Paenibacillus mutants and methods of using the same

Abstract

The present invention provides a composition comprising a biologically pure culture of a Paenibacillus sp. strain having reduced viscosity in liquid culture, the culture containing 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. The present invention also provides a method for identifying a Paenibacillus sp. mutant derivative strain having reduced viscosity in liquid culture compared to a parent Paenibacillus strain by visual screening for mutants with non-mucoid morphology. [Selected Figure] 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 strains of Paenibacillus sp. that have a relatively high level of antifungal activity and a reduced viscosity that facilitates downstream processing and concentration of the whole culture broth product of the strain.

[0002] Cross-reference to related applications: This application claims priority based on U.S. Provisional Patent Application No. 62 / 671,067 filed on May 14, 2018, the entire content of which is incorporated herein by reference.

[0003] Reference to electronically submitted sequence listing: A formal copy of the sequence listing is electronically submitted here simultaneously with this specification via EFS-Web by a 29 kilobyte file named "BCS169009_WO_ST25.txt" created on April 30, 2019 as an ASCII-formatted sequence listing. The sequence listing contained in this ASCII-formatted document is part of this specification and is incorporated herein by reference in its entirety.

Background Art

[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 antimicrobial substances, including non-ribosomal peptide classes such as fusaricidin and polymyxin. Fusaricidins are known to have antimicrobial activity against various plant pathogenic fungi and bacteria.

[0005] Many Paenibacillus species are prolific producers of exopolysaccharides (EPS). Microbial EPS are water-soluble biopolymers that adhere to the cell surface and are released into the extracellular medium. These polymers are commercially used as thickeners in a wide range of industries, including food, feed, packaging, cosmetics, and pharmaceutical industries, due to their physicochemical and rheological properties. EPS production results in an increase in the viscosity of the whole culture broth sample, especially for high molecular weight species. The increase in culture broth viscosity causes problems in bioreactor growth and downstream processing of the culture broth materials intended for live bacterial whole culture broth products. To remove EPS from large-scale fermentation liquid cultures before further processing, costly and labor-intensive procedures may be required.

Summary of the Invention

Problems to be Solved by the Invention

[0006] There is a need for a method to produce and identify strains of Paenibacillus sp. that exhibit increased fungicidal activity and processability by showing reduced viscosity and higher levels of fusaricidin and fusaricidin-like compounds.

Means for Solving the Problems

[0007] The present invention is directed towards strategies to enhance the fungicidal activity and processability of strains of Paenibacillus and its mutagenized 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 fermentation broth cultures to improve the large-scale growth and downstream processing of fungicidal mutagenized derivatives of Paenibacillus strains. Several Paenibacillus strains with improved fungicidal and processing characteristics were created and characterized.

[0008] In some embodiments, the present invention relates to a composition comprising a biologically pure culture of a strain of a Bacillus genus bacterium comprising a mutant DegU lacking a functional receptor 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 the mutant DegS result in a liquid culture of a strain of a Bacillus genus bacterium having a reduced viscosity as compared to a liquid culture of a strain of a Bacillus genus bacterium comprising wild-type DegU and wild-type DegS.

[0009] In certain aspects, the mutant DegU and / or the mutant DegS inhibit the formation of colonies of a strain of a Bacillus genus bacterium having a mucoid morphology.

[0010] In one embodiment, the mutant DegU and / or the mutant DegS are knockouts or are truncated as a result of a premature stop codon. In certain aspects, the premature stop codon results in a mutant DegU truncated at position 218 numbered according to its correspondence with 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 to an acidic residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a small residue at position 228 to a polar residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of an acidic residue at position 63 to a polar residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a polar residue at position 195 to a small residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a hydrophobic residue at position 204 to a small residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a polar residue at position 208 to a small residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a basic residue at position 212 to a small residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a hydrophobic residue at position 217 to a small residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a basic residue at position 207 to a small residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a polar residue at position 211 to a small residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a polar residue at position 214 to a small residue numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 2, and includes.

[0012] In one embodiment, the mutant DegU comprises SEQ ID NO: 2 having an amino acid substitution 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 a variant thereof having a conservative amino acid substitution.

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

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

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

[0016] In certain aspects, decreased 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. In other aspects, decreased amylase expression and / or enzymatic activity occurs in an α-amylase protein comprising a sequence having greater than about 95% sequence identity to SEQ ID NO:9 or SEQ ID NO:10, 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. 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 instances, the non-mutagenized parental strain is Paenibacillus sp. NRRL B-50972 or Paenibacillus sp. NRRL B-67129. In other instances, the non-mutagenized parental 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 aspect, 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 the 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 one embodiment, the fungicidal mutant has a genomic sequence having greater than about 90% sequence identity with the genomic sequence of Paenibacillus sp. strain NRRL B-67304, Paenibacillus sp. strain NRRL B-67306 or Paenibacillus sp. strain NRRL B-67615.

[0021] In other embodiments, the present invention relates to a method for identifying an induced mutant strain of a Paenibacillus bacterium having a reduced viscosity in liquid culture as compared to the parental strain of the Paenibacillus bacterium, the method comprising: mutagenizing a parental strain of a Paenibacillus bacterium to produce mutant isolates; culturing the mutant isolates and the parental strain of the Paenibacillus bacterium on a solid medium containing sugar at a concentration between about 1% (w / v) and about 40% (w / v), wherein the parental strain of the Paenibacillus bacterium has a mucoid form on the solid medium; and visually screening the mutant isolates on the solid medium to identify an induced mutant strain of the Paenibacillus bacterium having a non-mucoid form, which is an indicator of reduced viscosity in liquid culture.

[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 certain embodiments, 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 certain aspects, the solid medium is a solid agar medium.

[0024] In other embodiments, the method of the present invention further comprises culturing a mutant-derived strain of Bacillus 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-derived strain of Bacillus compared to the parental strain of Bacillus.

[0025] In certain embodiments, the method of the present invention further comprises identifying sequences encoding mutant DegU lacking a functional receptor domain or a functional DNA-binding domain and / or mutant DegS lacking a functional single-binding domain or a functional ATP-ase domain in a mutant-derived strain of Bacillus degU and / or degS including sequencing of.

[0026] In other embodiments, the method of the present invention further comprises determining the expression and / or enzyme activity of amylase in a mutant-derived strain of Bacillus and in a parental strain of Bacillus to determine whether its expression and / or enzyme activity is decreased in the mutant-derived strain of Bacillus. In certain aspects, the expression and / or enzyme activity of amylase in the mutant-derived strain of Bacillus 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 the parental strain of Bacillus. In other aspects, the expression and / or enzyme activity of amylase in the mutant-derived strain of Bacillus 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 the parental strain of Bacillus.

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

[0028] In one embodiment, the method of the invention further comprises quantifying the level of fusaricidin in a mutant isolate in order to identify a mutant isolate having an increased level of fusaricidin as compared to the parental strain of the genus Paenibacillus.

[0029] In one embodiment, the strain of the genus Paenibacillus is 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. brasiliensis, 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. 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. phoenici, 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.It is 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 certain aspects, the strain of Paenibacillus is a strain of Paenibacillus that produces fusaricidin.

[0032] Examples of strains of Paenibacillus 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-derived strain of Bacillus paenibacillus having a reduced viscosity in liquid culture as compared to the parental strain of Bacillus paenibacillus, the method comprising: mutagenizing a parental strain of Bacillus paenibacillus to produce a mutant isolate; culturing the mutant isolate and the parental strain of Bacillus paenibacillus on a solid medium containing sugar at a concentration between about 1% (w / v) and about 40% (w / v), wherein the parental strain of Bacillus paenibacillus has a mucoid form on the solid medium; visually screening the mutant isolate on the solid medium to identify a mutant-derived strain of Bacillus paenibacillus having a non-mucoid form, which is an indicator of reduced viscosity in liquid culture; and producing a fermentation product of the identified mutant-derived strain of Bacillus paenibacillus. In one aspect, the mutagenesis comprises chemical mutagenesis of the parental strain of Bacillus paenibacillus.

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

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

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

[0037] In some cases, plant diseases are caused by fungi. In some cases, the plant disease is powdery mildew or downy mildew. In other cases, 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 cases, plant diseases are caused by bacteria. In some cases, the bacteria are selected from the group consisting of Xanthomonas campestris, Pseudomonas syringae, and Erwinia carotovora.

[0039] In still other embodiments, the present invention relates to the use of the compositions disclosed herein or the fermentation products disclosed herein for controlling phytopathogenic organisms in useful plants.

Brief Description of the Drawings

[0040]

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Mode for Carrying Out the Invention

[0041] Unless otherwise specified, all of the microorganisms and specific strains described in this specification are isolated from nature and are grown under artificial conditions such as shaking flask culture or through scaled-up manufacturing processes such as bioreactors to maximize the production of bioactive metabolites. Growth under such conditions causes the "domestication" of the strain. Generally, such "domesticated" strains are different from their counterparts found in nature in that they are not subject to the selective pressures seen in the natural environment and are rather cultured as a homogeneous population that is susceptible to artificial selective pressures.

