Mutants of paenibacillus and methods for use thereof

Abstract

To provide methods for producing and identifying Paenibacillus sp. strains with enhanced fungicidal activity and processability.SOLUTION: The present invention provides a composition comprising a biologically pure culture of a Paenibacillus sp. strain comprising a mutant DegU lacking a functional receiver domain or a functional DNA binding domain and / or a mutant DegS lacking a functional single binding domain or a functional ATPase domain with decreased viscosity in a liquid culture. Also provided is a method of identifying a Paenibacillus sp. mutant derivative strain with decreased viscosity in a liquid culture compared to a Paenibacillus sp. parental strain with a visual screen for mutant with a non-mucoid morphology.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the field of bacterial strains and their ability to control plant diseases. In particular, the present invention is directed to a strain of Paenibacillus sp. that has a relatively high level of antifungal activity and reduced viscosity that facilitates downstream processing and concentration of the whole culture product of the strain.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS: This application claims priority to U.S. Provisional Patent Application No. 62 / 671,067, filed May 14, 2018, the entire contents of which are incorporated herein by reference.

[0003] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING: An official copy of the Sequence Listing is being submitted contemporaneously herewith electronically via EFS-Web in an ASCII format Sequence Listing entitled "BCS169009_WO_ST25.txt", a 29 kilobyte file created on April 30, 2019. The sequence listing contained in this ASCII document is a part of this specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Paenibacillus is a genus of low GC content, endospore-forming, Gram-positive bacteria (phylum Firmicutes). Bacteria belonging to this genus are prolific producers of industrially relevant extracellular enzymes and antibacterial substances, including non-ribosomal peptide classes such as fusaricidins and polymyxins. Fusaricidins are known to have antimicrobial activity against a variety of phytopathogenic fungi and bacteria.

[0005] Many Paenibacillus species are prolific producers of exopolysaccharides (EPS). Microbial EPS are water-soluble biopolymers that are attached to the cell surface and released into the extracellular medium. These polymers are used commercially as thickening agents in a wide range of industries, including food, feed, packaging, cosmetics and pharmaceutical industries, due to their physicochemical and rheological properties. The production of EPS leads to an increase in the viscosity of whole broth samples, especially as a result of high molecular weight species. The increase in broth viscosity poses problems for downstream processing of broth material intended for bioreactor growth and live bacterial whole broth products. Costly and labor-intensive procedures may be required to remove EPS from large-scale fermentation liquid cultures before further processing. Summary of the Invention [Problem to be solved by the invention]

[0006] There is a need for methods to produce and identify strains of Paenibacillus sp. that exhibit reduced viscosity and higher levels of fusaricidin and fusaricidin-like compounds, thereby increasing fungicidal activity and processability. [Means for solving the problem]

[0007] The present invention is directed to strategies to enhance the fungicidal activity and processing suitability of Paenibacillus strains and their mutant derivatives. A strain improvement strategy was devised to enhance the production of fusaricidin by successive rounds of chemical treatment and high-throughput screening. Furthermore, a visual screening was developed to reduce the viscosity of the fermentation broth culture to improve the large-scale growth and downstream processing of fungicidal mutant derivatives of Paenibacillus strains. Several Paenibacillus strains with improved fungicidal and processing properties were generated and characterized.

[0008] In some embodiments, the present invention relates to a composition comprising a biologically pure culture of a strain of Paenibacillus comprising a mutant DegU lacking a functional receiver domain or a functional DNA-binding domain and / or a mutant DegS lacking a functional single-binding domain or a functional ATPase domain, wherein the mutant DegU and / or mutant DegS results in a liquid culture of the Paenibacillus strain having reduced viscosity compared to a liquid culture of a Paenibacillus strain comprising a wild-type DegU and a wild-type DegS.

[0009] In certain aspects, the mutant DegU and / or mutant DegS inhibit colony formation of a strain of Paenibacillus having a mucoid morphology.

[0010] In one embodiment, the mutant DegU and / or mutant DegS are knockout or truncated as a result of a premature stop codon, hi one aspect, the premature stop codon results in a mutant DegU that is truncated at position 218, numbered according to the amino acid sequence of SEQ ID NO:2.

[0011] In other embodiments, the mutant DegU has an amino acid substitution of a small residue at position 109, numbered according to the amino acid sequence of SEQ ID NO:2, for an acidic residue; and / or an amino acid substitution of a small residue at position 228, numbered according to the amino acid sequence of SEQ ID NO:2, for a polar residue; and / or an amino acid substitution of an acidic residue at position 63, numbered according to the amino acid sequence of SEQ ID NO:2, for a polar residue; and / or an amino acid substitution of a polar residue at position 195, numbered according to the amino acid sequence of SEQ ID NO:2, for a small residue; and / or an amino acid substitution of a hydrophobic residue at position 204, numbered according to the amino acid sequence of SEQ ID NO:2, for a small residue; and / or an amino acid substitution of a hydrophobic residue at position 210, numbered according to the amino acid sequence of SEQ ID NO:2, for a small residue; and / or an amino acid substitution of a polar residue at position 208, numbered by correspondence with the amino acid sequence of SEQ ID NO:2, for a small residue; and / or an amino acid substitution of a basic residue at position 212, numbered by correspondence with the amino acid sequence of SEQ ID NO:2, for a small residue; and / or an amino acid substitution of a hydrophobic residue at position 217, numbered by correspondence with the amino acid sequence of SEQ ID NO:2, for a small residue; and / or an amino acid substitution of a basic residue at position 207, numbered by correspondence with the amino acid sequence of SEQ ID NO:2, for a small residue; and / or an amino acid substitution of a polar residue at position 211, numbered by correspondence with the amino acid sequence of SEQ ID NO:2, for a small residue; and / or an amino acid substitution of a polar residue at position 214, numbered by correspondence with the amino acid sequence of SEQ ID NO:2.

[0012] In one embodiment, the mutant DegU comprises SEQ ID NO:2 with amino acid substitutions of G109D and / or A228T and / or D63N and / or N195A and / or I204A and / or T208A and / or H212A and / or L217A and / or K207A and / or N211A and / or S214A, or variants thereof having conservative amino acid substitutions.

[0013] In another aspect, the mutant DegS comprises an amino acid substitution of the hydrophobic residue at position 99, numbered according to the amino acid sequence of SEQ ID NO:4, with an aromatic residue; and / or an amino acid substitution of the acidic residue at position 294, numbered according to the amino acid sequence of SEQ ID NO:4, with a basic residue; and / or an amino acid substitution of the polar residue at position 73, numbered according to the amino acid sequence of SEQ ID NO:4, with a small residue; and / or an amino acid substitution of the small residue at position 190, numbered according to the amino acid sequence of SEQ ID NO:4, with a hydrophobic residue.

[0014] In one embodiment, the mutant DegS comprises SEQ ID NO:4 having amino acid substitutions L99F and / or E294K and / or T73A and / or A190V, or a variant thereof having conservative amino acid substitutions.

[0015] In some embodiments, the Paenibacillus strain is a mutagenized derivative and exhibits increased fusaricidin levels compared to the non-mutagenized parent strain, hi other embodiments, the Paenibacillus strain is a mutagenized derivative and exhibits decreased amylase expression and / or enzyme activity compared to the non-mutagenized parent strain.

[0016] In certain aspects, reduced amylase expression and / or enzymatic activity occurs in α-amylase proteins that comprise a sequence having greater than about 90% sequence identity to SEQ ID NO:9 or SEQ ID NO:10. In other aspects, reduced amylase expression and / or enzymatic activity occurs in α-amylase proteins that comprise a sequence having greater than about 95% sequence identity, greater than about 96% sequence identity, greater than about 97% sequence identity, greater than about 98% sequence identity, or greater than about 99% sequence identity to SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the α-amylase protein comprises SEQ ID NO:9. In another embodiment, the α-amylase protein consists of SEQ ID NO:9. In one embodiment, the α-amylase protein comprises SEQ ID NO:10. In another embodiment, the α-amylase protein consists of SEQ ID NO:10.

[0017] In some examples, the non-mutagenized parent strain is Paenibacillus sp. NRRL B-50972 or Paenibacillus sp. NRRL B-67129. In other examples, the non-mutagenized parent strain is Paenibacillus sp. NRRL B-50972, Paenibacillus sp. NRRL B-67129, Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306, or Paenibacillus sp. NRRL B-67615.

[0018] In one embodiment, the strain of Paenibacillus is Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306, Paenibacillus sp. NRRL B-67615, or a fungicidal mutant thereof.

[0019] In another aspect, the composition comprises a fermentation product of Paenibacillus sp. strain NRRL B-67304, Paenibacillus sp. strain NRRL B-67306, Paenibacillus sp. strain NRRL B-67615, or a fungicidal mutant thereof.

[0020] In some embodiments, the fungicidal mutant strain has a genomic sequence that has greater than about 90% sequence identity to Paenibacillus sp. strain NRRL B-67304, Paenibacillus sp. strain NRRL B-67306, or Paenibacillus sp. strain NRRL B-67615.

[0021] In another embodiment, the present invention relates to a method for identifying a mutant derivative of Paenibacillus spp. having reduced viscosity in liquid culture compared to a parent strain of Paenibacillus spp., the method comprising: mutagenizing a parent strain of Paenibacillus spp. to produce a mutant isolate; culturing the mutant isolate and the parent strain of Paenibacillus spp. on a solid medium comprising a sugar at a concentration between about 1% (w / v) and about 40% (w / v), wherein the parent strain of Paenibacillus spp. has a mucoid morphology on the solid medium; and culturing a Paenibacillus isolate having a non-mucoid morphology indicative of reduced viscosity in liquid culture. This involves visually screening mutant isolates on solid media to identify natural mutant derivatives.

[0022] In one aspect, the sugar in the solid medium is at a concentration between about 5% (w / v) and about 20% (w / v).In another aspect, the sugar is selected from the group consisting of sucrose, starch, maltodextrin, corn syrup solids, fructose, glucose, galactose, lactose, maltose, xylose, xylitol, inulin, sorbitol, fucose, molasses, and combinations thereof.

[0023] In one embodiment, the carbon to nitrogen ratio in the solid medium is between about 10:1 and about 1000:1. In one aspect, the solid medium further comprises agar, agarose and / or gelatin. In a particular aspect, the solid medium is a solid agar medium.

[0024] In other embodiments, the method of the invention further comprises culturing the mutant derivative of Paenibacillus in a liquid medium to produce a liquid culture; and measuring the viscosity and / or packed cell volume of the liquid culture to confirm the reduced viscosity of the mutant derivative of Paenibacillus compared to the parent strain of Paenibacillus.

[0025] In a particular embodiment, the method further comprises the step of: detecting a mutant DegU that lacks a functional receiver domain or a functional DNA-binding domain and / or a mutant DegS that lacks a functional single-binding domain or a functional ATPase domain in a mutant derivative of Paenibacillus sp. degU and / or degS This includes sequencing.

[0026] In other embodiments, the method further comprises determining the expression and / or enzymatic activity of amylase in the mutant derivative strain of Paenibacillus and the parent strain of Paenibacillus to determine whether the expression and / or enzymatic activity is reduced in the mutant derivative strain of Paenibacillus. In certain aspects, the expression and / or enzymatic activity of amylase in the mutant derivative strain of Paenibacillus is less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of that of the parent strain of Paenibacillus. In other aspects, the expression and / or enzymatic activity of amylase in the mutant derivative strain of Paenibacillus is about 1% to about 90%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 20% to 90%, about 20% to 80%, about 20% to 70%, or about 20% to 60% of that of the parent strain of Paenibacillus.

[0027] In some embodiments, decreased amylase expression and / or enzymatic activity occurs in an α-amylase protein that comprises a sequence having greater than about 90% sequence identity to SEQ ID NO:9 or SEQ ID NO:10.

[0028] In one embodiment, the method of the invention further comprises quantifying fusaricidin levels in the mutant isolates to identify mutant isolates having increased levels of fusaricidin compared to the parent strain of Paenibacillus.

[0029] In some embodiments, P. agarexedens, P. agaridevorans, P. alginolyticus, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. assamensis, P. azoreducens, P. azotofixans, P. barcinonensis, P. boreali s. .glucanolyticus, P.glycanilyticus, P.gordonae, P.graminis, P.granivorans, P.hodogayensis, P.illinoisensis, P.jamilae, P.kobensis, P.koleovorans, P.koreensis, P.kribbensis, P.lactis, P.larvae, P.lautus, P.lentimorbus, P.macerans, P.macquariensis, P.massiliensis, P.mendelii, P.motobuensis, P.naphthalenovorans, P.nematophilus, P.nov.spec.epiphyticus, P.odorifer, P.pabuli, P.peoriae, P.phoenicis, P.phyllosphaerae, P.polymyxa, P.polymyxa ssp.polymyxa, P.polymyxa ssp.plantarum, P.popilliae, P.pulvifaciens, P.rhizosphaerae, P.sanguinis, P.stellifer, P.taichungensis, P.terrae, P.thiaminolyticus, P.timonensis, P.tylopili, P.turicensis, P.validus, P.vortex, P.vulneris, P.wynnii or P.xylanilyticus. .

[0030] In another embodiment, the strain of Paenibacillus is Paenibacillus polymyxa, Paenibacillus polymyxa ssp. polymyxa, Paenibacillus polymyxa ssp. plantarum, Paenibacillus nov. spec. epiphyticus, Paenibacillus terrae, Paenibacillus macerans or Paenibacillus alvei. In yet another embodiment, the strain of Paenibacillus is Paenibacillus terrae.

