Paenibacillus strains producing small amounts of exopolysaccharide

Abstract

Disclosed are Paenibacillus strains that contain reduced activity of flippase PepR, flippase PepH, mannose-1-phosphate guanylyltransferase, and / or levansucrase SacB. These Paenibacillus strains exhibit reduced viscosity when grown in liquid cultures as well as agricultural compositions comprising these strains and methods of making and using these strains.

Description

[Technical field]

[0001] The present invention relates to Paenibacillus strains comprising reduced activity of flippase PepR, flippase PepH, mannose-1-phosphate guanylyltransferase, and / or levansucrase SacB. These Paenibacillus strains exhibit reduced viscosity when grown in liquid culture. The present invention also relates to compositions comprising these strains, and methods of making and using these strains. [Background technology]

[0002] Paenibacillus is known for its ability to produce several types of exopolysaccharides (EPS) (Liang and Wang, Recent Advances in Exopolysaccharides from Paenibacillus spp.: Production, Isolation, Structure, and Bioactivities, Mar. Drugs 2015, 13, 1847 to 1863). The main components of EPS produced by Paenibacillus species described so far are as follows: 1) Levan, a polymer of fructose linked by β-(2→6) fructofuranoside bonds (Bezzate et al, Disruption of the Paenibacillus polymyxa levansucrase gene impairs its ability to aggregate soil in the wheat rhizosphere, Environmental Microbiology 2000 2(3),333 to 342), 2) Curdlan, a linear glucan composed only of (1→3)-d-glycosidic bonds and forming a coaxial triple helix (Rafigh et al., Optimization of culture medium and modeling of curdlan production from Paenibacillus polymyxa by RSM and ANN, International Journal of Biological Macromolecules 70(2014)463-473), and 3) Paenan, a heteropolysaccharide containing glucose, mannose, galactose, and glucuronic acid (Marius Ruetering, Exopolysaccharides by Paenibacilli: from Genetic Strain Engineering to Industrial Application, Dissertation, 2019).

[0003] Paenibacilli use several pathways to produce these EPS. In addition to the gene for levansucrase (SacB) required for the production of levan, they have several gene clusters. One known gene cluster consists of 29 genes that produce EPS (Marius Ruetering, Exopolysaccharides by Paenibacilli: from Genetic Strain Engineering to Industrial Application, Dissertation, 2019). In addition to the genes that produce the building blocks of EPS and the genes that code for the transport function of EPS through the peptidoglycan cell wall, this gene cluster consists of two flippases (pepH and pepR) that provide the function of transporting lipid-linked oligosaccharides through the cell membrane and two polymerases (pepE and pepG) that polymerize the transported oligosaccharides to form the final EPS (Marius Ruetering, Exopolysaccharides by Paenibacilli: from Genetic Strain Engineering to Industrial Application, Dissertation, 2019). The exact mechanism of curdlan production in Paenibacilli is still unknown. However, it is believed that paenan is produced via a membrane-located synthetase (Marius Ruetering, Exopolysaccharides by Paenibacilli: from Genetic Strain Engineering to Industrial Application, Dissertation, 2019).

[0004] The produced EPS performs important functions for Paenibacilli in natural environments, such as the formation of biofilms, but tends to be produced in excess when sugars are abundantly supplied, as in the artificial growth conditions in industrial production processes. This excess production of EPS results in a viscous culture broth that is difficult to stir and makes it difficult to optimally aerate and evenly supply all areas of the fermenter. If the production of EPS is not the purpose of the industrial process, its excess production not only represents a metabolic burden and an absorption of valuable nutrients, but often also causes additional problems in downstream processing and isolation of economically valuable products of interest, such as spores and biomass of Paenibacilli, enzymes, secondary metabolite compounds, or low-weight chemicals such as 2,3-butanediol.

[0005] To circumvent such problems, several approaches have been taken to reduce the EPS production ability of Paenibacilli. These approaches usually use the deletion of individual genes or large parts of the EPS production cluster to destroy the ability to produce one or more EPS. For example: Okonkwo et al. Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation, Applied and Environmental Microbiology, Volume 86 Issue 9, 2020, e00196-20 deleted the gene encoding levansucrase. These approaches included deletion of the levansucrase gene and an additional 16 genes in the Paenibacillus EPS gene cluster, including both genes for the EPS polymerase (pepE and pepG) and one of the two flippase genes (pepH). The same gene cluster is exemplified in He et al. Effects of an EPS Biosynthesis Gene Cluster of Paenibacillus polymyxa WLY78 on Biofilm Formation and Nitrogen Fixation under Aerobic Conditions, Microorganisms 2021, 9, 289. https: / / doi.org / 10.3390 / microorganisms9020289. He et al. deleted the entire gene cluster in two separate large deletions and found that the large deletion containing pepH resulted in a strain that produced low amounts of EPS, but that a later deletion of the large gene cluster containing pepR did not affect EPS production.

[0006] While these approaches have been successful in reducing EPS production during large-scale fermentation, there remains a need for less drastic approaches to reduce EPS production during large-scale fermentation, which have less severe effects on microbial metabolism, especially when high concentrations of glucose and / or saccharose are present in the growth medium. There is also a need for new strains that sufficiently reduce EPS production during large-scale fermentation, but still maintain the ability to produce sufficient amounts of EPS when grown under natural conditions to allow biofilm formation and interaction with plants.

[0007] Surprisingly, it was found that reducing the activity of one flippase in Paenibacilli was sufficient to strongly affect total EPS production. Equally surprising, it was found that mutant levansucrases could lead to a stronger reduction in total EPS production than deleting the entire levansucrase coding sequence. In both cases, the metabolic activity of the mutant strains was increased, as shown, for example, by the maximum carbon dioxide transfer rate (CTR) reached during the fermentation process. Summary of the Invention [Means for solving the problem]

[0008] In one aspect, provided herein is a strain of Paenibacillus sp., comprising: a) the absence or mutant PepR, or b) a mutant PepH, or c) the absence or mutant PepX, or d) a mutant ManC, or e) a mutant SacB, or f) a combination of at least two of a), b), c), d), or e), wherein the absence of PepR or the mutant PepR results in a reduced viscosity of a liquid culture of a strain of Paenibacillus sp. compared to a liquid culture of a strain of Paenibacillus sp. comprising wild-type PepR, the mutant PepH results in a reduced viscosity of a liquid culture of a strain of Paenibacillus sp. compared to a liquid culture of a strain of Paenibacillus sp. comprising wild-type PepH, and the absence of PepX or the mutant PepX results in a reduced viscosity of a liquid culture of a strain of Paenibacillus sp. compared to a liquid culture of a strain of Paenibacillus sp. comprising wild-type PepX. and wherein the mutant ManC results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC or compared to a liquid culture of a Paenibacillus sp. strain not containing ManC; A strain of Paenibacillus sp. is provided, wherein the mutant SacB results in a reduced viscosity of a liquid culture of the strain of Paenibacillus sp. compared to a liquid culture of the strain of Paenibacillus sp. that does not contain SacB.

[0009] In a further aspect there is provided herein an agricultural composition comprising a strain of Paenibacillus sp. comprising a) an absent or mutant PepR, or b) an absent or mutant PepH, or c) an absent or mutant PepX, or d) an absent or mutant ManC, or e) an absent or mutant SacB, or f) a combination of at least two of a), b), c), d), e), or f), the absence of PepR or a mutant PepR results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepR; the absence of PepH or mutant PepH results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepH; the absence of PepX or mutant PepX results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepX; the absence of ManC or mutant ManC results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC; the absence of SacB or mutant SacB results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain that does not contain SacB; The strains of Paenibacillus sp. containing the respective wild-type PepR, PepH, PepX, ManC, or SacB and used for comparison are of the same Paenibacillus sp. as the strains containing the absence or mutants of PepR, PepH, PepX, ManC, or SacB. An agricultural composition is provided.

[0010] In a further aspect there is provided herein a plant propagation material comprising a strain of Paenibacillus sp. comprising a) an absent or mutant PepR, or b) an absent or mutant PepH, or c) an absent or mutant PepX, or d) an absent or mutant ManC, or e) an absent or mutant SacB, or f) a combination of at least two of a), b), c), d), e), or f), the absence of PepR or a mutant PepR results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepR; the absence of PepH or mutant PepH results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepH; the absence of PepX or mutant PepX results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepX; the absence of ManC or mutant ManC results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC; the absence of SacB or mutant SacB results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain that does not contain SacB; The strains of Paenibacillus sp. containing the respective wild-type PepR, PepH, PepX, ManC, or SacB and used for comparison are of the same Paenibacillus sp. as the strains containing the absence or mutants of PepR, PepH, PepX, ManC, or SacB. Plant propagation material is provided.

[0011] In one aspect, provided herein is a method for inhibiting or preventing fungal infection in plants, comprising treating a fungus, its habitat or material or plant to be protected from fungal attack, or soil or plant propagation material with an effective amount of a strain of Paenibacillus sp. comprising a) the absence or mutant PepR of PepR, or b) the absence or mutant PepH of PepH, or c) the absence or mutant PepX of PepX, or d) the absence or mutant ManC of ManC, or e) the absence or mutant SacB of SacB, or f) a combination of at least two of a), b), c), d), e), or f), the absence of PepR or a mutant PepR results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepR; the absence of PepH or mutant PepH results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepH; the absence of PepX or mutant PepX results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepX; the absence of ManC or mutant ManC results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC; the absence of SacB or a mutant SacB results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain that does not contain SacB; or a) an absence or mutant PepR of PepR, or b) an absence or mutant PepH of PepH, or c) an absence or mutant PepX of PepX, or d) an absence or mutant ManC of ManC, or e) an absence or mutant SacB of SacB, or f) a combination of at least two of a), b), c), d), e), or f), the absence of PepR or a mutant PepR results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepR; the absence of PepH or mutant PepH results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepH; the absence of PepX or mutant PepX results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepX; the absence of ManC or mutant ManC results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC; the absence of SacB or mutant SacB results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain that does not contain SacB; The strains of Paenibacillus sp. containing the respective wild-type PepR, PepH, PepX, ManC, or SacB and used for comparison are of the same Paenibacillus sp. as the strains containing the absence or mutants of PepR, PepH, PepX, ManC, or SacB. A method is provided.