[0042] The microorganism of the present invention or its culture or isolate can be described as being in an "isolated" or "biologically pure" form. These terms mean that the microorganism has been separated from its environment or from one or more components, cells or other things and may be relevant when discovered in nature or by other means. The terms "isolated" or "biologically pure" should not be understood to indicate to what extent the microorganism has been purified. However, in one embodiment, an isolate or culture of the microorganism comprises the preponderant microorganism of the present invention.

[0043] As used herein in the specification and claims, the verb "comprising" and its conjugations are used in their non-limiting sense, including the items listed below the word but not excluding items not specifically mentioned. Further, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, except in contexts where it is clearly necessary to indicate that only one of the elements is present. Thus, generally, the indefinite article "a" or "an" means "at least one".

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

[0045] In some embodiments, a strain of a Bacillus genus bacterium comprising mutant DegU and / or mutant DegS produces a liquid culture having a reduced viscosity as compared to the liquid culture of a strain of a Bacillus genus bacterium comprising wild-type DegU and wild-type DegS. In certain aspects, the decrease in viscosity is measured by growing a strain of a Bacillus genus bacterium comprising mutant DegU and / or mutant DegS, and a strain of a Bacillus genus bacterium comprising wild-type DegU and wild-type DegS, separately in the same liquid culture medium until the stationary phase, and measuring the viscosity of each liquid culture. The 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 presented as SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and as SEQ ID NO: 3 and SEQ ID NO: 4.

[0046] As used herein, the terms "mucoid" and "mucoid form" refer to the phenotype of a microbial colony, which has a well-defined round edge and a shiny appearance under an optical microscope. Further, a microbial colony exhibiting a mucoid form is taller and tends to be three-dimensionally round. An example of a mucoid colony is shown in Figure 3.

[0047] As used herein, the terms "non-mucoid" and "non-mucoid form" refer to the phenotype of a microbial colony that has a less distinct, random-shaped edge and a dull appearance under an optical microscope. Non-mucoid colonies tend to be flat in three dimensions. Examples of non-mucoid colonies are also presented in Figure 3.

[0048] On solid agar media containing sugar at a concentration of about 1% (w / v) to about 40% (w / v), the mucoid form and the non-mucoid form are more easily distinguishable. In some aspects, the sugar concentration is between about 1% (w / v) and about 30% (w / v), about 1% (w / v) and about 20% (w / v), about 5% (w / v) and about 40% (w / v), about 5% (w / v) and about 30% (w / v), or about 5% (w / v) and 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, about 10:1 and about 750:1, about 10:1 and about 500:1, about 10:1 and about 250:1, about 10:1 and about 100:1, about 10:1 and about 75:1, about 10:1 and about 50:1, about 10:1 and about 25:1, about 1:1 and about 100:1, about 1:1 and about 75:1, about 1:1 and about 50:1, 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, about 10:1 and about 750:1, about 10:1 and about 500:1, about 10:1 and about 250:1, about 10:1 and about 100:1. In some aspects, the carbon-to-nitrogen ratio in the solid medium is between about 10:1 and about 1000:1.

[0050] In one embodiment, the solid medium and / or liquid medium used in the disclosed method for identifying mutant-derived strains of Bacillus spp. with a reduced viscosity in liquid culture compared to the parental strain of Bacillus spp. contains any sugar that aids in the growth of Bacillus spp. 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 embodiment, the sugar is selected from the group consisting of sucrose, maltodextrin, fructose, and combinations thereof. In yet another embodiment, the sugar is sucrose or maltodextrin.

[0052] In some embodiments, the present invention relates to a method for identifying mutant-derived strains of Bacillus paenibacillus having a reduced viscosity in liquid culture compared to the parental strain of Bacillus paenibacillus using visual screening. As used herein, the terms "visual screening" and "visual screening" mean any process of analyzing the size, shape, and / or gloss (i.e., sheen) of microbial colonies grown on solid media, whether performed manually or automatically by machine or robot. In some aspects, the solid medium is a solid agar medium.

[0053] In other embodiments, the present invention relates to a method for identifying mutant-derived strains of Bacillus paenibacillus having a reduced viscosity in liquid culture compared to the parental strain of Bacillus paenibacillus, the method comprising mutagenizing the parental strain of Bacillus paenibacillus to produce mutant isolates; culturing the mutant isolates and the parental strain of Bacillus paenibacillus in a liquid medium containing 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 isolates in the liquid medium to identify mutant-derived strains of Bacillus paenibacillus having a reduced viscosity in liquid culture compared to the parental strain of Bacillus paenibacillus.

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

[0055] In one aspect, the strain of the genus Paenibacillus of the present invention is selected from any one of the following: 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. brasiliensis, 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. 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. phoenici, 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 and P. xylanilyticus.

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

[0057] In one embodiment, mutant strains of Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306, or Paenibacillus sp. NRRL B-67615 are 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 identified (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 controls fungi, oomycetes, and / or bacteria as a functional identified characteristic, similar to at least its parent strains, Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306, or Paenibacillus sp. NRRL B-67615. 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.The mutant can be obtained by treating cells of Paenibacillus sp. NRRL B-67304 strain, Paenibacillus sp. NRRL B-67306 strain or Paenibacillus sp. NRRL B-67615 strain with a chemical substance or radiation, selecting natural mutants (such as phage-resistant or antibiotic-resistant mutants) from a cell population of Paenibacillus sp. NRRL B-67304 strain, Paenibacillus sp. NRRL B-67306 strain or Paenibacillus sp. NRRL B-67615 strain, performing genome shuffling as described below, or by other known methods practiced in the art.

[0058] Genome shuffling between Paenibacillus strains can be facilitated by using a process called protoplast fusion. This process begins with the formation of protoplasts from vegetative bacterial cells. Typically, the peptidoglycan cell wall is removed using lysozyme and an osmotic stabilizer, resulting in the formation of protoplasts. This process is visualized by the appearance of spherical cells under an optical microscope. The 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 then undergo redivision and are recovered on a solid growth medium. During the recovery process, the protoplasts reconstruct the peptidoglycan cell wall and return to the bacillus shape. See Schaeffer et al., PNAS USA, vol. 73, 6:2151-2155 (1976).

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

[0060] In certain aspects, mutant DegU and / or mutant DegS characteristic of the Paenibacillus strains of the present invention contain conservative amino acid substitutions. For example, conservative amino acid substitutions within the sequences of SEQ ID NOs: 1-4 are contemplated. 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 generally do not change the specific activity are known in the art and are described, for example, in H. Neurath and R. L. Hill, 1979, The Proteins, Academic Press, New York, which is hereby incorporated by reference in its entirety. Common conservative substitutions include Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Lys / Arg, Leu / Ile, and Leu / Val.

[0061] The present invention also encompasses a method of treating a plant to control a plant disease by applying to the plant or a plant part, such as a leaf, stem, flower, fruit, root or seed, or to the location where the plant or plant part grows, such as soil, a strain of a Paenibacillus bacterium disclosed herein or a mutant thereof, or a cell-free preparation or metabolite thereof.

[0062] In the method according to the present invention, a composition comprising a strain of a Paenibacillus bacterium disclosed herein or a fungicidal mutant thereof can be applied to any plant or any part of any plant grown in any type of medium (such as soil, vermiculite, shredded cardboard and water) used for growing the plant, or to a plant or a part of a plant growing in air, such as an orchid or a cactus. The compositions of the present invention can be applied, for example, by spraying, atomizing, vaporizing, broadcasting, dusting, flooding, spraying, watering, injecting or fumigating. As already indicated, application can be carried out at any desired location where the target plant is located, such as in agriculture, horticulture, forestry, afforestation, orchards, nurseries, organically grown crops, turf grass and urban environments.

[0063] The compositions of the present invention can be obtained by culturing a strain of a Paenibacillus bacterium disclosed herein or a fungicidal mutant (strain) derived therefrom according to methods well known in the art, which includes using the media and methods described in the following examples. Conventional large-scale microbial culture processes include submerged fermentation, solid-state fermentation or liquid surface culture. Towards the end of fermentation, as nutrients become depleted, the cells begin to transition from the growth phase to the sporulation phase. As a result, the final products of fermentation are mainly spores, metabolites and residual fermentation broth. Sporulation is part of the natural life cycle of Paenibacillus and is generally initiated by the cells in response to nutrient limitation. Fermentation is designed to obtain high levels of colony-forming units and to promote sporulation. The bacterial cells, spores and metabolites in the culture medium resulting from 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 contains a fermentation product. In some embodiments, the concentrated fermentation broth (broth) is washed, for example, via a diafiltration step to remove residual fermentation broth and metabolites. As used herein, the term "culture broth concentrate" refers to the entire culture broth (fermentation broth) that has been concentrated by conventional industrial methods as described above but remains in liquid form. As used herein, the term "fermentation solid" refers to the solid material remaining after the fermentation broth has been dried. As used herein, the term "fermentation product" refers to the entire culture broth, culture broth concentrate, and / or fermentation solid. The composition of the present invention contains a fermentation product.

