Method of treating fungal disease

Abstract

The present invention relates to a method for controlling or preventing infestation of Zymoseptoria tritici on a plant comprising treating the plant, plant propagation material and / or a locus thereof with a composition comprising a Streptomyces chrestomyceticus, wherein the Zymoseptoria tritici is resistant against SDHI, QoI and / or DMI fungicides.

Description

[0001] METHOD OF TREATING FUNGAL DISEASE

[0002] The present invention relates to a method for controlling or preventing infestation of Zymoseptoria tritici on a plant comprising treating the plant, plant propagation material and / or a locus thereof with a composition comprising a Streptomyces chrestomyceticus.

[0003] BACKGROUND

[0004] Zymoseptoria tritici (Septoria tritici) is a widespread fungus that causes septoria tritici blotch, a foliar disease of wheat which poses a significant threat to global food production. Zymoseptoria tritici is a difficult fungus to control because Zymoseptoria tritici populations contain extremely high levels of genetic variability. In addition, resistance to known chemical fungicides in Zymoseptoria tritici may develop easily.

[0005] Resistances or adaptations that are known to have been developed in Zymoseptoria tritici are against succinate dehydrogenase inhibitor fungicides, also called SDHI-fungicides, Quinone outer, “Qo” inhibitor (Qol) fungicides and demethylation inhibitor (DMI) fungicides (see also the webpage of the Fungicide Resistance Action Committee (FRAC)).

[0006] Resistance in Zymoseptoria tritici against quinone outside inhibitors (Qol) is caused by a G143A substitution in cytochrome b, and the occurrence of Qol resistance Zymoseptoria tritici is increasing. Cheval et al. (2017), Crop protection, Vol 92, 131-133, suggest to manage such Qol resistant strains with DMIs or SDHIs.

[0007] In addition, resistance against DMI’s and SDHI’s is also developing (Kildea et al, 12 October 2023, A note on the impact of CYP51 alterations and their combination in the wheat pathogen Zymoseptoria tritici on sensitivity to the azole fungicides epoxiconazole and metconazole Irish Journal of Agricultural and Food Research, 62(1), 44-50) and (Dooley et al, (2016) Detection of Zymoseptoria tritici SDHI-insensitive field isolates carrying the SdhC-H152R and SdhD-R41W substitutions. Pest Management Science,: 72: 2203-2207) and use of mixtures of chemical DMI’s and SDHI’s may loose its efficiency.

[0008] WQ2018 / 069114 discloses Zymoseptoria tritici strains having a T286I amino acid substitution in the SDH subunit B or a H152R, T79N, N86S, V166M or W80S amino acid substitution in the SDH subunit C, which showed a level of resistance against a number of SDHI fungicides, but which strains were still efficiently treated by the SDHI fungicide pydiflymetofen.

[0009] WQ2024 / 074637 discloses a method for controlling fungi on plants, for instance Zymoseptoria tritici, using a composition comprising a Streptomyces chrestomyceticus. WQ2024 / 074637 does not disclose whether the S. chrestomyceticus effectively controls Zymoseptoria tritici strains which have developed resistance against fungicides with different mode of action, such as to SDHI-fungicides, Qol fungicides and / or DMI fungicides.

[0010] There is a need for improved methods to control Zymoseptoria tritici diseases. SUMMARY

[0011] The present invention relates to a method for controlling or preventing infestation of Zymoseptoria tritici on a plant comprising treating the plant, plant propagation material and / or a locus thereof with a composition comprising a Streptomyces chrestomyceticus, wherein the Zymoseptoria tritici is resistant against SDHI, Qol and / or DMI fungicides.

[0012] DETAILED DESCRIPTION

[0013] The present invention relates to a method for controlling or preventing infestation of Zymoseptoria tritici on a plant comprising treating the plant, plant propagation material and / or a locus thereof with a composition comprising a Streptomyces chrestomyceticus, wherein the Zymoseptoria tritici is resistant against succinate dehydrogenase inhibitor (SDHI), Quinone outer inhibitor (Qol) and / or demethylation inhibitor (DMI) fungicides. It was surprisingly found that a composition comprising Streptomyces chrestomyceticus effectively controlled or prevented infestation of Zymoseptoria tritici that was resistant against SDHI, Qol and / or DMI fungicides.

