Cells of bacillus species and mutants thereof

Abstract

The present invention relates to mutant cells of Bacillus species, preferably mutant cells derived from Bacillus amylolyquefaciens, Bacillus subtilus, or Bacillus velezensis most preferably from Bacillus velezensis strain UMAF6639. Further aspects of the invention relate to the use of the Bacillus mutant cells for producing a fermentate. According to yet a further aspect the invention relates to a composition comprising a fermentate of Bacillus mutant cells according to the invention, preferably comprising viable cells of said mutant cells.

Description

[0001] CELLS OF BACILLUS SPECIES AND MUTANTS THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to Bacillus velezensis strain UMAF6639 and mutant cells derived therefrom. The invention furthermore relates to specific mutants derived from Bacillus velezensis strain UMAF6639 and mutants of other Bacillus species having similar mutations. Other aspects of the invention relate to uses of the cells of the invention, in particular for crop protection, and compositions, such as fermentates comprising the cells of the invention.

[0004] BACKGROUND

[0005] A major challenge for agriculture relies on the ability to produce food to feed a growing population but limiting the negative impact to the environment. Climate change, limitation of natural resources and the constant threat of crops by diverse abnormalities, biotic or abiotic, are motivating the development and implementation of agricultural practices capable of meeting production needs and sustainability [1]. Thus, effective alternatives are needed to face the abuse of chemicals in agriculture and its unfavorable ecological consequences besides the developing of treatment-resistant strains among plant pathogens [2]. Biological control agents (BCAs), naturally antagonistic to phytopathogens, are proposed as feasible and safe candidates to be implemented in more responsible agricultural programs, given their versatile modes of action and low environmental impact [3]. Bacillus spp. are promising BCAs, able to secrete a variety of bioactive compounds and to colonize plant tissues due to their ability to form communities known as biofilms [4]. The extracellular matrix (ECM) that embeds bacterial cells within the biofilm, has been recently demonstrated to mediate the beneficial interaction of Bacillus with other microorganisms or plants [5,6].A representative member of Bacillus BCAs is B. velezensis UMAF6639, which exhibits an exceptional ability to control plant diseases caused by pathogenic fungi (e.g., Podosphaera xanthii [7]), bacteria (e.g., Pectobacterium carotovorum [8]) or plant parasitic nematodes (e.g., Meloidogyne incognita [9]). This wide antagonistic activity is mostly associated to the production of a variety of secondary metabolites with diversified functioning mechanisms [7,10]. Beyond their antagonistic potential against phytopathogens, this biologically based small secondary metabolite factory showcases an impressive repertoire of capabilities that includes priming plant immune responses to enhance the plant resilience against pathogens, and synthesizing crucial plant hormones that foster further plant growth

[0011] . This multifaceted metabolic capability makes these strains promising candidates for harnessing their biotechnological potential in agricultural and environmental applications.

[0006] SUMMARY OF THE INVENTION

[0007] The invention according to a first aspect relates to Bacillus velezensis strain UMAF6639 as deposited at CECT with the accession number CECT 8237, compositions comprising this strain and the use of this strain for producing a mutant cell according to the invention. Cells from this strain are a valuable (intermediate) starting material for producing a mutant cell according to preferred embodiments of the invention, as is described below and in the experiments. Apart from utility in producing a mutant cell according to the invention, this strain, similar to the mutant cells of the invention, can be used in crop protection.

[0008] A further aspect of the invention is a mutant cell of a Bacillus species, preferably from Bacillus velezensis, Bacillus subtilis or Bacillus amylolyquefaciens, more preferably from Bacillus velezensis, most preferably from Bacillus velezensis strain UMAF6639, containing a first autologous gene coding for a protein comprising an amino acid sequence having at least 70%, such as at east 80%, at least 90% or at least 95% sequence similarity with the amino acid sequence presented in SEQ ID NO: 4. Said mutant cell comprises a number of mutations interfering with the formation of a fullyfunctional protein product of the first autologous gene, preferably by interfering with the translation of the gene to a fully functional gene product. The B. velezensis UMAF6639 mutant UV_EMS_26 is a specific embodiment of this aspect of the invention. The present invention also relates to different embodiments of a Bacillus mutant cell that shares a number of similar mutations with UMAF6639 mutant UV_EMS_26.

[0009] According to a further aspect the invention relates to the use of Bacillus cells of the invention, including mutant cells, preferably Bacillus velezensis, Bacillus subtilis or Bacillus amylolyquefaciens cells, more preferably Bacillus velezensis cells, for producing a fermentate. According to yet a further aspect the invention relates to a composition comprising a fermentate of Bacillus cells according to the invention, preferably comprising viable cells of said cells.

[0010] Yet a further aspect relates to the use of Bacillus cells according to the invention for crop protection, preferably crop protection against a fungal pathogen. The invention also relates to a method of producing a Bacillus mutant cell according to the invention.

[0011] OVERVIEW OF SEQUENCES SEQ ID NO: 1 presents the genomic DNA sequence of Bacillus velezensis strain UMAF6639.

[0012] SEQ ID NO: 2 presents the genomic DNA sequence of Bacillus velezensis mutant strain UV_EMS_26 derived from UMAF6639.

[0013] SEQ ID NO: 3 presents the DNA sequence of the UMAF6639 gene with ID:

[0014] U3P88_RS18425.

[0015] SEQ ID NO: 4 presents the amino acid sequence of the protein product of UMAF6639 gene with ID: U3P88_RS 18425.

[0016] SEQ ID NO: 5 presents the DNA sequence of the UMAF6639 gene with ID:

[0017] U3P88_RS01405.

[0018] SEQ ID NO: 6 presents the amino acid sequence of the protein product of UMAF6639 gene with ID: U3P88_RS01405.

[0019] SEQ ID NO: 7 presents the DNA sequence of primer srfAAFw.

[0020] SEQ ID NO: 8 presents the DNA sequence of primer srfAARv.DETAILED DESCRIPTION

[0021] Isolation of Bacillus velezensis strain UMAF6639 has been described by Romero et al. (2004). As described in this publication in the procedures leading to isolation of UMAF6639 bacteria potential antagonistic against plant pathogenic fungi and oomycetes were isolated from healthy leaf areas of cucurbit plants affected mainly by powdery mildew and rhizospheric soil samples from commercial or experimental plantations located in Malaga (Spain). For bacterial isolation, samples were processed according to protocols described previously by Cazorla et al. (1998). In the procedure isolated bacterial strains were routinely grown on nutrient agar (NA) at 28°C for 24 h. Bacterial isolates showing a broad spectrum of antifungal activity on PDA plate assays were identified according to biochemical and physiological tests. The tests carried out for preliminary characterization included Gram staining, endospore formation, nitrate reductase, and oxidase and catalase activities. Further characterization was performed using analytical profile index (API) 20E and API 20NE test strips. Further details on the isolation and characterization of UMA6639 are presented in the Materials and Methods of Romero et al. (2004) with the Results section presenting details of the results of the applied procedures. In the initial identification of the UMA6639 isolate at species level the results from API test strips was compared with the keys for the Bacillus genus published in Bergeys Manual of Determinative Bacteriology (Holt et al. 1994). UMA6639 showed liquefaction of gelatine, was P-glucosidase positive, capable of utilizing citrate as the sole carbon source and growing at pH 5.7 and 6.8. It was further negative for urease, E-arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase reactions, and was not capable of producing H2S. These characteristics are similar to those of Bacillus subtilis. Homology DNA studies carried out with the sequences corresponding to 16S-rDNA also showed that it was closely related to B. subtilis (93-97% homology). On the basis of these results, UMAF6639 was initially characterized as Bacillus subtilis. Eater it was characterized as Bacillus amyloliquefaciens and based on its deposit at CECT redesignated CECT 8237 (Magno-Perez-Bryan (2015)). Following the reclassification of certain Bacillus amyloliquefaciens subspecies (see e.g. Dunlap et al. (2016)) to Bacillus velezensis also CECT 8237 / UMAF6639 was further subjected to taxonomic characterization. Based on comparative genomic analysis (using 16S, rpoB (Vos et al. 2012), gyrA (Liu et al. 2022) as molecular markers and the B. velezens is- specific Bvel primer set described byDunlap (see Dunlap (2019))) CECT 8237 / UMAF6639 was shown to be genetically more related to Bacillus velezensis and thus was classified as such. In line with this the CECT8237 deposit was updated. It will be clear that earlier publications referring to CECT 8237 / UMAF6639 may still characterize this strain as Bacillus amyloliquefaciens or Bacillus subtilis. Although isolation of UMAF6639 and results of its activity as biocontrol agent (see e.g. Romero et al. (2004), Romero et al. (2007a), Romero et al. (2007b), Zeriouh et al. 2011, Garcia- Gutierrez et al. (2012)) have previously been disclosed in written form in various scientific publications, biomass of this proprietary strain so far has not been made publicly available.

[0022] The excellent performance of B. velezensis UMAF6639 and other non-domesticated Bacillus strains as BCAs however contrast with the difficulties to do large scale production of active cells at the required concentration for their biotechnological application. The inventors of the present invention conceived a random mutagenesis strategy to generate a derived strain of B. velezensis UMAF6639 optimized for large scale production but preserving the innate abilities for its biotechnological use as a safe alternative to the conventional agriculture. Their findings indicate that a selected mutant strain (UV_EMS_26) not only exhibits enhanced antimicrobial activity in planta, but also demonstrates superior characteristics for biotechnological applications.

[0023] In a random mutagenesis procedure, as detailed in the experiments described further in this description of the invention, the inventors have obtained mutant cells of Bacillus velezensis UMAF6639. The cells of this mutant strain obtained, designated UV_EMS_26, were genetically and physiologically characterized. The DNA sequence of the full genome of cells of the Bacillus velezensis parent strain UMAF6639 is presented in SEQ ID NO: 1. This sequence is also published in the NCBI databases with the ID: NZ_CP140297.1 (https: / / www.ncbi.nlm.nih.gOv / nuccore / NZ_CP140297.l). The mutations of UV_EMS_26 mutant cells relative to this parent genome are summarized in table 1 below. The DNA sequence of the full genome of the UV_EMS_26 mutant cells is presented in SEQ ID NO: 2. In table 1, the chromosome position of a certain mutation MID1-MID99 refers to the chromosome position on the parent strain UMAF6639 genomic sequence as presented in SEQ ID NO: 1 and the reference sequence connected to a certain mutation is the sequence of the parent strain UMAF6639, whereas the alternate sequence is the sequence of the cells of theUV_EMS_26 mutant. The Bacillus velezensis parent strain UMAF6639 has been deposited at the International Depository Authority of Coleccidn Espanola de Cultivos Tipo (CECT) by Universidad de Malaga, Avenida Cervantes, 2, 29071, Malaga, Spain, via its Departamento de Microbiologia, Vicerrectorado de Transferencia, Empresa y Transformacidn Digital, Parque Technoldgico de Andalucia, Calle Severe Ochoa, 29590, Campanillas, Malaga, Spain. This deposit received a deposit date of 28 November 2012 and the accession number CECT 8237. On 9 January 2025 this deposit was converted to a deposit under the Budapest treaty. Reference to Bacillus velezensis strain UMAF6639 is thus synonymous to Bacillus velezensis strain CECT 8237 and these terms are interchangeable for all aspects and embodiments of the present invention.

[0024] The phenotypic characterization of the UV_EMS_26 mutant strain has revealed that it shows accumulation of antimicrobial compounds. Connected to this is an improved antimicrobial activity, in particular an antifungal activity. Thus, the UV_EMS_26 strain will have benefits when used as a biocontrol agent, preferably an antifungal biocontrol agent, more preferably against plant infecting fungi such as Botrytis cinerea and similar fungi. The UV_EMS_26 mutant strain also shows a reduced foam formation. As foaming is a major problem in Bacillus industrial fermentations, Bacillus cells having a reduced foam formation have significant technological benefits. This benefit is not only relevant for fermentations of Bacillus species executed to produce biocontrol agents, but also for fermentations of Bacillus species executed for other purposes.

[0025] Based on the analysis of the UV_EMS_26 mutant, as described in the experimental section, the inventors of the present invention have inferred that the mutation MID93 presented in table 1 (located around chromosome position 3789419 of the parent chromosome (SEQ ID NO: 1)) can be linked to a mutation in a gene (gene ID:

[0026] U3P88_RS 18425) orthologous to IcfB in Bacillus subtilis sp. 168, encoding a protein responsible for the degradation of long-chain fatty acids, located at this position and that the observed improvement in antimicrobial activity of the UV_EMS_26 mutant can be linked to a disrupted functionality of this gene. The DNA sequence of this gene (gene ID: U3P88_RS 18425) is presented in SEQ ID NO: 3. The sequence of its protein product is presented in SEQ ID NO: 4. It may be expected that interference with the functionality of a similar gene in cells of other Bacillus velezensis strains or of other

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[0076] Bacillus species, such as Bacillus subtilis, Bacillus amylolyquefaciens, Bacillus siamensis, Bacillus licheniformis or Bacillus pumilus will result in similar effects as observed for the UV_EMS_26 mutant.

[0077] Based on the analysis of the UV_EMS_26 mutant, as described in the experimental section, the inventors of the present invention have also inferred that the mutation MID 10 presented in table 1 (located at chromosome position 323657 of the parent genomic sequence (SEQ ID NO: 1)) can be linked to a mutation in a gene (gene ID: U3P88_RS01405) encoding the extracellular matrix and biofilm formation transcriptional regulator RemA located at this position and that the observed reduced foaming characteristics of the UV_EMS_26 mutant can be linked to the altered functionality of the product of this gene. The DNA sequence of this gene (gene ID: U3P88_RS01405) is presented in SEQ ID NO: 5. The sequence of its protein product is presented in SEQ ID NO: 6. It may be expected that alteration of the functionality of the product of a similar gene in cells of other Bacillus velezensis strains or of other Bacillus species, such as Bacillus subtilis, Bacillus amylolyquefaciens, Bacillus siamensis, Bacillus licheniformis or Bacillus pumilus will result in similar effects as observed for the UV_EMS_26 mutant.

[0078] The invention according to an aspect relates to a mutant cell of a Bacillus species, and a bacterial culture thereof, containing a first autologous gene coding for a protein having at least 70%, such as at east 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97%, sequence similarity with the amino acid sequence presented in SEQ ID NO: 4, said mutant cell comprising a number of mutations interfering with the formation of a fully functional protein product of the first autologous gene. The term “a number of’ should be understood to mean “one or more”, or alternatively “at least one”, including one or a plurality.

[0079] The mutant cell of the invention is from a Bacillus species. The Bacillus species may be Bacillus velezensis, Bacillus subtilis, Bacillus amylolyquefaciens, Bacillus siamensis, Bacillus licheniformis or Bacillus pumilus. Preferably the Bacillus species is Bacillus velezensis, Bacillus subtilis or Bacillus amylolyquefaciens, more preferably from Bacillus velezensis, most preferably Bacillus velezensis strain UMAF6639. The cells ofthe Bacillus species from which the mutant cell is derived contain a gene, in particular an autologous gene, coding for a protein comprising an amino acid sequence having at least 70% sequence similarity with the amino acid sequence presented in SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 4 is the protein product coded by the gene of SEQ ID NO: 3. This gene is the Bacillus velezensis UMAF6639 gene (gene ID: U3P88_RS 18425) orthologous to IcfB in Bacillus subtilis sp. 168, for which a disrupted translation is linked to the observed improvement in antimicrobial activity of the UV_EMS_26 mutant.

[0080] The skilled person will understand that a mutant cell has a genetic alteration relative to its parent cell(s). It should be understood that although the present invention relates to individual mutant cells, microbial cells usually are obtained in plurality. Thus, whenever the reference to a singular cell is used, the inclusion of such a singular cell in a plurality of cells is also envisaged. Thus, the singular term “cell” is interchangeable with the plural term “cells” and vice versa, whenever used in the present invention, unless the specific context of its use would explicitly or implicitly indicate differently.

[0081] As the skilled person will understand, “sequence similarity” refers to the extent to which individual nucleotide or derived peptide sequences are alike. The extent of similarity between two sequences is based on the extent of identity combined with the extent of conservative changes. The percentage of "sequence similarity" is the percentage of amino acids or nucleotides which is either identical or conservatively changed viz. "sequence similarity" = (% sequence identity) + (% conservative changes). For the purpose of this invention "conservative changes" and "identity" are considered to be species of the broader term "similarity". Thus whenever, the term sequence "similarity" is used it embraces sequence "identity" and "conservative changes".

[0082] According to certain embodiments the conservative changes are disregarded, and the % sequence similarity refers to % sequence identity.