[0065] The fermentation broth or culture broth concentrate can be dried using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluidized bed drying, drum drying, or evaporation, with or without the addition of a carrier.

[0066] The resulting dried product can be further processed, such as by grinding or granulation, to achieve a specific particle size or physical form. A carrier described later 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 cell-free, but rather mostly cell-free or essentially cell-free. The resulting cell-free preparation can be dried and / or formulated with components that assist in its application to plants or plant growth media. The above concentration methods and drying techniques for fermentation broth are also applicable to cell-free preparations.

[0068] In one embodiment, the fermentation product is at least about 1×10 per mL of culture broth 4It contains microorganisms of colony forming units (CFU) (for example, Paenibacillus sp. NRRL B - 67304 strain, Paenibacillus sp. NRRL B - 67306 strain or Paenibacillus sp. NRRL B - 67615 strain or its fungicidal mutants). In another embodiment, the fermentation product contains at least about 1×10 5 CFU of microorganisms per 1 mL of the culture solution. In another embodiment, the fermentation product contains at least about 1×10 6 CFU of microorganisms per 1 mL of the culture solution. In yet another embodiment, the fermentation product contains at least about 1×10 7 CFU of microorganisms per 1 mL of the culture solution. In another embodiment, the fermentation product contains at least about 1×10 8 CFU of microorganisms per 1 mL of the culture solution. In another embodiment, the fermentation product contains at least about 1×10 9 CFU of microorganisms per 1 mL of the culture solution. In another embodiment, the fermentation product contains at least about 1×10 10 CFU of microorganisms per 1 mL of the culture solution. In another embodiment, the fermentation product contains at least about 1×10 11 CFU of microorganisms per 1 mL of the culture solution.

[0069] The composition of the present invention can be used as it is, or according to its specific physical and / or chemical properties, for example, aerosol, capsule suspension, cold fog concentrate, warm fog concentrate, encapsulated granules, fine granules, thick flowable for seed treatment, ready - to - use solution, powder, emulsifiable concentrate, oil - in - water emulsion, water - in - oil emulsion, macrogranules, microgranules, oil - dispersible powder, oil - miscible thick flowable, oil - miscible liquid, gas (under pressure), gas - generating product, foam, paste, pesticide - coated seeds, thick suspension, oil dispersion, thick SE agent, soluble concentrate, suspending agent, wettable powder, water - soluble powder, powder and granule, water - soluble and water - dispersible granules or tablets, water - soluble and water - dispersible powder for seed treatment, wettable powder infiltrated with the active ingredient, natural and synthetic substances, and microcapsules by high - molecular substances and coating materials for seeds, and can be used in the form of formulations such as ULV cold fog and warm fog formulations or usage forms prepared from such formulations.

[0070] In some embodiments, the composition of the present invention is a liquid formulation. Non-limiting examples of liquid formulations include concentrated suspensions and oil dispersions. In other embodiments, the composition of the present invention is a solid formulation. Non-limiting examples of solid formulations include lyophilized powders and spray-dried powders.

[0071] All plants and plant parts can be treated according to the present invention. In this regard, 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 biotechnology and recombinant methods, or combinations of these methods, including genetically modified plants and plant varieties that are or are not protected by plant breeder's rights. Plant parts are understood to mean all above-ground and underground parts and organs of the plant, such as shoots, leaves, flowers, roots, etc. For example, leaves, needles, petioles, stems, flowers, fruiting bodies, fruits and seeds, roots, tubers, rhizomes, etc. can be mentioned. Plant parts also include crop elements and vegetative propagation elements, reproductive propagation elements such as cuttings, tubers, rhizomes, scions and seeds.

[0072] As already mentioned, all plants and their parts can be processed according to the present invention. In a preferred embodiment, plant species and plant varieties that grow wild or are obtained by traditional biological breeding methods such as hybridization or protoplast fusion, and their parts are processed. In an even more preferred embodiment, genetically modified plants and plant varieties obtained by recombinant methods, and their parts are processed, optionally in combination with conventional methods (genetically modified organisms). The term "part" or "plant part" or "plant portion" has been described as above. Plants of plant varieties that are commercially available or used in each case are preferably processed in particular according to the present invention. A plant variety is understood to mean a plant having novel traits that has been bred by both traditional breeding, mutagenesis or recombinant DNA technology. These may take the form of varieties, races, biotypes and genotypes.

[0073] Treatment with the compositions of plants and plant parts according to the present invention is carried out directly, or with respect to the environment, habitat or storage space, for example by dipping, spraying, atomizing, irrigation, vaporization, dusting, smoking, broadcasting, foaming, coating, spreading, injection, drenching, drip irrigation and, in the case of reproductive elements (especially in the case of seeds), by general treatment methods such as dry seed treatment methods, wet seed treatment methods, slurry treatment methods, film formation, coating with one or more coatings, etc. Furthermore, it is also possible to apply the active substance by microspraying method or to inject the active substance preparation or the active substance itself into the soil.

[0074] A preferred direct treatment of plants is foliar spraying treatment. That is, the composition according to the present invention can be applied to the leaf surface, and the treatment frequency and application rate can be adjusted according to the infection pressure of the pathogen in question.

[0075] In the case of systemically active compounds, the compositions according to the invention reach the plants via the root system. In this case, the treatment of the plants is effected by enabling the compositions according to the invention to act on the plant environment. This can be done, for example, by drenching (i.e., impregnating the liquid form of the composition according to the invention into the growth site (e.g., soil or hydroponic system)) 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 growth site). In the case of rice cultivation, the composition according to the invention can also be effected by metering it in solid use form (e.g., in the form of granules) into the paddy field.

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

[0077] As used herein, "useful plants" refers to crops used as plants for obtaining food, feed, fuel or for industrial use.

[0078] Useful plants that can be treated and / or improved with the compositions and methods of the present invention include, for example, the following: turfgrass; grapes; grains such as wheat, barley, rye, oats, rice, corn and millet / sorghum; beets such as sugar beet and fodder beet; fruits such as pomaceous 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, olive, sunflower, coconut, sesame, cocoa and peanuts; cucurbits such as pumpkin / squash, cucumber and melons; fiber plants such as cotton, flax, hemp and jute; citrus fruits such as oranges, lemons, grapefruits and tangerines; vegetables such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, peppers; Lauraceae plants such as avocado, Cinnamomum zeylanicum, camphor; or other plants such as tobacco, nuts, coffee, eggplant, sugarcane, tea, pepper, grapes, hops, banana, rubber tree and ornamental plants such as flowers, shrubs, deciduous trees and conifers. This exemplification is not limiting.

[0079] The following plants are considered to be particularly suitable target crops for applying the compositions and methods of the present invention: cotton, eggplant, turfgrass, pomaceous fruits, stone fruits, soft fruits, corn, wheat, barley, cucumber, tobacco, grapes, rice, grains, pears, legumes, soybeans, rapeseed, tomatoes, peppers, melons, cabbages, potatoes and apples.

[0080] Examples of trees that can be improved according to the method of the present invention include 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., Populus sp.

[0081] Preferred trees that can be improved according to the method of the present 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. radiata, P. ponderosa, P. contorta, P. sylvestre, P. strobes, P. elliottii, P. montecola, P. albicaulis, P. resinosa, P. palustris, P. taeda, P. flexilis, P. jeffregi, P. baksiana from the tree species Pinus; E. grandis, E. globulus, E. camadentis, E. nitens, E. obliqua, E. regnans, E. pilularus from the tree species Eucalyptus.

[0082] Particularly preferred trees that can be improved according to the method of the present 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] Highly preferred trees that can be improved according to the method of the present invention are horse chestnut, Platanaceae, linden and maple.

[0084] The present invention can also be applied to any turfgrass, including cool-season turfgrasses and warm-season turfgrasses. Examples of cool-season turfgrasses are 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 bulbous bluegrass (Poa bulbosa L.); 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 spp. including Agrostis tenuis Sibth., Agrostis canina L. and Agrostis palustris Huds.) and redtop (Agrostis alba L.);

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

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

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

[0089] Examples of warm-season turfgrasses include Bermuda grass (Cynodon spp. L.C. Rich), 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), Kikuyu grass (Pennisetum clandestinum Hochst.ex Chiov.), buffalo grass (Buchloe dactyloids (Nutt.) Engelm.), Blue gramma (Bouteloua gracilis (H.B.K.) Lag.ex Griffiths), seashore paspalum (Paspalum vaginatum Swartz), and sideoats grama (Bouteloua curtipendula (Michx.Torr.)). Cool-season turfgrasses are generally preferred for use according to the present invention. Particularly preferred are bluegrass, bentgrass, and fescue, orchardgrass, and ryegrass. Bentgrass is particularly preferred.