[0031] In a particular aspect, the Paenibacillus strain is a fusaricidin-producing Paenibacillus strain.

[0032] Examples of Paenibacillus strains that produce fusaricidin include, but are not limited to, Paenibacillus polymyxa, Paenibacillus polymyxa ssp. polymyxa, Paenibacillus polymyxa ssp. plantarum, Paenibacillus nov. spec. epiphyticus, Paenibacillus terrae, Paenibacillus macerans, and Paenibacillus alvei.

[0033] In yet another embodiment, the present invention relates to a method for producing a mutant derivative of Paenibacillus having reduced viscosity in liquid culture compared to a parent strain of Paenibacillus, the method comprising: mutagenizing the parent strain of Paenibacillus to produce a mutant isolate; culturing the mutant isolate and the parent strain of Paenibacillus on a solid medium comprising a sugar at a concentration between about 1% (w / v) and about 40% (w / v), wherein the parent strain of Paenibacillus has a mucoid morphology on the solid medium; visually screening the mutant isolates on the solid medium to identify a mutant derivative of Paenibacillus having a non-mucoid morphology indicative of reduced viscosity in liquid culture; and producing a fermentation product of the identified mutant derivative of Paenibacillus. In one aspect, the mutagenizing comprises chemical mutagenesis of the parent strain of Paenibacillus.

[0034] In one embodiment, the invention provides a fermentation product comprising a mutant derivative of Paenibacillus identified by the disclosed method. In another aspect, the fermentation product comprises a broth concentrate of whole broth from a mutant derivative of Paenibacillus to increase its fungicidal and / or bactericidal activity.

[0035] In some embodiments, the present invention relates to a method of treating a plant to control a disease comprising applying an effective amount of a composition disclosed herein or a fermentation product disclosed herein to the plant, to a part of the plant and / or to the planting locus.

[0036] In one embodiment, the composition comprises about 1×10 per hectare. 4 ~Approx. 1×10 14 It is applied in colony forming units (CFU) or at about 0.1 kg to about 20 kg of fermentation solids per hectare.

[0037] In one aspect, the plant disease is caused by a fungus. In one aspect, the plant disease is powdery mildew or downy mildew. In another aspect, the fungus is selected from the group consisting of Alternaria alternata, Alternaria solani, Botrytis cinerea, Colletotrichum lagenarium, Erysiphe necator, Fusarium culmorum, Phaeosphaeria nodorum, Zymoseptoria tritici, Phytophthora cryptogea, Phytophthora infestans, Plasmopara viticola, Podosphaera leucotricha, Pseudoperonospora cubensis, Pythium ultimum, Magnaporthe oryzae, Sphaerotheca fuliginea, Thanatephorus cucumeris, Ustilago segetum var. avenae, Uromyces appendiculatus, and Puccinia triticina.

[0038] In other aspects, the plant disease is caused by a bacterium. In some aspects, the bacterium is selected from the group consisting of Xanthomonas campestris, Pseudomonas syringae, and Erwinia carotovora.

[0039] In yet another embodiment, the present invention relates to the use of the composition disclosed herein or the fermentation product disclosed herein for controlling phytopathogenic organisms in useful plants. [Brief description of the drawings]

[0040] [Figure 1]FIG. 1 shows the lineage of Paenibacillus sp. NRRL B-67129, Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67615 derived from Paenibacillus sp. NRRL B-50972. [Diagram 2] Figure 2 compares Paenibacillus spp. strains with a mucoid colony phenotype grown on solid agar medium containing sucrose to the same Paenibacillus spp. strains lacking the mucoid colony phenotype grown on solid agar medium without sucrose. The Paenibacillus spp. strains are: (1) Paenibacillus terrae strain A; (2) Paenibacillus brasilensis strain B; (3) Paenibacillus sp. NRRL B-50972; (4) Paenibacillus polymyxa strain C; (5) Paenibacillus polymyxa strain D; and (6) Paenibacillus peoriae strain E. [Diagram 3] FIG. 3 shows colonies from a mixed population of mucoid and nonmucoid isolates on solid agar medium supplemented with sucrose. [Figure 4] Figure 4 compares the mucoid colony phenotype of (1) Paenibacillus sp. NRRL B-50972 and (4) Paenibacillus sp. NRRL B-67129 on sucrose-containing solid agar medium with the nonmucoid phenotype of (2) Paenibacillus sp. NRRL B-67304 and (3) Paenibacillus sp. NRRL B-67306. All strains show a nonmucoid phenotype on the control solid agar medium lacking sucrose. [Diagram 5]Figure 5 shows the pelleting of fermentation broth after centrifugation for Paenibacillus sp. NRRL B-50972, Paenibacillus sp. NRRL B-67304 and Paenibacillus sp. NRRL B-67306. Strains exhibiting a non-mucoid phenotype on sucrose-containing solid agar medium tend to form denser pellets with smaller packed cell volumes (PCVs). [Figure 6A] FIG. 6A shows SNPs in the degS and degU genes identified in Paenibacillus strains with a nonmucoid colony phenotype. [Figure 6B] FIG. 6B shows an alignment of the DegU amino acid sequences from Bacillus subtilis strain 168 (SEQ ID NO:1) and Paenibacillus sp. strain NRRL B-50972 (SEQ ID NO:2) and the SNPs identified in the receiver and DNA binding domains of the proteins. [Figure 6C] FIG. 6C shows an alignment of the DegS amino acid sequences from Bacillus subtilis strain 168 (SEQ ID NO: 3) and Paenibacillus sp. strain NRRL B-50972 (SEQ ID NO: 4) and the SNPs identified in the single binding and ATPase domains of the proteins. [Figure 7] 7 shows the disruption of degS and degU resulting in a nonmucoid colony phenotype on sucrose-containing solid agar medium by the following Paenibacillus strains: (1) Paenibacillus sp. NRRL B-67129, (2) Paenibacillus sp. NRRL B-67306, (3) Paenibacillus sp. NRRL B-67129 degS::kanR, (4) Paenibacillus sp. NRRL B-67129 degSdegU:kanR, and (5) Paenibacillus terrae strain F. [Figure 8A]FIG. 8A shows viscosity (solid line) and fusaricidin A (dashed line) measurements in liquid cultures of Paenibacillus sp. NRRL B-67304 (parental strain) and NRRL B-67615 (progeny strain) grown at an agitation rate of 250 rpm for 72 hours. [Figure 8B] FIG. 8B shows viscosity (solid line) and fusaricidin A (dashed line) measurements in liquid cultures of Paenibacillus sp. NRRL B-67304 (parental strain) and NRRL B-67615 (progeny strain) grown at an agitation rate of 300 rpm for 72 hours. [Figure 9A] 9A and 9B show the relative protein expression of two α-amylases (i.e., "α-amylase #1" and "α-amylase #2") evaluated at 40 and 48 hours in liquid cultures of Paenibacillus sp. strains NRRL B-67304 (parental strain) and NRRL B-67615 (progeny strain). [Figure 9B] 9A and 9B show the relative protein expression of two α-amylases (i.e., "α-amylase #1" and "α-amylase #2") evaluated at 40 and 48 hours in liquid cultures of Paenibacillus sp. strains NRRL B-67304 (parental strain) and NRRL B-67615 (progeny strain). [Figure 10] FIG. 10 shows the release of glucose from polysaccharides in the medium as an indication of amylase activity in cell-free supernatants from liquid cultures of Paenibacillus sp. strains NRRL B-67304 (parent strain) and NRRL B-67615 (progeny strain). [Figure 11] FIG. 11 shows viscosity measurements of liquid cultures of Paenibacillus sp. strains NRRL B-67304 (parent strain) and NRRL B-67615 (progeny strain) that were added with 0 g / L glucose (i.e., control), 2 g / L glucose, 5 g / L glucose, or 10 g / L glucose at 40 hours and allowed to continue growing for 6 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] All of the microorganisms and specific strains described herein, unless otherwise specified, are isolated from nature and grown under artificial conditions, such as shake flask cultures or through scaled-up manufacturing processes, such as bioreactors, to maximize bioactive metabolite production. Growth under such conditions results in "domestication" of the strain. Generally, such "domesticated" strains differ from their counterparts found in nature in that they are not subject to the selection pressures found in the natural environment, but rather are cultivated as homogenous populations that are subject to artificial selection pressures.

[0042] The microorganism of the present invention or a culture or isolate thereof can be described as being in "isolated" or "biologically pure" form. These terms mean that the microorganism is separated from the environment or one or more components, cells or other things with which it may be found in nature or otherwise. The terms "isolated" or "biologically pure" should not be understood to indicate the degree to which the microorganism has been purified. However, in one embodiment, the microbial isolate or culture contains predominantly the microorganism of the present invention.

[0043] In this application, the verb "comprise" and its conjugations as used in the specification and claims are used in their open sense, including the items listed below, but not excluding any items not specifically mentioned. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that one or more of the element are present, except where the context clearly requires that one and only one of the element is present. Thus, the indefinite article "a" or "an" generally means "at least one."

[0044] As used herein, a "basic residue" is arginine, lysine or histidine; an "acidic residue" is glutamic acid or aspartic acid; a "polar residue" is serine, threonine, cysteine, glutamine or asparagine; a "hydrophobic residue" is methionine, proline, leucine, isoleucine or valine; an "aromatic residue" is phenylalanine, tryptophan or tyrosine; and a "small residue" is glycine or alanine.

[0045] In some embodiments, the strain of Paenibacillus containing mutant DegU and / or mutant DegS produces a liquid culture with reduced viscosity compared to a liquid culture of a strain of Paenibacillus containing wild-type DegU and wild-type DegS. In certain aspects, the reduced viscosity is measured by separately growing the strain of Paenibacillus containing mutant DegU and / or mutant DegS and the strain of Paenibacillus containing wild-type DegU and wild-type DegS in the same liquid culture medium to stationary phase and measuring the viscosity of each liquid culture. Viscosity can be measured by any method known in the art, including the method outlined in Example 2. Examples of wild-type DegU and wild-type DegS include the amino acid sequences provided as SEQ ID NO:1 and SEQ ID NO:2, and SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0046] As used herein, the terms "mucoid" and "mucoid morphology" refer to a phenotype of a microbial colony, which has clearly defined rounded edges and a shiny appearance under a light microscope. Additionally, microbial colonies exhibiting mucoid morphology tend to be taller and rounder in three dimensions. An example of a mucoid colony is shown in FIG. 3.

[0047] As used herein, the terms "nonmucoid" and "nonmucoid morphology" refer to a phenotype of a microbial colony that has poorly defined, randomly shaped edges and a dull appearance under a light microscope. Nonmucoid colonies tend to be flattened in three dimensions. An example of a nonmucoid colony is also provided in FIG. 3.

[0048] Mucoid and non-mucoid forms are more easily distinguished on solid agar medium containing sugar at a concentration of about 1% (w / v) to about 40% (w / v). In some aspects, the sugar concentration is between about 1% (w / v) to about 30% (w / v), about 1% (w / v) to about 20% (w / v), about 5% (w / v) to about 40% (w / v), about 5% (w / v) to about 30% (w / v), or about 5% (w / v) to about 20% (w / v). In one embodiment, the sugar in the solid agar medium is at a concentration of about 5% (w / v) to about 20% (w / v).

[0049] In some embodiments, the carbon to nitrogen ratio in the solid medium is between about 10:1 and about 1000:1, between about 10:1 and about 750:1, between about 10:1 and about 500:1, between about 10:1 and about 250:1, between about 10:1 and about 100:1, between about 10:1 and about 75:1, between about 10:1 and about 50:1, between about 10:1 and about 25:1, between about 1:1 and about 100:1, between about 1:1 and about 75:1, between about 1:1 and about 50:1, between about 1:1 and about 25:1. In another aspect, the carbon to nitrogen ratio in the solid medium is between about 10:1 and about 1000:1, between about 10:1 and about 750:1, between about 10:1 and about 500:1, between about 10:1 and about 250:1, between about 10:1 and about 100:1. In one aspect, the carbon to nitrogen ratio in the solid medium is between about 10:1 and about 1000:1.

[0050] In one embodiment, the solid and / or liquid media used in the disclosed method for identifying a mutant derivative of Paenibacillus having reduced viscosity in liquid culture compared to a parent strain of Paenibacillus comprises any sugar that supports the growth of Paenibacillus cells.

[0051] In certain aspects, the sugar is selected from the group consisting of sucrose, maltodextrin, starch, corn syrup solids, fructose, glucose, galactose, lactose, maltose, xylose, xylitol, inulin, sorbitol, fucose, molasses, and combinations thereof. In another aspect, the sugar is selected from the group consisting of sucrose, starch, corn syrup solids, maltodextrin, fructose, glucose, galactose, lactose, maltose, and combinations thereof. In another aspect, the sugar is selected from the group consisting of sucrose, maltodextrin, fructose, and combinations thereof. In yet another aspect, the sugar is sucrose or maltodextrin.

[0052] In some embodiments, the present invention relates to a method for identifying mutant derivatives of Paenibacillus that have reduced viscosity in liquid culture compared to a parent strain of Paenibacillus using visual screening. As used herein, the terms "visual screening" and "visual screening" refer to any process, whether performed manually or automatically by a machine or robot, that analyzes the size, shape, and / or sheen (i.e., shine) of microbial colonies grown on a solid medium. In some aspects, the solid medium is a solid agar medium.