[0012] There is also provided a method for producing a valuable product by fermentation, comprising the steps of: 1) in a culture medium under conditions such that a valuable product is produced; Cultivating a strain of Paenibacillus sp. comprising: a) an absent or mutant PepR; or b) an absent or mutant PepH; or c) an absent or mutant PepX; or d) an absent or mutant ManC; or e) an absent or mutant SacB; or f) a combination of at least two of a), b), c), d), e), or f), the absence of PepR or a mutant PepR results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepR; the absence of PepH or mutant PepH results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepH; the absence of PepX or mutant PepX results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepX; the absence of ManC or mutant ManC results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC; the absence of SacB or a mutant SacB results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain not containing SacB, and containing the respective wild-type PepR, PepH, PepX, ManC, or SacB, and the Paenibacillus sp. strain used for comparison is of the same Paenibacillus sp. as the strain containing the absence or mutants of PepR, PepH, PepX, ManC, or SacB. The process and 2) collecting the valuable product; A method is provided that includes: [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the change in viscosity over culture time for different mutants of Paenibacillus strain LU17007 and strains derived from Paenibacillus strain LU17007 by deleting the regions encoding PepR (herein referred to as exoT), SacB, and strain variants containing the G323S mutant of SacB. [Diagram 2] FIG. 2 shows the alignment of the amino acid sequences of PepR from Paenibacillus strain LU17007, Paenibacillus ottowii, Paenibacillus polymyxa DSM365, Paenibacillus terrae, Paenibacillus kripbbensis, and Paenibacillus sp. Aloe-11. [Diagram 3]FIG. 3 shows an alignment of the amino acid sequences of PepH from Paenibacillus strain LU17007, Paenibacillus ottowii, Paenibacillus polymyxa DSM365, Paenibacillus terrae, Paenibacillus kripbbensis, and Paenibacillus sp. Aloe-11. [Figure 4] FIG. 4 shows an alignment of the amino acid sequences of PepX from Paenibacillus strain LU17007, Paenibacillus ottowii, Paenibacillus polymyxa DSM365, Paenibacillus terrae, Paenibacillus kripbbensis, and Paenibacillus sp. Aloe-11. [Diagram 5] FIG. 5 shows an alignment of the amino acid sequences of ManC from Paenibacillus strain LU17007, Paenibacillus ottowii, Paenibacillus polymyxa DSM365, Paenibacillus terrae, Paenibacillus kripbbensis, and Paenibacillus sp. Aloe-11. [Figure 6]FIG. 6 shows an alignment of the amino acid sequences of SacB from Paenibacillus strain LU17007, Paenibacillus ottowii, Paenibacillus polymyxa DSM365, Paenibacillus terrae, Paenibacillus kripbbensis, and Paenibacillus sp. Aloe-11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] As used herein, a Paenibacillus strain is a) the absence of PepR or a mutant PepR, or b) a mutant PepH, or c) the absence of PepX or a mutant PepX, or d) a mutant ManC, or e) a mutant SacB, or f) a combination of at least two of a), b), c), d), or e); the absence of PepR or a mutant PepR results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepR; the mutant PepH results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepH; the absence of PepX or mutant PepX results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepX; the mutant ManC results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC or compared to a liquid culture of a Paenibacillus sp. strain not containing ManC; A strain of Paenibacillus sp. is described in which a mutant SacB results in a reduced viscosity of a liquid culture of the strain of Paenibacillus sp. compared to a liquid culture of the strain of Paenibacillus sp. that does not contain SacB. Preferably, the strain of Paenibacillus sp. that contains the respective wild-type PepR, PepH, PepX, ManC, or SacB and is used for comparison is of the same Paenibacillus sp. as the strain in which PepR, PepH, PepX, ManC, or SacB is absent or contains a mutant thereof. Even more preferably, the strain of Paenibacillus sp. used for comparison is a parent strain of Paenibacillus lacking PepR, PepH, PepX, ManC, or SacB or containing the respective mutants. Preferably, the strain that does not contain PepR, PepH, PepX, ManC, or SacB has a deletion in the respective gene sequence or a mutation leading to a premature stop codon.

[0015] As used herein, the term "Paenibacillus strain" is the same as the term "strain of Paenibacillus sp." and refers to bacterial strains of the Paenibacillus genus. The genus Paenibacillus includes all Paenibacillus spp.

[0016] These Paenibacillus strains disclosed herein exhibit reduced viscosity when grown in liquid culture when compared to Paenibacillus strains containing wild-type PepR, PepH, or SacB, respectively.

[0017] A Paenibacillus strain in which PepR is absent or which contains a mutant PepR is typically compared to a Paenibacillus strain which contains a wild-type PepR. Preferably, a Paenibacillus strain in which PepR is absent or which contains a mutant PepR is compared to a Paenibacillus strain which has been generated by mutation or transgenic or gene editing techniques, i.e. the parent strain.

[0018] Thus, the parent strains have identical genomic sequences to the respective Paenibacillus strains, except for the presence of the respective mutations. The same principle applies to Paenibacillus strains containing mutant PepH or mutant SacB.

[0019] The viscosity is preferably measured using the same method as described in Examples 2 and 3 herein, with either glucose or saccharose, preferably glucose, being used as the C source. Preferably, the liquid medium contains glucose as described in Example 2. A strain is considered to have a lower viscosity when grown in liquid culture if the viscosity [mPa·s] measured at 100 / s is lower than the viscosity [mPa·s] measured at 100 / s of a comparative strain after at least 12 hours, at least 24 hours, or at least 40 hours of culture time from the start of growth of the microorganism, preferably after at least 24 hours of culture time. A strain is considered to have a lower viscosity if the sum of the viscosities measured every hour from 8 hours to 40 hours of culture is lower than the sum of the viscosities measured every hour from 8 hours to 40 hours of culture of the comparative strain.

[0020] As used herein, the verb "comprise" and its conjugations used in the specification and claims are used in an open-ended sense meaning that the items following the term are included, but not excluding items not specifically mentioned. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that there is more than one element, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" generally means "at least one."

[0021] The term "strain" is intended to refer to a microbial cell that can be considered genetically homogeneous, taking into account the natural frequency of mutations during growth of the cell. Strains are often isolated from a single cell via the cultivation of a microbial colony.

[0022] A microbial culture grown from a single strain is considered a biologically pure culture.

[0023] As used herein, the term "biologically pure culture" refers to a culture of a strain that is essentially free of microorganisms of other species or strains. In a preferred embodiment, a "biologically pure culture" contains less than 1%, more preferably less than 0.1%, and even more preferably less than 0.01% cells of other species.

[0024] Strains can also be purposefully mixed with other strains to create co-cultures, which are considered to contain one or more biologically pure cultures of the individual strains at the same time.

[0025] The term mutant, when used in reference to a protein, refers to the situation where a genomic region encoding such a protein contains at least one nucleotide that results in an alteration in the amino acid sequence of the encoded protein compared to the wild-type amino acid sequence of such protein.

[0026] The term mutant, when used in reference to a gene for a protein, refers to a situation in which the polynucleotide sequence of the promoter or the encoded RNA sequence of such a protein differs from the wild-type sequence, resulting in an altered, preferably reduced, level of expression of the encoded protein.

[0027] In one embodiment of the invention, a Paenibacillus strain comprises mutant genes that result in reduced expression levels of PepR, PepH, PepX, ManC, and / or SacB in such Paenibacillus strain compared to a wild-type version of the Paenibacillus strain.

[0028] The Paenibacillus strains of the invention can be cultured continuously or discontinuously in a batch process or a fed-batch process or a repeated fed-batch process. An overview of known culture methods can be found in the textbooks by Chmiel (Bioprozesstechnik 1. Einfuehrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

[0029] The medium used for culturing Paenibacillus strains must be appropriately adapted to the requirements of the particular strain. Descriptions of media for various microorganisms can be found in the American Society for Bacteriology handbook "Manual of Methods for General Bacteriology" (Washington DC, USA, 1981). Further suitable media are disclosed in the art, for example in the documents cited herein.

[0030] Preferably, the Paenibacillus strain belongs to the following Paenibacillus species: Paenibacillus polymyxa, Paenibacillus jamilae, Paenibacillus ottowii, Paenibacillus terrae, or Paenibacillus kribensis.

[0031] Preferred strains suitable for constructing the strains of the present invention are P. polymyxa strain DSM365, P. polymyxa strain PKB1, P. polymyxa strain JB05-01-1, P. polymyxa strain AC-1, P. polymyxa strain HY96-2, Paenibacillus sp. Aloe-11, Paenibacillus sp. strains NRRL B-50972, NRRL B-67129, NRRL B-67304, NRRL B-67306, and NRRL B-67615, NRRL B-50374, NRRL B-67721, NRRL B-67723, NRRL B-67724, NRRL B-67725, NRRL B-67726, NRRL B-67727, NRRL B-67728, NRRL B-67729, NRRL B-67729, NRRL B-67720, NRRL B-67721, NRRL B-67722, NRRL B-67723, NRRL B-67724, NRRL B-67725, NRRL B-67726, NRRL B-67727, NRRL B-67728, NRRL B-67729 ... B-67724, P. polymyxa strain VMC10 / 96, Paenibacillus sp. strain 10.6D, Paenibacillus sp. strain 9.4E, Paenibacillus sp. strains Lu16774, Lu17007, and Lu17015, P. polymyxa strain M1, P. polymyxa strain SC2, P. polymyxa strain Sb3-1, and P. polymyxa strain E681. Particularly preferred strains are Lu17007 and DSM365.