[0090] The composition of the present invention has strong bactericidal activity and can be used for controlling 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 of the present invention is applied to phytopathogenic fungi, phytopathogenic bacteria and / or their habitats.

[0092] Fungicides can be used in crop protection for the control of phytopathogenic fungi. They are characterized by significant efficacy against a wide range of phytopathogenic fungi, including soil-borne pathogens, which are particularly members of the classes of Plasmodiophoromycetes, Peronosporomycetes (synonym 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 the pathogens of fungal diseases that can be treated according to the present invention include the following:

[0095] Diseases caused by powdery mildew pathogens, such as those caused by species of Blumeria, such as Blumeria graminis; species of Podosphaera, such as Podosphaera leucotricha; species of Sphaerotheca, such as Sphaerotheca fuliginea; species of Uncinula, such as Uncinula necator;

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

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

[0098] Leaf blotch disease and leaf wilt disease, for example, 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 form: synonymous with Drechslera, Helminthosporium), Cochliobolus miyabeanus; Colletotrichum species such as Colletotrichum lindemuthanium; Cycloconium species such as Cycloconium oleaginum; Diaporthe species such as Diaporthe citri; Elsinoe species such as Elsinoe fawcettii; Gloeosporium species such as Gloeosporium laeticolor; Glomerella species such as Glomerella cingulata; Guignardia species such as Guignardia 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 nodorum; Pyrenophora species, such as Pyrenophora teres, Pyrenophora tritici repentis; Ramularia species, such as Ramularia collo-cygni, Ramularia areola; Rhynchosporium species, such as Rhynchosporium secalis; Septoria species, such as Septoria apii, Septoria lycopersii; Typhula species, such as Typhula incarnata;Caused by Venturia species, such as Venturia inaequalis;

[0099] Root and stem diseases, such as those caused by Corticium species, such as Corticium graminearum; Fusarium species, such as Fusarium oxysporum; Gaeumannomyces species, such as Gaeumannomyces graminis; Rhizoctonia species, such as Rhizoctonia solani; Sarocladium disease, such as that caused by Sarocladium oryzae; Sclerotium disease, such as that caused by Sclerotium oryzae; Tapesia species, such as Tapesia acuformis; Thielaviopsis species, such as Thielaviopsis basicola;

[0100] Diseases of the ear (including the cob), e.g., Alternaria species, e.g., Alternaria spp.; Aspergillus species, e.g., Aspergillus flavus; Cladosporium species, e.g., Cladosporium cladosporioides; Claviceps species, e.g., Claviceps purpurea; Fusarium species, e.g., Fusarium culmorum; Gibberella species, e.g., Gibberella zeae; Monographella species, e.g., Monographella nivalis; Septoria species, e.g., Septoria nodorum; caused by

[0101] Diseases caused by smut fungi, e.g., 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 spoilage caused by, for example, 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] Corrosion, mold, withering, putrefaction, and damping-off disease mediated by seeds and soil, e.g., caused by Alternaria species such as Alternaria brassicicola; caused by Aphanomyces species such as Aphanomyces euteiches; caused by Ascochyta species such as Ascochyta lentis; caused by Aspergillus species such as Aspergillus flavus; caused by Cladosporium species such as Cladosporium herbarum; caused by Cochliobolus species such as Cochliobolus sativus; (conidial form: Drechslera, Bipolaris: synonymous with Helminthosporium); caused by Colletotrichum species such as Colletotrichum coccodes; caused by Fusarium species such as Fusarium culmorum; caused by Gibberella species such as Gibberella zeae; caused by Macrophomina species such as Macrophomina phaseolina; caused by Monographella species such as Monographella nivalis;Caused by Penicillium species, such as Penicillium expansum; caused by Phoma species, such as Phoma lingam; caused by Phomopsis species, such as Phomopsis sojae; caused by Phytophthora species, such as Phytophthora cactorum; caused by Pyrenophora species, such as Pyrenophora graminea; caused by Pyricularia species, such as Pyricularia oryzae; caused by Pythium species, such as Pythium ultimum; caused by Rhizoctonia species, such as Rhizoctonia solani; caused by Rhizopus species, such as Rhizopus oryzae; caused by Sclerotium species, such as Sclerotium rolfsii; caused by Septoria species, such as Septoria nodorum; caused by Typhula species, such as Typhula incarnata; caused by Verticillium species, such as Verticillium dahliae;

[0104] Caused by cancer tumors, knots and witches’ broom, e.g., Nectria species, e.g., Nectria galligena;

[0105] Wilt disease, e.g., caused by Monilinia species, e.g., Monilinia laxa;

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

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

[0108] Degenerative diseases of woody plants, e.g., Esca disease, e.g., caused by Phaeomoniella clamydospora, Phaeoacremonium aleophilum and Fomitiporia mediterranea;; Eutypa dieback, e.g., caused by Eutypa lata; Ganoderma disease, e.g., caused by Ganoderma boninense; Rigidoporus disease, e.g., caused by Rigidoporus lignosus;

[0109] Diseases of flowers and seeds, for example, caused by Botrytis species, such as Botrytis cinerea;

[0110] Diseases of plant tubers, for example, caused by Rhizoctonia species, such as Rhizoctonia solani; Helminthosporium species, such as Helminthosporium solani;

[0111] Clubroot, for example, caused by Plasmodiophora species, such as Plasmodiophora brassicae;

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

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

[0114] Fungal diseases of leaves, stems, pods and seeds, such as Alternaria leaf spot (Alternaria species atrans tenuissima), anthracnose (Colletotrichum gloeosporoides dematium var. truncatum), brown spot (Septoria glicines), cercospora leaf spot and blight (Cercospora kikuchii), choanephora leaf blight (synonymous with 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 glycinesglycines)), Rhizoctonia aerial, foliage, and web blight (Rhizoctonia solani), rust (Phakopsora pachyrhizi, Phakopsora meibomiae), scab (Sphaceloma glycines), Stemphylium leaf blight (Stemphylium botryosum), target spot (Corynespora cassiicola).

[0115] Fungal diseases of roots and the basal part of the stem, such as black root rot (Calonectria crotalariae), charcoal rot (Macrophomina phaseolina), red mold or Fusarium blight, wilt, root rot, and pod and collar rot (Fusarium oxysporum, Fusarium orthoceras, Fusarium semitectum, Fusarium equiseti), mycoleptodiscus root rot (Mycoleptodiscus 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), Thielaviopsis root rot (Thielaviopsis basicola).

[0116] The fungicidal composition of the present invention can be used for curative or protective / preventive control of phytopathogenic fungi. Accordingly, the present invention also relates to curative and protective methods for the control of phytopathogenic fungi by the use of the composition 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 composition is well tolerated by plants at the concentrations required to control plant diseases allows for the treatment of the aerial parts of plants, propagules and seeds, and soil.

[0118] According to the present invention, all plants and plant parts can be treated, including cultivars and plant varieties (regardless of whether they are 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 the use of doubled haploids, protoplast fusion, random and directed mutagenesis, molecular or genetic markers, or biotechnological and genetic engineering methods.

[0119] In certain aspects, the compositions of the invention are applied at about 1×10 4 to about 1×10 14 colony forming units (CFU) per hectare, about 1×10 4 to about 1×10 12 colony forming units (CFU) per hectare, about 1×10 4 to about 1×10 10 colony forming units (CFU) per hectare, about 1×10 4 to about 1×10 8 colony forming units (CFU) per hectare, about 1×10 6 to about 1×10 14 colony forming units (CFU) per hectare, about 1×10 6 to about 1×10 12 colony forming units (CFU) per hectare, about 1×10 6 to about 1×10 10 colony forming units (CFU) per hectare, about 1×10 6 to about 1×10 8 colony forming units (CFU) per hectare, about 1×10 8 to about 1×10 14 colony forming units (CFU) per hectare, about 1×10 8 to about 1×10 12 colony forming units (CFU) per hectare, about 1×10 8 to about 1×10 10 colony forming units (CFU).

[0120] In other aspects, the composition of the present invention is applied at about 1×10 6 to about 1×10 14 colony forming units (CFU) per hectare, and is applied at about 1×10 6 to about 1×10 12 colony forming units (CFU) per hectare, and is applied at about 1×10 6 to about 1×10 10 colony forming units (CFU) per hectare, and is applied at about 1×10 6 to about 1×10 8 colony forming units (CFU) per hectare. In yet other aspects, the composition of the present invention is applied at about 1×10 9 to about 1×10 13 colony forming units (CFU) per hectare. In certain aspects, the composition of the present invention is applied at about 1×10 10 to about 1×10 12 colony forming units (CFU) per hectare.

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

[0122] The composition of the present invention is suitable for protecting plants and plant organs, increasing the yield, and improving the quality of the harvested product when the plants show good tolerance, the toxicity to warm-blooded animals is also at a desirable level, and it shows good environmental compatibility. These are preferably used as crop protection compositions. These are active against normal susceptible and resistant species, and are further active against all or some growth stages.

