Bacillus isolate and uses thereof

Abstract

The present invention relates to a microbial strain having superior fungicidal and / or nematicidal activity, as well as its progeny and derivatives, compositions comprising the same, and methods of using the same, inter alia in agriculture. The strain, as well as its progeny and derivatives, is capable of increased fungicidal activity against at least one fungi selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia when compared to Bacillus velezensis strain GB03.

Description

[0001] BACILLUS ISOLATE AND USES THEREOF

[0002] The present invention relates to a novel microbial strain having superior fungicidal and / or nematicidal activity, as well as its progeny and derivatives, compositions comprising the same, and methods of using the same, inter alia in agriculture.

[0003] Background

[0004] Plant growth-promoting rhizobacteria (PGPR) colonize plant roots and have beneficial effects on plant growth and plant immunity.

[0005] Rhizobacteria strains of various genera are designated as PGPR. The species Bacillus velezensis is an example of a PGPR, and is gaining popularity as a biocontrol product in the field of agriculture. B. velezensis is an endospore-forming soil bacterium. B. velezensis and various strains thereof were previously classified as other Bacillus species such as B. subtilis and B. amyloliquefaciens. B. velezensis produces various secondary metabolites that act against different phytopathogenic microbes.

[0006] Bacillus velezensis strain GB03, previously also known as Bacillus subtilis strain GB03 or Bacillus amyloliquefaciens strain GB03, is a well-studied Bacillus strain. It has the ability to protect plants against foliar pathogens, soil-borne pathogens, and abiotic stress, while also being able to promote plant growth and increase crop yield.

[0007] B. velezensis GB03 colonizes plant roots and is known to compete with fungal pathogens in the soil. Analysis of the B. velezensis GB03 genome revealed 10 antibiotic biosynthetic gene clusters that produce three lipopeptides (surfactin, bacillomycin D, and fengycin), one siderophore (bacillibactin), three polyketides (macrolactin, bacillaene, and difficidin), two bacteriocins (mersacidin and amylocylicin), and one dipeptide (bacilysin).

[0008] Several B. velezensis strains have been commercialized by inclusion in several products. For example, B. velezensis QST71 is comprised in Serenade® (Bayer CropScience, e.g. Serenade® ASO: U.S. EPA Reg. No. 264-1152), B. velezensis GB03 is present in Kodiak® (Bayer CropScience, e.g. U.S. EPA Reg. No. 264-970), a formulation comprising GB03 spores to be applied to plant seeds resulting in suppression of soil-borne phytopathogens. B. velezensis GB03 is also comprised in Companion® (Growth Products, Inc.), and BioYield™ (Gustafson, Inc.) comprising B. velezensis GB03 and B. amyloliquefaciens IN937a. B. velezensis GB03 is currently regarded as the most effective fungicidal commercial PGPR.

[0009] Although current commercial applications of PGPRs are effective to a certain extent, there is a continued need in the art for products and methods that provide improved control of fungi.

[0010] Brief description of the invention

[0011] The present invention discloses a novel microbial strain. It was found to be capable of superior fungicidal and / or nematicidal activity, particularly when compared to other Bacillus velezensis strains, for example Bacillus velezensis strain GB03. As described in the examples, the inventors have isolated and identified a novel strain of the species Bacillus that is capable of highly effectively controlling the growth of fungi and / or nematodes. One aspect of the invention provides a novel Bacillus strain.

[0012] Another aspect of the invention provides a novel Bacillus strain deposited under deposit accession number DSM 33551 .

[0013] Another aspect of the invention provides a novel Bacillus strain, wherein the novel Bacillus strain is capable of nematicidal activity.

[0014] Another aspect of the invention provides a novel Bacillus strain, wherein the novel Bacillus strain is capable of increased fungicidal activity.

[0015] Another aspect of the invention provides a novel Bacillus strain, wherein the novel Bacillus strain is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03.

[0016] Another aspect of the invention provides a progeny or derivative of the novel Bacillus strain deposited under deposit accession number DSM 33551.

[0017] Another aspect of the invention provides a progeny or derivative of the novel Bacillus strain, wherein the novel Bacillus strain is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03.

[0018] Another aspect of the invention provides a progeny or derivative of the novel Bacillus strain deposited under deposit accession number DSM 33551 , wherein the progeny or derivative is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03 and wherein the progeny or derivative has at least the same fungicidal and / or nematicidal activity when compared to the novel Bacillus strain deposited under deposit accession number DSM 33551 .

[0019] Another aspect of the invention provides a progeny or derivative of the novel Bacillus strain, wherein the novel Bacillus strain is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03, wherein the progeny or derivative is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03 and wherein the progeny or derivative has at least the same fungicidal and / or nematicidal activity when compared to the novel Bacillus strain.

[0020] Yet another aspect of the invention provides an agricultural composition comprising: the novel Bacillus strain according to the invention, progeny, or derivative thereof; and an agriculturally acceptable carrier and / or adjuvant.

[0021] A further aspect of the invention provides an agricultural foliar or soil inoculant comprising: the novel Bacillus strain according to the invention, progeny, or derivative thereof; and optionally an agriculturally acceptable carrier and / or adjuvant.

[0022] Another aspect of the invention provides a coated plant propagation material, wherein the coating comprises the novel Bacillus strain according to the invention, progeny, or derivative thereof.

[0023] Yet another aspect of the invention provides a method of controlling or preventing phytopathogenic fungi and / or nematode damage on a plant, locus thereof, or on propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, the novel Bacillus strain according to the invention, progeny, or derivative thereof, or an agricultural composition comprising: the novel Bacillus strain according to the invention, the progeny, derivative thereof; and an agriculturally acceptable carrier and / or adjuvant, or an agricultural foliar or soil inoculant comprising: the novel Bacillus strain according to the invention, progeny, or derivative thereof.

[0024] Another aspect of the invention provides a use of the novel Bacillus strain according to the invention, progeny, or derivative thereof, or an agricultural composition comprising: the novel Bacillus strain according to the invention, progeny, or derivative thereof; and an agriculturally acceptable carrier and / or adjuvant, or an agricultural foliar or soil inoculant comprising: the novel Bacillus strain according to the invention, progeny, or derivative thereof, in agriculture or as a fungicide and / or nematicide.

[0025] Another aspect of the invention provides a use of the novel Bacillus strain according to the invention, progeny, or derivative thereof, or an agricultural foliar or soil inoculant comprising: the novel Bacillus strain according to the invention, progeny, or derivative thereof; and optionally an agriculturally acceptable carrier and / or adjuvant, in the manufacture of a composition for the control of phytopathogenic fungi and / or plant damaging nematodes.

[0026] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0027] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0028] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0029] The patent, scientific, and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published, and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail. Various aspects of the invention are described in further detail below.

[0030] Brief description of the drawings

[0031] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0032] Figure 1 shows confirmation of the presence of a plasmid in the strain according to the invention. Figure 2 shows a two-dimensional peak distribution diagram of the strain according to the invention. Figure 3 shows a gel view format displaying the mass spectrum profile of the strain according to the invention.

[0033] Figure 4 shows a gel view showing the mass spectrum profile of the strain according to the invention and a comparative strain Bacillus velezensis GB03 for visual evaluation of characteristic peaks.

[0034] Figure 5 shows ClinProTools peak statistics for all the species-specific peaks. Figure 6 shows average spectra of each discriminative peak between the strain according to the invention and a comparative strain Bacillus velezensis GB03.

[0035] Figure 7 shows discriminative peaks between the strain according to the invention and a comparative strain Bacillus velezensis GB03 classes in gel view.

[0036] Figure 8 shows second derivatives of FT-IR spectra in the polysaccharide region (1300-800 cm'1) for the strain according to the invention and a comparative strain Bacillus velezensis GB03.

[0037] Figure 9 shows a dendogram tree of the strain according to the invention and a comparative strain Bacillus velezensis GB03.

[0038] Figure 10 shows PCA of the strain according to the invention and a comparative strain Bacillus velezensis GB03 FT-IR spectra in the region 1300-800 cm'1wn.

[0039] Figure 11 shows similarity between the strain according to the invention and other B. velezensis strains. Figure 12 shows a maximum likelihood phylogenetic tree comprising the strain according to the invention (B. velezensis VMC 10 / 119).

[0040] Further details of the figures are disclosed in the examples below.

[0041] Detailed description of the invention

[0042] It was found that a novel Bacillus strain provided increased fungicidal and / or nematicidal effects, for example when compared to Bacillus velezensis strain GB03. B. velezensis strain GB03 was found to be the closest genetically related known Bacillus strain to the novel Bacillus strain according to the present invention.

[0043] The present invention relates to a novel Bacillus strain. Suitably, the novel Bacillus strain may be isolated. Suitably, the novel Bacillus strain is the strain that has been deposited under deposit accession number DSM 33551. Suitably, the novel Bacillus strain may be capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03.

[0044] The invention also relates to a novel Bacillus strain, wherein the novel Bacillus strain is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03.

[0045] Suitably, the novel Bacillus strain according to the invention is a Bacillus velezensis strain.

[0046] As used herein, the term "isolated strain" refers to a microbial strain that has been removed from its natural environment. Suitably, the novel Bacillus strain according to the invention as disclosed herein may be an isolated Bacillus strain.

[0047] Herein, the terms “novel Bacillus strain”, "Bacillus strain according to the invention”, and "strain according to the invention" are used interchangeably.

[0048] The novel Bacillus strain deposited under accession number DSM 33551 was tested in the examples and is also referred to herein as strain VMC 10 / 119. References to DSM 33551 and VMC 10 / 119 are used interchangeably. A nucleic acid sequence encoding a gene for 16S ribosomal RNA of the DSM 33551 strain that was tested is provided in SEQ ID NO: 1 .

[0049] The genome of strain DSM 33551 comprises two genomic scaffolds: a first scaffold which may suitably be considered a chromosome and a second scaffold which may suitably be considered a plasmid. A nucleic acid sequence of the first scaffold of strain DSM 33551 (Scaffold 01) is provided in SEQ ID NO: 3. A nucleic acid sequence of the second scaffold of strain DSM 33551 (Scaffold 02) is provided in SEQ ID NO: 2.

[0050] Another aspect of the invention provides a progeny or derivative of the novel Bacillus strain according to the invention.

[0051] Another aspect of the invention provides a progeny or derivative of a novel Bacillus strain deposited under deposit accession number DSM 33551.

[0052] Another aspect of the invention provides a progeny or derivative of a novel Bacillus strain, wherein the novel Bacillus strain is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03.

[0053] Yet another aspect of the invention provides a progeny or derivative of the novel Bacillus strain deposited under deposit accession number DSM 33551 , wherein the progeny or derivative is capable of increased fungicidal activity and / or nematicidal activity when compared to Bacillus velezensis strain GB03 and wherein the progeny or derivative has at least the same fungicidal and / or nematicidal activity when compared to the novel Bacillus strain deposited under deposit accession number DSM 33551 .

[0054] Another aspect of the invention provides a progeny or derivative of the novel Bacillus strain, wherein the novel Bacillus strain is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03, wherein the progeny or derivative is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03 and wherein the progeny or derivative has at least the same fungicidal and / or nematicidal activity when compared to the novel Bacillus strain.

[0055] Thus, also is provided a novel Bacillus strain, progeny, or derivative thereof, wherein the novel Bacillus strain is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03, and wherein the progeny or derivative has at least the same fungicidal activity when compared to the novel Bacillus strain.

[0056] Also is provided a novel Bacillus strain deposited under deposit accession number DSM 33551 , progeny, or derivative thereof, wherein the progeny or derivative has at least the same fungicidal and / or nematicidal activity when compared to the novel Bacillus strain, and preferably wherein the novel Bacillus strain is capable of increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03.

[0057] As used herein, the term "progeny" refers to the descendent(s) of the Bacillus strain according to the invention and encompasses both immediate offspring of the Bacillus strain according to the invention and any descendants thereof. In particular, a progeny of the strain according to the invention is fungicidally and / or nematicidally active. A progeny will have comparable pesticidal activity, preferably at least the same fungicidal and / or nematicidal activity, to the original Bacillus strain according to the invention. In particular, a progeny will elicit comparable effects, preferably at least the same effects, on the fungi and / or nematode to the effects shown in the examples, which may be identified according to the materials and methods described in the examples.

[0058] As used herein, the term "derivative" refers to a progeny of the Bacillus strain according to the invention or a strain cultured (subcloned) from the original Bacillus strain according to the invention. Suitably, a derivative of a strain according to the invention may be a modified strain. Suitably, the modified strain may be modified at the genetic level, preferably without ablating the biological activity, for example the fungicidal and / or nematicidal activity. In particular, a derivative strain according to the invention is fungicidally and / or nematicidally active. A derivative strain will have comparable pesticidal activity, preferably at least the same fungicidal and / or nematicidal activity, to the original Bacillus strain according to the invention. In particular, a derivative strain will elicit comparable effects, preferably at least the same effects, on the fungi and / or nematode to the effects shown in the examples, which may be identified according to the materials and methods described in the examples. The modification may be by design. The modification may be the result of targeted or untargeted genetic modification methods known in the art. The modification may be due to directed evolution by methods known in the art.

[0059] As used herein, the term "modified strain" refers to a strain that is modified from a strain isolated from nature. Modified strains may be produced by any suitable method(s), including, but not limited to: chemical or other forms of induced mutation to a polynucleotide within any genome within the strain; the insertion or deletion of one or more nucleotides within any genome within the strain, or combinations thereof; an inversion of at least one segment of DNA within any genome within the strain; a rearrangement of any genome within the strain; generalized or specific transduction of homozygous or heterozygous polynucleotide segments into any genome within the strain; introduction of one or more phage into any genome of the strain; transformation of any strain resulting in the introduction into the strain of stably replicating autonomous extrachromosomal DNA; any change to any genome or to the total DNA composition within the strain isolated from nature as a result of conjugation with any different microbial strain; and any combination of the foregoing. The term modified strains includes a strain with (a) one of more heterologous nucleotide sequences, (b) one or more non-naturally occurring copies of a nucleotide sequence isolated from nature (i.e., additional copies of a gene that naturally occurs in the strain from which the modified strain was derived), (c) a lack of one or more nucleotide sequences that would otherwise be present in the natural reference strain by for example deleting nucleotide sequence, and (d) added extrachromosomal DNA. In some embodiments, modified strains comprise a combination of two or more nucleotide sequences (e.g., two or more naturally occurring genes that do not naturally occur in the same microbial strain) or comprise a nucleotide sequence isolated from nature at a locus that is different from the natural locus.