[0053] In another embodiment, the present invention relates to a method for identifying a mutant derivative of Paenibacillus sp. that has reduced viscosity in liquid culture compared to a parent strain of Paenibacillus sp., the method comprising: mutagenizing a parent strain of Paenibacillus sp. to produce a mutant isolate; the parent strain of Paenibacillus in a liquid medium containing a sugar at a concentration of about 1% (w / v) to about 40% (w / v); and measuring the viscosity and / or packed cell volume of the mutant isolate in the liquid medium to identify a mutant derivative of Paenibacillus having reduced viscosity in liquid culture compared to the parent strain of Paenibacillus.

[0054] Paenibacillus sp. NRRL B-50972 and Paenibacillus sp. NRRL B-67129 were previously identified as producers of a unique group of fusaricidin and fusaricidin-like compounds with broad-spectrum antifungal activity (WO2016 / 154297).

[0055] One of these is a slightly less expensive dressing One of the following is 1:P.agarexede ns、P.agaridevorans、P.alginolyticus、P.alkaliterrae、P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. assamensis, P. azoreducens, P. azotofixans, and P. barcinonens is, P. borealis, P. brasiliensis, P. brassicae, P. campinasensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus P. cineris, P. cookie, P. curdlanolyticus, P. daejeonensis, P. dendritiformis, P. duram, P. ehimensis, P. elgii, P. favi spores, P. glucanolyticus, P. glycanilyticus, P. gordonae, P. graminis, P. granivorans, P. hodogayensis, P. illinoise nsis, P. jamilae, P. kobensis, P. koleovorans, P. koreensis, P. kribbensis, P. lactis, P. larvae, P. lautus, and P. lentimorbu s, P. macerans, P. macquariensis, P. masssiliensis, P. mendelii, P. motobuensis, P. naphthalenovorans, P. nematophilus P. nov.spec.epiphyticus, P. odorifer, P. pabuli, P. peoriae, P. phoenicis, P. phyllosphaerae, P. polymyxa, P. polymyxa spp.tylopili, P.turicensis, P.validus, P.vortex, P.vulneris, P.wynnii and P.xylanilyticus. .

[0056] In another aspect, the Paenibacillus strain of the invention is selected from any one of the following: P. terrae, P. brasilensis, P. polymyxa, or P. peoriae. In one embodiment, the Paenibacillus strain of the invention is P. terrae.

[0057] In one embodiment, a mutant strain of Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306, or Paenibacillus sp. NRRL B-67615 is provided. The "mutant" refers to a genetic variant derived from Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306, or Paenibacillus sp. NRRL B-67615. In one embodiment, the mutant has one or more or all of the identifying (functional) characteristics of Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306, or Paenibacillus sp. NRRL B-67615. In certain examples, the mutant or its fermentation product has, as a functional identifying characteristic, at least as good as its parent strain, Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306 or Paenibacillus sp. NRRL B-67615, control fungi, oomycetes and / or bacteria. Such mutants can be genetic variants having a genomic sequence with greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% sequence identity to Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306 or Paenibacillus sp. NRRL B-67615.Mutants can be obtained by treating cells of Paenibacillus sp. strain NRRL B-67304, Paenibacillus sp. strain NRRL B-67306 or Paenibacillus sp. strain NRRL B-67615 with chemicals or irradiation, by selecting spontaneous mutants (such as phage-resistant or antibiotic-resistant mutants) from a cell population of Paenibacillus sp. strain NRRL B-67304, Paenibacillus sp. strain NRRL B-67306 or Paenibacillus sp. strain NRRL B-67615, by genome shuffling as described below, or by other methods known and practiced in the art.

[0058] Genome shuffling between Paenibacillus strains can be facilitated by using a process called protoplast fusion. The process begins with the formation of protoplasts from vegetative bacterial cells. Protoplasts are formed by removing the peptidoglycan cell wall, typically with lysozyme and an osmotic stabilizer. This process is visualized by the appearance of spherical cells under a light microscope. Addition of PEG, polyethylene glycol, induces fusion between protoplasts, bringing the genetic contents of two or more cells into contact and promoting recombination and genome shuffling. The fused cells are then redivided and recovered on solid growth medium. During recovery, the protoplasts remodel the peptidoglycan cell wall and revert to a rod-shaped form. See Schaeffer et al., PNAS USA, vol. 73, 6:2151-2155 (1976).

[0059] Paenibacillus sp. NRRL B-67304, NRRL B-67306 or NRRL B-67615 and its mutants are active against a wide range of plant pathogens. In one aspect, the strain is active against fungi such as cucumber anthracnose, cucumber powdery mildew, wheat leaf rust, barley powdery mildew, Alternaria and Botrytis; Oomycetes such as tomato late blight, cucumber downy mildew and crucifer downy mildew; and / or bacteria such as Pseudomonas, Xanthomonas and Erwinia.

[0060] In a particular aspect, the mutant DegU and / or mutant DegS characteristic of the strain of Paenibacillus of the present invention comprises conservative amino acid substitutions. For example, conservative amino acid substitutions within the sequences of SEQ ID NOs: 1-4 are considered. Examples of conservative amino acid substitutions are within the groups of basic amino acids (i.e., arginine, lysine and histidine), acidic amino acids (i.e., glutamic acid and aspartic acid), polar amino acids (i.e., serine, threonine, cysteine, glutamine and asparagine), hydrophobic amino acids (i.e., methionine, proline, leucine, isoleucine and valine), aromatic amino acids (i.e., phenylalanine, tryptophan and tyrosine) and small amino acids (i.e., glycine and alanine). Amino acid substitutions that do not generally change the specific activity are known in the art and are described, for example, in H. Neurath and RL Hill, 1979, The Proteins, Academic Press, New York, which is incorporated herein by reference in its entirety. Commonly occurring conservative substitutions include Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Lys / Arg, Leu / Ile, and Leu / Val.

[0061] The invention also includes methods of treating plants to control plant diseases by applying the disclosed Paenibacillus strains or mutants thereof, or cell-free preparations or metabolites thereof to the plants or plant parts, such as leaves, stems, flowers, fruits, roots or seeds, or to the locus in which the plants or plant parts grow, such as the soil.

[0062] In the method according to the present invention, the composition comprising the disclosed strain of Paenibacillus or its fungicidal mutant can be applied to any plant or any part of any plant grown in any type of medium used for growing plants (e.g. soil, vermiculite, shredded cardboard and water), or to aerial growing plants or parts of plants, such as orchids or staghorn ferns. The composition of the present invention can be applied, for example, by spraying, spraying, vaporizing, scattering, dusting, flooding, fountaining, watering, injecting or fumigating. As already indicated, application can be carried out at any desired location where the target plant is located, such as agriculture, horticulture, forests, forestry, orchards, nurseries, organic crops, turfgrass and urban environments.

[0063] The compositions of the present invention can be obtained by culturing the disclosed strains of Paenibacillus or fungicidal mutants derived therefrom according to methods well known in the art, including using the media and methods described in the Examples below. Conventional large-scale microbial cultivation processes include submerged fermentation, solid-state fermentation or liquid surface culture. Towards the end of the fermentation, as nutrients are exhausted, the cells begin the transition from the growth phase to the sporulation phase. As a result, the end products of the fermentation are mainly spores, metabolic products and residual fermentation medium. Sporulation is part of the natural life cycle of Paenibacillus and is generally initiated by the cells in response to nutrient limitation. The fermentation is designed to obtain a high level of colony forming units and promote sporulation. The bacterial cells, spores and metabolic products in the culture medium resulting from the fermentation can be used directly or concentrated by conventional industrial methods such as centrifugation, tangential flow filtration, depth filtration and evaporation.

[0064] The composition of the present invention includes a fermentation product. In some embodiments, the concentrated fermentation broth is washed, for example, via a diafiltration process, to remove residual fermentation broth and metabolic products. The term "broth concentrate" as used herein refers to the whole broth (fermentation broth) that has been concentrated by conventional industrial methods as described above, but remains in liquid form. The term "fermentation solid" as used herein refers to the solid material remaining after the fermentation broth is dried. The term "fermentation product" as used herein refers to the whole broth, broth concentrate and / or fermentation solid. The composition of the present invention includes a fermentation product.

[0065] Fermentation broth or culture concentrates can be spray-dried, freeze-dried, or tray-dried. The drying may be accomplished using any conventional drying process or method, such as fluidized bed drying, drum drying, or evaporation, with or without the addition of a carrier.

[0066] The resulting dried product may be further processed, such as by milling or granulation, to achieve a particular particle size or physical form. Carriers, as described below, may be added after drying.

[0067] The cell-free preparation of the fermentation broth of the strain of the present invention can be obtained by any means known in the art, such as extraction, centrifugation and / or filtration of the fermentation broth. Those skilled in the art will recognize that depending on the technique used to remove cells (e.g., the speed of centrifugation), the so-called cell-free preparation may not be completely free of cells, but rather may be mostly cell-free or essentially cell-free. The resulting cell-free preparation may be dried and / or formulated with ingredients that aid in application to plants or plant growth media. The concentration methods and drying techniques described above for fermentation broth are also applicable to cell-free preparations.

[0068] In one embodiment, the fermentation product is at least about 1×10 4In another embodiment, the fermentation product comprises at least about 1×10 colony forming units (CFU) of the microorganism (e.g., Paenibacillus sp. strain NRRL B-67304, Paenibacillus sp. strain NRRL B-67306, or Paenibacillus sp. strain NRRL B-67615, or a fungicidal mutant thereof) per mL of culture medium. 5 In another embodiment, the fermentation product contains at least about 1×10 colony forming units (CFU) of microorganisms per mL of culture medium. 6 In yet another embodiment, the fermentation product contains at least about 1 x 10 CFU of microorganisms per mL of culture medium. 7 In another embodiment, the fermentation product contains at least about 1 x 10 CFU of microorganisms per mL of culture medium. 8 In another embodiment, the fermentation product contains at least about 1 x 10 CFU of microorganisms per mL of culture medium. 9 In another embodiment, the fermentation product contains at least about 1 x 10 CFU of microorganisms per mL of culture medium. 10 In another embodiment, the fermentation product contains at least about 1 x 10 CFU of microorganisms per mL of culture medium. 11 Contains CFU of microorganisms.

[0069] The compositions of the present invention can be used as they are or in the form of formulations or use forms prepared therefrom, such as, for example, aerosols, capsule suspensions, cold fog concentrates, hot fog concentrates, encapsulated granules, fine granules, concentrated flowables for seed treatment, ready-to-use solutions, dusts, emulsifiable concentrates, oil-in-water emulsions, water-in-oil emulsions, macrogranules, microgranules, oil-dispersible powders, oil-miscible concentrated flowables, oil-miscible liquids, gases (under pressure), gas generating products, foams, pastes, pesticide-coated seeds, concentrated suspensions, oil dispersions, concentrated SEs, soluble concentrates, suspensions, wettable powders, water-soluble powders, dusts and granules, water-soluble and water-dispersible granules or tablets, water-soluble and water-dispersible powders for seed treatment, wettable powders impregnated with active ingredients, microcapsules with natural and synthetic substances, and polymeric substances and seed coating materials, and ULV cold and hot fog formulations, depending on their specific physical and / or chemical properties.

[0070] In some embodiments, the composition of the present invention is a liquid formulation.Liquid formulations include, but are not limited to, concentrated suspensions and oil dispersions.In other embodiments, the composition of the present invention is a solid formulation.Solid formulations include, but are not limited to, freeze-dried powders and spray-dried powders.

[0071] All plants and plant parts can be treated according to the invention. In this context, plants are understood to mean all plants and plant populations, such as desirable and undesirable wild plants or crop plants (including naturally occurring crop plants). Crop plants are plants that can be obtained by traditional breeding and optimization methods or by biotechnological and recombinant methods or a combination of these methods, including genetically modified plants and plant varieties protected or not by plant breeder's rights. Plant parts are understood to mean all above-ground and underground parts and organs of a plant, such as shoots, leaves, flowers, roots, etc. For example, leaves, needles, stalks, stems, flowers, fruiting bodies, fruits and seeds, roots, tubers, rhizomes, etc. may be mentioned. Plant parts also include crop elements, such as cuttings, tubers, rhizomes, scions and seeds, as well as vegetative and generative propagation elements.

[0072] As already mentioned, all plants and their parts can be treated according to the invention. In a preferred embodiment, plant species and plant cultivars, which grow wild or which have been obtained by traditional biological breeding methods such as hybridization or protoplast fusion, and their parts are treated. In a further preferred embodiment, genetically modified plants and plant cultivars, which have been obtained by recombinant methods, if appropriate in combination with traditional methods (genetically modified organisms), and their parts are treated. The terms "parts" or "plant parts" or "plant parts" have been explained above. Plants of plant cultivars which are in each case commercially available or in use are particularly preferably treated according to the invention. Plant cultivars are understood to mean plants with novel traits, which have been bred both by traditional breeding, by mutagenesis or by recombinant DNA techniques. These may take the form of varieties, races, biotypes and genotypes.

