS 149411. By another example, the microbial strain may comprise a nucleotide sequence having at least 99.94% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity, or 100% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under the deposit number CBS 149411. By another example, the microbial strain may comprise a nucleotide sequence having at least 99.95% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity, or 100% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under the deposit number CBS 149411. By way of another example, the microbial strain may comprise a nucleotide sequence having at least 99.96% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity or 100% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under the accession number CBS149411.By another example, the microbial strain may comprise a nucleotide sequence having at least 99.97% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity, or 100% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under the deposit number CBS 149411. By another example, the microbial strain may comprise a nucleotide sequence having at least 99.98% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity, or 100% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under the deposit number CBS 149411. By another example, the microbial strain may comprise a nucleotide sequence having at least 99.99% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity, or 100% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under the deposit number CBS 149411. By another example, the microbial strain may comprise a nucleotide sequence having at least 100% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genomic sequence having at least 99.8%, 99.9% identity, or 100% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under the deposit number CBS 149411.

[0021] Suitably, the microbial strain may comprise a nucleotide sequence having at least 99.99% identity to SEQ ID NO: 1, and wherein the microbial strain comprises a genome sequence having at least 99.9% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under accession number CBS 149411. The term "isolated" in relation to a microbial strain means that the microbial strain has been separated from its native environment.

[0022] Surprisingly, it was found that the microbial strains according to the present invention exhibited pesticidal activity against plant pathogenic microorganisms found on various crops. Surprisingly, the microbial strains as disclosed herein produce one or more novel metabolites or metabolite combinations, which have pesticidal, preferably fungicidal activity. Surprisingly, the microbial strains as disclosed herein produce or are capable of producing at least one, at least two, at least three, at least four, at least five or all of one or more compounds selected from the group consisting of malenomycin, cyclothiazomycin C, and streptoglutarimide, oligosaccharide compounds according to compound I (the compound I comprises the oligosaccharide compounds according to C 53 H90 N2O 44 The molecular formula further characterized by the NMR spectra listed in Tables 1 and 2, preferably characterized by structural formula I), lipopeptides or salts thereof according to formula II (wherein R1 = CH3 or C2H5) and polyene compounds (which are characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0023] Preferably, the microbial strain as disclosed herein produces or is capable of producing malenomycin and at least one, at least two, at least three, at least four or at least five of the following compounds: cyclothiazolylmycin C, streptoglutarimide, an oligosaccharide compound according to compound I (the compound I comprises a molecule according to compound C 53 H 90 N2O 44 The molecular formula further characterized by the NMR spectra listed in Table 1 and Table 2, preferably characterized by structural formula I as disclosed herein), lipopeptides according to formula II as disclosed herein, or salts thereof (wherein R1=CH3 or C2H5) and polyene compounds (which are characterized by structural formula I according to C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0024] Preferably, the microbial strain as disclosed herein produces or is capable of producing at least one, at least two, or at least three of the following compounds: malenomycin, cyclothiazolylmycin C, and streptoglutarimide, and at least one, at least two, or at least three of the following compounds: an oligosaccharide compound according to compound I (the compound I comprising the oligosaccharide compound according to compound C 53 H 90 N2O 44 The molecular formula further characterized by the NMR spectra listed in Table 1 and Table 2, preferably characterized by structural formula I as disclosed herein), lipopeptides according to formula II as disclosed herein, or salts thereof (wherein R1=CH3 or C2H5) and polyene compounds (which are characterized by structural formula I according to C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0025] As used herein, the term "microbial strain, compound, metabolite or composition having pesticidal activity" or "pesticide" means a microbial strain, compound, metabolite or composition that controls, modifies or prevents the growth of pests. The term "pesticide effective amount" when used means the amount of such microbial strain, compound, metabolite or composition or combination of such compounds that can have an effect on the growth of pests. The effects of control or modification include all deviations from natural development, such as killing, blocking, etc., and prevention includes the formation of barriers or other defenses in or on plants to prevent pest infection.

[0026] As used herein, the term "microorganism strain, compound, metabolite or composition having fungicidal activity" or "fungicide" means a microorganism strain, compound, metabolite or composition that controls, modifies or prevents the growth of fungi. The term "fungicidal effective amount" when used means an amount of such a microorganism strain, compound, metabolite or composition or a combination of such compounds that is capable of producing an effect on the growth of fungi. The effects of control or modification 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.

[0027] Preferably, the microbial strain as disclosed herein produces or is capable of producing at least one compound selected from the group consisting of malenomycin, cyclothiazolylmycin C, streptoglutarimide, and at least one compound selected from the group consisting of an oligosaccharide compound according to Compound I (the compound I comprising the oligosaccharide compound according to Compound I 53 H 90 N2O 44 The molecular formula further characterized by the NMR spectra listed in Tables 1 and 2, preferably characterized by structural formula I), lipopeptides or salts thereof according to formula II (wherein R1 = CH3 or C2H5) and polyene compounds (which are characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0028] Suitably, the microbial strain as disclosed herein produces or is capable of producing malenomycin, cyclothiazolylmycin C, streptoglutarimide, an oligosaccharide compound according to Compound I (the compound I comprising the oligosaccharide compound according to Compound C 53 H 90 N2O 44 The molecular formula further characterized by the NMR spectra listed in Tables 1 and 2, preferably characterized by structural formula I), lipopeptides or salts thereof according to formula II (wherein R1 = CH3 or C2H5) and polyene compounds (which are characterized by the C 67 H 115 O25 N), wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0029] As used herein, the terms "percent identity" and "percent identity" 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) occurs in the two sequences to obtain 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 the "percent identity" is calculated with respect 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 for 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%.

[0030] In one embodiment, the microbial strain according to the present invention is Streptomyces coronavirinus, which is Streptomyces species Saigon 413 deposited in the Fungal Culture Collection Center with the deposit number CBS149411.

[0031] The present invention also relates to a composition comprising a Streptomyces species, preferably Streptomyces crown, and at least one of malenomycin and a compound selected from the group consisting of cyclothiazomycin C, and streptoglutarimide, an oligosaccharide compound according to compound I, the compound I comprising a 53 H 90 N2O 44 The molecular formula is further characterized by the NMR spectra listed in Table 2 and Table 3, preferably characterized by structural formula I,

[0032]

[0033] A lipopeptide according to formula II, or a salt thereof, wherein R1 = CH3 or C2H5,

[0034]

[0035] and a polyene compound, the polyene compound being obtained by 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10The optical absorption spectrum shown in FIG. has a molecular formula of C 67 H 115 NO 25 The polyene has a molecular weight of 1333.7758 g.

[0036] Suitably, the present disclosure relates to a composition comprising a strain of Streptomyces, preferably Streptomyces coronomicin, and at least one compound selected from the group consisting of: malenomycin, cyclothiazomycin C and streptoglutarimide, and at least one compound selected from the group consisting of: an oligosaccharide compound according to Compound I (the compound I comprising the oligosaccharide compound according to Compound C 53 H 90 N2O 44 , further characterized by the NMR spectra listed in Tables 2 and 3, preferably characterized by structural formula I), lipopeptides according to formula II or salts thereof (wherein R1 = CH3 or C2H5) and polyene compounds (which are characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0037] It was found that the compositions according to the invention have a surprising level of biological activity against phytopathogenic microorganisms, in particular against phytopathogenic fungi, for example Zymoseptoria, Puccinia, Mycorsphearella, Pyricularia, Rhizoctonia, Blumeria, Alternaria, Colletotrichum, Ramularia, Parastagonospora, Rhynchosporium, Oculimacula, Fusarium, Gaeumannomyces sp., Botrytis, or Sclerotinia, the phytopathogenic bacteria Xanthomonas and phytopathogenic oomycetes such as Aphanomyces sp. sp.)

[0038] Cyclothiazolycin C is a known compound. The structure of cyclothiazolycin C is disclosed on page 3 of WO 2015191789 and can be produced as disclosed in Example 4 of WO 2015 / 191789.

[0039] 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, which can be prepared according to the method disclosed in Example I of WO 2006 / 078939. Malonomicin can also be prepared according to the method disclosed in Examples IA and B of EP 1860939, or according to Law et al., 2018 (Nature Catalysts | Vol. 1 | Dec. 2018 | 977-984).

[0040] Streptoglutarimide is a known compound having formula III

[0041]

[0042] 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).

[0043] The composition according to the present invention preferably comprises malenomycin, cyclothiazolylmycin C and streptoglutarimide, and at least one, at least two or three of the compounds selected from the group consisting of: an oligosaccharide compound according to compound I (the compound I comprises a oligosaccharide compound according to compound C 53 H 90 N2O 44 The molecular formula further characterized by the NMR spectra listed in Tables 1 and 2, preferably characterized by structural formula I), lipopeptides or salts thereof according to formula II (wherein R1 = CH3 or C2H5) and polyene compounds (which are characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0044] The compositions disclosed herein are non-naturally occurring compositions.

[0045] Preferably, the composition according to the present invention comprises Streptomyces, preferably Streptomyces crown, or a microbial strain as disclosed herein, which produces or is capable of producing at least one, at least two, at least three, at least four, at least five or all of one or more compounds selected from the group consisting of malenomycin, cyclothiazomycin C, and streptoglutarimide, an oligosaccharide compound according to compound I (the compound I comprises a compound according to C 53 H 90 N2O 44 The molecular formula further characterized by the NMR spectra listed in Tables 1 and 2, preferably characterized by structural formula I), lipopeptides or salts thereof according to formula II (wherein R1 = CH3 or C2H5) and polyene compounds (which are characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0046] The lipopeptides according to Formula II disclosed herein include lipopeptides according to Formula II(a)

[0047]

[0048] The lipopeptide according to formula II(a) comprises a molecular formula C 55 H 85 N 11 O 19 and accurate mass 1203.602 g. The solubility of the lipopeptide according to formula I(a) in DMSO is higher than 10,000 ppm.

[0049] The lipopeptides according to formula II disclosed herein include lipopeptides according to formula II(b)

[0050]

[0051] The lipopeptide of formula II(b) comprises or has the molecular formula C 56 H 87 N 11 O 19 and accurate mass 1217.618 g. The solubility of the lipopeptide according to formula I(b) in DMSO is higher than 10,000 ppm.

[0052] Preferably, the composition comprises a strain of Streptomyces, wherein the strain is Streptomyces coronamicinus. Preferably, the composition comprises a strain of Streptomyces, suitably Streptomyces coronamicinus, comprising a genome sequence having at least 91%, 92%, 93%, 94%, preferably at least 95%, 96%, 97%, 98% or at least 99% identity with the whole genome of Streptomyces coronamicinus NRRL-3672 or with the whole genome of Streptomyces species Saigon413 deposited at the Fungal Culture Collection under the accession number CBS149411. In one embodiment, the composition comprises Streptomyces coronamicinus, comprising a genome sequence having 100% identity with the whole genome of Streptomyces species Saigon413 deposited at the Fungal Culture Collection under the accession number CBS149411. In one embodiment, the composition comprises Streptomyces coronamicinus, which is Streptomyces species Saigon413 deposited at the Fungal Culture Collection under the accession number CBS149411.

[0053] The microbial strain according to the present invention, or the Streptomyces strain as disclosed herein, preferably comprises 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: 71 to 115, preferably comprises 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: 91 and / or SEQ ID NO: 92.

[0054] In one embodiment, the microbial strain according to the present invention, or the Streptomyces strain as disclosed herein, comprises at least one nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to at least one of the nucleotide sequences of SEQ ID NOs: 2 to 46. The nucleotide sequences according to SEQ ID NOs: 2 to 46 comprise at least one nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to at least one of the nucleotide sequences of SEQ ID NOs: 2 to 46. Fig.11 The gene cluster shown in is used to produce the lipopeptide according to formula II.

[0055] Preferably, the microbial strain according to the present invention, and / or the Streptomyces strain as disclosed herein (e.g., Streptomyces coronamicinus) 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 NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID 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 ID NO: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 the one or more nucleotide sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto.Preferably, the microbial strain according to the present invention comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to at least one of the nucleotide sequences according to SEQ ID NO: 22 and / or 23. Suitably, the microbial strain according to the present invention comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence according to SEQ ID NO: 22. Suitably, the microbial strain according to the present invention comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence according to SEQ ID NO: 23.

[0056] In another embodiment, the microbial strain according to the present invention, and / or the Streptomyces strain as disclosed herein (e.g., Streptomyces coronamicinus) comprises 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 at least one of the amino acid sequences according to SEQ ID NOs: 116-139.

[0057] Preferably, the microbial strain according to the present invention and / or the Streptomyces strain (e.g. Streptomyces coronavirinus) comprises or contains at least one, preferably at least two, preferably at least three, preferably at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten, preferably at least eleven, preferably at least twelve, preferably at least thirteen, preferably at least fourteen, preferably at least fifteen, preferably at least sixteen, preferably at least seventeen, preferably at least eighteen, preferably at least nineteen, preferably at least twenty, preferably at least twenty-one, preferably at least twenty-two, preferably at least twenty-three, preferably all twenty-four of the following nucleotide sequences, which encode a protein having at least 80% identity, preferably at least 85%, preferably at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, preferably at least 99% identity, preferably 100% identity to the amino acid sequence according to SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138 and / or SEQ ID NO:139.

[0058] Preferably, the microbial strain according to the present invention, and / or the Streptomyces strain as disclosed herein (e.g., Streptomyces coronamicinus) comprises at least one nucleotide sequence encoding a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or having 100% identity to at least one of the amino acid sequences according to SEQ ID NO: 136 and / or SEQ ID NO: 137. Suitably, the microbial strain according to the present invention, and / or the Streptomyces strain as disclosed herein (e.g., Streptomyces coronamicinus) comprises a nucleotide sequence encoding a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or having 100% identity to the amino acid sequence according to SEQ ID NO: 136. Suitably, the microbial strain according to the present invention, and / or the Streptomyces strain as disclosed herein (e.g. Streptomyces coronavirinus) comprises a 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: 137.

[0059] In another embodiment, the microbial strain according to the present invention, and / or the Streptomyces strain as disclosed herein (e.g., Streptomyces coronavirinus) comprises at least one nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to at least one of the nucleotide sequences of SEQ ID NOs: 47 to 70. The nucleotide sequence according to SEQ ID NOs: 47 to 70 comprises Fig.12 The gene cluster shown in FIG. 1 is used to produce a gene having a C 67 H 115 NO 25 A polyene of the molecular formula wherein the polyene is further treated as Fig.10Preferably, the microbial strain according to the present invention, and / or the Streptomyces strain as disclosed herein (e.g., Streptomyces coronamicinus) comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of the nucleotide sequences according to SEQ ID NO: 67 and / or 68. Suitably, the microbial strain according to the present invention comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence according to SEQ ID NO: 67. Suitably, the microbial strain according to the present invention comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence according to SEQ ID NO: 68.

[0060] Preferably, the microbial strain according to the present invention and / or the Streptomyces strain (e.g., Streptomyces coronavirinus) 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 of the following nucleotide 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, 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 NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 or SEQ ID NO:70, or the one or more nucleotide sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto.

[0061] As disclosed herein, microbial strains or Streptomyces strains or Streptomyces crown can be naturally occurring microorganisms or recombinant microorganisms. Recombinant microorganisms can be produced by methods known to those skilled in the art. Recombinant microorganisms can be produced by transforming microorganisms with nucleotide sequences encoding proteins of amino acids according to SEQ ID NOs: 71 to 115 and / or encoding proteins of amino acids according to SEQ ID NOs: 116 to 139, preferably according to SEQ ID NOs: 2 to 46, preferably SEQ ID NOs: 22 and / or SEQ ID NOs: 23 nucleotide sequences and / or according to SEQ ID NOs: 47 to 70, preferably SEQ ID NOs: 67 and SEQ ID NOs: 68 nucleotide sequences to produce recombinant microorganisms.

[0062] Preferably, the composition of the present invention comprises an adjuvant, preferably an agriculturally acceptable adjuvant.The composition disclosed herein is preferably an agriculturally acceptable composition.

[0063] 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, humectants, repellents, attractants, feeding stimulants, compatibilizers, bactericides, antifreeze agents, defoamers, colorants, tackifiers and binders.

[0064] Suitable solvents and liquid vehicles include, for example, water, organic solvents, vegetable or animal oils, 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.

[0065] Suitable solid carriers include, for example, ammonium salts, talc, titanium dioxide, pyrophyllite clay, silica, attapulgite clay, kieselguhr, chalk, diatomaxeous earth, lime, calcium carbonate, bentonite, Fuller's earth, cottonseed hulls, wheat flour, soy flour, pumice, wood flour, walnut shell flour, and lignin.

[0066] Amounts of carriers typically range from 0.9% to 99.99% by weight of the composition.