[0123] Plants that can be processed according to the present 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., field mustard), and Brassica carinata, etc., Arecaceae sp. (e.g., oil palm, coconut), rice, wheat, sugar beet, sugarcane, rye, triticale, barley, millet, and sorghum, triticale, flax, nuts, grapes and grapevines, as well as various fruits and vegetables from various botanical classifications, such as Rosaceae sp. (e.g., pome fruits, such as apples and pears, etc., not only these but also stone fruits, such as apricots, cherries, almonds, plums, and peaches, etc., as well as berry fruits, such as strawberries, raspberries, blackberries, and loganberries and dewberries), 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 tree and plantation), Rubiaceae 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, chili pepper, eggplant, tobacco), Liliaceae sp.) Compositae plants (such as lettuce, artichoke, and root chicory, endive, or common chicory), Umbelliferae plants (such as carrot, parsley, celery, and celeriac), Cucurbitaceae plants (such as cucumber - gherkin, pumpkin, watermelon, gourd, and melon), Alliaceae plants (such as chives and onion), Cruciferae plants (such as white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, Chinese cabbage, kohlrabi, daikon, horseradish, cress, and pakchoi), Leguminosae plants (such as peanuts, peas, lentils, and beans - such as kidney beans and broad beans), Chenopodiaceae plants (such as spinach, fodder beet, spinach, and beetroot), Linaceae plants (such as flax), Cannabeacea plants (such as cannabis), Malvaceae plants (such as okra, cocoa), Papaveraceae (such as poppy), Asparagaceae (such as asparagus); plants useful in gardens and forests and ornamental plants such as turf, lawn, grass, and Stevia rebaudiana; and in each case genetically modified forms of these plants.

[0124] In certain aspects, the fermentation product further comprises formulation components. The formulation components may be wetting agents, bulking agents, solvents, self - promoting agents, emulsifiers, dispersants, cryoprotectants, thickeners, and / or adjuvants. In one embodiment, the formulation component is a wetting agent. In other aspects, the fermentation product is a lyophilized powder or a spray - dried powder.

[0125] The composition of the present invention may contain formulation ingredients added to the composition of the present invention to improve recovery rate, effectiveness or physical properties and / or to assist in processing, packaging and application. Such formulation ingredients may be added individually or in combination.

[0126] Formulation components 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 components include agriculturally acceptable carriers, inerting 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 composites derived biologically or by chemical synthesis. In some embodiments, the formulation component is a binder, adjuvant or adhesive that facilitates the attachment of the composition to plant parts such as leaves, seeds or roots. See, for example, Taylor, A.G. et al., “Concepts and Technologies of Selected Seed Treatments”, Annu. Rev. Phytopathol., 28:321-339 (1990). Stabilizers can be anti-caking agents, antioxidants, anti-settling agents, defoamers, desiccants, protectants or preservatives. Nutrients can be carbon, nitrogen and phosphorus sources such as sugars, polysaccharides, oils, proteins, amino acids, fatty acids and phosphates. Physical property modifiers can be bulking agents, wetting agents, thickeners, pH modifiers, rheology modifiers, dispersants, adjuvants, surfactants, film formers, hydrotropes, builders, antifreeze agents or colorants. In some embodiments, compositions containing cells, cell-free preparations and / or metabolites produced by fermentation can be used directly, with or without water as a diluent, without further formulation preparation. In certain embodiments, a wetting agent or dispersant is added to fermentation solids such as lyophilized or spray-dried powders. In some embodiments, the formulation inerting agent is added after concentration of the fermentation broth and / or during and / or after drying. Wetting agents increase spreading and penetration properties, and dispersants increase the dispersibility and solubility of the active ingredient (once diluted) when applied to the surface. Exemplary wetting agents are known to those skilled in the art and include sulfosuccinates and derivatives such as MULTIWET TMMO-70R (Croda Inc., Edison, NJ); siloxanes such as BREAK-THRU® (Evonik, Germany); nonionic 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) etc. are mentioned.

[0127] Deposit information: Samples of the Paenibacillus strains of the present invention have been deposited under the Budapest Treaty with the Agricultural Research Service Culture Collection, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604, USA (NRRL). 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 strains of those Paenibacillus bacteria have been deposited under conditions that guarantee access to the cultures during the term of this patent application to those determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. Section 1.14 and 35 U.S.C. Section 122. However, it should be understood that the availability of the deposited material does not limit the patent rights granted by government action and does not provide a license to practice the invention.

[0129] For purely illustrative and non-limiting purposes of the present invention, the following examples are given.

Example

[0130] Example 1. Enhancement of fusaricidin production by Paenibacillus spp. strain NRRL B - 67129 and mutagenized derivatives The Paenibacillus sp. NRRL B-67129 strain was treated with 1-methyl-3-nitro-1-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS) to introduce genetic mutations. Treatments that resulted in a 50 - 90% loss of colony-forming units (CFUs) were considered appropriate to obtain sufficient genetic mutations and viable cells for subsequent screening. Individual isolates from the chemically treated population were cultured in 96-well deep-well blocks and screened for increased production of four fusaricidin-like compounds, namely fusaricidin A (also known as "Fus A"), LiF08a, paenicillin A1 and B1 (also known as "M868" from their molecular weights); and paeniprolixin A2 and B2 (also known as "M938" from their molecular weights) as described in WO2016 / 154297.

[0131] Isolates with significantly improved fusaricidin levels from this initial screening were re-screened by technical replicates in 96-well deep well blocks and confirmed to be overproducers. The 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, chemically treated again, and screened for further improvement in fusaricidin production. This process was repeated several times.

[0132] The strains confirmed in this analysis were further evaluated to confirm fusaricidin production. Briefly, each strain was cultured in a soybean-based medium, and the lipophilic fraction of the whole broth was extracted. The total broth extract was analyzed by high-performance liquid chromatography (HPLC), and the presence of fusaricidin A was identified based on the HPLC profile prepared with a standard sample containing fusaricidin A. The heat-resistant spore-forming ability of the mutant strains was also evaluated.

[0133] In total, approximately 10,000 isolates derived from the Paenibacillus sp. NRRL B-67129 strain were screened in 96-well deep well block format. From this analysis, several isolates were obtained for which the relative levels of four fusaricidin-like compounds were characterized (see Table 1). These strains were extensively characterized, and it was confirmed that Paenibacillus sp. strain J exhibits high levels of fusaricidin production and favorable growth characteristics (e.g., sporulation) compared to the Paenibacillus sp. NRRL B-67129 strain. However, Paenibacillus sp. strain J produced a viscous fermentation broth that made processing difficult. From this observation, it became clear that mutant strains need to be screened for viscosity in liquid culture and for the production of fusaricidin-like compounds.

Table 1

[0134] Example 2. Reduction of fermentation broth viscosity by Paenibacillus spp. strain NRRL B - 67129 mutant derivatives The Paenibacillus sp. NRRL B-50972 strain and the Paenibacillus sp. NRRL B-67129 strain produced viscous fermentation broth cultures, similar to many of the mutants derived from the Paenibacillus sp. NRRL B-67129 strain. The physicochemical properties of these cultures presented challenges in fermentation and downstream processing. Therefore, it was desirable to find a method to identify mutant derivatives of the Paenibacillus sp. NRRL B-67129 strain that produce fermentation broth cultures with low viscosity.

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

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

[0137] Strains of several Paenibacillus species, including strains of P. terrae, P. brasilensis, P. polymyxa, and P. peoriae, produced a mucoid phenotype on solid agar media containing sucrose (see Figure 2). It was hypothesized that a rapid visual screening could be designed for non-mucoid colony isolates that would result in a fermentation broth culture 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 mutagenized derivatives, and strains of other Paenibacillus species in submerged culture. [Table 2]

[0138] The following protocol was developed and validated as a rapid visual screening to identify non-mucoid colony isolates. A liquid culture of a fungicidal mutagenized derivative of the Paenibacillus sp. NRRL B-67129 strain was chemically treated, then diluted and inoculated onto solid agar media supplemented with sucrose to obtain single colonies. Non-mucoid colonies were readily distinguishable from mucoid colonies by the naked eye (see Figure 3). Non-mucoid isolates were picked up and streaked onto fresh solid agar media supplemented with sucrose to confirm the phenotype.

[0139] Eight non-mucoid isolates were identified from a fusaricidin overproducing parental strain derived from Paenibacillus sp. NRRL B-67129. The non-mucoid 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 equal to or greater than those of their respective parental strains. Furthermore, 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 production factors are genetically separable.

[0140] The eight non-mucoid isolates were evaluated in larger scale cultures, and two non-mucoid isolates, Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304, were found to exhibit improved fusaricidin production and favorable growth characteristics in a soybean-based medium. The packed cell volume (%PCV) and the viscosity of these strains were analyzed.