[0060] Comparison of the 16S rRNA gene sequence is presently the standard means in the art for bacteria typing and differentiation on (sub-)species or strain level.

[0061] Suitably, the Bacillus strain according to the invention may comprise a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1.

[0062] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to SEQ ID NO: 1.

[0063] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99.05%, at least 99.1 %, at least 99.15%, at least 99.2%, at least 99.25%, at least 99.3%, at least 99.35%, at least 99.4%, at least 99.45%, at least 99.5%, at least 99.55%, at least 99.6%, at least 99.65%, at least 99.7%, at least 99.75%, at least 99.8%, at least 99.81 %, at least 99.82%, at least 99.83%, at least 99.84%, at least 99.85%, at least 99.86%, at least 99.87%, at least 99.88%, at least 99.89%, at least 99.9%, at least 99.91 %, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or 100% sequence identity to SEQ ID NO: 1 . Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99.9% sequence identity to SEQ ID NO: 1.

[0064] Suitably, the Bacillus strain may comprise a nucleic acid sequence of SEQ ID NO: 1 . Suitably, the nucleic acid sequence of SEQ ID NO: 1 may be a nucleic acid sequence encoding a gene for 16S ribosomal RNA.

[0065] In the context of sequences, “identical”, “percent identical”, “sequence identity” or “percent sequence identity” in the context of two or more nucleic acid sequences, refers to two or more nucleic acid sequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using sequence comparison or sequence alignment. For clarity, the percentage identity of a sequence; the “target sequence”, for example a sequence listed herein as a SEQ ID NO, is compared and aligned for maximum correspondence over a comparison window, as measured using sequence comparison or sequence alignment, wherein the length of the individually specified contiguous sequence of the comparison window of the target sequence, for example a sequence listed herein as a SEQ ID NO, is the same or substantially the same as the total length of the target sequence.

[0066] A percentage identity between any two nucleic acid sequences can be determined via sequence comparison or sequence alignment. Methods of sequence comparison and sequence alignment are well-known in the art and can be determined via manual alignment and visual inspection or an algorithm, which is suitably implemented on a computer. When performing sequence comparison or alignment one sequence is typically used as a reference sequence to which the other sequence is compared. The comparison occurs in a comparison window which is an individually specified contiguous sequence of each of the compared sequences. Additions or deletions relative from one sequence to the other may be included in any of the sequences, thereby introducing so-called gaps in the other sequence. The introduction of gaps can result in a better alignment between the two sequences. However, the number of gaps in an alignment should be kept to a minimum in order to create a useful alignment, because too many gaps can cause an alignment to become meaningless. To avoid a high sequence identity between two sequences because of the introduction of too many gaps it is known to a person skilled in the art to use a gap penalty in order to compensate. Gap penalties are used to adjust alignment scores based on the number and length of gaps. Examples of gap penalties are constant, linear, affine, convex, and profile-based gap penalties.

[0067] Strains closely related to the strain according to the invention, tested in the examples, or deposited under deposit accession number DSM 33551 are also expected to be effective as having fungicidal and / or nematicidal activity, preferably an increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03. Such strains are also aspects of the present invention and may suitably be identified via their genetic information.

[0068] Suitably, the Bacillus strain according to the invention may comprise a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to SEQ ID NO: 2. Suitably, the Bacillus strain according to the invention may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2.

[0069] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99.05%, at least 99.1 %, at least 99.15%, at least 99.2%, at least 99.25%, at least 99.3%, at least 99.35%, at least 99.4%, at least 99.45%, at least 99.5%, at least 99.55%, at least 99.6%, at least 99.65%, at least 99.7%, at least 99.75%, at least 99.8%, at least 99.81 %, at least 99.82%, at least 99.83%, at least 99.84%, at least 99.85%, at least 99.86%, at least 99.87%, at least 99.88%, at least 99.89%, at least 99.9%, at least 99.91 %, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or 100% sequence identity to SEQ ID NO: 2. Suitably, the Bacillus strain according to the invention may comprise a nucleic acid sequence having at least 99.5% sequence identity to SEQ ID NO: 2. Suitably, the Bacillus strain according to the invention may comprise a nucleic acid sequence having at least 99.9% sequence identity to SEQ ID NO: 2.

[0070] Suitably, the Bacillus strain may comprise a nucleic acid sequence of SEQ ID NO: 2.

[0071] Suitably, the above nucleic acid sequence may be comprised in a plasmid.

[0072] Suitably, the Bacillus strain according to the invention may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 2 across at least 80% of SEQ ID NO: 2. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2 across at least 80% of SEQ ID NO: 2.

[0073] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 2 across at least 85% of SEQ ID NO: 2. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2 across at least 85% of SEQ ID NO: 2.

[0074] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 2 across at least 90% of SEQ ID NO: 2. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2 across at least 90% of SEQ ID NO: 2.

[0075] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 2 across at least 95% of SEQ ID NO: 2. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2 across at least 95% of SEQ ID NO: 2.

[0076] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 2 across at least 99% of SEQ ID NO: 2. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2 across at least 99% of SEQ ID NO: 2.

[0077] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 2 across at least 99.5% of SEQ ID NO: 2. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2 across at least 99.5% of SEQ ID NO: 2.

[0078] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 2 across at least 99.9% of SEQ ID NO: 2. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 2 across at least 99.9% of SEQ ID NO: 2.

[0079] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 2 across at least 99.99% of SEQ ID NO: 2.

[0080] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 2 across SEQ ID NO: 2.

[0081] Suitably, the above nucleic acid sequence may be comprised in a plasmid.

[0082] Suitably, the Bacillus strain according to the invention may comprise a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3.

[0083] Suitably, the Bacillus strain invention may comprise a nucleic acid sequence having at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to SEQ ID NO: 3. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3.

[0084] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99.05%, at least 99.1 %, at least 99.15%, at least 99.2%, at least 99.25%, at least 99.3%, at least 99.35%, at least 99.4%, at least 99.45%, at least 99.5%, at least 99.55%, at least 99.6%, at least 99.65%, at least 99.7%, at least 99.75%, at least 99.8%, at least 99.81 %, at least 99.82%, at least 99.83%, at least 99.84%, at least 99.85%, at least 99.86%, at least 99.87%, at least 99.88%, at least 99.89%, at least 99.9%, at least 99.91 %, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or 100% sequence identity to SEQ ID NO: 3. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99.9% sequence identity to SEQ ID NO: 3. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99.99% sequence identity to SEQ ID NO: 3.

[0085] Suitably, the Bacillus strain may comprise a nucleic acid sequence of SEQ ID NO: 3. Suitably, the Bacillus strain according to the invention may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 3 across at least 80% of its whole genome. Suitably, substantially the whole genome may be sequenced.

[0086] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 3 across at least 85% of its whole genome. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3 across at least 85% of its whole genome.

[0087] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 3 across at least 90% of its whole genome. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3 across at least 90% of its whole genome.

[0088] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 3 across at least 95% of its whole genome. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3 across at least 95% of its whole genome.

[0089] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 3 across at least 99% of its whole genome. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3 across at least 99% of its whole genome.

[0090] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 3 across at least 99.5% of its whole genome. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3 across at least 99.5% of its whole genome.

[0091] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 3 across at least 99.9% of its whole genome. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3 across at least 99.9% of its whole genome.

[0092] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 3 across at least 99.99% of its whole genome. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 3 across at least 99.99% of its whole genome.

[0093] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to SEQ ID NO: 3 across its whole genome.

[0094] Suitably, the Bacillus strain according to the invention may comprise a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91 %, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, or 99.99% sequence identity to the corresponding sequence of the bacterium deposited under accession number DSM 33551.

[0095] Suitably, the above nucleic acid sequence may be comprised in a chromosome.

[0096] Combinations of the aforementioned sequences may suitably be comprised in the novel Bacillus strain according to the invention. Examples include, but are not limited to the following combinations.

[0097] Suitably, the Bacillus strain according to the invention may comprise: a. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 ; and b. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2.

[0098] Suitably, the Bacillus strain may comprise: a. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 ; and b. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3.

[0099] Suitably, the Bacillus strain may comprise: a. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2; and b. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3.

[0100] Suitably, the Bacillus strain may comprise: a. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 ; b. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2; and c. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3.

[0101] Several genes were identified that may suitably affect the superior fungicidal and / or nematicidal activity of the strain according to the invention.

[0102] Suitably, the Bacillus strain according to the invention may comprise a nucleic acid sequence selected from any one of SEQ ID NOs: 4 to 6. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 84%, at least 86%, at least 88%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. SEQ ID NO: 4 was identified as a gene encoding gsiB.

[0103] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 84% sequence identity to SEQ ID NO: 4. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 4. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 4.

[0104] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 56%, at least 58%, at least 70%, at least 72%, at least 75%, at least 80%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 5. SEQ ID NO: 5 was identified as a gene encoding a hypothetical protein 1 .

[0105] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 56% sequence identity to SEQ ID NO: 5. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 5. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 5.

[0106] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 71 %, at least 72%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 6. SEQ ID NO: 6 was identified as a gene encoding putative translation initiation factor 2.

[0107] Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 71 % sequence identity to SEQ ID NO: 6. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO: 6. Suitably, the Bacillus strain may comprise a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 6.

[0108] Combinations of the aforementioned sequences may suitably be comprised in the novel Bacillus strain according to the invention. Examples include, but are not limited to the following combinations.

[0109] Suitably, the Bacillus strain according to the invention may comprise: a. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 ; and b. a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 4.

[0110] Suitably, the Bacillus strain may comprise: a. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3; and b. a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 4.

[0111] The Bacillus strain according to the invention may comprise a mutation in its genome in a nucleic acid sequence encoding gsiB when compared to nucleotide 465206 to 465577 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03. Suitably, the Bacillus strain may comprise a mutation in its genome in a nucleic acid sequence encoding a hypothetical protein 1 when compared to nucleotide 735288 to 742211 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03.

[0112] Suitably, the Bacillus strain may comprise an additional mutation in the nucleic acid sequence encoding a hypothetical protein 1 when compared to nucleotide 735288 to 742211 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03.

[0113] Suitably, the Bacillus strain may comprise a mutation in its genome in a nucleic acid sequence encoding putative translation initiation factor 2 when compared to nucleotide 735288 to 742211 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03.

[0114] Suitably, the mutation may comprise a deletion.

[0115] Suitably, the Bacillus strain may comprise a mutation in its genome in a nucleic acid sequence encoding gsiB when compared to nucleotide 465206 to 465577 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03, the mutation comprising a deletion of at least 60 nucleotides. Suitably, the deletion may be a deletion comprising nucleotides 465395 to 465454 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03.

[0116] Suitably, the Bacillus strain may comprise a mutation in its genome in a nucleic acid sequence encoding a hypothetical protein 1 when compared to nucleotide 735288 to 742211 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03, the mutation comprising a deletion of at least 2132 nucleotides. For clarity, the deletion is determined between the sequences common to the nucleic acid sequence encoding the hypothetical protein 1 of the Bacillus strain and nucleotide 735288 to 742211 according to GenBank Accession Number CP049904.1 of the homologous region, excluding the terminal 5' and 3' regions of 338 and 439 nucleotides of nucleotide 735288 to 742211 according to GenBank Accession Number CP049904.1 of the homologous region. Suitably, the deletion may be a deletion comprising nucleotides 737013 to 739019 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03.

[0117] Suitably, the Bacillus strain may comprise a mutation in its genome in a nucleic acid sequence encoding a hypothetical protein 1 when compared to nucleotide 735288 to 742211 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03, the mutation comprising a deletion of at least 126 nucleotides. Suitably, the deletion may be a deletion comprising nucleotides 740913 to 741039 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03.

[0118] Suitably, the Bacillus strain may comprise a mutation in its genome in a nucleic acid sequence encoding putative translation initiation factor 2 when compared to nucleotide 742393 to 744897 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03, the mutation comprising a deletion of at least 738 nucleotides. Suitably, the deletion may be a deletion comprising nucleotides 743125 to 743862 according to GenBank Accession Number CP049904.1 of the homologous region in the genome of Bacillus velezensis strain GB03.

[0119] Strains closely related to the strain according to the invention, tested in the examples, or deposited under deposit accession number DSM 33551 are also expected to be effective as having fungicidal and / or nematicidal activity, preferably an increased fungicidal and / or nematicidal activity when compared to Bacillus velezensis strain GB03. Such strains are also aspects of the present invention and may suitably be identified via their physiological and / or biochemical characteristics. Preferably, such strains are genetically closely related.

[0120] Several expressed proteins were identified that may suitably affect the superior fungicidal and / or nematicidal activity of the strain according to the invention.

[0121] Suitably, the Bacillus strain according to the invention may comprise a protein A having a mass of 3075 Da, wherein the mass peak of the protein A is at least 50% reduced in a sample of the Bacillus strain when compared to the mass peak of the protein A comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). Suitably, the mass peak of the protein A may be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% reduced in a sample of the Bacillus strain when compared to the mass peak of the protein A comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS.

[0122] Suitably, the Bacillus strain may comprise a protein B having a mass of 6154 Da, wherein the mass peak of the protein B is at least 50% reduced in a sample of the Bacillus strain when compared to the mass peak of the protein B comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS. Suitably, the mass peak of the protein B may be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% reduced in a sample of the Bacillus strain when compared to the mass peak of the protein B comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS.

[0123] Suitably, the Bacillus strain may comprise a protein C having a mass of 6904 Da, wherein the mass peak of the protein C is at least 50% reduced in a sample of the Bacillus strain when compared to the mass peak of the protein C comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS. Suitably, the mass peak of the protein C may be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% reduced in a sample of the Bacillus strain when compared to the mass peak of the protein C comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS.

[0124] Suitably, the Bacillus strain may comprise a protein D having a mass of 7526 Da, wherein the mass peak of the protein D is at least 50% reduced in a sample of the Bacillus strain when compared to the mass peak of the protein D comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS. Suitably, the mass peak of the protein D may be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% reduced in a sample of the Bacillus strain when compared to the mass peak of the protein D comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS. The whole bacterial Fourier transform infrared spectroscopy (FT-IR) spectrum is composed of a complex of peaks broad generated by different biomolecules in the cell. In more detail, the FT-IR fingerprint-like pattern corresponds to a spectrum splitting into five subranges for bacterial typing and differentiation: the membrane amphiphile region (3000-2800 cm'1), the biological infrared-silent region (2800-1800 cm'1), the protein and peptide region (1500-800 cm'1), the protein and fatty acid region (1500-1200 cm'1), and the nucleic acid and polysaccharide region (1200-900 cm'1). The region corresponding to the wavenumbers from 1300-800 cm'1is recommended by many researchers for bacteria typing and differentiation on sub-species or strain level

[0125] Suitably, the Bacillus strain according to the invention may comprise at least one substantially different second derivative of a spectrum in the range of from 1020 cm'1to 980 cm'1in a sample of the Bacillus strain when compared to a second derivative of a spectrum in the range of from 1020 cm-1to 980 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by Fourier transform infrared spectroscopy (FT-IR).