[0083] The term "sequence identity" is known to the skilled person. In order to determine the degree of sequence identity shared by two amino acid sequences or by two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). Such alignment may becarried out over the full lengths of the sequences being compared. Alternatively, the alignment may be carried out over a shorter comparison length, for example over about 20, about 50, about 100 or more nucleic acids / bases or amino acids.

[0084] The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The degree of identity shared between sequences is typically expressed in terms of percentage identity between the two sequences and is a function of the number of identical positions shared by identical residues in the sequences (i.e., % identity = number of identical residues at corresponding positions / total number of positions x 100). Preferably, the two sequences being compared are of the same or substantially the same length.

[0085] The percentage of "conservative changes" may be determined similar to the percentage of sequence identity. However, in this case changes at a specific location of an amino acid or nucleotide sequence that are likely to preserve the functional properties of the original residue are scored as if no change occurred.

[0086] For amino acid sequences the relevant functional properties are the physico- chemical properties of the amino acids. A conservative substitution for an amino acid in a polypeptide of the invention may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity and hydrophilicity) can be substituted for another amino acid without substantially altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity (see, e.g., Watson, et al., Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p.

[0087] 224 -4th Edition 1987). For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain anegative charge; Ser for Thr and vice versa so that a free -OH is maintained; and Gin for Asn and vice versa to maintain a free -NH2. Exemplary conservative substitutions in the amino acid sequences referred to in connection to this invention can be made in accordance with those set forth below.

[0088] Exemplary Conservative Amino Acid Substitutions

[0089] Original residue Conservative substitution

[0090] Ala (A) Gly; Ser

[0091] Arg (R) Lys, His

[0092] Asn (N) Gin; His

[0093] Asp (D) Glu; Asn

[0094] Cys (C) Ser; Ala

[0095] Gin (Q) Asn

[0096] Glu (E) Asp; Gin

[0097] Gly (G) Ala

[0098] His (H) Asn; Gin

[0099] He (I) Leu; Vai

[0100] Leu (L) He; Vai

[0101] Lys (K) Arg; His

[0102] Met (M) Leu; lie; Tyr

[0103] Phe (F) Tyr; Met; Leu

[0104] Pro (P) Ala

[0105] Ser (S) Thr

[0106] Thr (T) Ser

[0107] Trp (W) Tyr; Phe

[0108] Tyr (Y) Trp; Phe

[0109] Vai (V) lie; Leu

[0110]

[0111] For nucleotide sequences the relevant functional property is mainly the biological information that a certain nucleotide carries within the open reading frame of the sequence in relation to the transcription and / or translation machinery. It is common knowledge that the genetic code has degeneracy (or redundancy) and that multiple codons may carry the same information in respect of the amino acid for which theycode. For example, in certain species the amino acid leucine is coded by UUA, UUG, CUU, CUC, CUA, CUG codons (or TTA, TTG, CTT, CTC, CTA, CTG for DNA), and the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU, AGC (or TCA, TCG, TCC, TCT, AGT, AGC for DNA). Nucleotide changes that do not alter the translated information are considered conservative changes.

[0112] The skilled person will be aware of the fact that several different computer programs, using different mathematical algorithms, are available to determine the identity between two sequences. For instance, use can be made of a computer program employing the Needleman and Wunsch algorithm (Needleman and Wunsch, Journal of Molecular Biolology, 48, 443-453). According to an embodiment the computer program is the GAP program in the Accelrys GCG software package (Accelrys Inc., San Diego U.S. A). Substitution matrices that may be used are for example a BLOSUM 62 matrix or a PAM250 matrix, with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

[0113] According to an embodiment the percent identity between two nucleotide sequences is determined using the GAP program in the Accelrys GCG software package (Accelrys Inc., San Diego U.S. A) A NWSgapdna CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 is used.

[0114] In another embodiment, the percent identity of two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Meyers and Miller, 1989, Bull. Math. Biol. 51, 5-37)..

[0115] For the present invention it is most preferred to use BLAST (Basic Local Alignment Tool) to determine the percentage identity and / or similarity between nucleotide or amino acid sequences.

[0116] Queries using the BLASTn, BLASTp, BLASTx, tBLASTn and tBLASTx programs of Altschul et al. (1990) may be posted via the online versions of BLAST accessible via http: / / www. ncbi.nlm.nih.gov. Alternatively, a standalone version of BLAST downloadable also via the NCBI internet site may be used. Preferably BLAST queriesare performed with the following parameters. To determine the percentage identity and / or similarity between amino acid sequences the preferred settings are: algorithm: blastp; word size: 3; scoring matrix: BLOSUM62; gap costs: Existence: 11, Extension: 1; compositional adjustments: conditional compositional score matrix adjustment; filter: off; mask: off. To determine the percentage identity and / or similarity between nucleotide sequences the preferred settings are: algorithm: blastn; word size: 11; max matches in query range: 0; match / mismatch scores: 2, -3; gap costs: Existence: 5, Extension: 2; filter: low complexity regions; mask: mask for lookup table only.

[0117] The percentage of "conservative changes" may be determined similar to the percentage of sequence identity with the aid of the indicated algorithms and computer programs. Some computer programs, e.g., BLASTp, present the number / percentage of positives (= similarity) and the number / percentage of identity. The percentage of conservative changes may be derived therefrom by subtracting the percentage of identity from the percentage of positives / similarity (percentage conservative changes = percentage similarity - percentage identity).

[0118] At least 70% sequence similarity may be at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97% sequence similarity and includes 100% sequence similarity. Sequence similarity according to preferred embodiments is sequence identity. Thus at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity or at least 97% sequence identity and 100% sequence identity is included according to the invention.

[0119] The mutant cell comprises a number of mutations interfering with the formation of a fully functional protein product from the gene coding for a protein comprising an amino acid sequence having at least 70%, such as at east 80%, at least 90%, at least 95% or at least 97% sequence similarity with the amino acid sequence presented in SEQ ID NO: 4.

[0120] The skilled person will understand how mutations can interfere with the formation of a fully functional protein product from a gene. For example, the mutation may be a mutation in a regulatory sequence, interfering with the transcription of the gene.

[0121] Alternatively, the mutation may be a mutation in the coding sequence of the geneinterfering with the transcription to a fully functional protein product, such as an insertion or deletion of a number of nucleotides and / or introduction of an additional interfere with the formation of the fully functional protein product of the first autologous gene codon in the sequence of the first autologous gene. Preferably the number of mutations comprises a frameshift mutation by incorporation or removal, preferably incorporation, of a number of nucleotide in the sequence of the mutated gene. As the skilled person will understand, insertion or deletion of a single nucleotide may be sufficient to introduce a frameshift mutation. The skilled person will further understand, that insertion or deletion of a higher number of nucleotides may also interfere with the formation of a functional protein product either by creating an untranslatable RNA transcript and / or by translating into an unfunctional protein product. When the parent strain of the mutant cell is Bacillus velezensis strain UMAF6639, as is preferred, the number of mutations according to certain preferable embodiments comprise a conversion of the sequence GAAAAAAGA (reference) to GAAAAAAAGA (mutant alternate) starting at position 3789419 (MID93) of the genomic sequence (SEQ ID NO: 1) of Bacillus velezensis strain UMAF6639. The skilled person will understand that introduction of an additional (premature) stop codon upstream of the native stop codon, will also result in interference with the formation of the fully functional protein product. The skilled person will know that the codons TAA, TGA and TAG in most organisms are designated as stop codons and that TAA for many bacterial species is the prevalent stop codon. According to preferred embodiments, interference with the formation of the fully functional protein product of the first autologous gene is disruption of the formation of the fully functional protein product of the first autologous gene. According to alterative preferred embodiments interreference with the formation of the fully functional protein product of the first autologous gene is blocking of the formation of the fully functional protein product of the first autologous gene. The skilled person will also understand that a nonstop mutation or stop-loss mutation, wherein the native stop codon is mutated such that it loses its stop function may also interfere with the formation of the fully functional protein product of the first autologous gene.

[0122] The mutant cell according to preferred embodiments further comprises a second autologous gene coding for a protein having an amino acid sequence having at least 70%, such as at east 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97%, sequence similarity with the amino acid sequence as presentedin SEQ ID NO: 6, wherein said mutant cell comprises a number of mutations interfering with the formation of a fully functional protein product of the second autologous gene, preferably by producing a full length protein product having an altered functionality. The amino acid sequence of SEQ ID NO: 6 is coded by the Bacillus velezensis UMAF6639 gene U3P88_RS01405. This gene encodes the extracellular matrix and biofilm formation transcriptional regulator RemA. The mutation of this gene is linked to the observed reduction in foam formation of the UV_EMS_26 mutant. The number of mutations interfering with the formation of a fully functional protein product of the second autologous gene, preferably by interfering with translation of the gene to a fully functional gene product, thus preferably result in an altered activity of transcriptional regulator RemA. When the parent strain is Bacillus velezensis strain UMAF6639, or a similar strain wherein the second autologous gene codes for a protein having at least 95%, most preferably at least 97%, sequence similarity with the amino acid sequence as presented in SEQ ID NO: 6 and having an Ala residue at position 74, as is preferred, the number of mutations preferably result in a number of amino acid substitutions between positions 72-76 on the protein sequence of SEQ ID NO: 6, that result in an altered activity of transcriptional regulator RemA . Preferably the number of amino acid substitutions including an Ala to Vai substitution at position 74. More preferably the number of amino acid substitutions is an Ala to Vai substitution at position 74 as a single amino acid substitution. Such an Ala to Vai substitution may be achieved by a C to T substitution on position 323657 (MID10) of the genomic sequence (SEQ ID NO: 1) of Bacillus velezensis strain UMAF6639.

[0123] According to preferred embodiments of the invention, the parent strain of the mutant cell is Bacillus velezensis strain UMAF6639 and said mutant, apart from the mutation MID93 in gene U3P88_RS 18425 of SEQ ID NO: 3 comprises a number of further mutations selected from MID1-MID92 and / or MID94-MID99 presented in table 1 above. These preferred embodiments specifically include the embodiment wherein the mutant cell comprises all mutations MID1-MID99 presented in table 1 above. Such a mutant cell comprises all mutations MID1-MID99 of the Bacillus velezensis UV_EMS_26 mutant.

[0124] The invention alternatively relates to a mutant cell of a Bacillus species, such as from Bacillus velezensis, Bacillus subtilis, Bacillus amylolyquefaciens, Bacillus siamensis,Bacillus licheniformis or Bacillus pumilus, preferably from Bacillus velezensis, Bacillus subtilis or Bacillus amylolyquefaciens, more preferably Bacillus velezensis, most preferably Bacillus velezensis strain UMAF6639, containing an autologous gene coding for a protein comprising an amino acid sequence having at least 70%, such as at east 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97% sequence similarity with a gene as presented in SEQ ID NO: 6, wherein said mutant cell comprises a number of mutations interfering with the formation of a fully functional protein product of the autologous gene, preferably by producing a full length protein product having an altered functionality. Preferably the parent strain of the mutant of this alternative embodiment is Bacillus velezensis strain UMAF6639, or a similar strain wherein the autologous gene codes for a protein having at least 95%, most preferably at least 97%, sequence similarity with the amino acid sequence as presented in SEQ ID NO: 6 and having an Ala residue at position 74, and the number of mutations result in a number of amino acid substitutions between positions 72-76 on the protein sequence of SEQ ID NO: 6, preferably including an Ala to Vai substitution at position 74, such as, when the parent strain is UMAF6639, by a C to T substitution at position 323657 (MID 10) of the UMAF6639 genomic sequence of SEQ ID NO: 1. Preferably, the mutant cell according to this alternative comprises a further autologous gene coding for a protein comprising an amino acid sequence having at least 70%, such as at east 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97% sequence similarity with a gene as presented in SEQ ID NO: 4, wherein said mutant cell comprises a number of mutations interfering with the formation of a fully functional protein product of the further autologous gene, preferably by interfering with the translation of the furtherautologous gene to a fully functional gene product. The number of mutations preferably interfere with the formation of the fully functional protein product of the further autologous gene by a deletion of part of the gene and / or a frameshift mutation in the further autologous gene, preferably a frameshift mutation by incorporation of a number of additional nucleotides in the sequence of the further autologous gene, wherein the further autologous gene most preferably is selected from a gene translating to the same protein product as the sequence of SEQ ID NO: 3, such as a gene having the sequence of SEQ ID NO: 3. When the parent strain is Bacillus velezensis strain UMAF6639 the number of mutations preferably comprise a conversion of the sequence GAAAAAAGA to GAAAAAAAGA at position 3789419 (MID93) of the UMAF6639 genomic sequence (SEQ ID NO: 1). For this alternative embodimentthe features of the further autologous gene may also be varied similar to the features of the first autologous gene described above.

[0125] When the parent strain for this alternative is Bacillus velezensis strain UMAF6639, the mutation in the gene coding for the protein comprising the amino acid sequence having similarity with the sequence of SEQ ID NO: 6, preferably is MID 10 of table 1. It is preferred that this mutant cell apart from the mutation MID10 in gene U3P88_RS01405 (SEQ ID NO: 5) coding for the amino acid sequence of SEQ ID NO: 6 comprises a number of further mutations selected from MID1-MID9 and / or MIDI 1-MID99 presented in table 1. These preferred embodiments specifically include the embodiment wherein the mutant cell comprises all mutations MID1-MID99 presented in table 1. Such a mutant cell comprises all mutations MID1-MID99 of the Bacillus velezensis UV_EMS_26 mutant.

[0126] A further aspect of the invention relates to the use of mutant cells according to the invention for producing a fermentate. The skilled person will understand that a fermentate is a fermentation broth obtained by fermentation with cells of a microorganism. Fermentates of different Bacillus species and their strains, fermentates are relevant products containing Bacillus biomass (cells) and often excreted metabolites. For example biocontrol compositions are prepared from fermentates of Bacillis species suitable as biocontrol agent, such as Bacillus velezensis. A fermentate of a Bacillus mutant cell according to the invention, preferably comprising viable mutant cells is a further aspect of the invention. Liquid fermentation cultures may be obtained in analogy with the procedures disclosed in the experimental section below or alternatively as disclosed by Romero et al. (2007a) or Romero et al. (2007b).

[0127] Yet a further aspect of the invention relates to the use of a Bacillus mutant cell according to the invention, in particular a bacterial culture of such cells, more particularly a biologically pure bacterial culture, for crop protection, in particular crop protection against a plant pathogen and / or plant parasite such as (i) a plant pathogenic fungus, such as selected from the genus Bipolaris, including Bipolaris may dis. from the genus Bolrylis. including Botrytis cine re a. from the genus Cercospora. including Cercospora kikuchii, Cercospora longipes, Cercospora zea-maydis, from the genus Cladosporium, including Cladosporium cladosporiodes, from the genusColletotrichum, including Colletotrichum falcatum, from the genus Corynespora, including Corynespora cassiicola, from the genus Erisiphe, including Erisiphe necator, from the genus Fusarium, including Fusarium oxysporum or Fusarium graminearium, from the genus Globodera, from the genus Eeveillula, including Eeveillula taurica, from the genus Meloidogyne, from the genus Mycosphaerella, including Mycosphaerella fijiensis, from the genus Phaeosphaeria, including Phaeosphaeria maydis, from the genus Phakopsora, including Phakopsora Pachyrhizi, from the genus Phyllosticta, including Phyllosticta citricarpa, from the genus Physopella, including Physopella zeae, from the genus Podosphaera, including Podosphaera fusca or Podosphaera xanthii, from the genus Pratylenchus, from the genus Puccinia, including Puccinia Kuehnii, Puccinia Melanocephala, Puccinia polysora or Puccinia sorghi, from the genus Sclerotinia, including Sclerotinia sclerotiorum, from the genus Septoria, including Septoria glycines, from the genus Sphaerotheca, including Sphaerotheca pannosa, or from the genus Uncinula, including Uncinula necatori, (ii) a plant pathogenic Oomycete, such as selected from the genus Phytophthora including Phytophthora capsici or Phytophthora infestans, or from the genus Plasmopara, including Plasmopara viticola (iii) plant pathogenic bacteria, such as selected from Pectobacterium species, for example P ectobacterium carotovorum, (iv) plant parasitic nematodes, such as a from the genus Meloidogyne, including Meloidogyne incognita, from the genus Globodera, including Globodera rostochiensis, or from the genus Pratylenchus. More preferably the crop protection is aimed against a plant pathogenic fungus. The crop protection preferably is aimed against a plant pathogenic fungus selected from Sordariomycetes, Eurotiomycetes, Dothideomycetes, Pucciniomycetes or Leotiomycetes, more preferably Sordariomycetes, Dothideomycetes, Pucciniomycetes, or Leotiomycetes. These fungal classes contain numerous plant pathogens causing various plant diseases on different hosts plants, including but not limited to powdery mildews, leaf spot diseases, rust diseases, leaf blight, fungal rot diseases. According to certain preferred embodiments, it is in particular preferred that the plant pathogenic fungus is selected from the genus Botrytis, including Botrytis cinerea, from the genus Phyllosticta, including Phyllosticta citricarpa, from the genus Phakopsora, including Phakopsora Pachyrhizi, from the genus Cercospora, including Cercospora kikuchii, Cercospora longipes, Cercospora zea-maydis, from the genus Erisiphe, including Erisiphe necator, from the genus Fusarium, including Fusarium oxysporum or Fusarium graminearium, from the genusPuccinia, including Puccinia Kuehnii, Puccinia Melanocephala, Puccinia polysora or Puccinia sorghi, from the genus Corynespora, including Corynespora cassiicola, from the genus Podosphaera, including Podosphaera fusca or Podosphaera xanthii, The use of Bacillus fermentates in crop protection is known to the skilled person.

