Use of streptomyces as nematicide

Abstract

The present invention relates to a method of controlling or reducing nematode damage to a plant comprising applying a composition comprising a Streptomyces chrestomyceticus to a seed material prior to planting and / or to a soil surrounding a planted seed or plant.

Description

[0001] USE OF STREPTOMYCES AS NEMATICIDE

[0002] The present invention relates to the use of Streptomyces and / or a fermentation product thereof for controlling or reducing plant parasitic nematodes. The invention also relates to methods of treating plants to reduce nematode damage by applying a seed and / or soil treatment are described.

[0003] BACKGROUND

[0004] Nematodes are microscopic unsegmented worms known to reside in virtually every type of environment (terrestrial, freshwater, marine). Of the over 80,000 known species, many are agriculturally significant and generally referred to as plant parasitic nematodes. One such species is the root knot nematode (Meloidogyne spp.) which attacks a broad range of plants, shrubs, and crops. These soil- born nematodes attack newly formed roots causing stunted growth, swelling or gall formation. Chemical nematicides such as soil fumigants or non-fumigants have been in use for many years to combat infestations. As the use of traditional chemical nematicides such as methyl-bromide and organophosphates continue to be phased out, a need for the development of alternative treatment options has arisen.

[0005] Such alternative treatment may be the use of a Streptomyces. WO93 / 18135 discloses that a Streptomyces dicklowii CR-43 cells or supernatant exhibited nematicidal activity against nematodes such as Meloidogyne incognita, Pratylenchus penetrans and Caenorhabiditis elegans.

[0006] Meidani C. et al (2020) Plants, 9, 699, 1-16. discloses that supernatants of a few Streptomyces species, in particular S. monomycin! and S. colombiensis and S. youssoufensis were found to show activity against Meloidogyne spp. such as M. incognita and Mjavanica on Arabidopsis plants.

[0007] KR20230142986 discloses a mutant strain Streptomyces sp. S-N87 with an increased production of spectinabilin with nematicidal activity.

[0008] There is a need for improved methods of controlling or reducing plant parasitic nematodes using biological solutions using Streptomyces.

[0009] SUMMARY

[0010] The present invention relates to a method of controlling or reducing nematode damage to a plant comprising applying a composition comprising a strain of Streptomyces chrestomyceticus, to a seed prior to planting and / or to a soil surrounding a planted seed or plant, wherein the Streptomyces comprises a Streptomyces chrestomyceticus.

[0011] In another aspect the present invention relates to the use of a composition comprising Streptomyces chrestomyceticus as a nematicide.

[0012] DETAILED DESCRIPTION

[0013] The present invention relates to a method of controlling or reducing nematode damage to a plant comprising applying a composition comprising a Streptomyces and / or a fermentation product thereof, to a seed prior to planting and / or to a soil surrounding a planted seed or plant, wherein the Streptomyces comprises or is a Streptomyces chrestomyceticus.

[0014] The invention also relates to the use of a composition comprising Streptomyces chrestomyceticus as a nematicide. In one embodiment said use of a composition comprising Streptomyces chrestomyceticus as a nematicide comprises controlling or reducing nematode damage to a plant comprising applying to a seed prior to planting and / or to a soil surrounding a planted seed or plant the composition comprising Streptomyces chrestomyceticus.

[0015] Surprisingly, it was found that a composition comprising Streptomyces chrestomyceticus, effectively controlled nematodes such as Heterodera sp. Meloidogyne sp. Pratylenchus sp., for instance Heterodera glycines, Meloidogyne incognita, Pratylenchus zeae. As used herein controlling or reducing nematode damage to a plant, is understood to involve control or reduction of nematode damage to a plant, such a from 1 % to 100% control or reduction of nematode damage, for instance 5% to 90%, for instance from 10% to 80%, for instance from 20% to 70%, for instance from 30% to 60% control or reduction of nematode damage to a plant. Damage to a plant may for instance be measured by root galling or the root length. Root galling as used herein is a malformation or swelling of the root.

[0016] The method and use according to the present invention excludes methods for the treatment of a human or animal body by surgery or therapy.

[0017] Applying a compositition comprising a Streptomyces chrestomyceticus as disclosed herein to a seed prior to planting and / or to a soil surrounding a planted seed or plant may be performed in any suitable way, for example by dipping, spraying fumigating, fogging, scattering, brushing on, in furrow application, drenching, drip irrigation and I or injecting. Applying a composition to a seed for instance comprises drenching a seed or coating a seed with one or more coats.

[0018] Preferably, Streptomyces chrestomyceticus comprises a nucleotide sequence which has at least 99.8 %, preferably at least 99.9%, preferably at least 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% identity to SEQ ID NO: 1. In one embodiment the Streptomyces chrestomyceticus comprises a nucleotide sequence which has 100% identity to SEQ ID NO: 1 . SEQ ID NO: 1 comprises the 16SRNA gene of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411 .

[0019] In another preferred embodiment the Streptomyces chrestomyceticus, comprises a genome sequence which has at least 91 %, 92%, 93%, 94%, 95% identity, preferably at least 96%, 97%, 98%, 99% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411 , or to the whole genome of Streptomyces chrestomyceticus NRRL B-3672. The Streptomyces chrestomyceticus may comprise a genome sequence which has at least 99.1 %, 99.2%, 99.3%. 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411 , or to the whole genome of Streptomyces chrestomyceticus NRRL B-3672. Preferably, the composition comprises a Streptomyces chrestomyceticus, which is Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411 .

[0020] 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.

[0021] The composition comprising Streptomyces chrestomyceticus as disclosed herein comprises a cell count of the Streptomyces from 1*10° to 1 *1014cfu / g dry weight, for instance from1*101to 1 *1013cfu / g dry weight, 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*104to 1*101° cfu / g dry weight, from 2*104to 2*101° cfu / 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.

