Novel paenibacillus-like strains, antifungal compounds, and methods of use thereof

Abstract

The present invention relates to compositions comprising a biologically pure culture of a fungicidal Paenibacillus sp. strain comprising a variant fusaricidin synthetase lacking a functional adenylation domain in the third module. The present invention also provides compositions comprising a biologically pure culture of a fungicidal Paenibacillus sp. strain or a cell-free extract thereof, the biologically pure culture or cell-free extract comprising at least one Paeniserine and at least one Paeniprolixin. Isolated compounds are also provided as well as methods of treating plants to control plant diseases with the disclosed compositions and compounds.

Description

[0001] This application is a divisional application of international application filed on March 23, 2016, with application number PCT / US2016 / 023760 and the invention title "Novel Bacillus strains, antifungal compounds and methods of using the same". The international application entered the Chinese national phase on November 24, 2017, with application number 201680030395.7.

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 138,765, filed March 26, 2015, and U.S. Provisional Patent Application No. 62 / 232,205, filed September 24, 2015, the contents of which are incorporated herein by reference in their entirety.

[0004] References to sequence lists submitted electronically

[0005] An official copy of the sequence list was submitted electronically via EFS-Web as a sequence list in ASCII format, named "BCS 159002WO_ST25.txt", created on March 21, 2016, and 68 kilobytes in size. This official copy of the sequence list was submitted together with the instruction manual. The sequence list contained in this ASCII format file is part of the instruction manual and is incorporated herein by reference in its entirety. Technical Field

[0006] This invention relates to the field of bacterial strains and their ability to control plant diseases. Specifically, this invention relates to Paenibacillus sp. strains with relatively high levels of broad-spectrum antifungal activity. Background Technology

[0007] Fungicides have numerous uses, including crop protection; as preservatives in food, feed, and cosmetics; and as therapeutic agents for human and veterinary applications. Crop yield reduction, foodborne diseases, and fungal infections in humans and animals are problems in both developed and developing countries.

[0008] Synthetic pesticides or fungicides are often non-specific, thus acting on organisms other than their target organisms, including other naturally occurring beneficial organisms. Due to their chemical properties, they can also be toxic and non-biodegradable. Consumers worldwide are increasingly aware of the potential environmental and health problems associated with chemical residues, particularly in food products. This has led to growing consumer pressure to reduce the use of chemical (i.e., synthetic) pesticides, or at least to reduce their quantities. Therefore, there is a need to manage food chain demands while still allowing for effective pest control.

[0009] Another problem arising from the use of synthetic insecticides or fungicides is that repeated and individual application of these agents often leads to the selection of resistant pathogens. Typically, such strains also exhibit cross-resistance to other active ingredients with the same mode of action. Subsequently, it becomes impossible to effectively control pathogens using these active compounds. However, active ingredients with novel mechanisms of action are difficult to develop and costly to develop.

[0010] The risk of resistance development in pathogen populations, along with concerns about the environment and human health, has fueled interest in identifying alternatives to synthetic insecticides and fungicides used to manage plant diseases. The use of biocontrol agents is one such alternative.

[0011] The antimicrobial properties of non-ribosomal peptides (such as fusaricidin) are well-established and have been used in crop protection. Due to their mode of action, they also have potential applications in biomedicine and other biotechnological fields. Fusaricidin can be isolated from Bacillus species and has a cyclic structure consisting of six amino acid residues and 15-guanidino-3-hydroxypentadecanoic acid. Fusarium oxysporins isolated from *Paenibacillus polymyxa* include LI-F03, LI-F04, LI-F05, LI-F07, and LI-F08 (Kurusu K, Ohba K, Arai T, and Fukushima K., *J. Antibiotics*, 40:1506-1514, 1987), and other fusarium oxysporins A, B, C, and D have been reported (Kajimura Y and Kaneda M., *J. Antibiotics*, 49:129-135, 1996; Kajimura Y and Kaneda M., *J. Antibiotics*, 50:220-228, 1997).

[0012] Some fusaric acid fungicides are known to have fungicidal activity against plant pathogenic fungi such as *Fusarium oxysporum*, *Aspergillus niger*, *Aspergillus oryzae*, and *Penicillium thomii*. Some fusaric acid fungicides also have fungicidal activity against Gram-positive bacteria, including *Staphylococcus aureus* (Kajimura Y and Kaneda M., *J. Antibiotics*, 49:129-135, 1996; Kajimura Y and Kaneda M., *J. Antibiotics*, 50:220-228, 1997). Furthermore, certain fusarinins have been found to possess antifungal activity against *Leptosphaeria maculans*, the bacterium that causes black root rot in Canadian rapeseed (Beatty PH and Jensen SE., Can. J. Microbiol., 48:159-169, 2002). Further characterization of fusarinin compounds and identification of *Bacillus* strains that produce fusarinins that provide broad-spectrum antifungal activity at relatively low application rates are needed.

[0013] Fusarium oxysporins and other antifungal metabolites are obtained through fermentation in *Bacillus* species. However, many *Bacillus* strains also produce antibiotics called polymyxins. Polymyxins are selectively toxic to Gram-negative bacteria and can have neurotoxic and nephrotoxic effects when administered to human patients. The increasing global problem of antimicrobial resistance and the relative toxicity of polymyxins necessitate the cautious use and administration of these antibiotics. For this reason, there is a strong need to develop *Bacillus* strains for agricultural use that express relatively high levels of fusarium oxysporins and do not express detectable polymyxins. Such strains pose little or no risk to workers and consumers. Furthermore, there is a need to identify *Bacillus* strains exhibiting broad-spectrum activity. There is a strong need to improve the efficacy of existing fungicides, particularly those less susceptible to the development of fungal resistance. Summary of the Invention

[0014] This invention relates to compositions comprising biopure cultures of fungicidal Bacillus species containing a variant fusarium synthase lacking a functional adenylated domain (FusA-A3) in a third module, wherein the deletion of the functional FusA-A3 inhibits the synthesis of fusarium containing tyrosine or phenylalanine at amino acid residue (3) compared to fusarium synthesis in Bacillus species containing wild-type fusarium synthase. In some aspects, the variant fusarium synthase contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10 substrate-specific amino acid residues deleted in FusA-A3. In other respects, the amino acid residues are selected from Asp235, Ala236, Ser239, Thr278, Leu299, Ala301, Ala / Gly322, Val330, Cys331, Lys517 or combinations thereof.

[0015] In one embodiment, the amino acid residues are located at positions 3203, 3204, 3207, 3246, 3267, 3269, 3290, 3298, 3299, and / or 3486 of SEQ ID NO:11. In another embodiment, the variant fusarium synthase contains deletions of Asp235, Ala236, Ser239, Thr278, Leu299, Ala301, Ala / Gly322, Val330, and Cys331 in FusA-A3. In some embodiments, the variant fusarium synthase comprises SEQ ID NO:10.

[0016] The present invention also provides a composition comprising a biopure culture of a fungicide Bacillus species or a cell-free extract thereof (including at least one Paeniserine and at least one Paeniprolixin).

[0017] In some respects, the at least one Paeniserine is selected from Paeniserine A1, Paeniserine A2, Paeniserine A3, Paeniserine A4, Paeniserine B1, Paeniserine B2, Paeniserine B3, Paeniserine B4, Paeniserine C1, Paeniserine C2 and Paeniserine C3.

[0018] In other aspects, the at least one Paeniprolixin is selected from Paeniprolixin A1, Paeniprolixin A2, Paeniprolixin B1, Paeniprolixin B2, Paeniprolixin C1, Paeniprolixin D1, Paeniprolixin E1, Paeniprolixin E2, Paeniprolixin F1, Paeniprolixin F2, Paeniprolixin G1 and Paeniprolixin G2.

[0019] In some embodiments, the composition includes Fusarium oxysporin A, LiF08a, Paeniserine A1, Paeniserine B1, Paeniprolixin A2, and Paeniprolixin B2.

[0020] In some embodiments, the composition does not include LiFO3a, LiFO3b, LiFO3c, LiFO3d, LiFO7a, LiFO7b, LiFO7c and / or LiFO7d. In other embodiments, the composition includes synergistically effective amounts of Paeniserine A1, Paeniserine B1, Paeniprolixin A2 and Paeniprolixin B2.

[0021] In some aspects, the present invention relates to compositions wherein the *Bacillus* species strain is *Bacillus* species strain NRRL B-50972, *Bacillus* species strain NRRL B-67129, or a fungicide mutant strain thereof. The composition may include fermentation products of *Bacillus* species strain NRRL B-50972, *Bacillus* species strain NRRL B-67129, or a fungicide mutant strain thereof.

[0022] In some embodiments, the genomic sequence of the fungicidal mutant strain shares greater than about 90% sequence identity with the *Bacillus* species strain NRRL B-50972. In other embodiments, the fungicidal mutant strain has fungicidal activity and / or levels of fumonisin, paeniserine, and / or paeniprolixin comparable to or better than those of the *Bacillus* species strain NRRL B-50972. In other embodiments, the fermentation product does not contain polymyxins.

[0023] In some aspects, the fermentation product is a liquid formulation. The liquid formulation may be a suspension concentrate or an oil dispersion. In one embodiment, the composition comprises at least about 1 × 10⁻⁶ 4 CFU strain / ml liquid formulation. In another embodiment, the composition comprises about 1% to about 25% fermentation solids.

[0024] In other respects, the present invention relates to compositions comprising synergistically effective amounts of: a) at least one fusarin; and b) at least one Paeniserine or at least one Paeniprolixin. In one embodiment, the Paeniserine is at least one selected from Paeniserine A1, Paeniserine A2, Paeniserine A3, Paeniserine A4, Paeniserine B1, Paeniserine B2, Paeniserine B3, Paeniserine B4, Paeniserine C1, Paeniserine C2, and Paeniserine C3. In another embodiment, the Paeniprolixin is at least one of Paeniprolixin A1, Paeniprolixin A2, Paeniprolixin B1, Paeniprolixin B2, Paeniprolixin C1, Paeniprolixin D1, Paeniprolixin E1, Paeniprolixin E2, Paeniprolixin F1, Paeniprolixin F2, Paeniprolixin G1, and Paeniprolixin G2.

[0025] Specifically, in one embodiment, the synergistic ratio of at least one fusarium spore-killing agent to at least one paeniserine or at least one paeniprolixin ranges from 1:1000 to 1000:1, preferably 1:500 to 500:1, more preferably 1:250 to 250:1. In another embodiment, the synergistic weight ratio of at least one fusarium spore-killing agent to at least one paeniserine or at least one paeniprolixin ranges from 1:100 to 100:1, preferably 1:100 to 10:1 or even 1:50 to 25:1. On one hand, the fusarium spore-killing agent is fusarium spore-killing agent A. On the other hand, the paeniserine is paeniserine A1. On the other hand, the paeniprolixin is paeniprolixin C1.

[0026] In other respects, the present invention relates to isolated compounds having structure (I), including their salts, hydrates, solvates, polymorphs, optical isomers, geometric isomers, enantiomers, diastereomers, acyclic analogs, and mixtures.

[0027]

[0028] in

[0029] R 1 and R 2 Each is independently -CH(CH3)2 or -CH(CH3)CH2CH3;

[0030] R 3 It is -CH2C(O)NH2 or -(CH2)2C(O)NH2; and

[0031] n is an integer between 13 and 20.

[0032] In some implementation schemes, R 3 It is -CH2C(O)NH2. In other embodiments, R 3 It is -(CH2)2C(O)NH2. On the one hand, R 1 It is -CH(CH3)2. On the other hand, R 1 It is -CH(CH3)CH2CH3. On the one hand, R 2 It is -CH(CH3)2. On the other hand, R 2 It is -CH(CH3)CH2CH3.

[0033] In other respects, the present invention relates to isolated compounds having structure (II), including their salts, hydrates, solvates, polymorphs, optical isomers, geometric isomers, enantiomers, diastereomers, acyclic analogs, and mixtures.

[0034]

[0035] in

[0036] R 1 It is -CH2OH or -CH(OH)CH3;

[0037] R 2 It is -CH2C(O)NH2 or -(CH2)2C(O)NH2; and

[0038] R 3 It is H or CH3;

[0039] The condition is if R 1 It is -CH2OH and R 2If it is -CH2C(O)NH2, then R 3 It's H.

[0040] In some implementation schemes, R 3 It is CH3. In other implementations, R 3 It's H. On the one hand, R 1 It is -CH2OH. On the other hand, R 1 It is -CH(OH)CH3. On the one hand, R 2 It is -CH2C(O)NH2. On the other hand, R 2 It is -(CH2)2C(O)NH2.

[0041] In one embodiment, the present invention relates to compositions comprising the isolated compounds disclosed herein and agriculturally acceptable carriers.

[0042] In some embodiments, the present invention relates to solutions comprising a compound comprising structure (I), wherein the concentration of said compound is at least 0.001 mg / ml, at least 0.01 mg / ml, or at least 0.1 mg / ml. In another embodiment, the present invention relates to solutions comprising a compound comprising structure (II), wherein the concentration of said compound is at least 0.001 mg / ml, at least 0.01 mg / ml, or at least 0.1 mg / ml. In some aspects, the disclosed solutions also comprise an agriculturally acceptable carrier.