[0128] The invention further relates to a method of producing a Bacillus mutant cell according to the invention. Such a mutant cell can be obtained with present day molecular biological techniques. The method comprises the steps of:

[0129] -providing viable cells of a Bacillus species, such as from Bacillus velezensis, Bacillus subtilis, Bacillus amylolyquefaciens, Bacillus siamensis, Bacillus licheniformis or Bacillus pumilus, preferably of Bacillus subtilis, Bacillus velezensis or Bacillus amylolyquefaciens, more preferably of Bacillus velezensis, most preferably Bacillus velezensis strain UMAF6639 deposited at CECT with the accession number CECT 8237;

[0130] -stably introducing a number of mutations interfering with the formation of a fully functional protein product of the gene coding for the protein comprising an amino acid sequence having at least 70% sequence similarity with SEQ ID NO: 4, preferably by interfering with the translation of the gene to a fully functional gene product, wherein when the Bacillus species cells are selected from UMAF6639, the number of mutations more preferably comprise mutation MID93;

[0131] -stably introducing a number of additional mutations , wherein when the cells of the Bacillus species are from Bacillus velezensis strain UMAF6639 the number of mutations preferably comprises a number of mutation resulting in a number of amino acid substitutions between positions 72-76 on the protein sequence of SEQ ID NO: 6, preferably including an Ala to Vai substitution at position 74, more preferably selected from an Ala to Vai substitution at position 74 as a single amino acid substitution, such as by a C to T substitution at position 323657 (MID 10) of the UMAF6639 genomic sequence of SEQ ID NO: 1.

[0132] Mutations that interfere with the formation of a fully functional protein product of the gene coding for the protein comprising an amino acid sequence having at least 70% sequence similarity with SEQ ID NO: 4 have been discussed in the present application.Mutation resulting in a number of amino acid substitutions between positions 72-76 on the protein sequence of SEQ ID NO: 6, preferably including an Ala to Vai substitution at position 74, more preferably selected from an Ala to Vai substitution at position 74 as a single amino acid substitution, such as by a C to T substitution at position 323657 (MID 10) of the UMAF6639 genomic sequence of SEQ ID NO: 1 have also been discussed in the present application. It is within the ambit of the knowledge of the skilled person to stably introduce such mutations in the genome of parent cells of a Bacillus species to obtain a mutant cell of the invention.

[0133] The skilled person will understand that it is possible to introduce multiple mutations, including up to all 99 mutations of mutant UV_EMS_26 as shown in table 1, in a Bacillius parent strain, such as a Bacillus velezensis strain, in particular UMAF6639. Thus a mutant cell having all mutations of mutant UV_EMS_26 can be artificially obtained starting from the genomic basis (SEQ ID NO: 1) of its parent UMAF6639 strain or a related strain. For this for example, single-guide RNAs (sgRNAs) targeting each characterized variant could be designed alongside donor DNA templates containing the desired mutations and homologous sequences to facilitate homology-directed repair (HDR)

[0075] . Alongside this technique, base editors such as cytosine base editors (CBEs) or adenine base editors (ABEs) can be utilized, allowing for specific base substitutions without creating double-strand breaks [76,77]. For modifications such as insertions or deletions, prime editing can also be employed, as it enables precise editing without relying on donor DNA or HDR

[0078] . For introducing a high number of variants, such as all 99 variants of mutant UV_EMS_26 as shown in table 1, a multiplex strategy can be employed, where multiple sgRNAs are introduced in parallel to target several loci simultaneously

[0079] . Such a multiplex process can be optimized to balance editing efficiency and minimize off-target effects, which can be addressed by using high-fidelity variants of Cas9, such as eCas9 or Cas9-HF1

[0080] . For bacterial systems like Bacillus, recombineering using homologous recombination pathways can also be applied to introduce mutations iteratively or in bulk, ensuring that sufficient homology is available around each target site

[0081] . Validation through genome sequencing (e.g., Illumina or PacBio platforms) after each editing cycle can be used to confirm the successful introduction of the desired modifications and to check for absence of off-target effects.According to a further aspect the invention relates to cells of Bacillus velezensis corresponding to strain UMAF6639 as deposited in biologically pure form at CECT with the accession number CECT 8237. Mutant cells derived from strain UMAF6639 comprising one or more mutations have already been discussed above and also form related aspects of the invention. The cells of the UMAF6639 strain most preferably are presented in a bacterial culture, preferably a pure bacterial culture. As the skilled person will understand a pure culture of a bacterial strain is biologically homogeneous by comprising only one type of microorganism, derived from a single colony. The cells of the Bacillus velezensis culture of the invention are in particular in a microbial culture, which similar to the material deposited for CECT 8237, is biologically pure. The cells of the biologically pure culture correspond to cells deposited for and in the deposit CECT 8237, meaning that they have the same biological characteristic, in particular the same genetic information, but may exist outside the physical perimeters of the CECT 8237 deposit. The skilled person will thus understand that the cells of the invention corresponding with strain UMAF6639 may thus have been directly or indirectly derived from the cells in the CECT 8237 deposit, but alternatively may have been derived from a different bacterial culture containing UMAF6639, e.g. the source from which the CECT 8237 deposit has been derived. Cells corresponding with the Bacillus velezensis strain UMAF6639 deposited at CECT with the accession number CECT 8237 can alternatively be expressed as cells from the Bacillus velezensis strain UMAF6639 deposited at CECT with the accession number CECT 8237.

[0134] Biomass, preferably viable biomass, of the Bacillus velezensis strain UMAF6639, or of a mutant derived therefrom, may be included in a composition, particularly in an agronomical composition, more particularly in a biocontrol composition, preferably together with a carrier, more preferably an agronomically acceptable carrier, most preferably an aqueous carrier. The biomass preferably is a biologically pure bacterial culture. The biomass furthermore preferably is in the form of a fermentate of the Bacillus velezensis strain UMAF6639, or of a mutant derived therefrom. Liquid fermentation cultures may be obtained in analogy with the procedures disclosed in the experimental section below or alternatively as disclosed by Romero et al. (2007a) or Romero et al. (2007b). As the skilled person will understand, an agronomical composition is a formulated composition designed and processed for an agricultural purpose. An agronomical composition of the present invention in particular has utilityas a biocontrol composition, in particular a composition for crop protection against a plant pathogen and / or plant parasite. Selection of agronomically acceptable carriers is within the ambit of the skills of the knowledge of the skilled person. For example, water such as in an aqueous solution may be selected as a suitable carrier. Optionally, one or more co-formulants such as one or more preservatives and / or one or more surfactants may further be added to the agronomical composition. The skilled person will understand that for an agronomical composition the water used for an aqueous carrier will normally be purified water, such as filtered, distilled or reverse osmosis water. The skilled person will further understand that for a biological formulation sterilized water is preferred, in more preferably sterilized purified water. The composition can suitably be formed by adjusting a (concentrated) fermentate of UMAF6639, or of a mutant derived therefrom, with water, preferably sterilized purified water, to a preset cell density of between about 1.0*104-5.0*10n, preferably 2.O*1O5-1.O*1O10, more preferably 1.0*107-9.0* 109CFU / ml, most preferably 1.0* 109- 9.0* 109CFU / ml. For use in crop protection, the composition can be used as a foliar application, e.g. by spraying the composition on the above ground parts, in particular the leafs, of a plant, and / or the root system of a plant can be contacted with the composition by drenching in the composition and / or by watering the growth medium of the plant with the composition. Compositions with 1.0*104-9.0*107CFU / ml, in particular 3.0*105-5.0*107CFU / ml are preferred for direct application to plants. Compositions with 9.0*106-9.0*1011CFU / ml, preferably 5.0*107-5.0* IO10CFU / ml, more preferably 1.0*109-9.0*109are suitable as concentrates that may be diluted with water before application to plants. It is common in the art to market Bacillus biocontrol liquid formulations as concentrates that must be diluted prior to application to plants. The prescribed application rate per hectare of such products commonly is presented based on the concentrated undiluted formulation. Calculated from the concentrated form prior to dilution, suitable application rates of the concentrated compositions of the invention for foliar application are between about 0.1-151 / ha, in particular between about 2-101 / ha, more particularly about 4-81 / ha. For certain applications about 0.1-0.51 / ha are suitable. For drenching and / or watering the concentrated formulations are suitably diluted between about 1 / 50-1 / 500, such as about 1 / 100 with water.

[0135] Apart from utility in producing mutant cells according to the invention, the UMAF6639 strain of the invention may also be used as biocontrol agent, in crop protection againstthe same plant pathogen and / or plant parasite from groups (i)-(iv) as described above for the mutant cells of the invention derived from UMAF6639, in particular in crop protection against (i) a plant pathogenic fungus. Thus, the description of the embodiments relating to the use as biocontrol agent of the mutant cells of the invention presented above, equally apply to the use of the UMAF6639 strain of the invention as a biocontrol agent. Embodiments wherein the plant pathogen is a plant pathogenic fungus selected from Sordariomycetes, Eurotiomycetes, Dothideomycetes, Pucciniomycetes or Leotiomycetes, preferably Sordariomycetes, Dothideomycetes, Pucciniomycetes, or Leotiomycetes or is a plant pathogenic Oomycete are also included for the UMAF6639 strain. Such uses of cells of the UMAF6639 strain are a further aspect of the invention. A fermentate of UMAF6639 may be used for biocontrol. Such a fermentate of UMAF6639, preferably a fermentate comprising viable cells of UMAF6639, is therefore a further aspect of the invention.

[0136] As will be clear for the skilled person from the above description, an agronomical composition for crop protection comprising viable biomass of UMAF6639 or of a mutant derived therefrom, most preferably in a biologically pure culture, in combination with an agronomically acceptable carrier, most preferably an aqueous carrier, is a particularly preferred embodiment of the invention. Such a composition in particular has ultility in biocontrol against plant pathogenic fungi or against a plant pathogenic Oomycete, such as from Sordariomycetes, Eurotiomycetes, Dothideomycetes, Pucciniomycetes or Leotiomycetes, preferably Sordariomycetes, Dothideomycetes, Pucciniomycetes, or Leotiomycetes, as previously described. The viable biomass most preferably is a biologically pure culture of UMAF6639.

[0137] Further embodiments of the invention of particular interest are defined in the following clauses:

[0138] 1. A mutant cell of a Bacillus species, such as from Bacillus velezensis, Bacillus subtilis, Bacillus amylolyquefaciens, Bacillus siamensis, Bacillus licheniformis or Bacillus pumilus, preferably from Bacillus subtilis, Bacillus amylolyquefaciens or Bacillus velezensis, more preferably from Bacillus velezensis, most preferably Bacillus velezensis strain UMAF6639 deposited at CECT with the accession number CECT 8237, containing a first autologousgene coding for a protein comprising an amino acid sequence having at least 70%, such as at east 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97% sequence similarity with the amino acid sequence presented in SEQ ID NO: 4, wherein said mutant cell comprises a number of mutations interfering with the formation of a fully functional protein product of the first autologous gene, preferably by interfering with the translation of the first autologous gene to a fully functional gene product.

[0139] The mutant cell according to clause 1, wherein the number of mutations interfere with the formation of the fully functional protein product of the first autologous gene by insertion or deletion of a number of nucleotides in the sequence of the first autologous gene a, introduction of an additional stop codon in the sequence of the first autologous gene, or a nonstop mutation, preferably a frameshift mutation by incorporation of a number of additional nucleotides in the sequence of the first autologous gene, wherein the first autologous gene most preferably is selected from a gene translating to the same protein product as the sequence of SEQ ID NO: 4, such as a gene having the sequence of SEQ ID NO: 3.

[0140] The mutant cell according to clause 2, wherein its parent strain is Bacillus velezensis strain UMAF6639 and the number of mutations comprise a conversion of the sequence GAAAAAAGA to GAAAAAAAGA at position 3789419 (MID93) of the UMAF6639 genomic sequence (SEQ ID NO: 1).

[0141] A mutant cell according to any of the clauses 1-3, further comprising a second autologous gene coding for a protein comprising an amino acid sequence having at least 70%, such as at east 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97% sequence similarity with an amino acid sequence as presented in SEQ ID NO: 6, wherein said mutant cell comprises a number of mutations interfering with the formation of a fully functional protein product of the second autologous gene, preferably by producing a full length protein product having an altered functionality.

[0142] The mutant cell according to clause 4, wherein its parent strain is Bacillus velezensis strain UMAF6639 and wherein the mutation results in an number ofamino acid substitutions between positions 72-76 on the protein sequence of SEQ ID NO: 6, preferably including an Ala to Vai substitution at position 74, such as by a C to T substitution at position 323657 (MID 10) of the UMAF6639 genomic sequence of SEQ ID NO: 1, more preferably selected from an Ala to Vai substitution at position 74 as a single amino acid substitution.

[0143] 6. A mutant cell according to any of the clauses 1-5, wherein its parent strain is Bacillus velezensis strain UMAF6639, said mutant comprising a number of mutations selected from mutations MID1-MID99 presented below, such as all mutations MID1-MID99 presented below:

[0144] Mutation Chromosome Reference Alternate Gene ID ID position

[0145] MIDI 21955 C I I I I I I ATGC C I I I I I I I ATGC U3P88_RS0011

[0146] 0

[0147] MID2 54418 GAAAAAAACCG GAAAAAAAACCG U3P88_RS0025

[0148] 5

[0149] MID3 186978 G A U3P88_RS0084

[0150] 5

[0151] MID4 244099 G A U3P88_RS0103

[0152] 0

[0153] MID5 253428 GAAAAAAGC GAAAAAAAAGC U3P88_RS0109

[0154] 5

[0155] MID6 263141 G l I I I I I I I Al G I G l I I I I I I I I A I G r U3P88_RS0111

[0156] 5

[0157] MID7 265073 GAAAAAAATAAGT GAAAAAAAATAAGT U3P88_RS0116

[0158] 5

[0159] MID8 290720 AGGGGGAGC AGGGGGGAGC U3P88_RS0125

[0160] 0

[0161] MID9 297721 C I I I I I I AC C I I I I I I I AC U3P88_RS0129

[0162] 0

[0163] MID10 323657 C T U3P88_RS0140

[0164] 5 MID11 361614 C T U3P88_RS0160

[0165] 5

[0166] MID12 371056 GAAAAACAA GAAAAAACAA U3P88_RS0165

[0167] 0

[0168] MID13 424464 C T U3P88_RS0193

[0169] 0

[0170] MID14 548493 TAAAAAAGACAAACG TAAAAAAAGACAAACG U3P88_RS0220

[0171] 0

[0172] MID15 549032 A I I I I I I I AGC A I I I I I I I I AGC U3P88_RS0224

[0173] 0

[0174] MID16 555993 GAAAAAGC GAAAAAAGC U3P88_RS0229

[0175] 0

[0176] MIDI 7 571603 G A U3P88_RS0236

[0177] 5

[0178] MID18 576876 G A U3P88_RS0238

[0179] 0

[0180] MID19 590411 G A U3P88_RS0244

[0181] 5

[0182] MID20 651865 G A U3P88_RS0280

[0183] 5

[0184] MID21 755755 A I I I I I I AAAA A I I I I I I I AAAA U3P88_RS0296

[0185] 5

[0186] MID22 825403 C I I I I I I AC C I I I I I I I AC U3P88_RS0334

[0187] 5

[0188] MID23 894721 GAAAAAACG GAAAAAAACG U3P88_RS0376

[0189]