[0126] Suitably, the Bacillus strain according to the invention may comprise at least one substantially different second derivative of a spectrum in the range of from 940 cm'1to 880 cm'1in a sample of the Bacillus strain when compared to a second derivative of a spectrum in the range of from 940 cm'1to 880 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0127] Suitably, the at least one substantially different second derivatives of the sample and the comparable sample are at the same respective wavenumbers.

[0128] Suitably, the at least one substantially different second derivative may be determined at one or more defined wavenumbers. Suitably, the one or more defined wavenumbers may be selected from about 1017 cm'1, about 1005 cm-1, about 992 cm-1, about 980 cm'1, about 935 cm'1, about 915 cm'1, about 900 cm'1, and about 887 cm-1. Suitably, the one or more defined wavenumbers may be selected from about 1017 cm'1, about 1005 cm'1, and about 992 cm'1. Suitably, the one or more defined wavenumbers may be selected from about 935 cm'1and about 900 cm-1. Suitably, the at least one substantially different second derivative may be decreased or increased in a sample of the Bacillus strain when compared to a comparable sample of Bacillus velezensis strain GB03.

[0129] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 1020 cm'1to 980 cm-1may be increased in the sample of the Bacillus strain by at least 0.00004 at about 1005 cm-1and / or by at least 0.00003 at about 980 cm'1, when compared to a second derivative of a spectrum at respectively about 1005 cm'1and / or about 980 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0130] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 1020 cm'1to 980 cm'1may be decreased in the sample of the Bacillus strain by at least 0.00004 at about 1017 cm'1and / or by at least 0.00004 at about 992 cm'1, when compared to a second derivative of a spectrum at respectively about 1017 cm'1and / or about 992 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0131] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 940 cm'1to 880 cm'1may be increased in the sample of the Bacillus strain by at least 0.00004 at about 935 crrr1and / or by at least 0.00003 at about 900 cm'1, when compared to a second derivative of a spectrum at respectively about 935 cm-1and / or about 900 cm-1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0132] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 940 cm'1to 880 cm'1may be decreased in the sample of the Bacillus strain by at least 0.00002 at about 915 cm'1and / or by at least 0.00002 at about 887 cm'1, when compared to a second derivative of a spectrum at respectively about 915 cm'1and / or about 887 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0133] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 1020 cm'1to 980 cm-1may be increased in the sample of the Bacillus strain by at least 0.00005 at about 1005 cm'1and / or by at least 0.00004 at about 980 cm'1, when compared to a second derivative of a spectrum at respectively about 1005 cm'1and / or about 980 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0134] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 1020 cm'1to 980 cm'1may be decreased in the sample of the Bacillus strain by at least 0.00005 at about 1017 cm'1and / or by at least 0.00005 at about 992 cm'1, when compared to a second derivative of a spectrum at respectively about 1017 cm'1and / or about 992 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0135] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 940 cm'1to 880 cm'1may be increased in the sample of the Bacillus strain by at least 0.00005 at about 935 cm'1and / or by at least 0.00004 at about 900 cm'1, when compared to a second derivative of a spectrum at respectively about 935 cm'1and / or about 900 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0136] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 940 cm'1to 880 cm'1may be decreased in the sample of the Bacillus strain by at least 0.00003 at about 915 cm'1and / or by at least 0.00003 at about 887 cm'1, when compared to a second derivative of a spectrum at respectively about 915 cm'1and / or about 887 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0137] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 1020 cm-1to 980 cm'1may be increased in the sample of the Bacillus strain by of from 0.00004 to 0.00007 at about 1005 cm'1and / or by of from 0.00003 to 0.00006 at about 980 cm-1, when compared to a second derivative of a spectrum at respectively about 1005 cm'1and / or about 980 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0138] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 1020 cm'1to 980 cm-1may be decreased in the sample of the Bacillus strain by of from 0.00004 to 0.00007 at about 1017 cm-1and / or by of from 0.00004 to 0.00007 at about 992 cm-1, when compared to a second derivative of a spectrum at respectively about 1017 cm'1and / or about 992 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0139] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 940 cm'1to 880 cm-1may be increased in the sample of the Bacillus strain by of from 0.00004 to 0.00007 at about 935 cm'1and / or by of from 0.00003 to 0.00006 at about 900 cm'1, when compared to a second derivative of a spectrum at respectively about 935 cm-1and / or about 900 cm-1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0140] Suitably, the at least one substantially different second derivate of a spectrum in the range of from 940 cm'1to 880 cm'1may be decreased in the sample of the Bacillus strain by of from 0.00002 to 0.00005 at about 915 cm-1and / or by of from 0.00002 to 0.00005 at about 887 cm'1, when compared to a second derivative of a spectrum at respectively about 915 cm'1and / or about 887 cm-1in a comparable sample of Bacillus velezensis strain GB03, as determined by FT-IR.

[0141] Combinations of the aforementioned sequences and expressed proteins or FT-IT spectra may suitably be comprised in the Bacillus strain according to the invention. Examples include, but are not limited to the following combinations.

[0142] Suitably, the Bacillus strain may comprise: a. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 ; and b. a protein A having a mass of 3075 Da, wherein the mass peak of the protein A is at least 50% reduced in a sample of the Bacillus strain when compared to the mass peak of the protein A comprised in a comparable sample of B. velezensis strain GB03, as determined by MALDI-TOF MS.

[0143] Suitably, the Bacillus strain may comprise: a. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 ; and b. at least one substantially different second derivative of a spectrum in the ranges of from 1020 cm'1to 980 cm'1and 940 cm'1to 880 cm-1in a sample of the Bacillus strain when compared to a second derivative of a spectrum in the ranges of from 1020 cm-1to 980 cm'1and of from 940 cm-1to 880 cm'1in a comparable sample of B. velezensis strain GB03, as determined by FT-IR.

[0144] Suitably, the Bacillus may comprise: a. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3; b. a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 4; and c. a protein B having a mass of 6154 Da, wherein the mass peak of the protein B is at least 50% reduced in a sample of the Bacillus strain when compared to the mass peak of the protein B comprised in a comparable sample of B. velezensis strain GB03, as determined by MALDI-TOF MS.

[0145] Suitably, the Bacillus strain may comprise: a. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 ; b. a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3; and c. at least one substantially different second derivative of a spectrum in the ranges of from

[0146] 1020 cm'1to 980 cm'1and of from 940 cm-1to 880 cm-1in a sample of the Bacillus strain when compared to a second derivative of a spectrum in the ranges of from 1020 cm'1to 980 cm'1and of from 940 cm'1to 880 cm-1in a comparable sample of B. velezensis strain GB03, as determined by FT-IR.

[0147] The fungicidal and / or nematicidal activity or increased fungicidal and / or nematicidal activity of the strain according to the invention, progeny, or derivative thereof may be effective against a broad range of fungi and / or nematode, preferably phytopathogenic fungi and / or plant damaging nematode. Suitably, the fungi and / or nematode may be soil-borne fungi.

[0148] Suitably, the fungicidal activity may comprise controlling, modifying, or preventing 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.

[0149] Suitably, the fungicidal activity of the Bacillus strain according to the invention, progeny, or derivative thereof may be against at least one fungi selected from the group consisting of: Ascomycetes, Basidiomycetes, Fungi imperfecti (also known as Deuteromycetes), and Oomycetes (also known as Peronosporomycetes; e.g. Phytophthora, Peronospora, Pseudoperonospora, Albugo, Bremia, Pythium, Pseudosclerospora, Plasmopara), preferably Ascomycetes, Basidiomycetes, and Fungi imperfecti, more preferably Ascomycetes and Basidiomycetes.

[0150] Suitably, the fungicidal activity may be against at least one fungi selected from the group consisting of: Leotiomycetes, Sordariomycetes, and Agaricomycetes.

[0151] Suitably, the fungicidal activity may be against at least one fungi selected from the group consisting of: Botrytis, Erysiphe, Monilinia, Podosphaera, Pseudopezicula, Sclerotinia, and Uncinula.

[0152] Suitably, the fungicidal activity may be against at least one fungi selected from the group consisting of: Colletotrichum, Fusarium, Magnaporthe, Phomopsis, and Pyricularia.

[0153] Suitably, the fungicidal activity may be against at least one fungi selected from the group consisting of: Phakopsora, Puccinia, Rhizoctonia, Tilletia, and Ustilago.

[0154] Suitably, the fungicidal activity may be against at least one fungi selected from the group consisting of: Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Corynespora, Erysiphe, Fusarium, Glomerella, Guignardia, Helminthosporium, Magnaporthe, Mycosphaerella, Monilinia, Penicillium, Phakopsora, Phoma, Phomopsis, Podosphaera, Pseudopezicula, Puccinia, Pyricularia, Pyrenophora, Ramularia, Rhizoctonia, Sclerotinia, Septoria, Tilletia, Uncinula, Ustilago, Venturia, and Zymoseptoria, preferably Botrytis, Colletotrichum, Erysiphe, Fusarium, Glomerella, Magnaporthe, Monilinia, Phomopsis, Podosphaera, Pseudopezicula, Pyricularia, Rhizoctonia, Sclerotinia, and Uncinula.

[0155] Suitably, the fungicidal activity of the Bacillus strain according to the invention, progeny, or derivative thereof may be against at least one fungi selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia. Suitably, the fungicidal activity may comprise fungicidal activity against at least two fungi selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia. Suitably, the fungicidal activity may comprise fungicidal activity against at least three fungi selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia.

[0156] Suitably, the fungicidal activity may comprise fungicidal activity against at least one fungi selected from the group consisting of: Botrytis cinerea, Fusarium culmorum, and Rhizoctonia solani. Suitably, the fungicidal activity may comprise fungicidal activity against at least two fungi selected from the group consisting of: Botrytis cinerea, Fusarium culmorum, and Rhizoctonia solani. Suitably, the fungicidal activity may comprise fungicidal activity against at least three fungi selected from the group consisting of: Botrytis cinerea, Fusarium culmorum, and Rhizoctonia solani. Suitably, the fungicidal activity may comprise a reduction in radial growth of fungi by at least 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or 45% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of fungi by at least 95% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of fungi by at least 65% when compared to an untreated control.

[0157] Suitably, the fungicidal activity may comprise a reduction in radial growth of fungi by at least 60% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of fungi by at least 55% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of fungi by at least 45% when compared to an untreated control.

[0158] Suitably, the fungicidal activity may comprise a reduction in radial growth of Botrytis cinerea by at least 45% when compared to an untreated control, a reduction in radial growth of Fusarium culmorum by at least 53% when compared to an untreated control, and / or a reduction in radial growth of Rhizoctonia solani by at least 60% when compared to an untreated control.

[0159] Suitably, the fungicidal activity may comprise a reduction in radial growth of Botrytis cinerea by at least 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 44% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Botrytis cinerea by at least 95% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Botrytis cinerea by at least 65% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Botrytis cinerea by at least 55% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Botrytis cinerea by at least 45% when compared to an untreated control.

[0160] Suitably, the fungicidal activity may comprise a reduction in radial growth of Fusarium culmorum by at least 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 53%, 50%, or 45% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Fusarium culmorum by at least 95% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Fusarium culmorum by at least 65% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Fusarium culmorum by at least 60% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Fusarium culmorum by at least 55% when compared to an untreated control.

[0161] Suitably, the fungicidal activity may comprise a reduction in radial growth of Rhizoctonia solani by at least 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or 45% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Rhizoctonia solani by at least 95% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Rhizoctonia solani by at least 70% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Rhizoctonia solani by at least 65% when compared to an untreated control. Suitably, the fungicidal activity may comprise a reduction in radial growth of Rhizoctonia solani by at least 60% when compared to an untreated control. Suitably, the fungicidal activity may be increased fungicidal activity.

[0162] Suitably, the nematicidal activity may comprise controlling, modifying, or preventing the damage to the plant or propagation material by the nematode. 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 nematode attack.

[0163] Suitably, the nematicidal activity may be against at least one nematode selected from the order Ascaridida, for example, Heterakis spp. from the order Rhabditida, for example, Bursaphelenchus spp., and Bursaphelenchus xylophilus; and from the order Tylenchida, for example, Anguina agrostis, Anguina pacificae, Anguina spp., Aphelenchoides besseyi, Aphelenchoides spp., Belonolaimus longicaudatus, Belonolaimus spp., Criconema spp., Criconemella spp., Criconemella sp, Criconemoides onoensis, Criconemoides spp., Ditylenchus angustus, Ditylenchus destructor, Ditylenchus dipsaci, Ditylenchus spp., Dolichodorus spp., Globodera rostochiensis, Globodera spp., Helicotylenchus spp., Heliocotylenchus multicinctus, Hemicriconemoides spp., Hemicycliophora spp., Heterodera avenae, Heterodera glycines, Heterodera leuceilyma, Heterodera schachtii, Heterodera spp., Heterodera trifolii, Hirschmanniella spp., Hirshmanniella spp., Hoploaimus spp., Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimus spp., Hypsoperine spp., Macroposthonia spp., Macropostonia sp, Melinius spp., Meloidogyne arenaria, Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica, Meloidogyne marylandi, Meloidogyne spp., Mesocriconema spp., Pratylenchus agilis, Pratylenchus alien!, Pratylenchus brachyurus, Pratylenchus curvitatus, Pratylenchus goodeyi, Pratylenchus neglectans, Pratylenchus neglectus thornei, Pratylenchus penetrans, Pratylenchus sefaensis, Pratylenchus spp., Punctodera spp., Quinisulcius spp., Rotylenchulus brachyurus, Rotylenchulus reniformis, Rotylenchulus robustus, Rotylenchulus spp., Rotylenchus reniformis, Rotylenchus spp., Scutellonema spp., and Subanguina spp..

[0164] Bacillus velezensis strain GB03, previously also known as Bacillus subtilis strain GB03 or Bacillus amyloliquefaciens strain GB03, is a commercialized Bacillus strain for use in agriculture. For example, B. velezensis GB03 is comprised in Kodiak®, a formulation comprising GB03 spores, in Companion®, and in BioYield™ together with B. amyloliquefaciens IN937a.