[0032] A Paenibacillus strain comprising the flippase PepR absent or a mutant flippase PepR or a mutant flippase PepH, the flippase PepX absent or a mutant flippase PepX, the mannose-1-phosphate guanylyltransferase ManC absent or a mutant mannose-1-phosphate guanylyltransferase ManC, or the mutant levansucrase SacB is preferably a strain in which the wild-type PepR has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 and the mutant PepR comprises amino acids different from the amino acids at the same positions as the 100% conserved amino acids in the alignment shown in FIG. 2, or a strain in which the wild-type PepH has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:8 and the mutant PepH contains amino acids different from the amino acids at the same positions as the 100% conserved amino acids in the alignment shown in Figure 3; a strain in which the wild-type PepX has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 15 and the mutant PepH contains amino acids different from the amino acids at the same positions as the 100% conserved amino acids in the alignment shown in Figure 4; a strain in which the wild-type ManC has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:22 and the mutant ManC contains amino acids different from the amino acids at the same positions as the 100% conserved amino acids in the alignment shown in Figure 5; A strain in which the wild-type SacB has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:29 and the mutant SacB contains an amino acid different from the amino acid at the same position as the 100% conserved amino acid in the alignment shown in Figure 6.

[0033] Flippase PepR is absent or a mutant flippase PepR or a mutant flippase PepH, flippase PepX is absent or a mutant flippase PepX, mannose-1-phosphate guanylyltransferase ManC is absent or a mutant mannose-1-phosphate guanylyltransferase ManC, or a Paenibacillus strain comprising a mutant levansucrase SacB is preferably a strain in which the wild-type PepR has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 and the mutant PepR comprises an amino acid sequence that is not 100% identical to any one of SEQ ID NOs: 1 to 7 or comprises a premature stop codon, or a strain in which wild-type PepH has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8, and mutant PepR comprises an amino acid sequence that is not 100% identical to any one of SEQ ID NOs:8-14, or comprises a premature stop codon; or a strain in which the wild-type PepX has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15, and the mutant PepX comprises an amino acid sequence that is not 100% identical to any one of SEQ ID NOs: 15 to 21, or comprises a premature stop codon; or a strain in which the wild-type ManC has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22, and the mutant ManC comprises an amino acid sequence that is not 100% identical to any one of SEQ ID NOs: 22-28, or comprises a premature stop codon; or A strain in which wild-type SacB has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29, and mutant SacB contains an amino acid sequence that is not 100% identical to any one of SEQ ID NOs:29-35 or contains a premature stop codon.

[0034] In one embodiment, the Paenibacillus strain is W224 * or a mutant PepR containing the S393F mutation.

[0035] In one embodiment, the Paenibacillus strain comprises a mutant PepH comprising an E96K mutation or an E163K mutation or an E96K and an E163K mutation.

[0036] In one embodiment, the Paenibacillus strain comprises a mutant PepH and an absent or mutant PepR.

[0037] In one embodiment, the Paenibacillus strain comprises a mutant PepH comprising an E96K mutation or an E163K mutation or an E96K and an E163K mutation, and a W224 * or a mutant PepR comprising an S393F mutation.

[0038] In one embodiment, the Paenibacillus strain comprises a mutant ManC comprising a P90S, E340K, or G433D mutation.

[0039] In one embodiment, the Paenibacillus strain comprises a mutant SacB that includes a G323S mutation.

[0040] Paenibacillus strains can be grown in culture broths and under culture conditions well known to those skilled in the art and disclosed, for example, in WO 16020371 (page 20, line 33 to page 22, line 29, which are incorporated herein by reference).

[0041] Paenibacilli can be used as a biopesticide to suppress or prevent pathogen infection of plants. Strains of Paenibacilli suitable for suppressing or preventing pathogen infection are disclosed, for example, in WO 2016 / 154297, WO 2019 / 221988, WO 2020 / 181053, WO 2019 / 155253, and WO 2018 / 195603. Many Paenibacillus strains that can suppress or prevent pathogen infection of plants produce Fusarididins. Thus, the strains disclosed herein are preferably made from Fusarididin-producing strains. Fusaricidins are a group of antibiotics isolated from Paenibacillus spp., a class of cyclic lipodepsipeptides, which often share the following structural features: a macrocyclic ring consisting of six amino acid residues (three of which are L-Thr, D-allo-Thr and D-Ala) and a 15-guanidino-3-hydroxypentadecanoic acid tail linked to the N-terminal L-Thr residue by an amide bond (ChemMedChem 7, 871-882, 2012; J. Microbiol. Meth. 85, 175-182, 2011, Table 1 herein). These compounds are cyclized by a lactone bridge between the N-terminal L-Thr hydroxyl group and the C-terminal D-Ala carbonyl group. The positions of the amino acid residues in the depsipeptide ring are usually numbered starting from the L-Thr as described above with the GHPD chain itself, and ending with the C-terminal D-Ala.Non-limiting examples of fusaricidins isolated from Paenibacillus are designated LI-F03, LI-F04, LI-F05, LI-F07 and LI-F08 (J. Antibiotics 40(11), 1506-1514, 1987; Heterocycles 53(7), 1533-1549, 2000; Peptides 32, 1917-1923, 2011) and fusaricidins A (also called LI-F04a), B (also called LI-F04b), C (also called LI-F03a) and D (also called LI-F03b) (J. Antibiotics 49(2), 129-135, 1996; J. Antibiotics 50(3), 220-228, 1997). The amino acid chain of fusaricidin is not produced by ribosomes, but by non-ribosomal peptide synthetases. The structural formulas of known fusaricidins are shown in Table 1 (Biotechnol Lett.34,1327-1334,2012; FIG. 1 thereof). The compounds named LI-F03a, LI-F03b to LI-F08a and LI-F08b and the fusaricidins of formula I and I.1 described herein are also called fusaricidins LI-F03a, LI-F03b to LI-F08a and LI-F08b due to their structure within the fusaricidin family (see, for example, Table 1).

[0042] Among the isolated fusaricidin antibiotics, fusaricidin A showed the most promising antibacterial activity against various clinically relevant fungi and Gram-positive bacteria such as Staphylococcus aureus (MIC values ​​range: 0.78-3.12 μg / mL) (ChemMedChem 7, 871-882, 2012). The synthesis of fusaricidin analogues containing 12-guanidino-dodecanoic acid (12-GDA) or 12-amino-dodecanoic acid (12-ADA) instead of the naturally occurring GHPD has been established, but replacement of GHPD with 12-ADA resulted in a complete loss of antibacterial activity, whereas replacement of GHPD with 12-GDA retained antibacterial activity (Tetrahedron Lett. 47, 8587-8590, 2006; ChemMedChem 7, 871-882, 2012).

[0043] [Table 1]

[0044] where the arrows define a single (amide) bond either between the carbonyl moiety of GHPD and the amino group of L-Thr (L-threonine) or between the carbonyl group of an amino acid and the amino group of an adjacent amino acid, with the arrowhead indicating the bond with the amino group of the amino acid L-Thr or of the adjacent amino acid, The single line without arrowhead defines a single (ester) bond between the carbonyl group of D-Ala (D-alanine) and the hydroxyl group of L-Thr; GHPD is 15-guanidino-3-hydroxypentadecanoic acid].

[0045] Fusaricidins A, B, C and D have also been reported to inhibit phytopathogenic fungi such as Fusarium oxysporum, Aspergillus niger, Aspergillus oryzae and Penicillium thomii (J. Antibiotics 49(2), 129-135, 1996; J. Antibiotics 50(3), 220-228, 1997). Fusaricidins such as Li-F05, LI-F07 and LI-F08 were found to have specific antifungal activity against various phytopathogenic fungi, such as Fusarium moniliforme, F. oxysporum, F. roseum, Giberella fujkuroi, Helminthosporium sesamum and Penicillium expansum (J.Antibiotics 40(11), 1506-1514, 1987). Fusaricidins also have antibacterial activity against gram-positive bacteria, including Staphylococcus aureus (J. Antibiotics 49, 129-135, 1996; J. Antibiotics 50, 220-228, 1997).Furthermore, fusaricidins have antifungal activity against Leptosphaeria maculans, which causes black root rot of rapeseed (Can. J. Microbiol. 48, 159-169, 2002). Furthermore, fusaricidins A and B, produced by certain Paenibacillus strains, and two related compounds in which D-allo-Thr is linked to an additional alanine via its hydroxyl group by means of an ester bridge, have been shown to induce resistance responses in cultured parsley cells and inhibit the growth of Fusarium oxysporum (WO 2006 / 016558; EP 1 788 074 A1).

[0046] WO 2007 / 086645 describes the fusaricidin synthetase enzyme and its encoding gene isolated from Paenibacillus polymyxa strain E681. Fusaricidin synthetase and its homologues in other Paenibacilli species are involved in the synthesis of fusaricidins A, B, C, D, LI-F03, LI-F04, LI-F05, LI-F07, and LI-F08.

[0047] The present invention therefore also provides an agricultural composition comprising: a) the absence of PepR or a mutant PepR, or b) the absence of PepH or a mutant PepH, or c) the absence of PepX or a mutant PepX, or d) the absence or mutation of ManC, or e) SacB is absent or a mutant SacB, or f) a strain of Paenibacillus sp. comprising a combination of at least two of a), b), c), d), e), or f); the absence of PepR or a mutant PepR results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepR; the absence of PepH or mutant PepH results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepH; the absence of PepX or mutant PepX results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepX; the absence of ManC or mutant ManC results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC; the absence of SacB or mutant SacB results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain that does not contain SacB; The agricultural compositions include those containing wild-type PepR, PepH, PepX, ManC, or SacB, respectively, and the strain of Paenibacillus sp. used for comparison is of the same Paenibacillus sp. as the strain in which PepR, PepH, PepX, ManC, or SacB is absent or contains a mutant thereof.