[0043] In another embodiment, the present invention relates to a method of treating plants to control diseases, wherein the method comprises applying an effective amount of the composition disclosed herein to the plant, a portion of the plant, and / or a location on the plant. In some aspects, the composition is a fermentation product of a Bacillus species NRRL B-50972, a Bacillus species NRRL B-67129, or a fungicide mutant strain thereof. In other aspects, the method comprises applying the composition to the leaf portion of the plant. In other aspects, the composition is applied at a concentration of about 1 × 10⁻⁶ per hectare. 10 To approximately 1×10 12 The composition is applied using Bacillus species NRRL B-50972, Bacillus species NRRL B-67129, or their fungicidal mutant strains, at colony-forming units (CFU). In one embodiment, the composition is applied at a rate of about 0.5 kg to about 5 kg of fermentation solids per hectare.

[0044] In some respects, plant diseases are caused by fungi. In others, plant diseases are mildew or rust. In one embodiment, the mildew is powdery mildew or downy mildew. In another embodiment, the rust is selected from wheat leaf rust, barley leaf rust, rye leaf rust, brown leaf rust, crown rust, and stem rust.

[0045] In some embodiments, the fungi are selected from Alternaria alternata, Alternaria solani, Botrytis cinerea, Colletotrichumlagenarium, Fusarium culmorum, Phaeosphaeria nodorum, Zymoseptoria tritici, Phytophthora cryptogea, Phytophthora infestans, Pythium ultimum, Magnaphortheoryzae, Thanatephorus cucumeris, Ustilago segetum var. avenae, Uromyces appendiculatus, and Puccinia triticina.

[0046] In other implementations, the plant disease is caused by bacteria. On one hand, the bacteria are selected from Xanthomonas campestris, Pseudomonas syringae, and Erwinia carotovora.

[0047] The present invention also relates to the use of the disclosed compositions for controlling plant pathogens in useful plants. In some aspects, the plant pathogens are selected from *Alternaria alternata*, *Alternaria solanacea*, *Botrytis cinerea*, *Cladosporium anthracis*, *Fusarium oxysporum*, *Syngonium graminearum*, *Cyclocarya paliurus*, *Phytophthora infestans*, *Pythium oxysporum*, *Pythium oryzae*, *Bacillus oryzae*, *Rhizoctonia solani*, *Ustilago maydis*, *Aureobasidium verruciformis*, and *Phytophthora leucosus*. In other aspects, the plant pathogens are selected from *Xanthomonas laurentii*, *Pseudomonas syringae*, and *Erwinia carotenoides*.

[0048] In other respects, the useful plants are selected from apples, bananas, citrus fruits, kiwifruit, melons, peaches, pears, pineapples, pome fruits, pomegranates, cabbage, cauliflower, cucumbers, gourds, tomatoes, potatoes, wheat, rice, and soybeans. Attached Figure Description

[0049] Figure 1The fungicidal activity of whole cultures of Bacillus species in plants against Phytin, Botrytis cinerea, and PUCCRT (a fungicide) was described.

[0050] Figure 2 The in vitro antifungal activity of fusarium extracts from whole cultures of Bacillus species was described against Altepal, Botrci, Fusacchari, Leptno, Sepptr, Phytophthora, Phytin, Pythul, Pyrior, Rhizoma Ricinus, smut fungus, and Uromaporifolia was described.

[0051] Figure 3 The ring-opening of LiF04a (also known as fusarium oxychloride A) is used to produce the acyclic analog LiF04c. Acyclic analogs of each fusarium oxychloride and fusarium oxychloride-like compound are produced in a similar manner.

[0052] Figure 4A The diagram depicts the structure of a known fusarium oxysporin, which has conserved amino acids at identified positions (1), (4), and (6) and different amino acids denoted as AA (amino acid). The tail of 15-guanidino-3-hydroxypentadecanoic acid (GHPD) forms an amide bond with the N-terminus of L-threonine at position (1). The C-terminus of D-alanine at position (6) forms an ester bond with the hydroxyl group of L-threonine at position (1), indicated by an arrow pointing to "O". Figure 4B This represents an HPLC / MS TOF chromatogram of an extract from a Bacillus species cell—in which known fusarium spores were identified. Figure 4C Known fusarinins detectable in cell extracts from Bacillus species NRRL B-50972 and / or their derivatives are described.

[0053] Figure 5A The diagram depicts the structure of Paeniserine. This class of compounds is similar to Fusarium oxysporins, except that one or both of the conserved threonine residues at positions (1) and (4) are replaced by serine residues. Figure 5B HPLC / MS TOF chromatograms of cell extracts from Bacillus species NRRL B-50972 and / or their derivatives—in which Paeniserine was identified. Figure 5CPaeniserine detectable in cell extracts from Bacillus species NRRL B-50972 and / or their derivatives is depicted. m / z values ​​and retention times (RT) for all detected compounds are shown.

[0054] Figure 6A Depicting from Figure 6B The chemical structure of Paeniserine A1 is shown in the UPLC / MS triple TOF spectrum.

[0055] Figure 7A Depicting from Figure 7B The chemical structure of Paeniserine B1 is shown in the UPLC / MS triple TOF spectrum.

[0056] Figure 8A This diagram depicts the structure of Paeniprolixin. This class of compounds is similar to fusarium oxysporins, except that the GHPD tail length changes from -(CH2). 12 -Extended to -(CH2) 14 -or-(CH2) 16 - Figure 8B The HPLC / MS TOF chromatograms of cell extracts from Bacillus species NRRL B-50972 and / or their derivatives are shown. Paeniprolixin was identified in these extracts. Figure 8C Paeniprolixin detectable in cell extracts from Bacillus species NRRL B-50972 and / or their derivatives is depicted. m / z values ​​and retention times (RT) for all detected compounds are shown.

[0057] Figure 9A Depicting from Figure 9B The chemical structure of Paeniprolixin C1 is shown in the UPLC / MS triple TOF spectrum.

[0058] Figure 10A Depicting from Figure 10B The chemical structure of Paeniprolixin D1 is shown in the UPLC / MS triple TOF spectrum.

[0059] Figure 11The Kirby-Bauer antibiotic disc diffusion assay depicts a spore-bearing grass of *Colletotrichum coccinea* on an agar plate, in which fusarium oxysporum A and B (“AB”), paeniserine A1 and B1 (“868”), paeniprolixin A2 and B2 (“938”), or a combination of 868 and 938 are applied. The diameter of each disc with a fungal growth inhibition zone is expressed in millimeters.

[0060] Figure 12A The chemical structure of Fusarium oxysporin A and a simplified description of the structure are shown. Figure 12B-12E A simplified description is provided of the combination of fusarin, paeniserine, and / or paeniprolixin produced by Bacillus spp. strain NRRL B-50972 and / or its derivatives. This combination (such as these) produces a synergistic antifungal effect and is the reason for the relatively high efficacy and broad-spectrum antifungal activity observed with Bacillus spp. strain NRRL B-50972 and its derivatives.

[0061] Figure 13Multiple sequence alignments representing a segment of the FusA fusarium synthase expressed by the following *Paenibacillus* strains: *Paenibacillus peoriae* A (SEQ ID NO:1); *Paenibacillus polymyxa* A (SEQ ID NO:2); *Paenibacillus polymyxa* PKB 1 (GenBank ABQ96384.2; SEQ ID NO:3); *Paenibacillus polymyxa* E681 (GenBank ADM67985.1; SEQ ID NO:4); *Paenibacillus polymyxa* B (SEQ ID NO:5); *Paenibacillus polymyxa* SQR (GenBank AHM63812.1; SEQ ID NO:6); *Paenibacillus polymyxa* C (SEQ ID NO:7); *Paenibacillus polymyxa* M1 (GenBank CCC83015.1; SEQ ID NO:8); *Paenibacillus polymyxa* SC2 (GenBank... ACA09733.2 (SEQ ID NO: 9); Bacillus subtilis strain NRLB-50972 (SEQ ID NO: 10) and Bacillus subtilis strain A (SEQ ID NO: 11). Substrate-specific amino acid residues were identified using black wireframes (see also Table 1). These amino acid residues are located at positions 3203, 3204, 3207, 3246, 3267, 3269, 3290, 3298, 3299, and 3486 in SEQ ID NO: 1-5 and 11, and at positions 3204, 3205, 3208, 3247, 3268, 3270, 3291, 3299, 3300, and 3487 in SEQ ID NO: 6-9.

[0062] Figure 14The fuscin gene clusters in *Bacillus* strain NRRL B-50972 and *Bacillus* strain A (“strain A”) are depicted. Arrows indicate individual genes within a gene cluster (i.e., fusG is indicated by a “G” arrow, fusF by a “F” arrow, etc.). The largest arrow indicates the fusA fuscin synthase gene, with the following abbreviations and symbols: A = adenylate domain (substrate recognition and activation); C = condensation domain (peptide bond formation); E = epimerization domain (substrate racemization); TE = thioesterase domain (product release); ellipse without a letter = thiolation (T) domain (peptide carrier protein). The fusA gene has six modules responsible for incorporating the amino acids indicated in the boxes above or below each gene cluster. Strain A possesses a typical fusarium oxysporin gene cluster, while the fusarium oxysporin gene cluster of Bacillus subtilis strain NRRL B-50972 lacks the functional A domain in module 3. Therefore, the fusarium oxysporin produced by Bacillus subtilis strain NRRL B-50972 lacks tyrosine and phenylalanine at position (3) and contains only valine or isoleucine.

[0063] Figure 15 The sequence alignment of the spo0A gene in Bacillus subtilis strains NRRL B-50972 (SEQ ID NO:12) and NRRL B-67129 (SEQ ID NO:13) was described.

[0064] Figure 16 Sequence alignments of Spo0A orthologs from bacteria that form endospores were depicted, showing that nucleotide changes in the coding sequence of Bacillus spp. strain NRRL B-67129 resulted in a single amino acid substitution in a conserved region. The sequence of the orthologs of Spo0A that were compared is as follows: Paenibacillus terrae Spo0A (SEQ ID NO:14), Paenibacillus terrae strain NRRL B-50972 Spo0A (SEQ ID NO:15), Paenibacillus terrae strain NRRL B-67129 Spo0A (SEQ ID NO:16), Paenibacillus polymyxa Spo0A (SEQ ID NO:17), Paenibacillus subtilis Spo0A (SEQ ID NO:18), Paenibacillus cereus Spo0A (SEQ ID NO:19), and Clostridium pasteurianum Spo0A (SEQ ID NO:20).

[0065] Figure 17The minimum inhibitory concentrations (MICs) of several fusarium spore-killing agents, paeniserine, and paeniprolixin against the fungal pathogens Alternaria solanacea (ALTSO) and Collla anthracnose (COLLLA) were described. Detailed Implementation

[0066] This invention provides a Bacillus genus strain NRRL B-50972 or a fungicidal mutant (strain) derived therefrom. Bacillus genus strain NRRL B-50972 has been found to possess broad-spectrum activity against plant pathogens.

[0067] The microorganisms and specific strains described herein, unless otherwise specified, are isolated from nature and grown under artificial conditions, such as in shake-flask cultures or through scale-up manufacturing processes (e.g., in bioreactors) to maximize, for example, the production of bioactive metabolites. Growth under such conditions results in the "domestication" of the strains. Typically, such "domesticated" strains differ from their naturally occurring counterparts in that the "domesticated" strains are cultured as homogeneous populations, subjected to artificial selection pressures rather than those present in the natural environment.

[0068] As used in this article, the term "isolated" refers to a compound that has been enriched or concentrated in the whole culture medium or fermentation product, or a compound that has been partially or substantially purified from the whole culture medium or fermentation product.

[0069] In one embodiment, a mutant strain of *Bacillus* strain NRRL B-50972 is provided. The term "mutant" refers to a genetic variant derived from *Bacillus* strain NRRL B-50972. In one embodiment, the mutant possesses one or more or all of the identifying (functional) characteristics of *Bacillus* strain NRRL B-50972. In a particular instance, the mutant or its fermentation product controls fungi, oomycetes, and / or bacteria (as identifying functional characteristics) at least as well as the parental *Bacillus* strain NRRL B-50972. The mutant can be a genetic variant whose genomic sequence shares greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% sequence identity with *Bacillus* strain NRRL B-50972. Mutants can be obtained by treating Bacillus subtilis strain NRRL B-50972 cells with chemicals or radiation, by selecting spontaneous mutants (e.g., phage-resistant or antibiotic-resistant mutants) from a population of Bacillus subtilis strain NRRL B-50972 cells, or by other methods known to those skilled in the art.

[0070] The Bacillus genus strain NRRL B-50972 and its mutants exhibit activity against a variety of plant pathogens. Specifically, the strain is active against fungi such as *Anthracnose of cucumber*, *Powdery mildew of cucumber*, *Leymus chinensis*, *P. chinensis*, *P. barley*, and *Botrytis*; oomycetes such as *Fusarium wilt* of tomato, *Downy mildew* of cucumber, and *Downy mildew* of brassicae; and / or bacteria such as *Pseudomonas*, *Xanthomonas*, and *Erwinia*.

[0071] In some respects, the DNA sequences contained in the Bacillus species strains show at least 75% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with SEQ ID NO:10.

[0072] In some aspects, the present invention relates to fermentation products comprising strains of the genus *Bacillus*, wherein the *Bacillus* strains produce paeniserine, paeniserine, and / or paeniprolixin. Paeniserine is a family of depsipeptides having a 15-guanidino-3-hydroxypentadecanoic acid (GHPD) tail, and their linear counterparts. Specific conserved features of paeniserine are the GHPD tail and three of the six amino acids in the sequence: (1) threonine, (4) threonine, and (6) alanine.