[0165] The full genomic sequence of B. velezensis GB03 has been reported by Choi et al. (Genome Announc. 2014 Sep-Oct; 2(5): e01092-14) and deposited at DDBJ / EMBL / GenBank under the accession no. AYTJ00000000, which has currently been superceded by the complete genome record(s) CP049904.1. GenBank is the National Institutes of Health (NIH) genetic sequence database, an annotated collection of all publicly available DNA sequences (Nucleic Acids Research, 2013 Jan;41 (D1):D36-42). GenBank is part of the International Nucleotide Sequence Database Collaboration, which comprises the DNA DataBank of Japan (DDBJ), the European Nucleotide Archive (ENA), and GenBank at the National Center for Biotechnology Information (NCBI).

[0166] Suitably, the B. velezensis strain GB03 may comprise or consist of the genomic sequence of

[0167] GenBank accession no. CP049904.1. Suitably, the B. velezensis strain GB03 may be comprised in a commercially available product. Suitably, the commercially available product may comprise Kodiak®, Companion®, or BioYield®, preferably Companion®, for example Companion® Liquid Microbial Inoculant.

[0168] Cultures

[0169] A Bacillus strain according to the invention, progeny, or derivative thereof may be cultured using any suitable method(s), including, but not limited to, the method(s) as provided in the examples, liquid-state fermentation, or solid-state fermentation. A strain according to the invention, progeny, or derivative thereof may be cultured using any suitable substrate(s), including, but not limited to the substrate(s) as provided in the examples. A strain according to the invention, progeny, or derivative thereof may be harvested during any suitable growth phase. Suitably, a strain according to the invention, progeny, or derivative thereof may be allowed to reach the stationary growth phase and harvested as vegetative cells. Suitably, a strain according to the invention, progeny, or derivative thereof may be harvested as spores. Suitably, one or more cells of the harvested strain, progeny, or derivative thereof may be used as a culture or to start a culture.

[0170] The present invention also provides a culture of the Bacillus strain according to the invention, progeny, or derivative thereof.

[0171] Suitably, the culture may comprise, consists essentially of, or consists of the Bacillus strain according to the invention. Suitably, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% of subcultures taken from the culture may exhibit a genotype that is 100% identical to that of the Bacillus strain according to the invention. Suitably, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% of subcultures taken from the culture may exhibit a genotype that is 99.99% identical to that of the Bacillus strain according to the invention. Suitably, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% of subcultures taken from the culture may exhibit a genotype that is 99.95% identical to that of the Bacillus strain according to the invention. Suitably, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% of subcultures taken from the culture may exhibit a genotype that is 99.9% identical to that of the Bacillus strain according to the invention. Suitably, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% of subcultures taken from the culture may exhibit a genotype that is 99% identical to that of the Bacillus strain according to the invention.

[0172] Suitably, a culture comprising the Bacillus strain according to the invention may comprise vegetative cells and / or spores, for example dormant spores. Suitably, at least 1 %, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of microbes in the culture may be present as vegetative cells. Suitably, at least 1 %, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of microbes in the culture may be present as spores.

[0173] Compositions

[0174] The Bacillus strain according to the invention, progeny, or derivative thereof may be useful in agriculture.

[0175] The present disclosure also provides an agricultural composition comprising: the Bacillus strain according to the invention; and an agriculturally acceptable carrier and / or adjuvant. The present disclosure further provides an agricultural composition comprising: a progeny or derivative of the Bacillus strain according to the invention; and an agriculturally acceptable carrier and / or adjuvant. The present disclosure further provides an agricultural composition comprising: the Bacillus strain according to the invention, progeny, or derivative thereof; and an agriculturally acceptable carrier and / or adjuvant.

[0176] As used herein, the term “composition” is understood to encompass the “agricultural composition” according to the disclosure, and can be used as a foliar, soil, or seed treatment composition.

[0177] Some compositions according to the disclosure may have a systemic action. A Bacillus strain of the present invention may be formulated into any suitable type of composition, including, but not limited to: seed coatings, foliar inoculants, and soil inoculants.

[0178] The present disclosure also provides an agricultural foliar or soil inoculant comprising: the Bacillus strain according to the invention; and optionally an agriculturally acceptable carrier and / or adjuvant. The present disclosure further provides an agricultural foliar or soil inoculant comprising: a progeny or derivative of the Bacillus strain according to the invention; and optionally an agriculturally acceptable carrier and / or adjuvant. The present disclosure further provides an agricultural foliar or soil inoculant comprising: the Bacillus strain according to the invention, progeny, or derivative thereof; and optionally an agriculturally acceptable carrier and / or adjuvant. Suitably, the agricultural foliar or soil inoculant may comprise a culture of the Bacillus strain according to the invention, progeny, or derivative thereof. Suitably, the agricultural foliar or soil inoculant may comprise a culture of the Bacillus strain according to the invention.

[0179] As used herein, the term “inoculant” is understood to encompass the “agricultural foliar or soil inoculant” according to the disclosure.

[0180] As used herein, the term “inoculant” refers to a composition capable of introducing at least one bacterium into a medium, for example a liquid medium, plant, propagation material of the plant (e.g. seed), locus of the plant, peat powder, or soil. Suitable inoculants and methods for producing inoculants are well known in the art. Those skilled in the art can select or produce suitable inoculants. The present disclosure also provides a coated plant propagation material, wherein the coating comprises the Bacillus strain according to the invention. The present disclosure further provides a coated plant propagation material, wherein the coating comprises a progeny or derivative of the Bacillus strain according to the invention. The present disclosure further provides a coated plant propagation material, wherein the coating comprises the Bacillus strain according to the invention, progeny, or derivative thereof.

[0181] The present disclosure also provides a coated plant propagation material, wherein the coating comprises the agricultural composition according to the disclosure.

[0182] The present disclosure also provides a coated plant propagation material, wherein the coating comprises the agricultural foliar or soil inoculant according to the disclosure; and optionally an agriculturally acceptable carrier and / or adjuvant.

[0183] The term "plant propagation material” as used herein denotes all generative parts of a plant, for example seeds or vegetative parts of plants such as cuttings and tubers. It includes seeds in the strict sense, as well as roots, fruits, tubers, bulbs, rhizomes, and parts of plants. Preferably, “plant propagation material” is understood to denote seeds. A preferred plant propagation material is a seed. In a further aspect, the present invention also relates to plant propagation material coated with the Bacillus strain according to the invention, progeny, or derivative thereof, or the composition according to the disclosure.

[0184] As used herein, the term "seed" denotes any resting stage of a plant that is physically detached from the vegetative stage of a plant and / or may be stored for prolonged periods of time and / or can be used to re-grow another plant individual of the same species. Here, the term "resting" refers to a state wherein the plant retains viability, within reasonable limits, in spite of the absence of light, water and / or nutrients essential for the vegetative (i.e. non-seed) state. In particular, the term refers to true seeds but does not embrace plant propagules such as suckers, corms, bulbs, fruit, tubers, grains, cuttings and cut shoots.

[0185] The compositions of the disclosure may comprise agriculturally acceptable carriers or adjuvants. Acceptable carriers or adjuvants for agricultural use are well known in the art. Those skilled in the art can select suitable carriers or adjuvants.

[0186] Methods and uses

[0187] A composition according to the disclosure may be effective against phytopathogenic fungi and / or damage caused by nematodes because of the fungicidal and / or nematicidal activity of the Bacillus strain according to the invention, progeny, or derivative thereof. The Bacillus strain according to the invention, progeny, or derivative thereof, or the composition according to the disclosure may be used to control plant diseases caused by a broad spectrum of fungal plant pathogens, for example the fungi disclosed above, and / or plant or propagation material damage caused by nematode, for example the nematodes disclosed above.

[0188] The present disclosure also provides a method of controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, the Bacillus strain according to the invention. The present disclosure further provides a method of controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, a progeny or derivative of the Bacillus strain according to the invention. The present disclosure further provides a method of controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, the Bacillus strain according to the invention, progeny, or derivative thereof.

[0189] The present disclosure also provides a method of controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, a composition according to the disclosure.

[0190] The present disclosure also provides a method of controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, an agricultural soil inoculant according to the disclosure.

[0191] The present disclosure also provides a method of controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof, preferably on a plant or locus thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, preferably on a plant or locus thereof, an agricultural foliar inoculant according to the disclosure.

[0192] The present disclosure also provides a method of controlling or preventing damage caused by nematodes on a plant or propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, the Bacillus strain according to the invention. The present disclosure further provides a method of controlling or preventing damage caused by nematodes on a plant or propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, a progeny or derivative of the Bacillus strain according to the invention. The present disclosure further provides a method of controlling or preventing damage caused by nematodes on a plant or propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, the Bacillus strain according to the invention, progeny, or derivative thereof.

[0193] The present disclosure also provides a method of controlling or preventing damage caused by nematodes on a plant or propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, a composition according to the disclosure.

[0194] The present disclosure also provides a method of controlling or preventing damage caused by nematodes on a plant or propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, an agricultural soil inoculant according to the disclosure.

[0195] The present disclosure also provides a method of controlling or preventing damage caused by nematodes on a plant or propagation material thereof, preferably on a plant or locus thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, preferably on a plant or locus thereof, an agricultural foliar inoculant according to the disclosure.

[0196] The term “plants” as used herein refers to all physical parts of a plant, saplings, stems, stalks, foliage, and fruits. The term "plant propagation material" is understood to denote all the generative parts of the plant, such as seeds, which can be used for the multiplication of the latter and vegetative plant material such as cuttings and tubers (for example, potatoes). There may be mentioned, e.g., the seeds (in the strict sense), roots, fruits, tubers, bulbs, rhizomes, parts of plants. 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 may be protected before transplantation by a total or partial treatment by immersion. In a preferred embodiment, plant propagation material is a seed.

[0197] The term “locus” as used herein is understood to mean 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. Preferably, the locus may be the soil.

[0198] As used herein, the term "plants" is to be understood as also including plants which have been so transformed by the use of recombinant DNA techniques that they are capable of synthesizing one or more selectively acting toxins, such as are known, for example, from toxin-producing bacteria. Examples of toxins which can be expressed include 6-endotoxins, vegetative insecticidal proteins (Vip), insecticidal proteins of bacteria colonizing nematodes, and toxins produced by scorpions, arachnids, wasps, and fungi.

[0199] Suitably, applying to the plant, locus thereof, or propagation material thereof, the Bacillus strain according to the invention, a progeny or derivative thereof, a composition according to the disclosure, or an inoculant according to the disclosurecomprises applying the Bacillus strain according to the invention, progeny, or derivative thereof, the composition according to the disclosure, or the inoculant according to the disclosure in a fungicidally effective amount.

[0200] Suitably, the phytopathogenic fungi may be selected from the group consisting of: Ascomycetes, Basidiomycetes, Fungi imperfecti (also known as Deuteromycetes), and Oomycetes (also known as Peronosporomycetes; e.g. Phytophthora, Peronospora, Pseudoperonospora, Albugo, Bremia, Pythium, Pseudosclerospora, Plasmopara), preferably Ascomycetes, Basidiomycetes, and Fungi imperfecti, more preferably Ascomycetes and Basidiomycetes.

[0201] Suitably, the phytopathogenic fungi may be selected from the group consisting of: Leotiomycetes, Sordariomycetes, and Agaricomycetes.

[0202] Suitably, the phytopathogenic fungi may be selected from the group consisting of: Botrytis, Erysiphe, Monilinia, Podosphaera, Pseudopezicula, Sclerotinia, and Uncinula.

[0203] Suitably, the phytopathogenic fungi may be selected from the group consisting of: Colletotrichum, Fusarium, Magnaporthe, Phomopsis, and Pyricularia.

[0204] Suitably, the phytopathogenic fungi may be selected from the group consisting of: Phakopsora, Puccinia, Rhizoctonia, Tilletia, and Ustilago.

[0205] Suitably, the phytopathogenic fungi may be selected from the group consisting of: Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Corynespora, Erysiphe, Fusarium, Glomerella, Guignardia, Helminthosporium, Magnaporthe, Mycosphaerella, Monilinia, Penicillium, Phakopsora, Phoma, Phomopsis, Podosphaera, Pseudopezicula, Puccinia, Pyricularia, Pyrenophora, Ramularia, Rhizoctonia, Sclerotinia, Septoria, Tilletia, Uncinula, Ustilago, Venturia, and Zymoseptoria, preferably Botrytis, Colletotrichum, Erysiphe, Fusarium, Glomerella, Magnaporthe, Monilinia, Phomopsis, Podosphaera, Pseudopezicula, Pyricularia, Rhizoctonia, Sclerotinia, and Uncinula.

[0206] Suitably, the phytopathogenic fungi may be selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia.

[0207] Suitably, the phytopathogenic fungi may be selected from the group consisting of: Botrytis cinerea, Fusarium culmorum, and Rhizoctonia solani.

[0208] Suitably, the nematode may be selected from the order Ascaridida, for example, Heterakis spp. from the order Rhabditida, for example, Bursaphelenchus spp., and Bursaphelenchus xylophilus; and from the order Tylenchida, for example, Anguina agrostis, Anguina pacificae, Anguina spp., Aphelenchoides besseyi, Aphelenchoides spp., Belonolaimus longicaudatus, Belonolaimus spp., Criconema spp., Criconemella spp., Criconemella sp, Criconemoides onoensis, Criconemoides spp., Ditylenchus angustus, Ditylenchus destructor, Ditylenchus dipsaci, Ditylenchus spp., Dolichodorus spp., Globodera rostochiensis, Globodera spp., Helicotylenchus spp., Heliocotylenchus multicinctus, Hemicriconemoides spp., Hemicycliophora spp., Heterodera avenae, Heterodera glycines, Heterodera leuceilyma, Heterodera schachtii, Heterodera spp., Heterodera trifolii, Hirschmanniella spp., Hirshmanniella spp., Hoploaimus spp., Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimus spp., Hypsoperine spp., Macroposthonia spp., Macropostonia sp, Melinius spp., Meloidogyne arenaria, Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica, Meloidogyne marylandi, Meloidogyne spp., Mesocriconema spp., Pratylenchus agilis, Pratylenchus alien!, Pratylenchus brachyurus, Pratylenchus curvitatus, Pratylenchus goodeyi, Pratylenchus neglectans, Pratylenchus neglectus thornei, Pratylenchus penetrans, Pratylenchus sefaensis, Pratylenchus spp., Punctodera spp., Quinisulcius spp., Rotylenchulus brachyurus, Rotylenchulus reniformis, Rotylenchulus robustus, Rotylenchulus spp., Rotylenchus reniformis, Rotylenchus spp., Scutellonema spp., and Subanguina spp..