[0048] Preferably, the pesticide composition comprises W224 * or a mutant PepR containing the S393F mutation.

[0049] Preferably, the pesticide composition comprises a mutant PepH comprising an E96K mutation or an E163K mutation or an E96K and an E163K mutation.

[0050] Preferably, the pesticide composition comprises mutant PepH and absent or mutant PepR.

[0051] Preferably, the pesticide composition comprises a mutant PepH comprising the E96K mutation or the E163K mutation or the E96K and E163K mutations, and a W224 * or a mutant PepR comprising an S393F mutation.

[0052] Preferably, the pesticide composition comprises a mutant ManC that includes a P90S, E340K, or G433D mutation.

[0053] Preferably, the pesticide composition comprises a mutant SacB which comprises a G323S mutation.

[0054] The agricultural compositions are preferably pesticide compositions of the conventional type, such as solutions, emulsions, suspensions, dusts, powders, pastes, granules, presses, capsules, and mixtures thereof. Examples of types of compositions are given in "Catalogue of pesticide formulation types and international coding system", Technical Monograph No. 2, 6 th (see also Ed. May 2008, CropLife International) are suspensions (e.g. SC, OD, FS), emulsifiable concentrates (e.g. EC), emulsifiable concentrates (e.g. EW, EO, ES, ME), capsules (e.g. CS, ZC), pastes, pastilles, wettable powders or dusts (e.g. WP, SP, WS, DP, DS), presses (e.g. BR, TB, DT), granules (e.g. WG, SG, GR, FG, GG, MG), insecticidal articles (e.g. LN) as well as gel formulations (e.g. GF) for the treatment of plant propagation materials such as seeds. The compositions are prepared by known methods, for example as described by Mollet and Grubemann, Formulation technology, Wiley VCH, Weinheim, 2001; or Knowles, New developments in crop protection product formulation, Agrow Reports DS243, T&F Informa, London, 2005. The present invention also relates to an agrochemical composition comprising a Paenibacillus strain of the present invention and an adjuvant.

[0055] Suitable auxiliaries are solvents, liquid carriers, solid carriers or fillers, surfactants, dispersants, emulsifiers, wetting agents, adjuvants, solubilizers, penetration enhancers, protective colloids, adhesives, thickeners, moisturizers, repellents, attractants, feeding stimulants, compatibilizers, bactericides, antifreeze agents, antifoaming agents, colorants, tackifiers and binders. Suitable solvents and liquid carriers are water and organic solvents, such as medium to high boiling mineral oil fractions, e.g. kerosene, diesel oil; oils of vegetable or animal origin; aliphatic, cyclic and aromatic hydrocarbons, e.g. toluene, paraffins, tetrahydronaphthalene and alkylated naphthalenes; alcohols, e.g. ethanol, propanol, butanol, benzyl alcohol, cyclohexanol, glycols; DMSO; ketones, e.g. cyclohexanone; esters, e.g. lactates, carbonates, fatty acid esters, γ-butyrolactone; fatty acids; phosphonates; amines; amides, e.g. N-methylpyrrolidone, fatty acid dimethylamides; and mixtures thereof.

[0056] Suitable solid carriers or fillers are mineral earths, such as silicates, silica gel, talc, kaolin, limestone, lime, chalk, clay, dolomite, diatomaceous earth, bentonite, calcium sulfate, magnesium sulfate, magnesium oxide; polysaccharides, such as cellulose, starch; fertilizers, such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea; products of plant origin, such as grain meal, bark meal, wood meal, nut shell meal and mixtures thereof.

[0057] Suitable surfactants are surface active compounds such as anionic, cationic, nonionic and amphoteric surfactants, block polymers, polyelectrolytes and mixtures thereof. Such surfactants can be used as emulsifiers, dispersants, solubilizers, wetting agents, penetration enhancers, protective colloids or adjuvants. Examples of surfactants are listed in McCutcheon's, Vol. 1: Emulsifiers & Detergents, McCutcheon's Directories, Glen Rock, USA, 2008 (International Ed. or North American Ed.).

[0058] Suitable anionic surfactants are the alkali, alkaline earth or ammonium salts of sulfonates, sulfates, phosphates, carboxylates and their mixtures.Examples of sulfonates are alkylarylsulfonates, diphenylsulfonates, α-olefinsulfonates, ligninsulfonates, sulfonates of fatty acids and oils, sulfonates of ethoxylated alkylphenols, sulfonates of alkoxylated arylphenols, sulfonates of condensed naphthalenes, sulfonates of dodecyl- and tridecylbenzenes, sulfonates of naphthalenes and alkylnaphthalenes, sulfosuccinates or sulfosuccinamates.Examples of sulfates are the sulfates of fatty acids, oils, ethoxylated alkylphenols, alcohols, ethoxylated alcohols or fatty acid esters.Examples of phosphates are phosphate esters.Examples of carboxylates are alkyl carboxylates and carboxylated alcohols or alkylphenol ethoxylates.

[0059] Suitable nonionic surfactants are alkoxylates, N-substituted fatty acid amides, amine oxides, esters, sugar surfactants, polymeric surfactants, and mixtures thereof. Examples of alkoxylates are compounds such as alcohols, alkylphenols, amines, amides, arylphenols, fatty acids or fatty acid esters alkoxylated with 1 to 50 equivalents. Ethylene oxide and / or propylene oxide, preferably ethylene oxide, can be used for the alkoxylation. Examples of N-substituted fatty acid amides are fatty acid glucamides or fatty acid alkanolamides. Examples of esters are fatty acid esters, glycerol esters or monoglycerides. Examples of sugar surfactants are sorbitan, ethoxylated sorbitan, sucrose and glucose esters or alkyl polyglucosides. Examples of polymeric surfactants are homopolymers or copolymers of vinylpyrrolidone, vinyl alcohol or vinyl acetate.

[0060] Suitable cationic surfactants are quaternary surfactants, such as quaternary ammonium compounds with one or two hydrophobic groups or salts of long-chain primary amines.Suitable amphoteric surfactants are alkylbetaines and imidazolines.Suitable block polymers are AB or ABA type block polymers containing blocks of polyethylene oxide and polypropylene oxide or ABC type block polymers containing alkanol, polyethylene oxide and polypropylene oxide.Suitable polyelectrolytes are polyacids or polybases.Examples of polyacids are alkali salts of polyacrylic acid or polyacid comb polymers.Examples of polybases are polyvinylamines or polyethyleneamines.

[0061] Suitable adjuvants are compounds that have negligible or no insecticidal activity themselves and improve the biological performance of compound I against the target. Examples are surfactants, mineral or vegetable oils and other auxiliaries. Further examples are described in Knowles, Adjuvants and additives, Agrow Reports DS256, T&F Informa UK, 2006, chapter 5.

[0062] Suitable thickening agents are polysaccharides (eg xanthan gum, carboxymethylcellulose), inorganic clays (organically modified or unmodified), polycarboxylates and silicates.

[0063] Suitable fungicides are bronopol and isothiazolinone derivatives, such as alkylisothiazolinones and benzisothiazolinones.

[0064] Suitable antifreeze agents are ethylene glycol, propylene glycol, urea and glycerin.

[0065] Suitable antifoaming agents are silicones, long chain alcohols and salts of fatty acids.

[0066] Suitable colorants (for example red, blue or green) are pigments with low water solubility and water soluble dyes, such as inorganic colorants (for example iron oxide, titanium oxide, iron hexacyano) and organic colorants (for example alizarin colorants, azo colorants and phthalocyanine colorants).

[0067] Suitable tackifiers or binders are polyvinylpyrrolidone, polyvinyl acetate, polyvinyl alcohol, polyacrylates, biological or synthetic waxes and cellulose ethers.

[0068] The agrochemical composition generally contains 0.01 to 95% by weight of cells or spores of a Paenibacillus strain, preferably 0.1 to 90%, more preferably 1 to 70%, particularly 10 to 60%.

[0069] The amount of these cells or spores is preferably 5% w / w to 50% w / w, 10% w / w to 50% w / w, 15% w / w to 50% w / w, 30% w / w to 50% w / w, or 40% w / w to 50% w / w, or 5% w / w to 40% w / w, 10% w / w to 40% w / w, 15% w / w to 40% w / w, 30% w / w to 40% w / w, or 10% w / w to 40% w / w, 15% w / w to 40% w / w, or 30% w / w to 40% w / w.

[0070] The cells or spores of Paenibacillus strains are usually present in the form of solid particles with an average particle size of 1-150 μm, or in ascending order of preference 1-100 μm, 1-75 μm, 1-50 μm, 1-25 μm, 1-10 μm, 1-8 μm (determined by light scattering in a dispersion liquid according to CIPAC method 187).

[0071] The density value of spores per mL can be determined by measuring the number of colony forming units (CFU) on an agar medium, such as potato dextrose agar, after several days of incubation at a temperature of about 20 to about 35° C. The amount of CFU / g of biomass used to prepare the agrochemical composition of the present invention is typically less than 1×10 8 CFU / g ~1×10 11CFU / g, or 1 x 10 8 CFU / g ~1×10 10 CFU / g, or 5 x 10 8 ~5×10 10 CFU / g, preferably 1×10 9 CFU / g ~1×10 10 CFU / g. The CFU / g of biomass will influence the amount of biomass used to prepare the formulation of the present invention. Biomass with a relatively high amount of CFU / g can be used to prepare a formulation with a relatively low amount of biomass.

[0072] The amount of biomass used to prepare the formulations of the invention is usually selected to match the amount of CFU per hectare that should be applied for the respective purpose.