[0073] The treatment of plants and plant parts with the compositions according to the invention is carried out directly or by applying to the environment, habitat or storage space using the usual treatment methods, such as, for example, soaking, spraying, atomizing, irrigating, vaporizing, dusting, fogging, scattering, foaming, painting, spreading, injecting, drenching, drip irrigation and, in the case of propagation elements (especially seeds), dry seed treatment methods, wet seed treatment methods, slurry treatment methods, encrustation, coating with one or more films, etc. It is also possible to apply the active substances by microspraying or to inject active substance formulations or the active substances themselves into the soil.

[0074] A preferred direct treatment of plants is a foliar spray treatment, ie the composition according to the invention is applied to the leaves, the frequency of treatment and the application rate being possible to adapt to the infection pressure of the pathogen in question.

[0075] In the case of systemically active compounds, the composition according to the invention reaches the plant via the root system. In this case, the plant is treated by allowing the composition according to the invention to act on the plant's environment. This can be done, for example, by drench treatment (i.e. impregnating the growing area (e.g. soil or hydroponic system) with a liquid form of the composition according to the invention for incorporation into the soil or nutrient solution) or by soil application (i.e. incorporating the composition according to the invention in solid form (e.g. in the form of granules) into the growing area). In the case of rice cultivation, the composition according to the invention can also be metered into the rice field in solid application form (e.g. in the form of granules).

[0076] Preferred plants are from the group of useful plants, ornamental plants, turf, trees generally employed as ornamental plants in the public and domestic sectors and forestry trees, which include trees for the production of wood, cellulose, paper and products made from parts of the trees.

[0077] The term "useful plants" as used herein refers to crops that are used to obtain food, feed, or fuel, or as industrial plants.

[0078] Useful plants which can be treated and / or improved with the compositions and methods of the invention include, for example, turfgrass; grapes; cereals such as wheat, barley, rye, oats, rice, maize and millet / sorghum; beets, such as sugar beet and fodder beet; fruits, such as pome fruits, stone fruits and soft fruits, such as apples, pears, plums, peaches, almonds, cherries and berries (e.g. strawberries, raspberries, blackberries); legumes, such as beans, lentils, peas, soybeans; oil crops, such as rapeseed, mustard, poppy, olives, sunflowers, coconuts, castor beans. , cacao and peanuts; melons such as pumpkin / squash, cucumber and melons; fiber plants such as cotton, flax, hemp and jute; citrus fruits such as oranges, lemons, grapefruit and mandarins; vegetables such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, peppers; lauraceae plants such as avocado, cinnamon, camphor; or other plants such as tobacco, nuts, coffee, eggplant, sugar cane, tea, pepper, grapes, hops, bananas, rubber trees and ornamentals such as flowers, shrubs, deciduous trees and conifers. This list is not limiting.

[0079] The following plants are considered to be particularly suitable target crops for application of the compositions and methods of the present invention: cotton, eggplant, turf, pome fruits, stone fruits, soft fruits, corn, wheat, barley, cucumber, tobacco, grapes, rice, cereals, pears, beans, soybeans, rapeseed, tomatoes, peppers, melons, cabbage, potatoes and apples.

[0080] Examples of trees which may be improved according to the method according to the invention are Abies sp., Eucalyptus sp., Picea sp., Pinus sp., Aesculus sp., Platanus sp., Tilia sp., Acer sp., Tsuga sp., Fraxinus sp., Sorbus sp., Betula sp., Crataegus sp., Ulmus sp., Quercus sp., Fagus sp., Salix sp., Poplar sp., sp.) are mentioned.

[0081] Preferred trees which can be improved according to the method according to the invention are A. hippocastanum, A. pariflora, A. carnea from the tree species Aesculus; P. aceriflora, P. occidentalis, P. racemosa from the tree species Platanus; P. abies from the tree species Picea; P. radiate, P. ponderosa, P. contorta, P. sylvestre, P. elliottii, P. montecola from the tree species Pinus. (P. montecola), P. albicaulis, P. resinosa, P. palustris, P. taeda, P. flexilis, P. jeffregi, P. baksiana, P. strobes; from the tree species Eucalyptus genus E. grandis, E. globulus, E. camadentis, E. nitens, E. obliqua, E. regnans, E. pilularus.

[0082] Particularly preferred trees which can be improved according to the method according to the invention are P. radiata, P. ponderosa, P. contorta, P. sylvestre, P. strobes from the tree species Pinus; E. grandis, E. globulus and E. camadentis from the tree species Eucalyptus.

[0083] Very good properties that can be improved according to the method of the present invention Preferred trees are the horse chestnut, Platanaceae, lime and maple.

[0084] The present invention may also be applied to any turf, including cool season and warm season turf. Examples of cool season grasses include bluegrass (Poa spp.) such as Kentucky bluegrass (Poa pratensis L.), rough bluegrass (Poa trivialis L.), Canada bluegrass (Poa compressa L.), annual bluegrass (Poa annua L.), upland bluegrass (Poa glaucantha Gaudin), wood bluegrass (Poa nemoralis L.), and sedge bluegrass (Poa spp.). bentgrass (Agrostis spp.), such as creeping bentgrass (Agrostis palustris Huds.), colonial bentgrass (Agrostis tenuis Sibth.), velvet bentgrass (Agrostis canina L.), South German Mixed Bentgrass (Agrostis tenuis Sibth., Agrostis canina L. and Agrostis palustris Huds.); Huds.) and redtop (Agrostis alba L.);

[0085] Fescue (Festuca spp.), e.g. red fescue (Festuca rubra L. spp. rubra), creeping fescue (Festuca rubra L.), chewing fescue (Festuca rubra commutata Gaud.), sheep fescue (Festuca ovina L.), hard fescue (Festuca longifolia Thuill.), hair fescue (Festuca capirata L.), Lam. (Festucu capillata Lam.), tall fescue (Festuca arundinacea Schreb.) and meadow fescue (Festuca elanor L.);

[0086] Ryegrass (Lolium spp.), such as annual ryegrass (Lolium multiflorum Lam.), perennial ryegrass (Lolium perenne L.) and Italian ryegrass (Lolium multiflorum Lam.);

[0087] Wheatgrass (Agropyron spp.), such as fairway wheatgrass (Agropyron cristatum (L.) Gaertn.), crested wheatgrass (Agropyron desertorum (Fisch.) Schult.), and western wheatgrass (Agropyron smithii Rydb.).

[0088] Further examples of cool season grasses are beachgrass (Ammophila breviligulata Fern.), smooth bromegrass (Bromus inermis Leyss.), cattails such as Timothy (Phleum pratense L.), sand cattail (Phleum subulatum L.), orchardgrass (Dactylis glomerata L.), weeping alkaligrass (Puccinellia distance L.), Parl. (Puccinellia distans (L.) Parl.) and crested dog's-tail (Cynosurus cristatus L.).

[0089] Examples of warm season grasses include bermuda grass (Cynodon spp. LCRich), zoysia grass (Zoysia spp. Willd.), St. Augustine grass (Stenotaphrum secundatum Walt Kuntze), centipede grass (Eremochloa ophiuroides Munro Hack.), carpet grass (Axonopus affinis Chase), bahia grass (Paspalum notatum flugge), and turf grass (Cynodon spp. LCRich). Flugge), Kikuyu grass (Pennisetum clandestinum Hochst. ex Chiov.), buffalo grass (Buchloe dactyloids (Nutt.) Engelm.), Blue gramma (Bouteloua gracilis (HBK) Lag. ex Griffiths), seashore paspalum (Paspalum vaginatum Swartz) and sideoats grama (Bouteloua carchipendula). curtipendula (Michx.Torr.). Cool-season grasses are generally preferred for use in accordance with the present invention. Particularly preferred are bluegrass, bentgrass and oak, fescue and ryegrass. Bentgrass is especially preferred.

[0090] The compositions of the present invention have strong fungicidal activity and can be used to control unwanted microorganisms such as fungi and bacteria in crop protection and material protection.

[0091] The present invention also relates to a method for controlling unwanted microorganisms, characterized in that the composition according to the invention is applied to phytopathogenic fungi, bacteria and / or their habitat.

[0092] Fungicides can be used in crop protection for the control of phytopathogenic fungi. They are characterized by their remarkable efficacy against a wide range of phytopathogenic fungi, including soil-borne pathogens, in particular members of the classes Plasmodiophoromycetes, Peronosporomycetes (also known as Oomycetes), Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes. Some fungicides are systemically active and can be used in plant protection as foliar, seed coating or soil fungicides. Furthermore, they are particularly suitable for combating fungi that parasitize the xylem or roots of plants.

[0093] Bactericides can be used in crop protection for the control of Pseudomonadaceae, Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceae and Streptomycetaceae.

[0094] Non-limiting examples of fungal disease pathogens that can be treated according to the present invention include:

[0095] diseases caused by powdery mildew pathogens, such as Blumeria species, e.g. Blumeria graminis; Podosphaera species, e.g. Podosphaera leucotricha; Sphaerotheca species, e.g. Sphaerotheca fuliginea; Uncinula species, e.g. Uncinula necator;

[0096] Diseases caused by rust disease pathogens, for example Gymnosporangium species, such as Gymnosporangium sabinae; Hemileia species, such as Hemileia vastatrix; Phakopsora species, such as Phakopsora pachyrhizi and Phakopsora meibomiae; Puccinia species, such as Puccinia recondite, P. triticina, P. graminis (P. graminis) or P. striiformis; Uromyces species, such as those caused by Uromyces appendiculatus;

[0097] Diseases caused by pathogens from the group of the Oomycetes, for example Albugo species, such as Algubo candida; Bremia species, such as Bremia lactucae; Peronospora species, such as Peronospora pisi or P. brassicae; Phytophthora species, such as Phytophthora infestans; Plasmopara species, such as Plasmopara viticola; Pseudoperonospora species, such as Pseudoperonospora humurii; humuli or Pseudoperonospora cubensis; Pythium species, such as those caused by Pythium ultimum;

[0098] Leaf blotch disease and leaf wilt disease, e.g. Alternaria species, such as Alternaria solani; Cercospora species, such as Cercospora beticola; Cladiosporium species, such as Cladiosporium cucumerinum; Cochliobolus species, such as Cochliobolus sativus (conidial morphology: Drechslera, synonymous with Helminthosporium), Cochliobolus miyabeanus, miyabeanus; Colletotrichum species, for example Colletotrichum lindemuthanium; Cycloconium species, for example Cycloconium oleaginum; Diaporthe species, for example Diaporthe citri; Elsinoe species, for example Elsinoe fawcettii; Gloeosporium species, for example Gloeosporium laeticolor; Glomerella species, for example Glomerella cingulata; Guignardia species, for example Guignardia bidwelli bidwelli);Leptosphaeria species, such as Leptosphaeria maculans, Leptosphaeria nodorum; Magnaporthe species, such as Magnaporthe grisea; Marssonia species, such as Marssonia coronaria; Microdochium species, such as Microdochium nivale; Mycosphaerella species, such as Mycosphaerella graminicola, M. arachidicola and M. fijiensis; Phaeosphaeria species, such as Phaeosphaeria species, for example, Phaeosphaeria nodorum; Pyrenophora species, for example, Pyrenophora teres, Pyrenophora tritici repentis; Ramularia species, for example, Ramularia collo-cygni, Ramularia areola; Rhynchosporium species, for example, Rhynchosporium secalis; Septoria species, for example, Septoria apii, Septoria lycopersii; Typhula species species, e.g. Typhula incarnata;Venturia species, such as those attributed to Venturia inaequalis;

[0099] Root and stem diseases, for example caused by Corticium species, for example Corticium graminearum; Fusarium species, for example Fusarium oxysporum; Gaeumannomyces species, for example Gaeumannomyces graminis; Rhizoctonia species, for example Rhizoctonia solani; Sarocladium disease, for example caused by Sarocladium oryzae; Sclerotium disease, for example caused by Sclerotium oryzae; Tapescia species species, e.g. Tapesia acuformis; Thielaviopsis species, e.g. those attributed to Thielaviopsis basicola;

[0100] Diseases of ear and panicle (including corn cobs), for example Alternaria species, for example Alternaria spp.; Aspergillus species, for example Aspergillus flavus; Cladosporium species, for example Cladosporium cladosporioides; Claviceps species, for example Claviceps purpurea; Fusarium species, for example Fusarium culmorum; Gibberella species, for example Gibberella zeae; Monographella species, for example Monographella nivalis; nivalis); those caused by Septoria species, e.g. Septoria nodorum;

[0101] diseases caused by smut fungi, for example those caused by Sphacelotheca species, e.g. Sphacelotheca reiliana; Tilletia species, e.g. Tilletia caries, T. controversa; Urocystis species, e.g. Urocystis occulta; Ustilago species, e.g. Ustilago nuda, U. nuda tritici;

[0102] fruit rot, for example caused by Aspergillus species, such as Aspergillus flavus; Botrytis species, such as Botrytis cinerea; Penicillium species, such as Penicillium expansum and P. purpurogenum; Sclerotinia species, such as Sclerotinia sclerotiorum; Verticilium species, such as Verticilium alboatrum;