[0209] Suitably, the plant according to the disclosure may be selected from the group consisting of: cereals, such as wheat, barley, rye, oats, rice, maize or sorghum; beet, such as sugar or fodder beet; fruit, for example pomaceous fruit, stone fruit or soft fruit, such as apples, pears, plums, peaches, almonds, cherries or berries, for example strawberries, raspberries or blackberries; leguminous crops, such as beans, lentils, peas or soya (Glycine max); oil crops, such as oilseed rape, mustard, poppies, olives, sunflowers, coconut, castor, cocoa or ground nuts; cucurbits, such as pumpkins, cucumbers or melons; fibre plants, such as cotton, flax, hemp or jute; citrus fruit, such as oranges, lemons, grapefruit or tangerines; vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes or bell peppers; Lauraceae, such as avocado, Cinnamonium or camphor; and also tobacco, nuts, coffee, eggplants, sugarcane, tea, pepper, grapevines, hops, the plantain family and latex plants. Suitably, the plant may be selected from maize and soya (Glycine max).

[0210] Suitably, the plant may be an ornamental and / or vegetable crop, including flowers, shrubs, broad-leaved trees and evergreens.

[0211] Suitably, the plant may be selected from the group consisting of: grains, fruits and tree nuts, vegetables, field crops, oil seed crops, forage crops, fiber crops, forest plants, horticulture crops, floriculture, greenhouse and nursery plants, propagative materials, culinary herbs and spices, and medicinal herbs.

[0212] Suitably, the plant may be selected from the group consisting of: apples, almonds, bananas, cherries, citrus, grapes, grapevines, peaches, nectarines, blueberries, caneberries, raspberries, strawberries, tomatoes, potatoes, cucurbits, cucumbers, eggplants, lettuce, beans, brassicas, peas, oilseed rapes, soybeans, sugar beets, sunflowers, rice, peanuts, coffee, ornamental plants, and turfgrass.

[0213] A Bacillus strain of the present invention, progeny, or derivative thereof may suitably be incorporated into a composition in any suitable amount, preferably in a fungicidally and / or nematicidally effective amount. The absolute value of the amount of the composition, the Bacillus strain of the present invention, progeny, or derivative thereof that is sufficient to cause a desired fungicidal and / or nematicidal effect may be affected by factors such as the subject of the treatment, for example plants, soil, or seeds; the type of treatment, for example spraying, dusting, or seed dressing; the purpose of the treatment, for example prophylactic or therapeutic; the type of fungi and / or nematode to be controlled; the application time; the type, size, and volume of material to which the composition will be applied; and storage conditions (e.g., temperature, relative humidity, duration). A person skilled in the art will understand how to select an effective amount using routine experiments. The term “fungicidally and / or nematicdally effective amount” as used herein is understood to mean the quantity of the strain (e.g. spores and / or vegetative cells) that is capable of producing an effect on the growth of fungi. Preferably, an “effective” amount herein refers to an amount of the active ingredient that shows sufficient control or fungicidal and / or nematicidal activity, e.g., at least 10 %, preferably at least 20%, more preferably at least 30%, yet more preferably at least 40%, even more preferably at least 50%, and again more preferably at least 70 % effectiveness, compared to a control.

[0214] The active ingredient (ai) of the present disclosure is the Bacillus strain of the present invention, progeny, or derivative thereof, preferably the Bacillus strain of the present invention.

[0215] With the compositions according to the disclosure, the Bacillus strain of the present invention, progeny, or derivative thereof it is possible to control phytopathogenic fungi which occur in plants or in parts of plants (e.g. fruit, blossoms, leaves, stems, tubers, roots), while at the same time the parts of plants which grow later are also protected from attack by phytopathogenic fungi. Suitably, the compositions according to the disclosure, the Bacillus strain of the present invention, progeny, or derivative thereof may be applied to the phytopathogenic fungi, the plants, the locus thereof, the propagation material thereof, storage goods, or technical materials threatened by fungi. Suitably, the compositions according to the disclosure, the Bacillus strain of the present invention, progeny, or derivative thereof may be applied before or after infection of the plants, the propagation material thereof, storage goods, or technical materials by the fungi.

[0216] The present disclosure also provides a use of the Bacillus strain according to the invention in agriculture or as a fungicide and / or nematicide. The present disclosure further provides a use of a progeny or derivative of the Bacillus strain according to the invention in agriculture or as a fungicide and / or nematicide. The present disclosure further provides a use of a composition according to the disclosure in agriculture or as a fungicide and / or nematicide. The present disclosure further provides a use of an inoculant according to the disclosure in agriculture or as a fungicide and / or nematicide.

[0217] Suitably, applying to the plant, the Bacillus strain according to the invention, a progeny or derivative thereof, a composition according to the disclosure, or an inoculant according to the disclosure comprises applying the Bacillus strain according to the invention, progeny, or derivative thereof, the composition according to the disclosure, or the inoculant according to the disclosure at a rate of from 1 e+3 to 9.1 e+13 cfu / ha, preferably of from 3.3e+3 to 4.55e+13 cfu / ha, especially of from 3e+7 to 5e+11 cfu / ha .(where cfu is colony-forming units).

[0218] Suitably, applying to the locus of the plant, the Bacillus strain according to the invention, a progeny or derivative thereof, or a composition according to the disclosure, comprises applying the Bacillus strain according to the invention, progeny, or derivative thereof, or the composition according to the disclosure, at a rate of from 4.4e+3 to 9.1 e+13 cfu / ha, preferably of from 3e+7 to 1 e+12 cfu / ha, especially of from 3e+7 to 5e+11 cfu / ha.

[0219] Suitably, applying to the propagation material thereof, the Bacillus strain according to the invention, a progeny or derivative thereof, comprises applying the Bacillus strain according to the invention, progeny, or derivative thereof, at a rate of from 2.2e+2 to 2.73e+13 cfu / 100kg seeds, preferably of from 2e+6 to 3e+11 cfu / 100 kg seeds, especially of from 2e+6 to 1 e+11 cfu / 100kg seeds.

[0220] Preferably, the Bacillus strain according to the invention, progeny, or derivative thereof may be applied at the aforementioned indicated rates. Preferably, the Bacillus strain according to the invention may be applied at the aforementioned indicated rates.

[0221] Preferably, the Bacillus strain according to the invention, progeny, or derivative thereof, the composition, or the inoculant may be applied to a plant or locus of the plant at the aforementioned indicated rates. Preferably, the Bacillus strain according to the invention, progeny, or derivative thereof may be applied to a plant or locus of the plant at the aforementioned indicated rates. Preferably, the Bacillus strain according to the invention may be applied to a plant or locus of the plant at the aforementioned indicated rates.

[0222] Preferably, the Bacillus strain according to the invention, progeny, or derivative thereof, the composition, or the inoculant may be applied to a plant propagation material, such as a seed, at the indicated rates. Preferably, the Bacillus strain according to the invention, progeny, or derivative thereof may be applied to a plant propagation material, such as a seed, at the aforementioned indicated rates. Preferably, the Bacillus strain according to the invention may be applied to a plant propagation material, such as a seed, at the aforementioned indicated rates

[0223] A preferred method according to the invention comprises a method of controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof by phytopathogenic fungi selected from the group consisting of: the group consisting of: Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Corynespora, Erysiphe, Fusarium, Glomerella, Guignardia,

[0224] Helminthosporium, Magnaporthe, Mycosphaerella, Monilinia, Penicillium, Phakopsora, Phoma, Phomopsis, Podosphaera, Pseudopezicula, Puccinia, Pyricularia, Pyrenophora, Ramularia,

[0225] Rhizoctonia, Sclerotinia, Septoria, Tilletia, Uncinula, Ustilago, Venturia, and Zymoseptoria, the method comprising applying to the plant, locus thereof, or propagation material thereof the Bacillus strain according to the invention, wherein the Bacillus strain is applied at a rate of at least 10 g ai / ha, preferably at least 50 g ai / ha, more preferably at least 100 g ai / ha. Preferably, the phytopathogenic fungi is selected from: the group consisting of: Botrytis, Colletotrichum, Erysiphe, Fusarium, Glomerella, Magnaporthe, Monilinia, Phomopsis, Podosphaera, Pseudopezicula, Pyricularia, Rhizoctonia, Sclerotinia, and Uncinula, more preferably from the group consisting of: Botrytis, Fusarium, and Rhizoctonia.

[0226] The Bacillus strain according to the invention, a progeny or derivative thereof, a composition according to the disclosure, or an inoculant according to the disclosure may be applied with any suitable application method, such as foliar, drench, spraying, atomizing, dusting, scattering, coating, or pouring, to be chosen in accordance with the intended objectives and the prevailing circumstances.

[0227] The Bacillus strain according to the invention, a progeny or derivative thereof, a composition according to the disclosure, or an inoculant according to the disclosure may suitably be applied to the plant, locus thereof, or propagation material thereof according to known methods by means of, for example, foliar application, soil application, in furrow application, drench or seed treatment application.

[0228] Suitably, the Bacillus strain, a progeny or derivative thereof, the composition, or the inoculant may be applied to the leaves of plants. Suitably, the Bacillus strain, a progeny or derivative thereof, the composition, or the inoculant may be applied to the soil in or on which the plants are growing and / or will grow. Suitably, the Bacillus strain, a progeny or derivative thereof, the composition, or the inoculant may be applied to a plant propagation material, such as a seed.

[0229] A suitable means is readily determined by the skilled person in the art, using known techniques and by observing results obtained under analogous circumstances. In determining the suitable means, a number of factors are considered including, but not limited to the type and / or age of plant or derived product to be applied; the fungus to be controlled; the particular composition applied; and other relevant circumstances.

[0230] The term “foliage” as used herein refers to those portions of a plant that normally grow above the ground, including, but not limited to, leaves, stalks, stems, flowers, fruiting bodies, nuts, and fruits.

[0231] The term “foliar application” as used herein refers to the application of an active ingredient to the foliage of a plant (e.g., to the leaves of the plant). Application may be effected by any suitable means, including, but not limited to, spraying the plant with a composition comprising the active ingredient. Suitably, the active ingredient may be applied to the leaves, stems and / or stalk of the plant, and not to the flowers, fruiting bodies, nuts, or fruits of the plant.

[0232] In an example, the Bacillus strain according to the invention, a progeny or derivative thereof, a composition according to the disclosure, or an inoculant according to the disclosure may be applied in the field to the foliage of the plant (foliar application), thus allowing to select frequency and rate of application to match the danger of phytopathogenicity with the fungus in question. Alternatively, the active ingredient can reach the plants via the root system (systemic action), by drenching the locus of the plants in the field with a liquid composition or by incorporating the active ingredient in solid form into the locus of the plants in the field, for example into the soil or furrow, such as in the form of granules. In the case of paddy rice crops, such granules can be metered into the flooded paddy-field.

[0233] Suitably, the method according to the invention may comprise applying to the plant, locus thereof, or propagation material thereof, the Bacillus strain according to the invention, a progeny or derivative thereof, by means of foliar application, soil application, in furrow application, drench or seed treatment application.

[0234] Suitably, the method according to the invention may comprise applying an inoculant or agricultural composition comprising the Bacillus strain according to the invention, a progeny or derivative thereof to the plant, locus thereof, or propagation material thereof by means of foliar application, soil application, in furrow application, drench or seed treatment application, optionally wherein the inoculant or composition further comprises an agriculturally acceptable adjuvant and / or carrier.

[0235] A preferred propagation material according to the method of controlling or preventing phytopathogenic fungi according to the disclosure may be seed.

[0236] Preferably, the Bacillus strain according to the invention, progeny, or derivative thereof may be applied to seed at the aforementioned indicated rates. Preferably, the Bacillus strain according to the invention may be applied to seed at the aforementioned indicated rates.

[0237] A preferred method according to the invention comprises a method of controlling or preventing phytopathogenic fungi on seed by phytopathogenic fungi selected from the group consisting of: the group consisting of: Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Corynespora, Erysiphe, Fusarium, Glomerella, Guignardia, Helminthosporium, Magnaporthe, Mycosphaerella, Monilinia, Penicillium, Phakopsora, Phoma, Phomopsis, Podosphaera, Pseudopezicula, Puccinia, Pyricularia, Pyrenophora, Ramularia, Rhizoctonia, Sclerotinia, Septoria, Tilletia, Uncinula, Ustilago, Venturia, and Zymoseptoria, the method comprising applying to the seed the Bacillus strain according to the invention, wherein the Bacillus strain is applied at a rate of at least 2.2e+2 cfu / 100kg seeds, preferably of from 2.2e+2 to 2.73e+13 cfu / 100kg seeds, more preferably of from 2e+6 to 3e+11 cfu / 100 kg seeds, especially of from 2e+6 to 1 e+11 cfu / 100kg seeds. Preferably, the phytopathogenic fungi is selected from: the group consisting of: Botrytis, Colletotrichum, Erysiphe, Fusarium, Glomerella, Magnaporthe, Monilinia, Phomopsis, Podosphaera, Pseudopezicula, Pyricularia, Rhizoctonia, Sclerotinia, and Uncinula, more preferably from the group consisting of: Botrytis, Fusarium, and Rhizoctonia.

[0238] In another aspect, the present invention provides a use of the Bacillus strain according to the invention, a progeny or derivative thereof, a composition according to the disclosure, for controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof according to the method of the invention.

[0239] In another aspect, the present invention provides a use of the Bacillus strain according to the invention, a progeny or derivative thereof, or a composition according to the disclosure, for controlling or preventing damage to a plant by a nematode according to the method of the invention.

[0240] In another aspect, the present invention provides a use of the Bacillus strain according to the invention, a progeny or derivative thereof, in the manufacture of a composition for controlling or preventing phytopathogenic fungi, preferably for controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof. In another aspect, the present invention provides a use of the Bacillus strain according to the invention, a progeny or derivative thereof, in the manufacture of a composition for controlling or preventing damage to a plant by a nematode.

[0241] Methods and means for manufacturing a suitable composition for the control of fungi are known in the art, examples and embodiments of which are set out in detail herein.

[0242] The methods and uses of the present invention do not comprise a method for treatment of the human or animal body by surgery or therapy or a diagnostic method practiced on the human or animal body. Thus, they exclude a method for treatment of the human or animal body by surgery or therapy and diagnostic methods practiced on the human or animal body.