[0073] Nakajima et al. (J. Antibiot. 1972, 25, 243-247) first discovered Fusarium oxysporine in the mid-1970s but did not characterize it. Kurusu et al. (J. Antibiot., 1987, 40, 1506-1514) described Fusarium oxysporine in the late 1980s. Kajimura et al. (J. Antibiot., 1996, 49, 129-135; J. Antibiot., 1997, 50, 220-228), Kuroda et al. (Heterocycles, 2000, 53, 1533-1549; J. Mass Spectrom., 2001, 36, 30-37), and Beatty et al. (Can. J. Microbiol., 2002, 48, 159-169) further investigated fusarinins from the mid-1990s to the early 21st century. During this period of extensive research, these compounds were named several times according to the authors (fusarin A was also known as LiF04a, Gatavalin, or KT-6291A). Although there are numerous published works on this subject, each record describes selected compounds from the same group of 24 known fusarinins.

[0074] Following a period of relative calm on the subject, Vater et al. (J. Am. Soc. Mass Spectrom., 2015, 26, 1130-1141) described the structural elucidation of fusarin by mass spectrometry and described several analogs of this family. Vater et al. identified a new class of fusarin-like compounds with 7 amino acids (i.e., in the peptide sequence, 0 additional alanine residues linked to (4) threonine residues). The term “acyclic analog” as used herein refers to compounds that correspond to fusarin or fusarin-like compounds (e.g., Paeniserine or Paeniprolixin) but lack the ester bond to produce a linear structure.

[0075] The amino acid chains of fusaricidins are linked together and modified by nonribosomal peptide synthases (NRPS). Multidomain NRPSs consist of up to 15,000 amino acids, thus being considered one of the longest proteins in nature (Schwarzer et al., (2003) Nonribosomal Peptides: From Genes to Products. Nat. Prod. Rep. 20, 275-287). NRPS incorporation is not limited to the 21 standard amino acids translated from ribosomes; this heterogeneity contributes to the enormous structural diversity and biological activity of nonribosomal peptides (Li and Jensen, (2008). Nonribosomal biosynthesis of fusaricidins by Paenibacillus polymyxa PKB1 involves direct activation of a d-amino acid. Chem. Biol. 15, 118-127).

[0076] In *Bacillus polymyxa* E68, the fusaricidin biosynthetic gene cluster (fusGFEDCBA) has been characterized, and the NRPS coding sequence (the largest coding DNA sequence (CDS) in this gene cluster) has been observed to encode a peptide of six modules (Choi et al., Identification and Functional Analysis of the Fusaricidin Biosynthetic Gene of *Paenibacillus polymyxa* E681. Biochem. Biophys. Res. Commun. 365, 89-95; Li and Jensen, Identification and Functional Analysis of the Fusaricidin Biosynthetic Gene of *Paenibacillus polymyxa* E681. Biochem. Biophys. Res. Commun. 365, 89-95; Li et al., (2013). Promoter Analysis and Transcription Regulation offus Gene Cluster Responsible for Fusaricidin Synthesis of *Paenibacillus polymyxa*). SQR-21. Appl. Microbiol. Biotechnol. 97, 9479-9489). The biosynthetic cluster includes other CDS responsible for the biosynthesis of lipid moieties, but does not contain transporter genes (Li and Jensen, (2008). Nonribosomal Biosynthesis of Fusaricidins by Paenibacillus polymyxa PKB1 Involves Direct Activation of a d-amino acid. Chem. Biol. 15, 118-127).In *Paenibacillus polymyxa*, the promoter of the fus operon was identified and demonstrated to bind to a transcriptional repressor (AbrB), which previous studies have suggested is a regulator of sporulation. This is significant because fusaricin was observed to be synthesized during sporulation, thus coordinating the microbial secondary metabolism with its life cycle (Li et al., (2013). Promoter Analysis and Transcription Regulation of fus Gene Cluster Responsible for Fusaricidin Synthesis of Paenibacillus polymyxa SQR-21. Appl. Microbiol. Biotechnol. 97, 9479-9489).

[0077] Allelic diversity is generally considered to be the cause of chemical diversity. However, an interesting feature of the fus cluster is that the diversity of fusaric acid (distinguished by the amino acids they incorporate (Tyr, Val, Ile, allo-Ile, Phe)) can be generated by a single allele of fusA; the underlying mechanism is that the NRPS A domain responsible for amino acid recognition relaxes substrate specificity (Han et al., (2012). Site-Directed Modification of the Adenylation Domain of the Fusaricidin Nonribosomal Peptide Synthetase for Enhanced Production of Fusaricidin Analogs. Biotechnol. Lett. 34, 1327-1334; Mousa et al., (2015) Biodiversity of Genes Encoding Anti-Microbial Traits within Plant Associated Microbes, Front Plant Sci. 2015; 6:231).

[0078] Using X-ray crystallography, the structure of the A domain responsible for substrate recognition and fusA gene activation has been determined from GrsA, and 10 amino acid residues (Asp235, Ala236, Trp239, Thr278, Ile299, Ala301, Ala322, Ile330, Cys331, and Lys517) that determine substrate specificity have been identified (Challis et al, (2000) Predictive, Structure-Based Model of Amino Acid Recognition by Nonribosomal Peptide Synthetase Adenylation Domains. Chem Biol 7:211-224; Stachelhaus et al, (1999) The Specificity Conferring Code of Adenylation Domains in Nonribosomal Peptide Synthetases. Chem Biol 6:493-505). These 10 marker residues can be divided into three subgroups based on their function at the substrate binding site. Asp235 and Lys517 interact with the carboxyl terminus and amino acid of the substrate, respectively, and sequence analysis revealed that their positions in the A domain of the NRPS are invariant. Ala236, Ala301, and Ile330 can vary appropriately in the A domain specifically targeting amino acid substrates with aliphatic side chains. Trp239, Thr278, Ile299, Ala322, and Cys331 are highly variable positions and are considered important in the differentiation and selection of different substrates (Challiset et al, (2000) Predictive, Structure-Based Model of Amino Acid Recognition by Nonribosomal Peptide Synthetase Adenylation Domains. Chem Biol 7:211-224; Stachelhaus et al, (1999) The Specificity Conferring Code of Adenylation Domains in Nonribosomal Peptide Synthetases. Chem Biol 6:493-505).Among the substrate-specific sequences, Ile299 is the most variable position (Stachelhaus et al., (1999) The Specificity Conferring Code of Adenylation Domains in Nonribosomal Peptide Synthetases. Chem Biol6:493-505).

[0079] The 10 amino acid residues that determine substrate specificity in the fusarium oxysporin synthase are shown in Table 1. The adenylated domain (A domain) of each of the six modules in the synthase is designated FusA-A1 in the first module, FusA-A2 in the second module, FusA-A3 in the third module, and so on. (The remaining text appears to be from...) Figure 13 These 10 amino acid residues were identified in multiple sequence alignment of FusA from various Bacillus species strains shown.

[0080] Table 1

[0081]

[0082] In some respects, fungicidal Bacillus strains express a variant of the fusarium synthase, which contains the deletion of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all ten substrate-specific amino acid residues in FusA-A3. In other respects, fungicidal Bacillus strains express the fusarium synthase, which has the deletion of at least one amino acid residue in FusA-A3, said amino acid residue being selected from Asp235, Ala236, Ser239, Thr278, Leu299, Ala301, Ala / Gly322, Val330, Cys331, Lys517, and combinations thereof.

[0083] The deletion in FusA-A3 disclosed herein affects the ability of fusarium synthase to incorporate a specific amino acid at amino acid position (3) of the peptide ring in fusarium or fusarium-like compounds. For example, the Bacillus spp. strain NRRL B-50972 contains a deletion in FusA-A3 and cannot produce fusarium compounds with a tyrosine or phenylalanine amino acid at amino acid position (3). Without wishing to limit itself to any theory, the deletion in FusA-A3 may deviate the metabolic pathway from the biosynthesis of typical fusarium and favor the biosynthesis of fusarium-like compounds such as Paeniserine and Paeniprolixin.

[0084] In some embodiments, the present invention relates to compositions comprising biopure cultures of fungicidal Bacillus species containing a variant of a fumonisin synthase lacking a functional adenylation domain in a third module (FusA-A3), the composition further comprising at least one paeniserine and at least one paeniprolixin. In some aspects, the at least one paeniserine and at least one paeniprolixin are isolated or enriched in the composition.

[0085] In some embodiments, the isolated compound or Paeniprolixin is:

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] In some embodiments, the isolated compound or Paeniserine is:

[0092]

[0093]

[0094]

[0095] In other aspects, the present invention relates to methods for identifying fungicidal Bacillus species and / or producing corresponding fermentation products. The methods include sequencing FusA-A3 in Bacillus species to characterize variant fusarium-killing synthases, and determining the fungicidal activity of Bacillus species. In some aspects, a method based on… Figure 13Primers for the sequences shown or more (i.e., SEQ ID NO: 1-11) are used to sequence FusA-A3. In some embodiments, cells are grown prior to screening and selected for cells having one or more of the following characteristics: reduced or undetectable levels of fusarium spores (e.g., LiF03a, LiF03b, LiF03c, LiF03d, LiF07a, LiF07b, LiF07c and / or LiF07d) having tyrosine or phenylalanine at amino acid residue (3) compared to quantified fusarium spores containing wild-type fusarium spore synthase (i.e., expressing functional FusA-A3); and / or elevated levels of Paeniserine (e.g., Paeniserine A1 and / or Paeniserine B1) and / or Paeniprolixin compared to those quantified in reference Bacillus spore strains containing wild-type fusarium spore synthase (i.e., expressing functional FusA-A3).

[0096] On one hand, the present invention covers a method for producing fermentation products with broad-spectrum antifungal activity, the method comprising culturing a Bacillus genus strain with a variant of Fusarium oxysporin synthase until spore formation.

[0097] In another embodiment, the present invention relates to a method for identifying fungicidal Bacillus species with broad-spectrum antifungal activity, the method comprising: a) sequencing FusA-A3 in the Bacillus species to characterize a variant fusarium synthase; b) determining the fungicidal activity of the Bacillus species containing the variant fusarium synthase; and c) selecting the fungicidal Bacillus species as having broad-spectrum antifungal activity if the Bacillus species contains the variant fusarium synthase and shows enhanced fungicidal activity compared to a reference Bacillus species containing wild-type fusarium synthase. The method further comprises quantifying the Paeniserine and / or Paeniprolixin produced by the Bacillus species, selecting the Bacillus species as having broad-spectrum antifungal activity if the levels of Paeniserine and / or Paeniprolixin produced by the Bacillus species are elevated compared to a reference Bacillus species containing wild-type fusarium synthase. On the other hand, the method also includes culturing the fungicidal Bacillus genus strain to produce a fungicidal fermentation product.

[0098] In one embodiment, the present invention relates to a method for producing an antifungal fermentation comprising a fungicide-containing *Bacillus* strain with broad-spectrum antifungal activity, the method comprising: a) sequencing FusA-A3 in the *Bacillus* strain to characterize a variant fusarium synthase; b) determining the fungicide activity of the *Bacillus* strain containing the variant fusarium synthase; c) selecting the fungicide-containing *Bacillus* strain as having broad-spectrum antifungal activity if the *Bacillus* strain contains the variant fusarium synthase and exhibits enhanced fungicide activity compared to a reference *Bacillus* strain containing wild-type fusarium synthase; and d) culturing the fungicide-containing *Bacillus* strain to produce a fungicide fermentation product.

[0099] In some embodiments, the variant fusarium synthase in FusA-A3 contains the deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10 substrate-specific amino acid residues. In other aspects, the variant fusarium synthase in FusA-A3 contains the deletion of at least 1 amino acid residue selected from Asp235, Ala236, Ser239, Thr278, Leu299, Ala301, Ala / Gly322, Val330, Cys331, Lys517, and combinations thereof.

[0100] The present invention also covers methods for treating plants to prevent plant diseases by administering Bacillus species NRRL B-50972 or its mutants or cell-free products or their metabolites to plants or plant parts (e.g., leaves, stems, flowers, fruits, roots or seeds) or by applying to the location where the plant or plant parts grow (e.g., soil).

[0101] In the method of this invention, a composition comprising Bacillus spp. strain NRRL B-50972 or a fungicidal mutant thereof may be applied to any plant or any part of any plant growing in any type of medium used for planting (e.g., soil, vermiculite, shredded cardboard, and water), or to epiphytic plants or plant parts (e.g., orchids or staghorn ferns). The composition may be applied, for example, by spraying, atomizing, vaporizing, scattering, dusting, watering, squirting, sprinkling, pouring, or fumigating. As described above, it can be applied wherever desired, such as in agriculture, horticulture, forestry, plantations, orchards, nurseries, organic crops, turf, and urban environments.

[0102] The compositions of the present invention are obtained by culturing *Bacillus* species strain NRRL B-50972 or fungicidal mutants (strains) derived therefrom, using methods known in the art, including the use of culture media and other methods described in the examples below. Conventional large-scale microbial culture methods include submerged fermentation, solid-state fermentation, or liquid surface culture. Near the end of fermentation, as nutrients are consumed, cells begin to transition from the growth phase to the sporulation phase, so the final fermentation products are mainly spores, metabolites, and residual fermentation medium. Sporulation is a natural part of the life cycle of *Bacillus* and is typically initiated by cells in response to nutrient limitation. Fermentation is set up to obtain high levels of colony-forming units and promote sporulation. The bacterial cells, spores, and metabolites in the fermentation-produced medium can be used directly or concentrated by conventional industrial methods, such as centrifugation, tangential flow filtration, submerged filtration, and evaporation.