[0141] The %PCV was quantified by centrifuging a 1 mL volume at 17,000 g for 3 minutes in a 2 mL microcentrifuge tube. The packed cell volume ratio was determined based on the scale of the tube with the initial 1 mL sample volume mark set as 100%.

[0142] Alternatively, approximately 10 mL of the total broth was placed into a 15 mL centrifuge tube, the weight of the total broth (“W wb ”) was recorded, the sample was centrifuged at 10,000 g for 10 minutes, the supernatant was poured off, and the weight of the supernatant (“W sup ”) was recorded. The following formula was used to calculate the %PCV: %PCV = 100×(W wb - W sup ) / (W wb )

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

[0144] The fermentation broths of Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304 strains produced fermentation broths with viscosities of 11.5 and 33.9 cP, respectively, compared to 56 centipoise (cP) of the fermentation broth of Paenibacillus sp. NRRL B-50972 strain (see Table 3). Further, Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304 strains had a smaller packed cell volume (PCV) compared to Paenibacillus sp. NRRL B-50972 strain (see Table 3 and Figure 5). These results demonstrated 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 the fermentation broths from Paenibacillus sp. NRRL B-67306 and Paenibacillus sp. NRRL B-67304 strains enabled enhanced processability of these non-mucoid strains as live microorganism-based products. Due to the low PCV and viscosity of the fermentation broth, the concentration of the total broth material can be increased to reduce the usage rate in agricultural applications.

Table 3

[0146] Example 3. Mutation analysis of strain - improved isolates The genomic sequences of several isolates with a non-mucoid phenotype were determined using standard sequencing methods. The single nucleotide polymorphisms (SNPs) of the isolates were compared. Surprisingly, 5 out of 8 non-mucoid strains derived from Paenibacillus sp. NRRL B-67129, including Paenibacillus sp. NRRL B-67304 strain and Paenibacillus sp. NRRL B-67306 strain, degS degU were found to have mutations in the protein coding sequences of the region (see Figure 6A). These mutations were within the receiver domain and DNA binding domain of DegU, as well as within the single binding domain and ATPase domain of DegS (see Figures 6B - 6C).

[0147] degS and degU The hypothesis was put forward that these mutations in and are related to the non-mucoid phenotype of these isolates on solid agar plates supplemented with sucrose. To test this, in the parental Paenibacillus sp. NRRL B-67129 strain, degS the gene was replaced with a kanamycin cassette, and degS and degU DNA constructs were made using standard molecular methods to replace the regions with a kanamycin cassette.

[0148] degS alone or degS and degUTargeting the substitution by kanamycin resistance (kanR), the gene encoding kanR was cloned into a conjugable Escherichia coli - Paenibacillus shuttle plasmid adjacent to the 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. Using the erythromycin resistance encoded by the plasmid backbone, those with successful plasmid introduction were selected. Double - crossover integrants were confirmed using kanamycin resistance, erythromycin sensitivity, and PCR validation. Paenibacillus sp. NRRL B - 67129 kanamycin - resistant marker replacement strain degS :: kanR strains and Paenibacillus sp. NRRL B - 67129 degSdegU :: kanR strains all mimicked the non - mucoid phenotype of previously selected isolates (see Figure 7). These results confirmed that mutations leading to non - functional gene products degS and degU give rise to the non - mucoid phenotype.

[0149] degS and degUOther mutations in were characterized and they resulted in non-functional gene products. The serine 76 residue of Bacillus subtilis strain 168 corresponds to threonine 73 of Paenibacillus sp. strain NRRL B-50972 and is a phosphorylation site that stimulates its kinase activity. When serine 76 was mutated to alanine, the enzymatic activity of DegS decreased significantly (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). The mutation of alanine 193 residue in Bacillus subtilis strain 168 corresponding to alanine 190 in Paenibacillus sp. strain 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] Substituting aspartic acid 56 corresponding to aspartic acid 63 in Paenibacillus sp. strain NRRL B-50972 with asparagine degU of the mutation inhibited the phosphorylation of DegU by DegS (see Dahl, M.K. et al., mentioned above). Alanine scanning of the DNA-binding domain of DegU revealed comK and aprE a drastic decrease in the binding of DegU to the promoter regions of and, and as a result, five common mutants that caused a decrease in the expression of these genes were identified. Furthermore aprEThree additional mutants were also identified that inhibit the binding of DegU to the promoter region, resulting in decreased expression of this gene (see the mutants in Table 4 reported by 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 gene or degS genes and degU genes replaced with an antibiotic resistance cassette, resulting in non-functional gene products including the above, based on the results degS or degU Any mutation of will result in a strain of Paenibacillus spp. showing a decrease in the viscosity of liquid culture and / or a non-mucoid colony morphology compared to a strain of Paenibacillus spp. containing wild-type DegU and wild-type DegS. It is concluded that

Table 4

Table 5

Table 6

[0152] Example 4. Further mutagenesis and screening of non - mucoid strains To further improve the titer of the fusaricidin-like compound, the chemical treatment of Paenibacillus sp. NRRL B-67304 strain was performed as described in Example 1. Samples from the culture broth produced in a 96-well block were analyzed for the relative level of fusaricidin A (see Table 7). Then, several isolates with increased fusaricidin production were selected and further tested after fermentation in a larger-scale culture. Samples from these larger-scale cultures were analyzed again for the fusaricidin A content (see Table 8), and their packed cell volumes were measured as described in Example 2 (see Table 9). The packed cell volume was evaluated only with samples from larger-scale cultures because sufficient volume for these measurements could not be obtained with cultures from the 96-well block.

[0153] Surprisingly, it was found that the Paenibacillus sp. NRRL B-67615 strain not only improved the level of the fusaricidin-like compound (see Tables 7 and 8), but also had a lower level of viscosity than the Paenibacillus sp. NRRL B-67304 strain (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 phylogenetic tree of the Paenibacillus sp. NRRL B-67615 strain and the Paenibacillus sp. NRRL B-50972 strain, Paenibacillus sp. NRRL B-67129 strain, Paenibacillus sp. NRRL B-67304 strain, and Paenibacillus sp. NRRL B-67306 strain is shown in Figure 1.

Table 7

Table 8

Table 9

[0154] The relative fusaricidin A levels, packed cell volumes, and viscosities of Paenibacillus sp. NRRL B-50972, Paenibacillus sp. NRRL B-67306, Paenibacillus sp. NRRL B-67304, and Paenibacillus sp. NRRL B-67615 were evaluated together, and the improvements achieved by multiple rounds of mutagenesis and screening by the disclosed method were confirmed. The results shown in Table 10 demonstrate that the disclosed screening method resulted in mutant-derived strains with a significant improvement in fusaricidin production and lower packed cell volumes and viscosities, enabling higher concentrations of the active compound in the fermentation broth.

Table 10

[0155] Example 5. Comparison of biological activities of Paenibacillus sp. strains NRRL B - 50972, NRRL B - 67306, NRRL B - 67304 and NRRL B - 67615 Paenibacillus sp. NRRL B-50972, Paenibacillus sp. NRRL B-67306, and Paenibacillus sp. NRRL B-67304 were cultured in a soybean-based medium to produce whole broth. The whole broth was diluted with a mixture of water and an 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 inoculum of Alternaria solani (ALTESO). A chemical fungicide was included in each assay as a positive control. A few days after exposure to the plant pathogen inoculum, each plant was scored as the percentage of pathogen control relative to untreated control plants. Each treatment was evaluated in triplicate, and the mean control percentage was recorded (see Table 11). 0% means the effectiveness corresponding to that of the untreated control, and 100% effectiveness means that no disease 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] This assay was repeated with Paenibacillus sp. strain NRRL B-67304 and Paenibacillus sp. strain NRRL B-67615 against the fungal pathogen Alternaria solani (ALTESO). This assay was performed as above, except that six replicates were evaluated instead of three replicates, and the total broth was applied at 1.25% or 0.625%. Table 12 shows the average control percentage by the treatment. Paenibacillus sp. strain NRRL B-67304 and Paenibacillus sp. strain NRRL B-67615 showed comparable antifungal activity in this assay.

Table 12

[0157] Example 6. Antifungal activities of Paenibacillus sp. strains NRRL B - 67306 and NRRL B - 67304 and NRRL B - 67615 against oomycete plant pathogens The Paenibacillus sp. NRRL B-67306 strain, Paenibacillus sp. NRRL B-67304 strain, and Paenibacillus sp. NRRL B-67615 strain were cultured in a soybean-based medium to produce whole broth. The whole broth was diluted with a mixture of water and an 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 inoculum of Pseudoperonospora cubensis (PSPECU), also known as cucumber downy mildew, or Phytophthora infestans (PHYTIN), also known as tomato blight. A chemical fungicide was included in each assay as a positive control. A few days after exposure to the inoculum of the plant pathogen, each plant was scored as the percentage of control of the pathogen relative to an untreated control plant. Each treatment was evaluated in triplicate, and the mean percentage of control was recorded (see Table 13 for the results of Pseudoperonospora cubensis and Table 14 for the results of Phytophthora infestans). 0% means the effectiveness corresponding to that of the untreated control, and 100% effectiveness means that no disease was observed. All three strains of Paenibacillus spp. showed consistent control of the two oomycete plant pathogens.