[0014] A Zymoseptoria tritici that is resistant against a fungicide, such as SDHI, Qol and / or DMI fungicides, as used herein means a Zymoseptoria tritici strain that has been adapted to a fungicide, for instance the Zymoseptoria tritici has been adapted such that the treatment of a fungicide is reduced with at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or at least 80%, or at least 90% as compared a Zymoseptoria tritici strain that is not resistant or that has not been adapted to the fungicide. A Zymoseptoria tritici strain that is resistant against a SDHI, Qol and / or DMI fungicides typically comprises one or more mutations in a gene encoding a succinate dehydrogenase (SDH) subunit, one or mutations, in particular one specific mutation, in a gene encoding cytochrome b (CytB) and / or one or more mutations in a gene encoding 14a-demethylase (Cyp51) as compared to a native Zymoseptoria tritici strain which does not contain said mutations. The nucleotide sequences encoding the succinate dehydrogenase subunits, cytochrome b and Cyp51 and of Zymoseptoria tritici and their corresponding amino acid sequences are known to a person skilled in the art and can for instance be found on the website of the national center of Biotechnology Information (NCBI).

[0015] It was found that Zymoseptoria tritici comprising an amino acid substitution N225T in the amino acid sequence of the succinate dehydrogenase B subunit, an amino acid substitution T79N, W80S, N86S, and / or H152R in the amino acid sequence of the succinate dehydrogenase C subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence, and / or an amino acid substitution V136A, V136C, 1381V, A379G, Y459G_Y460del, Y461 H, Y461 S, H513K, and I or S524T in the Cyp51 amino acid sequence was effectively controlled by a composition comprising Streptomyces chrestomyceticus as disclosed herein.

[0016] The person skilled in the art understands that a Zymoseptoria tritici in the method as disclosed herein comprising the amino acid substitution N225T in the amino acid sequence of the succinate dehydrogenase B subunit, an amino acid substitution T79N, W80S, N86S, and / or H152R in the amino acid sequence of the succinate dehydrogenase C subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence, and / or an amino acid substitution V136A, V136C, 1381V, A379G, Y459G_Y460del, Y461 H, Y461 S, H513K, and I or S524T in the Cyp51 amino acid sequence, comprises one or more mutation(s) in a gene encoding one or more of the amino acid substitutions. The amino acid substitutions indicated herein are substitutions compared to the native SDHB, SDHC, cytochrome b and Cyp51 proteins of a Zymoseptoria tritic strain known to a person skilled in the art, for instance disclosed on the NCBI website.

[0017] It was found that the Zymoseptoria tritici comprising an amino acid substitution N225T in the SDHB subunit, an amino acid substitution T79N, W80S, N86S, and / or H152R in the SDHC subunit showed decreased sensitivity against benzovindiflupyr (Solatenol™; Elatus Plus EC100). Surprisingly, the composition comprising Streptomyces chrestomyceticus as disclosed herein effectively controlled these SDHI sensitive Zymoseptoria tritici strains.

[0018] It was surprisingly found that Zymoseptoria tritici comprising an amino acid substitution G143A in the cytochrome b amino acid sequence, which confers high resistance against Qol fungicides, was effectively controlled by a composition comprising Streptomyces chrestomyceticus.

[0019] In addition, it was surprisingly found that Zymoseptoria tritici comprising an amino acid substitution V136A, V136C, 1381V, A379G, Y459G_Y460del, Y461 H, Y461 S, H513K, and I or S524T in the Cyp51 amino acid sequence, which confer resistance to DMI fungicides, was effectively controlled by a composition comprising Streptomyces chrestomyceticus as disclosed herein.

[0020] Suitably, the Zymoseptoria tritici comprises an amino acid substitution G143A at cytochrome b and an amino acid substitution A379G, Y459G_Y460del, 1381V and N513K in the Cyp51 amino acid sequence.

[0021] Suitably, the Zymoseptoria tritici comprises an amino acid substitution T79N in the amino acid sequence of the SDHC subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence and an amino acid substitution V136A, 1381V, Y461 H and S524T in the Cyp51 amino acid sequence.

[0022] Suitably, the Zymoseptoria tritici comprises an amino acid substitution H152R in the amino acid sequence of the SDHC subunit and an amino acid substitution A379G, Y459G_Y460del, V136C, 1381V, and S524T in the Cyp51 amino acid sequence.