[0073] For the treatment of plant propagation material, especially seeds, usually solutions for seed treatment (LS), suspoemulsions (SE), flowable concentrates (FS), powders for dry treatment (DS), water-dispersible powders for slurry treatment (WS), water-soluble powders (SS), emulsifiable concentrates (ES), emulsifiable concentrates (EC) and gels (GF) are employed. The subject compositions, after dilution 2-10 times, give active substance concentrations of 0.01-60% by weight, preferably 0.1-40%, in ready-to-use formulations. Application can be carried out before or during sowing. Methods for applying the mixtures, or agrochemical compositions containing the mixtures, to young plants and propagation material such as seedlings, rooted / unrooted cuttings, plants derived from cell cultures, respectively, include dressing, coating, pelleting, dusting, immersion and infallo application methods. Preferably, the mixture and the agrochemical composition thereof are applied to the seeds in a manner such that germination is not induced, for example by seed dressing, pelleting, coating, and dusting, respectively.

[0074] Various oils, wetting agents, adjuvants, fertilizers or micronutrients, and further pesticides (e.g. fungicides, growth regulators, herbicides, insecticides, safeners) may be added to the mixture or its pesticidal composition as a premix or may be left until just before use (tank mix). These agents may be incorporated into the mixture or pesticidal composition according to the invention in a weight ratio of 1:100 to 100:1, preferably 1:10 to 10:1.

[0075] Paenibacillus strains and agrochemical compositions containing these strains are usually applied in fungicidally active amounts. The term "fungicidal amount" means an amount of a composition or mixture that is sufficient to control harmful fungal plants and does not cause substantial damage to treated plants, young plants such as seedlings, rooted / unrooted cuttings, plants derived from cell cultures, or plant propagation materials such as seeds. Such amounts can vary within a wide range and depend on various factors, such as the fungal species to be controlled, the plant species to be treated, the climatic conditions, and the specific mixture used.

[0076] When the agrochemical composition is used for foliar treatment or application to soil, preferably for foliar treatment, the application rate for foliar treatment is usually 50 g / ha to 2000 g / ha, 100 g / ha to 2000 g / ha, 150 g / ha to 2000 g / ha, 600 g / ha to 2000 g / ha, or 800 g / ha to 2000 g / ha of fusaricidin containing cells or spores in its volume, or 50g / ha~1000g / ha, 100g / ha~1000g / ha, 150g / ha~1000g / ha, 600g / ha~1000g / ha or 800g / ha~1000g / ha, or 50g / ha to 800g / ha, 100g / ha to 800g / ha, 150g / ha to 800g / ha, 600g / ha to 800g / ha, or 150g / ha~1000g / ha, 300g / ha~1000g / ha, 600g / ha~1000g / ha, Aqueous spray solution is applied at 1000L / ha to 100L / ha, 600L / ha to 100L / ha, 400L / ha to 100L / ha, 200L / ha to 100L / ha, or 1000L / ha~600L / ha, 1000L / ha~400L / ha, or 1000L / ha~200L / ha, or 600L / ha~200L / ha, 600L / ha~400L / ha.

[0077] When the pesticide composition is used for seed treatment, e.g., seed coating, the application rate for plant propagation material is usually about 1×10 1 ~1×10 12 (or more) CFU / seed, preferably about 1×10 3 ~Approx. 1×10 10 CFU / seed, more preferably about 1×10 3 ~Approx. 1×10 6 Alternatively, the application rate for plant propagation material is preferably in the range of about 1 x 10 CFU / 100 kg of seeds. 7 ~1×10 16 (or more) CFU, preferably 1 × 10 per 100 kg of seeds 9 ~Approx. 1×10 15 CFU, more preferably 1 x 10 per 100 kg of seeds 11 ~Approx. 1×10 15 CFU range.

[0078] Paenibacillus strains and agrochemical compositions comprising these strains are suitable as fungicides effective against a wide range of phytopathogenic fungi, including soil-borne fungi, in particular those from the classes Plasmodiophoromycetes, Peronosporomycetes (synonymous with Oomycetes), Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes (synonymous with Fungi imperfecti), respectively. They can be used in crop protection as foliar fungicides, seed dressing fungicides and soil fungicides.

[0079] The mixtures and their agrochemical compositions are preferably applied to various cultivated plants, for example cereals, for example wheat, rye, barley, triticale, oats, or rice; beets, for example sugar beet or fodder beet; fruits, for example pome fruits (apples, pears, etc.), stone fruits (e.g. plums, peaches, almonds, cherries), or soft fruits, also called berries (strawberries, raspberries, blackberries, currants, etc.); legumes (e.g. lentils, peas, alfalfa, or soybeans); oil plants, for example rapeseed, mustard, olives, sunflowers, coconuts, cocoa beans, castor oil plants, oil palm, peanuts, or soybeans; cucurbits, for example pumpkin, cucumber, or melon; fiber plants, for example cotton, flax, cannabis, or horse mackerel; citrus fruits, for example oranges, raspberries, blackberries, currants, etc. for the control of phytopathogenic fungi in: apples, lemons, grapefruits, or mandarins; vegetables, such as spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes, melons, or peppers; lauraceae plants, such as avocado, cinnamon, or camphor; energy and raw material plants, such as corn, soybeans, rapeseed, sugarcane, or oil palm; corn; tobacco; nuts; coffee; tea plant; bananas; grapes (table grapes and grape juice grapes); hops; turfgrass; cypress (also called stevia); natural rubber plants; or ornamental and forest plants, such as flowers, shrubs, broadleaf trees, or evergreen trees (conifers, eucalyptus, etc.); plant propagation material, such as seeds; and crop material of these plants.

[0080] More preferably, the mixture and its agrochemical composition, respectively, are used to control fungi on agricultural crops such as potatoes, sugar beets, tobacco, wheat, rye, barley, oats, rice, corn, cotton, soybeans, rapeseed, legumes, sunflowers, coffee or sugarcane; fruits; grapes; wine grapes or fruit grapes, ornamental plants; or vegetables such as cucumbers, tomatoes, peppers, beans or pumpkins.

[0081] The term "plant propagation material" is understood to refer to all reproductive parts of plants, such as seeds, and viable plant material that can be used to propagate plants, such as cuttings and tubers (e.g., potatoes). This includes seeds, roots, fruits, tubers, bulbs, rhizomes, shoots, sprouts, and other parts of plants, including seedlings and young plants that are transplanted after germination or emergence from the soil.

[0082] Thus, one embodiment of the present invention is a plant propagation material comprising the mixture or comprising a coating of an agrochemical composition comprising the mixture. Preferably, the plant propagation material is a young plant, such as a seedling, a rooted / unrooted cutting, or a plant derived from a cell culture. Even more preferably, the plant propagation material is derived from a fruit or vegetable plant species, including grapes.

[0083] According to the present invention, all the above mentioned cultivated plants are understood to include all species, subspecies, varieties and / or hybrids belonging to the respective cultivated plants, for example, corn is also known as Indian corn or maize (Zea mays), including all kinds such as fodder corn and sweet corn. According to the invention, all maize or maize subspecies and / or varieties are included, in particular flower corn (Zea mays var. amylacea), popcorn (Zea mays var. everta), dent corn (Zea mays var. indentata), flint corn (Zea mays var. indurata), sweet corn (Zea mays var. saccharata and var. rugosa), waxy corn (Zea mays var. ceratina), amylomaize (high amylose Zea mays varieties), guinea pig or wild maize (Zea mays var. tunicata) and striped maize (Zea mays var. japonica). The skilled person will be aware of other plant species and varieties, such as sweet pepper, determinate or indeterminate soybean, etc.