[0067] The adjuvant may be a surfactant, a crystal inhibitor, a viscosity modifier, a suspending agent, a spray droplet modifier, a pigment, an antioxidant, a foaming agent, a defoamer, a light shielding agent, a compatibility agent, a masking agent, a neutralizing agent and a buffer, a corrosion inhibitor, a dye, a flavor enhancer, a spreading agent, a penetration aid, a micronutrient, an emollient, a lubricant and a fixative. The adjuvant may, for example, comprise an alkyl polyglucoside and / or polyoxyethylene (6) C9-C11 alcohol or methylcellulose.

[0068] Dispersants include, but are not limited to, surfactants and wetting agents. Typically, the one or more dispersants have low toxicity to the one or more microorganisms in the inoculant composition and the one or more plant parts to which the inoculant composition is to be applied.

[0069] The surfactant may be an ionic (cationic or anionic) or nonionic surfactant, such as an ionic or nonionic emulsifier, foam former known in the art, such as an acid (eg, polyacrylic acid), ester, ether, etc.

[0070] Suitable surfactants include polysorbates, for example polysorbate 20, such as 20. The composition as disclosed herein comprises the following amount of surfactant: 0.0005 wt / wt% to 0.5 wt / wt%, preferably 0.001 to 0.1 wt / wt%, preferably 0.002 to 0.08 wt / wt%, preferably 0.004 to 0.06 wt / wt%, preferably 0.005 to 0.04 wt / wt%, preferably 0.006 to 0.025% wt / wt% of polysorbate 20.

[0071] In one embodiment, the composition as disclosed herein is a fermentation broth, preferably a spray-dried fermentation broth or a freeze-dried fermentation broth, or a formulation. Spray-drying or freeze-drying of fermentation broth is known in the art.

[0072] The composition disclosed herein comprises the following cell count of Streptomyces coronarius: 1*10 2 Up to 1*10 13 cfu / g dry weight, for example 1*10 3 Up to 1*10 12 cfu / g dry weight, 2*10 3 Up to 2*10 11 cfu / g dry weight, 5*10 3 Up to 5*10 11 cfu / g dry weight, for example 1*10 4 Up to 1*10 10 cfu / g dry weight, 2*10 4 Up to 2*10 10 cfu / g dry weight, for example 1*10 5 Up to 1*10 9 cfu / g dry weight, 2*10 5 Up to 2*10 9 cfu / g dry weight, 5*10 5 Up to 5*10 9 cfu / g dry weight, 1*10 6 Up to 1*10 8cfu / g dry weight, e.g. 2*10 6 Up to 2*10 8 cfu / g dry weight.

[0073] Compositions disclosed herein include formulations. Thus, formulations include compositions disclosed herein. The formulations can be any suitable microbial strains, such as Streptomyces (e.g., Streptomyces coronarius) as disclosed herein, such as a composition of Streptomyces species Saigon 413 deposited in the Fungal Culture Collection Center under the accession number CBS149411.

[0074] Formulations of microbial strains are known in the art, for example, as disclosed in the Nouryon formulation toolbox of Croda CropCare and in: Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments (412 pages, December 6, 2012), ed. Burges HD, Springer, ISBN 978-94-011-4926-6. Preferably, the composition as disclosed herein is a formulation wherein the microbial strain as disclosed herein, e.g., Streptomyces coronamicinus, is formulated as an oil dispersion (OD), a non-aqueous dispersion (NAD) or a flowable formulation.

[0075] Formulations known in the art are, for example, emulsifiable concentrates, spreadable pastes, sprayable or dilutable solutions or suspensions, powders, dusts, granules and microcapsules.

[0076] Suspension concentrates are aqueous formulations in which finely divided solid particles of the active ingredient are suspended. Such formulations include anti-settling agents and dispersants, and may further contain wetting agents to enhance pesticidal activity, as well as defoamers and crystal growth inhibitors. When used, these concentrates are diluted in water and usually applied as a spray to the area to be treated. The amount of active ingredient may range from 0.5% to 95% of the concentrate.

[0077] Wettable powders are in the form of finely divided particles that disperse easily in water or other liquid carriers. These particles contain the active ingredient retained in a solid matrix. Typical solid matrices include Fuller's earth, kaolin, silica and other easily wet organic or inorganic solids. Wettable powders usually contain 5% to 95% of the active ingredient plus a small amount of a wetting agent, dispersant or emulsifier.

[0078] Emulsifiable concentrates are uniform liquid compositions dispersible in water or other liquids and may consist entirely of active ingredient liquid or solid emulsifiers, or may also contain liquid carriers such as xylene, heavy aromatic naphtha, isophorone and other non-volatile organic solvents. When used, these concentrates are dispersed in water or other liquids and are usually applied as a spray to the area to be treated. The amount of active ingredient may range from 0.5% to 95% of the concentrate.

[0079] Granular formulations include both extrudates and coarser granules and are usually applied to the area to be treated without dilution. Typical carriers for granular formulations include sand, fuller's earth, attapulgite clay, bentonite, montmorillonite, vermiculite, perlite, calcium carbonate, brick, pumice, pyrophyllite, kaolin, dolomite, plaster, wood flour, crushed corn cobs, crushed peanut shells, sugar, sodium chloride, sodium sulfate, sodium silicate, sodium borate, magnesium oxide, mica, iron oxide, zinc oxide, titanium oxide, antimony oxide, cryolite, gypsum, diatomaceous earth, calcium sulfate and other organic or inorganic materials that absorb active compounds or can be coated with active compounds. Granular formulations usually contain 5% to 25% active ingredients, which may include surfactants such as heavy aromatic naphtha, kerosene and other petroleum fractions, or vegetable oils; and / or adhesives such as dextrins, glues or synthetic resins.

[0080] Oil dispersions are solid active ingredients dispersed in oil, which is a carrier that is immiscible with water. Oil dispersions typically contain (by weight %) active ingredient (5%-60%) non-aqueous dispersant (1%-10% of solids) aqueous dispersant (1%-10% of solids) emulsifier (2%-10% of oil) rheology modifier (0.2%-5%) and oil carrier to constitute 100%. Suitable oils in oil dispersions can be mineral oil, paraffin oil, vegetable oil or methylated oil.

[0081] Suitable adjuvants, dispersants, emulsifiers and rheology modifiers in the oil dispersion depend on the type of oil and are known in the art. Suitable emulsifiers can be alcohol ethoxylates / alkoxylates, such as C16 / 18 ethoxylates, or C16 / C18 alkoxylates, or block copolymers. Adjuvants can be alkyl polyglucosides. Suitable rheology modifiers can be clay, hydrogenated castor oil and derivatives thereof, fumed silica polyamides, polyesters or agrilan ODS.

[0082] The oil dispersion may contain an adjuvant, wherein the adjuvant comprises an alkyl polyglucoside and / or a polyoxyethylene (6) C9-C11 alcohol. It has been found that in the curative treatment of Puccinia tritici and Puccinia recondita, the composition formulated as an oil dispersion and an adjuvant (e.g., an adjuvant comprising an alkyl polyglucoside and / or a polyoxyethylene (6) C9-C11 alcohol) according to the present invention resulted in improved reduction of fungal diseases.

[0083] Non-aqueous dispersions are liquid formulations in which solid ingredients are dispersed in a water-soluble vehicle. The solid ingredients are uniformly suspended in the vehicle but not dissolved.

[0084] Flowable formulations contain solid particles in a liquid, which is usually water. Flowable concentrates also include suspension concentrates. Flowable formulations typically contain (in wt%) active ingredient (5%-60%), dispersant / wetting agent (1%-10%), rheology modifier (0.1%-0.1%), biocide (0.1%), antifreeze (5%-10%), defoamer (0.2%), adjuvant (up to 25%) and water to make up 100%.

[0085] As disclosed herein, the isolated microbial strains or compositions of the present invention can advantageously be in the form of a soluble concentrate (SL), or in the form of a flowable concentrate (FS) for seed treatment, or in the form of a suspension concentrate (SC), and can more preferably be a seed treatment slurry that can be applied to seeds. Slurries for seed treatment applications are well known in the art. For example, a slurry can contain one or more active ingredients (e.g., isolated microbial strains or compositions of the present invention, in the form of a commercial product or a non-commercial product), which is mixed with water and optionally mixed with at least one polymer to optimize adhesion around plant propagation materials.

[0086] Each active ingredient can be applied to the plant propagation material separately from different compositions, or these active ingredients can be gathered in the same composition (premix composition). The composition can contain one or more active ingredients in an amount of about 0.001% to about 99% by weight of the total weight of the composition. Suitably, the composition contains one or more active ingredients in an amount of about 0.001% to about 60% by weight of the total weight of the composition.

[0087] The solid carrier can be a natural or synthetic solid material that is insoluble in water. Such carriers are usually inert and are acceptable in agriculture, particularly on treated seeds or other propagation materials. For example, it can be selected from clay, diatomaceous earth, natural or synthetic silicates, titanium dioxide, magnesium silicate, aluminum silicate, talc, pyrophyllite clay, silicon dioxide, attapulgite clay, diatomaceous earth (kieselguhr), chalk, limestone, calcium carbonate, calcium montmorillonite, bentonite, fuller's earth, cottonseed hulls, wheat flour, soy flour, pumice, wood flour, ground walnut shell powder, lignin, etc.

[0088] Thus, the isolated microbial strain and / or the one or more compositions according to the present invention may preferably be attached to a propagation material, such as a seed.

[0089] The compositions according to the present invention comprise coating compositions, which relate to liquid compositions for at least partially or completely covering and / or moistening plant propagation material, more preferably seeds.

[0090] The compositions according to the invention are particularly suitable for coating applications on plant propagation materials, especially seeds.

[0091] Typically, tank-mix formulations for seed treatment applications contain 0.25% to 80% by weight, especially 1% to 75% by weight, of one or more active ingredients, for example an isolated microbial strain or composition as disclosed herein, and 99.75% to 20% by weight, especially 99% to 25% by weight, of solid or liquid adjuvants (e.g. including solvents such as water), wherein these adjuvants may be surfactants, in an amount of 0 to 40% by weight, especially 0.5% to 30% by weight, based on the total weight of the tank-mix formulation.

[0092] Typically, premix formulations for seed treatment applications comprise 0.5% to 99.9%, especially 1% to 95% by weight of one or more active ingredients, for example an isolated microbial strain or composition as disclosed herein, and 99.5% to 0.1%, especially 99% to 5% by weight of solid or liquid adjuvants (e.g. including solvents such as water), wherein these adjuvants may be surfactants, in an amount of 0 to 50% by weight, especially 0.5% to 40% by weight, based on the total weight of the premix formulation.

[0093] Whereas commercial products will preferably be formulated as concentrates (eg, premix compositions (formulations)), the end user will typically use a diluted formulation (eg, a tank-mix composition).

[0094] Preferably, a composition, such as a preparation of Streptomyces coronamicin as disclosed herein, such as Streptomyces species Saigon 413 deposited in the Fungal Culture Collection under the accession number CBS149411, comprises 10 to 60 wt / wt% dry weight, preferably 20 to 50 wt / wt% dry weight of a spray-dried or freeze-dried fermentation broth of Streptomyces coronamicin (e.g. Streptomyces species Saigon 413 deposited in the Fungal Culture Collection under the accession number CBS149411).

[0095] In one embodiment, the isolated microbial strain and / or composition of the present invention further comprises at least one other active ingredient other than a Streptomyces strain (e.g., Streptomyces crownii) or an isolated microbial strain and composition as disclosed herein, and is used in the method of the present invention and is applied simultaneously or sequentially with the microbial strain and / or composition of the present invention. The active ingredient as defined herein has fungicidal and / or insecticidal and / or herbicidal activity or has activity as a plant growth regulator. The isolated microbial strain or composition of the present invention can be mixed with one or more other ingredients having pesticidal activity, and the other ingredients are, for example, fungicides, insecticides, herbicides, bactericides, acaricides, nematicides 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.

[0096] Other ingredients with pesticidal activity (e.g., fungicidal activity) can produce unexpected synergistic activity. Other ingredients with pesticidal activity and / or as plant growth regulators can be combined with microbial strains or compositions 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 for separate fungicidal, insecticidal or weeding applications, as part of a fungus, insect or grass control program that runs through part or the entire growing season.

[0097] At least one additional ingredient having pesticidal activity and / or being a plant growth regulator may be any suitable known fungicide, insecticide, herbicide and / or plant growth regulator. At least one additional ingredient having pesticidal activity and / or being a plant growth regulator may be from a chemical source or a biological source, for example from a plant or microbial source.

[0098] 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.

[0099] The isolated microbial strain or composition according to the invention can induce plant resistance. Priming is a mechanism leading to a physiological state that enables plants to respond more quickly and / or more effectively after exposure to biotic or abiotic stresses, as described, for example, in the review article: P. Aranega-Bou et al. Priming of plant resistance by natural compounds. Hexanoic acid as a model. Front. Plant. Sci. 1, October 2014.

[0100] The composition according to the invention and at least one further active ingredient are preferably in a mixing ratio of from 100:1 to 1:6000, in particular from 50:1 to 1:50, more particularly in a ratio of from 20:1 to 1:20, even more particularly from 10:1 to 1:10, very particularly from 5:1 and 1:5, particularly preferably in a ratio of from 2:1 to 1:2, and a ratio of from 4:1 to 2:1 is likewise preferred, in particular in a ratio of 1:1, or 5:1, or 5:2, or 5:3, or 5:4, or 4:1, or 4:2, or 4:3, Or 3:1, or 3:2, or 2:1, or 1:5, or 2:5, or 3:5, or 4:5, or 1:4, or 2:4, or 3:4, or 1:3, or 2:3, or 1:2, or 1:600, 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 ratio. Those mixing ratios are by weight. These mixtures may be used in a method of controlling pests which comprises applying to the pests or their environment a composition comprising a mixture as described above, excluding methods for treating the human or animal body by surgery or therapy and diagnostic methods performed on the human or animal body.

[0101] Compositions comprising an isolated microbial strain or composition of the invention and a mixture of one or more active ingredients as described above can be applied, for example, in a single "ready-to-use" form, in a combined spray mixture consisting of separate formulations of the single active ingredients (e.g., a "tank mix"), and in combination with the separate active ingredients when applied in a sequential manner (i.e., one after another within a suitably short period of time, e.g., hours or days).

[0102] On the other hand, the present invention also relates to a method for producing a microbial strain according to the present invention as disclosed above or a composition according to the present invention as disclosed above, the method comprising culturing the microbial strain or Streptomyces strain crown mycin Streptomyces in a suitable fermentation medium under suitable fermentation conditions, and optionally comprising a step of recovering the microbial strain or composition. Fermentation broth is usually produced during or when culturing a microbial strain or Streptomyces strain (e.g. crown mycin Streptomyces strain). Suitable fermentation conditions for culturing Streptomyces species are known to those skilled in the art.

[0103] Cultivating a microbial strain as disclosed herein, or a Streptomyces strain (e.g., a Streptomyces coronomicinus strain as disclosed herein) includes culturing the microbial strain under aerobic conditions at a temperature of 15 to 45 degrees Celsius, preferably a temperature of 20 to 35 degrees Celsius, preferably a temperature between 25 to 32 degrees Celsius, in the presence of a carbon source and a nitrogen source. A suitable carbon source can be molasses, such as beet or cane molasses, polysaccharides, flour, starch, sugar or glucose. A suitable nitrogen source can be casein hydrolyzate, tryptone, ammonium sulfate, ammonia, yeast extract, peptone or urea. The method for producing a microbial strain or a Streptomyces coronomicinus according to the present invention can be carried out in batch culture, fed-batch culture or continuous culture.