[0103] The compositions of the present invention include fermentation products. In some embodiments, the concentrated fermentation broth is washed (e.g., by percolation) to remove residual fermentation broth and metabolites. As used herein, the term "broth concentrate" refers to whole broth (fermentation liquid) that has been concentrated by conventional industrial methods as described above but is retained in liquid form. As used herein, the term "fermentation solids" refers to the solid material retained after the fermentation broth has been dried. As used herein, the term "fermentation product" refers to whole broth, broth concentrate, and / or fermentation solids. The compositions of the present invention include fermentation products.

[0104] Fermentation broth or broth concentrate can be dried using conventional drying processes or methods (e.g., spray drying, freeze drying, disc drying, fluidized bed drying, drum drying, or evaporation), with or without the addition of a carrier.

[0105] The resulting dried product can be further processed, for example by milling or granulation, to achieve a specific particle size or physical form. A carrier, as described below, may also be added after drying.

[0106] Cell-free products of the fermentation broth of the strains of the present invention can be obtained by any method known in the art (e.g., extraction, centrifugation, and / or filtration of the fermentation broth). Those skilled in the art will understand that a so-called cell-free product may not contain no cells, but rather be largely cell-free or substantially cell-free, depending on the technique used to remove the cells (e.g., centrifugation speed). The resulting cell-free product can be dried and / or formulated with components that facilitate its application to plants or plant growth media. The methods for concentrating and drying the fermentation broth described above are also applicable to cell-free products.

[0107] In one embodiment, the fermentation product contains at least about 1 × 10⁻⁶ 4 Colony forming unit (CFU) microorganisms (e.g., Bacillus spp. strain NRRL B-50972 or its fungicidal mutant strain) / mL fermentation broth. In another embodiment, the fermentation product contains at least about 1 × 10⁻⁶ microorganisms. 5 Colony forming unit (CFU) microorganisms (e.g., Bacillus spp. strain NRLB-50972 or its fungicidal mutant strain) / mL fermentation broth. In another embodiment, the fermentation product contains at least about 1 × 10⁻⁶ microorganisms. 6 CFU of microorganism (e.g., Bacillus spp. strain NRRL B-50972 or its fungicidal mutant strain) / mL fermentation broth. In another embodiment, the fermentation product contains at least about 1 × 10⁻⁶ microorganisms. 7 CFU of microorganism (e.g., Bacillus spp. strain NRRL B-50972 or its fungicidal mutant strain) / mL fermentation broth. In another embodiment, the fermentation product contains at least about 1 × 10⁻⁶ microorganisms. 8 CFU of microorganism (e.g., Bacillus spp. strain NRRL B-50972 or its fungicidal mutant strain) / mL fermentation broth. In another embodiment, the fermentation product contains at least about 1 × 10⁻⁶ microorganisms. 9 CFU of microorganism (e.g., Bacillus spp. strain NRRL B-50972 or its fungicidal mutant strain) / mL fermentation broth. In another embodiment, the fermentation product contains at least about 1 × 10⁻⁶ microorganisms. 10 CFU of microorganism (e.g., Bacillus spp. strain NRRL B-50972 or its fungicidal mutant strain) / mL fermentation broth. In another embodiment, the fermentation product contains at least about 1 × 10⁻⁶ microorganisms. 11 CFU microorganisms (e.g., Bacillus species NRRL B-50972 or its antifungal mutant strain) / mL fermentation broth.

[0108] The compositions of the present invention may be used in their own form or, depending on their specific physical and / or chemical properties, in their formulation form or in a form of use prepared therefrom, such as aerosols, capsule suspensions, cold atomized concentrates, hot atomized concentrates, capsule granules, fine granules, fluid concentrates for seed treatment, ready-to-use solutions, dusty powders, emulsion concentrates, oil-in-water emulsions, water-in-oil emulsions, large granules, small granules, oil-dispersible powders, oil-miscible fluid concentrates, oil-miscible liquids, gases (under pressure), gas-producing products, foaming agents, pastes, pesticide-coated seeds, suspension concentrates, oil dispersions, suspension emulsion concentrates, soluble concentrates, suspensions, wettable powders, soluble powders, powders, and granules. Granules), water-soluble and water-dispersible granules or tablets, water-soluble and water-dispersible powders for seed treatment, wettable powders, natural and synthetic products impregnated with active ingredients, microcapsules for seed polymer neutralization and coating materials, and ULV cold atomization and hot atomization formulations.

[0109] In some embodiments, the compositions of the present invention are liquid formulations. Non-limiting examples of liquid formulations include suspension concentrates and oil dispersions. In other embodiments, the compositions of the present invention are solid formulations. Non-limiting examples of liquid formulations include lyophilized powders and spray-dried powders.

[0110] The compositions of the present invention may include inert formulations added to compositions comprising cells, cell-free products, or metabolites to improve efficacy, stability, and availability and / or facilitate processing, packaging, and end-use applications. Such inert formulations and ingredients may include carriers, stabilizers, nutrients, or physical property modifiers, which may be added individually or in combination. In some embodiments, the carrier may include liquid materials such as water, oils, and other organic or inorganic solvents, and solid materials such as minerals, polymers, or polymer complexes obtained by biological methods or chemical synthesis. In some embodiments, the carrier is an adhesive or glue that promotes adhesion of the composition to plant parts (e.g., seeds or roots). See, for example, Taylor, AG, et al., "Concepts and Technologies of Selected Seed Treatments", Annu. Rev. Phytopathol. 28:321-339 (1990). Stabilizers may include anti-caking agents, antioxidants, desiccants, protective agents, or preservatives. Nutrients may include carbon, nitrogen, and phosphorus sources such as sugars, polysaccharides, oils, proteins, amino acids, fatty acids, and phosphates. Physical property modifiers may include fillers, wetting agents, thickeners, pH modifiers, rheology modifiers, dispersions, adjuvants, surfactants, antifreeze agents, and colorants. In some embodiments, water may be used as a diluent or water may not be used, allowing the direct use of compositions comprising cells, cell-free products, or metabolites produced by fermentation without any other formulations. In some embodiments, an inert formulation is added after concentrating the fermentation broth and during and / or after drying.

[0111] According to the present invention, all plants and plant parts can be processed. In the context of this invention, "plant" can be understood to mean all plants and plant populations, such as desired and unwanted wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants obtained through conventional breeding and optimization methods or through biotechnology and recombination methods, or a combination of these methods, including transgenic plants, and include plant varieties protected or not protected by plant breeders' rights. Plant parts can be understood to mean all above-ground and below-ground parts and organs of a plant, such as shoots, leaves, flowers, and roots; examples that may be mentioned include leaves, needles, culms, stems, flowers, fruiting bodies, fruits, and seeds, as well as roots, tubers, and rhizomes. Plant parts also include crop material as well as vegetative and sexual propagation material, such as cuttings, tubers, rhizomes, grafts, and seeds.

[0112] As mentioned above, all plants and their parts can be treated according to the present invention. In one preferred embodiment, wild or plant species and varieties obtained through conventional biological breeding methods (e.g., hybridization or protoplast fusion) and their parts are treated. In another preferred embodiment, transgenic plants and plant varieties (genetically modified organisms) obtained through recombinant methods (in combination with conventional methods, if appropriate) and their parts are treated. The terms "part" or "plant portion" or "plant fraction" have been explained above. Particularly preferred according to the present invention are plants that are commercially available or in use in each case. Plant variety is understood to mean a plant with novel traits, cultivated through conventional breeding, by mutagenesis, or by recombinant DNA technology. Plant varieties can be expressed in the form of varietal, race, biotype, and genotype.

[0113] Treatment of plants and plant parts using the compositions of the present invention can be carried out directly, or by applying them to the environment, habitat, or storage space using conventional treatment methods, such as dipping, spraying, atomizing, misting, evaporating, pulverizing, fogging, spreading, foaming, painting on, spreading, injecting, drenching, trickle irrigation, and in the case of propagation materials, especially seeds, by dry seed treatment, wet seed treatment, slurry treatment, by forming a shell, by coating with one or more coatings, etc. The active substance can also be applied by ultra-low volume methods, or by injecting the active substance product or the active substance itself into the soil.

[0114] A preferred direct plant treatment is foliar application, which involves applying the composition of the present invention to the leaves, allowing the treatment frequency and application rate to be matched with the infection pressure of the pathogen under discussion.

[0115] In the case of systemically active compounds, the compositions of the present invention reach the plant via the root system. In this case, the plant is treated by applying the compounds of the present invention to the plant environment. This can be done, for example, by infiltration, mixing into soil or nutrient solution, i.e., by irrigating the plant site (e.g., soil or solution culture system) with the liquid form of the compositions of the present invention, or by soil application, i.e., by incorporating the compositions of the present invention in solid form (e.g., granular form) into the plant site. In the case of rice cultures, this can also be achieved by adding the compositions of the present invention in solid application form (e.g., granular form) to the flooded rice paddy.

[0116] Preferred plants include useful plants, ornamental plants, lawns, trees commonly used as ornamental plants in public places and homes, and forestry trees. Forestry trees include trees used to produce timber, cellulose, paper, and products derived from tree parts.

[0117] In this context, “useful plants” refers to crop plants that are used to obtain food, feed, fuel, or for industrial purposes.

[0118] Useful plants that can be treated and / or improved using the compositions and methods of the present invention include, for example, the following plant types: turf, vines, cereals such as wheat, barley, rye, oats, rice, corn, and millet / sorghum; beets such as sugar beets and forage beets; fruits such as pome fruits, drupes, and berries such as apples, pears, plums, peaches, almonds, cherries, and berries such as strawberries, raspberries, and blackberries; legumes such as beans, lentils, peas, and soybeans; oil crops such as rapeseed, mustard, poppies, olives, sunflowers, coconuts, castor oil plants, cocoa, and peanuts; and gourds such as pumpkins. (pkin / squash), cucumbers 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, cabbage species, carrots, onions, tomatoes, potatoes and bell peppers; Lauraceae, such as avocados, Cinnamomum, camphor, or other plants such as tobacco, nuts, coffee, eggplant, sugarcane, tea, peppers, grapevines, hops, bananas, latex plants, and ornamental plants such as flowering plants, shrubs, deciduous trees and conifers. This list is non-limiting.

[0119] The following plants are considered to be target crops for which the compositions and methods of the present invention are particularly suitable: cotton, eggplant, turf, pome, stone fruit, berry, corn, wheat, barley, cucumber, tobacco, vines, rice, cereals, pear, beans, soybean, rapeseed, tomato, bell pepper, melon, cabbage, potato and apple.

[0120] Examples of trees that can be improved by the method of the present invention include: species of the genera *Abies*, *Eucalyptus*, *Picea*, *Pinus*, *Aesculus*, *Platanus*, *Tilia*, *Acer*, *Tsuga*, *Fraxinus*, *Sorbus*, *Betula*, *Crataegus*, *Ulmus*, *Quercus*, *Fagus*, *Salix*, and *Populus*.

[0121] Preferred trees that can be improved by the method of the present invention are: species from the genus *Aesculus*: *Aesculus hippocastanum*, *Aesculus pariflora*, and *Aesculus carnea*; species from the genus *Platanus*: *Platanus aceriflora*, *Platanus occidentalis*, and *Platanus racemosa*; species from the genus *Picea*: *Picea abies*; and species from the genus *Pinus*: *Picea radiata*, *Picea ponderosa*, *Picea contorta*, *Picea sylvestre*, and *Picea slashea*. lliottii), white pine (P. montecola), American white pine (P. albicaulis), resinous pine (P. resinosa), longleaf pine (P. palustris), stolonifera (P. taeda), flexible pine (P. flexilis), blackwood pine (P. jeffregi), North American shortleaf pine (P. baksiana), North American tall pine (P. strobus); from Eucalyptus species: giant eucalyptus (E. grandis), blue eucalyptus (E. globulus), E. camadentis, bright-fruited eucalyptus (E. nitens), obliqua eucalyptus (E. obliqua), king eucalyptus (E. regnans), pilularus eucalyptus.

[0122] The particularly preferred trees that can be improved by the method of the present invention are: trees from the genus *Pinus*: radiata pine, western yellow pine, twisted-leaf pine, European red pine, North American pine; and trees from the genus *Eucalyptus*: giant eucalyptus, blue eucalyptus, *E. camadentis*.

[0123] The most particularly preferred trees that can be improved by the method of the present invention are: horse chestnut, sycamore, linden, and maple.

[0124] This invention can also be applied to any turfgrass, including cool-season and warm-season turfgrasses. Examples of cool-season turfgrasses include *Poa* spp., such as *Poapratensis* L., *Poa trivialis* L., *Poa compressa* L., *Poa annua* L., *Poa glaucantha* Gaudin, *Poanemoralis* L., and *Poa bulbosa* L.; and *Agrostis* spp., such as *Agrostis palustris* Huds., *Agrostis tenuis* Sibth., and *Agrostis canina*. L.), mixed creeping bentgrass of southern Germany (creeping bentgrass species, including weak creeping bentgrass, hairy creeping bentgrass and creeping bentgrass), and Agrostis alba L.;

[0125] Fescue (species of the genus Festuca spp.), such as red fescue (Festuca rubra L.spp.rubra), creeping fescue (Festuca rubra L.), red fescue (Festuca rubra commutata Gaud.), fescue (Festuca ovina L.), hard fescue (Festuca longifolia Thuill.), fine-leaved fescue (Festuca capillata Lam.), tall fescue (Festuca arundinacea Schreb.), and cowtail (Festuca elanor L.);

[0126] Ryegrass (genus *Lolium* spp.), such as annual ryegrass (*Lolium multiflorum* Lam.), perennial ryegrass (*Lolium perenne* L.), and Italian ryegrass (*Lolium multiflorum* Lam.);

[0127] And also Agropyron species (Agropyron spp.), such as Agropyron cristatum (L.) Gaertn., Agropyron desertorum (Fisch.) Schult., and Agropyron smithii Rydb.