[0243] Deposit of biological materials and expert solution

[0244] The Bacillus strain of the present invention was isolated from a rhizosphere soil sample collected in Italy and was deposited with the name Bacillus velezensis VMC 10 / 119 on 22 June 2020 by Valagro SpA, Via Cagliari 1 , CAP 66041 , Atessa (CH), Italy, at the Leibniz-lnstitut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7 B, D-38124, Braunschweig, Germany. The deposited strain was assigned deposit number DSM 33551 . This deposit of biological materials was made under the terms and conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure

[0245] The deposit represents a pure culture of the deposited strain. The deposit is available as required by patent laws in countries wherein counterparts of the present application or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the present invention in derogation of patent rights granted by governmental action.

[0246] As regards the respective patent offices of the respective designated states, the applicant requests that a sample of the deposited microorganism stated above only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

[0247] General definitions

[0248] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper

[0249] Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined herein are more fully described by reference to the specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. As used herein, the term “substantially” refers to a great extent or degree. The exact allowable degree may depend on the specific context. For example, the term “substantially” in the context of substantially different second derivates of spectra as disclosed herein would mean that they may have a value that is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, or at least 20% different (i.e. the value may be less or greater).

[0250] Detailed description of the figures

[0251] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0252] Figure 1 shows confirmation of the presence of a plasmid in the strain according to the invention. Figure 1A shows agarose gel electrophoresis of a first PCR protocol to confirm the circularity structure of the Scaffold 02. Lane: M) GeneRuler DNA ladder mix (Thermo Scientific), 1) B. velezensis VMC 10 / 119, 2) B. velezensis B-41580T, 3) PCR negative control. Figure 1 B shows agarose gel electrophoresis of a second PCR protocol to confirm the presence of genes carried by Scaffold 02 only for B. velezensis VMC 10 / 119 (MB157). Lane: M) GeneRuler DNA ladder mix (Thermo Scientific), 1) B. velezensis VMC 10 / 119, 2) B. velezensis B-41580T, 3) PCR negative control.

[0253] Figure 2 shows a two-dimensional peak distribution diagram of the strain according to the invention. Mass data annotation (m / z) is reported at the top of the peaks.

[0254] Figure 3 shows a gel view format displaying the mass spectrum profile of the strain according to the invention.

[0255] Figure 4 shows a gel view showing the two generation classes (VMC 10 / 136 on the top, from 37 to 71 spectrum; VMC 10 / 119 on the bottom, from 1 to 36 spectrum) for visual evaluation of characteristic peaks. Selected spectra and mass values (m / z) are reported on y-axis and x-axis, respectively.

[0256] Figure 5 shows ClinProTools peak statistics for all the species-specific peaks. DAve, difference between the maximal and the minimal average peak area / intensity of VMC 10 / 119 (Ave2) and GB03 (Ave1); PWKW, p value of Wilcoxon / KruskaleWallis test (preferable for non-normally distributed data); PAD, p value of AndersoneDarling test, which gives information about normal distribution (p-value AD <0.05, non-normally distributed; p-value AD >0.05, normally distributed). Standard deviation (StdDev) refers to each discriminative pick between the two classes (VMC10 / 119 and GB03).

[0257] Figure 6 shows average spectra of each discriminative peak between the two classes (VMC 10 / 119 on the bottom (black line); VMC 10 / 136 on the top (gray line)).

[0258] Figure 7 shows discriminative peaks between the two classes in gel view (VMC10 / 119 from 1 to 36 spectrum; VMC 10 / 136 (B. velezensis GB03) from 37 to 71 spectrum): 3075 m / z (Figure 7A), 6904 and 6154 m / z (Figure 7B), and 7526 m / z (Figure 7C).

[0259] Figure 8 shows second derivative values (y-axis) of FT-IR spectra in the polysaccharide region (1300- 800 cm'1) for VMC 10 / 119 (light gray line; indicated by the arrows X) and VMC 10 / 136 (8. velezensis GB03) (dark gray line; indicated by the arrows Y). The two dashed circles highlight the differences between the second derivatives of the spectra of from about 1020 cm'1to about 980 cm'1and of from about 940 cm'1to about 880 cm'1). Figure 9 shows a dendogram tree of VMC 10 / 119 and VMC 10 / 136 (B. velezensis GB03) FT-IR spectra in the region 1300-800 cm-1wn (wavenumber) (8 replicates per sample). The distance or the similarity among the spectra is calculated by the linkage average metric euclidean exploration method name euclideanaveragejinkage. 0.454 is the cut-off applied for the technical variance of replicates. The two strains cluster separately, meaning that they are different. Each cluster is pure (consists of one isolate) and all technical replicates are within one cluster (coherence).

[0260] Figure 10 shows PCA of VMC 10 / 119 and VMC 10 / 136 (B. velezensis GB03) FT-IR spectra in the region 1300-800 cm'1wn (wavenumber) (8 replicates per sample; number of principal components used: 6; 96% variance). The two strains group separately (PC1 ; variance 75.6%), meaning that they are different. Each cluster is pure (consists of one isolate) and all technical replicates are within one cluster (coherence).

[0261] Figure 11 shows similarity between the strain according to the invention and other 8. velezensis strains. MLST according to Dunlap (Dunlap C.A., 2019, Taxonomy of registered Bacillus spp. strains used as plant pathogen antagonists. Biological Control. 134, 82-86) was performed. Percentage sequence identity of genes of the different strains with VMC 10 / 119 is indicated.

[0262] Figure 12 shows a maximum likelihood phylogenetic tree comprising the strain according to the invention (8. velezensis VMC 10 / 119). The tree was reconstructed from the concatenated sequence of the genes 16S, groEL, gyrA, polC, purH and rpoB retrieved from the reference genomes reported in Table 4 and from the genome sequence of the strain 8. velezensis VMC 10 / 119 described herein and using the indicated genomic coordinates of the gene sequences in Table 4. The tree was determined using the Tamura-Nei model. Bootstrap values (1000 replicates) are shown as a percentage at the branching points. The scale bar corresponds to 0.02 nucleotide substitutions per site.

[0263] Further details of the figures are disclosed in the examples below.

[0264] Examples

[0265] Aspects of the invention are demonstrated by the following non-limiting examples.

[0266] Example 1

[0267] MATERIALS AND METHODS

[0268] Next Generation Sequencing, de-novo assembly and annotation

[0269] Quality analysis of FASTQ sequence reads

[0270] FASTQ sequence files from Illumina reads were generated using bcl2fastaq2 version 2.18. Initial quality assessment was based on data passing the Illumina Chastity filtering. Subsequently, reads containing PhiX control signal were removed using an in-house filtering protocol. In addition, reads containing (partial) adapters were clipped (up to minimum read length of 50 bp). The second quality assessment was based on the remaining reads using the FASTQC quality control tool version 0.11.5. Finally, the Illumina data has been subsampled, in order to avoid a too high coverage which results in a negative impact on the assembly quality according to the bioinformatics pipeline.

[0271] Quality analysis of PacBio sequence reads The data collected from the PacBio Sequel instrument were processed and filtered using the SMRT Analysis software suite. Subreads shorter than 50 bp were discarded.

[0272] De-novo hybrid assembly (PacBio and Illumina)

[0273] The quality of the Illumina FASTQ sequences was enhanced by trimming off low-quality bases using the program bbduk, which is part of the BBMap suite version 36.77. The quality-filtered sequence reads were puzzled into a number of contig sequences. The analysis has been performed using ABySS version version 2.0.2 (Jackman SD, Vandervalk BP, Mohamadi H, Chu J, Yeo S, Hammond SA, Jahesh G, Khan H, Coombe L, Warren RL, Birol I. 2017. ABySS 2.0: resource-efficient assembly of large genomes using a Bloom filter. Genome Res. 27, 768-777).

[0274] The contigs were linked and placed into super-scaffolds based on the alignment of the PacBio CLR reads. Alignment has been performed with BLASR (Chaisson M and Tesler G. 2012. Mapping single molecule sequencing reads using Basic Local Alignment with Successive Refinement (BLASR): Theory and Application. BMC Bioinformatics. 13, 238). From the alignment, the orientation order and distance between the contigs has been estimated. This analysis has been performed using the SSPACE-LongRead scaffolder version 1.0 (Boetzer M and Pirovano W. 2014. SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information. BMC Bioinformatics. 15, 211).

[0275] The gapped regions within the super-scaffolds are (partially) closed in an automated manner using GapFiller version 1.10 (Boetzer M and Pirovano W. 2012. Toward almost closed genomes with GapFiller. Genome Biol. 13, R56). The method takes advantage of the insert size between the Illumina paired-end reads. Finally, assembly errors and the nucleotide disagreements between the Illumina reads and scaffold sequences were corrected using Pilon version 1.21 (Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 9, e112963).

[0276] Annotation pipeline

[0277] Genome annotation has been performed on the assembled Consensus sequences using a pipeline which is based on the Prokka Prokaryotic Genome Annotation System, more in detail: Prokaryote gene prediction by Prodigal 2.6; rRNA prediction using barrnap 0.6; tRNA prediction by Aragorn 1.2.36. MALDI-TOF mass spectrometry bacterial identification

[0278] VMC 10 / 119 was characterized by using a MicroflexTM MALDI-TOF mass spectrometer (Bruker Daltonics, Leipzig, Germany). Initial manual / visual estimation of the mass spectra was performed using the FlexAnalysis 2.4 software (Bruker Daltonik GmbH, Germany). For automated data analysis, raw spectra were processed using the MALDI BioTyper 1.1 software (Bruker Daltonik GmbH, Germany) with default settings. The smoothing, normalization, baseline subtraction and peak picking was carried out by the software, thereby creating a list of the most significant peaks of a spectrum (m / z values with a given intensity). Samples were prepared according to manufacturers’ instructions.

[0279] Briefly, after 24 hours of cultivation on Nutrient agar (NA) at 30°C, a single colony was picked up and inactivated by resuspension in 300 pL dH2O, then mixed by inversion with 900 pL absolute ethanol. After mixing the solution, the bacterial culture was harvested by centrifugation (16000 x g, 2 min), and air-dried at room temperature after the removal of the supernatant. The cells were resuspended in 50 pL 70% formic acid, vortexed for 1 minute, and resuspended in an equal volume of pure acetonitrile. Samples were centrifuged (16000 x g, 2 min) and one pL of supernatant was spotted onto a MALDI steel target plate, forming a thin film and leave to dry at room temperature. Subsequently, one pL of the matrix solution (i.e., 50% acetonitrile containing 1 % a-cyano-4-hydroxycinnamic acid and 2.5% trifluoroacetic acid) was introduced on the film again. The sample-matrix was dried at room temperature before analyzing it through MS for data acquisition. Mass spectrum analysis was performed using a MicroFlex LT mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) with linear positive model, and the analytic region was 2000-20000 Da. 240 laser shots (at frequency of 20 Hz) were collected, and a Bruker Daltonics Bacterial test standard (Bruker Daltonik GmbH, Bremen, Germany) was used for calibration and as the control with the linear positive model. The results of mass spectrum from the MALDI Biotyper 3.1 software (Bruker Daltonik GmbH, Bremen, Germany) were compared with those in the database and confirmed the belonging to Bacillus velezensis species with a score>2.3 (meaning reliable species identification). Moreover, once the raw spectra were acquired, the quality and relationships of each spectra were checked, screened, and estimated using FlexAnalysis software version 3.4 (Bruker Daltronics GmbH, Bremen, Germany) and a composite correlation index (CCI). The best quality spectra that remained were chosen and transferred to create a specific single main spectrum profile (MSP) for database expansion.

[0280] To assess the reproducibility of MALDI-TOF-MS identification, strains were tested in triplicate (analyses were performed on three different days and starting from different cultures to avoid a possible batch effect).

[0281] ClinProTools for bacterial comparison

[0282] ClinProTools is a software developed by Bruker (Bruker Daltonik GmbH, Bremen, Germany) for the visualization and statistical analysis of mass spectrometry data. This software allows to view statistical parameters describing each peak of a spectra and data are summarized in a Peak Statistic Table. This approach allows to discover significant biomarkers able to distinguish closely related species and even, isolates at the strain level. Biomarkers in bacteria are mainly ribosomal proteins and include a few housekeeping proteins, which is comparable with the multilocus sequence analysis of the housekeeping genes used to determine higher level taxonomy and phylogeny (Welker and Moore, 2011 , Applications of whole-cell matrix-assisted laser-desorption / ionization time-of-flight mass spectrometry in systematic microbiology. Syst. Appl. Microbiol. 34 2-11. 10.1016 / j.syapm.2010.11 .013).

[0283] This software was used to identify potential biomarkers able to discriminate VMC 10 / 119 and the most related species (according to the results at MALDI-TOF mass spectrometry bacterial identification), Bacillus velezensis GB03, also referred to herein as VMC 10 / 136. For the analysis, a standard ethanol / formic acid extraction protocol and mass data acquisition for both bacteria were performed by following the method described above for MALDI-TOF mass spectrometry bacterial identification. For each strain, the extraction was done from three different biological replicates, and three analyses were conducted on different days to avoid a possible batch effect. A total of 36 replicates for each strain were performed, and the obtained spectra were analyzed and screened using FlexAnalysis software. To determine the relationships among replicates, the CCI indicators between spectra were calculated. High CCI values (near 1) indicated almost identical spectra. At least 24 good-quality spectra of each strain were loaded into a database and used for peak signal analysis by ClinProTools™ software version 3.0.

[0284] The imported raw spectra of each investigated strain were processed in ClinProTools using the following parameters: baseline subtraction using top hat with minimal baseline width of 10%, normalization using total ion current, recalibration, average spectrum calculation using a resolution of 800, average pick list calculation using a signal to-noise-threshold of 3 and pick list normalization.

[0285] Acquired spectra were loaded in two classes, one for VMC 10 / 119 and one for VMC 10 / 136, and the average spectrum for each class was calculated (Figure 4). A detailed spectra analysis of each species was performed in the region between 2000 and 12000 m / z that concentrated the bulk of mass peaks, and the characteristic peaks between the two classes were selected and sorted through the following statistic tests: the Wilcoxon or Kruskal-Wallis (W / KW) test and the Anderson-Darling (AD) test. A P-value of 0.05 was set as the cutoff. If P was <0.05 in the AD test, a characteristic peak was selected if the corresponding value of P in the W / KW test was also <0.05. The characteristic peaks were sorted using the corresponding P-values obtained in the W / KW test because the AD test revealed P<0,05.