[0104] Preferably, the method according to the present invention comprises culturing a microbial strain or a Streptomyces strain as disclosed herein (suitably Streptomyces coronomicin), wherein the microbial strain or the Streptomyces strain (suitably Streptomyces coronomicin) produces malenomycin and at least one, at least two, at least three, at least four or at least five compounds selected from the group consisting of cyclothiazomycin C, streptoglutarimide, an oligosaccharide compound according to compound I (the compound I comprising the oligosaccharide compound according to compound C 53 H 90 N2O 44The molecular formula further characterized by the NMR spectra listed in Table 1 and Table 2, preferably wherein compound I is further characterized by structural formula I) and a lipopeptide or a salt thereof according to formula II (wherein R1=CH3 or C2H5) and a polyene compound (which is characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectra shown in

[0105] Suitably, the method according to the present invention comprises culturing a microbial strain or a Streptomyces strain (e.g., Streptomyces coronamicinus), wherein the microbial strain or the Streptomyces strain produces at least one, at least two or at least three of the compounds selected from the group consisting of: malenomycin, cyclothiazomycin C, streptoglutarimide, and at least one, at least two or at least three of the compounds selected from the group consisting of: an oligosaccharide compound according to Compound I (the compound I comprising the oligosaccharide according to Compound C 53 H 90 N2O 44 The molecular formula further characterized by the NMR spectra listed in Table 1 and Table 2, preferably wherein compound I is further characterized by structural formula I) and a lipopeptide or a salt thereof according to formula II (wherein R1=CH3 or C2H5) and a polyene compound (which is characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 Preferably, the microbial strain or Streptomyces strain (e.g., Streptomyces crown mycin) produces a compound selected from the group consisting of: malenomycin, cyclothiazomycin C, streptoglutarimide and a compound according to compound I (the compound I comprises a compound according to C 53 H 90 N2O 44 The molecular formula further characterized by the NMR spectra listed in Table 1 and Table 2, preferably wherein compound I is further characterized by structural formula I) and a lipopeptide or a salt thereof according to formula II (wherein R1=CH3 or C2H5) and a polyene compound (which is characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0106] The method according to the present invention may further include a step of recovering the microbial strain or composition according to the present invention. Recovering the microbial strain or composition of the present invention may include centrifuging or filtering the fermentation broth. Preferably, the recovery includes a step of drying the fermentation broth, for example by spray drying or freeze drying. Spray drying or freeze drying are methods known to those skilled in the art. The composition comprises the compounds malenomycin, cyclothiazomycin C, streptoglutarimide and an oligosaccharide compound according to compound I (the compound I comprises the oligosaccharide compound according to C 53 H 90 N2O 44 The molecular formula further characterized by the NMR spectra listed in Tables 1 and 2, preferably wherein Compound I is further characterized by structural formula I) and a lipopeptide or a salt thereof according to Formula II (wherein R1 = CH3 or C2H5) and / or a polyene compound (which is characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 These compounds can be recovered by suitable methods known in the art, such as via crystallization or chromatography (eg, HPLC).

[0107] The microbial strains of the present invention and the Streptomyces strains as disclosed above, and the examples such as Streptomyces coronamicinus, are also applicable to the microbial strains, Streptomyces strains and Streptomyces coronamicinus in the processes and methods of the present invention.

[0108] The method according to the invention may further comprise the step of formulating the microbial strain or composition according to the invention into a suitable formulation or composition as defined above.

[0109] In another aspect, the present invention relates to a method for controlling or preventing plants, plant propagation materials and / or harvested food, non-food and non-feed crops from being infected by phytopathogenic microorganisms, the method comprising treating the plant, plant propagation material and / or harvested food crops by applying an effective amount of Streptomyces coronamicin, a microbial strain of the present invention or a composition to the plant, to a part thereof or its location, the plant propagation material and / or the harvested food crops. Surprisingly, it was found that Streptomyces coronamicin (e.g., a microbial strain according to the present invention) or a composition according to the present invention is very effective in treating phytopathogenic microorganisms on plants. Streptomyces coronamicin (e.g., a microbial strain according to the present invention) or a composition according to the present invention surprisingly has insecticide activity and activity against fungi, bacteria and oomycetes.

[0110] The method according to the invention does not include methods of treatment of the human or animal body by surgery or therapeutic and diagnostic methods performed on the human or animal body.

[0111] 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.

[0112] The term "plant propagation material" should be understood to mean the reproductive parts of the plant, such as seeds, which can be used for the propagation of the plant; and vegetative materials, such as cuttings or tubers (e.g., potatoes), roots, fruits, bulbs, rhizomes or parts of plants. Before sowing or planting the plant propagation material, the material can be treated with the isolated microbial strains or compositions of the present invention. Alternatively, during sowing or planting, the plant propagation material can be treated with the isolated microbial strains or compositions of the present invention. In addition, before or during the planting of the previously treated propagation material, the isolated microbial strains or compositions of the present invention can be applied to the propagation material. The isolated microbial strains or compositions of the present invention can be applied during sowing. The isolated microbial strains can also be used for plant propagation materials derived from plants grown in greenhouses and / or during transplanting. More preferably, the plant propagation material is a plant seed.

[0113] The term plant refers to "useful plants" or "crops". The words "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 grasses and zoysias; herbs such as basil, borage, chives, cilantro, lavender, lovage, mint, oregano, parsley, rosemary, sage and thyme; legumes such as beans, lentils, peas and soybeans; nuts such as almonds, cashews, groundnuts, hazelnuts, peanuts, pecans, pistachios and walnuts; palms such as oil palms; ornamental plants such as flowers, shrubs and trees; other trees such as cocoa, coconut, olive and rubber trees; vegetables such as asparagus, eggplant, broccoli, cabbage, carrots, cucumbers, garlic, lettuce, zucchini, melon, okra, onions, peppers, potatoes, pumpkins, rhubarb, spinach and tomatoes; and vines such as grapes. The term "plant" also includes woody crops such as pine or woody plants.

[0114] 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 such as primisulfuron, prosulfuron and trifloxysulfuron, EPSPS (5-enol-pyruvyl-shikimate-3-phosphate-synthase) inhibitors, GS (glutamine synthetase) inhibitors or PPO (protoporphyrinogen oxidase) inhibitors as a result of conventional breeding or genetic engineering methods.

[0115] 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.

[0116] 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.

[0117] Any suitable plant, plant propagation material or food or fodder crop can all be handled in the method according to the present invention as defined herein.Preferably, plant, plant propagation material or food crop comprise following or are following: wheat, barley, rice, corn, soybean, beet, banana, tomato, cucumber and / or groundnut.

[0118] Phytopathogenic microorganisms affected by the isolated microbial strain or composition according to the invention are fungi and fungal vectors of diseases and phytopathogenic bacteria and viruses. The phytopathogenic microorganisms in the method according to the invention include the following fungi and fungal vectors of diseases and phytopathogenic bacteria:

[0119] Absidia corymbifera, Albugo candida, Altemaria 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, Botryosphaeria spp., including B. cinerea, Bremia lactucae, Cadophora gregata, Candida spp., including C. albicans, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, C. tropicalis, Cephaloascus fragrans, Ceratocystis spp., Cercospora spp.), including 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 cassiicola, Cryptococcus neoformans, Diaporthe spp., Dickeya zeae), Didymella spp., Drechslera spp., Elsinoe spp., Epidermophyton spp., Eremothecium gossypiim, Erwinia spp., including E. amylovora, E. carotovora, Erysiphe spp. spp.), including E. cichoracearum, E. necator, Eutypalata, Fusarium species, including F. culmorum, F. graminearum, F. langsethiae, F. moniliforme, F. oxysporum, F. po ae) Fusarium proliferatum, F. pseudograminearum, F. sacchari, F. sambucinum, F. subglutinans, F. solani, F. sporotrichioides, F. 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, Gymnosporangium juniperi-virginianae, Helminthosporium spp., Hemileia spp., Histoplasma spp.), including H. capsulatum, Hyaloperonospora parasitica, Kabatiella zeae, Laetisaria fuciformis, Leptographium lundbergii, Leveillula taurica, Lophodermium seditiosum, Microdochium majus, Microdochium nivale, Microsporum spp., Monilinia spp., including M. fructicola, Monographella spp., including M. nivalis, Mucor spp. spp.), Mycosphaerella spp., including M. arachidis, M. fijiensis, M. graminicola, M.pomi, Nakataea oryzae, Neopseudocercosporella spp., Oncobasidium theobromaeon, Ophiostoma spp., Pantoea stewartia, Paracoccidioides spp., Parastagonosporanodorum, 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. destructor, Phaeosphaeria nodorum, Phakopsora pachyrhizi, Phellinus igniarus, Phialophora spp., Phlyctema vagabunda, Phoma spp., Phomopsis viticola, Phyllactospora 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), Polymyxagraminis, Polymyxa betae, Pseudocercosporafijiensis, Pseudocercosporella herpotrichoides, Pseudomonas spp., including P. syringae, Pseudoperonospora spp., including P. cubensis, P. humuli, Pseudopeziza tracheiphila), Pseudopyrenochaetalycopersici, Puccinia species, including P. hordei, P. recondita, P. striiformis, P. triticina, Pyrenopeziza spp., Pyrenophora spp., including P. oryzae, Pythium spp., including P. ultimum, Ralstonia solanacearum, Pythium species, Rathayibacter spp., Remotididymella destructiva, Rhizoctonia species, Rhizomucor microsporus pusillus, Rhizopusarrhizus, Rhizosporium species, Robbsia andropogonis, Sarocladium oryzae, Scedosporium spp., including S. apiospermum and S. prorifans, Schizothyrium pomi, Sclerophthora macrospora, Sclerotinia species, including S. sclerotiorum, Sclerotium spp., Septoria spp., including S. nodorum, S.tritici), Setosphaeria turcica, Sphaerotheca macularis, Sphaerotheca fusca (Sphaerotheca fuliginea), Spiroplasma kunkelii, Porothorix spp., Stagonospora nodorum, Stagonosporopsis cucurbitacearum, Stemphylium spp., Stenocarpella macrospora, Stereum hirsutum, Streptomyces spp., Thanatephorus cucumeris, Thielaviopsis basicola), Tilletiaspp, Tranzschelia discolor, Trichoderma spp., including T. harzianum, T. pseudokoningii, T. viride, Trichophyton spp., Typhula spp., Uncinulanecator, Urocystis spp., Uromyces spp., Ustilago spp., Venturia spp., including V. inaequalis, Verticillium spp. spp), Wilsonia carpophilus, or Xanthomonas species, including Xanthomonas oryzae and Xanthomonas campestris, Xylella spp, Psoralea coli.

[0120] The phytopathogenic microorganisms found to be unexpectedly affected by the Streptomyces coronomicinus, the microbial strains or the compositions according to the invention are fungi, for example fungi belonging to the genera: Puccinia graminis, Puccinia spp., Mycosphaeria spp., Pyrospora spp., Rhizoctonia spp., Graminella graminis, Alternaria spp., Colletotrichum lagenarium, Paraconidia spp., Rhizoctonia spp., Eye spot fungus spp., Fusarium spp., Acrocystis spp., Botrytis spp., or Sclerotinia spp., preferably fungi belonging to the following species: Puccinia graminis, Puccinia occidentalis, Puccinia spp. stripe, Mycosphaeria fijiensis, Mycosphaeria peanut, Magnaporthe oryzae, Rhizoctonia solani, Blumeria graminis f.sp. tritici, Alternaria solani, Colletotrichum lagenarium, Ramularia spp. collo-cygni), Triticum aestivum, Rhynchosporium secalis, Oculimacula yallandae, Fusarium avenaceum, Fusarium graminearum, Fusarium leucoderma, Fusarium pseudosporioides, Fusarium submyxomorpha, Fusarium pseudograminearum, F. verticillioides, F. fujikuroi, Fusarium submyxomorpha, Fusarium oxysporum, for example, F. oxysporum f. sp. cubense, F. oxysporum f. sp. melonis, F. oxysporum f. sp. vasinfectum, F. oxysporum tomato oryzae pv. oryzae, or oomycetes, for example, from the genus Aphanomyces, preferably Aphanomyces cochlioides.

[0121] Control or prevention means reducing infestation by plant pathogenic microorganisms, especially fungi, until improvement occurs.

[0122] Suitably, the Streptomyces coronomicinus, or isolated microbial strains or compositions of the invention are applied preventively (meaning before the development of the disease) or curatively (meaning after the development of the disease). It has been surprisingly found that both preventive and curative application of the compositions according to the invention lead to a reduction in infection by phytopathogenic microorganisms.

[0123] A preferred method for controlling or preventing plants from being infected by phytopathogenic microorganisms (especially fungi or insects) includes foliar application of Streptomyces coronamicin as disclosed herein or isolated microbial strains or compositions according to the present invention. The frequency of application and the rate of application will depend on the risk of infection by the corresponding pathogen or insect. However, Streptomyces coronamicin as disclosed herein or isolated microbial strains or compositions according to the present invention can also penetrate plants through roots via the soil (systemic action) in the following manner: saturating the plant site with a liquid formulation, or applying Streptomyces coronamicin as disclosed herein or isolated microbial strains or compositions according to the present invention to the soil in solid form (e.g., in granular form) (soil application). In rice crops, such granules can be applied to irrigated paddies.

[0124] The phytopathogenic microorganisms which have been found to be unexpectedly affected by Streptomyces coronatus, for example in foliar application, the microbial strains or compositions according to the invention are fungi belonging to the genera Psoralea, Puccinia, Mycosphaeria, Pyrospora, Rhizoctonia, Graminus, Alternaria, Colletotrichum, Cylindrospermum, Parapolyspora, Rhizoctonia, Ophiocordyceps, Fusarium, Acrocystis species, Botrytis, or Sclerotinia, preferably belonging to the genera The following fungi: wheat leaf blight pathogen, concealed stem rust, stripe stem rust, fiji mycosphaeria, peanut mycosphaeria, rice blast pathogen, solani, oryzae, rice yellow monosporus pathogenic variant, wheat-specific type of powdery mildew of Poaceae, solanum chain spore, cucurbit anthracnose pathogen, swan neck column septum, wheat polyspora leaf blight pathogen, rye beak spore, Aaron's eyespot pathogen, avenae Fusarium, graminearum Fusarium, or bacteria belonging to the genus Xanthomonas, preferably Xanthomonas oryzae.

[0125] In one embodiment, a method for controlling or preventing plants, plant propagation materials and / or harvested food crops from being infected by plant pathogenic microorganisms is disclosed, wherein an effective amount of Streptomyces coronamicinus, a microbial strain of the present invention, or a composition as disclosed herein is applied to the plant, a part thereof, or a site thereof, the plant propagation material and / or the harvested food crop, wherein the plant pathogenic microorganisms are fungi belonging to the following genera: Psoralea corylifolia, Puccinia spp., Mycosphaeria spp., Pyrospora spp., Rhizoctonia spp., Xanthomonas spp., Graminaceae spp., Alternaria spp., Colletotrichum spp., Columnar The fungus is preferably a fungus belonging to the genus Septoria, Paraspora, Rhizoctonia, Eyespot Psora, Fusarium, Acrocystis species, Botrytis, or Sclerotinia, preferably a fungus belonging to the following: Psoralea tritici, Puccinia occidentalis, Puccinia streak, Mycosphaeria fijiensis, Mycosphaeria peanut, Magnaporthe grisea, Rhizoctonia solani, Xanthomonas oryzae pv. oryzae, Bryonia graminearum, Alternaria solani, Colletotrichum cucurbitae, Swan neck column Septoria, Psoralea tritici, Rhizoctonia solani, Aaron's Eyespot Psora, Fusarium avenae, or Fusarium graminearum, and wherein the plant comprises wheat, barley, rice, corn, soybean, sugar beet, banana, tomato, cucumber, and / or peanut.

[0126] In one embodiment, the method for controlling or preventing plants from being infected by plant pathogenic microorganisms comprises applying the Streptomyces coronamicin as disclosed herein or the isolated microbial strain or composition according to the present invention, wherein the plant is wheat, and the plant pathogenic microorganisms are fungi belonging to the following genera: Puccinia spp., Puccinia graminis, Puccinia graminis, Puccinia graminis, Puccinia graminis, preferably Puccinia graminis, ...

[0127] In one embodiment, the method for controlling or preventing infection of plants by phytopathogenic microorganisms comprises applying Streptomyces coronamicinus as disclosed herein or an isolated microbial strain or composition according to the invention, wherein the plant is barley and the phytopathogenic microorganisms are fungi belonging to the following: Cylindrospermum species, Rhynchosporium species, preferably Cylindrospermum swan-necked or Rhynchosporium secalis.

[0128] In one embodiment, the method of controlling or preventing infection of plants by phytopathogenic microorganisms comprises applying Streptomyces coronamicinus as disclosed herein or an isolated microbial strain or composition according to the invention, wherein the plant is banana and the phytopathogenic microorganisms are fungi belonging to the genus Mycosphaeria, preferably Mycosphaeria fijiensis.

[0129] In one embodiment, the method for controlling or preventing plants from being infected by phytopathogenic microorganisms comprises applying Streptomyces coronamicin as disclosed herein or an isolated microbial strain or composition according to the present invention, wherein the plant is rice and the phytopathogenic microorganisms are fungi belonging to the genus Pyricularia, Rhizoctonia, preferably Magnaporthe oryzae, Rhizoctonia solani, or bacteria belonging to the genus Xanthomonas, preferably Xanthomonas oryzae pv. oryzae.

[0130] In one embodiment, the method of controlling or preventing infection of plants by phytopathogenic microorganisms comprises applying the Streptomyces coronamicinus as disclosed herein or the isolated microbial strain or composition according to the invention, wherein the plant is tomato and the phytopathogenic microorganisms are fungi belonging to the genus Alternaria, preferably Alternaria solani.

[0131] In one embodiment, the method for controlling or preventing infection of plants by phytopathogenic microorganisms comprises applying the Streptomyces coronamicin as disclosed herein or the isolated microbial strain or composition according to the present invention, wherein the plant is cucumber and the phytopathogenic microorganisms are fungi belonging to the following: Colletotrichum species, preferably Colletotrichum cucurbitae.

[0132] In one embodiment, the method for controlling or preventing infection of plants by phytopathogenic microorganisms comprises applying Streptomyces coronamicinus as disclosed herein or an isolated microbial strain or composition according to the invention, wherein the plant is peanut and the phytopathogenic microorganisms are fungi belonging to the genus Mycosphaeria, preferably Mycosphaeria peanut.