[0022] A Streptomyces chrestomyceticus, may be cultivated in a suitable fermentation medium under suitable fermentation conditions and optionally comprising a step of recovering the Streptomyces chrestomyceticus known to a person skilled in the art. Usually, a fermentation broth is produced during cultivation of or when cultivating a Streptomyces chrestomyceticus. Suitable fermentation conditions for cultivating Streptomyces sp. are known to a person skilled in art.

[0023] Cultivating Streptomyces chrestomyceticus, as disclosed herein, comprises cultivating the microbial strain under aerobic conditions at a temperature of from 15 degrees Celsius to 45 degrees Celsius, preferably a temperature of from 20 to 35 degree Celsius, preferably a temperature of between 25 to 32 degrees Celsius, in the presence of a carbon source and a nitrogen source. A suitable carbon source may be molasses, such as beet or cane molasses, polysaccharides, flour, starch, sugar or glucose. A suitable nitrogen source may be casein hydrolysate, tryptone, ammonium sulphate, ammonia, yeast extract, peptone or urea. The process for cultivating a Streptomyces as disclosed herein may be performed in a batch, fed-batch or continuous culture.

[0024] A composistion comprising Streptomyces chrestomyceticus disclosed herein may be a supernatant of a fermentation broth of the Streptomyces chrestomyceticus disclosed herein. A supernatant of a fermentation broth of a Streptomyces chrestomyceticus as disclosed herein can be produced by known methods in the art, for instance by centrifugation or filtration of the fermentation broth.

[0025] In one embodiment the composition comprising Streptomyces chrestomyceticus further comprises a metabolite. A metabolite is preferably produced by the Streptomyces chrestomyceticus disclosed herein.

[0026] Preferably, the composition comprises a metabolite, wherein the metabolite comprises or is malonomicin.

[0027] Preferably, the composition comprising Streptomyces chrestomyceticus further comprises a metabolite, such as at least one, at least two, at least four, or at least five of the metabolite(s), selected from the group consisting of cyclothiazomycin C, streptimidone, an oligosaccharide compound according to compound I, comprising a molecular formula according to C53H90N2O44, further characterised by the NMR spectra listed in Table 2 and Table 3, and further characterized by a structural Formula I, Formula (I), a lipopeptide according to Formula II, or a salt thereof wherein R1 = CH3 or C2H5

[0028] Formula (II) and a polyene compound characterized by a molecular formula according to C67H115NO25, wherein 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. The polyene is further characterized by the spectrum of light absorption as shown in Figure 10. Preferably, the fermentation product further comprises a metabolite selected from the group consisting of cyclothiazomycin C, streptimidone, an oligosaccharide compound according to compound I, comprising a molecular formula according to C53H90N2O44, further characterised by the NMR spectra listed in Table 2 and Table 3, and further characterized by a structural Formula I, Formula (I), a lipopeptide according to Formula II, or a salt thereof wherein R1 = CH3 or C2H5

[0029] Formula (II).

[0030] The composition comprising a Streptomyces chrestomyceticus in the use or method as disclosed herein comprises a fermentation broth comprising the Streptomyces 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.

[0031] A composition comprising Streptomyces chrestomyceticus as disclosed herein also includes a formulation comprising the Streptomyces chrestomyceticus and / or a fermentation product thereof.

[0032] A composition as disclosed herein is an agricultural acceptable composition. Preferably the composition according to the present invention comprises an auxiliary, preferably 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.

[0033] A formulation as disclosed herein can be in the form of a soluble concentrate (SL), or a flowable concentrate for seed treatment (FS), or a suspension concentrate (SC), and can be more preferably a seed treatment slurry to be applied onto seeds. Slurries for seed treatment applications are well-known in the art.

[0034] A composition according to the present invention comprises a dressing composition, which relates to a liquid composition useful for covering and / or wetting a plant propagation material, and more preferably a seed, at least in part or in totality.

[0035] A seed dressing formulation is applied in a manner known per se to the seeds employing the combination of the invention and a diluent in suitable seed dressing formulation form, e.g. as an aqueous suspension or in a dry powder form having good adherence to the seeds. Such seed dressing formulations are known in the art.

[0036] A flowable formulation contains solid particles in a liquid which is usually water. A flowable concentration also comprises a suspension concentrate. A flowable formulation commonly comprises (in wt%) an active ingredient (5-60%), dispersants / wetting agent (1-10%), rheology modifiers (0.1-0.1 %), biocides (0.1 %), antifreezers (5-10%), antifoam (0.2%), adjuvants (up to 25%) and water to make up 100%.

[0037] The composition according to the present invention is particularly suited for dressing applications on plant propagation material, especially on seeds.

[0038] A composition as disclosed herein comprises from 10 to 60 wt / wt% dry weight, preferably from 20 to 50 wt / wt% dry weight of spray-dried or freeze-dried fermentation broth of the Streptomyces chrestomyceticus as disclosed herein.

[0039] The nematodes which can be targeted according to the method or use according to the invention include but are not limited to, for example Aphelenchoides spp, Bursaphelenchus spp., Criconemella spp., Ditylenchus spp., Dolichodorus spp., Heterodera spp., Hoplolaimus spp., Globodera spp., Longidorus spp., Meloidogyne spp., Paratrichodorus spp., Pratylenchus spp., Radopholus spp. Rotylenchulus spp, Trichodorus spp., Tylenchorhynchus spp., Scutellonema spp., Trichostrongylus spp., Xiphinema spp. for example the nematode species Aphelenchoides ritzemabosi, Belonolaimus longicaudatus, Bursaphelenchus xylophilus, Caenorhabditis elegans, Ditylenchus dipsaci, Helicotylenchus multicinctus, Haemonchus contortus, Meloidogyne incognita Mesocriconema xenoplax, Pratylenchus coffeae, Radopholus similis, Rotylenchulus reniformis, Tylenchulus semipenetrans, Xiphinema americanum.