[0023] Suitably, the Zymoseptoria tritici comprises an amino acid substitution G143A in the cytochrome b amino acid sequence and an amino acid substitution A379G, V136A, 1381V, Y461 S, and S524T in the Cyp51 amino acid sequence.

[0024] Suitably, the Zymoseptoria tritici comprises an amino acid substitution T79N in the amino acid sequence of the SDHC subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence and an amino acid substitution 1381V and Y461 H in the Cyp51 amino acid sequence.

[0025] Suitably, the Zymoseptoria tritici comprises an amino acid substitution G143A in the cytochrome b amino acid sequence and an amino acid substitution Y459G_Y460del, 1381V and N512K in the Cyp51 amino acid sequence.

[0026] Suitably, the Zymoseptoria tritici comprises an amino acid substitution N225T in the amino acid sequence of the SDHB subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence and an amino acid substitution V136A, and 1381V, Y461 H in the Cyp51 amino acid sequence.

[0027] Suitably, the Zymoseptoria tritici comprises an amino acid substitution T79N in the amino acid sequence of the SDHC subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence and an amino acid substitution V136A, 1381V, and Y461 H in the Cyp51 amino acid sequence. Suitably, the Zymoseptoria tritici comprises an amino acid substitution N86S in the amino acid sequence of the SDHC subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence and an amino acid substitution A379G, Y459G_Y460del, V136A, 1381V, and S524T in the Cyp51 amino acid sequence.

[0028] Suitably, the Zymoseptoria tritici comprises an amino acid substitution T79N at the SDHC subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence and an amino acid substitution V136A, 1381V, Y461 S, and S524T in the Cyp51 amino acid sequence.

[0029] Suitably, the Zymoseptoria tritici comprises an amino acid substitution H152R at the SDHC subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence and an amino acid substitution A379G, Y459G_Y 460del, V136A, 1381V and S524T in the Cyp51 amino acid sequence.

[0030] Suitably, the Zymoseptoria tritici comprises an amino acid substitution W80S at the SDHC subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence and an amino acid substitution Y459G_Y460del, 1381V and N513K in the Cyp51 amino acid sequence.

[0031] The protein sequences of the native SDHB, SDHC, cytochrome b and in the Cyp51 can be found in the public database, for instance NCBI: The positions of the amino acids which confer resistance or adaptation in Zymoseptoria tritici can be found on the FRAC website, which is known to a person skilled in the art.

[0032] The composition comprising Streptomyces chrestomyceticus has fungicidal activity or is a fungicide, which means a composition that controls, modifies, or prevents the growth of fungi. The term “fungicidally effective amount” where used means the quantity of such composition that is able of producing an effect on the growth of fungi. Controlling or modifying effects include all deviation from natural development, such as killing, retardation and the like, and prevention includes barrier or other defensive formation in or on a plant to prevent fungal infection.

[0033] In one embodiment, the composition comprising a Streptomyces chrestomyceticus further comprises a metabolite, preferably a metabolite produced by the Streptomyces chrestomyceticus.

[0034] Preferably, the metabolite comprises malonomicin, cyclothiazomycin C, streptimidone, an oligosaccharide which comprises a molecular formula according to C53H90N2O44, suitably characterized by a structural Formula I,

[0035]

[0036] Formula (I), a lipopeptide according to Formula (II), or a salt thereof, wherein Ri = CH3 or C2H5, and / or a polyene compound characterized by a molecular formula according to C67H115NO25, wherein the polyene is further characterized by the spectrum of light absorption with absorbance maxima at a wavelength of 235.5 nm, 301.1 nm, 315.8 nm, 330.9 nm and 348.3 nm when measured in an aqueous acetonitrile solution.

[0037] Cyclothiazomycin C is a known compound. The structure of cyclothazomycin C is disclosed on p. 3 of WO2015191789 and can be produced as disclosed in Example 4 of WO2015 / 191789.

[0038] Malonomicin (sometimes spelt ‘malonomycin’) is {[(2S)-2-amino-3-hydroxypropanoyl]amino} {2- [(5S)-5-(aminomethyl)-4-hydroxy-2-oxo-2,5-dihydro-1 H-pyrrol-3-yl]-2-oxoethyl}malonic acid can be produced as disclosed in Example I of W02006 / 078939. Malonomicin may also be prepared according to the method disclosed in Example I A and B in EP 1860939. or according to Law et al, 2018 (Nature Catalysis | VOL 1 | DECEMBER 2018 | 977-984).