[0103] Seed and soil borne decay, mould, wilt, rot and damping-off diseases, e.g. caused by Alternaria species, e.g. Alternaria brassicicola; Aphanomyces species, e.g. caused by Aphanomyces euteiches; Ascochyta species, e.g. caused by Ascochyta lentis; Aspergillus species, e.g. caused by Aspergillus flavus; Cladosporium species, e.g. caused by Cladosporium herbarum; Cochliobolus species, e.g. Cochliobolus sativus; (conidiomorph: Drechslera, Bipolaris: synonymous with Helminthosporium); Colletotrichum species, e.g. Colletotrichum caused by Colletotrichum coccodes; Fusarium species, e.g. caused by Fusarium culmorum; Gibberella species, e.g. caused by Gibberella zeae; Macrophomina species, e.g. caused by Macrophomina phaseolina; Monographella species, e.g. caused by Monographella nivalis; Penicillium species, e.g. caused by Penicillium expansum; Phoma species, e.g. caused by Phoma lingam; Phomopsis species species, e.g. those caused by Phomopsis sojae; Phytophthora species, e.g. those caused by Phytophthora cactorum; Pyrenophora species, e.g. those caused by Pyrenophora graminea; Pyricularia species, e.g. those caused by Pyricularia oryzae; Pythium species, e.g. those caused by Pythium ultimum; Rhizoctonia species, e.g. those caused by Rhizoctonia solani; Rhizopus species, e.g. those caused by Rhizopus oryzae;Sclerotium species, e.g. caused by Sclerotium rolfsii;Septoria species, e.g. caused by Septoria nodorum;Typhula species, e.g. caused by Typhula incarnata;Verticillium species, e.g. caused by Verticillium dahliae;

[0104] Canker, galls and witches' broom, for example caused by Nectria species, such as Nectria galligena;

[0105] Wilt diseases, for example caused by Monilinia species, such as Monilinia laxa;

[0106] Leaf blister disease or leaf curl disease, for example caused by Exobasidium species, e.g. Exobasidium vexans;

[0107] Taphrina species, e.g. Taphrina deformans;

[0108] Degenerative diseases of woody plants, for example Esca disease, for example caused by Phaemoniella clamydospora, Phaeoacremonium aleophilum and Fomitiporia mediterranea;Eutypa dyeback, for example caused by Eutypa lata;Ganoderma disease, for example caused by Ganoderma boninense;Rigidoporus disease, for example caused by Rigidoporus lignosus;

[0109] Flower and seed diseases, for example those caused by Botrytis species, such as Botrytis cinerea;

[0110] Diseases of plant tubers, for example those caused by Rhizoctonia species, e.g. Rhizoctonia solani; Helminthosporium species, e.g. Helminthosporium solani;

[0111] Root galls, for example those caused by Plasmodiophora species, e.g. Plamodiophora brassicae;

[0112] Diseases caused by bacterial pathogens, such as Xanthomonas species, e.g. Xanthomonas campestris pv. oryzae; Pseudomonas species, e.g. Pseudomonas syringae pv. lachrymans; Erwinia species, e.g. Erwinia amylovora.

[0113] The following soybean diseases can be effectively controlled:

[0114] Fungal diseases of leaves, stems, pods and seeds, such as Alternaria leaf spot (Alternaria sp. atrans tenuissima), anthracnose (Colletotrichum gloeosporoides dematium var. truncatum), brown spot (Septoria glicines), cercospora leaf spot and blight (Cercospora kikuchii), choanephora leaf blight (Choanephora infundibulifera trispora), dactuliophora leaf spot (Dactuliophora glycines), downy mildew (Peronospora manshurica), drechslera blight (Drechslera glycini), frogeye leaf spot (Cercospora sojina), leptosphaerulina leaf spot (Leptosphaerulina trifolii), phyllostica leaf spot (Phyllosticta sojaecola), pod and stem blight (Phomopsis sojae), powdery mildew (Microsphaera diffusa), pyrenochaeta leaf spot (Pyrenochaeta grisea),glycines), rhizoctonia aerial, foliage, and web blight (Rhizoctonia solani), rust (Phakopsora pachyrhizi, Phakopsora meibomiae), scab (Sphaceloma glycines), stemphyllium leaf blight (Stemphylium botryosum), and target spot (Corynespora cassiicola).

[0115] Fungal diseases of the roots and stem bases, such as black root rot (Calonectria crotalariae), charcoal rot (Macrophomina phaseolina), fusarium blight or wilt, root rot, and pod and collar rot (Fusarium oxysporum, Fusarium orthoceras, Fusarium semitectum, Fusarium equiseti), mycoleptodiscus root rot (Mycoleptodiscus terrestris), terrestris), neocosmospora (neocosmospora vasinfecta), pod and stem blight (Diaporthe phaseolorum), stem canker (Diaporthe phaseolorum var.caulivora), phytophthora rot (Phytophthora megasperma), brown stem rot (Phialophora gregata), pythium rot (Pythium aphanidermatum, Pythium irregulare, Pythium debaryanum, Pythium myriotylum, Pythium ultimum), rhizoctonia root rot, stem decay, and damping-off. (Rhizoctonia solani), sclerotinia stem decay (Sclerotinia sclerotiorum), sclerotinia southern blight (Sclerotinia rolfsii), and Thielaviopsis root rot (Thielaviopsis basicola).

[0116] The fungicidal compositions of the present invention can be used for the curative or protective / preventive control of phytopathogenic fungi.The present invention therefore also relates to curative and protective methods for the control of phytopathogenic fungi by using the compositions of the present invention applied to seeds, plants or plant parts, fruits or the soil in which the plants grow.

[0117] The fact that the compositions are well tolerated by plants at the concentrations required to control plant diseases makes it possible to treat the above-ground parts of plants, the propagules and seeds, and the soil.

[0118] According to the invention, all plants and plant parts can be treated, including cultivars and plant varieties (whether or not protectable by plant variety or plant breeder's rights). Cultivars and plant varieties can be plants obtained by conventional propagation and breeding methods that can be assisted or supplemented by one or more biotechnological methods, such as by the use of double haploids, protoplast fusion, random and directed mutagenesis, molecular or genetic markers, or by biotechnological and genetic engineering methods.

[0119] In certain aspects, the compositions of the present invention provide an amount of about 1×10 per hectare 4 ~Approx. 1×10 14 of colony forming units (CFU), approximately 1 × 10 per hectare 4 ~Approx. 1×10 12 of colony forming units (CFU), approximately 1 × 10 per hectare 4 ~Approx. 1×10 10 of colony forming units (CFU), approximately 1 × 10 per hectare 4 ~Approx. 1×10 8 of colony forming units (CFU), approximately 1 × 10 per hectare 6 ~Approx. 1×10 14 Colony forming units (CFU), approximately 1 × 10 per hectare 6 ~Approx. 1×10 12 of colony forming units (CFU), approximately 1 × 10 per hectare 6 ~Approx. 1×10 10 of colony forming units (CFU), approximately 1 × 10 per hectare 6 ~Approx. 1×10 8 of colony forming units (CFU), approximately 1 × 10 per hectare 8 ~Approx. 1×10 14 of colony forming units (CFU), approximately 1 × 10 per hectare 8 ~Approx. 1×10 12 of colony forming units (CFU), approximately 1 × 10 per hectare 8 ~Approx. 1×10 10 The formulation is administered in colony forming units (CFU).

[0120] In another aspect, the composition of the present invention has an average yield of about 1×10 per hectare. 6 ~Approx. 1×10 14 It is applied in colony forming units (CFU), approximately 1 × 10 per hectare. 6 ~Approx. 1×10 12 Colony forming units (CFU), approximately 1 × 10 per hectare 6 ~Approx. 1×10 10 Colony forming units (CFU), approximately 1 × 10 per hectare 6 ~Approx. 1×10 8 In yet another embodiment, the compositions of the present invention are applied in colony forming units (CFU) at about 1 x 10 per hectare. 9 ~Approx. 1×10 13 In one aspect, the compositions of the present invention are applied at about 1 x 10 colony forming units (CFU) per hectare. 10 ~Approx. 1×10 12 The formulation is administered in colony forming units (CFU).

[0121] In certain embodiments, the compositions of the present invention are applied at about 0.1 kg to about 20 kg of fermentation solids per hectare. In some embodiments, the compositions of the present invention are applied at about 0.1 kg to about 10 kg of fermentation solids per hectare. In other embodiments, the compositions of the present invention are applied at about 0.25 kg to about 7.5 kg of fermentation solids per hectare. In yet other embodiments, the compositions of the present invention are applied at about 0.5 kg to about 5 kg of fermentation solids per hectare. The compositions of the present invention may also be applied at about 1 kg or about 2 kg of fermentation solids per hectare.

[0122] The compositions of the present invention are suitable for protecting plants and plant organs, for increasing yields and for improving the quality of crops, provided that the plants are well tolerated, have a desirable toxicity to homeotherms and are well suited to the environment.They are preferably used as crop protection compositions.They are active against normally sensitive and resistant species, and are also active against all or some of the developmental stages.

[0123] Plants which can be treated according to the invention include the following major crop plants: corn, soybean, alfalfa, cotton, sunflower, Brassica oilseeds such as Brassica napus (e.g. canola, rapeseed), Brassica rapa, B. juncea (e.g. mustard (field mustard)) and Brassica carinata, Arecaceae sp. (e.g. oil palm, coconut), rice, wheat, sugar beet, sugar cane, oats, rye, barley, millet and sorghum, triticale, flax, nuts, grapes and vines, as well as various fruits and vegetables from various botanical classes, such as Rosaceae. sp.) (e.g. pome fruits, such as apple and pear, but also stone fruits, such as apricot, cherry, almond, plum and peach, and berries, such as strawberry, raspberry, red and black currant and gooseberry), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp. (e.g. olive), Actinidaceae sp., Lauraceae sp. (e.g. avocado, cinnamon, camphor), Musaceae sp. (e.g. banana trees and plantations), Rubiaceae sp. sp. (e.g. coffee), Theaceae sp. (e.g. tea), Sterculiceae sp., Rutaceae sp. (e.g. lemon, orange, mandarin and grapefruit); Solanaceae sp. (e.g. tomato, potato, pepper, chilli, eggplant, tobacco), Liliaceae sp.), Compositiae sp. (e.g. lettuce, artichoke and chicory - root chicory, endive or common chicory, etc.), Umbelliferae sp. (e.g. carrot, parsley, celery and celeriac), Cucurbitaceae sp. (e.g. cucumber - gherkin, pumpkin, watermelon, gourd and melon, etc.), Alliaceae sp. (e.g. leek and onion), Cruciferae sp. (e.g. white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, bok choy, kohlrabi, radish, horseradish, watercress and Chinese cabbage), Leguminosae sp. (e.g. peanuts, peas, lentils and legumes - e.g. kidney beans and broad beans), Chenopodiaceae sp. (e.g. chard, fodder beet, spinach, beetroot), Linaceae sp. (e.g. hemp), Cannabeacea sp. (e.g. cannabis), Malvaceae sp. (e.g. okra, cocoa), Papaveraceae (e.g. poppy), Asparagaceae (e.g. asparagus); useful and ornamental plants in gardens and forests, such as turf, lawns, grasses and Stevia rebaudiana; and in each case genetically modified versions of these plants.

[0124] In certain aspects, the fermentation product further comprises a formulation component. The formulation component may be a wetting agent, a bulking agent, a solvent, an autogenous promoter, an emulsifier, a dispersing agent, a cryoprotectant, a thickener, and / or an adjuvant. In one embodiment, the formulation component is a wetting agent. In other aspects, the fermentation product is a freeze-dried or spray-dried powder.

[0125] The compositions of the present invention may include formulation ingredients added to the compositions of the present invention to improve recovery, efficacy or physical properties, and / or to aid in processing, packaging and application. Such formulation ingredients may be added individually or in combination.

[0126] Formulation ingredients can be added to compositions containing cells, cell-free preparations, isolated compounds and / or metabolites to improve efficacy, stability and physical properties, usability, and / or to facilitate processing, packaging and end use. Such formulation ingredients include agriculturally acceptable carriers, inert agents, stabilizers, preservatives, nutrients, or physical property modifiers, which can be added individually or in combination. In some embodiments, the carriers can include liquid materials such as water, oils, and other organic or inorganic solvents, and solid materials such as minerals, polymers, or polymer complexes derived biologically or by chemical synthesis. In some embodiments, the formulation ingredients are binders, adjuvants, or adhesives that facilitate attachment of the composition to plant parts such as leaves, seeds, or roots. See, e.g., Taylor, AG, et al., "Concepts and Technologies of Selected Seed Treatments," Annu. Rev. Phytopathol., 28:321-339 (1990). The stabilizing agent may be an anti-caking agent, an antioxidant, an anti-settling agent, an anti-foaming agent, a desiccant, a protectant, or a preservative. The nutrients may be carbon, nitrogen, and phosphorus sources, such as sugars, polysaccharides, oils, proteins, amino acids, fatty acids, and phosphates. The physical property modifier may be a bulking agent, a wetting agent, a thickening agent, a pH modifier, a rheology modifier, a dispersant, an adjuvant, a surfactant, a film former, a hydrotrope, a builder, an antifreeze agent, or a colorant. In some embodiments, the composition comprising the cells, the cell-free preparation, and / or the metabolic products produced by fermentation may be used directly, with or without water as a diluent, without other formulation preparation. In certain embodiments, a wetting agent or dispersing agent is added to the fermentation solid, such as a freeze-dried or spray-dried powder. In some embodiments, the formulation inert agent is added after concentrating the fermentation broth and / or during and / or after drying. The wetting agent increases the spreading and penetration properties, and the dispersing agent increases the dispersibility and solubility of the active ingredient (once diluted) when applied to a surface. Exemplary wetting agents are known to those of skill in the art and include sulfosuccinates and derivatives such as MUL TIWETTM MO-70R (Croda Inc., Edison, NJ); siloxanes such as BREAK-THRU® (Evonik, Germany); non-ionic compounds such as ATLOX TM 4894 (Croda Inc., Edison, NJ); alkyl polyglucosides such as TERWET® 3001 (Huntsman International LLC, The Woodlands, Texas); C12-C14 alcohol ethoxylates such as TERGITOL® 15-S-15 (The Dow Chemical Company, Midland, Michigan); phosphate esters such as RHODAFAC® BG-510 (Rhodia, Inc.); and alkyl ether carboxylates such as EMULSOGEN TM LS (Clariant Corporation, North Carolina) is an example.