[0040] Particularly, a nematode belongs to Meloidogyne spp., Helicotylenchus spp. Heterodera spp., Globodera spp., Rotylenchus spp. or Pratylenchus spp., preferably a nematode belongs to a nematode species Meloidogyne incognita, Meloidogyne hapla, Meloidogyne arenaria, Meloidogyne javanica, Globodera rostochiensis, Globodera pallida, Heterodera avenae, Heterodera glycines, or Heterodera schachtii, and / or Pratylenchus zeae. Preferably the nematode species includes Heterodera glycines Meloidogyne incognita, and / or Pratylenchus zeae.

[0041] Seeds as disclosed herein are generative parts of plants.

[0042] The terms "plants", "crop", "crops", "useful plants", "crop plants", "agricultural plants", "food plants" are used interchangeably herein.

[0043] According to the invention "useful plants" to which a composition as disclosed herein can be applied, typically comprise the following species of plants: grape vines; cereals, such as wheat, barley, rye or oats; beet, such as sugar beet or fodder beet; fruits, such as pomes, stone fruits or soft fruits, for example apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries or blackberries; leguminous plants, such as beans, lentils, peas or soybeans; oil plants, such as rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans or groundnuts; cucumber plants, such as marrows, cucumbers or melons; fibre plants, such as cotton, flax, hemp or jute; citrus fruit, such as oranges, lemons, grapefruit or mandarins; vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cucurbits or paprika; lauraceae, such as avocados, cinnamon or camphor; maize; tobacco; nuts; coffee; sugar cane; tea; vines; hops; durian; bananas; natural rubber plants; turf or ornamentals, such as flowers, shrubs, broad-leaved trees or evergreens, for example conifers.

[0044] Preferably, a plant comprises soy or soybean, potato, sugarbeet, sugarcance, rice, coffee, peas, corn, cotton, fruit and / or vegetables, preferably the plant comprises cucumber, tomato, soybean and I or corn.

[0045] Useful plants and I or target crops in accordance with the invention include conventional as well as genetically enhanced or engineered varieties such as, for example, insect resistant (e.g. Bt. and VIP varieties) as well as disease resistant, herbicide tolerant (e.g. glyphosate- and glufosinate-resistant maize varieties) and nematode tolerant varieties. Useful plants include plants transformed by the use of recombinant DNA techniques, for example, to be capable of synthesising one or more selectively acting toxins, such as are known, for example, from toxin-producing bacteria. Examples of toxins which can be expressed include 8-endotoxins, vegetative insecticidal proteins (Vip), insecticidal proteins of bacteria colonising nematodes, and toxins produced by scorpions, arachnids, wasps and fungi.

[0046] The plants in a method or use of the invention are plants in need of protection from nematodes.

[0047] A method for controlling or reducing nematode damage to a plant comprises applying a composition comprising Streptomyces chrestomyceticus at a suitable rate. Typical rates of concentration are between 0.1 and 1000 ppm, preferably between 0.1 and 500 ppm, of active ingredient. The rate of application per hectare is preferably 1 g to 2000 g of active ingredient per hectare, more preferably 10 to 1000 g / ha, most preferably 10 to 600 g / ha. When used as seed drenching agent, convenient dosages are from 10mg to 1g of active substance per kg of seeds. When the composition of the present invention is used for treating seed, rates of 0.001 to 50 g of the composition per kg of seed, preferably from 0.01 to 10g per kg of seed are generally sufficient.

[0048] As used herein the active ingredient comprises a composition comprising Streptomyces chrestomyceticus.

[0049] In one embodiment, the method comprises applying an effective amount of of the composition comprising Streptomyces chrestomyceticus as disclosed herein above, wherein the 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 from 5*102to 5*1012, from 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 Streptomyces chrestomyceticus, per hectare.

[0050] An effective amount of the composition comprising Streptomyces chrestomyceticus as disclosed herein above, comprises from 1 g to 10 kg / per hectare (ha), such as from 5 g to 5 kg, such as from 10 g to 1 kg / ha, such as from 50 g to 800 g / ha, such as from 100 g to 700 g / ha, such as from 200 to 600 g / ha. The weight in g and kg is dry weight of the Streptomyces chrestomyceticus.

[0051] In one embodiment the method according to the present invention comprises applying an effective amount of the composition comprising Streptomyces chrestomyceticus to seed wherein the effective amount comprises from 5x102to 5x1015, from 2x103to 5x1014, from 5x103to 5x1013, from 2x105to 5x1012, preferably from 5*102to 5*1012, from 1*103to 5*1011, from 5*103to 1*1011, from 1*104to 5*101°, 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 Streptomyces chrestomyceticus as disclosed herein per kg of seed.

[0052] An effective amount of the composition comprising Streptomyces chrestomyceticus and / or a fermentation product thereof as disclosed herein above, may also comprise from 0.001 g to 100 g / per kg of seeds, such as from 0.005 g to 80 g / kg seeds, such as from 0.01 g to 50 g / kg seeds, such as from 0.5 g to 10 g / kg seeds, The weight in g dry weight of component A comprising Streptomyces chrestomyceticus per kg dry weight of seeds.

[0053] Suitably, the composition comprising Streptomyces chrestomyceticus can be applied either preventative, meaning prior to nematode development or curative, meaning after nematode development.