Table 13

Table 14

[0158] Example 7. Comparison of Paenibacillus strains in a potato field trial infected with Alternaria solani A field test was conducted on potato plants exposed to the naturally occurring summer blight (Alternaria solani). The liquid fermentation products of Paenibacillus sp. NRRL B-50972 and Paenibacillus sp. NRRL B-67306 were prepared by culturing the strains in a soybean-based medium and concentrating the resulting whole broth through centrifugation and supernatant collection. As outlined in Table 16, during the growth stages from BBCH65 to BBCH70, between July 20 and August 4, the fermentation products were applied to the plants at 10 liters / hectare and 20 liters / hectare. The average disease incidence was approximately 13% in the untreated plants. The disease control percentages shown in Table 15 are the results of visual observations of the disease symptoms conducted 7 days after the final application. 0% means the effectiveness corresponding to that of the untreated control, and 100% effectiveness means no disease was observed.

Table 15

Table 16

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

[0160] Example 8. Comparison of Paenibacillus strains in a strawberry field trial infected with Botrytis cinerea A field trial was conducted with strawberry plants exposed to naturally occurring gray mold (Botrytis cinerea). The liquid fermentation products of Paenibacillus sp. NRRL B-50972 and Paenibacillus sp. NRRL B-67304 were prepared by culturing the strains in a soybean-based medium and concentrating the resulting whole broth through centrifugation and separation of the supernatant. As outlined in Table 18, during the growth stages from BBCH67 to BBCH87, between March 31 and April 18, the fermentation products were applied to the plants at 10 liters / hectare and 20 liters / hectare. The average disease incidence was approximately 22% in the untreated plants. The disease control percentages shown in Table 17 are the results of visual observation of the disease symptoms conducted two days after the final application. 0% means the effectiveness corresponding to that of the untreated control, and 100% effectiveness means no disease was observed.

Table 17

Table 18

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

[0162] Example 9. Comparison of Paenibacillus strains in a pepper field trial infected with Colletotrichum capsici A field test was conducted with pepper plants exposed to naturally occurring anthracnose (Colletotrichum capsici). The liquid fermentation products of Paenibacillus sp. NRRL B-50972 strain and Paenibacillus sp. NRRL B-67306 strain were prepared by culturing the strains in a soybean-based medium and concentrating the resulting whole broth through centrifugation and supernatant separation. As outlined in Table 20, at the BBCH75 growth stage, between December 28 and January 2, the fermentation products were applied to the plants at 10 liters / hectare and 20 liters / hectare. The average disease incidence was approximately 60% in the untreated plants. The disease control percentage shown in Table 19 is the result of visual observation of the disease symptoms conducted 2 days after the final application. 0% means the effectiveness corresponding to that of the untreated control, and 100% effectiveness means no disease was observed.

Table 19

Table 20

[0163] The results in Table 19 clearly show that in this field trial, the observed activity of Paenibacillus sp. NRRL B-67306 strain is superior compared to Paenibacillus sp. NRRL B-50972.

[0164] Example 10. Identification of growth conditions for viscosity divergence in Paenibacillus sp. strains NRRL B - 67304 and NRRL B - 67615 As shown in Figure 1, the Paenibacillus sp. NRRL B-67615 strain was generated by chemical mutagenesis of the Paenibacillus sp. NRRL B-67304 strain. As a result of this chemical mutagenesis, the Paenibacillus sp. NRRL B-67615 strain had a significantly reduced viscosity, while the concentration of fusaricidin A was relatively high (see Table 10). To determine the point at which the viscosities of the two strains diverged in liquid culture, each strain was grown in a soybean-based medium for a period of 72 hours. One group of cultures was stirred at 250 rpm and another group was stirred at 300 rpm. Samples of each liquid culture were taken at 24 hours, 32 hours, 40 hours, 48 hours, 56 hours, and 72 hours. The viscosity and relative levels of fusaricidin A of each sample were measured as outlined in Example 4. The mean values and standard deviations (n = 4) were determined for the 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 equivalent and increased at a similar rate over 72 hours. Spore formation was visually evaluated microscopically in all samples and no spores were present at any time point. A significant increase in viscosity was observed in the Paenibacillus sp. NRRL B-67304 strain, while the viscosity of the Paenibacillus sp. NRRL B-67615 strain was relatively constant at a low level during this time (compare the solid lines indicating viscosity in Figures 8A and 8B). Therefore, the time points of 40 hours and 48 hours were selected for future experiments. The liquid culture grown while stirring at 300 rpm was also selected for future experiments because its viscosity values were more consistent.

[0166] Example 11. Proteome analysis of liquid cultures of Paenibacillus sp. strains NRRL B - 67304 and NRRL B - 67615 Using Paenibacillus sp. NRRL B-67304 strain (parent strain) and Paenibacillus sp. NRRL B-67615 strain (descendant strain), exploratory proteomics and pathway analysis methods were performed to gain insights into the viscous phenotype at the molecular level. The strains were grown in shaking flasks in a soybean-based medium for 40 hours and 48 hours to obtain the divergent viscosity phenotypes of the two strains. For Paenibacillus sp. NRRL B-67304 strain and NRRL B-67615 strain, six replicates per condition were grown for a total of 24 samples. At harvest, the samples were quickly frozen at -80 °C to stop growth and then sample preparation was performed in a batch manner. Protein extraction was performed on the whole fermentation broth to obtain secreted and vegetative cell proteins. The total protein samples were reduced, alkylated and digested with trypsin to create a total peptide pool for proteome analysis. The total peptide samples were separated by liquid chromatography and analyzed on a SCIEX 4600 TRIPLETOF (registered trademark) mass spectrometer, continuously in data-dependent (IDA) and data-independent (SWATH) acquisition modes, enabling the creation of an ion library and relative quantification across the whole peptide pool.

[0167] To create the ion library, first the IDA runs were analyzed with SCIEX's Protein Pilot (5.0.1.0,4895) software and run in thorough ID mode with false discovery rate (FDR) analysis. Next, using the SWATH Micro App (2.0.1.2133), an ion library was created in SCIEX's PeakView (2.2.0.11391) software with a 1% global protein FDR. Data analysis with the SWATH Micro App was continued and relative quantification of the SWATH runs was performed with a 99% peptide confidence level and a 1% FDR threshold. Next, the protein regions calculated from the total intensity of six transitions per peptide and six peptides per protein were exported for downstream analysis. The threshold for the protein regions was set at 50,000.

[0168] The primary interest was the identification of proteins whose expression varied at a single time point (40 or 48 hours) between the Paenibacillus sp. NRRL B-67304 strain (parent strain) and the Paenibacillus sp. NRRL B-67615 strain (descendant strain). The aim was to elucidate differences in protein levels between the strains, which was hypothesized to contribute to exopolysaccharide (EPS) production and different viscosity phenotypes. Statistical analysis was first performed using SCIEX's MarkerView (1.2.1) software. There were no major data anomalies in the exploratory data analysis including mean 1 vs mean 2 and Log (expression variation) vs p-value, and principal component analysis grouped samples by strain and time. To determine different protein expression between strains, a t-test was performed in MarkerView, and then the p-values were adjusted for multiple comparisons in R (FDR / BH correction). Proteins were considered to have expression variation at P(FDR / BH correction) < 0.05 and a minimum expression variation fold = 1.5. Of the 442 proteins detected at 40 hours, 54 proteins met the expression variation criteria (see Table 21). Of the 422 proteins detected at 48 hours, 94 proteins were found to have expression variation (see Table 22).

[0169] Bacterial exopolysaccharides have diverse structures, consist of various constituent units, and are synthesized via various pathways. Expression also varies depending on the strain and the environment (such as the fermentation process). For example, different strains of Paenibacillus are characterized as producing curdlan-type and levan-type EPS composed of glucose, or glucose and fructose, respectively. From this initial proteome analysis, it was suggested that none of the proteins identified as having expression variation in the Paenibacillus sp. NRRL B-67304 strain (parent strain) and the Paenibacillus sp. NRRL B-67615 strain (descendant strain) were directly involved in EPS synthesis, as identified by homology to proteins described in the literature.