[0039] Streptimidone is a known compound of Formula III (Formula III)

[0040] Streptimidone can be synthesised following the method disclosed in Kondo, H., Oritani, T., and Kiyota, H. Synthesis and antifungal activity of the four stereoisomers of streptimidone, a glutarimide antibiotic from Streptomyces rimosus forma paromomycinus. Eur. J. Org. Chem. (20), 3459-3462 (2000).

[0041] The oligosaccharide which comprises a molecular formula according to C53H90N2O44, suitably characterized by a structural Formula I, is further disclosed and characterized in Example 2 of WO204 / 074627.

[0042] The lipopeptide as disclosed herein comprises a compound according to Formula 11(a) and / or a compound according to Formula 11(b) or a salt thereof and is disclosed and characterized in Example 2 of WO2024 / 074628.

[0043] A lipopeptide compound according to Formula 11(a), or a salt thereof, comprises structural formula

[0044] Formula 11(a).

[0045] The compound according to Formula 11(a) comprises a molecular formula C55H85NH O19 and an exact mass of 1203.602 g. The compound according to Formula ll(a) has a solubility in DMSO of above 10,000 ppm. The compound according to Formula ll(a) comprises or is a lipopeptide.

[0046] A compound according to Formula 11 (b) , or a salt thereof, comprises structural formula

[0047] Formula 11(b),

[0048] The compound of Formula 1(b) comprises or has a molecular formula CseHa / NnOig and an exact mass of 1217.618 g.

[0049] The polyene as disclosed herein is disclosed in WO2024 / 074637.

[0050] The composition comprises a Streptomyces chrestomyceticus, wherein the Streptomyces chrestomyceticus comprises a nucleotide sequence which has at least 99.8 % identity to SEQ ID NO: 1 . Preferably, wherein S. chrestomyceticus comprises a nucleotide sequence which has at least 99.9%, 99.91 %, 99,92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99% or which has 100% identity to SEQ ID NO: 1 .

[0051] In one embodiment the S. chrestomyceticus comprises a genome sequence which has at least 95% identity, preferably at least 96%, 97%, 98%, 99%% identity preferably 100% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411 .

[0052] For example, the S. chrestomyceticus comprises a nucleotide sequence which has at least 99.8 % identity to SEQ ID NO: 1 and a genome sequence which has at least 96% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411 . For example, the S. chrestomyceticus comprises a nucleotide sequence which has at least 99.81 % identity to SEQ ID NO: 1 and a genome sequence which has at least 96.1 % identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411. For example, the S. chrestomyceticus comprises a nucleotide sequence which has at least 99.82 % identity to SEQ ID NO: 1 and a genome sequence which has at least 96.2% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS14941 1. For example, the S. chrestomyceticus comprises a nucleotide sequence which has at least 99.83% identity to SEQ ID NO: 1 and a genome sequence which has at least 96.3% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411. For example, the S. chrestomyceticus comprises a nucleotide sequence which has at least 99.84 % identity to SEQ ID NO: 1 and a genome sequence which has at least 96.4% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS14941 1. For example, the S. chrestomyceticus comprises a nucleotide sequence which has at least 99.85% identity to SEQ ID NO: 1 and a genome sequence which has at least 96.5% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411. For example, the S. chrestomyceticus comprises a nucleotide sequence which has at least 99.86 % identity to SEQ ID NO: 1 and a genome sequence which has at least 96.6% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS14941 1 . For example, the S. chrestomyceticus comprises a nucleotide sequence which has at least 99.87 % identity to SEQ ID NO: 1 and a genome sequence which has at least 96.7% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411 .

[0053] The Streptomyces chrestomyceticus as disclosed herein, preferably comprises at least one nucleotide sequence that encodes a protein that has at least 90%, 95%, 96%, 97%, 98%, 99% identity or has 100% identity to an amino acid sequence according to SEQ ID NO: 71 to 1 15, preferably at least one nucleotide sequence that encodes a protein that has at least 90%, 95%, 96%, 97%, 98%, 99% identity or has 100% identity to an amino acid sequence according to SEQ ID NO: 91 and / or SEQ ID NO: 92, which are involved in the production of a lipopeptide according to Formula II.