[0128] Other examples of cool-season turfgrasses include: Ammophila breviligulata Fern., Bromus inermis Leyss., Phleum pratense L., Phleum subulatum L., Dactylis glomerata L., Puccinellia distans (L.) Pari., and Cynosurus cristatus L.

[0129] Examples of warm-season turfgrasses include: Cynodon spp. L. Crich, Zoysia spp. Willd., Stenotaphrum secundatum Walt Kuntze, Eremochloa ophiuroides Munro Hack., Axonopus affinis Chase, Paspalum notatum Flugge, Pennisetum clandestinum Hochst. ex Chiov., Buchloe dactyloids (Nutt.) Engelm., Boutelouagracilis (HBK) Lag. ex Griffiths, Paspalum vaginatum Swartz, and sideoats grama (Bouteloua curtipendula (Michx. Torr.). Cool-season turfgrasses are generally preferred for use according to the invention. Kentucky bluegrass, creeping bentgrass, and sedge, fescue, and ryegrass are particularly preferred. Creeping bentgrass is especially preferred.

[0130] The compositions of the present invention have effective antimicrobial activity and can be used in crop protection and material protection to control unwanted microorganisms, such as fungi and bacteria.

[0131] The present invention also relates to a method for controlling unwanted microorganisms, characterized in that the composition of the present invention is applied to plant pathogenic fungi, plant pathogenic bacteria and / or their habitats.

[0132] Fungicides are used in crop protection to control plant pathogenic fungi. They are characterized by significant efficacy against a broad spectrum of plant pathogens, including soil-borne pathogens, particularly members of the classes Plasmodiophoromycetes, Peronosporomycetes, Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes. Some fungicides possess systemic activity and can be used as foliar, seed dressing, or soil fungicides for plant protection. Furthermore, they are suitable for controlling fungi, especially those that infest the wood or roots of plants.

[0133] Bactericides can be used in crop protection to control Pseudomonadaceae, Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceae, and Streptomycetaceae.

[0134] Non-limiting examples of pathogens of fungal diseases that can be treated according to the present invention include:

[0135] Diseases caused by powdery mildew pathogens, such as species of the genus *Blumeria*, for example, *Blumeria graminis* (Grass family); species of the genus *Podosphaera*, for example, *Podosphaera leucotricha* (apple); species of the genus *Sphaerotheca*, for example, *Sphaerotheca fuliginea* (cucumber); and species of the genus *Uncinula*, for example, *Uncinulanecator* (grape powdery mildew).

[0136] Diseases caused by rust pathogens, such as species of the genus *Gymnosporangium*, for example, *Gymnosporangium sabinae*; species of the genus *Hemileia*, for example, *Hemileia vastatrix*; species of the genus *Phakopsora*, for example, *Phakopsora pachyrhizi* and *Phakopsora meibomiae*; species of the genus *Puccinia*, for example, *Puccinia recondite*, *P. triticina*, *P. graminis*, or *P. striiformis*; and species of the genus *Uromyces*, for example, *Uromyces appendiculatus*.

[0137] Diseases caused by pathogens belonging to the class Oomycetes, such as species of the genus *Albugo*, for example, *Albugo candida*; species of the genus *Bremia*, for example, *Bremialactcae*; species of the genus *Peronospora*, for example, *Peronospora pisi* or *P. brassicae*; species of the genus *Phytophthora*, for example, *Phytophthorainfestans*; species of the genus *Plasmopara*, for example, *Plasmopara viticola*; and species of the genus *Pseudoperonospora*, for example, *Pseudoperonospora humuli* or *Pseudoperonospora*. cubensis); species of the genus Pythium, such as Pythium ultimum;

[0138] Leaf spot and leaf wilt diseases can be caused by pathogens such as: *Alternaria* species, such as *Alternaria solani*; *Cercospora* species, such as *Cercospora beticola*; *Cladiosporium* species, such as *Cladiosporium cucumerinum*; *Cochliobolus* species, such as *Cochliobolus sativus* (conidia: *Drechslera*, synonym: *Helminthosporium*) and *Cochliobolus miyabeanus*; and *Colletotrichum* species, such as *Colletotrichum beanus*. Species of the genera *Leptosphaeria* (e.g., *Cycloconium oleaginum*); species of the genus *Diaporthe* (e.g., *Diaporthecitri*, the causal agent of citrus brown stem rot); species of the genus *Elsinoe* (e.g., *Elsinoe fawcettii*, the causal agent of scab); species of the genus *Gloeosporium* (e.g., *Gloeosporium laeticolor*, the causal agent of peach tree rot); species of the genus *Glomerella* (e.g., *Glomerella cingulata*, the causal agent of anthracnose); species of the genus *Guignardia* (e.g., *Guignardia bidwelli*); and species of the genus *Leptosphaeria* (e.g., *Leptosphaeria maculans*, a member of the Brassicaceae family) and *Leptosphaeria maculans*. 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, Mycosphaerella arachidicola, and Mycosphaerella fijiensis.*Phaeosphaeria* species, such as *Phaeosphaerianodorum*, the causal agent of wheat leaf blight; *Pyrenophora* species, such as *Pyrenophora teres* and *Pyrenophora tritici repentis*; *Ramularia* species, such as *Ramularia collo-cygni* and *Ramularia areola*, the causal agent of leaf spot; *Rhynchosporium* species, such as *Rhynchosporium secalis*, the causal agent of barley cloudiness; *Septoria* species, such as *Septoria apii* and *Septoria lycopersii*; *Typhula* species, such as *Typhula sarcosporium*. Incarnata); species of the genus Venturia, such as *Venturia inequalis*.

[0139] Root and stem diseases caused by pathogens such as: 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 diseases caused by Sarocladium oryzae; Sclerotium diseases caused by Sclerotium oryzae; Tapesia species, such as Tapesia acuformis; Thieviopsis species, such as Thieviopsis basicola.

[0140] Diseases of the spadix and panicle (including the corn cob) caused by pathogens such as: *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 nigricans*. nivalis); species of the genus Septoria, such as Septoria nodorum;

[0141] Diseases caused by smut fungi, such as species of the genus *Sphacelotheca*, for example, *Sphacelotheca reiliana*; species of the genus *Tilletia*, for example, *Tilletia caries* and *T. controversa*; species of the genus *Urocystis*, for example, *Urocystis occulta*; and species of the genus *Ustilago*, for example, *Ustilago nuda* and *U. nuda tritici*.

[0142] Fruit rot can be caused by pathogens such as: Aspergillus species, such as Aspergillus flavus; Botrytis species, such as Botrytis cinerea; Penicillium species, such as Penicillium expansum and Penicillium purpurogenum; Sclerotinia species, such as Sclerotinias clerotiorum; and Verticilium species, such as Verticilium alboatrum.

[0143] Species-borne and soil-borne diseases of rot, mold, wilting, decay, and damping-off caused by pathogens such as: *Alternaria* species, for example, *Alternaria brassicicola*; *Aphanomyces* species, for example, *Aphanomyces euteiches*; *Ascochyta* species, for example, *Ascochyta lentis*; *Aspergillus* species, for example, *Aspergillus flavus*; *Cladosporium* species, for example, *Cladosporium herbarum*; and *Cochliobolus* species, for example, *Cochliobolus gracilis*. Caused by *Sativus*; (Conidial forms: *Drechslera*, *Bipolaris*, synonym: *Helminthosporium*)); *Colletotrichum* species, such as those caused by *Colletotrichum coccodes*; *Fusarium* species, such as those caused by *Fusarium culmorum*; *Gibberella* species, such as those caused by *Gibberella zeae*; *Macrophomina* species, such as those caused by *Macrophomina phaseolina*; *Monographella* species, such as those caused by *Monographella nivalis*; *Penicillium* species, such as those caused by *Penicillium expansum*. Caused by: *Phoma* species, such as *Phoma lingam*; *Phomopsis* species, such as *Phomopsis sojae*; *Phytophthora* species, such as *Phytophthora cactorum*; *Pyrenophora* species, such as *Pyrenophora graminea*; *Pyricularia* species, such as *Pyricularia oryzae*.Species of the genus *Pythium*, such as *Pythium ultimum*; species of the genus *Rhizoctonia*, such as *Rhizoctonia solani*; species of the genus *Rhizopus*, such as *Rhizopus oryzae*; species of the genus *Sclerotium*, such as *Sclerotium rolfsii*; species of the genus *Septoria*, such as *Septoria nodorum*; species of the genus *Typhula*, such as *Typhula incarnata*; species of the genus *Verticillium*, such as *Verticillium dahliae*.

[0144] Cancer, gall, and witches' broom diseases caused by pathogens such as the following: species of the genus *Nectria*, such as *Nectria galligena*.

[0145] Wilting diseases caused by pathogens such as the following: species of the genus *Monilinia*, such as *Monilinia laxa*;

[0146] Leaf blister or leaf curl diseases caused by pathogens such as the following species of the genus Exobasidium, such as Exobasidium vexans;

[0147] Species of the genus *Taphrina*, such as *Taphrina deformans*;

[0148] Degenerative diseases of woody plants: Esca caused by fungi such as *Phaemoniella clamydospora*, *Phaeoacremonium aleophilium*, and *Fomitiporia mediterranea*; grape vine rot caused by fungi such as *Eutypa lata*; Ganoderma diseases caused by fungi such as *Ganoderma boninense*; and Rigidoporus diseases caused by fungi such as *Rigidoporus lignosus*.

[0149] Diseases of flowers and seeds caused by pathogens such as those in the genus *Botrytis*, such as *Botrytis cinerea*.

[0150] Plant tuber diseases caused by pathogens such as: Rhizoctonia species, such as Rhizoctonia solani; Helminthosporium species, such as Helminthosporium solani.

[0151] Clubroot disease is caused by pathogens such as Plamodiophora species, for example Plamodiophora brassicae.

[0152] Diseases caused by bacterial pathogens, such as species of Xanthomonas, for example Xanthomonas campestris pv. oryzae; species of Pseudomonas, for example Pseudomonas syringaepv. lachrymans; and species of Erwinia, for example Erwinia amylovora.

[0153] Preferably, it can prevent and control the following diseases of soybeans:

[0154] Fungal diseases affecting leaves, stems, pods, and seeds, caused by pathogens such as Alternaria speciosum leaf spot (Alternaria speciosum atrans tenuissima), anthracnose (Colletotrichum gloeosporoides dematium var. truncatum), brown spot (Septoria glycines), Cercospora leaf spot and blight (Cercospora kikuchii), Choanephora leaf blight (Choanephora infundibulifera trispora), Dactuliophora leaf spot (Dactuliophoraglycines), downy mildew (Peronospora manshurica), and Drechslera wilt. Glycini, Frog-eye leaf spot (Cercospora sojina), Leptosphaerulina leaf spot (Leptosphaerulina trifolii), Phyllostica leaf spot (Phyllosticta sojaecola), Pod and stem wilt (Phomopsis sojae); Powdery mildew (Microsphaeradiffusa), Pyrenochaeta glycines, Rhizoctonia solani (Rhizoctonia solani), Rust (Phakopsora pachyrhizi, Phakopora pachyrhizi). Meibomiae), scab (Sphacelomaglycines), leaf blight (Stemphylium botryosum), target spot (Corynespora cassiicola).

[0155] Fungal diseases affecting the roots and stem base caused by pathogens such as Calonectria crotalariae, anthracnose (Macrophomina phaseolina), Fusarium wilt or wilting, root rot, and pod and root collar rot (Fusarium oxysporum, Fusarium orthoceras, Fusarium semitectum, Fusarium equiseti), Mycoleptodiscus terrestris, Neocosmospora (Neocosmopspora vasinfecta), pod and stem blight (Diaporthe phaseolorum), and stem canker (Diaporthe...). Phytophthora var. caulivora), Phytophthora megasperma, Phyalophora gregata, Pythium aphanidermatum, Pythium irregulare, Pythium debaryanum, Pythium myriotylum, Pythium ultimum, Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotinia rolfsii, and Thieviopsis basicola.

[0156] The compositions of the present invention can be used for therapeutic or protective / preventive control of plant pathogenic fungi. Therefore, the present invention also relates to therapeutic and protective methods for controlling plant pathogenic fungi by using the compositions of the present invention, wherein the compositions of the present invention are applied to seeds, plants or plant parts, fruits or soil in which plants grow.

[0157] The composition is well tolerated by plants at the concentrations required for the control of plant diseases, which enables it to be used on the above-ground parts of plants, propagated rhizomes and seeds, as well as the soil.

[0158] The present invention can process all plants and plant parts, including cultivars and plant varieties (whether or not protected by plant variety or plant breeder rights). Cultivars and plant varieties can be plants obtained through conventional propagation and breeding methods, which can be aided or supplemented by one or more biotechnological methods, such as the use of double haploids, protoplast fusion, random and directed mutagenesis, molecular or genetic markers, or through bioengineering and genetic engineering methods.