[0190] 0MID24 933974 GCCCCCTG GCCCCCCTG U3P88_RS0403

[0191] 5

[0192] MID25 953105 G A U3P88_RS0415

[0193] 0

[0194] MID26 974779 C I I I I I I I ATTT C l I I I I I I I A I I I U3P88_RS0426

[0195] 0

[0196] MID27 974909 G A U3P88_RS0426

[0197] 0

[0198] MID28 982901 G I I I I I I CG G I I I I I I I CG U3P88_RS0431

[0199] 0

[0200] MID29 1050955 TAAAAAGAT TAAAAAAGAT U3P88_RS0468

[0201] 5

[0202] MID30 1058917 C I I I I I I AT C I I I I I I I AT U3P88_RS0473

[0203] 5 MID31 1086450 G A U3P88_RS0478

[0204] 5

[0205] MID32 1165722 C I I I I I I CCA C I I I I I I I CCA U3P88_RS0503

[0206] 5

[0207] MID33 1194879 G I I I I I I I GCG G I I I I I I I I GCG U3P88_RS0521

[0208] 0

[0209] MID34 1223227 GAAAAAAGC GAAAAAAAGC U3P88_RS0539

[0210] 0

[0211] MID35 1300760 C I I I I I I AA C I I I I I I I AA U3P88_RS0580

[0212] 5

[0213] MID36 1315959 C I I I I I I CTT C I I I I I I I CTT U3P88_RS0592

[0214] 0

[0215] MID37 1317627 TAAAAAAATC TAAAAAAAATC U3P88_RS0591

[0216] 0

[0217] MID38 1350766 G I I I I I I CA G I I I I I I I I CA U3P88_RS0607

[0218] 5

[0219] MID39 1418506 G I I I I I I I CTTCTGG G I I I I I I I I C I I CTGG U3P88_RS0643

[0220] 5

[0221] MID40 1493690 GAAAAAATG GAAAAAAATG U3P88_RS0677

[0222] 5 MID41 1501789 GAAAAAACAG GAAAAAAACAG U3P88_RS0680

[0223] 5

[0224] MID42 1512192 C I I I I I I CTGG C I I I I I I I CTGG U3P88_RS0685

[0225] 0

[0226] MID43 1519544 GAAAAAGAAGAC GAAAAAAGAAGAC U3P88_RS0690

[0227] 0

[0228] MID44 1522776 CAAAAAAACCT CAAAAAAAACCT U3P88_RS0689

[0229] 5

[0230] MID45 1697791 C I I I I I CTTG C I I I I I I CTTG U3P88_RS0780

[0231] 5

[0232] MID46 1824328 CAAAAAACCCGGCCGCCT CAAAAAAACCCGGCCGCCT U3P88_RS0850

[0233] 5

[0234] MID47 2108280 G I I I I I I I CA G I I I I I I I I CA U3P88_RS1001

[0235] 0

[0236] MID48 2150719 GAAAAAAGC GAAAAAAAGC U3P88_RS1020

[0237] 5

[0238] MID49 2244923 C I I I I I I CTTGC C l I I I I I I I C I IGC U3P88_RS1064

[0239] 5

[0240] MID50 2274038 CGG I I I I I I GA CGG I I I I I I I GA U3P88_RS1081

[0241] 0 MID51 2276351 ATTATATAT AATATATAT U3P88_RS1079

[0242] 5

[0243] MID52 2276375 CAAAAAAAGC CAAAAAAAAGC U3P88_RS1079

[0244] 5

[0245] MID53 2278670 GAAAAAAGAACG GAAAAAAAGAACG U3P88_RS1083

[0246] 5

[0247] MID54 2301282 TAAAAAAAGGGCCGCT TAAAAAAAAGGGCCGCT U3P88_RS1094

[0248] 5

[0249] MID55 2350723 C T U3P88_RS1124

[0250] 5

[0251] MID56 2354978 GAAAAAAACAGC GAAAAAAAAACAGC U3P88_RS1125

[0252] 5

[0253] MID57 2385130 CAAAAAAAAAACAAAAAAG CAAAAAAAAACAAAAAAGC U3P88_RS1142

[0254] C 0

[0255] MID58 2463623 GAAAAAACCG GAAAAAAACCG U3P88_RS1184

[0256] 5

[0257] MID59 2524689 C T U3P88_RS1213

[0258] 0

[0259] MID60 2536320 C T U3P88_RS1221

[0260]

[0261] 0MID61 2554028 GAAAAAAGC GAAAAAAAGC U3P88_RS1224

[0262] 0

[0263] MID62 2620111 TCCCCCCAT TCCCCCCCAT U3P88_RS1257

[0264] 5

[0265] MID63 2631739 C I I I I I OTA C I I I I I I CTA U3P88_RS1262

[0266] 5

[0267] MID64 2673611 CAAAAAAACG CAAAAAAAACG U3P88_RS1284

[0268] 5

[0269] MID65 2820184 AGGGGTTT AGGGGGTTT U3P88_RS1367

[0270] 0

[0271] MID66 2879940 C T U3P88_RS1403

[0272] 0

[0273] MID67 2910278 C T U3P88_RS1417

[0274] 0

[0275] MID68 3009889 CGGGGGGAT CGGGGGGGAT U3P88_RS1452

[0276] 5

[0277] MID69 3062344 GAAAAAGC GAAAAAAGC U3P88_RS0133

[0278] 0

[0279] MID70 3076436 C T U3P88_RS1485

[0280] 5 MID71 3093519 GAAAAAAGT GAAAAAAAGT U3P88_RS1491

[0281] 5

[0282] MID72 3134669 GAAAAAGGA GAAAAAAGGA U3P88_RS1500

[0283] 5

[0284] MID73 3139160 CAAAAAAATG CAAAAAAAATG U3P88_RS1504

[0285] 5

[0286] MID74 3170749 TAAAAAAGG TAAAAAAAGG U3P88_RS1521

[0287] 0

[0288] MID75 3210489 GAAAAAAGGG GAAAAAAAGGG U3P88_RS1540

[0289] 5

[0290] MID76 3222026 CAAAGAA CAAAAGAA U3P88_RS1544

[0291] 0

[0292] MID77 3224459 G I I I I I I I CCA G I I I I I I I I CCA U3P88_RS1549

[0293] 5

[0294] MID78 3230395 G A U3P88_RS1553

[0295] 5

[0296] MID79 3234632 GAAAAAACG GAAAAAAACG U3P88_RS1555

[0297] 0

[0298] MID80 3324252 TAAAAAAGC TAAAAAAAGG U3P88_RS1607

[0299] 0 MID81 3411709 TAAAAAGAGG TAAAAAAGAGG U3P88_RS1653

[0300] 0

[0301] MID82 3449492 TAAAAAAAGCAG TAAAAAAAAGCAG U3P88_RS1670

[0302] 0

[0303] MID83 3466318 GAAAAATA GAAAAAATA U3P88_RS1680

[0304] 5

[0305] MID84 3552374 G I I I I I I I GCTG G l I I I I I I IGC IG U3P88_RS1716

[0306] 0

[0307] MID85 3565764 GAAAAAACG GAAAAAAACG U3P88_RS1724

[0308] 0

[0309] MID86 3583264 AGGGGCT AGGGGGGCT U3P88_RS1732

[0310] 0

[0311] MID87 3608213 GAAAAAAATAAC GAAAAAAAATAAC U3P88_RS1743

[0312] 0

[0313] MID88 3614717 GAAAAAATAAATG GAAAAAAAATAAATG U3P88_RS1744

[0314] 0

[0315] MID89 3633943 GCCCCCTCA GCCCCCCTCA U3P88_RS1765

[0316] 0

[0317] MID90 3702507 GAAAAAAGGAAT GAAAAAAAAGGAAT U3P88_RS1797

[0318] 0 MID91 3770864 AGGGGGCCT AGGGGGGCCT U3P88_RS1834

[0319] 5

[0320] MID92 3785078 GAAAAAGT GAAAAAAGT U3P88_RS1838

[0321] 5

[0322] MID93 3789419 GAAAAAAGA GAAAAAAAGA U3P88_RS1842

[0323] 5

[0324] MID94 3809856 C I I I I I I I CG C I I I I I I I I CG U3P88_RS1851

[0325] 0

[0326] MID95 3833258 G A U3P88_RS1861

[0327] 0

[0328] MID96 3903890 CAAAAAAGC GAAAAAAAGC U3P88_RS1900

[0329] 0

[0330] MID97 3967786 AGGGAA AGGGGAA U3P88_RS1941

[0331]

[0332] 0MID98 4018847 C I I I I I I CC C I I I I I I I CC U3P88_RS1973

[0333] 0 MID99 4030406 C I I I I I I CTTT C I I I I I I I CTTT U3P88_RS1982

[0334]

[0335] 0

[0336] 7. A mutant cell according to any of the clauses 1-6 wherein the percentage sequence similarity is percentage sequence identity.

[0337] 8. A mutant cell according to any of the clauses 6-7 comprising a number of mutation selected from MID38, MID7, MID21, MID50 or MID78 as defined in clause 6, preferably all mutations selected from MID38, MID7, MID21, MID50 and MID78.

[0338] 9. Use of mutant cell according to any of the clauses 1-8 for producing a fermentate.

[0339] 10. A fermentate of Bacillus mutant cells according to any of the clauses 1-8, preferably comprising cell material, most preferably viable mutant cells.

[0340] 11. Use of a population of Bacillus mutant cells according to any of the clauses 1-8 as a bio control agent, preferably a biocontrol agent for crop protection, more preferably crop protection against a plant pathogen and / or plant parasite selected from (i) a plant pathogenic fungus, such as selected from the genus Bipolaris, including Bipolaris may dis, from the genus Botrytis, including Botrytis cinerea, from the genus Cercospora, including Cercospora kikuchii, Cercospora longipes, Cercospora zea-maydis, from the genus Cladosporium, including Cladosporium cladosporiodes, from the genus Colletotrichum, including Colletotrichum falcatum, from the genus Corynespora, including Corynespora cassiicola, from the genus Erisiphe, including Erisiphe necator, from the genus Fusarium, including Fusarium oxysporum or Fusarium graminearium, from the genus Globodera, from the genus Eeveillula, including Eeveillula taurica, from the genus Meloidogyne, from the genus Mycosphaerella, including Mycosphaerellafijiensis, from the genus Phaeosphaeria, including Phaeosphaeria maydis, from the genus Phakopsora, including Phakopsora Pachyrhizi, from the genus Phyllosticta, including Phyllosticta citricarpa, from the genus Physopella, including Physopella zeae, from the genus Podosphaera, including Podosphaera fusca or Podosphaeraxanthii, from the genus Pratylenchus, from the genus Puccinia, including Puccinia Kuehnii, Puccinia Melanocephala, Puccinia polysora or Puccinia sorghi, from the genus Sclerotinia, including Sclerotinia sclerotiorum, from the genus Septaria, including Septaria glycines, from the genus Sphaerotheca, including Sphaerotheca pannosa, or from the genus Uncinula, including Uncinula necatori, (ii) a plant pathogenic Oomycete, such as selected from the genus Phytophthora including Phytophthora capsici or Phytophthora infestans, or from the genus Plasmopara, including Plasmopara viticola (iii) plant pathogenic bacteria, such as selected from P ectobacterium species, for example P ectobacterium carotovorum, (iv) plant parasitic nematodes, such as a from the genus Meloidogyne, including Meloidogyne incognita, from the genus Globodera, including Globodera rostochiensis, or from the genus Pratylenchus, wherein most preferably the crop protection is against (i) a plant pathogenic fungus.

[0341] Use according to clause 11 wherein the plant pathogen is a plant pathogenic fungus selected from Sordariomycetes, Eurotiomycetes, Dothideomycetes, Pucciniomycetes or Leotiomycetes, preferably Sordariomycetes, Dothideomycetes, Pucciniomycetes, or Leotiomycetes or is a plant pathogenic Oomycete.

[0342] Method of producing a number of Bacillus mutant cells according to any of the clauses 1-8 comprising:

[0343] -providing viable cells of a Bacillus species, such as from Bacillus velezensis, Bacillus subtilis, Bacillus amylolyquefaciens, Bacillus siamensis, Bacillus licheniformis or Bacillus pumilus, preferably of Bacillus velezensis, Bacillus subtilis, or Bacillus amylolyquefaciens, more preferably from Bacillus velezensis, most preferably Bacillus velezensis strain UMAF6639 deposited at CECT with the accession number CECT 8237;

[0344] -stably introducing a number of mutations interfering with the formation of a fully functional protein product of the gene coding for the protein comprising an amino acid sequence having at least 70%, such as at east 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97%, sequencesimilarity with SEQ ID NO: 4, preferably by interfering with the translation of the gene to a fully functional gene product, wherein when the Bacillus species cells are selected from UMAF6639, the number of mutations more preferably comprise mutation MID93;

[0345] -optionally, stably introducing a number of additional mutations, wherein when the cells of the Bacillus species comprise a gene coding for the protein sequence of SEQ ID NO: 6, such as cells from Bacillus velezensis strain UMAF6639, the number of additional mutations preferably comprises a number of mutation resulting in a number of amino acid substitutions between positions 72-76 on the protein sequence of SEQ ID NO: 6, preferably including an Ala to Vai substitution at position 74, more preferably selected from an Ala to Vai substitution at position 74 as a single amino acid substitution, such as, when the cells of the Bacillus species are selected from strain UMAF6639, by a C to T substitution at position 323657 (MID 10) of the UMAF6639 genomic sequence of SEQ ID NO: 1.

[0346] 14. Method according to clause 13, wherein, when the cells of the Bacillus species are selected from strain UMAF6639, the number of optional additional mutations are selected from MID1-MID92 and / or MID94-MID99 as defined in clause 6, preferably a number of mutations selected from MID 10, MID38, MID7, MID21, MID50 or MID78, preferably a number of mutations comprising mutation MID 10, more preferably all mutations selected from MID 10, MID38, MID7, MID21, MID50 and MID78.

[0347] 15. Method according to clause 13, wherein a number of mutant cells according to clause 2 or clause 3 are produced.

[0348] 16. Bacillus velezensis strain as deposited at CECT with the accession number CECT 8237.

[0349] 17. A composition comprising biomass, preferably viable biomass, of a Bacillus velezensis strain as deposited at CECT with the accession number CECT 8237, preferably together with a carrier, more preferably an agronomically acceptable carrier, most preferably an aqueous carrier.18. Use of cells of the Bacillus velezensis strain UMAF6639 according to clause 16, for producing a mutant cell according to any of the clauses 1-8, preferably in a method according to any of the clauses 13-15.

[0350] EXPERIMENTS

[0351] Materials and methods

[0352] Microorganisms and growth conditions.

[0353] Bacillus velezensis UMAF6639 (CECT8237), isolated from the phylosphere of distinct cucurbit plants

[0012] , was obtained from our laboratory strain collection. Bacterial cultures were grown at 28 °C and 150 rpm (when agitation needed) from frozen stocks in Lysogeny Broth (LB: 5 g / L NaCl, 5 g / L yeast extract, 10 g / L tryptone, 15 g / L agar). For bio film experiments MSgg medium was used, (100 mM pH 7 morpholinepropane sulphonic acid (MOPS), 0.5 % glycerol, 0.5 % glutamate, 5 mM potassium phosphate (pH 7), 50 pg / mL tryptophan, 50 pg / mL phenylalanine, 2 mM MgCh, 700 pM CaCh, 50 pM FeCh, 50 pM MnCh, 2 pM thiamine, 1 pM ZnCh)

[0013] . The necrotrophic fungus Botrytis cinerea Bc05 strain was grown from frozen stocks into potato dextrose agar (PDA) plates and maintained until sporulation of the culture to perform the corresponding experiments.

[0354] Random mutagenesis and candidate screening

[0355] Short wavelength ultraviolet radiation (254 nm) was used for a first round of random mutagenesis of B. velezensis UMAF6639 as previously described elsewhere

[0014] . Overnight-grown bacterial cultures were diluted in LB media and transferred to sterile plates for UV exposure. 1 mL aliquots were taken at different timepoints within a 30 min treatment and were further inoculated onto LB plates to calculate the survival rate of the culture. 15-25 % of survival rate was considered to ensure the occurrence of relevant mutations. For the second round of random mutagenesis ethyl methanesulphonate (EMS) was used over aliquots corresponding to previously UV exposed samples following a previously described procedure

[0015] . For EMS mutagenesis, 2 ml of diluted bacterial suspension was treated with different concentrations of EMS and incubated at 28 °C in an incubator shaker, each for 0, 10, 20, 30 and 40 min. Screening and selection of potential mutants were done based on their ability to produce a bigger inhibition zone corresponding to an increasedantifungal activity. For this purpose, 5 pL of bacterial suspensions corresponding to randomly selected mutants were inoculated onto PDA plates containing a concentration of 300 B. cinerea spores per mL and incubated for 5-7 days at 25 °C. The selected mutants were further screened for higher antifungal activity on the supernatant by studying the minimal inhibitory concentration (MIC) of a cell-free supernatant (CFS) of each bacterial culture. Serial * dilutions of the CFS were inoculated with 100 spores of B. cinerea into 96-well plates, reaching a final volume of 200 pL and incubated at 25 °C for 48 h. Optical density at 600 nm (ODeoo) was measured to determine fungal growth.