[0054] Suitably, the Streptomyces chrestomyceticus as disclosed herein, comprises at least one nucleotide sequence which has at least 90%, 95%, 96%, 97%, 98%, 99% identity or has 100% identity to at least one of the nucleotide sequence(s) of SEQ ID NO: 2 to 46. The nucleotide sequences according to SEQ ID NO: 2 to 46 comprise a gene cluster for the production of a lipopeptide according to Formula II. Suitably, the Streptomyces chrestomyceticus comprises a nucleotide sequence which has at least 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to at least one of the nucleotide sequences according to SEQ ID NO: 22 and I or 23. As disclosed in WO2024 / 074637 these genes were essential in the production of the lipopeptide.

[0055] Suitably, the Streptomyces chrestomyceticus, as disclosed herein 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 nucleotide sequences of 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

[0056] NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID

[0057] NO: 16; SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID

[0058] 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

[0059] 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 which nucleotide sequence(s) has / have at least 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0060] In another embodiment, the Streptomyces chrestomyceticus as disclosed herein comprises at least one nucleotide sequence which encodes a protein which has at least 90%, 95%, 96%, 97%, 98%, 99% identity or which has 100% identity to at least one of the amino acid sequences according to SEQ ID NO: 116-139, which are involved in the production of the polyene which has a molecular formula according to C67H115NO25 and which is disclosed herein.

[0061] Preferably, the Streptomyces chrestomyceticus, 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 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 nucleotide sequences which encode a protein that has / have 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 sequences according to SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 1 18, 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.

[0062] Preferably, the Streptomyces chrestomyceticus as disclosed herein comprises at least one nucleotide sequence which encodes a protein which has at least 90%, 95%, 96%, 97%, 98%, 99% identity or which has 100% identity to at least one of the amino acid sequences according to SEQ ID NO: 136 and / or SEQ ID NO: 137.

[0063] Preferably the Streptomyces chrestomyceticus, as disclosed herein comprises at least one nucleotide sequence which has at least 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to at least one of the nucleotide sequence(s) of SEQ ID NO: 47 to 70. The nucleotide sequences according to SEQ ID NO: 47 to 70 comprise a gene cluster involved in the production of a polyene which has a molecular formula according to C67H115NO25. Suitably, the Streptomyces chrestomyceticus as disclosed herein comprises a nucelotide sequence which has at least 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity to one of the nucleotide sequences according to SEQ ID NO: 67 and / or 68. As disclosed in WO2024 / 074637 these genes were essential in the production of the polyene.

[0064] Suitably, the Streptomyces chrestomyceticus, 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 nucleotide sequences of 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, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70 or which nucleotide sequence(s) has / have at least 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0065] As used herein, the terms "percent identity," and "percent identical" refer to the relatedness of two or more nucleotide or amino acid sequences, which may be calculated by (i) comparing two optimally aligned sequences over a window of comparison, (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100 percent to yield the percent identity. If the "percent identity" is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present invention, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the "percent identity" for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100 percent.

[0066] In one embodiment, the Streptomyces chrestomyceticus is a Streptomyces chrestomyceticus deposited at the Westerdijk Institute under accession number CBS149411 .

[0067] The method according to the present invention does not include a method for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body.

[0068] The method for controlling or preventing infestation of Zymoseptoria tritici on a plant comprises treating any suitable plant, plant propagation material and / or a locus thereof. The plant preferably comprises or is a cereal plant, preferably wheat.

[0069] The term “plant” refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage, and fruits. Germinated plants and young plants which are to be transplanted after germination or after emergence from the soil, may also be mentioned. These young plants can be protected before transplantation by a total or partial treatment by immersion.

[0070] The term “plant propagation material” is understood to denote generative parts of the plant, such as seeds, which can be used for the multiplication of the latter, and vegetative material, such as cuttings or tubers, (for example potatoes), roots, fruits, bulbs, rhizomes or parts of plants. The plant propagation material can be treated with a composition comprising S. chrestomyceticus before the material is sown or planted, or during sowing or planting.