[0133] It has also been surprisingly found that Streptomyces coronamicinus or an isolated microbial strain or composition according to the invention as disclosed herein can move throughout the stem or leaves and exhibit fungicidal activity on other parts of the plant than where it is applied.

[0134] It is also possible to use the isolated microbial strains and / or compositions according to the present invention as a coating agent for treating plant propagation materials (e.g., seeds, such as fruits, tubers or grains) or plant cuttings, for protection from fungal infections and from plant pathogenic harmful organisms present in the soil. Propagation materials can be treated with isolated microbial strains and / or compositions according to the present invention before planting: for example, seeds can be covered before sowing. It is also possible to apply the isolated microbial strains and / or compositions according to the present invention to grains (coating) by dipping seeds in liquid formulations or by coating them with solid formulations. It is also possible to apply the isolated microbial strains and / or compositions according to the present invention to the planting site when planting propagation materials, for example, to the furrows of seeds during sowing. This paper discloses such methods for treating plant propagation materials and the plant propagation materials so treated.

[0135] The Streptomyces coronamicins as disclosed herein or the isolated microbial strain or composition according to the present invention can also be applied to seeds by impregnating the seeds or tubers with a liquid formulation, or coating them with a solid formulation. Surprisingly, it was found that the Streptomyces coronamicins as disclosed herein or the isolated microbial strain or composition according to the present invention effectively reduced fungal and oomycete infections on seeds.

[0136] In one embodiment, the method for controlling or preventing plants from being infected by plant pathogenic microorganisms comprises treating seeds, wherein an effective amount of Streptomyces coronamicinus, a microbial strain of the present invention, or a composition as disclosed herein is applied to the seeds, wherein these plant pathogenic microorganisms are selected from Fusarium solani, Fusarium graminearum, Fusarium graminearum, Fusarium pseudosporangium, Fusarium submyxocarpus, Fusarium pseudograminearum, Fusarium verticillium, Fusarium fujikura, Fusarium submyxocarpus, Fusarium oxysporum, for example, Fusarium oxysporum Cuban specialization type, Fusarium oxysporum melon specialization type, Fusarium oxysporum wilt specialization type, Fusarium oxysporum tomato specialization type, Aspergillus graminearum, Botrytis cinerea, or Sclerotinia sclerotiorum and oomycetes, for example, oomycetes belonging to the genus Aphanomyces, preferably Aphanomyces spiralis.

[0137] Preferably, the seeds are from barley, wheat, corn, sugar beet or soybean. The terms edamame, soybean or soya bean are used interchangeably herein.

[0138] For the purpose of germination and growth of plants, seed treatment can occur on unsown seeds, and the term "unsown seeds" is meant to include seeds at any period between the harvest of seeds and the sowing of seeds in the land. Treatment of unsown seeds is not intended to include those practices in which the microbial strains or compositions of the present invention are applied to the soil, but rather includes any application practice that targets seeds during the sowing / planting process.

[0139] In one aspect, the present invention relates to plants or plant propagation material treated with an isolated microbial strain or a composition according to the invention.

[0140] The treated plant propagation materials of the present invention can be handled in the same manner as conventional plant propagation materials. These treated plant propagation materials can be stored, handled, sown and cultivated in the same manner as any other pesticide-treated materials.

[0141] Although it is believed that the method of the present invention can be applied to seeds in any physiological state, it is preferred that the seeds are in a sufficiently persistent state that the state does not cause damage during the treatment process. Typically, the seeds are seeds that have been harvested from the field; removed from the plant; and separated from any cobs, stems, husks, and surrounding pulp or other non-seed plant materials. The seeds are also preferably biologically stable to the extent that the treatment does not cause biological damage to the seeds. It is believed that the treatment can be applied to seeds at any time between the harvesting of the seeds and the sowing of the seeds or at any time during the sowing process (seed-directed application).

[0142] Seed treatment application techniques are well known to those skilled in the art, and they can be readily employed in the context of the present invention.

[0143] A method of applying the composition according to the invention comprises spraying or wetting the plant propagation material with the aqueous liquid formulation, or mixing the plant material with such a liquid formulation. In addition, prior to application, the composition according to the invention can be diluted with water by simple mixing at ambient temperature to prepare a farm seed treatment formulation.

[0144] In one embodiment, the method comprises administering an effective amount of Streptomyces coronamicinus, or an isolated microbial strain, or a composition according to the present invention as disclosed above, wherein the effective amount comprises 2*10 2 Up to 5*10 17 , 3*10 2 Up to 5*10 16 , 5*10 2 Up to 5*10 15 , 2*10 2 Up to 5*10 14 , 2*10 2 Up to 5*10 13 , preferably 5*10 2 Up to 5*10 12 , 1*10 3 Up to 5*10 11 , 5*10 3 Up to 1*10 11 , 1*10 4 Up to 5*10 10 , 5*10 4 Up to 1*10 10 , 1*10 5 Up to 5*10 9 , 5*10 5 Up to 1*10 9 , 1*10 6 Up to 5*10 8 , 5*10 6 Up to 1*10 8colony forming units (cfu) of the Streptomyces coronomicinus or the isolated microbial strain or composition per hectare.

[0145] The effective amount of the Streptomyces coronomicins or isolated microbial strains or compositions according to the present invention as disclosed above comprises 0.1 g to 10 kg / hectare (ha), such as 0.5 g to 5 kg, such as 1 g to 1 kg / ha, such as 5 g to 500 g / ha, such as 10 g to 200 g / ha, such as 50 to 100 g / ha. The weights in g and kg are the dry weight of the Streptomyces coronomicins, microbial strains or compositions.

[0146] In one embodiment, the method according to the invention comprises treating plant propagation material, wherein the plant propagation material is seeds and the effective amount comprises 5 x 10 2 Up to 5x10 15 , 2x10 3 Up to 5x10 14 , 5x10 3 Up to 5x10 13 , 2x10 5 Up to 5x10 12 , preferably 5*10 2 Up to 5*10 12 , 1*10 3 Up to 5*10 11 , 5*10 3 Up to 1*10 11 , 1*10 4 Up to 5*10 10 , 5*10 4 Up to 1*10 10 , 1*10 5 Up to 5*10 9 , 5*10 5 Up to 1*10 9 , 1*10 6 Up to 5*10 8 , 5*10 6 Up to 1*10 8 Colony forming units (cfu) of Streptomyces coronamicinus or isolated microbial strains or compositions according to the present invention.

[0147] When the plant propagation material is seeds, the effective amount of the Streptomyces coronamicins or the isolated microbial strain or composition according to the present invention as disclosed above can also include 0.0001g to 100g / kg of seeds, for example 0.0005g to 80g / kg of seeds, for example 0.001g to 50g / kg of seeds, for example 0.005g to 10g / kg of seeds, the weight being g dry weight of the Streptomyces coronamicins or the microbial strain or composition / kg dry weight of seeds.

[0148] In another aspect, the present invention relates to the use of Streptomyces coronamicinus or an isolated microbial strain or a composition according to the present invention as a pesticide, preferably as a fungicide.

[0149] In another aspect, the present invention relates to the use of the microbial strain of the present invention, or Streptomyces, which has at least 91% identity with the whole genome of Streptomyces crown mycelium NRRL B-3672, or with the whole genome of Streptomyces species Saigon 413 deposited in the Fungal Culture Collection under the deposit number CBS149411 as disclosed herein, for producing malenomycin and at least one, at least two, at least three, at least four or at least five of the compounds selected from the group consisting of cyclothiazolin C, streptomyces glutarimide, an oligosaccharide compound according to compound I (the compound I comprises a compound according to C 53 H 90 N2O 44 , further characterized by the NMR spectra listed in Tables 2 and 3, preferably characterized by structural formula I), lipopeptides according to formula II or salts thereof (wherein R1 = CH3 or C2H5) and polyene compounds (which are characterized by the C 67 H 115 NO 25 wherein the polyene is further characterized by Fig.10 The optical absorption spectrum characterization is shown in .

[0150] The features associated with the isolated microbial strains, the compositions according to the invention, the Streptomyces strains and the Streptomyces coronarimi are as disclosed above and are applicable to their use. BRIEF DESCRIPTION OF THE DRAWINGS

[0151] Figure 1 .1D of compound I in D2O 1 H NMR spectrum (600 MHz)

[0152] Figure 2 .1D of compound I in D2O 13 C NMR spectrum (600MHz)

[0153] Figure 3 .2D Dept edited 1H-13C HSQC NMR spectrum of compound I in D2O at 600 MHz, showing positive (CH) signal

[0154] Figure 4 .2D Dept edited 1H-13C HSQC NMR spectrum of compound I in D2O at 600 MHz, showing negative (CH2) signal

[0155] Figure 5 . According to formula II (a), formula II (b) lipopeptide or lipopeptide antibiotic (Lipopeptin) A light absorption spectrum (UV-VIS) 200-400nm

[0156] Figure 6 . Precursor of lipopeptide of formula II(a) 1204.6m / z(M+H) + The LC-ESI-MS / MS spectrum of the peptide depicts fragmentation peaks consistent with the following amino acids: aspartic acid, hydroxy-glutamine, serine, methyl-asparagine, methyl-phenylalanine

[0157] Figure 7 LC-ESI-MS / MS / MS spectrum of the precursor of the lipopeptide of Formula II(a) at m / z 294.2, depicting a peak consistent with the molecule C14H25-OH2-C4H5ON

[0158] Figure 8 1D of the lipopeptide according to formula II (a) at 600 MHz in CD3OD 1 High-field region of the H NMR spectrum

[0159] Fig. 9 1D of the lipopeptide according to formula II (a) at 600 MHz in CD3OD 1 Low-field region of the H NMR spectrum

[0160] Fig.10 .Light absorption spectrum of polyene compounds (UV-VIS) 200-500nm

[0161] Fig.11 Graphical representation of the lipopeptide gene cluster

[0162] Fig.12 Graphical representation of the polyene gene cluster

[0163] Examples

[0164] Example 1

[0165] Example 1.1. Source and fermentation

[0166] Fermentation of Streptomyces species

[0167] Streptomyces species were ordered from the culture collections disclosed in Table 1 .

[0168] Streptomyces sp. Saigon 413 has been deposited with the Central Collection of Fungal Cultures 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.

[0169] Streptomyces species were cultured in Erlenmeyer flasks containing a liquid medium consisting of (g / I) 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.

[0170] Large-scale fermentation

[0171] For large-scale production, fed-batch fermentation is used to culture Streptomyces species, such as Streptomyces coronomicinus NRRL 3672 and Streptomyces coronomicinus CBS 149411, to high cell density using standard procedures. After harvesting, the culture broth is spray-dried or freeze-dried according to methods known to those skilled in the art.

[0172] The cell count of the final product (TGAI) after spray or freeze drying is 1*10 6 Up to 1*10 13 CFU / g dry mass. TGAI: Technical grade active ingredient (unformulated product).

[0173] Example 1.2. Formulation

[0174] To prepare different formulations of the spray-dried or freeze-dried products (microorganisms), the Nouryon formulator toolbox from Croda Crop Care and the general formulation techniques disclosed in the following literature were used: Formulation of Microbial Biopesticides: Beneficialmicroorganisms, nematodes and seed treatments (412 pages, December 6, 2012), ed. Burges HD, Springer, ISBN 978-94-011-4926-6.

[0175] For foliar treatment of diseases on different crops (Example 3), whole fermentation broths of Streptomyces coronomicinus NRRL B-3672 and Streptomyces sp. Saigon 413 were tested, as well as 10 5 Up to 10 10 cfu / g TGAI of spray-dried fermentation broth of Streptomyces Saigon 413 TGAI, which was formulated as oil dispersion (OD) and non-aqueous dispersion (NAD).

[0176] In some cases, the surfactant polyethylene glycol sorbitan monolaurate ( 20).

[0177] OD 40% w / w of 40% dry fermentation broth is prepared in vegetable oil containing an emulsifier (eg surfactant). A UV protectant such as lauryl gallate may be added.

[0178] NAD 35% or NAD 40% formulations were prepared containing 35% or 40% w / w% of dry material, respectively, in alcohol (e.g. dipropylene glycol methyl ether) and rheology modifier (organoclay AEROSIL KLUCEL G IND-cellulose 200)

[0179] For seed treatment (Example 4), spray-dried or freeze-dried Streptomyces coronomicinus CBS149411 (Streptomyces Saigon 413) TGAI was added at 10 7 Up to 10 10 cfu / g TGAI was used as such or formulated as a flowable concentrate (FS) or oil dispersion (OD).

[0180] FS300 is a flowable concentrate containing 30% w / w% spray-dried TGAI in water, a surfactant (such as a silicon-based defoamer) and a dispersant (such as styrene / methacrylic acid).

[0181] The OD400 formulation contained 40% w / w of spray-dried TGAI.

[0182] Example 1.3. Isolation and species identification of 16S rDNA and genomic DNA

[0183] 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.

[0184] The species of strain Streptomyces species Saigon 413 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 from the Streptomyces species genome (based on the genome classification database GTDB (Parks, DH, et al. (2021). GTDB: Nucleic Acids Research [GTDB: Nucleic Acids Research], 50: D785-D794)) using barrnap v0.9. Based on this comparative analysis, Streptomyces species Saigon 413 was identified as the species Streptomyces coronamicinus. The sequence identity between the 16S rRNA sequences of Streptomyces species Saigon 413 and Streptomyces coronamicinus NRRL-3672 was determined to be 99.87% using Muscle v3.8.31 and the R package Seqinr v4.2-16.

[0185] 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 refined with Pilon using Illumina reads. Genomic DNA was also extracted from Streptomyces crassa CBS 492.64, Streptomyces crassa CBS 570.66, Streptomyces crassa CBS 569.66, Streptomyces coronomicinus DSM 41224, Streptomyces crassa subsp. crassa DSM 40673, and Streptomyces crassa subsp. crassa DSM 41057 using methods 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).

[0186] After assembling the genome from 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 percent identity (ANI) between the Streptomyces species Saigon 413 genome and the Streptomyces crown NRRL B-3672 genome was 96.9%.

[0187] Using 16S RNA sequence identity and ANI score (%), strains CBS 596.66, CBS570.66 and DSM41429 were also found to be Streptomyces coronamicinus strains, rather than Streptomyces rimosis or Streptomyces paromomycinus strains as specified by the depository. In Table 1, the identity percentages of the whole genome and 16S RNA sequences of Streptomyces species Saigon 413 and Streptomyces coronamicinus NRRL B-3672 are shown.

[0188] Table 1. Sequence identity of the 16S RNA and whole genome of Streptomyces sp. Saigon 413 with the 16S RNA and genomes of other closely related Streptomyces species that are publicly available.

[0189]

[0190]

[0191] CBS Fungal Collection or Westerdijk Fungal Diversity Institute: Uppsala Boulevard 8, 3584 CT, Utrecht, The Netherlands https: / / wi.knaw.nl /

[0192] DSMZ German Collection of Microorganisms: Inhoffenstraße 7B, 38124 Braunschweig, Germany - www.dsmz.de

[0193] ARS ARS Culture Collection (NRRL), 1815 North University Street, Peoria, IL 61604, USA - https: / / nrrl.ncaur.usda.gov

[0194] Example 1.4. Analysis of streptoglutarimide, cyclothiazolylmycin C and malenomycin

[0195] The structure of cyclothiazolycin C is disclosed on page 3 of WO 2015191789 and can be extracted and analyzed according to the method disclosed in Wang et al. (2010) Appl. Environmental Microbiology, Vol. 76, No. 7, p. 2336. Malenomycin can be extracted and analyzed according to the method disclosed in Example I (B) of WO 2006 / 078939. Streptomycin glutarimide can be extracted and isolated according to the method disclosed in Lee et al. J. of Antibiotics (2020) 73: p. 184-188 (including supplementary information).

[0196] Example 1.5. Purification of Oligosaccharide Compound I

[0197] The whole fermentation broth of the Streptomyces species shown in Table 1 (e.g., Streptomyces crown mycelium CBS149411 (Streptomyces species Saigon 413)) was centrifuged to produce an aqueous extract and a precipitate. The aqueous extract was freeze-dried. The material was resuspended in a minimum volume of water and partitioned with ethyl acetate to remove lipophilic components. The aqueous suspension was retained and freeze-dried, resuspended in a minimum volume of water, and then applied to an activated carbon column.

[0198] The column was washed with water and eluted with water:acetone (50:50).

[0199] Compound I was further purified by hydrophilic interaction liquid chromatography (HILIC) using mass-directed fractionation and ELSD detection, for example, using a Waters XBridge Amide, 5 micron, 30x100 mm, using a gradient of acetonitrile and 10 mM ammonium acetate.

[0200] Characterization of Oligosaccharide Compound I

[0201] Compound I in the purified fermentation broth was determined according to the method disclosed below.