[0356] Bioassays on melon leaves

[0357] Bacterial strains were grown in liquid LB at 28 °C overnight. The cells in the cultures were washed twice with sterile distilled water. The bacterial cell suspensions were adjusted to the same ODeoo, and 1 mL was sprayed onto leaves of 4- to 5-week-old melon plants. Once the leaf surface was completely dried, 5 pL of a 105spore / mL suspension of B. cinerea prepared in grape juice diluted to 50 % in water to promote the infection was inoculated twice in each leaf. The plants were placed in a growth chamber at 25 °C with a 16-h photoperiod, 3800 lux, and 85% Relative Humidity (RH). The severity of the symptoms in melon leaves was evaluated by measuring the lesion size using Fiji image software

[0016] analysis and pictures of infected leaves. To evaluate the persistence, sporulation, and colonization of B. velezensis strains on plants, melon seeds (Cucumis melo cv. Rochet Panal, Fito, Barcelona, Spain) were surface sterilized with 1 % bleach for 3 minutes and washed for three times with distilled water. Seeds were placed in Petri plates with wet filter paper and germinated for 5 days at 25 °C with 60 % of RH in dark. After germination, seedlings were transferred to soil and grown for 3 weeks at 25 °C with a 16 h light / 8 h dark photoperiod and 60 % RH. For leaf inoculation, 1 mL of a bacterial suspension in distilled water at a concentration of 108CFU mL'1was spread onto the adaxial axis of second and third leaves. Bacterial persistence and sporulation were evaluated at 0 h, 48 h, and 10 days post inoculation (dpi). Leaves were placed individually into sterile plastic stomacher bags with 10 mL of distilled water and further homogenized for 3 min in a stomacher homogenizer (Interscience, France). 10-fold serial dilutions were prepared from leaf extracts and plated onto LB agar plates incubated at 28 °C for 24 h. Spore count was carried out by incubating the dilutions at 80 °C for 10 min to get rid of vegetative cells, and spores were subsequently plated and incubated as previously described for vegetative cells.Scanning Electron Microscopy (SEM) was used to evaluate the colonization dynamics. For this purpose, leaf disks were taken 10 dpi with a sterile cork borer and fixed in 0.1 M sodium cacodylate and 2 % glutaraldehyde overnight at 4 °C. Three washes were performed with 0.1 M sodium cacodylate and 0.1 M sucrose followed by ethanol dehydration in a series of ethanol solutions from 50 % to 100 %. A final drying with hexamethyldisilazane was performed as indicated

[0017] . The dried samples were coated with a thin layer of iridium using an Emitech K575x turbo sputtering coater before viewing in a Helios Nanolab 650 Scanning Electron Microscope and Focus Ion Beam (SEM-FIB) with a Schottky-type field emission electron gun.

[0358] Phenotypic characterization of selected mutant

[0359] For the in vitro antifungal activity, PDA plates mixed with B. cinerea spores (300 spores / mL) were prepared and inoculated with B. velezensis strains. Plates were grown at 28 °C for 5 days and the antifungal activity was determined by measuring the inhibition zone diameter. Data were normalized by dividing the distance by the bacterial colony diameter. For the biofilm characterization two approaches were taken: i) studying the air-liquid interphase pellicle formation, by inoculating 48-well plates with 500 pL of MSgg medium with 5 pL of a bacterial suspension (ODeoo value previously adjusted to 1) incubating at 28 °C under static conditions for 48 h, and ii) analyzing the colony architecture and EPS production by inoculating MSgg plates with the same bacterial suspension and under the same incubation conditions. In this case, plates were supplemented with Congo Red and Coomassie Brilliant Blue G at final concentrations of 20 pg / mL and 10 pg / mL respectively. For the motility assays, LB plates with 0,3 % and 0,7 % agar (for swimming and swarming motility respectively) were inoculated with 5 pL of a ODeoo = 1 bacterial suspension and incubated at 28 °C for 24 h (swimming) and 48 h (swarming).

[0360] Aggregation assays

[0361] Aggregation assays were carried out as previously described

[0018] . Flasks containing 25 mL of LB medium were inoculated with B. velezensis strains and incubated with agitation at 150 rpm at 28 °C overnight. The cultures were divided into two fractions. The ODeoo values of the 10 mL culture aliquots for each strain were adjusted to 3 using the supernatant obtained from centrifugation of the other fraction. Auto-aggregation was measured using a final point measurement and a kinetic method. The tubes were thenincubated vertically at room temperature without agitation or movement. At each sampling time, 10 pL aliquots were carefully taken from the air-liquid interface of the medium and diluted in 90 p L of LB medium to measure the ODeoo values in a plate reader (FLUOstar Omega reader, BMG LabTech).

[0362] Genome sequencing

[0363] For the DNA isolation, bacterial cultures were grown in LB medium overnight at 28 °C and 150 rpm agitation. 500 pL of culture were transferred to 1.5 mL tube for genomic extraction with the JetFlex™ Genomic Purification Kit (Invitrogen) following the provided instructions. Genomic samples were resuspended in 50 pL of pre-heated Mili-Q water (55 °C) and stored at -20 °C until further use. The purity of extracted DNA was determined using a NanoDrop spectrophotometer by measuring the A260 / A280 and A260 / A230 ratios. DNA integrity and quantity were determined using an Agilent 2200 TapeStation and Qubit 3.0 assay, respectively. Two sets of sequencing runs were made to complete the reference genome, one using the long-read sequencing of Oxford Nanopore Technologies (ONT) and one using the short reads sequencing of Illumina. ONT sequencing libraries were prepared using a PCR-free method of multiplexing samples with the Rapid Barcoding kit (SQK-RBK004, ONT), and sequencing of the barcoded DNA was performed on a single R9.4 / FLO-MIN106 ONT flow cell on the MinlON (version MklB, ONT) for 48 h. Base calling of Nanopore reads was performed using Guppy v6.4.2

[0019] , with — min_qscore 8. Libraries were demultiplexed using guppy_barcoder from Guppy v6.4.2 and adapters were removed using the same software. NanoFilt v2.8.0 from NanoPack

[0020] was used for further quality filtering. Read quality was examined with NanoStat vl.2.0 from NanoPack. We used Flye v2.9.1 assembler

[0021] with Oxford Nanopore reads, and two rounds of polishing using Pilon vl.24

[0022] with Illumina reads to obtain a single, circularized chromosome. For the Illumina library preparation, a ready-to- sequence library was constructed using Nextera XT DNA Library Preparation Kit (Illumina) that was sequenced through a mid-output run with 300 cycles (2 xl50 paired ends) with the Illumina NextSeq 550 platform (Illumina, San Diego, CA, U.S.A. For variant calling the UV_EMS_26 genome was sequenced only with the Illumina platform with the same specifications previously mentioned. Obtained circular reference genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP; V6.7)

[0023] , with the best-placed reference protein set as annotation method. The new version of the genome can befound under the BioProject Accession: PRJNA224116 (BioSample: SAMN38777322; NCBI Reference Sequence: NZ_CP 140297.1).

[0364] Variant calling and annotation

[0365] Prior to variant calling, fastq files containing the raw reads corresponding to UV_EMS_26 Illumina sequencing were trimmed using SeqtrimNext v2.1.3

[0023] to remove low quality and low complexity sequences, adapters, and contaminant sequences. Then processed reads were mapped to the obtained reference genome using “bwa mem” vO.7.5 algorithm

[0024] . The resulting BAM files were indexed with samtools vl.3.1

[0025] . Generated files were used as input for FreeBayes

[0026] variant caller (vl.3.6) where ploidy was set to 1, using default settings for the rest of parameters. Variant filtering was performed with vcflib tools

[0027] , setting the vcffilter flag to discard variants with QU AL values below 20. Finally, variants were annotated with the SNPEff program

[0028] , also predicting the potentially effects of mutations over the genome of UV_EMS_26. Genes presenting variants were classified according to the predicted effect and were used to identify the KEGG Ontology functional categories affected due to random mutagenesis using the function enricher from the clusterProfiler v4.0 software

[0029] .

[0366] Exopolysaccharide purification

[0367] Exopolysaccharides were extracted following procedures previously described with modifications

[0030] . B. velezensis UMAF6639 and UV_EMS_26 were grown by inoculating ImL of liquid MSgg medium in 24- well plates and incubated at 28 °C for 5 days with no agitation. Pellicles in the air-liquid interphase were harvested with a sterile loop and washed with distilled water. After centrifugation at 8000 x g for 10 min, cell pellets were resuspended in PBS buffer and mildly sonicated (3 pulses at 30 % amplitude for 1 minute with 10 seconds of pause between intervals) to separate the EPS from the cells. Then, the mixture was centrifuged, and supernatants were subjected to protein precipitation with Sevag's method

[0031] . Then cold isopropanol was added in a 4:1 ratio with the supernatant volume and incubated at 4 °C overnight for EPS precipitation. EPS crude was collected by centrifugation (10000 x g, 20 min) and the resulting pellets were dialyzed against distilled water overnight (Spectra / Por® dialysis membrane molecular weight cutoff 3.5 kDa) and the resulting solution was filtered and applied onto ionic exchange chromatography column (HiTrap™ DEAE FF 5ml, Cytiva)eluting the EPSs with MiliQ water using an increasing gradient of IM NaCl from 0 % to 100 %. Aiming the sugar presence into the fractions, aliquots of each fraction were analyzed by the phenol- sulphuric acid method and monitored the absorption at 490 nm in a microplate reader (FLUOstar Omega reader, BMG LabTech). Once fractions containing sugars were detected, samples were lyophilized for further analysis. EPSs were again resuspended in distilled water and loaded into a size exclusion chromatography column (Hiprep™ 16 / 60 Sephacryl™ S-300HR) eluting with MQ water and analyzed again with the phenol- sulphuric acid method. Fractions containing carbohydrates were pooled and lyophilized for further analysis.

[0368] Monosaccharide composition analysis (GC-MS)

[0369] To analyze sugar composition in the extracted samples, 1-2 mg of EPS were hydrolyzed in 3 mF reacti-vials (ThermoFisher Scientific) with 600 p L of HCl / MeOH 3 N (Sigma Aldrich) incubated at 80 °C for 24 h. Solvent was evaporated under a nitrogen stream at 50 °C using an evaporator-concentrator (Stuart BlocK Heater, SBH200D / 3). In order to remove excess acid, the residue was washed three times with methanol and then dried again. Samples underwent derivatization through silylation with 300 pL of Tri-Sil reagent (Pierce, ThermoFisher Scientific) for 1 hour at 80 °C. Subsequently, the reagent was removed using a nitrogen stream, reconstituted with 500 pF of hexane, and centrifuged for 15 minutes. Supernatant was filtered, evaporated, and reconstituted in 150 pF of hexane (EC-MS grade, Sigma) in a chromatography vial. For the Gas Chromatography (GC), a Zebron ZB-5 column (30 m x 0,25 mm ID x 0,25 p df, Phenomenex) was used. Injection volume was set to 1 pF in Splitless mode and carrier gas flow rate to 1.2 mE / min with an injector temperature of 250 °C. Initial oven temperature was 80 °C for 2 minutes, followed by a ramp of 5 °C / min up to 155 °C, then a ramp of 0.5 °C / min up to 158 °C, and finally a ramp of 5 °C / min up to 230 °C. For the Mass Spectrometry (MS) analysis an Electronic Impact (El) ionization mode was used at 70 eV. Full scan was set between 50-600 m / z and Single Ion Monitoring (SIM) was set between 204 and 217 m / z. The identification of monosaccharides in polysaccharide samples was carried out by comparing retention times (RTs) and mass spectra with previously analyzed standards of monosaccharides under identical conditions (glucose, galactose, mannose, arabinose, xylose, rhamnose, fucose, and galacturonic acid). The obtained mass spectra were compared with the NIST mass spectra library.Liquid chromatography -tandem mass spectrometry (LC-MS / MS)

[0370] Non-targeted metabolomics was performed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) with an UHPLC coupled to a Q-Exactive HF mass spectrometer as previously described

[0032] . In brief, the UHPLC separation was performed using a C18 core-shell column (Kinetex, 50 x 1 mm, 1.7 pm particle size, 100 A pore size, Phenomenex, Torrance, USA). The mobile phases used were solvent (A) Water (LC / MS grade, Fisher Scientific) + 0.1 % Formic Acid (FA) and solvent (B) ACN (LC / MS grade, Fisher Scientific) + 0.1 % FA. After the sample injection, a linear gradient method of 5 minutes was used for elution of small molecules, the flowrate was set to 150 p L / min (microflow). The following separation conditions were set in the time range 0-4 min from 5 % to 50 % solvent (B) was used, 4— 5 min from 50 % to 99 % B, followed by 2 min washout phase at 99 % B and 3 min re-equilibration phase at 5 % B. The measurements were conducted in positive mode, the Heated Electrospray Ionization (HESI) parameters included a sheath gas flow rate of 30 L / min, auxiliary gas flow rate of 10 L / min, and sweep gas flow rate of 2 L / min. The spray voltage was set to 3.50 kV and the inlet capillary temperature to 250 °C, while the S-lens RF level to 50 V and the auxiliary gas heater temperature to 200 °C. Full MS survey scan acquisition range was set to 120-1,800 m / z with a resolution 45,000, automatic gain control (AGC) of 1E6, maximum injection time of 100 ms with one micro-scan. DDA MS / MS spectra acquisition was performed in data dependent acquisition (DDA) mode with TopN set to 5, as consequence the five most abundant precursor ions of the survey MS scan were destined to MS / MS fragmentation. The resolution of the MS / MS spectra was set to 15,000, the AGC target to 5E5 and the maximum injection time to 50 ms. The quadrupole precursor selection width was set to 1 m / z. Normalized collision energy was applied stepwise at 25, 35, and 45. MS / MS scans were triggered with apex mode within 2-15 s from their first occurrence in a survey scan. Dynamic precursor exclusion was set to 5 s.

[0371] Metabolomic data analysis and MS / MS network analysis

[0372] After LC-MS / MS acquisition, raw spectra were converted to mzML files using MSconvert (ProteoWizard). MSI and MS / MS feature extraction was performed with Mzmine3

[0033] . For MSI spectra, an intensity threshold of 1E5 was used, and for MS / MS spectra, an intensity threshold of 1E3 was used. For MSI chromatogram building, a 10 ppm mass accuracy and a minimum peak intensity of 5E5 was set.Extracted ion chromatograms (XICs) were deconvolved using the baseline cut-off algorithm at an intensity of 1E5. After chromatographic deconvolution, XICs were matched to MS / MS spectra within 0.02 m / z and 0.2-minute RT windows. Isotope peaks were grouped and features from different samples were aligned with 10 ppm mass tolerance and 0.1 -minute RT tolerance. MSI features without MS2 features assigned were filtered out the resulting matrix as well as features that did not contain isotope peaks and that did not occur in at least three samples. After filtering, gaps in the feature matrix were filled with relaxed RT tolerance at 0.2 minute but also 10 ppm mass tolerance. Finally, the feature table was exported as a .csv file, and corresponding MS / MS spectra exported as .mgf files. Contaminate features observed in blank samples were filtered, and only those with a relative abundance ratio blank to average lower than 30 % were considered for further analysis. For feature-based molecular networking and spectrum library matching, the mgf file was uploaded to GNPS

[0034] . For molecular networking, the minimum cosine score was set to 0.7. The precursor ion mass tolerance was set to 0.01 Da, and the fragment ion mass tolerance was set to 0.01 Da. Minimum matched fragment peaks were set to 6, minimum cluster size was set to 1 (MS Cluster off), and library search minimum matched fragment peaks were set to 6. When analog searches were performed, the cosine score threshold was 0.7 and the maximum analog search mass difference was 100 m / z. Molecular networks were visualized with Cytoscape version 3.9.1

[0035] . Mirror plots were done using GNPS and https: / / metabolomics-usi.ucsd.edu / , comparing mzspec of the selected features and the metabolites recorded in MS / MS databases (Fig. S6). Annotations were done according to guidelines in references. Statistical analysis of metabolomic datasets was performed by Metaboanalyst v.5.0 after data filtering by interquartile range (IQR)

[0036] . The automatic workflow for the analysis of UMAF6639 and UV_EMS_26 (MSV000089885) can be accessed at: https: / / massive.ucsd.edu / ProteoSAFe / dataset.jsp?task=3dceab4f74784102ac6eb384124 32014; feature-based molecular networking: https: / / gnps.ucsd.edu / ProteoSAFe / status.jsp?task=694924d8e01c44c8a27c27e4f20ecde 1.