[0084] The mixtures of the present invention and their agrochemical compositions are particularly suitable for controlling the causative agents of the following plant diseases: Albugo spp. (white rust) in ornamentals, vegetables (e.g., A. candida) and sunflowers (e.g., A. tragopogonis); Alternaria spp. spp. (black spot disease) in vegetables (e.g. A. dauci or A. porri), in rapeseed (e.g. A. brassicicola or A. brassicae), in sugar beet (A. tenuis), in fruit (e.g. A. grandis), in rice, soybean, potato and tomato (e.g. A. solani, A. grandis or A. alternata), in tomato (e.g. A. solani or A. alternata) and in wheat (e.g. A. triticina); Aphanomyces spp. spp. in sugar beet and vegetables; Ascochyta spp. in cereals and vegetables, for example A. tritici (anthracnose) in wheat and A. hordei in barley; Aureobasidium zeae in maize (synonymous Kapatiella zeae); Bipolaris and Drechslera spp. (teleomorph: Cochliobolus spp.), for example brown spot (D. maydis) or brown spot (B. zeicola) in maize, for example spot disease (B. sorokiniana) in cereals, for example B. oryzae (B. zeicola) in rice and turfgrass.oryzae; Blumeria (formerly Erysiphe) graminis (powdery mildew) in cereals (e.g. wheat or barley); Botrytis cinerea (teleomorph: Botrytinia fuckeliana: grey mold) on fruits and berries (e.g. strawberries), vegetables (e.g. lettuce, carrots, celery and cabbage); B. squamosa or grey rot (B. allii) on onion, rapeseed, ornamentals (e.g. B eliptica), grapes, forest plants and wheat; Bremia lactucae on lettuce lactucae (downy mildew); Ceratocystis (syn. Ophiostoma) spp. (root rot or dieback disease) on deciduous and evergreen trees, e.g. C. ulmi (Dutch elm disease) on elms; Cercospora spp. spp.) (Cercospora leaf spot) in maize (e.g. gray leaf spot: C. zeae-maydis), rice, sugar beet (e.g. C. beticola), in sugarcane, vegetables, coffee, soybean (e.g. C. sojina or C. kikuchii) and rice; Cladobotryum (synonymous with Dactylium) species in mushrooms (e.g. C. mycophilum. (formerly Dactylium dendroides, teleomorph: Nectria albertinii, Nectria rosella (synonymous with Hypomyces rosellus); Cladosporium spp. in tomato (e.g. C. fulvum: leaf mold) and in cereals, e.g. C. herbarum (black spot) in wheat; Claviceps purpurea in cereals purpurea (grass); Cochliobolus (anamorph: Helminthosporium in Bipolaris) species (leaf spot) in corn (C. carbonum), cereals (e.g., C. sativus, anamorph: B. sorokiniana) and rice (e.g., C. miyabeanus, anamorph: H. oryzae); Colletotrichum (teleomorph: Glomerella) species (anthracnose) in cotton (e.g., C. gossypii), In corn (e.g. C. graminicola: anthracnose root rot), soft fruits, in potato (e.g. C. coccodes: black spot), in legumes (e.g. C. lindemuthianum), soybean (e.g. C. truncatum or C. gloeosporioides), vegetables (e.g. C. lagenarium or C. capsici), fruits (e.g. C. acutatum), coffee (e.g. C. coffeeum or C. kahawae) and in various crops C.gloeosporioides); Corticium spp. in rice, e.g. C. sasakii (sheath blight); Corynespora cassiicola (spot disease) in soybean, cotton and ornamentals; Cycloconium spp., e.g. C. oleaginum in olive; Cylindrocarpon spp. (e.g. fruit canker or young grapevine decline, teleomorph: Nectria or Neonectria spp.) in fruit trees, in grapes (e.g. C. liriodendri, teleomorph: Neonectria liriodendri, liriodendri, black leg disease) and ornamental plants; Dematophora (teleomorph: Roselinia) and necatrix (root and stem rot) in soybean; Diaporthe species in soybean spp., such as D. phaseolorum (seedling damping-off); Drechslera (synonym Helminthosporium, teleomorph: Pyrenophora) species in maize, in cereals such as barley (e.g. D. teres, net blotch) and in wheat (e.g. D. tritici-repentis: tan spot), in rice and turfgrass; in grapes, Formitiporia (synonym Phellinus) punctata, F. mediterranea (F.mediterranea, Phaeomoniella chlamydospora (previously Phaeoacremonium chlamydosporum), Phaeoacremonium aleophilum and / or Esca disease (canker, apoplexy) caused by Botryosphaeria obtusa; Elsinoe spp. in pome fruits (E. pyri) and soft fruits (E. veneta: anthracnose) and in grapes (E. ampelina: anthracnose); Entyloma oryzae in rice; oryzae (leaf mildew); Epicoccum spp. (black mold) on wheat; Erysiphe spp. (powdery mildew) on sugar beet (E. betae), on vegetables (e.g. E. pisi), for example on cucurbits (e.g. E. cichoracearum) and on cabbage, rapeseed (e.g. E. cruciferarum); Eutypa lata (Eutypa canker or blight, anamorph: Cytosporina lata, Libertella blepharis) on fruit trees, grapes and ornamentals. blepharis); Exserohilum (synonymous with Helminthosporium) species in maize (e.g. E. turcicum); Fusarium (teleomorph: Gibberella) species in various plants (damage, root or stem rot), such as F. graminearum or F. culmorum (root rot, scab or red mold) in cereals (e.g. wheat or barley), F. oxysporum (F.oxysporum, F. solani (sp. glycines, now synonymous with F. virguliforme) and F. tucumaniae and F. brasiliense, each of which causes sudden death syndrome in soybean, and F. verticillioides in maize; Gaeumannomyces graminis (damaging disease) in cereals (e.g. wheat or barley) and maize; Gibberella spp. in cereals (e.g. G. zeae) and in rice (e.g. G. fujikuroi, bakanae disease); Glomerella cingulata in grapes, pome fruits and other plants and G. gossypii in cotton; grain stain complex in rice; Guignardia bidwellii (black rot) in grapes; Gymnosporangium spp. in roses and junipers. spp., e.g. G. sabinae (rust) on pear; Helminthosporium spp. (syn. Drechslera, teleomorph: Cochliobolus) on maize, cereals, potato and rice; Hemileia spp., e.g. H. vastatrix (coffee leaf rust) on coffee; Isariopsis clavispora (syn. Cladosporium vitis) on grape; Macrophomina phaseolina on soybean and cotton. phaseolina (synonymous with phaseoli) (root and stem rot); Microdochium (synonymous with Fusarium) nivale (pink snow mold) in cereals (e.g. wheat or barley); Microsphaera diffusa (powdery mildew) in soybeans; Monilinia spp., e.g. M. laxa, M. fructicola and M. fructigena (Monilia spp.) in stone fruits and other Rosaceae plants.synonymous with: flower blight and branch blight, brown rot; Mycosphaerella spp. in cereals, bananas, soft fruits and groundnuts, e.g. M. graminicola in wheat (anamorph: Zymoseptoria tritici, formerly Septoria tritici: Septoria leaf spot) or M. fijiensis in bananas (synonymous with Pseudocercospora fijiensis: black Sigatoka disease) and M. musicola, M. arachidicola (M. arachidis or Cercospora arachidis) arachidis), M. berkeleyi in groundnut, M. pisi in pea and M. brassiciola in cruciferous plants; Peronospora spp. (downy mildews) in cabbage (e.g. P. brassicae), rapeseed (e.g. P. parasitica), onion (e.g. P. destructor), tobacco (P. tabacina) and soybean (P. manshurica); Phakopsora pachyrhizi in soybean; pachyrhizi and P. meibomiae (soybean rust); Phialophora spp., for example in grapes (e.g. P. tracheiphila and P. tetraspora) and in soybeans (e.g. P. gregata: stem rot); Phoma lingam (Leptosphaeria biglobosa and L. macranthus) in rapeseed and cabbage;maculans (synonymous with Phyllostica zeae) in sugar beet (root rot, leaf spot and seedling damping-off) and P. zeae-maydis (synonymous with Phyllostica zeae) in maize; Phomopsis spp. in sunflower, grape (e.g. P. viticola: stem and leaf spot) and soybean (e.g. stem rot: P. phaseoli, teleomorph: Diaporthe phaseolorum); Physoderma maydis (brown spot) in maize; Phytophthora spp. in various plants. spp. (damage, root, leaf, fruit and stem rot) in peppers and cucurbits (e.g. P. capsici), in soybeans (e.g. P. megasperma, synonymous with P. sojae), in potatoes and tomatoes (e.g. P. infestans: leaf rot) and in deciduous trees (e.g. P. ramorum: oak death); Plasmodiophora brassicae (club root disease) in cabbage, rapeseed, radish and other plants; Plasmopara spp. spp., such as P. viticola on grapes (downy mildew) and P. halstedii on sunflower; Podosphaera spp. (powdery mildew) on roses, hops, pome fruits and soft fruits (e.g. P. leucotricha on apples) and on cucurbits (P. xanthii); Polymyxa spp., such as P. graminis on cereals such as barley and wheat (P. graminis) and on sugar beet (P. bethae).betae) and the viral diseases transmitted by it; cereals, such as wheat or barley, with Pseudocercosporella herpotrichoides (syn. Oculimacula yallundae, O. acuformis: eyespot disease, teleomorph: Tapesia yallundae); various plants, such as Pseudoperonospora (downy mildew), such as P. cubensis on cucurbits or P. humili on hops; grapes, with Pseudopezicula tracheifila. tracheiphila (red fireworks or Rotbrenner, anamorph: Phialophora); Puccinia species on various plants spp. (rusts), in cereals such as wheat, barley or rye with P. triticina (brown rust or leaf rust), P. striiformis (stripe rust or yellow rust), P. hordei (stunt rust), P. graminis (stem rust or black rust) or P. recondita (brown rust or leaf rust), in sugarcane with P. kuehnii (orange rust) and in asparagus with P. asparagi; in rapeseed with Pyrenopeziza spp. spp., such as P. brassicae; Pyrenophora (anamorph: Drechslera)-tritici-repentis (tan spot) in wheat or P. teres (net blotch) in barley; Pyricularia spp., such as P. oryzae (teleomorph: Magnaporthe grisea: rice blast) in rice and P. in turfgrass and cereals.Pythium spp. (seedling damping off) in turfgrass, rice, corn, wheat, cotton, rapeseed, sunflower, soybean, sugar beet, vegetables and various other plants (e.g. P. ultimum or P. aphanidermatum) and P. oligandrum in mushrooms; Ramularia spp., e.g. R. collo-cygni (ramularia leaf spot, physiological leaf spot) in barley, R. areola (teleomorph: Mycosphaerella areola) in cotton. areola) and R. beticola in sugar beet; Rhizoctonia spp. in cotton, rice, potato, turfgrass, corn, rapeseed, potato, sugar beet, vegetables and various other plants, such as R. solani (root and stem rot) in soybean, R. solani (sheath blight) in rice or R. cerealis (Rhizoctonia spring blight) in wheat or barley; Rhizopus stolonifer (black mold, soft rot) in strawberry, carrot, cabbage, grapes and tomato; Rhynchosporium secalis and R. commune (fire blight) in barley, rye and triticale; Sarocladium oryzae and S. attenuatum (sheath rot) in rice; Sclerotinia species in vegetables (S. minor and S. sclerotiorum) and field crops. spp. (stem rot or white mold disease) in e.g. rapeseed, sunflower (e.g. S. sclerotiorum) and soybean, S. rolfsii (synonymous Athelia rolfsii) in soybean, peanut, vegetables, maize, cereals and ornamentals; Septoria spp. in various plants, e.g. S. glycines (brown spot disease) in soybean, S. tritici in wheat (Zymoseptoria tritici, Septoria leaf spot) and S. (synonymous with Stagonospora) nodorum (Stagonaspora leaf spot) in cereals; Uncinula (synonymous with Erysiphe) necator (powdery mildew, anamorph: Oidium tuckeri) in grapes; Setosphaeria spp. (black leaf blight) in maize (e.g. S. turcicum, synonymous with Helminthosporium turcicum) and in turfgrass; Sphacelotheca spp. (sooty mildew) in maize (e.g. S. leiliana, S.reiliana (synonym: Ustilago reiliana: smut) in millet and sugarcane; Sphaerotheca fuliginea (synonym: Podosphaera xanthii: powdery mildew) in cucurbits; Spongospora subterranea (powdery scab) and the viral diseases it transmits in potatoes; Stagonospora spp. in cereals, e.g. S. nodorum (Stagonaspora spot, teleomorphs: Leptosphaeria [synonym: Phaeosphaeria] nodorum, Septoria nodorum (Septoria nodorum); Synchytrium endobioticum in potato (potato wart); Taphrina spp., e.g. T. deformans in peach (leaf curl) and T. pruni in plum (pocket plum); Thielaviopsis spp. (black root rot) in tobacco, pome fruit, vegetables, soybean and cotton, e.g. T. basicola (synonymous with Chalara elegans); Tilletia spp. in cereals spp. (common or smut), e.g. T. tritici (synonymous with T. caries, net smut) and T. controversa (stunt smut) in wheat; Trichoderma harzianum in mushrooms; Typhula incarnata (grey snow mold) in barley or wheat; Urocystis spp., e.g. U. occulta (striped sooty mold) in rye; Uromyces spp. (rust) in vegetables, e.g. in legumes (e.g. U.U. appendiculatus (syn. U. phaseoli), in sugar beet (e.g. U. betae or U. beticola) and legumes (e.g. U. vignae, U. pisi, U. viciae-fabae and U. fabae); Ustilago spp. (naked smut) in cereals (e.g. U. nuda and U. avaenae), in maize (e.g. U. maydis: maize sooty mold) and in sugar cane; Venturia spp. spp. (black spot) in apples (e.g. V. inaequalis) and in pears; and Verticillium spp. (damping off) in various plants, such as fruit and ornamental plants, grapes, soft fruits, vegetables and field crops, e.g. V. longisporum in rapeseed, V. dahliae in strawberry, rapeseed, potato and tomato, and V. fungicola in mushrooms; Zymoseptoria tritici in cereals.