[0202] Molecular composition and total molecular mass

[0203] The molecular composition and total molecular mass are C 53 H 90 N2O 44 and 1458.487 g, which were determined using MS-MS and NMR spectroscopy as disclosed below.

[0204] Solubility

[0205] The solubility of oligosaccharide Compound I in water (pH 7.01) is >10'000 ppm and the solubility in DMSO is >9772 ppm.

[0206] MS-MS spectroscopy and liquid chromatography

[0207] 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, purge gas (Arb): 1, ion transfer tube temperature: 350°C, evaporator temperature: 350°C). Scan parameters were as follows;

[0208] Experiment 1: MS OT (Orbitrap resolution: 60,000, Scan range (m / z): 200 to 2000, RF lens (%): 60, AGC target: standard, Max injection time mode: automatic, Micro scan: 1, Data type: spectral, Polarity: both),

[0209] Experiment 2: tMS2 OT CID (MSn level (n): 2, isolation window (m / z): 1.6, activation type: CID, CID collision energy (%): 30, detector type: Orbitrap, Orbitrap resolution: 30,000, RF lens (%): 60, polarity: negative). The mass spectrometer was connected to a Vanquish Flex UHPLC from Thermo Fisher Scientific using a Vanquish split sampler FT, Vanquish binary pump F, Vanquish column oven H, Vanquish diode array detector FG, and Vanquish charged aerosol detector. Liquid chromatography conditions included: Thermo Fisher Scientific Hypercarb TM Porous graphite carbon column 5μm4.6x50mm, PN35005-054630. Temperature: 40°C, DAD wavelength range: 250 to 260nm, solvent gradient: solvent A: H2O with 0.1% formic acid, solvent B: CH3CN with 0.1% formic acid, gradient: 0min 1% B, 99% A; 4.00min 50% B, 50% A; 4.25min 100% B; 4.50min 100% B; 4.95min 1% B, 99% A; 6.00min 1% B, 99% A, flow rate: 1.0ml / min, injection volume: 2uL, total run time: 6.0min.

[0210] NMR spectroscopy

[0211] 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 (equipped with Z gradient) using a standard Bruker pulse sequence. The samples were dissolved in D2O and spectra were recorded at 300°K and referenced to 2.225 ppm (for 1 H) and 31.07ppm (for 13 C) at acetone. Figures 1 to 4 The NMR spectra of the compounds of the present invention are shown.

[0212] single bond 1 H- 13 The C-related spectrum contains peaks corresponding to 1 methyl (CH3) and 40 methine (CH) groups (listed in Table 1) and 9 methylene (CH2) groups (listed in Table 2).

[0213] In addition, 1D 13The C spectrum contains signals from three quaternary carbons at 104.7, 159.3 and 175.2 ppm (±0.1).

[0214] Table 2. Single bonds of oligosaccharide compound I 1 H- 13 Methyl and methine signals in C-correlation spectra and 1D 1 The multiplicity information of the protons resolved in the H spectrum.

[0215]

[0216]

[0217] Table 3. Single bonds of compound I 1 H- 13 Methylene signals in C-correlation spectra and 1D 1 The multiplicity information of the protons resolved in the H spectrum.

[0218] <![CDATA[d 13 C ppm(±0.1)]]> <![CDATA[d 1 H ppm(±0.05)]]> <![CDATA[ 1 H Multiplicity]]> 69.2 4.23 69.2 3.85 63.5 4.07 63.5 3.37 dd(10.5,11.5,1H) 63.4 3.79 63.4 3.66 63.3 3.81 63.3 3.72 63.0 3.85 63.0 3.67 62.0 3.82 62.0 3.78 61.6 3.93 61.6 3.77 61.2 3.89 61.2 3.77 60.9 3.85 60.9 3.79

[0219] Example 1.6. Lipopeptide compounds having formula II

[0220] Purification of lipopeptides according to Formula II (Formula II (a) and Formula II (b))

[0221] Mycelia from fermentation broths of Streptomyces species listed in Table 1 (e.g., Streptomyces species Saigon 413) 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). A gradient of 60% aqueous to 40% aqueous in the above solvent conditions allowed the separation of lipopeptide compounds according to Formula II (a) and Formula II (b).

[0222] By UV-VIS( Figure 5 )、Mass Spectrometry( Figure 6 and 7 ) and NMR spectroscopy ( Figure 8 and 9 ) test compounds.

[0223] Lipopeptide antibiotic A

[0224] Lipopeptide antibiotic A was purchased from Fundación MEDINA, Centro de Excelencia en Investigatión de Medicamentos Innovadores en Andalucía, Avda. del Conocimiento 34, Edificio Centro de Desarrollo Farmacéutico y Alimentario, Parque Tecnológico de Ciencias de la Salud, 18016 Granada (Spain).

[0225] Characterization of the lipopeptide having formula II

[0226] Liquid chromatography and high-resolution mass spectrometry

[0227] 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, purge gas (Arb): 1, ion transfer tube temperature: 350°C, evaporator temperature: 350°C). Scan parameters were as follows;

[0228] 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: spectral, Polarity: both),

[0229] Experiment 2: tMS2 OT CID (MSn level (n): 2, isolation window (m / z): 1.0, activation type: CID, CID collision energy (%): 30, detector type: Orbitrap, Orbitrap resolution: 30,000, RF lens (%): 60, polarity: positive),

[0230] Experiment 3: tMS2 OT HCD (MSn level (n): 2, isolation window (m / z): 1.0, activation type: HCD, HCD collision energy (%): 30, detector type: Orbitrap, Orbitrap resolution: 30,000, RF lens (%): 60, polarity: positive), Experiment 4: tMS3 OT HCD (MSn level (n): 3, isolation window (m / z): 1.6, activation type: HCD, HCD collision energy (%): 30, MS2 isolation window (m / z): 2, MS2 activation type: HCD, MS2 HCD collision energy (%): 30, detector type: Orbitrap, Orbitrap resolution: 30,000, RF lens (%): 60, polarity: positive). The mass spectrometer was connected to a Vanquish Flex UHPLC from Thermo Fisher Scientific using a Vanquish split sampler FT, a Vanquish binary pump F, a Vanquish column oven H, a Vanquish diode array detector FG and a Vanquish charged aerosol detector.

[0231] 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. The purified fermentation broth as described above was injected.

[0232] Figure 6 and 7 The LC-ESI-MS / MS / MS spectrum of the compound of formula II(a) is shown.

[0233] NMR spectroscopy

[0234] NMR spectra were recorded on a Bruker AVIII 600 NMR spectrometer equipped with a 5 mm Bruker ( 1 H / 19 F) / 13 C / 15N TCI cryoprobe (equipped with Z gradient) using standard Bruker pulse sequence. Samples were dissolved in CD3OD and spectra were recorded at 300°K and referenced to 3.31 ppm (for 1 H) Residual solvent signal. Figure 8 and Fig. 9 In each case half of the 1H NMR spectrum of the compound according to formula II(a) is covered.

[0235] Molecular composition and mass

[0236] The molecular composition and mass of the lipopeptides according to formula II (a) and formula II (b) were determined using the results of liquid chromatography and high-resolution mass spectrometry as disclosed above. The lipopeptide compounds of formula II (a) and II (b) have the following compositions. Formula II (a) Lipopeptide 1204: Molecular composition is C55H85N11O19, and the accurate mass is 1203.602. Formula II (b) Lipopeptide 1218: Molecular composition is C56H87N11019, and the accurate mass is 1217.618.

[0237] The reference lipopeptide antibiotic A has the following composition C54H84N10O19 and exact mass 1176.591421.

[0238] Solubility

[0239] Determine the solubility of compounds having formula II (a), formula II (b) and lipopeptide antibiotic A in water and DMSO:

[0240]

[0241] Identification of the biosynthetic gene cluster for the production of the lipopeptide according to Formula II in Streptomyces sp. Saigon 413

[0242] To identify genes involved in the production of lipopeptides according to Formula II, the assembled genome (see Example 1.4) was run through AntiSMASH (version 5.1.1, Blin et al., Nucleic Acids Res (2019) doi: 10.1093 / nar / gkz310), a commonly used tool to assist in the identification of biosynthetic gene clusters responsible for the production of secondary metabolites.

[0243] By identifying the lipopeptide compound as a lipopeptide family (see above) and AntiSMASH output, we were able to infer that the lipopeptide compound was 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 was based on structural analysis of the 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 ( Fig.11 ), which gene cluster is capable of incorporating amino acid precursors (including asparagine, aspartic acid, glutamic acid, phenylalanine, serine and threonine) and is therefore involved in the production of lipopeptide compounds having formula II (a) and II (b).

[0244] The NRPS biosynthetic gene cluster contains 45 coding sequences, including two NRPS gene coding sequences, a regulator coding sequence and a coding sequence responsible for the biosynthesis of a precursor incorporated into the lipopeptide of Formula II ( Fig.11 and Table 4).

[0245] Table 4. Coding sequences present in the NRPS biosynthetic gene cluster responsible for the production of lipopeptides according to Formula II. The annotations provided are based on pBLAST searches performed in the National Center for Biotechnology Information database using non-redundant protein sequences.

[0246]

[0247]

[0248]

[0249] 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

[0250] 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 pBCon2192 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).

[0251] Plasmid pBCon2192 was used to transform E. coli ET12567 / pUZ8002 using standard electroporation methods and then introduced into Streptomyces species Saigon 413 by hyphal conjugation (T. Kieser et al., Practical Streptomyces Genetics, 2000, John Innes Foundation, Norwich). Thiostrepton resistant colonies were plated on ISP-4 agar medium supplemented with 40 μg / ml thiostrepton and 25 μg / ml nalidixic acid. These plates were initially incubated at 28°C for 6 days to allow plasmid replication. After incubation at 28°C for 6 days, the strains were re-plated 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 incubation at 37°C for 6 days, the obtained strains were transferred to ISP-4 solid agar medium without selection and incubated at 28°C for 15 days to allow secondary exchange.

[0252] After 15 days of growth, strains were harvested in 20% glycerol. 100 μl of the cell suspension was used to inoculate fresh plates and serial dilutions were performed until 10 -10 Then 100 μl of 10 -8 Up to 10 -10 Inoculate onto ISP-4 agar plates. Incubate plates at 28°C until single colonies are observed.

[0253] Individual colonies were plated in parallel on non-selective and thiostrepton selective ISP-4 agar media. Sensitive patches (representing secondary recombination) were then screened via PCR using gDNA isolated with the FastSpin Soil Kit (MP Biomedicals) to identify correct colonies.

[0254] 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 outside 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 Illumina PCR-independent sequencing confirmed that no other changes had occurred in the genome.

[0255] Cultivation of Streptomyces sp. Saigon 413Δ7318-7319 and analysis of strain extracts confirmed that the lipopeptide compound according to formula II(a) was no longer produced by this strain, confirming that SEQ ID NO:22 and SEQ ID NO:23 are essential for the production of the lipopeptide compound according to formula II(a)

[0256] Example 1.7. Polyene compounds

[0257] Purification of polyene compounds

[0258] The spray-dried sample of Streptomyces crown mycin CBS149411 (Streptomyces species Saigon 413) culture was washed with water. The solid residue was extracted twice with isopropanol, and the isopropanol was removed. The gained solid was purified by acetonitrile: water gradient by preparative reversed phase (C18) HPLC. It was further purified by preparative reversed phase HPLC, wherein using Zorbax C8 column and acetonitrile: water gradient elution.

[0259] The compounds were detected by UV-VIS ( Fig.10 ).

[0260] Characterization of polyene compounds

[0261] Liquid chromatography and high-resolution mass spectrometry

[0262] 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, purge gas (Arb): 1, ion transfer tube temperature: 350°C, evaporator temperature: 350°C). Scan parameters were as follows;

[0263] 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: spectral, Polarity: both),

[0264] Experiment 2: tMS2 OT CID (MSn level (n): 2, isolation window (m / z): 1.0, activation type: CID, CID collision energy (%): 30, detector type: Orbitrap, Orbitrap resolution: 30,000, RF lens (%): 60, polarity: positive),

[0265] Experiment 3: tMS2 OT HCD (MSn level (n): 2, isolation window (m / z): 1.0, activation type: HCD, HCD collision energy (%): 30, detector type: Orbitrap, Orbitrap resolution: 30,000, RF lens (%): 60, polarity: positive), Experiment 4: tMS3 OT HCD (MSn level (n): 3, isolation window (m / z): 1.6, activation type: HCD, HCD collision energy (%): 30, MS2 isolation window (m / z): 2, MS2 activation type: HCD, MS2 HCD collision energy (%): 30, detector type: Orbitrap, Orbitrap resolution: 30,000, RF lens (%): 60, polarity: positive).

[0266] The mass spectrometer was connected to a Vanquish Flex UHPLC from Thermo Fisher Scientific using a Vanquish split sampler FT, a Vanquish binary pump F, a Vanquish column oven H, a Vanquish diode array detector FG and a Vanquish charged aerosol detector.

[0267] 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.

[0268] Inject the purified fermentation broth as described in section 1.8.1.

[0269] The key peaks observed are:

[0270] Negative ion: C67H114NO25[MH] - Expected value: 1332.7685, Observed value: 1332.7679

[0271] Positive ion: C67H114NO24[M-H2O+H] + Expected value: 1316.7725, Observed value: 1316.7709

[0272] Positive ion: C67H115NO24[M-H2O+2H] 2+ Expected value: 658.8899, Observed value: 658.8895

[0273] Positive ion: C67H113NO23[M-2(H2O)+2H] 2+ Expected value: 649.8846, Observed value: 649.8843

[0274] Positive ion: C67H111NO22[M-3(H2O)+2H] 2+ Expected value: 640.8793, Observed value: 640.8790

[0275] Molecular composition and mass

[0276] The molecular composition and mass of the polyene compound were determined using the results of liquid chromatography and high-resolution mass spectrometry as disclosed above. The molecular composition of the polyene compound is C 67 H 115 NO 25 , the exact mass is 1333.7758.

[0277] Solubility

[0278] Determine the solubility of a compound in DMSO:

[0279]

[0280] Identification of a biosynthetic gene cluster for the production of polyene compounds in Streptomyces sp. Saigon 413

[0281] To identify genes involved in the production of polyene compounds identified in 1.8.2 above, the assembled genome (see Example 1.4 above) was run through AntiSMASH (version 5.1.1, Blin et al., Nucleic Acids Res (2019) doi: 10.1093 / nar / gkz310), a commonly used tool to assist in the identification of biosynthetic gene clusters responsible for the production of secondary metabolites.

[0282] By identifying polyene compounds (see above) and AntiSMASH output, we were able to infer that the polyenes of the compounds were produced by modular type I polyketide synthase (PKS) gene clusters. The identification of modular type I PKS gene clusters responsible for the biosynthesis of polyene compounds was based on the analysis of biosynthetic gene clusters associated with the production of characterized polyenes, such as filipin produced by Streptomyces filipinensis, amphotericin produced by Streptomyces nodosus, and thailandins A and B produced by Actinokineosporabangkokensis 44EHW. In Streptomyces species Saigon 413, a large modular type I PKS biosynthetic gene cluster was identified ( Fig.12 ), and are therefore relevant to the production of the polyene compounds according to the present invention.

[0283] The modular type I PKS biosynthetic gene cluster contains 24 coding sequences, including eight modular type I PKS gene coding sequences, regulator coding sequences, and coding sequences responsible for the biosynthesis of precursors incorporated into polyene compounds ( Fig.12 Table 5: Coding sequences present in the type I PKS biosynthetic gene cluster responsible for the production of polyene compounds. The annotations provided are based on pBLAST searches performed in the National Center for Biotechnology Information database using non-redundant protein sequences.

[0284]

[0285]

[0286] Deletion of the genomic region including CDS_31 (SEQ ID NO: 67) and CDS_32 (SEQ ID NO: 68) from Streptomyces sp. Saigon 413 and phenotypic analysis

[0287] To confirm that the identified biosynthetic gene cluster is related to the production of polyene compounds, a region containing two polyketide synthase genes encoded by CDS_31 and CDS_32 (SEQ ID NO: 67 and SEQ ID NO: 68) was deleted from Streptomyces species Saigon 413. To generate Streptomyces species Saigon 413A941-942, plasmid p073-031 was used. Plasmid p073-031 was prepared from pRAR017 and contains regions homologous to either side of the region to be deleted from the strain (facilitating primary and secondary crossover).

[0288] Plasmid p073-031 was used to transform E. coli ET12567 / pUZ8002 using standard electroporation methods and then introduced into Streptomyces species Saigon 413 by hyphal conjugation (T. Kieser et al., Practical Streptomyces Genetics, 2000, John Innes Foundation, Norwich). Thiostrepton resistant colonies were plated on ISP-4 agar medium supplemented with 40 μg / ml thiostrepton and 25 μg / ml nalidixic acid. These plates were initially incubated at 28°C for 6 days to allow plasmid replication. After incubation at 28°C for 6 days, the strains were re-plated 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 incubation at 37°C for 6 days, the obtained strains were transferred to ISP-4 solid agar medium without selection and incubated at 28°C for 15 days to allow secondary exchange.