[0373] Surfactin isoforms purification and characterization

[0374] Surfactin isoforms from B. velezensis UMAF6639 were extracted by acid precipitation of 6 E-CFS by adding HC1 6N upon reaching pH 2, followed by a 24 h incubation at 4°C to ensure lipopeptide precipitation. The mixture was centrifuged 20 min at 10.000 rpm to recover the crude lipopeptide extract and incubated in methanol for 5 h. Concentration of the extract was performed at 45 °C and 200 rpm using a rotary evaporator. After evaporation, the crude extract was reconstituted in 20 % Acetonitrile (ACN). A first extraction was performed by Solid Phase Extraction (SPE) in a Strata C18-U column (200 mg, Phenomenex™) previously activated with 1 column volume (CV) of methanol. Columns were pre-conditioned with 1 CV of water and samples were loaded in a 3 mL volume using aqueous solutions of increasing concentration of ACN to wash the columns from impurities. For surfactins elution, ACN concentration was increased up to 75 %- 100 %. Isoform purification was done by semi-preparative HPLC. All analytical methods were performed with an Eclipse plus C18 5 pm, 4.6 x 250 mm (Agilent™) and preparative methods with a Ecliplse XDB C18 5 pm, 9.4 x 250 mm (Agilent ™). Surf actin isoforms were detected by absorbance measurement at 210 nm. The mobile phase consisted in 0.1 % trifluoroacetic acid (TFA) water (A) and ACN (B). The crude extract obtained from SPE was initially separated to enrich the sample in surfactins. An analytical method was developed to determine the RT of Surfactins in the mixture. For this purpose, a gradient strategy was employed using a flow rate of 1 mL / min, with the percentage of ACN increasing at the following time points (min): TO: 100 % A, Tl: 20 % A - 80 % B, T8: 60 % A - 40 % B, T25: 60 % A - 40 % B, T35: 40 % A - 60 % B, T50: 40 % A - 60 % B, T60: 20 % A - 80 % B, T100: 20 % A - 80 % B. For the preparative method, 50 pL of sample were injected successively, and the same conditions were utilized with the flow rate of the mobile phase changed to 2 mL / min and using the same gradient at time points 0 min, 1.5 min, 11.5 min, 35.5 min, 50 min, 71 min, 85 min, and 140 min. To determine RTs corresponding to surfactin isoforms, the purified mixture was subjected to analytical separation under the same aforementioned conditions. Finally, a preparative method was used to purify each isoform for further analysis. To determine the physiological concentration of each isoform, calibration curves were obtained by running different concentrations in analytical HPLC with the same conditions as used with the preparative method, setting the flow rate to 1 mL / min.

[0375] Mass spectrometry

[0376] The dried droplet method was used to prepare the samples for MALDI analysis. Briefly, samples were mixed in an Eppendorf tube at a 1:1 ratio with 2,5-dihydroxybenzoic acid(DHB), a-Cyano-4-hydroxycinnamic acid (a-CHCA) matrix or CHCA_DHB matrix mixture (7:3). Each matrix was prepared at 15 mg mL-1 concentration and dissolved in TA50 (50% [v / v] acetonitrile, 0.1% [v / v] trifluoroacetic acid in distilled water). Then, a 2 p L volume of the sample-matrix mixture was spotted on a stainless steel sample plate and allowed to dry for 10 min at room temperatura. The experiments were conducted using an ultrafleXtreme MALDI-TOF MS instrument (Bruker Daltonics, Germany) equipped with a 337 nm pulsed nitrogen laser and operated in reflectron positive mode with the flexControl software (version 3.4; Bruker Daltonics). The laser power was manually adjusted until obtaining optimum signal-to-noise ratio and each acquired spectrum resulted from the accumulation of a minimum of 3000 laser shots.

[0377] MALDI-TOF MS / MS coupled with LIFT mode in the same spectrometer was used to analyze the fragment ions of the selected precursor ions. Spectra were analysed using the Flex Analysis software (Bruker Daltonics).

[0378] RNA extraction and RT-qPCR

[0379] For RNA extraction, a previously described protocol was followed, albeit with several modifications

[0037] . Bacterial strains were cultivated in LB for 72 h at 150 rpm. Biomass was harvested by centrifugation at 12.000 rpm for 5 min and washed twice with 1 mL of PBS. Cells were disrupted by adding lysozyme (10 mg / mL) and further incubation at 37 °C for 30 min. After disruption, the mixture was centrifuged for 1 min at 16.000 x g and pellets were resuspended in 900 pL TRLReagent (Merck™) previously heated at 60 °C. Total RNA extraction was performed as instructed by the manufacturer. Genomic DNA removal was carried out using TURBO™DNAse (ThermoFisher Scientific™) following the instructions of the manufacturer. The integrity and quality of the total RNA was assessed by gel electrophoresis and with Qubit 3.0 assay.

[0380] Quantitative real-time (RT-qPCR) was performed using the iCycler-iQ system and the iQ SYBR Green Supermix Kit from Bio-Rad. The primer pairs used to amplify the target genes were designed using the Primer3 software (http: / / bioinfo.ut.ee / primer3 / ) and, maintaining the parameters described elsewhere

[0038] . For the RT-qPCR assays, the RNA concentration was adjusted to 100 ng / p L. Next, 1 pg of DNA-free total RNA was retro-transcribed into cDNA using the SuperScript III reverse transcriptase (Invitrogen) and random primers in a final reaction volume of 20 pL according to the instructions provided by the manufacturer. The RT-qPCR cycle was: 95 °C for 3 min, followed by PCR amplification using a 40-cycle amplification program (95 °C for 20 s, 60 °C for30 s, and 72 °C for 30 s), followed by a third step of 95 °C for 30 s. To normalize the data, the rpsJ gene, encoding the 30S ribosomal protein S10, was used as a reference gene

[0039] . The target gene srfAA, encoding Surfactin synthetase A, was amplified using the primer pair srfAAFw (5’-AAGGAAACATCGTCACACAT-3’) and srfAARv (5’-TTTAACAGCGAACCGAACAT-3’). The relative transcript abundance was estimated using the AA Cyclethreshold (Ct) method

[0040] . Transcriptional data was normalized to the rpsJ gene and shown as the fold-changes in the expression levels of the target genes in each B. velezensis strain. The RT-qPCR analyses were performed three times (technical replicates) using three independent RNA isolations (biological replicates).

[0381] Antifungal activity assay

[0382] B. cinerea spores were harvested from sporulated plates with distilled water and filtered through a 0.45 pM pore membrane to avoid mycelial contamination. 96-well plates containing 100 p L of PDB with 100 spores per well were used, with the corresponding isoform concentration to be analyzed for antifungal activity. 10 mM H2O2 was used as positive control inducing fungal death. Plates were incubated for 24 h with agitation (150 rpm) at 25 °C. After incubation, ODeoo values of the inoculated plates was measured in a plate reader (FLUOstar Omega reader, BMG LabTech™) to evaluate fungal growth within the treatments.

[0383] Fermentation conditions

[0384] To perform the first steps in the upscaling of the fermentation of B. velezensis UMAF6639 and UV_EMS_26 strains for the industrial use a 3-L bioreactor (Applikon Bio 3 L, Getinge™) with temperature, pH, stir, Dissolved Oxygen (DO) and foaming control was used. Medium Optimal for Lipopeptide Production (MOLP; 30 g / L peptone, 20 g / L sucrose, 7 g / L yeast extract, 1.9 g / L KH2PO4, 0.001 mg / L CuSO4, 0.005 mg / L FeC13«6H2O, 0.004 mg / L Na2MoO4, 0.002 mg / L KI, 3.6 mg / L MnSO4*H2O, 0.45 g / L MgSO4, 0.14 mg / L ZnSO4*7H2O, 0.01 mg / L H3BO3, and 10 mg / L CeHsO?)

[0041] with slight modifications was used for these experiments. The pH was adjusted to 7 with 35 % NaOH before the sterilization. The working volume was set to 2.0 L, the temperature was 30°C and aeration rate was 0.2 v / v per min, maintaining the fermentation until the culture started to sporulate (48 h). Inoculation was performed with a sterile syringe with 20 mL of an overnight grown culture of each strain in LB liquid medium. Stirring was set in cascade mode between 800 rpm and1500 rpm to be increased when DO was under 20 % to promote gas exchange. To control foam formation, the level sensor was placed 2 cm above the liquid, pumping antifoam into the vessel when detecting a level increase. The reactor was initially adapted with a collection vessel in the air-exhaust line to trap the possible foam overflow. All fermentations were performed as triplicates.

[0385] Results

[0386] The VV_EMS_26 mutant strain is improved in antifungal activity.

[0387] The strain B. velezensis UMAF6639 has been widely described as a powerful biocontrol agent [7,10]. In order to improve large scale fermentation for further commercialization, the strain was consecutively subjected to rounds of random mutagenesis (radiation and chemical mutagenesis). Short wavelength UV radiation treatment was initially chosen for the first round of random mutagenesis. The duration of the treatment was set up when the mortality rate of the culture reached 85% (Figure SI A), ensuring a sufficient occurrence of genomic variations and theoretically, enabling the observation of significant phenotypic changes. Selected mutants were further subjected to a second round of chemical mutagenesis with Ethyl Methanesulphonate (EMS). The dose of the mutagen was stablished when 85% mortality rate was achieved (Figure SIB). Serial dilutions of cell-free supernatants (CFS) from selected mutants were tested for antifungal activity against the fungal plant necrotrophic pathogen Botrytis cinerea. The CFS of the mutant strain UV_EMS_26 inhibited fungal growth even up to four times more efficiently than the wild-type strain (Figure 1A). Negative effect on the growth rate of UV_EMS_26 was disregarded, indicating that vital genetic and physiological traits were not neglected in the consecutive rounds of random mutagenesis strategy (Figure SIC).

[0388] The VV_EMS_26 mutant strain retains essential traits for efficient plant colonization and persistence.

[0389] In vitro tests were initially performed to explore intrinsic traits related to bacterial social behavior and with implications in plant colonization. UV_EMS_26 formed a more relaxed pellicle in the air- liquid interphase and showed a less intricate colony architecture in solid medium, all indicative of variations in the genetic pathways involved in this bacterial developmental program. As biofilm, swarming, a kind of bacterial social motility coordinated by the action of flagella and water tensionreduction inflicted by surfactants [42,43], was also diminished compared to the wild type strain (Figure IB). In accordance with these phenotypes, UV_EMS_26 exhibited substantially reduced cellular auto-aggregation under static conditions, and the inability to form long bacterial chains or aggregates (Figure 1C). Optical density values of samples taken from the interphase at different timepoints confirmed the low selfaggregation of UV_EMS_26 cells compared to UMAF6639 (Figure SID). The antifungal activity exerted by the parental strain UMAF6639 has been associated to efficient plant surface colonization and biofilm formation. Based on the previously presented phenotypes, we hypothesized a loss of fitness of UV_EMS_26 on the phylloplane. To confirm this, cell density, evaluated as CFU counts, was measured and showed no statistically significant differences in comparison to UMAF6639. Similarly, Scanning Electron Microscopy revealed that the colonization pattern was not hindered, as could be expected by the defect in swarming motility displayed by UV_EMS_26 (Figure ID, top). Moreover, the sporulation rate on the same plant surface at different time points was examined, and no differences with the parental strain were recorded (Figure ID, bottom). The antifungal activity of the selected strain in comparison to the parental strain UMAF6639 was also tested in planta against the fungal pathogen B. cinerea, confirming that the enhanced biocontrol activity of the strain was maintained on the leaf surface (Figure IE, IF). Taken together these findings suggest that the differences observed in vitro in the social behavior of UV_EMS_26 are not resting fitness capacity of this strain in the melon phylloplane.

[0390] Mutant genome sequencing and variant analysis reveal mutations in key genes involved in motility, biofilm formation and ECM production.

[0391] To gain insight into the genetic bases affected by the random mutagenesis strategy, the UMAF6639 reference genome was re-sequenced with Illumina (Nextseq550) and Oxford Nanopore Technologies (ONT) to obtain a more accurate reference genome for variant calling. First round of Illumina sequencing yielded 40,552,218 paired reads. After ON sequencing and genome polishing, the obtained circular genome consisted in one single chromosome of 4,036,959 bp. Genome Annotation Summary showed 3,851 CDSs, 86 tRNAs, 27 rRNAs, 5 ncRNAs and 126 pseudogenes. The genome of the mutant strain UV_EMS_26 was sequenced by Illumina (Nextseq550) and rendered 34,221,892 paired reads. Given that random mutagenesis strategy was employed, a highly restrictive quality threshold used for variant calling was set to ensure thepresence of genuine mutations and to eliminate potential artifacts (see Materials and Methods), which identified 99 variants, including 20 SNPs and 79 InDeis (table 1) evenly dispersed throughout the entire genome (Figure 2A, top). All these mutations were subjected to analysis with SnpEff software

[0044] to predict their effect over the genome, highlighting the prediction in majority of upstream and downstream gene variants, potentially impacting the corresponding gene expression levels (Figure 2A, bottom). Among the variants found in UV_EMS_26 genome, KEGG Orthology (KO) term enrichment was performed to obtain an overview of molecular functions potentially altered by mutagenesis. Functional enrichments were performed over three mutation sets previously classified according to the predicted mutation impact over the genome and top 20 most significative results were represented, corresponding to high (Figure 2B), moderate (Figure 2C) or modifier (Figure 2D) impact mutations. Within high impact mutations, genes related to penicillin binding proteins (PBPs) harbored frameshift variants which might lead to the impairment of normal protein function. U3P88_RS06075, U3P88_RS01165 and U3P88_RS02965 genes were found as orthologs of PBPs according to KEGG Orthology database. These proteins are involved in the cell wall peptidoglycan biosynthesis, mainly by catalyzing the canonical 4-3 cross-links in this polymeric structure

[0045] . Considering the defects in motility, it was worth noting the frameshift variant in U3P88_RS10810 gene, which encodes a hypothetical protein that shares a remarkable 66% of query-cover and 100% of sequence identity with FlgE, a well-characterized flagellar basal-body rod protein of Bacillus subtilis sp. 168, known for its pivotal role in flagellum assembly and swarming motility

[0046] . In relation to the accumulation of potentially antifungal compounds, fatty acid metabolism-related gene U3P88_RS 18425, annotated as a degradative long-chain fatty-acid-CoA ligase, showed a frameshift variant also characterized as a high impact mutation. Regarding the moderate impact variants found in the UV_EMS_26 genome, which primarily consist of mis sense variants potentially altering protein function, the U3P88_RS 15535 gene stands out. This gene is annotated as a histidine kinase belonging to the CitB family of two-component system regulators (MalK, DctS, and CitS according to KO terms). The same subset of genes included U3P88_RS01405, which encodes the extracellular matrix and biofilm formation transcriptional regulator RemA. In solution, RemA forms an octamer in which each subunit assembles in a donut- like structure. Depending on protein concentration, these octamers can assemble onto a dimeric superstructure with a positively charged outer surface due to three arginines oneach monomer of the structure. This conformation allows the interaction with DNA through the positively charged outer surface on the lateral side of the octameric ring

[0047] activating the expression of tapA and eps operons, the products of which are main components of the ECM

[0048] . Finally, mutations classified as to produce a modifier impact (non-coding variants or variants affecting non-coding genes), or synonymous variants (no amino acid change) are listed in table 1.

[0392] EPS monosaccharide composition and polymer size vary significantly in the mutant strain

[0393] UV_EMS_26 has been shown to form a more relaxed biofilm compared to the parental strain, and genomic analysis revealed mutations in RemA as the most probable cause for the down-expression of ECM components. Among the structural component of the ECM, EPSs are known to provide with mechanical and biological functions all indispensable for the multifunctionality of the ECM. EPSs from both strains were extracted from the air-liquid interphase pellicle and separated by ionic exchange chromatography (lEx) which rendered 3 fractions (Figure S2A). These fractions were then resolved in size exclusion chromatography (SEC) to analyze the polymer size composition. Only two fractions were recovered from UMAF6639 (Figure S2B), and six fractions were collected from UV_EMS_26 (Figure S2C). Monosaccharide composition corresponding to each polymer size sample was analyzed by hydrolysis and subsequent derivatization prior further separation in gas chromatography and Mass Spectrometry. Glucose and fucose were found to be the major component of UMAF6639 extracts (Figure 3A), which coincided with the first two SEC fractions of UV_EMS_26 (Figure 3B). For the remaining fractions of the mutant, a significant shift in the relative monosaccharide composition was observed in comparison to UMAF6639, with a notable increase in the proportion of galactose, D-arabinose, rhamnose, xylose, galacturonic acid and mannose. These results were consistent with the differential binding of Congo Red in colony staining (Figure 1A) and somehow indicated that, as aforementioned, although B. velezensis UMAF6639 can produce a heteropolysaccharide, the strain UV_EMS_26 can still produce a completely different EPS without any apparent ecological disadvantage (Figure IE).