[0054] FIGURES

[0055] Figure 1. 1 D1H NMR spectrum of the compound I in D2O at 600 MHz

[0056] Figure 2. 1 D13C NMR spectrum of the compound I in D2O at 600 MHz

[0057] Figure 3. 2D Dept Edited 1 H-13C HSQC NMR Spectrum of the compound I in D2O.at 600 MHz showing the positive (CH) signals

[0058] Figure 4. 2D Dept Edited 1 H-13C HSQC NMR Spectrum of the compound I in D2O.at 600 MHz showing the negative (CH2) signals

[0059] Figure 5. Spectrum of light absorption (UV-VIS) 200-400nm of a lipopeptide according to Formula II (a), Formula II (b) or Lipopeptin A

[0060] Figure 6. LC-ESI-MS / MS spectrum of precursor 1204.6 m / z (M+H)+for a lipopeptide of Formula ll(a) depicting fragment peaks consistent with amino acids: aspartic acid, hydroxy-glutamine, serine, methylasparagine, methyl-phenylalanine

[0061] Figure 7. LC-ESI-MS / MS / MS spectrum of precursor 294.2 m / z for a lipoeptide of Formula ll(a) depicting peaks consistent with the molecule C14H25-OH2-C4H5ON

[0062] Figure 8. The upfield region of the 1 D1H NMR spectrum of a lipopeptide according to Formula ll(a) in CD3OD at 600 MHz Figure 9. The downfield region of the 1 D1H NMR spectrum of a lipopeptide according to Formula ll(a) in CD3OD at 600 MHz

[0063] Figure 10. Spectrum of light absorption (UV-VIS) 200-500nm of polyene compound

[0064] EXAMPLES

[0065] 1. Source, fermentation

[0066] 1 .1 Fermentation of Streptomyces sp.

[0067] Streptomyces sp. Saigon413, which was isolated in Vietnam before 1961 , was deposited at the Westerdijk institute under accession number CBS149411. The deposit was made by Syngenta Ltd., Jealott’s Hill Research International Centre, Bracknell, Berkshire, RG42 6EY, UK under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0068] Streptomyces chrestomyceticus NRRL B-3672 was obtained from ARS (NRRL) culture collection.

[0069] The Streptomyces was cultivated in Erlenmeyer flasks with a liquid medium consisting of (g / 1) casein hydrolysate 10, glucose 40, K2HPO4 1 .25, soytone 2, tryptone, 8 and incubated at 28°C in an incubator shaking 150 rpm with 25 mm throw for 4 days.

[0070] Large scale fermentations

[0071] For large scale production, standard procedures were applied for cultivating Streptomyces sp., such as Streptomyces chrestomyceticus CBS149411 to high cell density using fed-batch fermentation. After harvesting, the broth was spray dried or freeze dried according to methods known to a person skilled in the art.

[0072] The final product (TGAI) after spray- or freeze drying had a cell count of 1*106to 1*1013cfu / g dry mass. TGAI: technical grade active ingredient (unformulated product).

[0073] Comparison Streptomyces chrestomyceticus CBS149411 and NRRL B-3672.

[0074] The Streptomyces chrestomyceticus strain CBS149411 and NRRL B-3672 were cultivated in three different fermentation media (Soya Flour Mannitol (SFM), Kings and Tryptic Soy Broth (TSB)), knwon to a person skilled in the art. 40 ml of culture medium were dispensed into a 125ml flask. 130 pl of cell bank suspension were used as inoculum. The inoculated flasks and media controls were incubated at 28°C, shaking at 180 rpm for 4 days. The incubation was stopped on day 4 of cultivation and samples taken and stored at -80°C until the start of the test. The fermentation broth produced from the different media was tested in their activity against soybean cyst nematodes using the following method. The results are shown in Example 2.

[0075] 1.2 Isolation of 16S rDNA and whole genome sequencing

[0076] Genomic DNA was isolated from Streptomyces sp. Saigon413 using the method described in Kutchma et al. (1998) Biotechniques 24(3):452-457. The 16S rRNA gene was amplified using universal 16S primers and sequenced using Sanger sequencing. The 16S rRNA of Streptomyces sp. Saigon413 is shown in SEQ ID NO: 1 .

[0077] Whole genome sequencing, using the genomic DNA from Streptomyces sp. Saigon413, was completed using both Pacific Biosciences and Illumina sequencing technologies. The genome was assembled using HFAP4 and polished with Pilon using the Illumina reads.

[0078] In Table 1 , the percentage identity of the whole genome and the 16SRNA sequence of Streptomyces sp. Saigon 413 and Streptomyces chrestomyceticus NRRL B-3672 is shown.

[0079] Table 1.

[0080] CBS Westerdijk fungal diversity institute: Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands https / / wi. knaw.nl /

[0081] ARS ARS Culture Collection (NRRL), 1815 N. University Street, Peoria, IL 61604, USA - https: / / nrrl.ncaur.usda.gov

[0082] 1.3. Formulation

[0083] A flowable concentrate formulation of the spray-dried or freeze-dried product (microorganism) was prepared using formulation technology 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 978-94-011-4926-6.

[0084] The flowable concentrate (FS300) formulation used in the experiments contained 30% spray dried or freeze dried TGAI.

[0085] 1.4. Analysis of Streptimidone, cyclothiazomycin C and malonomicin

[0086] The structure of cyclothiazomycin C is disclosed on p. 3 of WO2015191789 and can be extracted and analysed according to the method discosed in Wang et al. (2010) Appl. Environmental Microbiology, Vol. 76, No. 7 p.2336. Malonomicin can be extracted and analysed according to the method disclosed in Example I (B) of W02006 / 078939. Streptimidone can be extracted and isolated according to the method as disclosed in Lee et al. J. of Antibiotics (2020) 73: p. 184-188, including the supplementary information. 1.5. Oligosaccharide compound I

[0087] Purification of the oligosaccharide compound I

[0088] Whole broth Streptomyces chrestomyceticus CBS149411 (Streptomyces sp. Saigon413), was centrifuged to produce an aqueous extract and a pellet. The aqueous extract was freeze dried. The material was resuspended in a minimal volume of water and partitioned with ethyl acetate to remove lipophilic components. The aqueous suspension was retained and freeze dried and resuspended in a minimal volume of water before being applied to an activated charcoal column.