[0289] After 15 days of growth, strains were harvested in 20% glycerol. 100 μl of the cell suspension was used to inoculate fresh plates and serial dilutions were performed until 10 -10 Then 100 μl of 10 -8 Up to 10 -10 Inoculate onto ISP-4 agar plates. Incubate plates at 28°C until single colonies are observed.

[0290] Individual colonies were plated in parallel on non-selective and thiostrepton selective ISP-4 agar media.Susceptible patches (representing secondary recombination) were then screened via PCR using gDNA isolated with the FastSpin Soil Kit (MP Biomedicals).

[0291] To confirm that the region containing SEQ ID NO:67 and SEQ ID NO:68 had been removed from the strain, a primer pair that binds outside the deleted region was used. Sanger sequencing of the PCR product and alignment with the Streptomyces sp. Saigon 413 genome confirmed the deletion of the genomic region containing SEQ ID NO:67 and SEQ ID NO:68. In addition, whole genome analysis using Illumina PCR-independent sequencing confirmed that no other changes had occurred in the genome.

[0292] Cultivation of Streptomyces sp. Saigon 413Δ941-942 and analysis of strain extracts confirmed that polyene compounds were no longer produced by this strain. SEQ ID NO: 67 and SEQ ID NO: 68 were confirmed to be essential for the production of the polyene compounds disclosed herein.

[0293] Example 2.

[0294] 2.1. Determination of metabolites malenomycin, CtmC and streptoglutarimide in fermentation broths of several Streptomyces strains

[0295] The presence of the following metabolites was measured in the fermentation broth of Streptomyces species Saigon 413 and other Streptomyces species: malenomycin, cyclothiazomycin C and streptoglutarimide, oligosaccharide compounds according to compound I, lipopeptides having formula II, and polyene compounds. These metabolites were determined by separation and purification according to the methods described in Sections 1.4 to 1.7.

[0296] Table 6A shows that the Streptomyces coronomicin strain produced at least the metabolites malenomycin, CtmC, streptoglutarimide, and at least one of the molecular formula C in fermentation broths of Streptomyces sp. Saigon 413 and several other Streptomyces strains. 53 H 90 N2O 44 The oligosaccharide compound I, the lipopeptide according to formula II, and the polyene C 67 H 115 NO 25

[0297] Table 6A. Metabolites of malenomycin, CtmC, streptoglutarimide, oligosaccharides of Compound I, lipopeptides according to Formula II, and polyene C in fermentation broths of Streptomyces sp. Saigon 413 and several other Streptomyces strains. 67 H 115 NO 25 Existence

[0298]

[0299]

[0300] nd: not detected; +: metabolites present in fermentation broth

[0301] 2.2. Efficacy of several Streptomyces strains against Puccinia tritici and Puccinia occidentalis on wheat

[0302] The strains listed in Table 6A were tested for their ability to control Psoralea corylifolia and Puccinia recondita on wheat. The strains were fermented in conical flasks as described above. After fermentation, samples of the culture broth were frozen at -80°C until used in greenhouse trials. On the day of application, the samples were thawed, mixed, and diluted in water supplemented with Tween 20 (at 0.025% v / v) in a range of dilutions from 10% (broth in water, v / v) to 0.3%. The spray volume was 400 liters / hectare and the application time was one day before infection. The affirmative statement (yes) reported in Table 6B indicates that the Streptomyces fermentation broth controlled or reduced the severity of the disease on the treated leaves at any of the multiple dilution ratios tested.

[0303] For infection with Psoralea corylifolia (EPPO code: SEPTTR), one day after application, test plants of the wheat cv. Riband were inoculated by spraying them with a spore suspension (1,5 Mio spores / ml in water supplemented with 0.01% Tween 20). After an incubation period of 4 days at 22°C / 21°C (day / night) and 95% rh, the inoculated test plants were kept in a greenhouse at 22°C / 21°C (day / night) and 70% rh. The efficacy was assessed visually when an appropriate level of disease appeared on the untreated control plants (16-19 days after application).

[0304] For infection with Puccinia recondita (EPPO code: PUCCRE), one day after application, test plants of wheat cv. Arina were inoculated by spraying them with a spore suspension (spore suspension, 80,000 spores / ml in water supplemented with 0.1% Tween 20). After an incubation period of 1 day at 20° C. and 95% rh, the inoculated test plants were kept at 20° C. and 60% rh in a greenhouse. When an appropriate level of disease appeared on untreated control plants (9-12 days after infection), the percentage of leaf area covered by disease was assessed visually.

[0305] in conclusion

[0306] The results in Table 6B show that the fermentation broth from the Streptomyces coronamicinus strain has the ability to control the development of the fungal pathogens Puccinia recondita and Puccinia tritici on wheat. Streptomyces species CBS492.64 has the ability to control only Puccinia recondita, while S. albofaciens and S. fissurea are unable to control any of the diseases tested.

[0307] Table 6B: Ability of several Streptomyces strains to control Puccinia recondita or Psoralea tritici infections on wheat plants in the greenhouse.

[0308]

[0309] Example 3. Foliar treatment using a Streptomyces coronomicinus (Streptomyces species Saigon 413) composition to control fungal infections

[0310] 3.1. Treatment of wheat infected with Puccinia tritici and Puccinia recondita with fermentation broth of Streptomyces coronarensis

[0311] Streptomyces isolate CBS149411 (deposited at the Fungal Culture Collection: 8 Uppsala Avenue, 3584 CT, Utrecht, the Netherlands) and NRRL B-3672 (a type strain of Streptomyces coronarius, deposited at the ARS Culture Collection (NRRL), 1815 North University Street, Peoria, IL 61604, USA) were fermented in a 200 L fermentor in the same manner as disclosed above. The fermentation endpoint solution was stored frozen at -20°C until used for the assay. Seedlings of wheat variety Riband (for wheat leaf blight test) or variety Arina (for cryptic rust test) were grown in a greenhouse until 14 days after sowing. One day before infection, the fermentation endpoint solution (supplemented with 0.025% Plants were treated with 400 L / ha of 1% chloramphenicol (diluted in 20% water). In Table 3, the time points for disease assessment are expressed as days after infection (DAI). The efficacy of the treatments is expressed as the percentage of symptom reduction compared to the untreated control.

[0312] For infection with Psoralea corylifolia (EPPO code: SEPTTR), one day after application, the test plants were inoculated by spraying a spore suspension (1.5 Mio spores / ml in water supplemented with 0.01% Tween 20) on them. After an incubation period of 4 days at 22°C / 21°C (day / night) and 95% rh, the inoculated test plants were kept in a greenhouse at 22°C / 21°C (day / night) and 70% rh.

[0313] Efficacy was assessed directly when appropriate levels of disease appeared on untreated control plants (16-19 days after application).

[0314] For infection with Puccinia reconditatis (EPPO code: PUCCRE), one day after application, the test plants were inoculated by spraying them with a spore suspension (spore suspension, 80,000 spores / ml in water supplemented with 0.1% Tween 20). After an incubation period of 1 day at 20°C and 95% rh, the inoculated test plants were kept at 20°C and 60% rh in a greenhouse. When the appropriate level of disease appeared on the untreated control plants (9-12 days after infection), the percentage of leaf area covered by disease was visually assessed.

[0315] Table 7. Reduction of disease symptoms in wheat infected with brown rust (Puccinia reconditatis) or wheat leaf blight (Puccinia tritici) after treatment with Streptomyces sp. Saigon 413 and Streptomyces coronomids NRRL B-3672.

[0316]

[0317] in conclusion

[0318] The results in Table 7 show that both Streptomyces strains CBS 149411 and NRRL B-3672 provided control of brown rust (Puccinia recondita) and wheat leaf blight (Psora tritici) in wheat seedlings when applied as fermentation endpoints. Further dilutions of the fermentation broth indicated that the Streptomyces CBS 149411 derived fermentation broth was more potent against brown rust and wheat leaf blight than Streptomyces NRRL B-3672, as shown by, for example, a higher level of control at a 0.16% dilution.

[0319] 3.2. Preventive and curative treatment of fungal infections caused by Puccinia tritici and Puccinia recondita on wheat using Streptomyces spp. Saigon 413 and Solatenol

[0320] Seedlings of wheat cv. Riband (for tests with Puccinia tritici) or cv. Arina (for tests with Puccinia recondita) were grown in a greenhouse until 14 d after sowing. One day before infection (1 d preventive assay) or 3 d after infection (3 d curative assay), plants were treated with different dilutions of OD40% formulations of Streptomyces Saigon 413, CBS149411. The spray volume was 200 L / ha. Tank mix adjuvants (spreader and retention aid at typical use rates) were added to some treatments. The efficacy of the CBS149411 derived formulations was compared with a commercial reference fungicide (Elatus TM Plus, with SOLATENOL TM , from Syngenta, Basel, Switzerland) were compared.

[0321] For infection with Psoralea corylifolia (EPPO code: SEPTTR), the test plants were inoculated by spraying a spore suspension (1.5 Mio spores / ml in water supplemented with 0.01% Tween 20). After a 4-day incubation period at 22°C / 21°C (day / night) and 95% rh, the inoculated test plants were kept in a greenhouse at 22°C / 21°C (day / night) and 70% rh. Efficacy was directly assessed when appropriate levels of disease appeared on untreated control plants (16-19 days after infection (DAI)).

[0322] For infection with Puccinia reconditatis (EPPO code: PUCCRE), the test plants were inoculated by spraying them with a spore suspension (spore suspension, 80,000 spores / ml in water supplemented with 0.1% Tween 20). After an incubation period of 1 day at 20°C and 95% rh, the inoculated test plants were kept at 20°C and 60% rh in a greenhouse. When the appropriate level of disease appeared on the untreated control plants (9-12 days after infection (DAI)), the percentage of leaf area covered by disease was visually assessed.

[0323] Table 8. Preventive and curative treatment of fungal infections caused by Puccinia tritici and Puccinia recondita on wheat using Streptomyces coronatrix CBS149411 and benzovindiflupyr

[0324]

[0325] (1) Alkyl polyglucoside polyoxyethylene (6) C9-C11 alcohol

[0326] in conclusion

[0327] The results in Table 8 show that Streptomyces sp. Saigon 413, CBS 149411 (OD 40% formulation) provided control of brown rust and wheat leaf blight in wheat similar to conventional fungicides such as SOLATENOL TM Streptomyces coronatus CBS149411 OD40% provided effective disease control when used as a preventive or curative spray. The use of tank mix adjuvants alkyl polyglucosides and / or polyoxyethylene (6) C9-C11 alcohols improved the efficacy of OD40% formulations against brown rust and wheat leaf blight in preventive and curative treatments.

[0328] 3.3. Treatment of wheat infected with Puccinia striata with Streptomyces species Saigon 413 and CBS149411

[0329] Wheat seedlings of the variety Loft were grown in a greenhouse until 14 days after sowing. One day before infection (1 day preventive assay) or 4 days after infection (4 day curative assay), plants were treated with one dilution of the OD40% formulation. The spray volume was 200 L / ha. Tank mix adjuvants (spreader and retention aid at typical use rates) were added. The efficacy of Streptomyces coronaeum CBS was compared with a commercial reference fungicide (Elatus TM Plus, with SOLATENOL TM , from Syngenta, Basel, Switzerland) were compared.

[0330] Wheat seedlings were inoculated with Puccinia fasciata (EPPO code: PUCCST) by spraying with a spore suspension (spore suspension, 80,000 spores / ml in water supplemented with 0.1% Tween 20). After a 48-hour incubation period at 11°C and 95% rh, the inoculated test plants were kept at 17°C and 60% rh in a greenhouse. When appropriate levels of disease appeared on untreated control plants (16-25 days after infection (DAI)), the percentage of leaf area covered by disease was visually assessed.

[0331] Table 9. Reduction of disease symptoms caused by wheat yellow rust (Puccinia striata) after treatment of plants with formulated samples at the time of preventive or curative spraying.

[0332]

[0333] in conclusion

[0334] The results in Table 9 show that Streptomyces coronatus CBS149411 formulated at OD40% provided control of wheat yellow rust in both preventive and curative treatments similar to conventional fungicides such as SOLATENOL TM .

[0335] Example 3.4. Treatment of black leaf tip blight (Mycosphaeria fijiensis) on bananas with Streptomyces sp. Saigon 413

[0336] The spray-dried powder of Streptomyces coronatus CBS149411 was mixed with spray oil ( 796) and an emulsifier (Emulsogen M) were applied to banana plants. Banana plants (dwarf Cavendish variety, grown in a greenhouse for 6 weeks from commercial in vitro explants) were treated twice with the triple mixture, 8 days and 1 day before infection. The efficacy of CBS149411 powder was compared with a treatment using only spray oil plus emulsifier at the same spraying time. Fungicide Bold(with fungicide from Syngenta, Basel, Switzerland), suspension concentrate (SC200) mixed with the same spray oil and emulsifier was used as a positive control in the experiment and sprayed once a day before infection. 796 is an agricultural spray oil (a product from Calumet specialty products partners, Indianapolis, USA). Emulsogen M is a mineral oil emulsifier (a product from Clariant International AG, Muttenz, Switzerland) and contains oleyl alcohol polyglycol ether.

[0337] Banana plants were inoculated with Mycosphaeria fijiensis (EPPO code: MYCOFI) by spraying with a spore suspension (spore suspension, 100,000 spores / ml in water supplemented with 0.1% Tween20). After a 48-hour incubation period under a hood maintaining high relative humidity (rh), the inoculated test plants were kept in a greenhouse at 24°C and 90% rh until symptoms were evaluated. When appropriate levels of disease appeared on untreated control plants (7-10 weeks post infection, (expressed as days post infection (DAI))), the percentage of leaf area covered by disease was visually assessed.

[0338] in conclusion

[0339] The results in Table 10 show that treatment of banana plants with Streptomyces coronamicinus CBS149411 dust in combination with spray oil and emulsifier provided control of banana black leaf tip blight.

[0340] Table 10. Reduction of disease symptoms caused by black tip blight (Mycosphaeria fijiensis) in banana after treatment of plants with Streptomyces coronavirinus CBS149411 at the time of preventive spraying.

[0341]

[0342] Example 3.5 Using Streptomyces saigonii 413 to treat fungal diseases on rice

[0343] OD40% or NAD40% preparation of Streptomyces sp. Saigon 413, CBS149411 and 0.05% The 20 combination was sprayed on rice plants one or two days before infection at a spray volume of 400 L / ha.

[0344] For infection with Magnaporthe oryzae (EPPO code: PYRIOR), 14-day-old rice plants cv. Koshihikari were planted in pots. One day after spraying the product, the plants were inoculated with a spore suspension (approximately 85'000 spores per ml) in 0.04% Tween 20 in brushing water. Prior to evaluation, the plants were kept at 23°C, 14 hours of light and high relative humidity (>80%). When appropriate levels of disease appeared on untreated control plants (approximately 8 days after infection (DAI)), the percentage of leaf area covered by disease was visually evaluated.

[0345] For infection with Rhizoctonia solani (EPPO code: RHIZSO), 14-day-old rice plants of the variety Koshihikari were planted in pots. One day after spraying the product, the plants were inoculated with a mycelial suspension in brushing water plus 0.1% Tween 20 (mycelials impregnated with a blender and filtered through a sieve). Prior to evaluation, the plants were kept at 23°C, 14 hours of light and high relative humidity (>80%). When appropriate levels of disease appeared on untreated control plants (approximately 7 days after infection (DAI)), the percentage of leaf area covered by disease was visually evaluated.

[0346] For infection with Xanthomonas oryzae pv. oryzae (EPPO code: XANTOR), 3-week-old rice cv. Koshihikari are treated with the formulated test compounds in a spray chamber. Two days after application, rice plants are infected by cutting off the upper ends of the leaves with scissors previously dipped in the bacterial suspension. After an incubation period of 14 days at 23°C and high relative humidity (>80%), the disease level is assessed visually.

[0347] product (containing the fungicide propiconazole, from Syngenta, Basel, Switzerland) and (containing the fungicide azoxystrobin, from Syngenta, Basel, Switzerland) was used as a positive control in the experiments.

[0348] in conclusion:

[0349] The results in Table 11 show that Streptomyces coronatus CBS149411 formulated at OD40% or NAD40% provided control of rice blast (PYRIOR), sheath blight (RHIZSO) and bacterial blight (XANTOR) in rice.