[0286] Fourier transform infrared spectroscopy (FT-IR) biotyper

[0287] The process for FT-IR bacterial spectra acquisition comprises the following steps: bacterial sample preparation, instrument operation, spectra collection, spectra preprocessing, and mathematical data analysis (PCA and HCA). To obtain stable results and avoiding variability among samples to be analyzed, the cultivation was started from bacteria of the same age and on the same medium (Nutrient agar - NA). All samples were subcultured on NA for three subsequent generations, at 30°C and for 24 hours to adapt the microorganism to the growth medium.

[0288] For each bacterium, a 10 pl loopful of an overnight culture was transferred into a 1.5 ml vial and mix with 70% ethanol to prepare a homogenous suspension. Four technical replicates (15 pl) of each sample suspension, prepared in duplicate, were transferred on a silicon 96-microtiter sample plate and leave to dry at room temperature. In summary, eight distinct spectra acquisitions were made for VMC 10 / 119 and B. velezensis GB03 (internal code VMC 10 / 136).

[0289] Once dried, the target plate was inserted in the IR Biotyper for FT-IR spectra acquisition according to the manufacturer parameters. Spectra were recorded in transmission mode in a spectral range of 4000-400 cm'1(mid-IR) using an IR spectrometer (Bruker Optics-Daltonics GmbH). Spectra were acquired, visualized, and processed by OPUS v7.5 software (Bruker Optics GmbH) through the following steps: spectrum subtracting, baseline correction, derivatization, normalization and analytical window selection of the area of polysaccharides (1300-800 cm'1). Next, the second derivative analysis was used to amplify differences between strains. The second derivative transformation is a mathematical tool that can resolve the broad bands overlapping raw spectra, reduce replicate variability, correct baseline shift, and amplify spectral variations. The second derivative spectra are most commonly used for bacterial classification, typing and identification (Yang et al., 2020, Bacterial Typing and Identification Based on Fourier Transform Infrared Spectroscopy; Protocol Exchange; https: / / doi.Org / 10.21203 / rs.2.23337 / v1).

[0290] Fungicidal activity

[0291] VMC 10 / 119 was grown overnight on SMC1 (Skim Milk medium), TSB (Tryptic Soy Broth), or NYB (Nutrient Yeast Extract Broth) medium. Assessment of fungicidal activity was performed on PDA (Potato Dextrose Agar medium). A drop of 10 pL (107CFU / mL) was spotted on PDA medium, and a 5 mm mycelium-agar disc taken from an actively growing culture of fungal pathogens was placed 30 mm away from the point of bacterium inoculation. Control plates consisted of fungal pathogen only. The experiment was performed in triplicate. Plates were incubated at 28 °C and 3 days after the inoculation the mycelial growth was measured the direction of the bacterial isolate. Subsequently, the inhibition was calculated according the formula: (Rc-Ri / Rc)x100, where Rc is the radial growth of mycelia in the control plate, Ri is the radial growth of mycelia towards the antagonist in the dual culture plate.

[0292] RESULTS

[0293] A Bacillus strain according to the invention (herein also referred to as VMC 10 / 119) was isolated from a rhizosphere soil sample from Italy. Specifically, the rhizosphere soil was collected in Castel Guelfo (BO), Emilia Romagna, Italy. The strain has been deposited in the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures on 22nd June 2020, with the following name Bacillus velezensis VMC 10 / 119 having the deposit number DSM 33551 .

[0294] Genome determination and comparison

[0295] The full genomic sequence of VMC 10 / 119 was determined. Two consensus scaffolds were identified: Scaffold 01 (SEQ ID NO: 3) with a size of 3878732 nucleotides and Scaffold 02 (SEQ ID NO: 2) with a size of 215523 nucleotides.

[0296] The application of the multilocus sequence typing (MLST) scheme based on the gene coding for the 16S rRNA and the coding genes groEL, gyrA, polC, purH, and rpoB described by Dunlap (Dunlap C.A., 2019, Taxonomy of registered Bacillus spp. strains used as plant pathogen antagonists. Biological Control. 134, 82-86), allowed to confirm the identification of the strain VMC 10 / 119 as a member of the species Bacillus velezensis.

[0297] Strain VMC 10 / 119 was compared with other B. velezensis strains using the same MLST scheme as described by Dunlap 2019. The respective National Institutes of Health (NIH) GenBank Gene Acc. No. of genes of other strains were compared with the positions within the reference genome of strain VMC 10 / 119, which are all listed in Table 4. The results are presented in Figure 11 and show that B. velezensis GB03 was the only strain with 100% identity with strain VMC 10 / 119 across five of the six MLST genes.

[0298] Next, a phylogenetic tree was reconstructed from the concatenated sequence of the genes 16S, groEL, gyrA, polC, purH and rpoB retrieved from the reference genomes reported in Table 4 and from the genome sequence of the strain B. velezensis VMC 10 / 119 described in Table 4. The tree was determined using the Tamura-Nei model and is presented in Figure 12. 8. velezensis GB03 was found to be the closest related to strain VMC 10 / 119.

[0299] The whole genome comparison between the strain VMC 10 / 119 and reference genome sequence of the strain B. velezensis GB03 (GenBank Accession Number CP049904.1 , released on January 3rd, 2022), also referred to herein as VMC 10 / 136, demonstrated a substantial similarity between the two genomes. The latest release of the 8. velezensis GB03 genome was obtained with Oxford Nanopore MinlON as sequencing technology using the bacteria and source DNA available from Choon-Min Ryu. The assembly method UniCycler_hybrid v. vO.4.8 The annotation was added by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).

[0300] For clarity, the whole genome comparison of the strain according to the invention was performed both against the reference genome of the strain 8. velezensis GB03 as disclosed by GenBank Accession Number CP049904.1 , released on January 3rd, 2022, and against a (re- )sequencing of the full genome of 8. velezensis GB03 using the bacteria and source DNA available from Choon-Min Ryu, one of the authors of Choi et al. (Genome Announc. 2014 Sep-Oct; 2(5): e01092- 14). The reference genome of the strain B. velezensis GB03 as disclosed by GenBank Accession Number CP049904.1 and the re-sequenced genome were confirmed to be identical.

[0301] The largest difference between VMC 10 / 119 and B. velezensis GB03 is represented by Scaffold 02 (SEQ ID NO: 2) of the strain VMC 10 / 119, which did not find any similarity in the available genome sequence of the strain 8. velezensis GB03 deposited under the GenBank Accession Number CP049904.1.

[0302] Scaffold 02 has a length of 215.5 Kb and carries 300 coding sequences, of which 33 were annotated as genes coding for protein with specific function.

[0303] Since the genome comparative analysis highlighted the potential correlation of Scaffold 02 with a plasmid element, two PCR protocols were developed aimed to prove the circularity structure of Scaffold 02 and to confirm the physical presence of genes carried by Scaffold 02 within the strain VMC 10 / 119. The application of both PCR protocols to the strain VMC 10 / 119 and 8. velezensis B-41580Tproduce positive results only for the strain VMC 10 / 119 confirming that: the genes carried by Scaffold 02 are physically present only in the strain VMC 10 / 119 compared to the type strain B. velezensis B- 41580T(Figure 1A); and Scaffold 02 of VMC 10 / 119 is characterized by a circular structure (Figure 1 B).

[0304] In addition, the two PCR products obtained for the strain VMC 10 / 199 were sequenced through Sanger platform and their nucleotide sequence was compared with that obtained for Scaffold 02. This analysis confirmed that the two PCR products originated from Scaffold 02 of the strain VCM 10 / 119, since they shared with Scaffold 02 an identity of 100%.

[0305] Multiple nucleotide differences between Scaffold 01 (SEQ ID NO: 3) of the strain VMC 10 / 119 and the available genome sequence of the strain B. velezensis GB03 deposited under the GenBank Accession Number CP049904.1 were found.

[0306] The whole genome sequence analysis performed on the strain VMC 10 / 119 revealed the presence of 9 gene clusters involved in non-ribosomal and ribosomal synthesis of secondary metabolites with putative biocontrol action promoting the growth of plants. In addition, the genome sequence of the strain VMC 10 / 119 carries genes for the synthesis of volatile organic compounds (VOCs) and encoding for extracellular enzymes that are able to hydrolyse polysaccharides, proteins, and other compounds available in the rhizosphere, which improve the interaction with plants. Regarding biofertilization properties, the results obtained from the genome sequence analysis of the strain VMC 10 / 119 support its capability to form biofilm and spores. The assessment of the toxigenic potential for the strain VMC 10 / 119 did not highlight the presence of any complete gene cluster involved in the synthesis of toxins, confirming the safety of the strain VMC 10 / 119.

[0307] Other remarkable differences are related to other three genetic loci, for which the strain VMC 10 / 119 shows deletions compared to the strain GB03, namely:

[0308] - one deleted region in the locus VMC10 / 119_03441 (Scaffold 01), namely gsiB, coding for the gene glucose starvation-inducible protein, compared to the homologous region in the genome of B. velezensis GB03 (Accession Number: CP049904.1 , from nucleotide 465206 to 465577);

[0309] - two deleted regions within the locus VMC10 / 119_03156 (Scaffold 01), coding for a hypothetical protein 1 , compared to the homologous region in the genome of B. velezensis GB03 (Accession Number: CP049904.1 , from nucleotide 735288 to 742211 , wherein the two deleted regions are comprised respectively from 737013 to 739019 and from 740913 to 741039);

[0310] - one deleted region within the locus VMC10 / 119_03154 (Scaffold 01), coding for putative translation initiation factor 2, compared to the homologous region in the genome of B. velezensis GB03 (Accession Number: CP049904.1 , from nucleotide 742393-744897).

[0311] The 16S rRNA gene sequence of the strain VMC 10 / 119 was determined by direct sequencing of PCR-amplified 16S rDNA and resulted in SEQ ID NO: 1. For the amplification primers consisting of SEQ ID NOs: 7 and 8 were used.

[0312] MALDI-TOF mass spectrometry bacterial identification

[0313] To better identify and characterize the strain, MALDI-TOF MS was used. Indeed, in recent years Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. In this case, intact cells were tested.

[0314] The results of mass spectrum from the MALDI Biotyper 3.1 software (Bruker Daltonik GmbH, Bremen, Germany) were compared with those in the database and confirmed the belonging to Bacillus velezensis species with a score>2.3 (meaning reliable species identification).

[0315] Figures 2 and 3 show MALDI-TOF MS mass spectra of VMC 10 / 119 in two-dimensional peak distribution diagram and in a gel view format, respectively. Each peak (Figure 2) or bar (Figure 3 in the graphs represents a different protein expressed by the microorganism. Peaks height (Figure 2) and the grayscale intensity of the bars (Figure 3) represent the concentration of the proteins within the microbial cell. Since proteins are a direct expression of genome and genome of each strains is unique, thus the proteomic profile of the strain is unique and can be used as fingerprint. The most and unique representative signal for the strain is in the range from 2 to about 12 KDa using spectrum m / z 103.

[0316] Figure 4 shows MALDI-TOF MS mass spectra of VMC 10 / 119 (bottom, spectra / rows 1-36) and GB03 (VMC 10 / 10-136) (top, spectra / rows 37-71) in a gel view format. ClinProTools for bacterial comparison

[0317] MALDI-TOF MS can discriminate at both the genus and species levels using proteomics-based identification and the taxonomic resolution can reach the subspecies and strain levels when the method is combined with specific tools, such as ClinProTools. This software was used to identify potential biomarkers able to discriminate VMC 10 / 119 and the most related species (according to the results above), Bacillus velezensis GB03, also referred to herein as GB03 or VMC 10 / 136.

[0318] In all three experiments, four peaks (6154, 6903, 7526, and 3075 Da) revealed P<0.000001 in the Wilcoxon or Kruskal-Wallis test, thus indicating that they were informative and discriminative peaks in the two classes (VMC 10 / 119 and GB03). All the four peaks are present in both strains but display a different expression of intensity (Figure 5 and 6). High expression levels of m / z 6154, 6903, 7526, and 3075 indicated the fingerprinting of GB03 (VMC 10 / 136); on the contrary low level of the same biomarkers indicated the fingerprinting of VMC 10 / 119 (Figure 5, 6, and 7). Based on these results, the four selected peaks can be considered good biomarkers to discriminate the two strains.

[0319] Fourier transform infrared spectroscopy (FT-IR) biotyper

[0320] Fourier transform infrared (FT-IR) spectroscopy is a label-free and highly sensitive technique that provides complete information on the chemical composition of biological samples. The bacterial FT-IR signals are extremely specific and highly reproducible fingerprint-like patterns, making FT-IR an efficient tool for differentiating very closely related groups of microorganisms (Yang et al., 2020). Bacteria have specific cell-wall composition that can produce a fingerprint-like pattern on the infrared signals. The whole bacterial FT-IR spectrum is composed of a complex of peaks broad generated by different biomolecules in the cell. In detail, the FT-IR fingerprint-like pattern corresponds to a spectrum splitting into five subranges for bacterial typing and differentiation: the membrane amphiphile region (3000-2800 cm'1), the biological infrared-silent region (2800-1800 cm'1), the protein and peptide region (1500-800 cm'1), the protein and fatty acid region (1500-1200 cm'1), and the nucleic acid and polysaccharide region (1200-900 cm'1). The region corresponding to the wavenumbers from 1300-800 cm'1is recommended by many researchers for bacteria typing and differentiation on sub-species or strain level.

[0321] VMC 10 / 119 and B. velezensis GB03 (VMC 10 / 136) were analyzed by the IR Biotyper (Bruker Daltonics GmbH) to see if any difference, able to distinguish the two strains, was present in the FT-IR spectra.

[0322] The second derivative transformation is a mathematical tool that can resolve the broad bands overlapping raw spectra, reduce replicate variability, correct baseline shift, and amplify spectral variations. The second derivative spectra are most commonly used for bacterial classification, typing and identification. Figure 8 shows the second derivative of the spectra in the polysaccharide region (1300-800 cm'1) for VMC 10 / 119 and VMC 10 / 136. The two FT-IR spectra differ in two regions: from 1020 to 980 cm'1and from 940 to 880 cm'1.

[0323] The FT-IR spectra of VMC 10 / 119 and VMC 10 / 136) in the polysaccharide region were analyzed and compared to each other by a multivariate statistical approach to identify the differences among them. The comparison between two spectra results in a spectral distance value: - the better two spectra match, the smaller the spectral distance;

[0324] - two spectra with a spectral distance of 0 are entirely identical;

[0325] - the higher the difference between the two spectra, the higher the spectral distance is.

[0326] The visualization of the spectral differences is realized by a Dendrogram Tree (Figure 9) and Principal Component Analysis (PCA; Figure 10). Both graphical visualizations show that the two strains cluster separately. More specifically, they differ by the region 1300-800 cm-1(wavenumber), meaning that they do not share the same polysaccharide profile.