[0089] The column was washed with water and eluted with water:acetone (50:50).

[0090] The compound I was further purified by Hydrophilic Interaction Liquid Chromatography (HILIC) using Mass guided fractionation and ELSD detector. Using for example Waters XBridge Amide, 5 micron, 30x100mm using a gradient of acetonitrile and 10mM Ammonium Acetate.

[0091] Characterisation of oligosaccharide compound I

[0092] The compound I was determined in the purified fermentation broth according to the methods disclosed below.

[0093] Molecular composition and total molecular mass

[0094] The molecular composition and total molecular mass were C53H90N2O44, and 1458.487 g, respectively which were determined using MS-MS and NMR spectroscopy as disclosed below.

[0095] Solubility

[0096] The solubility of the oligosaccharide compound I in water, pH 7.01 , was >10’000 ppm, and in DMSO was > 9772 ppm.

[0097] MS-MS Spectrometry and liquid chromatrography

[0098] Spectra were recorded on an Orbitrap ID-X Tribrid Mass Spectrometer from Thermo Scientific equipped with an OptaMax NG Heated Electrospray Source (Spray Voltage: Static, Polarity Ion (V): 3400 (Positive ion mode) & 2400 (Negative ion mode), Sheath Gas (Arb): 40, Aux Gas (Arb): 5, Sweep Gas (Arb): 1 , Ion Transfer Tube Temperature: 350 °C, Vaporizer Temperature: 350 °C). The Scan Parameters were as follows;

[0099] Experiment 1 : MS OT (Orbitrap Resolution: 60,000, Scan Range (m / z): 200 to 2000, RF Lens (%): 60, AGO Target: Standard, Maximum Injection Time Mode: Auto, Microscans: 1 , Data Type: Profile, Polarity: Both),

[0100] 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 Scientific using a Vanquish Split Sampler FT, Vanquish Binary Pump F, Vanquish Column Compartment H, Vanquish Diode Array Detector FG and Vanquish Charged Aerosol Detector. Liquid Chromatography Conditions included: Thermo Scientific Hypercarb™ Porous Graphitic Carbon column 5pm 4.6x50mm, P.N. 35005-054630. Temp: 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: 0 min 1 % B, 99% A; 4. OOmin 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 .Oml / min, Injection volume: 2 uL, Total run time: 6.0min.

[0101] NMR Spectroscopy NMR spectra were recorded on a Bruker AVIII 600 NMR spectrometer, equipped with a 5 mm Bruker (1H / 19F) / 13C / 15N TCI cryoprobe fitted with Z gradients, using standard Bruker pulse sequences. Samples were dissolved in D2O, and the spectra were recorded at 300° K and referenced to acetone at 2.225 ppm for1H and 31.07 ppm for13C. Figures 1 to 4 show NMR spectra of the compound of the present invention. The one bond1H-13C correlation spectrum contains peaks corresponding to 1 methyl (CH3) and 40 methine (CH) groups (listed in Table 2) and 9 methylene (CH2) groups (listed in Table 3).

[0102] In addition, the 1 D13C spectrum contains signals from 3 quaternary carbons at 104.7, 159.3 and 175.2 ppm (± 0.1). Table 2. Methyl and methine signals in the one bond1H-13C correlation spectrum of the oligosaccharide compound I together with multiplicity information for protons resolved in the 1 D1H spectrum.

[0103] Table 3. Methylene signals in the one bond1H-13C correlation spectrum of the compound I together with multiplicity information for protons resolved in the 1 D1H spectrum.

[0104] 1.6. Lipopeptide compound of Formula II

[0105] Purification of a lipopeptide according to Formula II (Formula ll(a) and Formula ll(b))

[0106] The mycelia from fermentation broth from Streptomyces sp. Saigon 413, was separated via centrifugation and the supernatant was treated with butanol. The butanol was removed and the extract partitioned between water and ethyl acetate. The lipopeptides were purified from the ethyl acetate fraction by preparative reverse phase (C18) HPLC. The lipopeptides were relatively aploar and elute in the higher organic fraction in a gradient system with 0.1 % formic acid and acetonirile (0.1 % formic acid). A gradient of 60% Aqueous to 40% Aqueous with the above solvents allowed separation of a lipopeptide compound according to Formula I l(a) and Formula I l(b).

[0107] Compounds were detected by UV-VIS (Figure 5), mass spectrometry (Figures 6 and 7) and NMR spectrometry (Figures 8 ad 9).

[0108] Characterisation of a lipopeptide of Formula II

[0109] Liquid Chromatography and High-Resolution Mass Spectrometry

[0110] Spectra were recorded on an Orbitrap ID-X Tribrid Mass Spectrometer from Thermo Scientific equipped with an OptaMax NG Heated Electrospray Source (Spray Voltage: Static, Polarity Ion (V): 3400 (Positive ion mode) & 2400 (Negative ion mode), Sheath Gas (Arb): 40, Aux Gas (Arb): 5, Sweep Gas (Arb): 1 , Ion Transfer Tube Temperature: 350 °C, Vaporizer Temperature: 350 °C). The Scan Parameters were as follows;

[0111] Experiment 1 : MS OT (Orbitrap Resolution: 50,000, Scan Range (m / z): 200 to 2000, RF Lens (%): 60, AGO Target: Standard, Maximum Injection Time Mode: Auto, Microscans: 1 , Data Type: Profile, Polarity: Both),