[0085] The mixture and the agrochemical composition thereof are particularly suitable for controlling the following plant pathogenic fungal genera: Alternaria, Botrytis, Venturia, Leptosphaeria, Fusarium, Rhizoctonia, Phytophthora, Pythium, Colletotrichum, Pyricularia, Sclerotinia, and Zymoseptoria.

[0086] The mixture and the pesticide composition thereof are each effective for inhibiting Alternaria solanum, Alternaria alternata, Alternaria brassicae, Alternaria brassicicola, Alternaria citri, Alternaria mali, Botrytis cinerea, Botrytis allii, Botrytis fabae, Botrytis squamosa, Venturia inaequalis, Venturia effusa, Venturia carpophila, Venturia pirina, Venturia serrata ... pyrina, Leptosphaeria maculans, Leptosphaeria nodorum, Fusarium oxysporum, Fusarium graminearum, Fusarium verticillioides, Rhizoctonia solani, Phytophthora infestans, Phytophthora capsici, Phytophthora fragariae, Phytophthora nicotianae, Phytophthora sojae, Pythium ultimum ultimum, Pythium acanthicum, Pythium deliense, Pythium graminicola, Pythium heterothalicumIt is particularly suitable for controlling phytopathogenic fungi such as Alternaria solani, Botrytis cinerea, Venturia inaequalis, Leptosphaeria maculans, Leptosphaeria nodorum, Fusarium oxysporum, Fusarium graminearum, Rhizoctonia solani, Phytophthora infestans, Pythium ultimum, Colletotrichum orbiculare, Pyricularia oryzae, Sclerotinia sclerotiorum, and Zymoseptoria tritici.

[0087] The mixture and its pesticidal composition are each particularly suitable for controlling phytopathogenic fungi such as Alternaria solanum, Botrytis cinerea, Venturia inaequalis, Leptosphaeria maculans, Leptosphaeria nodorum, Fusarium oxysporum, Fusarium graminearum, Rhizoctonia solani, Phytophthora infestans, Pythium ultimum, Colletotrichum orbiculare, Pyricularia oryzae, Sclerotinia sclerotiorum, and Zymoseptoria tritici.

[0088] The mixture and the agrochemical composition thereof are particularly suitable for controlling the phytopathogenic fungi Alternaria solanum, Botrytis cinerea, Leptosphaeria nodorum, Colletotrichum orbiculare, Pyricularia oryzae, Sclerotinia sclerotiorum, and Zymoseptoria tritici, respectively.

[0089] The present invention therefore also relates to a method for inhibiting or preventing fungal infection in plants, comprising the steps of: a) the absence of PepR or a mutant PepR, or b) the absence of PepH or a mutant PepH, or c) the absence of PepX or a mutant PepX, or d) the absence or mutation of ManC, or e) SacB is absent or a mutant SacB, or f) treated with an effective amount of a strain of Paenibacillus sp., comprising a strain of Paenibacillus sp. comprising a combination of at least two of a), b), c), d), e), or f); the absence of PepR or a mutant PepR results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepR; the absence of PepH or mutant PepH results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepH; the absence of PepX or mutant PepX results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepX; the absence of ManC or mutant ManC results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC; the absence of SacB or mutant SacB results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain that does not contain SacB; The method includes a method comprising: (a) detecting a strain of Paenibacillus sp. containing a respective wild-type PepR, PepH, PepX, ManC, or SacB, and used for comparison, the strain of Paenibacillus sp. being of the same Paenibacillus sp. as the strain containing no PepR, PepH, PepX, ManC, or SacB or a mutant thereof.

[0090] Paenibacillus strains produce plant growth products such as indole acetic acid. Thus, a) PepR is absent or a mutant PepR; b) the absence of PepH or a mutant PepH, or c) the absence of PepX or a mutant PepX, or d) the absence or mutation of ManC, or e) SacB is absent or a mutant SacB, or f) a combination of at least two of a), b), c), d), e), or f); the absence of PepR or a mutant PepR results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepR; the absence of PepH or mutant PepH results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepH; the absence of PepX or mutant PepX results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepX; the absence of ManC or mutant ManC results in a decrease in viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC; the absence of SacB or mutant SacB results in a decrease in the viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain that does not contain SacB; The strains of Paenibacillus sp. containing the respective wild-type PepR, PepH, PepX, ManC, or SacB and used for comparison are of the same Paenibacillus sp. as the strains containing the absence or mutants of PepR, PepH, PepX, ManC, or SacB. These Paenibacillus strains can also be used in methods of promoting plant growth by treating the plants, seedlings, seeds, or the soil adjacent to the plants or seeds.

[0091] Paenibacillus strains are also known to solubilize plant nutrients, therefore the strains disclosed herein can also be used to mobilize plant nutrients provided by organic or inorganic fertilizers.

[0092] In another aspect, the present invention also includes a method for producing a valuable product by fermentation of a strain of Paenibacillus sp. comprising a) an absent or mutant PepR, or b) an absent or mutant PepH, or c) an absent or mutant PepX, or d) an absent or mutant SacB, or a combination of at least two of a), b), c), or d).

[0093] The valuable product may be any product of commercial value that can be produced by fermentation of Paenibacilli. Non-limiting examples of such products are enzymes used in industrial applications such as food or feed processing, detergents or chemical synthesis. Examples of such enzymes are phytases, chitinases, proteases, mannanases, xylanases, cellulases, laccases or lipases. These enzymes may be enzymes that are also expressed by wild-type Paenibacillus strains or enzymes that are heterologous to the respective Paenibacillus strain and expressed from a transgene. Other examples are proteins (without enzymatic activity), for example for use as replacements for animal proteins such as milk or meat substitutes.

[0094] Further examples of valuable products are secondary metabolites such as vitamins or other chemicals of commercial value such as 2,3-butanediol, lactic acid or acetoin. When the valuable product is 2,3-butanediol, lactic acid or acetoin, the strain of Paenibacillus sp. preferably comprises a mutant or wild-type PepH and / or a mutant or wild-type SacB.

[0095] Further examples of valuable products are antibacterial compounds such as polymyxins, octapeptins, polypeptins, pergipeptins, fusaricidins or lantibiotics.

[0096] An additional example of a valuable product is EPS produced by a strain of Paenibacillus sp. comprising a) absent or mutant PepR, or b) absent or mutant PepH, or c) absent or mutant PepX, or d) absent or mutant SacB, or a combination of at least two of a), b), c), or d), wherein the composition of the EPS differs from that of a wild-type strain comprising wild-type PepR, PepH, PepX, and SacB.

[0097] The technical teachings of the present invention are expressed herein using linguistic means, in particular by using scientific and technical terms. However, those skilled in the art will understand that the linguistic means, however detailed and precise they may be, may only approximate the complete content of the technical teachings, if only because there are multiple ways of expressing the teachings, each of which is necessarily impossible to fully express all conceptual connections, since each expression must necessarily be complete. With this in mind, those skilled in the art will understand that the subject matter of the present invention is the sum of the individual technical concepts shown herein or necessarily expressed in a pulse-prototype manner by the inherent constraints of the present specification. In particular, those skilled in the art will understand that the expression of the individual technical concepts is made herein as a shorthand for detailing each possible combination of the concepts as far as technically practical, so that, for example, the disclosure of three concepts or embodiments A, B and C is a shorthand for the concepts A+B, A+C, B+C, A+B+C. In particular, alternatives regarding features are described herein with reference to a list that aggregates the alternatives or examples. Unless otherwise stated, the invention described herein includes any combination of such alternatives. Selection of more or less preferred elements from such lists is part of the invention and is subject to the preference of one skilled in the art to realize to the minimum extent the advantages conveyed by each feature. Such multiple combined embodiments represent suitably preferred forms of the invention. EXAMPLES

[0098] Example 1 Generation of mutants: The list of strains used for targeted integration of point mutations by CRISPR Cas9 in P. polymyxa is shown in Table 1. Cloning and propagation of plasmids were performed in either E. coli DH5α from NEB (New England Biolabs, USA). Transformation of P. polymyxa was performed by conjugation via E. coli S17-1 (DSMZ). Strains were grown in LB or TSB medium. 1.5% agar was used for plating. When necessary, media was supplemented with 50 μg / mL neomycin and / or 20 μg / L polymyxin. P. polymyxa was grown at 30 °C and 250 rpm, while E. coli was at 37 °C and 250 rpm.