[0327] Fun icidal activity

[0328] The antagonistic activity of the strain VMC 10 / 119 and comparative strains Bacillus velezensis GB03 and Bacillus subtilis QST 713 was tested by dual cultures assays using the fungal plant pathogens: Rhizoctonia solani, Fusarium culmorum, and Botrytis cinerea. SERENADE® MAX, Bayer CropScience S.r.l. (based on B. subtilis strain QST713) and COMPANION® Liquid Microbial Inoculant (based on B. velezensis GB03), Growth Products, Inc., White Plains, New York, USA, were included as controls.

[0329] COMPANION® MAXX Liquid Biological Fungicide, Growth Products, Inc., White Plains, New York, USA (currently continued by DPH Biologicals, Princeton, Illinois, USA) (based on B. velezensis GB03) were included as controls. COMPANION® MAXX Liquid Biological Fungicide may be indicated to comprise Bacillus amyloliquefaciens strain ENV503 as its active ingredient. Bacillus amyloliquefaciens strain ENV503 is genetically identical to Bacillus subtilis strain GB03 according to the US Environmental Protection Agency as indicated in its ruling disclosed in document 83 FR 58506 retrievable from the US

[0330] SERENADE® is a biological fungicide based on B. subtilis QST 713 which interferes with the development of fungal pathogens, competes for nutritional resources, and induces a plant resistance mechanism to the aggression of the pathogens themselves.

[0331] Results are summarized in Table 1 and show a stronger inhibition activity of VMC 10 / 119 against fungal growth compared to the controls and comparative examples.

[0332] Table 1 : Growth inhibition of selected fungi in the presence of the strain according to the invention and controls

[0333] Control: Untreated control, Example 1 : VMC 10 / 119 CELLS - TSB (tryptic soy broth), Example 2: VMC 10 / 119 CELLS - NYB (nutrient yeast extract broth), Example 3: VMC 10 / 119 CELLS - SMC1 (skim milk medium), Comparative Example 1 : Serenade®, Comparative Example 2: Companion®.

[0334] Nematicidal activity

[0335] Soybean seeds were treated and sown in a pot infested with Pratylenchus sp. Plants are grown for 39 days. Soil from roots of each pot was removed, and the roots rinsed and placed on a wet paper towel. Separate top and roots, blot dry and scan assessing root volume in cm3, root length (cm) and surface root area (cm2) - see Table 2.

[0336] Table 2:

[0337] Seeds were treated and sown in a pot infested with H. glycines. Plants are grown for 13 days. Roots are carefully washed, bleached in NACIOH and are stained in acid fuchsin and stored in glycerin.

[0338] On a plate the penetrated nematodes are counted under the microscope, only juveniles J2 were counted, see Table 3.

[0339] Table 3: Table 4 -Accession number and genomic coordinates of the gene sequences included in the MLST scheme described by Dunlap (2019) and used for the reconstruction of the phylogenetic tree. For the strain B. velezensis VMC 10 / 119 and B. velezensis GB03 the coordinates reported refer to the complete gene sequence of the six genes, while for the other strains refer to the specific fragment used for the generation of the concatenated sequence.

[0340]

[0341]

[0342]

[0343] Embodiments

[0344] Particular embodiments of the present invention are described in the following paragraphs.

[0345] Embodiment 1 . A novel Bacillus strain, progeny, or derivative thereof, wherein the novel Bacillus strain, progeny, or derivative is capable of increased fungicidal activity against at least one fungi selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia, particularly when compared to Bacillus velezensis strain GB03 comprising the genomic sequence of GenBank accession no. CP049904.1 , and wherein the progeny or derivative has at least the same fungicidal activity against at least one fungi selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia when compared to the novel Bacillus strain.

[0346] Embodiment 2. A novel Bacillus strain, progeny, or derivative thereof of, wherein the novel Bacillus strain is deposited under deposit accession number DSM 33551 .

[0347] Embodiment 3. The novel Bacillus strain, progeny, or derivative thereof of embodiment 1 or 2, wherein the novel Bacillus strain is a Bacillus velezensis strain.

[0348] Embodiment 4. The novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 3, wherein the novel Bacillus strain comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 1 .

[0349] Embodiment 5. The novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 4, wherein the novel Bacillus strain comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2.

[0350] Embodiment 6. The novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 5, wherein the novel Bacillus strain comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3.

[0351] Embodiment 7. The novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 6, wherein the novel Bacillus strain comprises: a. a nucleic acid sequence having at least 84% sequence identity to SEQ ID NO: 4; and / or b. a nucleic acid sequence having at least 56% sequence identity to SEQ ID NO: 5; and / or c. a nucleic acid sequence having at least 67% sequence identity to SEQ ID NO: 6.

[0352] Embodiment 8. The novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 7, wherein the novel Bacillus strain comprises: a. a protein A having a mass of 3075 Da, wherein the mass peak of the protein A is at least 50% reduced in a sample of the novel Bacillus strain when compared to the mass peak of the protein A comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS); and / or b. a protein B having a mass of 6154 Da, wherein the mass peak of the protein B is at least 50% reduced in a sample of the novel Bacillus strain when compared to the mass peak of the protein B comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS; and / or c. a protein C having a mass of 6904 Da, wherein the mass peak of the protein C is at least 50% reduced in a sample of the novel Bacillus strain when compared to the mass peak of the protein C comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS; and / or d. a protein D having a mass of 7526 Da, wherein the mass peak of the protein D is at least 50% reduced in a sample of the novel Bacillus strain when compared to the mass peak of the protein D comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS.

[0353] Embodiment 9. The novel Bacillus strain, progeny, derivative thereof according to any one of embodiments 1 to 8, wherein the novel Bacillus strain comprises at least one substantially different second derivative of a spectrum in the ranges of from 1020 cm-1to 980 cm-1and of from 940 cm-1to 880 cm'1in a sample of the Bacillus strain when compared to a second derivative of a spectrum in the ranges of from 1020 cm'1to 980 cm'1and of from 940 cm'1to 880 cm'1in a comparable sample of Bacillus velezensis strain GB03, as determined by Fourier transform infrared spectroscopy (FT-IR), wherein the at least one substantially different second derivatives of the sample and the comparable sample are at the same respective wavenumbers.

[0354] Embodiment 10. An agricultural composition comprising: the novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 9; and an agriculturally acceptable carrier and / or adjuvant.

[0355] Embodiment 11 . An agricultural foliar or soil inoculant comprising: the novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 9; and optionally an agriculturally acceptable carrier and / or adjuvant. Embodiment 12. A coated plant propagation material, wherein the coating comprises the novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 9, the composition according to embodiment 10, or the inoculant according to embodiment 11 .

[0356] Embodiment 13. A method of controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, the novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 9, the composition according to embodiment 10, or the inoculant according to embodiment 11 .

[0357] Embodiment 14. A method of controlling or preventing nematode damage on a plant, or on propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, the novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 9, the composition according to embodiment 10, or the inoculant according to embodiment 11.

[0358] Embodiment 15. The method according to embodiment 13, wherein the phytopathogenic fungi are selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia.

[0359] Embodiment 16. The method according to any one of embodiments 13 to 15, wherein the plant is selected from: cereals, beet, fruit, leguminous crops, oil crops, cucurbits, fibre plants, citrus fruit, vegetables, Lauraceae, tobacco, nuts, coffee, eggplants, sugarcane, tea, pepper, grapevines, hops, the plantain family, latex plants, and ornamental and / or vegetable crops.

[0360] Embodiment 17. The method according to any one of embodiments 13 to 16, wherein the plant is selected from maize and soya (Glycine max). Embodiment 18. The method according to any one of embodiments 13 to 17, comprising applying the Bacillus strain, progeny, or derivative thereof, the composition, or the inoculant in a fungicidally and / or nematicidally effective amount.

[0361] Embodiment 19. The method according to any one of embodiments 13 to 18, comprising applying the Bacillus strain, progeny, or derivative thereof, the composition, or the inoculant to the plant at a rate of from 1 e+3 to 9.1 e+13 cfu / ha, preferably of from 3.3e+3 to 4.55e+13 cfu / ha, especially of from 3e+7 to 5e+11 cfu / ha.

[0362] Embodiment 20. The method according to any one of embodiments 13 to 1618, comprising applying the Bacillus strain, progeny, or derivative thereof, or the composition, to seed at a rate of from 2.2e+2 to 2.73e+13 cfu / 100kg seeds, preferably of from 2e+6 to 3e+11 cfu / 100 kg seeds, especially of from 2e+6 to 1 e+11 cfu / 100kg seeds.

[0363] Embodiment 1921. The method according to any one of embodiments 13 to 18, comprising applying the Bacillus strain, progeny, or derivative thereof, the composition, or the inoculant to the locus of the plant at a rate of from 4.4e+3 to 9.1 e+13 cfu / ha, preferably of from 3e+7 to 1 e+12 cfu / ha, especially of from 3e+7 to 5e+11 cfu / ha.

[0364] Embodiment 22. Use of the novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 9, or the composition according to embodiment 10, or the inoculant according to embodiment 11 in agriculture, or as a fungicide and / or nematicide.

[0365] Embodiment 24. Use of the novel Bacillus strain, progeny, or derivative thereof according to any one of embodiments 1 to 9, in the manufacture of a composition for the control of phytopathogenic fungi and / or for controlling or preventing damage to a plant by a nematode.

Claims

Claims1. A novel Bacillus strain, progeny, or derivative thereof, wherein the novel Bacillus strain, progeny, or derivative is capable of increased fungicidal activity against at least one fungi selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia when compared to Bacillus velezensis strain GB03 comprising the genomic sequence of GenBank accession no. CP049904.1 , and wherein the progeny or derivative has at least the same fungicidal activity against at least one fungi selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia when compared to the novel Bacillus strain.

2. A novel Bacillus strain, progeny, or derivative thereof according to claim 1 , wherein the novel Bacillus strain is deposited under deposit accession number DSM 33551 .

3. The novel Bacillus strain, progeny, or derivative thereof according to claim 1 or 2, wherein the novel Bacillus strain is a Bacillus velezensis strain.

4. The novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 3, wherein the novel Bacillus strain comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 1.

5. The novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 4, wherein the novel Bacillus strain comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2.

6. The novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 5, wherein the novel Bacillus strain comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 3.

7. The novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 6, wherein the novel Bacillus strain comprises: a. a nucleic acid sequence having at least 84% sequence identity to SEQ ID NO: 4; and / or b. a nucleic acid sequence having at least 56% sequence identity to SEQ ID NO: 5; and / or c. a nucleic acid sequence having at least 71 % sequence identity to SEQ ID NO: 6.

8. The novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 7, wherein the novel Bacillus strain comprises: a. a protein A having a mass of 3075 Da, wherein the mass peak of the protein A is at least 50% reduced in a sample of the novel Bacillus strain when compared to the mass peak of the protein A comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS); and / or b. a protein B having a mass of 6154 Da, wherein the mass peak of the protein B is at least 50% reduced in a sample of the novel Bacillus strain when compared to the masspeak of the protein B comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS; and / or c. a protein C having a mass of 6904 Da, wherein the mass peak of the protein C is at least 50% reduced in a sample of the novel Bacillus strain when compared to the mass peak of the protein C comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS; and / or d. a protein D having a mass of 7526 Da, wherein the mass peak of the protein D is at least 50% reduced in a sample of the novel Bacillus strain when compared to the mass peak of the protein D comprised in a comparable sample of Bacillus velezensis strain GB03, as determined by MALDI-TOF MS.

9. The novel Bacillus strain, progeny, derivative thereof according to any one of claims 1 to 8, wherein the novel Bacillus strain comprises at least one substantially different second derivative of a spectrum in the ranges of from 1020 cm-1to 980 cm-1and of from 940 cm-1to 880 cm-1in a sample of the Bacillus strain when compared to a second derivative of a spectrum in the ranges of from 1020 cm-1to 980 cm-1and of from 940 cm-1to 880 cm-1in a comparable sample of Bacillus velezensis strain GB03, as determined by Fourier transform infrared spectroscopy (FT-IR), wherein the at least one substantially different second derivatives of the sample and the comparable sample are at the same respective wavenumbers.

10. An agricultural composition comprising: the novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 9; and an agriculturally acceptable carrier and / or adjuvant.11 . An agricultural foliar or soil inoculant comprising: the novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 9; and optionally an agriculturally acceptable carrier and / or adjuvant.

12. A coated plant propagation material, wherein the coating comprises the novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 9, the composition according to claim 10, or the inoculant according to claim 11.

13. A method of controlling or preventing phytopathogenic fungi on a plant, locus thereof, or on propagation material thereof, the method comprising applying to the plant, locus thereof, or propagation material thereof, the novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 9, the composition according to claim 10, or the inoculant according to claim 11 .

14. The method according to claim 13, wherein the phytopathogenic fungi are selected from the group consisting of: Botrytis, Fusarium, and Rhizoctonia.

15. The method according to claim 13 or 14, wherein the plant is selected from: cereals, beet, fruit, leguminous crops, oil crops, cucurbits, fibre plants, citrus fruit, vegetables, Lauraceae, tobacco,nuts, coffee, eggplants, sugarcane, tea, pepper, grapevines, hops, the plantain family, latex plants, and ornamental and / or vegetable crops.

16. The method according to any one of claims 13 to 15, comprising applying the Bacillus strain, progeny, or derivative thereof, the composition, or the inoculant in a fungicidally effective amount.

17. The method according to any one of claims 13 to 16, comprising applying the Bacillus strain, progeny, or derivative thereof, the composition, or the inoculant at a rate of from 1 e+3 to 9.1 e+13 cfu / ha, preferably of from 3.3e+3 to 4.55e+13 cfu / ha, especially of from 3e+7 to 5e+11 cfu / ha .

18. The method according to any one of claims 13 to 16, comprising applying the Bacillus strain, progeny, or derivative thereof, the composition, or the inoculant to seed at a rate of from 2.2e+2 to 2.73e+13 cfu / 100kg seeds, preferably of from 2e+6 to 3e+11 cfu / 100 kg seeds, especially of from 2e+6 to 1 e+11 cfu / 100kg seeds .

19. Use of the novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 9, the composition according to claim 10, or the inoculant according to claim 11 in agriculture or as a fungicide.

20. Use of the novel Bacillus strain, progeny, or derivative thereof according to any one of claims 1 to 9, or the inoculant according to claim 11 in the manufacture of a composition for the control of phytopathogenic fungi.

Images