[0071] The term plants involve “useful plants” or “crops”. The term "useful plants" is to be understood as also including useful plants which have been so transformed by using recombinant DNA techniques that they are capable of synthesising one or more selectively acting toxins, such as are known, for example, from toxin-producing bacteria, especially those of the genus Bacillus.

[0072] The term “locus” as used herein means fields in or on which plants are growing, or where seeds of cultivated plants are sown, or where seed will be placed into the soil. It includes soil, seeds, and seedlings, as well as established vegetation. The composition comprising S. chrestomyceticus preferably is an agricultural acceptable composition. An agricultural acceptable composition typically comprises an agricultural acceptable auxiliary. Suitable auxiliaries are known in the art and include for example solvents, liquid carriers, solid carriers or fillers, surfactants, dispersants, emulsifiers, wetters, adjuvants, solubilizers, penetration enhancers, protective colloids, adhesion agents, thickeners, humectants, repellents, attractants, feeding stimulants, compatibilizers, bactericides, anti-freezing agents, anti-foaming agents, colorants, tackifiers and binders.

[0073] The composition as disclosed herein usually comprises a fermentation broth of the S. chrestomyceticus, preferably a spray-dried fermentation broth or a freeze-dried fermentation broth. Spray-drying or freeze-drying of a fermentation broth is known in the art.

[0074] The composition comprising S. chrestomyceticus also includes a formulation, such as a formulation comprising a fermentation broth of S. chrestomyceticus as disclosed herein. A formulation may be any suitable composition for formulating Streptomyces chrestomyceticus.

[0075] Formulations of microbial strains are known in the art for instance as disclosed in Croda Crop Care, the Nouryon formulator toolbox and in: Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments (412 p., 6 December 2012) eds. Burges H.D., Springer, ISBN 978-94-01 1-4926-6. Preferably, a composition as disclosed herein comprises a formulation, wherein the composition comprising Streptomyces chrestomyceticus is formulated as an oil dispersion (OD), a non-aqueous dispersion (NAD) or a flowable formulation.

[0076] The composition as disclosed herein comprises a cell count of the Streptomyces chrestomyceticus, from 1*102to 1 *1013cfu / g dry weight, for instance from 1* 103to 1*1012cfu / g dry weight, from 2*103to 2*1011cfu / g dry weight, from 5*103to 5*1011cfu / g dry weight, for instance from 1*10410-|*1 oioCfu / g dry weight, from 2*104to 2*1010cfu / g dry weight, such as from 1*105to 1*109cfu / g dry weight, from 2*105to 2*109cfu / g dry weight, from 5*105to 5*109cfu / g dry weight, from 1 *106to 1 *108cfu / g dry weight, such as from 2*106to 2*108cfu / g dry weight .

[0077] In one embodiment the method according to the present invention comprises applying a fungicidally effective amount of the composition comprising Streptomyces chrestomyceticus as disclosed herein.

[0078] A fungicidally effective amount comprises from 2*102to 5*1017, from 3*102to 5*1016, from 5*102to 5*1015, from 2*102to 5*1014, from 2*102to 5*1013, preferably 5*102to 5*1012, 1*103to 5*1011, from 5*103to 1*1011, from 1*104to 5*1010, from 5*104to 1*1010, from 1*105to 5*109, from 5*105to 1*109, from 1*106to 5*108, from 5*106to 1*108colony forming unit (cfu) of the composition comprising Streptomyces chrestomyceticus per hectare.

[0079] An effective amount of a composition comprising Streptomyces chrestomyceticus as disclosed herein above, comprises from 0.1 g to 10 kg / per hectare (ha), such as from 0.5 g to 5 kg, such as from 1 g to 1 kg / ha, such as from 5 g to 500 g / ha, such as from 10 g to 400 g / ha. The weight in g and kg is dry weight of the composition comprising Streptomyces chrestomyceticus composition.

[0080] In one embodiment the method according to the present invention comprises treating plant propagation material wherein the plant propagation material is seed and the effective amount comprises 5x102to 5x1015, 2x103to 5x1014, from 5x103to 5x1013, from 2x105to 5x1012, preferably 5*102to 5*1012, 1*103to 5*1011, from 5*103to 1 *1011, from 1 *104to 5*1010, from 5*104to 1*1010, from 1 *105to 5*109, from 5*105to 1 *109, from 1*106to 5*108, from 5*106to 1*108colony forming unit (cfu) of the composition comprising the Streptomyces chrestomyceticus as disclosed herein per kg of seed.