[0394] A on-targeted metabolomics reveal overaccumulation of antimicrobial compounds in the mutant CFSMetabolites from the CFS of 72 h cultures corresponding to the wild-type and mutant strains were extracted with ethyl acetate and were analyzed by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS) non-targeted metabolomics in search for the metabolites responsible for the enhanced antifungal activity of UV_EMS_26. A total of 73 metabolites were detected to be differentially accumulated in the mutant strain CFS. Among them, we focused on the molecules accumulated with a log2 Fold Change above 2 (Figure 4A). UV_EMS_26 CFS contained differential accumulation of metabolites annotated as Surfactin analogs, as shown by the peak abundance of compounds with ID 4987, 5251 and 5258 (Figure S5A). Considering the absence of mutations in genes related to Surfactin biosynthetic machinery in the mutant strain, the expression levels of srfAA (the first gene of the operon) were studied to determine the presence of alterations in its biosynthesis, thereby justifying the higher accumulation in the supernatant of UV_EMS_26. RT-qPCR revealed no statistically significant differences in the expression of srfAA in UV_EMS_26 (Figure 4B), confirming that the Surfactin biosynthetic machinery was not altered. However, the differential accumulation of Surfactin analogs detected by non-targeted metabolomics led us to confirm whether these annotated metabolites were indeed Surf actins. Considering the variability of isoforms produced by different Bacillus strains and their range of activity, the complete Surfactin isoform profile produced by UMAF6639 was characterized to determine their contribution to the increased antifungal activity observed in UV_EMS_26. Surfactin isoforms were purified with SPE chromatography followed by HPLC separation and were further identified by Tandem Mass Spectrometry (MALDI-TOF-TOF). From CFS extraction, 8 fractions were purified (Figure S6) corresponding to the peaks detected in the HPLC chromatogram. After MS / MS, 5 Surfactin isoforms were identified (Figure 4C). UMAF6639 fractions were identified as C12, C13, C14, C15 and Ci6-Surfactin corresponding to m / z values from MS / MS fragmentation (Figure S7), according to previous characterization

[0049] . Each isoform physiological concentration, listed in Table 1, was calculated based on the calibration curves performed and was used for the treatment of the pathogen B. cinerea to study their antifungal effect, as well as a higher concentration range to determine the potential antagonistic nature of these molecules against the fungal pathogen. Biological assays with the differentially accumulated isoforms showed no reduction of the fungal growth at physiological concentrations. In fact, for Ci6 isoform an increase in the ODeoo values of the fungal cultures was detected.When concentration was increased, C16 isoforms showed a strong antifungal activity, as well as the C12 isoform at the highest concentration tested (Figure 4D). In addition to this, lipidic molecules as Monomyristin (MM) and Stearic Acid analogs (SA) were also found to be overrepresented in the CFS of the mutant strain, as well as Dipalmitoyl cephalin, also known as l,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (Figure S5B), one of the most accumulated metabolites.

[0395] UV_EMS_26 present biotechnological advantages for the first steps of the industrial fermentation

[0396] Given the phenotypical and metabolomic changes observed in UV_EMS_26 which could be potentially interesting biotechnological traits, we aimed at exploring the possible benefits of this strain for industrial fermentation. UMAF6639 or UV_EMS_26 were cultured in 3-L bioreactors with temperature, DO, level, aeration and stirring controlled, as a first step in the upscaling for the industrial fermentations. Bacterial growth and sporulation were measured by CFU counts and were found to be similar in the mutant strain compared to the wild type (Figure 5A). DO levels during the bioprocess were very similar in both strains, revealing that oxygen consumption was occurring approximately at the same rate between UMAF6639 and UV_EMS_26 (Figure 5B). This correlates with the similar growth and sporulation rate observed for both fermentations confirming that random mutagenesis does not imply any modification or generates disadvantage in the normal dynamic of UV_EMS_26. Based on our previous studies, we expected foam formation to be lower in UV_EMS_26. Foaming appears as a major problem in Bacillus industrial fermentations, thus, differences in foam formation were analysed by measuring the accumulative active time of the antifoam pump in response to the detection of an increase in the culture level by the corresponding sensor. Although the antifoam addition dynamics were slightly different between runs, the antifoam pump was active for longer periods of time in all the fermentations corresponding to UMAF6639 (Figure 5C), which is directly associated with higher volumes of antifoam needed to maintain the foam level under control during the whole process when compared to the mutant strain culture.

[0397] Discussion

[0398] The incorporation of biocontrol agents arises as a key strategy to be considered in the implementation of sustainable agriculture. The use of random mutagenesis for theenhancement of a bacterial strain with industrial interest implies the emergence of different phenotypic variations, emphasizing the need to assess not only its effectiveness in biocontrol but also its potential implications for a broader range of biotechnological applications. It is well known that plant growth promoting rhizobacteria (PGPR), as those belonging to the Bacillus genus, must effectively colonize and persist on the plant surfaces to exert their beneficial activities, which may rely on their ability to form biofilms

[0050] and to sporulate as a resistance and defense mechanism in the interaction with co-habitant competitors

[0051] . The operons eps and tapA-sipW-tasA of B. subtilis and related species encode two of the main components of the ECM, and their expression is coordinately inhibited by the master regulator of biofilm formation SinR or activated by the regulatory protein RemA

[0052] . In our study, the strain UV_EMS_26 was found to accumulate a substitution of an alanine for a valine at position 74 in RemA, which we propose may compromise the correct assembly of the RemA structure, impeding the proper interaction with the promoter of the biofilm related operons eps and tasA-sipW-tapA. Structural analysis of a RemA variant (RemAR-18W32) demonstrated that a single amino acid substitution may modify the quaternary structure of this protein leading to an impairment of the protein functionality and thus, the inability to form biofilms correctly

[0047] . Protein modeling using Alphafold software

[0053] of the assembled RemA octamer in the UMAF6639 and UV_EMS_26 strain revealed a slight conformational change in the quaternary structure, potentially affecting the position of the two highly conserved arginines R50 and R51, described to be crucial for the DNA binding of the regulatory protein complex and to successfully complete biofilm formation in B. subtilis (Figure S8)

[0054] .

[0399] The reduction in swimming and swarming motility of UV_EMS_26 (in terms of motility in 0,3 % and 0,7 % LB-agar plates respectively) could also be initially conceived as an ecological disadvantage for the derivative strain when colonizing or developing biofilms to effectively persist over time on plants. Mutants lacking the functional product of the flagellar basal-body rod proteins have been described to exhibit a pronounced defect in swimming and swarming motility, a phenotype attributed to their inability to construct a fully functional flagellar structure

[0055] . The presence of a frameshift variant in U3P88_RS10810 gene, encoding a hypothetical protein similar to the flagellar protein FlgE would reasonably explain the reduced motility of UV_EMS_26. Nevertheless, despite the phenotypic alterations observed in the mutant, the robust behavior of the strain in the phylloplane highlights the complexity of theprocesses involved in Bacillus fitness, as well as the multitude of factors that must be coordinated to enable the successful establishment of the bacteria in this environment and to provide the host with their benefits. This finding, on the other hand, is not unprecedented and is in alignment to a previous report that showed how defect in swarming motility of a srf mutant, involved in Surfactin production, did not correlate with a complete defect in the colonization pattern of the phylloplane

[0010] . We thus hypothesize on alternative mechanisms used by UV_EMS_26 to compensate the lack of flagellar- associated motility and the deficiency in biofilm formation in planta. In this scenario, it is noteworthy the distinction in ECM composition between the two strains. The fact that the remA gene (encoding the biofilm regulator RemA responsible for the expression of eps operon) presents a missense mutation leads us to reason on the impairment of EPS production in UV_EMS_26. The pronounced disparity in size and composition of EPS between these two strains highlights the requirement for an alternative biosynthetic machinery to address the perturbation in EPS production driven by epsA-0 operon. Prior investigations have demonstrated the presence of a gene cluster, the ydaJKLMN operon, involved in the synthesis of an alternative EPS, suggesting that its production is probably restricted to stress conditions, helping bacteria to protect themselves from unsafe conditions

[0056] . UV_EMS_26 carries orthologs for this operon, thus it is conceivable that the pleiotropic effects of random mutagenesis might potentially induce stress, thereby increasing the biosynthesis of these uncharacterized EPSs. This interconnected regulatory pathway might be conceived as a compensatory mechanism to mitigate possible ecological disadvantages that could result from the down production of the main EPS during colonization and persistence on plant surfaces. The differences in the composition of EPSs within the mutant hold profound significance given that EPSs not only influence ecological interactions, but also have substantial implications for the biotechnological industry. In addition to this, it has been shown that the presence of rare monosaccharides such as L-arabinose, which is present in higher proportion in the mutant strain, is an essential structural feature that influences the biological activity of EPSs as an antitumor agent, as well as the presence of glucose, galactose and rhamnose, increasing the anti-inflammatory capacity against macrophages and inhibiting the secretion of TNF- a [57,58]. These findings might position UV_EMS_26 as a valuable source of biotechnologically significant materials beyond its contribution as a biocontrol agent.In relation to the increased antagonistic activity against phytopathogens, non-targeted metabolomics revealed increased accumulation of different metabolites in the mutant strain supernatant. The higher concentration of Surfactins raised the need to investigate their potential role in the observed phenotypes of UV_EMS_26. Surfactins have been widely described to exert crucial activities in Bacillus social behaviour [43,59] and also, to promote plant health by the induction of the plant immune system [7,60]. Biofilm and motility impairment in UV_EMS_26 could be initially conceived as a consequence of a reduction in surfactant production. Consistently with this thought, metabolomic studies revealed that the mutant strain CFS possessed less concentration of Ci2-Surf actin, one of the most produced in the parental strain. However, the fact that C -Surfactin and Ci6-Surfactin were found to be considerably over- accumulated in UV_EMS_26 CFS led us to speculate that these isoforms could be countering the repression in Ci2-Surfactin production. Prior investigations revealed how Bacillus Surfactin defective mutants showed less biocontrol activity, showcasing the importance of these molecules in the antagonism to plant pathogens

[0061] . These contribution to the biocontrol activity of Bacillus strains might be attributed to the facilitation of plant surface colonization and biofilm formation. However, the lack of swarming motility observed in the mutant, the aberrant biofilm architecture and the absence of significant differences in the persistence and colonization pattern over melon leaves, prompted further investigation into the direct role of Surfactin isoforms in the antagonistic activity against B. cinerea. Biological assays conducted with Surfactin isoforms at various concentrations revealed their ability cause a significant decrease in fungal growth (Figure 4D). Notably, the Ci6 isoform, despite being the less produced by UMAF6639 under normal conditions, exhibited exceptional efficacy in arresting the pathogen development at all concentrations tested. Similar effects were observed with the C12 isoform at the highest concentration assayed. Delving deeper into the metabolomic profile of the mutant strain, the increased accumulation Ci6 in UV_EMS_26 CFS, which showed high antifungal activity at their physiological concentrations, could explain the increase of antifungal activity of this strain. Although antifungal activity of surfactin produced by Bacillus species has been previously reported

[0062] and is consistent with our results, this study provides a more comprehensive approach by studying the isoform-specific activity. Furthermore, in connection with the aforementioned compounds, some fatty acids have been described to exert antimicrobial activity by affecting a wide variety of cellular functions including protein synthesis, metabolism and topoisomerase activity, beingbiological membrane interruption their main target

[0063] . Monomyristin variants, as 1-Monomyristin and 2-Monomyristin, exhibited antibacterial activity against Escherichia coli, Staphylococcus aureus and Aggregatibacter actinomycetemcomitans, and were also effective against Candida albicans, highlighting their potential to be used as antifungal agents

[0064] . These findings support the fact that the accumulation of Monomyristin analogues might be reasonably contributing to the improved antifungal activity of UV_EMS_26. Several studies also report stearic acid as one of the main fatty acids in plant oil extracts

[0065] and microorganism derived metabolites

[0066] , with potential use as antifungal molecules. Even that no antimicrobial activity has been described for Dipalmitoyl cephalin, this long-chain fatty acid can be speculated to contribute to the enhanced antifungal activity of the mutant strain due to the predicted tendence to interact with biological membranes. As most of the detected compounds did not match with annotated molecules, further investigation is still needed to determine their real contribution to the augmented antifungal activity. The predominance of lipidic molecules among the annotated metabolites produced in higher concentrations in the mutant strain CFS indicated the possibility that the increase of these molecules could be related to the mutation detected in U3P88_RS 18425 gene. This gene is the ortholog to IcfB in Bacillus subtilis sp. 168, encoding a protein responsible for the degradation of long-chain fatty acids. The regulation of this gene occurs via catabolite repression system

[0067] , which implies that in the presence of carbohydrates, mainly glucose, its expression is repressed to avoid fatty acid catabolism when preferred carbon sources are available

[0068] . Focusing on the frameshift variant affecting this gene in UV_EMS_26, we could speculate that the protein product would be altered and therefore, long-chain fatty acid degradation would be abolished or at least diminished in the mutant strain, causing the accumulation of the lipidic molecules detected in non-targeted metabolomics. In this context, the regulation of this repression could be compared to a situation of continuous glucose availability, even when bacteria had reached the stationary phase and consumed the nutrients from the medium, leading to the constant repression of alternative catabolic pathways and thereby resulting in an increase of the long-chain fatty acids present in the CFS of UV_EMS_26.

[0400] As Bacillus strains are recognized as small factories of secondary metabolites with interesting biological activities [69,70], there is a substantial motivation in the optimization of the industrial fermentation processes to produce these molecules in a costly effective manner. At this juncture, uncontrolled foam formation is a bottleneckfor the successful bioprocess due to operational problems associated to the blocking of exiting filters, potential contamination, and the subsequent reduction in productivity

[0071] . The less foam formation detected during fermentation of UV_EMS_26 represent a compelling advantage for the industrial production of either cells or different bioactive compounds. Two non-exclusive explanations to this improvement are the reduction in the amount of certain Surfactin isoforms, as they are known to trigger foam formation because of their surface-active properties in aerated and stirred bioreactors

[0072] , or the change in the EPS composition, which could lead to changes in the rheology of the media in the bioreactor with the consequent alteration of the hydrodynamics governing the complex network of air-liquid interfaces conforming foam

[0073] . A parallel positive operational consequence of the decrease in foam formation, is the reduction in the use of antifoam agents which are known to be toxic to bacteria, to modify bacterial metabolism and to reduce the oxygen transfer efficiency and in general the gas-liquid dispersion performances of the bioreactor

[0074] .

[0401] In summary, the combinational use of two mutagens to accelerate the generation of mutations have been proven effective to obtain an improved version of the biotechnologically relevant strain UMAF6639. Besides preserving the intrinsic traits defining the antimicrobial production profile and ecological fitness, the relaxation in biofilm formation driven by downregulation of ECM components and changes in the chemical composition of the EPS seem to be an effective strategy that improves operational fermentation in bioreactors, an important bottleneck in the production of commercially available formulations. Moreover, by unraveling the specific roles of over-accumulated molecules within the intricate network of metabolites relevant to plant disease biocontrol, our findings underscore their potential as targeted strategies for enhancing the effectiveness of future biocontrol agents or to guide the development of new products for sustainable agricultural practices.

[0402] References

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[0495]

[0496] 1 The indications made below relate to

[0497] the deposited microorganism(s) or

[0498] other biological material referred to in

[0499] the description on:

[0500] 1-1 page 6

[0501] 1-2 line 7

[0502] 1-3 Identification of deposit

[0503] 1-3-1 Name of depositary institution CECT Coleccion Espanola de Cultivos Tipo 1-3-2 Address of depositary institution Coleccion Espanola de Cultivos Tipo (CECT) Edificio 3 CUE . Parc Cientific Universitat de Valencia

[0504] Catedratico Agustin Escardino , 9

[0505] 46980 Paterna (Valencia)

[0506] Spain

[0507] 1-3-3 Date of deposit 09 January 2025 (09.01.2025)

[0508] 1-3-4 Accession Number CECT 8237

[0509] 1-4 Additional Indications

[0510] 1-5 Designated States for Which All designations

[0511] Indications are Made

[0512] 1-6 Separate Furnishing of Indications

[0513] These indications will be submitted to the

[0514]

[0515] International Bureau later

[0516] FOR RECEIVING OFFICE USE ONLY

[0517] 0-4 This form was received with the

[0518] international application: yes

[0519] (yes or no)

[0520]

[0521] 0-4-1 Authorized officer Kuiper-Cristina, Nathalie

[0522] FOR INTERNATIONAL BUREAU USE ONLY

[0523] 0-5 This form was received by the

[0524] international Bureau on:

[0525] 0-5-1 Authorized officer

[0526]

Claims

Claims1. A culture of Bacillus velezensis cells, in particular a biologically pure culture, corresponding with strain UMAF6639 as deposited at CECT with the accession number CECT 8237 or of mutant cells derived therefrom comprising one or more mutations.