[0350] Table 11. Reduction of disease symptoms caused by rice blast (Pyricularia oryzae), sheath blight (Rhizoctonia solani) or bacterial leaf blight (Xanthomonas oryzae pv. oryzae) after treatment of plants with a Streptomyces coronomids CBS149411 OD 40% and NAD 40% formulation and chemical fungicides in preventive treatments

[0351]

[0352] Example 3.6. Using Streptomyces sp. Saigon 413 and SOLATENOL TM Treatment of powdery mildew on wheat (Blumeria graminicola type 1)

[0353] Streptomyces coronaeum CBS149411 TGAI powder was applied to wheat plants in combination with a tank mix adjuvant (methylcellulose (3%) in water) to improve retention and spread. The spray volume was 200 L / ha. The efficacy of the CBS149411 derived formulation was compared with a commercial reference fungicide (Elatus TM Plus, with SOLATENOL TM , from Syngenta, Basel, Switzerland) were compared.

[0354] 14-day-old wheat seedlings (cultivar Arina) were inoculated with Blumeria graminis, wheat-specific (EPPO code: ERYSGT) by dusting spores from infected plants with good sporulation (from the disease nursery). The inoculated test plants were kept at 20°C and 60% rh in a greenhouse. When appropriate levels of disease appeared on untreated control plants (about 7 days after infection (DAI)), the percentage of leaf area covered by disease was visually assessed.

[0355] in conclusion

[0356] The results in Table 12 show that Streptomyces coronamicinus CBS149411 TGAI in combination with a tank-mix adjuvant provided control of powdery mildew Blumeria graminearum in wheat.

[0357] Table 12. Use of Streptomyces coronaeum CBS149411 and SOLATENOL TM Reduction of disease symptoms caused by wheat powdery mildew (Blumeria graminicola type 1 in Gramineae) after treatment of plants

[0358]

[0359] Example 3.7. Using Streptomyces sp. Saigon 413 or SOLATENOL TM Treating Fungal Infections in Tomatoes, Cucumbers, and Peanuts

[0360] One day before infection, the OD40% preparation of Streptomyces coronomicinus CBS 149411 was mixed with 0.025% 20 combinations sprayed on tomato, cucumber and peanut plants at a spray volume of 400L / ha. Contains SOLATENOL TM Elatus TM Plus (Syngenta, Basel, Switzerland) was used as the reference fungicide

[0361] Alternaria solani (EPPO code: ALTESO)

[0362] Four-week-old tomato plants of the cv. Roter Gnom are sprayed with the formulated test compound diluted in water in a spray chamber. Two days after application, the test plants are inoculated by spraying them with a spore suspension. These inoculated test plants are incubated in a greenhouse at 22°C / 18°C (day / night) and 95% rh (relative humidity), and the percentage leaf area covered by disease is assessed when appropriate levels of disease appear on untreated check plants (5-7 days after application).

[0363] Cucurbit Anthracnose (EPPO code: COLLLA)

[0364] One week old cucumber plants cv. Wisconsin were treated with formulated test compounds in a spray chamber. One day after application, the plants were inoculated by spraying a spore suspension (1 x 105 conidia / ml) on the test plants. After a 1 day incubation period at 25°C and 100% rh, the plants were kept in a greenhouse at 22°C and 70% rh for 6 days. The incidence was assessed 7 days after inoculation (DAI).

[0365] Mycosphaeria arachidica (EPPO code: MYCOAR)

[0366] 3-week-old peanut plants cv. Florunner were treated with the formulated test compounds in a spray chamber. One day after application, the plants were inoculated by spraying a spore suspension (3 x 105 conidia / ml) on the test plants. After a 4-day incubation period at 24°C and 95% rh, the plants were kept in a greenhouse at 26°C and 70% rh for 7 days. The incidence was assessed 11 days after inoculation (DAI). Rh: relative humidity

[0367] in conclusion

[0368] Table 13 shows that an OD40% formulation of Streptomyces coronamicinus CBS149411 provided control of early blight (Alternaria solani) on tomato, anthracnose (Colletotrichum cucumerinas) on cucumber, and early leaf spot (Mycosphaeria arachis) on peanut.

[0369] Table 13. Use of Streptomyces coronaensis CBS149411 OD40% formulation and SOLATENOL TM Reduction of disease symptoms caused by early blight (Alternaria solani) on tomatoes, anthracnose (Colletotrichum cucumeris) on cucumbers, or early leaf spot (Mycosphaeria arachis) on peanuts after treatment of plants

[0370]

[0371] Example 3.8. Treatment of fungal infections of barley and wheat with Streptomyces coronatus CBS149411 (Streptomyces species Saigon 413) and chemical fungicides

[0372] One day before infection, OD40% and NAD40% formulations from Streptomyces coronatus CBS149411 were sprayed on barley and wheat plants at a spray volume of 200 L / ha. TM Elatus TM Plus (Syngenta, Basel, Switzerland) was used as the reference fungicide

[0373] Swan neck columnar septoria (EPPO code: RAMUCC)

[0374] Twelve-day-old barley plants cv. Venture were treated with the formulated test compounds in a spray chamber. One day after application, the test plants were sprayed with a spore suspension (3 x 10 5 The plants were inoculated with 100 conidia / ml). After a 2-day incubation period under a hood, the plants were kept in a greenhouse at 21°C / 19°C (day / night) and 80% relative humidity for 15 days. The disease incidence was assessed 17 days after inoculation (DAI).

[0375] Triticum truncatum (EPPO code: LEPTNO)

[0376] Thirteen-day-old wheat plants cv. Riband were treated with the formulated test compounds in a spray chamber. One day after application, the test plants were sprayed with a spore suspension (4 x 10 5 Conidia / ml) were used to inoculate plants. After a 2-day incubation period at high relative humidity (>95%), the plants were kept in a greenhouse at 21°C / 19°C (day / night) and 70% relative humidity for 5 days. Disease incidence was assessed 7 days after inoculation (DAI).

[0377] Rhynchophora ryegrassii (EPPO code: RHYNSE)

[0378] Fourteen-day-old barley plants cv. Pasadena were treated with the formulated test compounds in a spray chamber. One day after application, the spore suspension (4 x 10 5 Conidia / ml) were used to inoculate plants. After a 2-day incubation period at high relative humidity (>95%), the plants were kept in a greenhouse at 21°C / 19°C (day / night) and 70% relative humidity for 13 days. The disease incidence was assessed 15 days after inoculation (DAI).

[0379] in conclusion

[0380] The results in Table 14 show that the OD40% or NAD40% formulations of Streptomyces coronomids CBS148411 provided control of barley septoria leaf spot (Swan neck septoria), wheat glumen blight (Aspergillus tritici) or barley wilt (Septoria secalis)

[0381] Table 14. Reduction of disease symptoms caused by schizontella leaf spot (Schizontella swannensis), wheat glume blight (Aspergillus tritici) or barley wilt (Schizontella secalis) after treatment of plants with OD40% or NAD40% formulations of Streptomyces coronomids

[0382]

[0383] Example 3.9. Treatment of wheat eyespot disease (Aaronella syringae) with Streptomyces coronomids CBS149411 (Streptomyces species Saigon 413) and cyprodinil

[0384] One day before infection, the NAD 40% formulation of Streptomyces coronomicin CBS149411 was sprayed on the plants at a spray volume of 200 L / ha. WG750 (Syngenta, Basel, Switzerland) was used as the reference fungicide

[0385] For infection with Aaron's eye spot bacteria (EPPO code: PSDCHE)

[0386] 11-day-old wheat plants cv. Arina were treated with the formulated test compounds in a spray chamber. One day after application, the spore suspension (6 x 10 5 Conidia / ml) were inoculated into the plants, especially targeting the base of the plants. After a 2-day incubation period under a hood, the plants were kept in a greenhouse at 11°C / 10°C (day / night) and 80% relative humidity. When the untreated controls fully developed symptoms (about 7 to 12 weeks after inoculation), the incidence was assessed.

[0387] in conclusion

[0388] The results in Table 15 show that Streptomyces coronomicin CBS 149411 formulated with NAD 40% provides an effective means of controlling wheat eye spot disease (Aaronella eye spot). Table 15. Reduction of disease symptoms caused by wheat yellow rust (Aaronella eye spot) with Streptomyces coronomicin CBS 149411 and cyprodinil

[0389]

[0390] Example 3.10. Treatment of wheat fungal diseases with Streptomyces coronaeum CBS149411, beyond the treatment area

[0391] One day before infection, a NAD 40% formulation of Streptomyces coronatus CBS149411 was applied on leaf segments at a volume of approximately 400 L / ha. The efficacy of the CBS149411 derived formulation was compared with a commercial reference fungicide (Elatus TM Plus, with SOLATENOL TM , Syngenta AG, Switzerland) were compared.

[0392] Seedlings of wheat variety Riband (for wheat leaf blight test) or variety Arina (for hidden stem rust test) 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 approximately equal-sized sections: leaf base, leaf middle, and leaf tip. The compound to be tested is diluted in water at the indicated concentration (in ppm). Use a regular cotton swab to apply the diluted compound to the middle section of the second leaf; soak the cotton swab in the diluted compound and rub it several times on the adaxial leaf surface between the two marks. One day later, use a paint brush to inoculate the entire plant with a fungal spore suspension. Apply the spore suspension until it is about to flow down.

[0393] For infection with Psoralea corylifolia (EPPO code: SEPTTR), one day after application, the test plants were inoculated by spraying a spore suspension (1.5 Mio spores / ml in water supplemented with 0.01% Tween 20) on them. After an incubation period of 4 days at 22°C / 21°C (day / night) and 95% rh, the inoculated test plants were kept in a greenhouse at 22°C / 21°C (day / night) and 70% rh. Efficacy was evaluated directly when an appropriate level of disease appeared on untreated control plants (16-19 days after application (DAI)).

[0394] For infection with Puccinia reconditatis (EPPO code: PUCCRE), one day after application, the test plants were inoculated by spraying them with a spore suspension (spore suspension, 80,000 spores / ml in water supplemented with 0.1% Tween 20). After an incubation period of 1 day at 20°C and 95% rh, the inoculated test plants were kept at 20°C and 60% rh in a greenhouse. When an appropriate level of disease appeared on untreated control plants (9-12 days after infection (DAI)), the percentage of leaf area covered by disease was visually assessed.

[0395] in conclusion

[0396] The results in Table 16 show that Streptomyces coronomicinus CBS149411 was able to protect the leaf area of ​​the treated wheat plants from infection with wheat leaf blight (Psoralea corylifolia) and brown rust (Puccinia recursa). The level of protection achieved by Streptomyces coronomicinus CBS149411 was comparable to that achieved by conventional fungicides (e.g., SOLATENOL TM ) is comparable to SOLATENOL TM Similarly, the apical portion of the untreated 2nd leaf was also well protected from both diseases. To our surprise, the basal portion of the 2nd leaf was also highly protected from the disease (>88%) when the 2500 ppm assay rate was used. Similarly, the systemic 3rd leaf was also protected by applying the product on the 2nd leaf. Protection of the basal portion of the 2nd leaf as well as the systemic 3rd leaf was found to be dependent on the application rate.

[0397] Table 16. Reduction of disease symptoms caused by Septoria tritici (Triticum oleraceum) and brown rust (Puccinia recondita) on treated and systemic (untreated) leaves after plants were treated with Streptomyces cerevisiae CBS149411 formulated with NAD 35% at the time of preventive application.

[0398]

[0399] For leaf 3 (*), a large part of the leaf was new growth since the time of infection, so even in the untreated control there was only partial infection.

[0400] Example 3.11. Treatment of wheat with Fusarium head blight using Streptomyces coronatus CBS149411 (Streptomyces species Saigon 413)

[0401] One day before infection, a NAD 35% formulation of Streptomyces coronatus CBS149411 was applied to the leaf segments in the presence of a tank mix adjuvant at a spray volume of approximately 220 L / ha. TM Miravis TM Plus (Syngenta, Switzerland) was used as the reference fungicide.

[0402] Fusarium solani (EPPO code: GIBBAV)

[0403] Flowering wheat plants cv. Monsun were treated with the formulated test compounds in a spray chamber. One day after application, the spores were sprayed on the ears with a spore suspension (1 x 10 5The plants were inoculated with 1000 conidia / ml). After a 2-day incubation period with water mist under high relative humidity (>95%) and without light, the plants were kept in a greenhouse at 21°C / 19°C (day / night) and 70% relative humidity for 12 days. The incidence of the ears was evaluated 14 days after inoculation. The inoculum consisted of a triple mix of three different isolates (ratio of approximately 1:1:1), each of which has been shown to produce a different mycotoxin specific to the species (enfusin A, enfusin A1, enfusin B, enfusin B1, moniliformin).

[0404] Fusarium graminearum (EPPO code: GIBBZE)

[0405] Flowering wheat plants cv. Monsun were treated with the formulated test compounds in a spray chamber. One day after application, the spores were sprayed on the ears with a spore suspension (1 x 10 5 The plants were inoculated with 100 conidia / ml). After a 2-day incubation period with water mist at high relative humidity (>95%) and without light, the plants were kept in a greenhouse at 21°C / 19°C (day / night) and 70% relative humidity for 5 days. The incidence of the ears was evaluated 7 days after inoculation. The inoculum consisted of a triple mix of three different isolates (ratio of approximately 1:1:1), each of which has been shown to produce a different mycotoxin specific to the species (DON = deoxynivalenol, 3- or 15-acetyldeoxynivalenol, zearalenone).

[0406] in conclusion

[0407] The results in Table 17 show that Streptomyces coronomicin CBS 149411 formulated with NAD 35% provides an effective means of controlling Fusarium head blight (Fusarium graminearum or Fusarium avenae) of wheat. Fusarium species with different chemotypes (DON producers and enfusarin producers) were controlled

[0408] Table 17. Reduction of disease symptoms caused by Fusarium head blight infection of wheat (Fusarium graminearum or Fusarium avenae) after plants were treated with formulated samples at the time of preventive spraying

[0409]

[0410] Example 4. Seed treatment with Streptomyces coronaeum CBS149411

[0411] Semi-field platform method as used in Examples 4.1A and 4.1B

[0412] The semi-field platform consists of a water basin system containing basins in which the soil temperature is controlled. A cooling device allows the water temperature to be controlled according to the requirements of the disease establishment, independent of external weather conditions. Plastic basins (volume 60L) are filled with 45L of freshly sieved soil

[0413] Seed treatment

[0414] Before sowing, using Turbula mixer, according to standard procedures known to those skilled in the art, for corn and wheat, 5g and 10g of slurry (containing test compound) were used for treating seeds per kg of seed, respectively. Treated seeds were allowed to dry in open bottles at room temperature for 24h and then stored in paper bags at room temperature until sowing.

[0415] Example 4.1A. Effect of Streptomyces graminearum Saigon 413 on soil-borne Fusarium graminearum in maize under semi-field conditions.

[0416] In this semi-field experiment, the topsoil layer was mixed with a pre-inoculated substrate of the test fungus (i.e., Fusarium graminearum). A total of 100 seeds of maize cultivar Arma were sown per pot. A completely randomized block design was used with 4 replicates (pots) per treatment. The pots were placed in a water tray filled with cold water (10°C). The water temperature was maintained at about 10°C for the first three weeks and then at 15°C to 20°C for the next two weeks.

[0417] Corn seeds were treated using the standard procedures disclosed above as follows:

[0418] Spray-dried culture broth, 200 g TGAI / 100 kg seeds of Streptomyces spp. Saigon 413

[0419] Freeze-dried supernatant, 200 g TGAI Streptomyces saigon 413 / 100 kg seeds

[0420] Standard (reference treatment), fludioxonil (product: Celest, FS025), 2.5 gai / 100 kg seeds

[0421] Two control treatments were included in this test, an infected control and an uninfected control.

[0422] The activity (%) of each seed treatment was calculated based on the plant emergence rate (number of plants) (%) at the final seedling stage compared with that of the infected control.

[0423] result:

[0424] Disease pressure in the experiment was high, i.e. final plant establishment was reduced by 72% in the infected control treatment. The reference treatment provided 97% activity. The spray-dried culture fluid provided 84% activity. The freeze-dried supernatant provided 69% activity.

[0425] Example 4.1B. Effect of seed-applied Streptomyces spp. Saigon 413 on soil-borne infection of Fusarium graminearum in corn and Fusarium solani in wheat under semi-field conditions.

[0426] In this semi-field experiment, the topsoil layer was mixed with a pre-inoculated matrix of the test fungus, either Fusarium graminearum (K-6102) or Fusarium solani. A total of 100 seeds of maize cv. Andromeda or 100 seeds of wheat cv. Taifun were sown per pot. A completely randomized block design was used with 4 replicates (pots) per treatment. The pots were placed in a water tray filled with cold water (12°C).

[0427] Corn and wheat seeds were treated as follows using the standard procedures disclosed above:

[0428] o Treat wheat seeds with a flowable concentrate of Streptomyces spp. Saigon 413 (FS300) at 200 g TGAI / 100 kg seeds

[0429] o Treatment of corn seeds with OD400 of an oil dispersion of Streptomyces Saigon 413 at 200 g TGAI / 100 kg seeds

[0430] o Standard (reference treatment), Fludioxonil (Product: Celest, FS025)

[0431] o 2.5g ai / 100kg seeds, used in corn experiments

[0432] o 5g ai / 100kg seeds, used in wheat experiments

[0433] Two control treatments were included in this test, an infected control and an uninfected control.