[0127] Deposit Information: Samples of the Paenibacillus strains of the present invention have been deposited under the Budapest Treaty at the Agricultural Research Service Culture Collection, Agricultural Utilization Research Center (NRRL), United States Department of Agriculture, 1815 North University Street, Peoria, Illinois, 61604, USA. Paenibacillus sp. NRRL B-50972 was deposited on August 28, 2014. Paenibacillus sp. NRRL B-67129 was deposited on September 1, 2015. Paenibacillus sp. NRRL B-67304 and Paenibacillus sp. NRRL B-67306 were both deposited on July 22, 2016. Paenibacillus sp. NRRL B-67615 was deposited on May 3, 2018.

[0128] The Paenibacillus strains have been deposited under conditions which ensure that access to such cultures will be available during the pendency of this patent application to any person determined by the Commissioner of the Patent and Trademark Office to be entitled thereto under 37 CFR Section 1.14 and 35 USC Section 122. It should be understood, however, that the availability of a deposit does not constitute a license to practice the invention in derogation of any patent rights granted by governmental action.

[0129] The following examples are given purely for illustrative and non-limiting purposes of the present invention. EXAMPLES

[0130] Example 1. Enhanced fusaricidin production by Paenibacillus sp. NRRL B-67129 strain and mutant derivatives Paenibacillus sp. strain NRRL B-67129 was treated with 1-methyl-3-nitro-1-nitroguanidine (NTG) or ethyl methanesulfonate (EMS) to introduce genetic mutations. Treatments resulting in 50–90% loss of colony forming units (CFU) were considered adequate to obtain sufficient genetic mutations and viable cells for subsequent screening. Individual isolates from the chemically treated populations were cultured in 96-deep well blocks and screened for increased production of four fusaricidin-like compounds, namely fusaricidin A (also known as "Fus A"), LiF08a, paenicellins A1 and B1 (also known as "M868" due to their molecular weight); and paeniprolixins A2 and B2 (also known as "M938" due to their molecular weight) as described in WO2016 / 154297.

[0131] Isolates with clearly improved fusaricidin levels from this initial screen were rescreened in technical replicates of 96-deep well blocks and confirmed to be overproducers. Confirmed isolates were then scaled up and analyzed for fusaricidin production and growth characteristics, including the ability to produce heat-resistant bacterial spores. From the first round of screening, confirmation, and scale-up, several overproducing isolates were identified, re-chemically treated, and screened for further improvement in fusaricidin production. This process was repeated several times.

[0132] Strains identified in this analysis were further evaluated to confirm fusaricidin production. Briefly, each strain was grown in a soy-based medium and the lipid-soluble fraction of the whole broth was extracted. The whole broth extract was analyzed by high performance liquid chromatography (HPLC) and the presence of fusaricidin A was identified based on the HPLC profile generated by a standard sample containing fusaricidin A. The mutants were also evaluated for their ability to produce heat-resistant spores.

[0133] In total, approximately 10,000 isolates derived from Paenibacillus sp. NRRL B-67129 were screened in a 96-well deep-well block format. This analysis yielded several isolates that were characterized for their relative levels of four fusaricidin-like compounds (see Table 1). These strains were extensively characterized, and Paenibacillus sp. strain J was identified as exhibiting high levels of fusaricidin production and favorable growth characteristics (e.g., sporulation) compared to Paenibacillus sp. NRRL B-67129. However, Paenibacillus sp. strain J produced a viscous fermentation broth that made processing difficult. This observation highlighted the need to screen mutant strains for viscosity and fusaricidin-like compound production in liquid culture. [Table 1] TIFF2025060869000003.tif62152

[0134] Example 2. Fermentation Broth Viscosity Reduction of Paenibacillus sp. NRRL B-67129 Strain Mutant Derivatives Paenibacillus sp. NRRL B-50972 and Paenibacillus sp. NRRL B-67129 produced viscous fermentation broth cultures, as did many of the mutants derived from Paenibacillus sp. NRRL B-67129. The physicochemical properties of these cultures presented challenges in fermentation and downstream processing. It was therefore desirable to find a way to identify mutant derivatives of Paenibacillus sp. NRRL B-67129 that would produce fermentation broth cultures with reduced viscosity.

[0135] Sucrose is often used as a carbon source for the production of exopolysaccharides (EPS) by Paenibacillus sp., and it has been reported that the use of sucrose can produce high molecular weight levan-type EPS in significant yields (Liang and Wang. Mar. Drugs 2015, 13, 1847-1863). High molecular weight EPS polymers are commercially used as thickening agents. In addition to sucrose, other oligosaccharides and polysaccharides are used as carbon sources for EPS production by Paenibacillus sp.

[0136] A distinct mucoid colony phenotype was observed when Paenibacillus sp. strain NRRL B-67129 and mutant derivatives were grown on solid agar medium supplemented with sucrose at final concentrations of 0.25-0.5 M (see recipe in Table 2). A mucoid colony phenotype also appeared on similar solid agar medium containing 200 g / L maltodextrin.

[0137] Several Paenibacillus strains, including strains of P. terrae, P. brasilensis, P. polymyxa, and P. peoriae, produced a mucoid phenotype on sucrose-containing solid agar medium (see Figure 2). We hypothesized that a rapid visual screen could be designed for non-mucoid colony isolates that would result in fermentation broth cultures with reduced viscosity and enhanced physical properties. Without wishing to be bound by any theory, the mucoid colony phenotype may correspond to the production of EPS by Paenibacillus sp. NRRL B-67129, its mutant derivatives, and other Paenibacillus strains in submerged culture. [Table 2]

[0138] The following protocol was developed and validated as a rapid visual screen to identify nonmucoid colony isolates. Liquid cultures of fungicidal mutant derivatives of Paenibacillus sp. strain NRRL B-67129 were chemically treated, then diluted and inoculated onto solid agar medium supplemented with sucrose to obtain single colonies. Nonmucoid colonies were easily distinguishable by the naked eye from mucoid colonies (see Figure 3). Nonmucoid isolates were picked and streaked onto fresh solid agar medium supplemented with sucrose to confirm the phenotype.

[0139] We identified eight nonmucoid isolates from the fusaricidin-overproducing parent strain derived from Paenibacillus sp. NRRL B-67129. The nonmucoid isolates included Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304 (see Figure 4). Six of the eight isolates produced fusaricidin biomarkers at levels comparable to or higher than their respective parent strains. In addition, five of the eight isolates were able to produce heat-resistant spores at levels similar to those produced by Paenibacillus sp. NRRL B-67129 under the same conditions. These observations indicated that the cellular processes associated with fusaricidin production, sporulation, and viscosity-producing factors are genetically separable.

[0140] Eight nonmucoid isolates were evaluated in larger scale cultures and two nonmucoid isolates, Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304, were found to exhibit improved fusaricidin production and favorable growth characteristics in soy-based media. These strains were analyzed for their packed cell volume (%PCV) and their viscosity.

[0141] The %PCV was determined by centrifuging a 1 mL volume at 17,000 g for 3 minutes in a 2 mL microcentrifuge tube. The percent packed cell volume was calculated based on the tube graduations, with the initial 1 mL sample mark being taken as 100%.

[0142] Alternatively, place approximately 10 mL of whole broth into a 15 mL centrifuge tube and measure the weight of the whole broth ("W wb The weight of the supernatant ("W") was recorded, the sample was centrifuged at 10,000 g for 10 minutes, the supernatant liquid was poured off, and the weight of the supernatant ("W") was recorded. sup The %PCV was calculated using the following formula: %PCV = 100 × (W wb -W sup ) / (W wb )

[0143] The viscosity of the fermentation broth was tested with a viscometer at 50 rpm and values ​​were reported in centipoise.

[0144] Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304 produced fermentation broths with viscosities of 11.5 and 33.9 centipoise (cP), respectively, compared to 56 cP for the fermentation broth of Paenibacillus sp. NRRL B-50972 (Table 3). Additionally, Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304 had smaller packed cell volumes (PCVs) compared to Paenibacillus sp. NRRL B-50972. These results validated the hypothesis that rapid visual screening on solid media containing high levels of polysaccharides (e.g., sucrose or maltodextrin) can identify non-mucoid colony isolates that result in fermentation broth cultures with reduced viscosity and enhanced physical properties.

[0145] The improved physical properties of fermentation broths from Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304 allowed for enhanced processability of these non-mucoid strains as live microbial-based products. The lower PCV and viscosity of the fermentation broths allowed for increased concentration of the whole broth material for reduced use rates in agricultural applications. [Table 3]

[0146] Example 3. Mutation analysis of strain-improved isolatesThe genome sequences of several isolates with a nonmucoid phenotype were determined using standard sequencing methods. Single nucleotide polymorphisms (SNPs) of the isolates were compared. Surprisingly, five of the eight nonmucoid strains derived from Paenibacillus sp. NRRL B-67129, including Paenibacillus sp. NRRL B-67304 and Paenibacillus sp. NRRL B-67306, were found to be mutated ... degS degU The regions were found to have mutations in the protein codon sequences (see Figure 6A), which were located within the receiver and DNA-binding domains of DegU and within the single-binding and ATPase domains of DegS (see Figures 6B-6C).

[0147] degS and degU It was hypothesized that these mutations in are associated with the nonmucoid phenotype of these isolates on solid agar plates supplemented with sucrose. To test this, the parent Paenibacillus sp. NRRL B-67129 strain degS The gene was replaced with a kanamycin cassette, as well as degS and degU A DNA construct was made using standard molecular methods to replace the region with a kanamycin cassette.

[0148] degS Alone or degS and degUTo target the replacement of α-kanamycin with kanamycin resistance (kanR), the gene encoding kanR was cloned into a conjugative E. coli-Paenibacillus shuttle plasmid flanked by a 1 kbp region upstream of the gene encoding DegS and 1 kbp downstream of the gene encoding DegS or DegU. This plasmid was first introduced into an E. coli strain by electroporation and subsequently transferred to the Paenibacillus sp. NRRL B-67129 strain by conjugation. Successful plasmid transfers were selected using erythromycin resistance encoded in the plasmid backbone. Kanamycin resistance, erythromycin sensitivity and PCR validation were used to confirm double-crossover integrants. Kanamycin resistance marker replacement strain Paenibacillus sp. NRRL B-67129 degS :: kanR strain and Paenibacillus sp. NRRL B-67129 degS degU :: kanR All of the strains mimicked the nonmucoid phenotype of the previously selected isolates (see Figure 7). These results suggest that the expression of a non-functional gene product may be a contributing factor to the development of a novel genomic DNA fragment. degS and degU We confirmed that the mutation results in a nonmucoid phenotype.

[0149] degS and degUOther mutations in DegS have been characterized that resulted in non-functional gene products. The serine 76 residue in Bacillus subtilis strain 168, which corresponds to threonine 73 in Paenibacillus sp. strain NRRL B-50972, is the phosphorylation site that stimulates its kinase activity, and mutating serine 76 to alanine significantly reduced the enzymatic activity of DegS (see Jers, C. et al., "Bacillus subtilis Two-Component System Sensory Kinase DegS is Regulated by Serine Phosphorylation in Its Input Domain," PLoS ONE (2011) 6(2):e14653). Mutation of the alanine 193 residue in Bacillus subtilis strain 168, which corresponds to alanine 190 in Paenibacillus sp. NRRL B-50972, to valine essentially abolished the kinase activity of DegS (see Dahl, M. K. et al., "The Phosphorylation State of the DegU Response Regulator Acts as a Molecular Switch Allowing Either Degradative Enzyme Synthesis or Expression of Genetic Competence in Bacillus subtilis," J. Biol. Chem. (1992) 267(20):14509).

[0150] Substitution of aspartic acid 56, which corresponds to aspartic acid 63 in Paenibacillus sp. NRRL B-50972 strain, with asparagine degU Mutations in the 2′-terminal cleavage domain of DegU prevented phosphorylation of DegU by DegS (see Dahl, MK et al., supra). Alanine scanning of the DNA-binding domain of DegU revealed that comK and aprE We identified five common mutants that cause severely reduced DegU binding to the promoter regions of genes and, consequently, reduced expression of these genes. aprEThree additional mutants were also identified that inhibited the binding of DegU to the promoter region of Bacillus subtilis and consequently reduced expression of this gene (Shimane et al., "Mutational Analysis of the Helix-Turn-Helix Region of Bacillus subtilis Response Regulator DegU, and Identification of cis-Acting Sequences for DegU in the aprE and comK Promoters”, J. Biochem. (2004) 136(3):387-397.)