[0112] 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),

[0113] Experiment 3: tMS2 OT HCD (MSn Level (n): 2, Isolation Window (m / z): 1.0, Activation Type: HCD, HCD Collision Energy (%): 30, Detector Type: Orbitrap, Orbitrap Resolution: 30,000, RF Lens (%): 60, Polarity: Positive), Experiment 4: tMS3 OT HCD (MSn Level (n): 3, Isolation Window (m / z): 1.6, Activation Type: HCD, HCD Collision Energy (%): 30, MS2 Isolation Window (m / z): 2, MS2 Activation Type: HCD, MS2 HCD Collision Energy (%): 30, Detector Type: Orbitrap, Orbitrap Resolution: 30,000, RF Lens (%): 60, Polarity: Positive). The mass spectrometer was connected to a Vanquish Flex UHPLC from Thermo Scientific using a Vanquish Split Sampler FT, Vanquish Binary Pump F, Vanquish Column Compartment H, Vanquish Diode Array Detector FG and Vanquish Charged Aerosol Detector.

[0114] Liquid Chromatography Conditions included: Waters ACQUITY UPLC C18 column 1.7pm 3.0x50mm, P.N. 186004660. Temp: 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: 0 min 10% B, 90% A; 4. OOmin 90% B, 10% A; 4.25min 90% B, 10% A; 4.50min 10% B, 90% A; 5. OOmin 10% B, 90% A, Flow rate: 1.0ml / min, Injection volume: 2 uL, Total run time: 5.0min. A purified fermentation broth as described above was injected.

[0115] Figures 6 and 7 show LC-ESI-MS / MS / MS spectra of a compound of Formula II (a).

[0116] NMR Spectroscopy

[0117] NMR spectra were recorded on a Bruker AVIII 600 NMR spectrometer, equipped with a 5 mm Bruker (1H / 19F) / 13C / 15N TCI cryoprobe fitted with Z gradients, using standard Bruker pulse sequences. Samples were dissolved in CD3OD, and the spectra were recorded at 300° K and referenced to the residual solvent signal at 3.31 ppm for1H. Figures 8 and 9 each cover half of the 1 H NMR spectrum of a compound according to Formula 11 (a) .

[0118] Molecular composition and mass

[0119] The molecular composition and mass of a lipopeptide according to Formula I l(a) and Formula I l(b) was determined using the results of liquid chromatography and high-resolution mass spectrometry as disclosed above. The lipopeptide compounds of Formula ll(a) and ll(b) have the following composition. Formula II (a) Lipopeptide 1204: Molecular composition C55H85N11 O19 and exact mass of 1203.602. Formula II (b) Lipopeptide 1218: Molecular composition C56H87N11 O19 and exact mass of 1217.618. Solubility

[0120] The solubility of a compound of Formula 11 (a) , Formula II (b) and Lipopeptin A was determined in water and DMSO:

[0121] Compound solvent (pH) solubility (ppm)

[0122] Formula II (a) Lipopeptide 1204 water (2.08) 21 .4

[0123] Formula II (a) Lipopeptide 1204 water (5.55) >10’000

[0124] Formula II (a) Lipopeptide 1204 DMSO >10’000

[0125] Formula II (b) Lipopeptide 1218 DMSO >10’000

[0126] 1.7. Polyene compound

[0127] Purification of the polyene compound

[0128] A spray dried sample from a culture of Streptomyces sp. Saigon 413 was washed with water. The solid residue was extracted twice with isopropanol and the isopropanol was removed. The resulting solid was purified by preparative reverse phase (C18) HPLC using an acetonitrile:water gradient. Further purification was conducted by preparative reverse phase HPLC using a Zorbax C8 column and eluting with an acetonitrile:water gradient.

[0129] The polyene compound was detected by UV-VIS (Figure 1).

[0130] Other polyene type compounds

[0131] Filipin complex isolated from Streptomyces filipinensis (CAS: 11078-21-0) was purchased from a commercial vendor. It is a macrolide antibiotic and used as a dye in microscopy. The sample is a mixture of 8 isomers, with Filipin III as a main component. Filipin III has a mass of 654.83 and a molecular composition of C35H58O11

[0132] Amphotericin B is a macrolide antibiotic isolated from Streptomyces nodosum (CAS1397-89-3) was purchased from a commercial vendor. It has a mass of 924.08 and a molecular composition of C47H73NO17.

[0133] Liquid Chromatography and High-Resolution Mass Spectrometry

[0134] Spectra were recorded on an Orbitrap ID-X Tribrid Mass Spectrometer from Thermo Scientific equipped with an OptaMax NG Heated Electrospray Source (Spray Voltage: Static, Polarity Ion (V): 3400 (Positive ion mode) & 2400 (Negative ion mode), Sheath Gas (Arb): 40, Aux Gas (Arb): 5, Sweep Gas (Arb): 1 , Ion Transfer Tube Temperature: 350 °C, Vaporizer Temperature: 350 °C). The Scan Parameters were as follows;

[0135] Experiment 1 : MS OT (Orbitrap Resolution: 50,000, Scan Range (m / z): 200 to 2000, RF Lens (%): 60, AGC Target: Standard, Maximum Injection Time Mode: Auto, Microscans: 1 , Data Type: Profile, Polarity: Both),

[0136] 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),

[0137] 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).

[0138] The mass spectrometer was connected to a Vanquish Flex UHPLC from Thermo Scientific using a Vanquish Split Sampler FT, Vanquish Binary Pump F, Vanquish Column Compartment H, Vanquish Diode Array Detector FG and Vanquish Charged Aerosol Detector.