[0081] When the plant propagation material is seed, an effective amount of the composition comprising Streptomyces chrestomyceticus as disclosed herein above, may also comprise from 0.0001 g to 100 g / per kg of seeds, such as from 0.0005 g to 80 g / kg seeds, such as from 0.001 g to 50 g / kg seeds, such as from 0.005 g to 10 g / kg seeds, The weight in g dry weight of the composition comprising Streptomyces chrestomyceticus per kg dry weight of seeds.

[0082] EXAMPLES

[0083] MATERIALS AND METHODS

[0084] Streptomyces sp. Saigon413, was deposited at the Westerdijk institute Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands, under accession number CBS149411 as disclosed in WO2024 / 074637. The Streptomyces chrestomyceticus was cultivated and formulated as disclosed in Examples 1.1 and 1 .2 of WO2024 / 074637.

[0085] Zymoseptoria tritici strains

[0086] Zymoseptoria tritici strains were isolated from different locations in European countries such as France, Germany, Ireland, Great Britain. These Zymoseptoria tritici strains were found to show a level of adaptation or resistance to SDHI, Qol and / or DMI fungicides.

[0087] Sequencing Zymoseptoria tritici strains

[0088] After growth on V8-Agarplate, Zymoseptoria tritici mycelium was scraped and lyophilized and DNA extracted using QIAGEN Mag Attrakt Plant Core Kit.

[0089] The DNA of the relevant genes (CYP51 CYTB, SDHB, SDHC), was amplified in a Multiplex PCR. A primer pair for each Gene, GoTaq G2 Polymerase Master mix (include Buffer, Polymerase, dNTPs and MgCI2) and additional MgCI2. The Amplification was checked by an Agarosegel. For each sample amplified genes were barcoded (Native Barcoding Kit 96 V14 (SQK-NBD114.96) Version: NBA_9170_v1 14_revN_15Sep2022), pooled and sequenced via Oxford Nanopore Technology.

[0090] The sequences were aligned with known native sequences of Zymoseptoria tritici in the NCBI database, such as the SDHB subunit according to Genbank accession number: AER08704.1 ; the SDHC subunit according to GenBank accession number: AFV73767.1 ; the Cyp51 according to Genbank accession number AAU43734.1 , and the cytochrome b protein according to Genbank accession number AZP56842.1 .

[0091] The amino acid substitution referred to as G143A in the cytochrome b protein is typically at position 143 of the cytochrome b protein according to AZP56842.1 , which is known to a person skilled in the art.

[0092] The amino acid subsititutions SDHB, SDHC, CYTB and CYP51 proteins are shown in Table 1 . EXAMPLE 1 : Treatment of wheat plants infected with resistant Zymoseptoria tritici strains with a composition comprising S. chrestomyceticus

[0093] Streptomyces chrestomyceticus CBS 149411 was fermented in a 200L fermenter in suitable media such as disclosed in Example 1.1 of WO2024 / 074637.

[0094] End of fermentation broth was dried to powder and formulated as OD40% (oil dispersion with 40% content of active ingredients). Wheat seedlings of the variety Riband were grown in the glasshouse until 14d after seeding. For infection with the resistant Zymoseptoria tritici strains (EPPO code: SEPTTR), the test plants were inoculated by spraying a spore suspension (1.5Mio spores per ml in water supplemented with 0.01 % Tween20) one day after treating plants with a test compound. After an incubation period of 3 days at 22°C / 21 °C (day / night) and 95% relative humidity (rh), the inoculated test plants were kept at 22°C / 21 °C (day / night) and 70% rh in a greenhouse. Efficacy was assessed directly when an appropriate level of disease appeared on untreated check plants (16 - 19 days after infection).

[0095] CONCLUSION

[0096] The results in Table 1 show that a composition comprising Streptomyces chrestomyceticus effectively controlled Zymoseptoria tritici strains which express fungicide resistance to at least one of fungicides from the mode of action group 7 (SDHI fungicides), group 11 (Qol fungicides) and group 3 (DMI fungicides) according the FRAC mode of action classification. Disease control provided by the treatment with S. chrestomyceticus was >30% at 120g / ha, >56% at 360g / ha and >83% at 533g / ha.