[0099] [Table 2]

[0100] Lu17007 was isolated from a German acreage and deposited at the German Microbial Control Agency (DSMZ) under the Budapest Treaty on February 20, 2013 under the accession number DSM26970.

[0101] Ruetering M, Cress BF,Schilling M,Ruehmann B,Koffas MAG,Sieber V,Schmid J.Tailor-made exopolysaccharides-CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa.Synth Biol(Oxf).2017 Dec 21;2(1):ysx007.doi:10.1093 / synbio / ysx007. Conjugation was performed between P. polymyxa (recipient strain) and E. coli S17-1 (donor strain) carrying the plasmid of interest according to the CRISPR Cas9 procedure described in. Verification of correct conjugants was performed by colony PCR and sequencing of DNA fragments. Curing of the plasmid was performed by subculturing positive mutants 1:100 in LB liquid medium at 37C.

[0102] Modification or knockout of the pepR and sacB genes was achieved by the CRISPR-Cas9 mediated system established by Ruetering M, Cress BF, Schilling M, Ruehmann B, Koffas MAG, Sieber V, Schmid J. Tailor-made exopolysaccharides-CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa. Synth Biol (Oxf). 2017 Dec 21; 2(1): ysx007. doi: 10.1093 / synbio / ysx007. The selected gRNA sequences were chosen based on their closest position to the target position within the pepR and sacB genes. As adjacent homologues, 1000 bp of adjacent genes from pepR or sacB, respectively, were used. The pCasPP vector system was used to generate the following 1000 mutations (Table 2):

[0103] [Table 3]

[0104] Example 2 Fermentation conditions and characterization of mutants: Characterization of the mutants was performed in a 21 L bioreactor (Techfors, Infors) filled with 12 L of exopolysaccharide production medium adapted from Ruetering M, Cress BF, Schilling M, Ruehmann B, Koffas MAG, Sieber V, Schmid J. Tailor-made exopolysaccharides-CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa. Synth Biol(Oxf). 2017 Dec 21;2(1):ysx007. doi:10.1093 / Synbio / ysx007. The composition of the fermentation medium is listed in Table 3.

[0105] Table 3: Composition of exopolysaccharide production medium with storage (room temperature (RT) or 4 °C) and sterilization method (sterile filtration / autoclave, s / a) specifications for stock solution.

[0106] [Table 4]

[0107] Fermentation was carried out for 40 h at 30° C., pH was set to 6.8 and adjusted with H3PO4 (25%) and NaOH (1 M). As precultures, all mutants were grown in 1 L baffled shake flasks containing 100 mL of modified TSB medium (30 g / L TSB, 3 g / L yeast extract, 20.9 g / L MOPS, 10 g / L glucose from Becton Dickenson Art. Nr. 211825) at 33° C. and 150 rpm / 2.5 cm shake diameter for 24 h.

[0108] In the bioreactor, a target dissolved oxygen level was set above 30% with a stirrer-gas flow cascade. To prevent detachment of the produced exopolysaccharides, agitation was limited to 300–600 rpm while using a stirring setup consisting of two propellers and one Rushton, the latter placed close to the stirring shaft. To maintain oxygen supply, aeration was performed at 5–30 L / min at a pressure of 0.5 bar. Struktol J673 (Schill+Seilacher “Struktol” GmbH, Germany) was used as an antifoam agent. Culture samples were taken every 4 h for rheological viscosity analysis and further offline analysis.

[0109] Example 3 Rheological analysis of culture broth viscosity: Rheological analysis of broth viscosity was performed every 4 hours during fermentation using an Anton Paar MCR302 rheometer in double slide geometry (Measuring Cup: C-DG26.7 / SS / Air, Temperature: 30°C, Sample Volume: 5 mL of total culture broth). Samples were preconditioned in a preshear experiment for 100 seconds at a constant shear rate of 10 s-1. Ten data points were recorded every 10 seconds. After preconditioning, viscosity was measured as a function of shear rate. Thus, the shear rate was logarithmically increased from 1 s-1 to 100 s-1, with a total of 25 data points recorded. Culture broth viscosity measured over the fermentation time is illustrated in Figure 1.

Claims

1. A strain of Paenibacillus sp., a) absent or mutant PepR, or b) mutant PepH, or c) absent or mutant PepX, or d) mutant ManC, or e) a mutant SacB, or f) a combination of at least two of a), b), c), d), or e). Including, the absence of PepR or the mutant PepR results in a decreased viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepR; the variant PepH results in a reduced viscosity of a liquid culture of a strain of Paenibacillus sp. compared to a liquid culture of a strain of Paenibacillus sp. comprising wild-type PepH; the absence of PepX or the mutant PepX results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type PepX; the mutant ManC results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain containing wild-type ManC or compared to a liquid culture of a Paenibacillus sp. strain not containing ManC; the mutant SacB results in a reduced viscosity of a liquid culture of a Paenibacillus sp. strain compared to a liquid culture of a Paenibacillus sp. strain that does not contain SacB; the Paenibacillus sp. strain containing wild-type PepR, PepH, PepX, ManC, or SacB and used for comparison is of the same Paenibacillus sp. as the strain not containing PepR, PeppH, PepX, ManC, or SacB or a mutant thereof; A strain of Paenibacillus sp.

2. a) the wild-type PepR has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, and the mutant PepR contains amino acids that are different from the amino acids at the same positions as the 100% conserved amino acids in the alignment shown in Figure 2; or b) the wild-type PepH has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, and the variant PepH contains amino acids that differ from the amino acids at the same positions as the 100% conserved amino acids in the alignment shown in Figure 3; or c) the wild-type PepX has an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15, and the mutant PepX contains amino acids that are different from the amino acids at the same positions as the 100% conserved amino acids in the alignment shown in Figure 4; or d) the wild-type ManC has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22, and the mutant ManC contains amino acids that differ from the amino acids at the same positions as the 100% conserved amino acids in the alignment shown in Figure 5; or e) the wild-type SacB has an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, and the mutant SacB contains amino acids that differ from the amino acids at the same positions as those that are 100% conserved in the alignment shown in Figure 6; 2. The strain of Paenibacillus sp. according to claim 1.

3. a) PepR is absent or contains a mutant PepR, preferably W224 ＊ or PepR with the S393F mutation, or b) comprising a mutated PepH, preferably comprising PepH with the E96K or E163K or E96K and E163K mutations, or c) PepR is absent or comprises a mutant PepR, preferably a truncated PepR, or d) comprising a mutated ManC, preferably comprising a ManC with a P90S, E340K, or G433D mutation; or e) containing a truncated SacB or a SacB with a G323S mutation; 2. The strain of Paenibacillus sp. according to claim 1.

4. 2. The Paenibacillus sp. strain of claim 1, which is free of PepR.

5. 2. The Paenibacillus sp. strain of claim 1, which is free of PepX.

6. 2. The Paenibacillus sp. strain of claim 1, wherein the Paenibacillus species is selected from Paenibacillus polymyxa, Paenibacillus jamilae, Paenibacillus ottowii, Paenibacillus terrae, and Paenibacillus kribensis.

7. The parent strain of the Paenibacillus sp. strain is P. polymyxa strain DSM365, P. polymyxa strain PKB1, P. polymyxa strain JB05-01-1, P. polymyxa strain AC-1, P. P. polymyxa strain HY96-2, Paenibacillus sp. Aloe-11, Paenibacillus sp. strains NRRL B-50972, NRRL B-67129, NRRL B-67304, NRRL B-67306, and NRRL B-67615, NRRL B-50374, NRRL B-67721, NRRL B-67723, NRRL B-67724, P. P. polymyxa strain VMC10 / 96, Paenibacillus sp. strain 10.6D, Paenibacillus sp. strain 9.4E, Paenibacillus sp. strains Lu16774, Lu17007, and Lu17015, Paenibacillus polymyxa strain M1, Paenibacillus polymyxa strain SC2, Paenibacillus.

2. The strain of Paenibacillus sp. according to claim 1, which is selected from the group of strains comprising Paenibacillus polymyxa strain Sb3-1, Paenibacillus polymyxa strain Sb3-2, and Paenibacillus polymyxa strain E681.

8. 10. An agricultural composition comprising the strain of Paenibacillus sp. according to claim 1.

9. 9. Plant propagation material comprising the strain of Paenibacillus sp. according to claim 1 or coated with the agricultural composition according to claim 8.

10. A method for suppressing or preventing fungal infection in plants, wherein the fungus, its habitat, or material or plant to be protected from fungal attack, or soil or plant propagation material, is treated with an effective amount of a strain of Paenibacillus sp. described in claim 1 or an agricultural composition described in claim 8.

11. 1. A method for producing a valuable product by fermentation, comprising: 1) culturing the strain of Paenibacillus sp. according to claim 1 in a culture medium under conditions in which the valuable product is produced; 3) collecting said valuable product; A method comprising:

12. said valuable product being a) an enzyme or protein, or b) a metabolite of Paenibacillus or c) 2,3-butanediol, lactic acid, or acetoin, or d) The EPS produced by this strain.

12. A method for producing a product of value according to claim 11.

13. 9. Use of the strain of Paenibacillus sp. according to claim 1 in an agricultural composition according to claim 8.

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