[0139] Liquid Chromatography Conditions included: Waters ACQUITY UPLC C18 column 1.7pm 3.0x50mm, P.N. 186004660. Temp: 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: 0 min 10% B, 90% A; 4. OOmin 90% B, 10% A; 4.25min 90% B, 10% A; 4.50min 10% B, 90% A; 5. OOmin 10% B, 90% A, Flow rate: 1.0ml / min, Injection volume: 2 uL, Total run time: 5.0min.

[0140] A purified fermentation broth as described under section 1.3 was injected.

[0141] The key peaks observed were:

[0142] Negative ion: C67H114NO25 [M-H]’ Expected: 1332.7685, Observed: 1332.7679

[0143] Positive ion: C67H114NO24 [M-H2O+H]+Expected: 1316.7725, Observed: 1316.7709

[0144] Positive ion: C67H115NO24 [M-H2O+2H]2+Expected: 658.8899, Observed: 658.8895

[0145] Positive ion: C67H113NO23 [M-2(H2O)+2H]2+Expected: 649.8846, Observed: 649.8843

[0146] Positive ion: C67H111 NO22 [M-3(H2O)+2H]2+Expected: 640.8793, Observed: 640.8790

[0147] In a second experiment, Liquid Chromatography Conditions included: Kinetex Polar C18 column 100A 4.6x100mm, P.N. H17-055453. Temp: 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: Omin 10% B, 90% A; 1 min 10% B, 90% A; 6.50min 95% B, 5% A; 8.00min 95% B, 5% A; 9.00min 10% B, 90% A; 10. OOmin 10% B, 90% A, Flow rate: 1.0ml / min, Injection volume: 5 uL, Total run time: 10. Omin.

[0148] Under these conditions, the polyene compound had a retention time of 5.55-5.57 minutes.

[0149] The observed mass of the polyene compound was the same as in the previous Liquid chromatography run (results not shown).

[0150] Molecular composition and mass

[0151] The molecular composition and mass of polyene compound was determined using the results of liquid chromatography and high-resolution mass spectrometry as disclosed in 2.3. The polyene compound has the following composition.

[0152] Molecular composition C67H115NO25 and exact mass of 1333.7758.

[0153] Solubility

[0154] The solubility of a compounds was determined in DMSO:

[0155] Compound solvent (pH) solubility (ppm) Polyene of compound DMSO >10’000

[0156] Philipin complex DMSO >10’000

[0157] Amphotericin B DMSO >10’000

[0158] The presence of the polyene compound with a mass of 1333.7758 g in the fermentation broth of several Streptomyces species, was determined by isolating and purifying according to the method described section 2. The results in Table 2 show that the polyene compound with a molecular formula of C67H115NO25 was produced by Streptomyces chrestomyceticus species.

[0159] EXAMPLE 1. Use of a composition of Streptomyces chrestomyceticus against Melodoigyne incognita

[0160] Materials

[0161] Majestene® was obtained from a commercial vendor

[0162] A FS300 formulation of Streptomyces chrestomyceticus CBS149411 was prepared as described above.

[0163] Method 1 : Cucumber seeds

[0164] Cucumber seeds were sown in 200ml pots and infested with 3000 eggs of Melodoigyne incognita. At the same day a drench application of FS300 formulation of CBS149411 TGAI was carried out with a volume of 10ml. After 14 days the plant roots were washed out and a root galling assessment was carried out following an adapted Zeck index (1 .) and total root length of roots were measured using WhinRhizo Pro 2005 software.

[0165] Table 4.

[0166] Method 2: Tomato seeds

[0167] Tomato seeds were sown in 45ml pots and 7d after sowing infested with 1 '000 second stage juveniles (J2's) of Meloidogyne incognita. At the same day as the infestation a drench application of the FS300 formulation of CBS149411 was carried out with a volume of 1 ml in combination with a sticker at a ratio 1 :1 . The sticker was tested alone at various rates to test for effects of the sticker alone. At 23 days after infestation the root gall ratings (adapted Zeck index (1)) were assessed. The test was carried out under greenhouse conditions. Table 5

[0168] *Polymei7Sticker alone was also tested at various rates (1 ,5 - 41 mg / pot) with no observable effects on nematode / plant

[0169] Method 3

[0170] Penetration Assay with Pratylenchus zeae on corn

[0171] The seeds of corn (variety Falkone) were treated with the given rates of the products tested. Treated seeds were sown in potting soil in pots. Three days after sowing the pots were infested with the 1000 juveniles of Pratylenchus zeae Graham. Seven days after the infestation the plants were cut, the roots removed from the soil and gently cleaned. The roots were bleached and stained with acid fuchsin following the method from Bybd DW et al 1983. Finally, the root samples were checked under the dissecting microscope and the number of nematodes counted. The efficacy of the treatments was calculated by using the Abott's formula.

[0172] Table 6

[0173] Method 4. Penetration Assay with Meloidogyne incognita on soybean

[0174] The seeds of soybean (variety Toliman) were treated with the given rates of the products tested. Treated seeds were sown in potting soil in pots. Pots were infested with 20000 eggs of Meloidogyne incognita Chitwood at the day of sowing. 14 days after infestation the plants were cut, the roots removed from the soil and gently cleaned. The roots were bleached and stained with acid fuchsin following the method from Bybd DW et al 1983. Finally, the root samples were checked under the dissecting microscope and the number of nematodes counted. The efficacy of the treatments was calculated by using the Abott's formula.

[0175] Table 7. The results in Tables 4 to 7 show that a composition of Streptomyces chrestomyceticus CBS 149411 reduced root damage by the nematode Melodoigyne incognita on cucumber, tomato and soybean seeds I roots. In addition, Streptomyces chrestomyceticus CBS 149411 reduced root damage by the nematode Pratylenchus zeae on corn seeds / roots.