[0159] In some aspects, the compositions of the present invention are at a concentration of about 1 × 10⁻⁶ per hectare. 8 To approximately 1×10 14 The fungicidal Bacillus spp. strain NRRL B-50972 or a fungicidal mutant strain thereof, with colony-forming units (CFU), are applied. In other aspects, the compositions of the present invention are applied at approximately 1 × 10⁻⁶ per hectare. 9 To approximately 1×10 13 The fungicidal Bacillus spp. strain NRRL B-50972 or a fungicidal mutant strain thereof, with colony-forming units (CFU), are applied. In other aspects, the compositions of the present invention are applied at approximately 1 × 10⁻⁶ per hectare. 10 To approximately 1×10 12 The fungicidal Bacillus genus strain NRRLB-50972 or its fungicidal mutant strain, containing colony forming units (CFU), is applied.

[0160] In some embodiments, the composition of the present invention is applied at a rate of about 0.1 kg to about 10 kg of fermentation solids per hectare. In other embodiments, the composition of the present invention is applied at a rate of about 0.25 kg to about 7.5 kg of fermentation solids per hectare. In other embodiments, the composition of the present invention is applied at a rate of about 0.5 kg to about 5 kg of fermentation solids per hectare. The composition of the present invention can also be applied at a rate of about 1 kg to about 2 kg of fermentation solids per hectare.

[0161] When well tolerated by plants, the compositions of the present invention exhibit favorable homeothermic animal toxicity and are well tolerated by the environment, making them suitable for protecting plants and plant organs to increase yield and improve the quality of harvested material. They are preferably used as crop protection compositions. They are active against generally sensitive and resistant species and against all or some developmental stages.

[0162] Plants that can be processed according to this invention include the following major crops: corn, soybean, alfalfa, cotton, sunflower, Brassica oilseeds (e.g., European rapeseed (e.g., canola), brassica rapa, mustard (e.g., field mustard) and Ethiopian mustard (Brassica carinata)), Arecaceae species (e.g., oil palm, coconut), rice, wheat, sugar beets, sugarcane, oats, rye, barley, millet and sorghum, triticale, flax, nuts, grapes and vines, as well as various fruits and vegetables from various plant taxonomic units, such as Rosaceae species (e.g., pome fruits (e.g., apples and pears), drupe fruits (e.g., apricots, cherries, almonds, plums and peaches), and berry fruits (e.g., strawberries, raspberries, red currants and black currants and round currants)), Ribesioidae species. Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp. (e.g., olive), Actinidaceae sp., Lauraceae sp. (e.g., avocado, cinnamon, camphor), Musaceae sp. (e.g., banana trees and banana plantations), Rubiaceae sp. (e.g., coffee), Theaceae sp. (e.g., tea), Sterculiaceae sp., Rutaceae sp. (e.g., lemon, orange, tangerine, and grapefruit); Solanaceae sp. (e.g., tomato, potato, pepper, chili, eggplant, tobacco), Liliaceae sp., Compositae sp. (e.g., lettuce, artichoke, and chicory - including root chicory, chicory, or common chicory), Umbelliferae sp. (e.g., carrot, parsley, celery, and root celery), Cucurbitaceae sp. (e.g., cucumber - including pickled cucumbers, squash, watermelon, gourd, and cantaloupe), Alliaceae sp. (e.g., leek, onion), and Cruciferae sp.(e.g., white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, bok choy, kohlrabi, radish, wasabi, watercress, and Chinese cabbage); Leguminosae sp. (e.g., peanuts, peas, lentils, and beans – such as sword beans and broad beans); Chenopodiaceae sp. (e.g., romaine lettuce, forage beets, spinach, beetroot); Linaceae sp. (e.g., hemp); Cannabeacea sp. (e.g., Indian hemp); Malvaceae sp. (e.g., okra, cocoa); Papaveraceae (e.g., poppy); Asparagaceae (e.g., asparagus); useful and ornamental plants in horticulture and forestry, including meadows, lawns, pastures, and stevia (Steviarebaudiana); and in each case, genotypes of these plants.

[0163] In some aspects, the fermentation product also includes formulation components. These formulation components may be wetting agents, fillers, solvents, spontaneous growth promoters, emulsifiers, dispersions, antifreeze agents, 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.

[0164] The compositions of the present invention may include formulation ingredients that are added to the compositions of the present invention to improve recovery, efficacy, or physical properties and / or aid in processing, packaging, and administration. Such formulation ingredients may be added alone or in combination.

[0165] The formulation ingredient may be added to compositions comprising cells, cell-free products, isolated compounds and / or metabolites to improve efficacy, stability, physical properties, usability and / or facilitate processing, packaging and end-use applications. Such formulation ingredients may include agriculturally acceptable carriers, inert substances, stabilizers, preservatives, nutrients or physical property modifiers, which may be added alone or in combination. In some embodiments, the carrier may include liquid materials such as water, oil and other organic or inorganic solvents, and solid materials such as minerals, polymers or polymer complexes obtained by biological methods or chemical synthesis. In some embodiments, the formulation ingredient is an adhesive, adjuvant or glue that promotes adhesion of the composition to plant parts (e.g., leaves, seeds or roots). See, for example, Taylor, AG, et al., "Concepts and Technologies of Selected Seed Treatments", Annu. Rev. Phytopathol. 28:321-339 (1990). Stabilizers may include anti-caking agents, antioxidants, anti-settling agents, defoamers, desiccants, protective agents or preservatives. Nutrients may include carbon, nitrogen, and phosphorus sources, such as sugars, polysaccharides, oils, proteins, amino acids, fatty acids, and phosphates. Physical property modifiers may include fillers, wetting agents, thickeners, pH modifiers, rheology modifiers, dispersions, adjuvants, surfactants, film-forming agents, water-soluble agents, builders, antifreeze agents, and colorants. In some embodiments, water may be used as a diluent or not, allowing direct use of compositions comprising cells, cell-free products, or metabolites produced by fermentation without any other formulation. In a particular embodiment, a wetting agent or dispersion is added to the fermentation solid, such as a lyophilized or spray-dried powder. When applied to a surface, the wetting agent increases the diffusion and permeation properties of the active ingredient, or the dispersion increases the dispersibility and solubility of the active ingredient (once diluted). Exemplary wetting agents are known to those skilled in the art and include sulfosuccinates and their derivatives, such as MULTIWET. TM MO-70R (Croda Inc., Edison, NJ); siloxanes, for example (Evonik, Germany); nonionic compounds, such as ATLOX TM 4894 (Croda Inc., Edison, NJ); Alkyl polyglucan, for example 3001 (Huntsman International LLC, The Woodlands, Texas); C12-C14 alcohol ethoxylates, for example 15-S-15 (The Dow Chemical Company, Midland, Michigan); phosphate ester, for example BG-510 (Rhodia, Inc.); and alkyl ether carboxylic esters, such as EMULSOGEN. TM LS (Clariant Corporation, North Carolina).

[0166] Preservation Information

[0167] Samples of the *Bacillus* genus strain of the present invention were deposited on August 28, 2014, in accordance with the Budapest Treaty, at the Agricultural Research Service Culture Collection, located at 1815 North University Street, Peoria, 61604, USA, National Center for Agricultural Applied Research (NRRL), Agricultural Research Service, USDA, NRRL 61604, and assigned the following accession number: NRRL B-50972.

[0168] A sample of a Bacillus species, NRRL B-50972, exhibiting stable colony morphology, was deposited on September 1, 2015, under the Budapest Treaty, at the Agricultural Research Institute Culture Collection, 1815 North University Street, Peoria, Île-de-France, USDA, National Center for Agricultural Applied Research (NRRL), 61604, USA, and assigned the following accession number: NRRL B-67129.

[0169] The *Bacillus* species strain is preserved under conditions that ensure, during the examination of this patent application, that a qualified person, as determined by the Patent and Trademark Commissioner pursuant to 37 C. FR § 1.14 and 35 U. SC § 122, can obtain the culture. However, it should be understood that obtaining the deposit does not permit the practice of the invention in violation of the patent rights granted by the government.

[0170] The embodiments given below are for illustrative and non-limiting purposes only.

[0171] Example

[0172] Example 1. Selection of Bacillus species NRRL B-50972

[0173] The genomes of several Bacillus species were sequenced. Genomic data were analyzed to identify strains possessing the fusarium biosynthesis gene cluster but lacking the polymyxin synthase gene cluster. Similar to the polymyxin synthase gene cluster, the gene cluster responsible for fusarium biosynthesis (fusA) had previously been identified and characterized. See, for example, Li et al., "Nonnbosomal Biosynthesis of Fusaricidins by Paenibacillus polymyxa PKB lInvolves Direct Activation of a D-Amino Acid," Chemistry & Biology, 15:1 18-127 (2008); Li et al., "Promoter Analysis and Transcription Regulation of fus GeneCluster Responsible for Fusaricidin Synthesis of Paenibacillus polymyxa SQR-21," Applied Microbiol Biotechnol, 97:9479-9489 (2013); and Choi et al, "Identification of a Polymyxin Synthetase Gene Cluster of Paenibacilluspolymyxa and Heterologous Expression of the Gene in Bacillus subtilis" Journalof Bacteriology, 191(10):3350-3358(2009).

[0174] The strains identified using this analysis were further evaluated to confirm the production of fusarium oxysporine. In short, each strain was cultured in a soybean-based medium, and the lipophilic fraction of the whole culture was extracted. The whole culture extract was analyzed by high-performance liquid chromatography (HPLC), and the presence of fusarium oxysporine A was identified based on the HPLC chromatograms generated using a standard sample containing fusarium oxysporine A.

[0175] Example 2. Intraphytic antifungal activity of whole culture broth of Bacillus species

[0176] Selected Bacillus species (including Bacillus species NRRL B-50972) were grown in soybean-based media to produce complete broth cultures. Distilled water was added to each complete broth to achieve a final dilution of 10%.

[0177] Diluted whole culture solutions were applied to the leaves of young plants, which were then exposed to fungal inoculums of Phytin (Tomato Late Wilt), Botrytis cinerea (Botrytis cinerea), or Puccort (Wheat Leaf Rust). An untreated control was included in each assay for comparison. Several days after exposure to the fungal inoculum, the percentage of pathogen control for each plant relative to the untreated control was scored. Each treatment was evaluated three times, and the mean percentage of control from the whole culture solution for each Bacillus species was calculated as follows: Figure 1 As shown.

[0178] Among the 23 strains tested for antifungal activity against PHYTIN, BOTRCI, and PUCCRT, Bacillus spp. strain NRRL B-50972 was one of the strains that exhibited relatively high levels of activity against all three fungal pathogens.

[0179] Example 3. In vitro bioefficacy of the fusarium extract from Bacillus subtilis strain NRRL B-50972.

[0180] Whole-culture media of several *Bacillus* species (including *Bacillus* strain NRRL B-50972) were prepared using soybean-based media. Lipophilic fractions containing fusarium spores were extracted from the whole cultures. Three independent fractions (fractions 1, 2, and 3) containing various fusarium spores and antifungal metabolites were prepared from the whole-culture extracts of the first-order *Bacillus* species. The extract from *Bacillus* strain NRRL B-50972 was not further isolated.

[0181] Fractions containing fusarium oxysporins from each of the following 12 fungal pathogens were tested: *Alternaria alternata*, *Botrytis cinerea*, *Fusarium oxysporum*, *Syngonium leptonis*, *Sepptr*, *Phytophthora cryptoges*, *Phytophthora virosa*, *Pythulium oxysporum*, *Pyrior oryzae*, *Rhizoctonia solani*, *USTIAV*, and *Uromapora spp.* var. *oatella*. The inhibitory effects of different fractions on fungal cell growth were evaluated in soybean-based media and compared with the growth of untreated controls. Eight doses of each fraction, ranging from 0.005 ppm to 100 ppm, were tested. A 50% inhibition (ED) was achieved. 50 ) and 80% inhibition (ED) 80 The effective dose of ) is recorded in Figure 2 In the table.

[0182] The fraction of *Bacillus* strain NRRL B-50972 containing fumonisin showed broad-spectrum antifungal activity in 12 assays, which was not observed when fractions from other *Bacillus* strains were used. In the assays, the *Bacillus* strain NRRL B-50972 fraction also showed significantly higher activity than observed when fractions from other *Bacillus* strains were used (see [link to assay]). Figure 2 ).

[0183] Example 4. In vivo prophylactic test on tomatoes infected with Phytophthora.

[0184] In this plant pathogen greenhouse assay, the fermentation product of the Bacillus spp. strain NRRL B-50972 was tested and compared with three other Bacillus spp. strains (which had shown relatively high antifungal activity in previous screening assays). To produce a suitable compound product, 1 part by weight of spray-dried powder from the complete culture broth of each strain (cultured on a soybean-based medium) was mixed with water and 0.1 part by weight of emulsifier (alkylaryl polyethylene glycol ether), and then diluted with water to the desired concentration.

[0185] To test preventative activity, young plants were sprayed with the compound at a specified application rate. After the spray coating dried, the plants were inoculated with an aqueous spore suspension of Phytophthora infestans. The plants were then placed in an incubator at approximately 20°C and 100% relative humidity.

[0186] Evaluation was conducted 3 days after inoculation. 0% indicates efficacy corresponding to the untreated control, while 100% efficacy indicates no disease was observed.

[0187] Table 2: In vivo prophylactic test against Phytophthora (tomato)

[0188]

[0189] Example 5. In vivo prophylactic test on grapevines infected with *Monoaxillium*.

[0190] In this plant pathogen greenhouse assay, the fermentation product of the Bacillus spp. strain NRRL B-50972 was tested and compared with three other Bacillus spp. strains (which had shown relatively high antifungal activity in previous screening assays). To produce a suitable compound product, 1 part by weight of the spray-dried powder prepared as described in Example 5 was mixed with water and 0.1 part by weight of an emulsifier (alkylaryl polyethylene glycol ether), and then diluted with water to the desired concentration.