[0170] Further analysis of the proteome analysis data was necessary to explain the difference in the viscous phenotype between Paenibacillus sp. NRRL B-67304 strain and NRRL B-67615 strain. To contextually understand the protein-level differences seen by proteome analysis, the proteins were further annotated in KEGG (BLASTKOALA algorithm) and mapped to KEGG pathways. In Paenibacillus sp. NRRL B-67615 strain (descendant strain), it was observed that several proteins involved in the glycolysis system and tricarboxylic acid (TCA) cycle were significantly increased at the 48-hour time point (see the underlined proteins below "Upregulation in descendants" in Table 22). From this, it is suggested that in Paenibacillus sp. NRRL B-67615 strain (descendant strain), an increase in sugar metabolism occurs compared to Paenibacillus sp. NRRL B-67304 strain (parent strain). EPS production depends on the same hexose monomers (such as glucose and fructose) as primary metabolism. Thus, an increase in primary metabolism would lead to a decrease in the concentration of the starting substrate and, as a result, a decrease in EPS production and viscosity in Paenibacillus sp. NRRL B-67615 strain (descendant strain).

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

[0172] Using samples taken at the 40 - hour and 48 - hour time points, the relative expression of two α - amylases (“α - amylase #1” and “α - amylase #2”) was quantified and shown in Figures 9A and 9B. The relative quantification of the protein indicates that the Paenibacillus sp. NRRL B - 67304 strain (parent strain) consistently expresses significantly more α - amylase than the Paenibacillus sp. NRRL B - 67615 strain (descendant strain). Without wishing to be bound by any theory, the soybean - based culture medium in which the strains were grown contains polysaccharides that these amylases convert into hexose monomers necessary for EPS production. Subsequently, a large amount of substrate promotes EPS production and may increase the viscosity in the liquid culture of the Paenibacillus sp. NRRL B - 67304 strain (parent strain). [Table 21] TIFF0007695788000025.tif248168 [Table 22] TIFF0007695788000027.tif248161TIFF0007695788000028.tif253156TIFF0007695788000029.tif75167 [Table 23]

[0173] Example 12. Confirmation of increased amylase activity in Paenibacillus sp. strain NRRL B - 67304 (parent strain) The liquid cultures of Paenibacillus sp. NRRL B-67304 (parent strain) and Paenibacillus sp. NRRL B-67615 (descendant strain) were grown in a soybean-based medium, and samples were taken at 40 and 48 hours to confirm that the level of amylase activity was higher in the former than in the latter. The samples were centrifuged, and the supernatants were sterile-filtered to remove all cells from the medium, leaving cell proteins containing amylase and polysaccharides that had not been exhausted. The amylase in the supernatants continued to break down the polysaccharides to produce glucose. The glucose content in these cell-free supernatants was measured at the beginning and 5 hours after incubation at 28°C.

[0174] The glucose measurement results shown in Figure 10 indicate that Paenibacillus sp. NRRL B-67304 (parent strain) has a higher amylase activity level than Paenibacillus sp. NRRL B-67615 (descendant strain) when the strains are grown under similar conditions.

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

[0176] In all cultures except those with 10 g / L glucose added, the residual glucose concentration was nearly 0 g / L, indicating that the added glucose was consumed by the cells. The measured viscosities of the cultures are shown in Figure 11. Addition of glucose to the Paenibacillus sp. NRRL B-67304 strain (parent strain) culture had little effect on viscosity, probably because of the high level of amylase activity of this strain and, as a result, the abundant glucose obtained from the culture medium. In contrast, in the glucose-added culture of the Paenibacillus sp. NRRL B-67615 strain (descendant strain), its viscosity increased due to the increased viscosity resulting from the increased amount of added glucose (see the right bar graph in Figure 11).

[0177] Overall, the above experimental results indicate that the expression and activity of amylase are lower in the Paenibacillus sp. NRRL B-67615 strain (descendant strain) than in the Paenibacillus sp. NRRL B-67304 strain (parent strain), and for this reason, there is less simple sugar (such as glucose) for EPS production, resulting in 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 hereby incorporated by reference in their entirety for all purposes.

[0179] It is understood that the disclosed invention is not limited to the specific methodologies, protocols, and materials described herein as these may 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 invention, which will be limited only by the appended claims.

[0180] One of ordinary skill in the art can appreciate and confirm many equivalents to the specific embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be encompassed by the appended claims.

Claims

1. A composition comprising a culture of a strain of Paenibacillus sp. comprising a non-functional mutant DegU lacking a functional receptor domain or a functional DNA binding domain and / or a non-functional mutant DegS lacking a functional single binding domain or a functional ATPase domain, wherein the non-functional mutant DegU and / or the non-functional mutant DegS result in a liquid culture of a strain of Paenibacillus sp. having a reduced viscosity compared to a liquid culture of a strain of Paenibacillus sp. comprising wild-type DegU and wild-type DegS.

2. The composition according to claim 1, wherein the non-functional mutant DegU and / or the non-functional mutant DegS inhibit the formation of colonies of a strain of Paenibacillus sp. having a mucoid morphology.

3. The composition according to claim 1 or claim 2, wherein the non-functional mutant DegU and / or the non-functional mutant DegS are knockouts or are truncated as a result of a premature stop codon.

4. The composition according to claim 3, wherein the premature stop codon results in a non-functional mutant DegU truncated at position 218 numbered according to its correspondence with the amino acid sequence of SEQ ID NO:

2.

5. The non-functional mutant DegU is an amino acid substitution of a small residue acidic residue at position 109 numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a small residue polar residue at position 228 numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of an acidic residue at position 63 to a polar residue numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2; and / or an amino acid substitution of a polar residue at position 195 to a small residue numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2; and / or Amino acid substitution of the hydrophobic residue at position 204, numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2, to a small residue; and / or Amino acid substitution of the polar residue at position 208, numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2, to a small residue; and / or Amino acid substitution of the basic residue at position 212, numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2, to a small residue; and / or Amino acid substitution of the hydrophobic residue at position 217, numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2, to a small residue; and / or Amino acid substitution of the basic residue at position 207, numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2, to a small residue; and / or Amino acid substitution of the polar residue at position 211, numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2, to a small residue; and / or Amino acid substitution of the polar residue at position 214, numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 2, to a small residue, The composition according to any one of claims 1 to 4, comprising

6. The non-functional mutant DegU comprises the amino acid substitution 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 of SEQ ID NO: 2, or a mutant thereof having a conservative amino acid substitution, the composition according to any one of claims 1 to 5.

7. The non-functional mutant DegS is Amino acid substitution of the hydrophobic residue at position 99, numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 4, to an aromatic residue; and / or Amino acid substitution of the acidic residue at position 294, numbered according to its correspondence with the amino acid sequence of SEQ ID NO: 4, to a basic residue; and / or The composition according to any one of claims 1 to 6, comprising an amino acid substitution of the polar residue at position 73 numbered according to the correspondence with the amino acid sequence of SEQ ID NO: 4 to a small residue.

8. The composition according to any one of claims 1 to 7, wherein the non-functional mutant DegS comprises SEQ ID NO: 4 having L99F and / or E294K and / or T73A, or a mutant thereof having a conservative amino acid substitution.

9. The composition according to any one of claims 1 to 8, wherein the strain of the genus Paenibacillus is a mutagen-induced derivative strain and exhibits an increased fusaricidin concentration as compared to the parent strain.

10. The composition according to any one of claims 1 to 9, wherein the strain of the genus Paenibacillus is a mutagen-induced derivative strain and exhibits a decreased expression and / or enzyme activity of amylase as compared to the parent strain.

11. The composition according to claim 10, wherein the decreased expression and / or enzyme activity of amylase occurs in an α-amylase protein comprising a sequence having more than 90% sequence identity with SEQ ID NO: 9 or SEQ ID NO:

10.

12. The composition according to any one of claims 9 to 11, wherein the parent strain is Paenibacillus sp. NRRL B-50972 strain, Paenibacillus sp. NRRL B-67129 strain, Paenibacillus sp. NRRL B-67304 strain, Paenibacillus sp. NRRL B-67306 strain, or Paenibacillus sp. NRRL B-67615 strain.

13. The composition according to any one of claims 1 to 8, wherein the strain of the genus Paenibacillus is Paenibacillus sp. NRRL B-67304 strain, Paenibacillus sp. NRRL B-67306 strain, or Paenibacillus sp. NRRL B-67615 strain.

14. The composition according to claim 13, comprising a fermentation product of Paenibacillus sp. NRRL B-67304, Paenibacillus sp. NRRL B-67306, or Paenibacillus sp. NRRL B-67615.

15. A method for treating a plant to control a disease, the method comprising applying an effective amount of the composition according to any one of claims 1 to 14 to the plant, a part of the plant, and / or the planting site.

16. 1 × 10 per hectare ４ ~1 × 10 １４ The method according to claim 15, wherein the composition is applied in colony forming units (CFU) or in fermented solids in an amount of 0.1 kg to 20 kg per hectare.

17. The method according to claim 15 or 16, wherein the plant disease is caused by a fungus.

18. The method according to claim 17, wherein the plant disease is powdery mildew or downy mildew.

19. The method according to claim 17, wherein 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. **Claim 20** The method according to claim 15 or 16, wherein the plant disease is caused by bacteria. **Claim 21** The method according to claim 20, wherein the bacteria is selected from the group consisting of Xanthomonas campestris, Pseudomonas syringae and Erwinia carotovora.

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