2. The culture according to claim 1, wherein the cells are mutant cells of Bacillus velezensis strain UMAF6639 deposited at CECT with the accession number CECT 8237, said mutant cells containing a first autologous gene coding for a protein comprising an amino acid sequence having at least 70%, such as at east 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97% sequence similarity with the amino acid sequence presented in SEQ ID NO: 4, wherein said mutant cell comprises at least one mutation interfering with the formation of a fully functional protein product of the first autologous gene, preferably by interfering with the translation of the first autologous gene to a fully functional gene product.

3. The culture according to claim 2, wherein the at least one mutation interferes with the formation of the fully functional protein product of the first autologous gene by insertion or deletion of a number of nucleotides in the sequence of the first autologous gene, introduction of an additional stop codon in the sequence of the first autologous gene, or a nonstop mutation, preferably a frameshift mutation by incorporation of a number of additional nucleotides in the sequence of the first autologous gene, wherein the first autologous gene most preferably is selected from a gene translating to the same protein product as the sequence of SEQ ID NO: 4, such as a gene having the sequence of SEQ ID NO: 3.

4. The culture according to any of the claims 1-3, wherein the at least one mutation comprises a conversion of the sequence GAAAAAAGA to GAAAAAAAGA at position 3789419 (MID93) of the UMAF6639 genomic sequence (SEQ ID NO:

5. The culture according to any of the claims 2-4, further comprising a second autologous gene coding for a protein comprising an amino acid sequence having at least 70%, such as at east 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97% sequence similarity with an amino acid sequence as presented in SEQ ID NO: 6, wherein said mutant cell comprises at least one mutation interfering with the formation of a fully functional protein product of the second autologous gene, preferably by producing a full length protein product having an altered functionality.

6. The culture according to claim 5, wherein the mutation results in one or more amino acid substitutions between positions 72-76 on the protein sequence of SEQ ID NO: 6, preferably including an Ala to Vai substitution at position 74, such as by a C to T substitution at position 323657 (MID 10) of the UMAF6639 genomic sequence of SEQ ID NO: 1, more preferably selected from an Ala to Vai substitution at position 74 as a single amino acid substitution.

7. The culture according to any of the claims 1-5, wherein said mutant cell comprises at least one mutation selected from mutations MID1-MID99 presented below, such as all mutations MID1-MID99 presented below:Mutation Chromosome Reference Alternate Gene ID ID positionMIDI 21955 C I I I I I I ATGC C I I I I I I I ATGC U3P88_RS00110 MID2 54418 GAAAAAAACCG GAAAAAAAACCG U3P88_RS00255 MID3 186978 G A U3P88_RS00845 MID4 244099 G A U3P88_RS01030 MID5 253428 GAAAAAAGC GAAAAAAAAGC U3P88_RS01095 MID6 263141 G l I I I I I I I Al G I G l I I I I I I I I A I G r U3P88_RS01115 MID7 265073 GAAAAAAATAAGT GAAAAAAAATAAGT U3P88_RS01165 MID8 290720 AGGGGGAGC AGGGGGGAGC U3P88_RS01250 MID9 297721 C I I I I I I AC C I I I I I I I AC U3P88_RS01290 MID10 323657 C T U3P88_RS01405 MID11 361614 C T U3P88_RS01605 MID12 371056 GAAAAACAA GAAAAAACAA U3P88_RS01650 MID13 424464 C T U3P88_RS01930 MID14 548493 TAAAAAAGACAAACG TAAAAAAAGACAAACG U3P88_RS02200 MID15 549032 A I I I I I I I AGC A I I I I I I I I AGC U3P88_RS02240 MID16 555993 GAAAAAGC GAAAAAAGC U3P88_RS02290 MIDI 7 571603 G A U3P88_RS02365 MID18 576876 G A U3P88_RS02380MID19 590411 G A U3P88_RS02445 MID20 651865 G A U3P88_RS02805 MID21 755755 A I I I I I I AAAA A I I I I I I I AAAA U3P88_RS02965 MID22 825403 C I I I I I I AC C I I I I I I I AC U3P88_RS03345 MID23 894721 GAAAAAACG GAAAAAAACG U3P88_RS03760 MID24 933974 GCCCCCTG GCCCCCCTG U3P88_RS04035 MID25 953105 G A U3P88_RS04150 MID26 974779 C I I I I I I I ATTT C l I I I I I I I A I I I U3P88_RS04260 MID27 974909 G A U3P88_RS04260 MID28 982901 G I I I I I I CG G I I I I I I I CG U3P88_RS04310 MID29 1050955 TAAAAAGAT TAAAAAAGAT U3P88_RS04685 MID30 1058917 C I I I I I I AT C I I I I I I I AT U3P88_RS04735 MID31 1086450 G A U3P88_RS04785 MID32 1165722 C I I I I I I CCA C I I I I I I I CCA U3P88_RS05035 MID33 1194879 G I I I I I I I GCG G I I I I I I I I GCG U3P88_RS05210 MID34 1223227 GAAAAAAGC GAAAAAAAGC U3P88_RS05390 MID35 1300760 C I I I I I I AA C I I I I I I I AA U3P88_RS05805 MID36 1315959 C I I I I I I CTT C I I I I I I I CTT U3P88_RS05920 MID37 1317627 TAAAAAAATC TAAAAAAAATC U3P88_RS05910 MID38 1350766 G I I I I I I CA G I I I I I I I I CA U3P88_RS06075 MID39 1418506 G I I I I I I I CTTCTGG G I I I I I I I I C I I CTGG U3P88_RS06435 MID40 1493690 GAAAAAATG GAAAAAAATG U3P88_RS06775 MID41 1501789 GAAAAAACAG GAAAAAAACAG U3P88_RS06805 MID42 1512192 C I I I I I I CTGG C I I I I I I I CTGG U3P88_RS06850 MID43 1519544 GAAAAAGAAGAC GAAAAAAGAAGAC U3P88_RS06900 MID44 1522776 CAAAAAAACCT CAAAAAAAACCT U3P88_RS06895 MID45 1697791 C I I I I I CTTG C I I I I I I CTTG U3P88_RS07805 MID46 1824328 CAAAAAACCCGGCCGCCT CAAAAAAACCCGGCCGCCT U3P88_RS08505 MID47 2108280 G I I I I I I I CA G I I I I I I I I CA U3P88_RS10010 MID48 2150719 GAAAAAAGC GAAAAAAAGC U3P88_RS10205 MID49 2244923 C I I I I I I CTTGC C l I I I I I I I C I IGC U3P88_RS10645 MID50 2274038 CGG I I I I I I GA CGG I I I I I I I GA U3P88_RS10810 MID51 2276351 ATTATATAT AATATATAT U3P88_RS10795 MID52 2276375 CAAAAAAAGC CAAAAAAAAGC U3P88_RS10795 MID53 2278670 GAAAAAAGAACG GAAAAAAAGAACG U3P88_RS10835 MID54 2301282 TAAAAAAAGGGCCGCT TAAAAAAAAGGGCCGCT U3P88_RS 10945 MID55 2350723 C T U3P88_RS11245 MID56 2354978 GAAAAAAACAGC GAAAAAAAAACAGC U3P88_RS11255 MID57 2385130 CAAAAAAAAAACAAAAAAG CAAAAAAAAACAAAAAAGC U3P88_RS11420C MID58 2463623 GAAAAAACCG GAAAAAAACCG U3P88_RS11845 MID59 2524689 C T U3P88_RS12130 MID60 2536320 C T U3P88_RS12210 MID61 2554028 GAAAAAAGC GAAAAAAAGC U3P88_RS12240 MID62 2620111 TCCCCCCAT TCCCCCCCAT U3P88_RS12575 MID63 2631739 C I I I I I CTA C I I I I I I CTA U3P88_RS12625 MID64 2673611 CAAAAAAACG CAAAAAAAACG U3P88_RS12845 MID65 2820184 AGGGGTTT AGGGGGTTT U3P88_RS13670 MID66 2879940 C T U3P88_RS14030 MID67 2910278 C T U3P88_RS14170 MID68 3009889 CGGGGGGAT CGGGGGGGAT U3P88_RS14525MID69 3062344 GAAAAAGC GAAAAAAGC U3P88_RS01330 MID70 3076436 C T U3P88_RS14855 MID71 3093519 GAAAAAAGT GAAAAAAAGT U3P88_RS14915 MID72 3134669 GAAAAAGGA GAAAAAAGGA U3P88_RS15005 MID73 3139160 CAAAAAAATG CAAAAAAAATG U3P88_RS15045 MID74 3170749 TAAAAAAGG TAAAAAAAGG U3P88_RS15210 MID75 3210489 GAAAAAAGGG GAAAAAAAGGG U3P88_RS15405 MID76 3222026 CAAAGAA CAAAAGAA U3P88_RS15440 MID77 3224459 G I I I I I I I CCA G I I I I I I I I CCA U3P88_RS15495 MID78 3230395 G A U3P88_RS15535 MID79 3234632 GAAAAAACG GAAAAAAACG U3P88_RS15550 MID80 3324252 TAAAAAAGC TAAAAAAAGG U3P88_RS16070 MID81 3411709 TAAAAAGAGG TAAAAAAGAGG U3P88_RS16530 MID82 3449492 TAAAAAAAGCAG TAAAAAAAAGCAG U3P88_RS16700 MID83 3466318 GAAAAATA GAAAAAATA U3P88_RS16805 MID84 3552374 G I I I I I I I GCTG G l I I I I I I IGC IG U3P88_RS17160 MID85 3565764 GAAAAAACG GAAAAAAACG U3P88_RS17240 MID86 3583264 AGGGGCT AGGGGGGCT U3P88_RS17320 MID87 3608213 GAAAAAAATAAC GAAAAAAAATAAC U3P88_RS17430 MID88 3614717 GAAAAAATAAATG GAAAAAAAATAAATG U3P88_RS17440 MID89 3633943 GCCCCCTCA GCCCCCCTCA U3P88_RS17650 MID90 3702507 GAAAAAAGGAAT GAAAAAAAAGGAAT U3P88_RS17970 MID91 3770864 AGGGGGCCT AGGGGGGCCT U3P88_RS18345 MID92 3785078 GAAAAAGT GAAAAAAGT U3P88_RS18385 MID93 3789419 GAAAAAAGA GAAAAAAAGA U3P88_RS18425 MID94 3809856 C I I I I I I I CG C I I I I I I I I CG U3P88_RS18510 MID95 3833258 G A U3P88_RS18610 MID96 3903890 CAAAAAAGC CAAAAAAAGC U3P88_RS19000 MID97 3967786 AGGGAA AGGGGAA U3P88_RS19410 MID98 4018847 C I I I I I I CC C I I I I I I I CC U3P88_RS19730 MID99 4030406 C I I I I I I CTTT C I I I I I I I CTTT U3P88_RS198208. The culture according to any of the claims 2-7 wherein the percentage sequence similarity is percentage sequence identity.

9. The culture according to any of the claims 7-8 comprising at least one mutation selected from MID38, MID7, MID21, MID50 or MID78 as defined in claim 7, preferably all mutations selected from MID38, MID7, MID21, MID50 and MID78.

10. Use of mutant cells of a culture according to any of the claims 1-9 for producing a fermentate, preferably a fermentate having biocontrol activity, in particular in crop protection against a plant pathogen and / or plant parasite.

11. A fermentate of Bacillus cells of a culture according to any of the claims 1-8, preferably a fermentate comprising cell material of said bacillus cells, most preferably viable cells, wherein the fermentate preferably has biocontrol activity, in particular in crop protection against a plant pathogen and / or plant parasite.

12. A composition, in particular an agronomical composition, comprising biomass, preferably viable biomass, corresponding to Bacillus velezensis strain UMAF6639 as deposited at CECT with the accession number CECT 8237 or of a mutant strain derived therefrom, said composition preferably further comprising a carrier, more preferably an agronomically acceptable carrier, most preferably an aqueous carrier.

13. Use of a population of Bacillus cells from a culture according to any of the claims 1-9 as a biocontrol agent, preferably a biocontrol agent for crop protection, more preferably crop protection against a plant pathogen and / or plant parasite selected from (i) a plant pathogenic fungus, such as selected from the genus Bipolaris, including Bipolaris may dis, from the genus Botrytis, including Botrytis cinerea, from the genus Cercospora, including Cercospora kikuchii, Cercospora longipes, Cercospora zea-maydis, from the genus Cladosporium, including Cladosporium cladosporiodes, from the genus Colletotrichum, including Colletotrichum falcatum, from the genus Corynespora, including Corynespora cassiicola, from the genus Erisiphe, including Erisiphe necator, from the genus Fusarium, including Fusarium oxysporum or Fusarium graminearium, from the genus Globodera, from the genus Eeveillula, including Eeveillula taurica, from the genus Meloidogyne, from the genus Mycosphaerella, including Mycosphaerellafijiensis, from the genus Phaeosphaeria, including Phaeosphaeria maydis, from the genus Phakopsora, including Phakopsora Pachyrhizi, from the genus Phyllosticta, including Phyllosticta citricarpa, from the genus Physopella, including Physopella zeae, from the genus Podosphaera, including Podosphaera fusca or Podosphaera xanthii, from the genus Pratylenchus, from the genus Puccinia, including Puccinia Kuehnii, Puccinia Melanocephala, Puccinia polysora or Puccinia sorghi, from the genus Sclerotinia, including Sclerotinia sclerotiorum, from the genus Septoria, including Septoria glycines, from the genus Sphaerotheca,including Sphaerotheca pannosa, or from the genus Uncinula, including Uncinula necatori, (ii) a plant pathogenic Oomycete, such as selected from the genus Phytophthora including Phytophthora capsici or Phytophthora infestans, or from the genus Plasmopara, including Plasmopara viticola (iii) plant pathogenic bacteria, such as selected from P ectobacterium species, for example P ectobacterium carotovorum, (iv) plant parasitic nematodes, such as a from the genus Meloidogyne, including Meloidogyne incognita, from the genus Globodera, including Globodera rostochiensis, or from the genus Pratylenchus, wherein most preferably the crop protection is against (i) a plant pathogenic fungus.

14. Use according to claim 13 wherein the plant pathogen is a plant pathogenic fungus selected from Sordariomycetes, Eurotiomycetes, Dothideomycetes, Pucciniomycetes or Leotiomycetes, preferably Sordariomycetes, Dothideomycetes, Pucciniomycetes, or Leotiomycetes or is a plant pathogenic Oomycete.

15. Method of producing Bacillus mutant cells preferably cells according to any of the claims 1-9, said method comprising:-providing viable cells of a Bacillus species, such as from Bacillus velezensis, Bacillus subtilis, Bacillus amylolyquefaciens, Bacillus siamensis, Bacillus licheniformis or Bacillus pumilus, preferably of Bacillus velezensis, Bacillus subtilis, or Bacillus amylolyquefaciens, more preferably from Bacillus velezensis, most preferably from Bacillus velezensis strain UMAF6639 deposited at CECT with the accession number CECT 8237;-stably introducing a at least one mutations interfering with the formation of a fully functional protein product of the gene coding for the protein comprising an amino acid sequence having at least 70%, such as at east 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97%, sequence similarity with SEQ ID NO: 4, preferably by interfering with the translation of the gene to a fully functional gene product, wherein when the Bacillus species cells are selected from UMAF6639, the at least one mutation more preferably comprise mutation MID93, as defined in claim 7;-optionally, stably introducing at least one additional mutations, wherein when the cells of the Bacillus species comprise a gene coding for the protein sequence of SEQ ID NO: 6, such as cells from Bacillus velezensis strain UMAF6639, the at least one additional mutation preferably comprises a number of mutation resulting in a number of amino acid substitutions between positions 72-76 on the protein sequence of SEQ ID NO: 6, preferably including an Ala to Vai substitution at position 74, more preferably selected from an Ala to Vai substitution at position 74 as a single amino acid substitution, such as, when the cells of the Bacillus species are selected from strain UMAF6639, by a C to T substitution at position 323657 (MID 10) of the UMAF6639 genomic sequence of SEQ ID NO: 1.

16. Method according to claim 15, wherein, when the cells of the Bacillus species are selected from strain UMAF6639, the at least one optional additional mutationis selected from MID1-MID92 and / or MID94-MID99 as defined in claim 6, preferably at least one mutation selected from MID 10, MID38, MID7, MID21, MID50 or MID78, preferably at least one mutation comprising mutation MID 10, more preferably all mutations selected from MID 10, MID38, MID7, MID21, MID50 and MID78.

17. Method according to claim 15, wherein a number of mutant cells according to any of the claims 2-4 are produced.

18. Use of cells corresponding with the Bacillus velezensis strain UMAF6639 deposited at CECT with the accession number CECT 8237, for producing a mutant cell according to any of the claims 1-9, preferably in a method according to any of the claims 15-17.

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