[0191] To test preventative activity, young plants were sprayed with the compound at a specified application rate. After the spray coating dried, the plants were inoculated with an aqueous spore suspension of *Botrytis cinerea*, and then placed in an incubator at approximately 20°C and 100% relative humidity for one day. The plants were then placed in a greenhouse at approximately 21°C and approximately 90% relative humidity for four days. Finally, the plants were covered with a light mist and placed in an incubator for one day.

[0192] Evaluation was conducted 6 days post-inoculation. 0% indicates efficacy corresponding to the untreated control, while 100% efficacy indicates no disease was observed.

[0193] Table 3: In vivo prophylactic test against *Monoaxillary* species (grapevine)

[0194]

[0195] Example 6. In vivo prophylactic test on legumes infected with Uromyces spp.

[0196] In this plant pathogen greenhouse assay, the fermentation product of the Bacillus spp. strain NRRL B-50972 was tested and compared with three other Bacillus spp. strains (which had shown relatively high antifungal activity in previous screening assays). To produce a suitable compound product, 1 part by weight of the spray-dried powder prepared as described in Example 5 was mixed with water and 0.1 part by weight of an emulsifier (alkylaryl polyethylene glycol ether), and then diluted with water to the desired concentration.

[0197] To test preventative activity, young plants were sprayed with the compound at a specified application rate. After the spray coating dried, the plants were inoculated with an aqueous spore suspension of the bean rust pathogen (Aureobasidium verruciformis), and then the plants were kept in an incubator at approximately 20°C and 100% relative humidity for 1 day.

[0198] The plants were then placed in a greenhouse at approximately 21°C and a relative humidity of approximately 90%.

[0199] Evaluation was conducted 10 days post-inoculation. 0% indicates efficacy corresponding to the untreated control, while 100% efficacy indicates no disease was observed.

[0200] Table 4: In vivo prophylactic test against monosporous rust fungi (legumes)

[0201]

[0202] Example 7. Comparison of Bacillus strains in a field trial of zucchini infected with powdery mildew (Cucumber powdery mildew).

[0203] Two field trials were conducted on zucchini plants artificially inoculated with cucumber powdery mildew. Five treatments of spray-dried powder, consisting of complete cultures of each Bacillus species (cultured on soybean-based media), were resuspended in water at an application volume of 1000 L / ha and applied to the plants between July 15th and August 8th, during the growth period of BBCH59 to BBCH72, at intervals of 4 to 8 days as shown in Table 6. The percentages of disease control shown in Table 5 are the results of a final assessment conducted 10 days after the final application, performed by visual observation of disease symptoms. 0% indicates efficacy corresponding to the untreated control, while 100% efficacy indicates no disease observed.

[0204] Table 5

[0205]

[0206]

[0207] Table 6

[0208] A July 15 59 B July 23 65 C July 30 71 D August 4 72 E August 8 72

[0209] The results shown in Table 4 clearly demonstrate that the observed Bacillus species strain NRRL B-50972 exhibited higher activity than other strains tested in this field trial, which had previously shown relatively high antifungal activity in screening assays.

[0210] Example 8. Comparison of Bacillus strains in field trials on grapevines infected with powdery mildew (grape powdery mildew).

[0211] Two field trials were conducted using grapevines naturally infected with grape powdery mildew. Six treatments using the spray-dried powder described in Example 8 were resuspended in water at an application volume of 1000 L / ha and applied to the plants between June 3rd and July 1st, during the growing season from BBCH57 to BBCH75, at intervals of 5 to 7 days as shown in Table 8. The percentages of disease control shown in Table 7 are the results of a final assessment conducted 15 days after the final application, performed by visual observation of disease symptoms. 0% indicates efficacy corresponding to the untreated control, while 100% efficacy indicates no disease observed.

[0212] Table 7

[0213]

[0214] Table 8

[0215] A June 3 57 B June 10 60 C June 16 64 D June 21 71 E June 26 73 F July 1 75

[0216] The results in Table 7 clearly demonstrate that the observed Bacillus species strain NRRL B-50972 exhibited higher activity than other strains tested in this field trial, which had previously shown relatively high antifungal activity in screening assays.

[0217] Example 9. Comparison of Bacillus strains in a field trial of tomatoes infected with early wilt (Alternaria solanacea).

[0218] Two field trials were conducted using tomato plants artificially inoculated with *Alternaria solanacea*. Three treatments using the spray-dried powder described in Example 8 were resuspended in water at an application volume of 1000 L / ha and applied to the plants between June 26 and July 10, during the growth periods of BBCH51 to BBCH59, at intervals of 6 to 8 days as shown in Table 10. The disease control percentages shown in Table 9 are the results of a final evaluation conducted 8 days after the final application, performed by visual observation of disease symptoms. 0% indicates efficacy corresponding to the untreated control, while 100% efficacy indicates no disease observed.

[0219] Table 9

[0220]

[0221] Table 10

[0222] A June 26 51 B July 2 53 C July 10 59

[0223] The results in Table 9 clearly demonstrate that the observed Bacillus species strain NRRL B-50972 exhibited higher activity than other strains tested in this field trial, which had previously shown relatively high antifungal activity in screening assays.

[0224] Example 10. Comparison of Bacillus strains in a potato field trial infected with early wilt (Alternaria solanacea).

[0225] Field trials were conducted using potato plants artificially inoculated with *Alternaria solanacea*. Five treatments using the spray-dried powder described in Example 8 were resuspended in water at an application volume of 500 L / ha and applied to the plants between June 26 and July 19, during the growth period of BBCH37 to BBCH55, at intervals of 4 to 8 days as shown in Table 12. The percentages of disease control shown in Table 11 are the results of a final assessment conducted 6 days after the final application, performed by visual observation of disease symptoms. 0% indicates efficacy corresponding to the untreated control, while 100% efficacy indicates no disease observed.

[0226] Table 11

[0227]

[0228] Table 12

[0229] A June 26 37 B July 2 47 C July 10 51 D July 15 55 E July 19 55

[0230] The results in Table 11 clearly demonstrate that the observed Bacillus species strain NRRL B-50972 exhibited higher activity than other strains tested in this field trial, which had previously shown relatively high antifungal activity in screening assays.

[0231] Example 11. Comparison of Bacillus strains in a potato field trial infected with early wilt (Alternaria solanacea).

[0232] Field trials were conducted using potato plants artificially inoculated with *Alternaria solanacea*. Three treatments using the spray-dried powder described in Example 8 were resuspended in water at an application volume of 500 L / ha and applied to the plants between July 24th and August 5th, during the growth periods of BBCH37 to BBCH51, at 6-day intervals as shown in Table 14. The disease control percentages shown in Table 13 are the results of a final assessment conducted 6 days after the final application, performed by visual observation of disease symptoms. 0% indicates efficacy corresponding to the untreated control, while 100% efficacy indicates no disease observed.

[0233] Table 13

[0234]

[0235] Table 14

[0236] A July 24 37 B July 30 40 C August 5 51

[0237] The results in Table 13 clearly demonstrate that the observed Bacillus species strain NRRL B-50972 exhibited higher activity than other strains tested in this field trial, which had previously shown relatively high antifungal activity in screening assays.

[0238] Example 12. Identification of the fusA variant in Bacillus subtilis strain NRRL B-50972

[0239] To further characterize the *Bacillus* strain NRRL B-50972, the genomic sequence of the FusA gene encoding the fusarium oxysporin synthase FusA was determined using standard sequencing methods, and the relevant amino acid sequence was identified. The amino acid sequence of FusA expressed by *Bacillus* strain NRRL B-50972 was compared with the amino acid sequences of several other *Bacillus* strains (including those described in the following publications):

[0240] Li S., et al, (2014). "Complete Genome Sequence of Paenibacillus polymyxa SQR-21, a Plant Growth-Promoting Rhizobacterium with Antifungal Activity and Rhizosphere Colonization Ability," Genome Announc, 2(2):HASH(0x743db288);

[0241] Niu B., et al, (2011). "The Genome of the Plant Growth-Promoting Rhizobacterium Paenibacillus polymyxa M-1 Contains Nine Sites Dedicated to Nonribosomal Synthesis of Lipopeptides and Polyketides," J. Bacteriol. 193(20): 5862-3;

[0242] Ma M., et al., (2011) "Complete Genome Sequence of Paenibacillus polymyxa SC2, A Strain of Plant Growth-Promoting Rhizobacterium with Broad-Spectrum Antimicrobial Activity," J. Bacteriol. 193(1): 311-2; and

[0243] Li and Jensen, (2008). Nonribosomal Biosynthesis of Fusaricidins by Paenibacillus polymyxa PKB1 Involves Direct Activation of a d-amino Acid. Chem. Biol. 15, 118-127.

[0244] Figure 13The comparison shown reveals significant deletions in the FusA fusarium synthase variant expressed by Bacillus subtilis species NRRL B-50972. The first deletion extends from position 3009 to position 3037 of the corresponding sequence (SEQ ID NO: 11) in Bacillus subtilis strain A. The second deletion extends from position 3047 to position 3317 of the corresponding sequence (SEQ ID NO: 11) in Bacillus subtilis strain A. Both deletions fall within the A domain (i.e., FusA-A3) of the third module of the FusA fusarium synthase.

[0245] As explained above, each A domain contains 10 conserved amino acid residues responsible for substrate recognition and activation (see Table 1). These conserved amino acid residues... Figure 13 The comparison shown is described. The deletion identified in the variant FusA fusarium synthase expressed by the Bacillus species NRLB-50972 removes all amino acid residues except the last conserved amino acid residue (i.e., Lys517 at position 3486 of SEQ ID NO:11).

[0246] These two deletions in the FusA fusarium synthase variant are present in strains derived from the Bacillus species NRRL B-50972, including a variant strain with a stable colony morphology (designated in this paper as Bacillus species NRRL B-67129). Random mutant strains derived from Bacillus species NRRL B-50972 will typically retain the deletions in the FusA-A3 variant because, due to the extensive nature of these deletions, reversal to wild-type FusA-A3 is extremely unlikely.

[0247] Example 13. Comparison of Fusarium-killing production in Bacillus subtilis strain NRRL B-50972 and Bacillus subtilis strain A

[0248] To determine the role of variant FusA-A3, a group of fusarin and paeniserine were quantified in Bacillus subtilis strain NRRL B-50972 (expressing variant FusA-A3) and Bacillus subtilis strain A (expressing wild-type FusA-A3) using the method described in Example 14. The identity of each compound was determined by its unique retention time and mass. The relative signal intensity of each peak in the spectrum is shown in Table 15. Absolute quantification could not be performed in the absence of purified standards. However, similar amounts of each cell extract were injected, and the relative amounts of the compounds were assessed from the resulting signal intensities.

[0249] Table 15

[0250]

[0251]

[0252] In the wild-type FusA fusarium synthase, FusA-A3 is responsible for incorporating L-Tyr, L-Phe, L-Val, L-Ile, or L-allo-Ile into the fusarium compound at amino acid position (3) (see Table 1). The variant FusA-A3 in the *Bacillus* strain NRRL B-50972 resulted in the absence of any detectable fusarium C, fusarium D, LiF07a, or LiF07b in the extract. Fusarium C and fusarium D both have a tyrosine residue at amino acid position (3), while LiF07a and LiF07b both have a phenylalanine residue at amino acid position (3). These experimental data demonstrate that the genetic variation in FusA-A3 expressed by *Bacillus* strain NRRL B-50972 inhibits the biosynthesis of fusarium compounds with either a tyrosine or phenylalanine residue at amino acid position (3) (see Table 1). Figure 14 ).

[0253] Therefore, the *Bacillus* strain NRRL B-50972 and mutant strains derived from it cannot produce detectable amounts of fusaric acid or fusaric acid-like compounds (e.g., fusaric acid C and D or LiF07a and LiF07b) containing tyrosine or phenylalanine at amino acid position (3). Analysis of variant FusA-A3 in *Bacillus* strain NRRL B-50972 indicates that this strain and its mutants are genetically incapable of producing fusaric acid or fusaric acid analogs containing a peptide ring containing tyrosine or phenylalanine at amino acid position (3).

[0254] Of the two paeniserines analyzed, only one was detectable in *Bacillus* strain A, and its signal intensity was less than half that of the corresponding signal intensity observed in the extract of *Bacillus* strain NRRL B-50972. Without wishing to limit itself to any theory, it appears that one or more of the first nine conserved amino acids in FusA-A3 (i.e., Asp235, Ala236, Ser239, Thr278, Leu299, Ala301, Ala / Gly322, Val330, and Cys331) are responsible for the recognition and activation of tyrosine and phenylalanine at position (3) in fusaric compounds. Furthermore, the variant FusA-A3 expressed by *Bacillus* strain NRRL B-50972 may shift the production of metabolic intermediates from certain fusaric compounds to the biosynthesis of a wider range of fusaric compound-like compounds (e.g., paeniserine).

[0255] Example 14. Comparison of the bioactivity of Bacillus subtilis strain NRRL B-50972 and Bacillus subtilis strain A

[0256] Bacillus species NRRL B-50972 (expressing variant FusA-A3) and Bacillus species A (expressing wild-type FusA-A3) were cultured in soybean-based media to produce complete cultures. The complete cultures were diluted to concentrations of 10%, 5%, 2.5%, and 1.25% in a mixture of water and organic solvents. The diluted complete cultures were applied to young plantlets, which were then exposed to inoculum of *Pucc. var. chinensis*, *Botrytis cinerea*, or *Phytophthora*. Several days after exposure to the plant pathogen inoculum, the percentage of pathogen control for each treatment relative to untreated controls was scored. Each treatment was evaluated three times, and the mean percentage of control was reported (see Tables 16-18).