[0151] degS Genes or degS Genes and degU Gene replacement with an antibiotic resistance cassette results in a non-functional gene product, including those listed above. degS or degU It is concluded that any mutation in would result in a Paenibacillus strain that exhibits reduced liquid culture viscosity and / or non-mucoid colony morphology compared to a Paenibacillus strain containing wild-type DegU and wild-type DegS. [Table 4] [Table 5] [Table 6] TIFF2025060869000009.tif132169TIFF2025060869000010.tif131167

[0152] Example 4. Further mutagenesis and screening of non-mucoid strainsTo further improve the titer of fusaricidin-like compounds, chemical treatment of Paenibacillus sp. NRRL B-67304 strain was performed as described in Example 1. Samples from the culture broths produced in the 96-well blocks were analyzed for relative levels of fusaricidin A (see Table 7). Several isolates with increased fusaricidin production were then selected for further testing after fermentation in larger-scale cultures. Samples from these larger-scale cultures were again analyzed for fusaricidin A content (see Table 8) and their packed cell volumes were measured as described in Example 2 (see Table 9). Packed cell volumes were only evaluated in samples from the larger-scale cultures because the cultures from the 96-well blocks did not yield sufficient volumes for these measurements.

[0153] Surprisingly, it was found that Paenibacillus sp. NRRL B-67615 not only had enhanced levels of fusaricidin-like compounds (see Tables 7 and 8), but also had a lower level of viscosity than Paenibacillus sp. NRRL B-67304 (see Table 9). These results further confirmed that the cellular processes related to fusaricidin biosynthesis, sporulation and production of viscosity-producing factors are genetically separable. The phylogeny of Paenibacillus sp. NRRL B-67615 with Paenibacillus sp. NRRL B-50972, Paenibacillus sp. NRRL B-67129, Paenibacillus sp. NRRL B-67304 and Paenibacillus sp. NRRL B-67306 is shown in Figure 1. [Table 7] [Table 8] [Table 9]

[0154] The relative Fusaricidin A levels, packed cell volume and viscosity of Paenibacillus sp. NRRL B-50972, Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304, and Paenibacillus sp. NRRL B-67615 were evaluated together to confirm the improvements achieved by multiple rounds of mutagenesis and screening according to the disclosed methods. The results, shown in Table 10, are consistent with those of the disclosed methods. It has been demonstrated that the screening methodology used resulted in mutant derivatives with lower packed cell volume and viscosity, allowing for significant improvements in fusaricidin production and higher concentrations of the active compound in the fermentation broth. [Table 10]

[0155] Example 5. Comparison of biological activities of Paenibacillus sp. NRRL B-50972, Paenibacillus sp. NRRL B-67306, Paenibacillus sp. NRRL B-67304 and Paenibacillus sp. NRRL B-67615 Paenibacillus sp. NRRL B-50972, Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304 were cultivated in soy-based medium to produce whole broth. The whole broth was diluted with a mixture of water and organic solvent to concentrations of 2.5%, 1.25%, 0.625% and 0.312%. The diluted whole broth was applied to young plants, which were then exposed to an inoculum of Alternaria solani (ALTESO). A chemical fungicide was included in each assay as a positive control. After several days of exposure to the inoculum of the plant pathogen, each plant was scored as the percentage control of the pathogen relative to untreated control plants. Each treatment was evaluated in triplicate, and the average percentage control was recorded (see Table 11). 0% means efficacy corresponding to that of the untreated control, and 100% efficacy means that no disease damage was observed. Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304 had superior antifungal activity compared to Paenibacillus sp. NRRL B-50972. [Table 11]

[0156] The assay was repeated with Paenibacillus sp. NRRL B-67304 and Paenibacillus sp. NRRL B-67615 against the fungal pathogen Alternaria solani (ALTESO). The assay was performed similarly to above, except six replicates were evaluated instead of three, and whole broth was applied at 1.25% or 0.625%. Table 12 shows the average percentage control of the treatments. Paenibacillus sp. NRRL B-67304 and Paenibacillus sp. NRRL B-67615 showed similar antifungal activity in the assay. [Table 12]

[0157] Example 6. Antifungal activity of Paenibacillus sp. NRRL B-67306, Paenibacillus sp. NRRL B-67304 and Paenibacillus sp. NRRL B-67615 against oomycete plant pathogensPaenibacillus sp. NRRL B-67306, Paenibacillus sp. NRRL B-67304 and Paenibacillus sp. NRRL B-67615 were cultivated in soy-based medium to produce whole broth. The whole broth was diluted with a mixture of water and organic solvent to concentrations of 10%, 5%, 2.5%, 1.25% and 0.625%. The diluted whole broth was applied to young plants, which were then exposed to an inoculum of Pseudoperonospora cubensis (PSPECU), also known as cucumber downy mildew, or Phytophthora infestans (PHYTIN), also known as tomato late blight. A chemical fungicide was included in each assay as a positive control. After several days of exposure to the inoculum of the plant pathogen, each plant was scored as the percentage of pathogen control relative to untreated control plants. Each treatment was evaluated in triplicate and the average percentage control was recorded (see Table 13 for results for Pseudoperonospora cubensis and Table 14 for results for Phytophthora infestans). 0% means efficacy equivalent to that of the untreated control and 100% efficacy means no disease observed. All three Paenibacillus strains showed consistent control of the two oomycete plant pathogens. [Table 13] [Table 14]

[0158] Example 7. Comparison of Paenibacillus strains in field trials on potatoes infected with Alternaria solaniField trials were carried out with potato plants exposed to naturally occurring Alternaria solani. Liquid fermentation products of Paenibacillus sp. NRRL B-50972 and Paenibacillus sp. NRRL B-67306 strains were prepared by cultivating the strains in a soy-based medium and concentrating the resulting whole broth via centrifugation and fractionation of the supernatant. As outlined in Table 16, plants were applied with 10 and 20 liters / ha of fermentation product between July 20 and August 4 at the BBCH65 to BBCH70 growth stages. The average disease incidence was about 13% in untreated plants. The disease control percentages shown in Table 15 are the result of an assessment by visual observation of the disease condition carried out 7 days after the last application. 0% means an efficacy corresponding to that of the untreated control, 100% means that no disease was observed. [Table 15] [Table 16]

[0159] The results in Table 15 clearly show the superiority of the observed activity of Paenibacillus sp. NRRL B-67306 strain compared to Paenibacillus sp. NRRL B-50972 in this field trial.

[0160] Example 8. Comparison of Paenibacillus strains in field trials of strawberries infected with Botrytis cinereaField trials were conducted with strawberry plants exposed to naturally occurring gray mold (Botrytis cinerea). Liquid fermentation products of Paenibacillus sp. NRRL B-50972 and Paenibacillus sp. NRRL B-67304 strains were prepared by cultivating the strains in a soy-based medium and concentrating the resulting whole broth via centrifugation and fractionation of the supernatant. As outlined in Table 18, plants were applied with 10 and 20 liters / ha of fermentation product between March 31 and April 18 at growth stages BBCH67 to BBCH87. The average disease incidence was about 22% in untreated plants. The disease control percentages shown in Table 17 are the result of an assessment by visual observation of the disease condition carried out 2 days after the last application. 0% means an efficacy corresponding to that of the untreated control, and 100% means that no disease was observed. [Table 17] [Table 18]

[0161] The results in Table 17 clearly show that the observed activity of Paenibacillus sp. NRRL B-67304 strain is superior to that of Paenibacillus sp. NRRL B-50972 in this field trial.

[0162] Example 9. Comparison of Paenibacillus strains in field trials on peppers infected with Colletotrichum capsiciField trials were conducted with pepper plants exposed to naturally occurring anthracnose (Colletotrichum capsici). Liquid fermentation products of Paenibacillus sp. NRRL B-50972 and Paenibacillus sp. NRRL B-67306 strains were prepared by cultivating the strains in a soy-based medium and concentrating the resulting whole broth via centrifugation and fractionation of the supernatant. As outlined in Table 20, plants were applied with 10 and 20 liters / ha of fermentation product between December 28 and January 2 at the BBCH75 growth stage. The average disease incidence was about 60% in untreated plants. The disease control percentages shown in Table 19 are the result of an assessment by visual observation of the disease condition carried out 2 days after the last application. 0% means an efficacy corresponding to that of the untreated control, and 100% means that no disease was observed. [Table 19] [Table 20]

[0163] The results in Table 19 clearly demonstrate the superiority of the observed activity of Paenibacillus sp. NRRL B-67306 strain compared to Paenibacillus sp. NRRL B-50972 in this field trial.

[0164] Example 10. Identification of growth conditions that cause viscosity discrepancy between Paenibacillus sp. NRRL B-67304 and NRRL B-67615 strains As shown in Figure 1, Paenibacillus sp. NRRL B-67615 strain was generated by chemical mutagenesis of Paenibacillus sp. NRRL B-67304 strain. The results of this chemical mutagenesis NRRL B-67615 strain had a significantly reduced viscosity, but relatively high levels of fusaricidin A (see Table 10). To determine the time point at which the viscosities of the two strains diverged in liquid culture, each strain was grown in soy-based medium for a period of 72 hours. One group of cultures was agitated at 250 rpm and the other at 300 rpm. Samples of each liquid culture were removed at 24, 32, 40, 48, 56, and 72 hours. The viscosity and relative levels of fusaricidin A of each sample were measured as outlined in Example 4. The mean and standard deviation (n=4) were determined for cultures grown at 250 rpm and 300 rpm and are shown in Figures 8A and 8B, respectively.

[0165] The relative levels of Fusaricidin A produced by each strain were comparable and increased at a similar rate over 72 hours. Spore production was visually assessed under a microscope in all samples, and no spores were present at any time point. A significant increase in viscosity was observed for Paenibacillus sp. NRRL B-67304, whereas the viscosity of Paenibacillus sp. NRRL B-67615 remained low and relatively constant over this time period (compare the solid lines showing viscosity in Figure 8A and Figure 8B), so the 40 and 48 hour time points were selected for future experiments. Liquid cultures grown with agitation at 300 rpm were more consistent in their viscosity values, so this agitation speed was also selected for future experiments.

[0166] Example 11. Proteome analysis of liquid cultures of Paenibacillus sp. NRRL B-67304 and NRRL B-67615Exploratory proteomics and pathway analysis approaches were performed to gain insight into the viscosity phenotype at the molecular level using Paenibacillus sp. NRRL B-67304 (parental strain) and Paenibacillus sp. NRRL B-67615 (progeny strain). The strains were grown in shake flasks in soy-based medium for 40 and 48 h, resulting in divergent viscosity phenotypes for the two strains. A total of 24 specimens were grown for Paenibacillus sp. NRRL B-67304 and NRRL B-67615, with six replicates per condition. At harvest, specimens were rapidly frozen at -80°C to stop growth, followed by batch-based sample preparation. Protein extraction was performed on all fermentations to obtain secreted and vegetative cell proteins. Total protein samples were reduced, alkylated, and tryptic digested to generate total peptide pools for proteomic analysis. Total peptide samples were separated by liquid chromatography and analysis on a SCIEX 4600 TRIPLETOF® mass spectrometer was performed sequentially in data-dependent (IDA) and data-independent (SWATH) acquisition modes, allowing the creation of ion libraries and relative quantification across the entire peptide pool.

[0167] To create an ion library, the IDA runs were first analyzed with SCIEX Protein Pilot (5.0.1.0,4895) software in exhaustive ID mode with false discovery rate (FDR) analysis. Then, the SWATH microapp (2.0.1.2133) was used to generate an ion library in SCIEX PeakView (2.2.0.11391) software with 1% global protein FDR. Continuing with data analysis with the SWATH microapp, relative quantification of the SWATH runs was performed with 99% peptide confidence and 1% FDR threshold. Protein areas calculated from the summed intensities of six transitions per peptide and six peptides per protein were then exported for downstream analysis. The protein area threshold was set to 50,000.

[0168] Of primary interest was the identification of differentially expressed proteins at a single time point (40 or 48 h) between Paenibacillus sp. NRRL B-67304 (parental strain) and Paenibacillus sp. NRRL B-67615 (progeny strain). The aim was to elucidate differences in protein levels between the strains, which were hypothesized to contribute to exopolysaccharide (EPS) production and distinct viscosity phenotypes. Statistical analysis was first performed using MarkerView (1.2.1) software from SCIEX. Exploratory data analysis including mean 1 vs mean 2 and Log(expression change) vs p-values ​​showed no significant data anomalies, and principal component analysis showed samples grouped by strain and time. To determine differential protein expression between strains, t-tests were performed in MarkerView, followed by adjustment of p-values ​​for multiple comparisons in R (FDR / BH correction). Proteins were considered differentially expressed with P(FDR / BH corrected)<0.05 and a minimum fold change of 1.5. Of the 442 proteins detected at 40 hours, 54 proteins met the differential expression criteria (see Table 21). Of the 422 proteins detected at 48 hours, 94 proteins were differentially expressed (see Table 22).

[0169] Bacterial exopolysaccharides are structurally diverse, composed of different building blocks, and synthesized by various pathways. Expression also varies with strain and environment (e.g., fermentation process). For example, different strains of Paenibacillus have been characterized as producing curdlan-type and levan-type EPS composed of glucose or glucose and fructose, respectively. This initial proteomic analysis suggested that none of the proteins identified as differentially expressed in Paenibacillus sp. NRRL B-67304 (parental strain) and Paenibacillus sp. NRRL B-67615 (progeny strain) are directly involved in EPS synthesis, as identified by homology to proteins described in the literature.