[0434] The activity (%) of each seed treatment was calculated based on the plant emergence (%) at the final plant emergence compared with the infected control.

[0435] result:

[0436] Disease pressure in the trials was moderate, i.e. final plant establishment in the infected control treatment was reduced by 19% for wheat and 34% for maize. The reference treatment provided 100% activity in the maize trial and 93% activity in the wheat trial.

[0437] In the wheat experiment using Fusarium oxysporum, Streptomyces Saigon 413 provided 92% activity at 200 g TGAI.

[0438] In a corn experiment using Fusarium graminearum, Streptomyces Saigon 413 provided 99% activity at 200 g TGAI.

[0439] The results showed that both the oil dispersion formulation and the flowable concentrate formulation produced over 90% activity.

[0440] Example 4.1C. Effect of Streptomyces Saigon 413 applied to corn seeds on soil-borne Fusarium graminearum under controlled conditions in a greenhouse.

[0441] Maize cv. Andromeda seeds were treated with a slurry amount of 5 g / kg seed using a Turbula mixer according to standard procedures known to those skilled in the art. The treated seeds were dried in an open bottle at room temperature for 24 h. Four replicates (soil trays) were used, each containing 25 seeds. Prior to sowing, the soil was incubated at 18° C. for 7 days with fungal spores of the test pathogen (i.e., Fusarium graminearum).

[0442] The following seed application treatments were compared:

[0443] Streptomyces spp. Saigon 413TGAI, formulated as FS300, 200g TGAI / 100kg seeds

[0444] Standard (reference treatment), fludioxonil (product: Celest, FS025), 2.5 gai / 100 kg seeds

[0445] Two control treatments were included in this test, an infected control and an uninfected control.

[0446] The activity (%) of each seed treatment was calculated based on the plant emergence (%) at the final seedling stage compared with the infected control.

[0447] result:

[0448] Disease pressure in the experiment was moderately high, i.e. final plant establishment was reduced by 45% in the infected control treatment. The reference treatment (fludioxonil) provided 96% activity at 2.5 g ai. Streptomyces coronatus Saigon 413 TGAI provided 82% activity at 200 g TGAI.

[0449] Example 4.2. Effect of seed-applied Streptomyces cladoceronis Saigon 413 on soil-borne infection of Fusarium sporotrichioides in soybean under controlled conditions in a greenhouse.

[0450] Soybean cv. Toliman seeds were treated using the standard procedure disclosed above. Five replicates (pots) were used, each containing 5 seeds. The soil was mixed with a matrix pre-inoculated with the test fungus (i.e., Fusarium sporotrichioides).

[0451] The following seed application treatments were compared:

[0452] Streptomyces spp. Saigon 413TGAI, formulated as FS300, 200g TGAI / 100kg seeds

[0453] · Standards (reference treatment), (Active ingredient: Fluopicolide; FS500), 40 gai / 100 kg seeds

[0454] Two control treatments were included in this test, an infected control and an uninfected control.

[0455] Calculation of activity (%) of the seed treatments was based on a disease index derived from the severity of symptoms on the leaves compared with infected controls.

[0456] result:

[0457] The disease pressure in the experiment was high, i.e., the incidence rate was 80%. Provides 89% activity at 40 g ai. Streptomyces crotonii Saigon 413TGAI provides 58% activity at 200 g ai.

[0458] Example 4.3. Effect of seed-applied Streptomyces spp. Saigon 413 on soil-borne Aphanomyces spp. (oomycete) in sugar beet under controlled conditions in a greenhouse.

[0459] Seeds of sugar beet cv. Jagger were treated according to standard procedures known in the art as disclosed above. Five replicates (pots) were used, each containing 25 seeds. Seven days after sowing, a mycelial suspension of the plant pathogen Aphanomyces spirochetes strain K-9164 was added to each pot.

[0460] The following seed application treatments were compared:

[0461] Streptomyces Saigon 413TGAI, formulated as FS300, TGAI g ai / 1 million seeds

[0462] Standard (reference treatment), oxazolidinone (product Tachigaren, WS070), TGAI g ai / 1 million seeds

[0463] Two control treatments were included in this test, an infected control and an uninfected control.

[0464] The activity (%) of each seed treatment was calculated based on damping-off symptoms (dead plant counts) compared with infected controls.

[0465] result:

[0466] The disease pressure in the experiment was moderately high, ie 56% of the plants died in the infected controls. The reference treatment (hymexazol) provided 74% activity. Streptomyces Saigon 413TGAI provided 53% activity.

[0467] Example 4.4. Effect of seed-applied Streptomyces graminearum Saigon 413 on soil-borne infection of Thiothiophorum graminearum in wheat under controlled conditions in a greenhouse

[0468] Wheat cv. Arina seeds were treated according to the standard procedure disclosed above. Four replicates (pots) were used, each containing 15 seeds. At sowing, the soil substrate was mixed with the fungal inoculum.

[0469] The following seed application treatments were tested:

[0470] Streptomyces coronaeum (Streptomyces Saigon 413) TGAI, as FS300 (FS: flowable concentrate for seed treatment), at 100 g ai / 100 kg seeds

[0471] Two control treatments were included in this test, an infected control and an uninfected control.

[0472] The activity (%) of the seed treatments was calculated based on the root health (severity of disease symptoms) compared with the control treatments.

[0473] result:

[0474] The disease pressure in the experiment was high, ie 76% of the roots in the infected controls were unhealthy. Streptomyces Saigon 413 TGAI provided 25% activity (efficacy).

[0475] The results of Experiments 4.1 to 4.4 are summarized in Table 18.

[0476] in conclusion

[0477] The results of experiments 4.1 to 4.4 as shown in Table 18 show that Streptomyces coronomicinus CBS149411 effectively controls diseases caused by several fungal infections (Fusarium and Acrocystis) and oomycete infections on seeds.

[0478] Table 18. Summary of the activity of Streptomyces coronomicinus CBS149411 on seeds against several Fusarium species on different crops, against the oomycete Aphanomyces spirulina on sugar beet and against Thiophyta graminearum on wheat

[0479]

[0480] Example 5. Activity of Streptomyces spp. Saigon 413 against a broad spectrum of Fusarium species using an in vitro bioassay

[0481] In an in vitro bioassay, various Fusarium species were tested using petri dishes (9 cm diameter) containing Luria broth agar growth medium and different concentrations of spray-dried Streptomyces coronarius Saigon 413 (formulated to OD400) (see Table 19). Three replicates (petri dishes) were included for each treatment concentration and for the control. Agar plugs were removed from colonies growing Fusarium and one plug was placed in the center of each agar plate. The fungi were incubated at 22°C in the dark. After incubation, mycelial growth (diameter) was measured to calculate efficacy and half maximal effective concentration, EC-50.

[0482] Table 19. EC50 values ​​of spray-dried Streptomyces serovar Saigon 413TGAI against a broad spectrum of Fusarium species using in vitro bioassays

[0483] Fusarium species EC-50(ppm) Fusarium graminearum 180 Pseudofusorium graminearum 240 Fusarium scaly 210 Fusarium avenae 380 Fusarium sporotrichioides 400 Fusarium verticillium 200 Fusarium fujikura 490 Fusarium submyces 230 Fusarium oxysporum Cuban 350 Fusarium oxysporum melon 360 Fusarium oxysporum wilt type 340 Fusarium oxysporum tomato-specific 350

[0484] Example 6. Efficacy of Streptomyces spp. Saigon 413TGAI against Botrytis cinerea and Sclerotinia sclerotiorum under micro-profiling screening

[0485] Botrytis cinerea (Botrytis cinerea): Fungal conidia from frozen storage were mixed directly into nutrient broth (Vogel's minimal medium). DMSO solutions of the test compounds were placed in microtiter plates (96-well format) and the nutrient broth containing the fungal spores was added thereto. The test plates were incubated at 24° C. and the inhibition of growth was determined photometrically after 72 hours.

[0486] Sclerotinia(cotton rot, white rot, etc.): Mycelial fragments of the fungus prepared from fresh liquid culture were directly mixed into nutrient broth (PDB potato dextrose broth). DMSO solutions of the test compounds were placed in microtiter plates (96-well format) and nutrient broth containing fungal spores was added thereto. The test plates were incubated at 24°C and after 72 hours the inhibition of growth was determined photometrically at 620 nm.

[0487] result:

[0488] Botrytis cinerea :37ppm (mg / L) is 100% effective

[0489] Sclerotinia :333ppm (mg / L) is 100% effective

[0490] PCT

[0491] (Electronic original)

[0492] (This form is not part of, and does not count as, the international application form)

[0493]

[0494] For receiving office use only

[0495] For International Bureau use only

Claims

1. An isolated microbial strain comprising a genome sequence having at least 99.8% identity to the whole genome of Streptomyces sp. Saigon 413 deposited at the Fungal Culture Collection under the accession number CBS149411.

2. An isolated microbial strain, optionally as claimed in claim 1, wherein the strain comprises a nucleotide sequence having at least 99.9% identity with SEQ ID NO:

1.

3. The microbial strain according to claim 1 or 2, wherein the microbial strain is Streptomyces coronavirinus, preferably Streptomyces species Saigon 413 deposited in the Fungal Culture Collection Center with the accession number CBS149411.

4. A composition comprising a strain of Streptomyces, and malenomycin, and at least one compound selected from the group consisting of cyclothiazolylmycin C, streptoglutarimide, an oligosaccharide compound according to compound I, wherein compound I comprises a compound according to C 53 H 90 N2O 44 The molecular formula is further characterized by the NMR spectra listed in Table 2 and Table 3, preferably characterized by structural formula I, A lipopeptide according to formula II, or a salt thereof, wherein R1 = CH3 or C2H5 and a polyene compound, wherein the polyene compound is prepared by 67 H 115 NO 25 The polyene is characterized by a molecular formula of , wherein the polyene is further characterized by a light absorption spectrum as shown in FIG10 .

5. The composition of claim 4, further comprising an adjuvant.

6. A composition as described in claim 4 or 5, wherein the Streptomyces strain comprises a genome sequence that is at least 91% identical to the whole genome of Streptomyces crown-mycin NRRLB-3672, or a genome sequence that is at least 91% identical to the whole genome of Streptomyces species Saigon 413 deposited in the Fungal Culture Collection under the accession number CBS149411.

7. The composition of any one of claims 4 to 6, wherein the Streptomyces strain comprises a nucleotide sequence that is at least 99% identical to SEQ ID NO:

1.

8. The composition of any one of claims 4 to 7, wherein the composition is a fermentation broth, preferably a spray-dried fermentation broth or a freeze-dried fermentation broth, or a formulation.

9. The composition of any one of claims 4 to 8, wherein the composition comprises 10 2 Up to 10 12 cfu / g dry weight of the Streptomyces strain or microbial strain.

10. The isolated microbial strain of any one of claims 1 to 3, or the composition of any one of claims 4 to 9, wherein the microbial strain or the Streptomyces strain comprises at least one nucleotide sequence encoding a protein having at least 80% identity with an amino acid sequence of SEQ ID NO:71 to SEQ ID NO:115, preferably SEQ ID NO:91 and / or SEQ ID NO:92, preferably at least 80% identity with at least one of the nucleotide sequences of SEQ ID NO:2 to 46, preferably at least 80% identity with at least one nucleotide sequence of SEQ ID NO:22 or 23.

11. An isolated microbial strain as described in any one of claims 1 to 3 or 10, or a composition as described in any one of claims 4 to 10, wherein the isolated microbial strain or the Streptomyces strain comprises at least one nucleotide sequence encoding a protein having at least 80% identity with an amino acid sequence of SEQ ID NO: 116 to SEQ ID NO: 139, preferably an amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 137, preferably at least one nucleotide sequence having at least 80% identity with at least one of the nucleotide sequences of SEQ ID NO: 47 to 70, preferably at least one nucleotide sequence having at least 80% identity with at least one nucleotide sequence of SEQ ID NO: 67 or 68.

12. A method for producing a microbial strain as described in any one of claims 1 to 3, 10 or 11 or a composition as described in any one of claims 4 to 11, comprising culturing the microbial strain or the Streptomyces strain in a suitable fermentation medium under suitable fermentation conditions, and optionally comprising the step of recovering the microbial strain or the composition.

13. The method of claim 12, wherein the microbial strain or the Streptomyces strain produces at least one of malenomycin and a compound selected from the group consisting of cyclothiazolylmycin C, streptoglutarimide, an oligosaccharide compound according to Compound I, wherein Compound I comprises a molecule according to Compound C. 53 H 90 N2O 44 The molecular formula is further characterized by the NMR spectra listed in Table 2 and Table 3, preferably wherein Compound I is further characterized by structural formula I, And a lipopeptide according to formula II, or a salt thereof, wherein R1 = CH3 or C2H5 and a polyene compound, wherein the polyene compound is prepared by 67 H 115 NO 25 wherein the polyene is further characterized by a light absorption spectrum as shown in FIG1 .

14. A method for controlling or preventing plants, plant propagation materials and / or harvested food crops from being infected by plant pathogenic microorganisms, the method comprising treating the plants, plant propagation materials and / or harvested food crops by applying an effective amount of Streptomyces coronamycin, a microbial strain as described in any one of claims 1 to 3, 10 or 11, or a composition as described in any one of claims 4 to 11 to the plants, parts thereof or their locations, the plant propagation materials and / or harvested food crops.

15. The method of claim 14, wherein the plant pathogenic microorganism is a fungus, preferably a fungus belonging to the following genera: Puccinia repens, Mycosphaeria, Pyrospora, Rhizoctonia, Xanthomonas, Graminus graminicola, Alternaria, Colletotrichum, Cylindrospermum, Paracoelenteroides, Rhizoctonia, Eye spot fungus, Fusarium, Acrocystis species, Botrytis or Sclerotinia, preferably a fungus belonging to the following genera: Puccinia repens, Puccinia repens stripe, Mycosphaeria fijiensis, Mycosphaeria arachis, Magnaporthe grisea, Rhizoctonia solani, Xanthomonas oryzae pv. oryzae, Bryophyte graminicola, Gramineae powdery mildew ... The plant pathogenic microorganism is an oomycete, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium graminearum, a type of Fusarium oxysporum, for example, a type of Fusarium oxysporum Cuban, a type of Fusarium oxysporum melon, a type of Fusarium oxysporum wilt, a type of Fusarium oxysporum tomato, a type of Aspergillus graminearum, a type of Botrytis cinerea or a type of Sclerotinia sclerotiorum, or wherein the plant pathogenic microorganism is an oomycete belonging to the genus Aphanomyces, preferably a type of Aphanomyces spiralis.

16. The method of claim 14 or 15, wherein the effective amount comprises 2 x 10 2 Up to 5x 10 17 Colony forming units (cfu) / ha, or 0.1 g to 10 kg of the Streptomyces coronamicinus, or the isolated microorganisms of claims 1 to 3, 10, 11, or the composition of any one of claims 4 to 11 / hectare.

17. The method of claim 14 or 15, wherein the plant propagation material is a seed and the effective amount comprises 2 x 10 2 Up to 5x 10 15 (cfu), or 0.0001 g to 100 g of Streptomyces coronamicinus, or the isolated microbial strain of any one of claims 1 to 3, 10, 11, or the composition of any one of claims 4 to 11 / kg of seeds.

18. The method of any one of claims 14 to 17, wherein the plant comprises wheat, barley, rice, corn, soybean, sugar beet, banana, tomato, cucumber and / or peanut.

19. A plant or plant propagation material treated with a microbial strain as claimed in claims 1 to 3, 10 or 11 or a composition as claimed in any one of claims 4 to 11.

20. Use of the microbial strain according to any one of claims 1 to 3, 10 or 11, Streptomyces coronamicinus or the composition according to any one of claims 4 to 11 as a pesticide, preferably as a fungicide.

21. Use of a microbial strain or a Streptomyces strain as claimed in any one of claims 1 to 3, 10 or 11, wherein the Streptomyces strain has at least 91% identity with the whole genome of Streptomyces coronomicinus NRRL B-3672, or has at least 91% identity with the whole genome of Streptomyces species Saigon 413 deposited in the Fungal Culture Collection under the accession number CBS149411, for producing at least one of malenomycin and a compound selected from the group consisting of cyclothiazolinyl sulfadiazine; streptomyces glutarimide; an oligosaccharide compound according to compound I, wherein compound I comprises the oligosaccharide compound according to compound C 53 H 90 N2O 44 The molecular formula is further characterized by the NMR spectra listed in Tables 2 and 3, preferably characterized by structural formula I; a lipopeptide according to formula II, or a salt thereof, wherein R1 = CH3 or C2H5; and a polyene compound, which is characterized by the formula C 67 H 115 NO 25 The polyene is characterized by a molecular formula of , wherein the polyene is further characterized by a light absorption spectrum as shown in FIG10 .

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