[0097] able 1. Efficacy of a composition comprising Streptomyces chrestomyceticus and Solatenol® against Zymoseptoria tritici strains having amino acid substitutionsn the SDHB or SDHC subunit, CytB and / or Cyp51 proteins which confer resistance to SDHI, Qol, and / or DMI fungicides, respectively (n.d. = not determined).

Claims

CLAIMS1 . A method for controlling or preventing infestation of Zymoseptoria tritici on a plant comprising treating the plant, plant propagation material and / or a locus thereof with a composition comprising a Streptomyces chrestomyceticus, wherein the Zymoseptoria tritici is resistant against SDHI, Qol and / or DMI fungicides.

2. The method according to claim 1 , wherein the Zymoseptoria tritici comprises an amino acid substitution N225T in the amino acid sequence of the succinate dehydrogenase B subunit, an amino acid substitution T79N, W80S, N86S, and / or H152R in the amino acid sequence of the succinate dehydrogenase C subunit, an amino acid substitution G143A in the cytochrome b amino acid sequence, and / or an amino acid substitution V136A, V136C, 1381V, A379G, Y459G_Y460del, Y461 H, Y461 S, H513K, and I or S524T in the Cyp51 amino acid sequence.

3. The method according to claim 1 or 2, wherein the composition further comprises a metabolite, preferably a metabolite produced by the Streptomyces chrestomyceticus.

4. The method according to claim 3, wherein the metabolite comprises malonomicin, cyclothiazomycin C, streptimidone, an oligosaccharide which comprises a molecular formula according to C53H90N2O44, a lipopeptide according to Formula (II),Formula (II) or a salt thereof, wherein Ri = CH3 or C2H5, and / or a polyene compound characterized by a molecular formula according to C67H115NO25, wherein the polyene is further characterized by the spectrum of light absorption with absorbance maxima at a wavelength of 235.5 nm, 301 .1 nm, 315.8 nm, 330.9 nm and 348.3 nm when measured in an aqueous acetonitrile solution.

5. The method according to any one of the claims 1 to 4, wherein the Streptomyces chrestomyceticus comprises a nucleotide sequence which has at least 99.8 % identity to SEQ ID NO: 1.

6. The method according to any one of the claims 1 to 5 wherein the Streptomyces chrestomyceticus comprises a genome sequence which has at least 95% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411.

7. The method according to anyone of the claims 1 to 6, wherein the Streptomyces chrestomyceticus comprises at least one nucleotide sequence which encodes a protein which has at least 90% to an amino acid sequence of SEQ ID NO: 71 to SEQ ID NO: 1 15, preferably SEQ ID NO: 91 and / or SEQ ID NO: 92, preferably at least 90% identity to at least one of the nucleotide sequences of SEQ ID NO: 2 to 46, preferably to at least one nucleotide sequence of SEQ ID NO: 22 or 23.

8. The method according to any one of the claims 1 to 7, wherein Streptomyces chrestomyceticus comprises at least one nucleotide sequence which encodes a protein which has at least 90% to an amino acid sequence of SEQ ID NO:1 16 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 which has at least 90% identity to at least one of the nucleotide sequence(s) of SEQ ID NO: 47 to 70, preferably to at least one nucleotide sequence of SEQ ID NO: 67 or 68.

9. The method according to any one of the claims 1 to 8, wherein the Streptomyces chrestomyceticus is a Streptomyces chrestomyceticus deposited at the Westerdijk Institute under accession number CBS149411 .

10. The method according to any one of the claims 1 to 9, wherein the plant comprises a cereal plant, preferably wheat.

11. The method according to any one of the claims 1 to 10, wherein the composition is an agricultural acceptable composition.

12. The method according to any one of the claims 1 to 11 , wherein the composition comprises 2x102to 5x1017colony forming unit (cfu) / ha of the Streptomyces chrestomyceticus, or 0.1 g to 10 kg of the composition comprising Streptomyces chrestomyceticus, per hectare.

13. The method according to any one of the claims 1 to 11 , wherein the plant propagation material is seed and the composition comprises 2x102to 5x1015(cfu) of Streptomyces chrestomyceticus per kg of seeds, or 0.0001 g to 100 g of the composition comprising Streptomyces chrestomyceticus per kg of seed.

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