[0170] Further analysis of the proteomic data was required to explain the difference in viscosity phenotype between Paenibacillus sp. NRRL B-67304 and NRRL B-67615 strains. To contextualize the protein level differences seen by proteomic analysis, proteins were further annotated in KEGG (BLASTKOALA algorithm) and mapped to KEGG pathways. Several proteins involved in glycolysis and tricarboxylic acid (TCA) cycle were observed to be significantly elevated in Paenibacillus sp. NRRL B-67615 (progeny strain) at 48 hours (see underlined proteins under "Up-regulated in progeny" in Table 22). This suggests that an increase in sugar metabolism occurs in Paenibacillus sp. NRRL B-67615 (progeny strain) compared to Paenibacillus sp. NRRL B-67304 (parental strain). EPS production is dependent on the same hexose monomers (e.g., glucose and fructose) as primary metabolism. Thus, an increase in primary metabolism would lead to a decrease in the concentration of starting substrates and a resultant decrease in EPS production and viscosity in Paenibacillus sp. NRRL B-67615 (progeny strain).

[0171] Conversely, where carbohydrate resources are in excess and starting substrates are abundant, EPS production and viscosity will increase. Consistent with this idea, two α-amylase proteins were significantly elevated at 40 and 48 hours in Paenibacillus sp. NRRL B-67304 (parental strain) (see underlined proteins under "Up-regulated in parent" in Tables 21 and 22). The amino acid sequences of the two amylases are shown in Table 23. These two amylases have protein domains characteristic of the "α-amylase family", which is glycoside hydrolase family 13 (see Cockburn et al., Biologia 69(6):705-712, 2014).

[0172] The relative expression of two α-amylases ("α-amylase #1" and "α-amylase #2") was quantified using samples taken at 40 and 48 hours and is shown in Figures 9A and 9B. The relative quantification of the proteins indicates that Paenibacillus sp. NRRL B-67304 (parental strain) consistently expresses significantly more α-amylase than Paenibacillus sp. NRRL B-67615 (progeny strain). Without wishing to be bound by any theory, the soy-based culture medium in which the strains were grown contains polysaccharides that these amylases convert into hexose monomers required for EPS production. The high amount of substrate may then promote EPS production and increase viscosity in liquid cultures of Paenibacillus sp. NRRL B-67304 (parental strain). [Table 21] TIFF2025060869000026.tif248168 [Table 22] TIFF2025060869000028.tif248161TIFF2025060869000029.tif253156TIFF2025060869000030.tif75167 [Table 23]

[0173] Example 12. Confirmation of increased amylase activity in Paenibacillus sp. NRRL B-67304 strain (parent strain)To confirm that liquid cultures of Paenibacillus sp. NRRL B-67304 (parent strain) have higher levels of amylase activity than Paenibacillus sp. NRRL B-67615 (progeny strain), both strains were grown in soy-based medium and samples were taken at 40 and 48 hours. The samples were centrifuged and the supernatants sterile filtered to remove all cells from the medium, leaving behind cellular proteins including amylase and unconsumed polysaccharides. The amylase in the supernatants continues to break down polysaccharides and produce glucose. The glucose content in these cell-free supernatants was measured initially and after 5 hours of incubation at 28°C.

[0174] The glucose measurement results shown in Figure 10 show that Paenibacillus sp. NRRL B-67304 (parent strain) produced significantly more glucose than Paenibacillus sp. NRRL B-67615 (progeny strain) when the strains were grown under similar conditions. also show high levels of amylase activity.

[0175] Example 13. Addition of glucose to liquid cultures of Paenibacillus sp. NRRL B-67304 and NRRL B-67615 If the availability of hexose monomers (e.g., glucose, fructose) in liquid cultures of Paenibacillus strains limits EPS production, which contributes to the viscosity of these cultures, then the addition of glucose to the culture should result in increased EPS production and viscosity, an effect that would be most pronounced for Paenibacillus sp. NRRL B-67615 (progeny). To test this hypothesis, liquid cultures of Paenibacillus sp. NRRL B-67615 (progeny) and Paenibacillus sp. NRRL B-67304 (parent) were supplemented with 0 g / L glucose (i.e., control), 2 g / L glucose, 5 g / L glucose, or 10 g / L glucose at 40 hours. The control and glucose-supplemented liquid cultures were allowed to continue growing for 6 hours, at which point the viscosity and residual glucose concentration in each culture were measured.

[0176] In all cultures except for the one supplemented with 10 g / L glucose, the residual glucose concentration was near 0 g / L, indicating that the added glucose was consumed by the cells. Viscosity measurements of the cultures are shown in FIG. 11. The addition of glucose to the Paenibacillus sp. NRRL B-67304 (parent) culture had little effect on the viscosity, likely due to the high level of amylase activity of the strain and the resulting abundant glucose available from the culture medium. In contrast, the glucose-supplemented culture of Paenibacillus sp. NRRL B-67615 (progeny) had a higher viscosity due to the increased viscosity caused by the increased amount of added glucose (see the right bar graph in FIG. 11).

[0177] Overall, the above experimental results indicate that amylase expression and activity are lower in Paenibacillus sp. NRRL B-67615 (progeny) compared to Paenibacillus sp. NRRL B-67304 (parent), resulting in less simple sugars (e.g., glucose) for EPS production and a lower viscous phenotype.

[0178] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patents, and patent publications cited are incorporated herein by reference in their entirety for all purposes.

[0179] It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described as these can vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0180] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the appended claims.

Claims

1. 1. A method for identifying a mutant derivative of Paenibacillus having reduced viscosity in liquid culture compared to the parent Paenibacillus strain, the method comprising: generating a mutant isolate by mutagenesis of the parent Paenibacillus strain; culturing the mutant isolate and the parent Paenibacillus strain on a solid medium containing a sugar at a concentration of about 1% (w / v) to about 40% (w / v), wherein the parent Paenibacillus strain has a mucoid morphology on the solid medium; and visually screening the mutant isolates on the solid medium to identify mutant derivatives of Paenibacillus having a non-mucoid morphology, indicative of reduced viscosity in liquid culture.

2. 10. The method of claim 1, wherein the sugar in the solid medium is at a concentration of about 5% (w / v) to about 20% (w / v).

3. 3. The method of claim 1 or 2, wherein the sugar is selected from the group consisting of sucrose, maltodextrin, starch, corn syrup solids, fructose, glucose, galactose, lactose, maltose, xylose, xylitol, inulin, sorbitol, fucose, molasses, and combinations thereof.

4. 4. The method of claim 1, wherein the carbon to nitrogen ratio in the solid medium is from about 10:1 to about 1000:1, and / or the solid medium further comprises agar, agarose, and / or gelatin.

5. The method of any one of claims 1 to 4, further comprising culturing a mutant derivative of Paenibacillus in a liquid medium to produce a liquid culture; and measuring the viscosity and / or packed cell volume of the liquid culture to determine the reduced viscosity of the mutant derivative of Paenibacillus compared to the parent strain of Paenibacillus.

6. 6. The method of any one of claims 1 to 5, further comprising sequencing degU and / or degS in a mutant derivative strain of Paenibacillus to identify sequences encoding a mutant DegU lacking a functional receiver domain or a functional DNA-binding domain and / or a mutant DegS lacking a functional single-binding domain or a functional ATPase domain.

7. 7. The method of any one of claims 1 to 6, further comprising measuring amylase expression and / or enzymatic activity in the mutant derivative of Paenibacillus and the parent Paenibacillus strain, and determining whether said expression and / or enzymatic activity is reduced in the mutant derivative of Paenibacillus.

8. 8. The method of claim 7, wherein the reduced amylase expression and / or enzymatic activity occurs in an α-amylase protein comprising a sequence having greater than about 90% sequence identity to SEQ ID NO:9 or SEQ ID NO:

10.

9. The method of any one of claims 1 to 8, further comprising quantifying the amount of fusaricidin in mutant isolates to identify mutant isolates having increased levels of fusaricidin compared to the parent strain of Paenibacillus.

10. The aforementioned P. agarexedens, P. agaridevorans, P. alginolyticus, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. assamensis, P. azoreducens, P. azotofixans, P. barcinonensis, P. borealis, P. br asiliensis, P. brassicae, P. campinasensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P. cineris, P. cookie, P. curdlanolyticus, P. daejeonensis, P. dendritiformis, P. durum, P. ehimensis, P. elgii, P. favisporus, P. glu canolyticus, P. glycolyticus, P. gordonae, P. graminis, P. granivorans, P. hodogayensis, P. illinoisensis, P. jamilae, P. kobensis, P. coleovorans, P. koreensis, P. kribbensis, P. lactis, P. larvae, P. lautus, P. lentimorbus, P. ma cerans, P. macquariensis, P. massiliensis, P. mendelii, P. motobuensis, P. napthalenovorans, P. nematophilus, P. nov. spec. epiphyticus, P. odorifer, P. pabuli, P. peoriae, P. phoenicis, P. phyllosphaerae, P. polymyxa, P. polymyxa ssp. polymyxa, P. polymyxaAesthetic fiction、Asceticism、S Emotional emotion、Advice emotion、S aesthetics、aesthetics、aesthetics Aesthetics、Aesthetics、Aesthetics The snow、theatricals、theatricals、was Aesthetics、Aesthetics、Aesthetics、 Emotionally、Emotionally Thanksgiving is the best part of the 1990s.

11. 1. A method for producing a mutant derivative of Paenibacillus having reduced viscosity in liquid culture compared to the parent strain of Paenibacillus, the method comprising: mutagenizing the parent strain of Paenibacillus to produce a mutant isolate; culturing the mutant isolate and the parent strain of Paenibacillus on a solid medium containing a sugar at a concentration of about 1% (w / v) to about 40% (w / v), wherein the parent strain of Paenibacillus has a mucoid morphology on the solid medium; visually screening the mutant isolates on the solid medium to identify mutant derivatives of Paenibacillus having a non-mucoid morphology indicative of reduced viscosity in liquid culture; and producing a fermentation product of the identified mutant derivative of Paenibacillus.

12. 12. The method of claim 11, wherein the mutagenesis comprises chemical mutagenesis of a parent strain of Paenibacillus.

13. 13. The method of claim 11 or 12, wherein the sugar in the solid medium is at a concentration of about 5% (w / v) to about 20% (w / v).

14. 14. The method of any one of claims 11 to 13, wherein the sugar is selected from the group consisting of sucrose, maltodextrin, starch, corn syrup solids, fructose, glucose, galactose, lactose, maltose, xylose, xylitol, inulin, sorbitol, fucose, molasses, and combinations thereof.

15. The method of any one of claims 11 to 14, further comprising culturing the mutant derivative of Paenibacillus in a liquid medium to produce a liquid culture; and measuring the viscosity and / or packed cell volume of the liquid culture to determine the reduced viscosity of the mutant derivative of Paenibacillus compared to the parent strain of Paenibacillus.

16. 16. The method of any one of claims 11 to 15, further comprising sequencing degU and / or degS in a mutant derivative strain of Paenibacillus to identify sequences encoding a mutant DegU lacking a functional receiver domain or a functional DNA-binding domain and / or a mutant DegS lacking a functional single-binding domain or a functional ATPase domain.

17. 17. The method of any one of claims 11 to 16, further comprising measuring amylase expression and / or enzymatic activity in the mutant derivative of Paenibacillus and the parent Paenibacillus strain to determine whether said expression and / or enzymatic activity is reduced in the mutant derivative of Paenibacillus.

18. 18. The method of claim 17, wherein the reduced amylase expression and / or enzymatic activity occurs in an α-amylase protein comprising a sequence having greater than about 90% sequence identity to SEQ ID NO:9 or SEQ ID NO:

10.

19. The aforementioned P. agarexedens, P. agaridevorans, P. alginolyticus, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. assamensis, P. azoreducens, P. azotofixans, P. barcinonensis, P. borealis, P. br asiliensis, P. brassicae, P. campinasensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P. cineris, P. cookie, P. curdlanolyticus, P. daejeonensis, P. dendritiformis, P. durum, P. ehimensis, P. elgii, P. favisporus, P. glu canolyticus, P. glycolyticus, P. gordonae, P. graminis, P. granivorans, P. hodogayensis, P. illinoisensis, P. jamilae, P. kobensis, P. coleovorans, P. koreensis, P. kribbensis, P. lactis, P. larvae, P. lautus, P. lentimorbus, P. ma cerans, P. macquariensis, P. massiliensis, P. mendelii, P. motobuensis, P. napthalenovorans, P. nematophilus, P. nov. spec. epiphyticus, P. odorifer, P. pabuli, P. peoriae, P. phoenicis, P. phyllosphaerae, P. polymyxa, P. polymyxa ssp. polymyxa, P. polymyxaAesthetic fiction、Asceticism、S Emotional emotion、Advice emotion、S aesthetics、aesthetics、aesthetics Aesthetics、Aesthetics、Aesthetics Yes、Christianity、Christianity、S scientific、scientific、scientific、. snowflakes The 1118th episode of the 1118th episode of the 11th century.

20. A fermentation product comprising a mutant derivative of Paenibacillus sp. identified by the method of any one of claims 1 to 10.

21. 21. The fermentation product of claim 20, wherein the fermentation product comprises a whole culture broth concentrate from a mutant derivative of Paenibacillus sp. for increased fungicidal and / or bactericidal activity.

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