[0176] EXAMPLE 2. Activity of S. chrestomyceticus strains against soybean cyst nematodes

[0177] The seeds of soybean (variety Toliman) were treated with the given rates of the products tested. Treated seeds were sown in potting soil in pots. Pots were infested with 2000 eggs of Heterodera glycines (Ichinohe) at the day of sowing. 14 days after infestation the plants were cut, the roots removed from the soil and gently cleaned. The roots were bleached and stained with acid fuchsin following the method from Bybd DW et al 1983. Finally, the root samples were checked under the dissecting microscope and the number of nematodes counted. The efficacy of the treatments was calculated by using the Abott's formula.

[0178] The results in Table 7 and Table 8 show that both Streptomyces chrestomyceticus CBS149411 and NRRL B-3672 cultivated on Soy Four Mannitol (SFM), Kings and Tryptic Soy Broth (TSB) medium as disclosed under section 1.1 , are active against nematodes, such as Heterodera glycines on soybean roots

[0179] Table 8. Activity of Streptomyces chrestomyceticus CBS149411 and NRRL B-3672 are active against Heterodera glycines on soybean roots Table 9. Activity of Streptomyces chrestomyceticus CBS149411 and NRRL B-3672 are active against Heterodera glycines on soybean roots References:

[0180] (1) Abbott WS. A method of computing the effectiveness of an insecticide. J Econ Entomol (1925) 18:265-267

[0181] (2) Bybd DW, Kirkpatrick T, Barker KR. An improved technique for clearing and staining plant tissues for detection of nematodes. J Nematol. 1983 Jan;15(1):142-3. PMID: 19295781 ; PMCID: PMC2618249.

[0182] (3) Zeck, W.M., 1971 . A rating scheme for field evaluation of root-knot infestations. Pflanzenschutz- Nachr. Bayer AG 24, 141-144

[0183] (Original in Electronic Form)

[0184] (This sheet is not part of and does not count as a sheet of the international application)

[0185] FOR RECEIVING OFFICE USE ONLY

[0186] FOR INTERNATIONAL BUREAU USE ONLY

Claims

22CLAIMS1. Method of controlling or reducing nematode damage to a plant comprising applying a composition comprising a Streptomyces chrestomyceticus thereof to a seed prior to planting and / or to a soil surrounding a planted seed or plant.

2. Use of a composition comprising Streptomyces chrestomyceticus as a nematicide.

3. Use according to claim 2, wherein said use comprises controlling or reducing nematode damage to a plant comprising applying to a seed prior to planting and / or to a soil surrounding a planted seed or plant the composition comprising the Streptomyces chrestomyceticus.

4. The method according to claim 1 , or the use according to claims 2 or 3, wherein the Streptomyces chrestomyceticus comprises a nucleotide sequence which has at least 99.8 % identity to SEQ ID NO: 1 , and I or wherein the Streptomyces chrestomyceticus has at 91 % identity to the whole genome of Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411 .

5. The method according to any one of the claims 1 to 4, or the use according to any one of the claims 2 to 4, wherein the Streptomyces chrestomyceticus is Streptomyces sp. Saigon413 deposited with the Westerdijk Institute under accession number CBS149411 .

6. The method according to claims 1 or 5, or the use according to any one of the claims 2 to 5, wherein the composition comprises a cell count of the Streptomyces chrestomyceticus of 10° to 1014cfu / g dry weight.

7. The method according to any one fo the claims 1 , 3 to 6, or the use according to any one of the claims 2 to 6, wherein the composition further comprises a metabolite.

8. The method or use according to claim 7, wherein the metabolite comprises malonomicin.

9. The method according to any one of the claims 1 , 3 to 8, or the use according to any one of the claims 2 to 8, wherein the composition further comprises at least one metabolite selected from the group consisting of cyclothiazomycin C, streptimidone, an oligosaccharide compound according to compound I, comprising a molecular formula according to C53H90N2O44, further characterised by the NMR spectra listed in Table 2 and Table 3, and further characterized by a structural Formula I,Formula (I), a lipopeptide according to Formula II, or a salt thereof wherein R1 = CH3 or C2H5Formula (II) and 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.

10. The method according to any one of the claims 1 to 3 to 9, or the use according to any one of the claims 2 to 9, wherein the composition comprises a fermentation broth, preferably a spray dried fermentation broth or a freeze-dried fermentation broth, or a formulation.

11. The method according to claim 10, or the use according to claim 9, wherein the formulation comprises a flowable concentrate.

12. The method according to any one of the claims 1 , 3 to 11 , or the use according to any one of the claims 2 to 11 , wherein the nematode belongs to Meloidogyne spp., Heterodera spp., Helicotylenchus spp., Globodera spp, Rotylenchus spp. or Pratylenchus spp., preferably the nematode belongs to a nematode species Meloidogyne incognita, Meloidogyne hapla, Meloidogyne arenaria, Meloidogyne javanica, Globodera rostochiensis, Globodera pallida, Heterodera glycines, Heterodera avenae or Heterodera schachtii, or Pratylenchus zeae.

13. The method according to any one of the claims 1 , 3 to 12, or the use of claims 2 to 12, wherein the plant comprises soybean, potato, sugarbeet, sugarcance, rice, coffee, peas, corn, cotton, fruit and / or vegetables, preferably wherein the plant comprises cucumber, tomato, soybean and I or corn.

14. The method according to any one of the claims 1 , 3 to 13, or the use according to any one of the claims 2 to 13, wherein the composition comprising Streptomyces chrestomyceticus is applied at a rate of 1 g to 2000 g of the composition comprising Streptomyces chrestomyceticus per hectare, or at a rate of 0.001 to 50 g of the composition comprising Streptomyces chrestomyceticus per kg of seed.

15. The method or use according to any one of the previous claims wherein the composition is applied preventative or curative.

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