[0257] In each assay, *Bacillus* strain NRRL B-50972 showed superior control compared to *Bacillus* strain A. These experimental data indicate that alterations in the biosynthesis of the variant fusarium synthase and the resulting fusarium and fusarium-like compounds enhance the control of plant pathogens by *Bacillus* strain NRRL B-50972.

[0258] Table 16. Control of wheat leaf rust (PUCCRT) using Bacillus subtilis strain NRRL B-50972 and Bacillus subtilis strain A at dilution rates of 10%, 5%, 2.5%, and 1.25%.

[0259]

[0260]

[0261] Table 17. Control of Botrytis cinerea (BOTRCI) using Bacillus strain NRRL B-50972 and Bacillus strain A at dilution rates of 10%, 5%, 2.5%, and 1.25%.

[0262]

[0263] Table 18. Control of Phytophthora infestans achieved using Bacillus species NRRL B-50972 and Bacillus species A at dilution rates of 10%, 5%, 2.5%, and 1.25%.

[0264]

[0265] Example 15. Identification of Fusarium oxysporin in Bacillus spp. cell extracts

[0266] Bacillus species NRRL B-50972 and / or its derivatives were grown in soybean-based media until they reached a stationary phase. At this point, the whole culture was collected and extracted with organic solvents to produce cell extracts.

[0267] Develop a chromatographic method using high-performance liquid chromatography / time-of-flight mass spectrometry (HPLC / MS TOF) to isolate numerous fusarium-like molecules from cell extracts: Column: YMC TM Basic 4.6x250mm, 5μm; water (0.1% FA) and acetonitrile (0.1% formic acid (FA)); gradient (%B): 0-9min 28-30%; 9-14min 30-33%; 14-34min 33-50%; wash.

[0268] Chromatograms of cell extracts (in which known fusarium spores were identified) are shown below. Figure 4B As shown. The general structure of Fusarium oxysporin is as follows. Figure 4A As shown. Each cyclic fusarium oxysporin has a corresponding acyclic analogue.

[0269] All detectable fusarium spores in cell extracts were identified based on their retention time and m / z value (see [link to relevant documentation]). Figure 4C Interestingly, fusarium oxychloride C and D, as well as other fusarium oxychloride with tyrosine or phenylalanine at position (3), could not be detected in the cell extracts.

[0270] Example 16. Characterization of Paeniserine in Bacillus spp. cell extracts

[0271] To identify other compounds in cell extracts of Bacillus spp. strain NRRL B-50972 and / or its derivatives, a chromatographic method using ultra-high performance liquid chromatography / triple time-of-flight mass spectrometry (UPLC / MS triple TOF) was developed to fragment many fusarium-like molecules: Column: ZORBAX TM Eclipse Plus, 2.1x100mm, 1.8μm; water (0.1% FA) and acetonitrile (0.1% FA); gradient (% B): 0-5min 10-95%; cleaning.

[0272] Using this method, the applicant can check the information from AB SCIEX TRIPLE. Mass fragmentation patterns obtained by mass spectrometry and characterization of a new Paeniserine family of fusarium spores by comparing chromatograms with published literature were used. The applicant named this new family Paeniserine. Representative UPLC / MS triple TOF fragmentation patterns and the corresponding chemical structures of Paeniserine A1 and Paeniserine B1 are shown in Figures 5 and 6, respectively. Similar analyses were performed on each Paeniserine detected in cell extracts.

[0273] Paeniserine is named as such because it has one or more serine substitutions compared to the fusarium skeletal backbone (see Figure 5). According to the records, for a peptide sequence to be considered fusarium skeletal, it must contain three conserved amino acids: (1) threonine; (4) threonine; and (6) alanine. However, Paeniserine exhibits novel substitutions, where one or both of the (1) and (4) threonine residues are replaced by serine. In the Paeniserine characterized by the applicant, the amino acids at positions (2) and (3) are both valine. Figure 5B The chromatogram shown identifies the peak corresponding to Paeniserine.

[0274] The applicant also characterized the family of fusarium-like compounds with this serine substitution in cell extracts based on their retention times and m / z values ​​(see [link to relevant documentation]). Figure 5C Although Paeniserine C4 could not be detected, its production could be reasonably expected based on the previously characterized structures of fusarium oxysporins. For fusarium oxysporins, each cyclic Paeniserine has a corresponding acyclic analogue.

[0275] Importantly, it should be noted that although the Paeniserine characterized by the applicant has valine amino acids at residues (2) and (3), compounds with variations at these positions are still possible. These possible variations are similar to those of Fusarium oxysporin / LiF analogues, where residues (2) and (3) are amino acids such as isoleucine, phenylalanine, and tyrosine. Furthermore, although the GHPD tail has been described above, compounds with variations in tail length, similar to that of the Paeniprolixin family, are still possible (see Example 17).

[0276] Example 17. Characterization of Paeniprolixin in Bacillus spp. cell extracts

[0277] Cell extracts of Bacillus species NRRL B-50972 and / or its derivatives were further analyzed using the chromatographic method described in Example 14. This was achieved by examining the AB SCIEX TRIPLE. Mass fragmentation patterns obtained by mass spectrometry and characterization of the new family of fusarium spore-killing agents by comparing chromatograms with published literature were used. The applicant named this new family Paeniprolixin. Representative UPLC / MS triple TOF fragmentation patterns and the corresponding chemical structures of Paeniserine C1 and Paeniserine D1 are shown in Figures 8 and 9, respectively. Similar analyses were performed on each Paeniprolixin detected in cell extracts.

[0278] The name Paeniprolixin comes from the Latin word *prolix* (meaning long), due to another important difference in the aliphatic tail compared to the fusarium skeleton: Paeniprolixin has a longer tail than fusarium. According to the record, only fusarium has been observed to possess a specific GHPD tail. This has been shown to be consistent even in recent publications on the subject (i.e., Vater et al., J. Am. Soc. Mass Spectrom., 2015, 26, 1130-1141), where the authors claim that "this finding [strictly conserved GHPD tail] differs from many other lipopeptides reported in the literature where the fatty acid moiety is the primary target of structural variation, [e.g., in] subtilisin, iturin, and fengycin." The applicant identified a family of fusarium-like compounds with longer tails (i.e., 17-guanidino-3-hydroxypentadecanoic acid or GHPD+2CH2 and 19-guanidino-3-hydroxypentadecanoic acid or GHPD+4CH2) in cell extracts of Bacillus species NRRL B-50972 (see [link to relevant documentation]). Figure 8AUnlike Paeniserine, Paeniprolixin retains the conserved amino acid residue L-threonine at position (1) and D-allo-threonine at position (4).

[0279] Importantly, it should be noted that although the Paeniprolixin characterized by the applicant has valine or isoleucine amino acids at residues (2) and (3), compounds with variations at these positions are still possible. These possible variations are similar to those of Fusarium oxysporin / LiF analogues, such as other combinations of valine, isoleucine, or other amino acids (e.g., phenylalanine and tyrosine) as residues (2) and (3). Furthermore, hybrid combinations with the aforementioned Paeniserine having a longer tail length are possible.

[0280] Chromatogram as shown Figure 8B As shown, peaks corresponding to Paeniprolixin were identified. This family of fusarium-like compounds with longer GHPD tails was also characterized based on their retention times and m / z values ​​(see [link to relevant documentation]). Figure 8C Although Paeniprolixin C2 and D2 could not be detected, their production could be reasonably expected based on the previously characterized structures of fusarium oxysporins. For fusarium oxysporins, each cyclic Paeniprolixin has a corresponding acyclic analogue.

[0281] Example 18. Antifungal bioactivity characteristics of Paeniserine, Paeniprolixin and other Fusarium oxysporins

[0282] The samples shown in Table 19 were isolated from Bacillus spp. cells. The entire fermentation broth was centrifuged to remove the supernatant. The resulting precipitate was then extracted with methanol. The extracts were fractionated using reversed-phase medium-pressure liquid chromatography. The fractions were then further purified using reversed-phase preparative high-performance liquid chromatography.

[0283] Table 19

[0284]

[0285]

[0286] In vitro antifungal 96-well plate assay using a resazurin-based cell viability reagent It serves as an indicator of fungal growth. Starting from fungal spores, the assay measures the efficacy of a sample in inhibiting fungal spore germination and / or fungal cell growth. This assay was performed using three agriculturally relevant fungal diseases: Alternaria solanacea (ALTESO), Collla anthracnose (COLLLA), and Botrytis cinerea (BOTRCI).

[0287] All samples shown in Table 19 demonstrated activity against agriculturally relevant fungal diseases (see Table 20 for the 80% minimum inhibitory concentration (MIC80) values ​​for each sample, in ppm). Interestingly, some compounds appeared to exhibit varying activity against specific diseases. For example, while the asparagine analog in Sample 3 appeared important for controlling ALTESO, the glutamine counterpart of the same type of compound in Sample 4 was more involved in controlling COLLLA. The longer-tailed analog in Sample 6 was the most potent COLLLA inhibitor. This suggests that while all compounds are active individually, the combination of these compounds is important for the final efficacy and disease control range of the final product.

[0288] Table 20

[0289]

[0290] Example 19. Antibacterial bioactivity characteristics of Paeniserine, Paeniprolixin and other Fusarium oxysporins

[0291] The in vitro antibacterial 96-well plate assay uses absorbance as an indicator of bacterial growth. This assay measures the efficacy of a sample in inhibiting bacterial growth by comparing the absorbance of untreated wells with that of the sample wells. The final dilution / concentration that inhibits bacterial growth is called the MIC (minimum inhibitory concentration), which can be used to compare the efficacy of different samples. This assay was evaluated against three agriculturally relevant bacterial diseases: Xanthomonas laurentii (XANTAV), Pseudomonas syringae (PSDMTM), and Erwinia carotene soft rot (ERWICA).

[0292] The samples shown in Table 19 were applied to antibacterial assays to determine the MIC80 value for each bacterial pathogen. The results are shown in Table 21. Samples 1-5 were demonstrated to have activity against agriculturally relevant bacterial diseases. Interestingly, some compounds appeared to exhibit varying activity against specific diseases. For example, Paeniserine effectively compensated for the shortcomings of Fusarium oxysporine A, as Paeniserine is effective against PSDMTM, which is a weakness of Fusarium oxysporine A. On the other hand, Fusarium oxysporine A compensated for the shortcomings observed when using Paeniserine in controlling ERWICA. For fungal assays, it was shown that although all compounds are active on their own, the combination of these compounds is important for the final efficacy and disease control range of the final product.

[0293] Table 21

[0294]

[0295] NDR*: No detectable result (i.e., no inhibition of bacterial growth at the highest test concentration)** The sample is insoluble in microbial culture medium and cannot be tested.

[0296] Example 20. Using the Kirby-Bauer antibiotic disc diffusion assay to indicate synergistic effects

[0297] To obtain a preliminary assessment of the synergistic effects among multiple fusarium-like compounds, a bioassay was performed using the plant pathogen COLLLA. This bioassay was the classic Kirby-Bauer antibiotic disc diffusion assay performed on agar (Bauer, AW, et al, 1966 Am. J. Clin. Pathol. 36:493-496). In short, blank sterile discs containing similar amounts of various samples were placed on Piper dishes inoculated with COLLLA spores. The Piper dishes were incubated, and the activity was recorded as the diameter of the inhibition zone observed around each disc. Results are as follows: Figure 11 As shown.

[0298] Preliminary results indicate a synergistic effect when certain Paeniserine and Paeniprolixin are administered together. In this assay, Paeniserine A1 and B1 (“868”) or Paeniprolixin A2 and B2 (“938”) administered alone showed relatively small areas of inhibition. However, their combination (“868 / 938”) showed the largest and most complete area of ​​inhibition, exceeding the results obtained by administering 868, 938, or Fusarium oxysporins A and B (“AB”). For the AB, 868, and 938 samples, approximately 0.1 mg of total substance was administered to each sterile dish. Dishes containing both 868 and 938 samples contained approximately 0.05 mg of each sample, resulting in a total substance of approximately 0.1 mg on the 868 / 938 dish.

[0299] The limitation of this assay is that the fusarium compound must diffuse through agar to inhibit fungal growth. An in vitro antifungal assay in liquid culture will be used to further evaluate this preliminary indication of synergistic effect.

[0300] Example 21. In vitro antifungal assay demonstrating the synergistic effect of the fusarium oxysporin combination.

[0301] In addition to the combinations of fusarium spore-killing agents described in Example 17, in vitro antifungal assays in liquid culture media were performed to demonstrate the expected synergistic effect produced by the application of the combinations of fusarium spore-killing agents and / or fusarium spore-like compounds shown in Figure 12. Each group shown in Figure 12 was evaluated individually to first assess structural characteristics, and then they were combined to illustrate the synergistic effect. Binary and ternary mixtures were evaluated.

[0302] Although individual compounds may exhibit drawbacks in terms of antifungal activity, combinations will have more activity than the simple sum of their activities.

[0303] When the fungicidal activity of a combination of active compounds exceeds the sum of the activities of the active compounds when applied alone, a synergistic effect of fungicides always exists.

[0304] The expected activity of a given combination of two or three active compounds can be calculated as follows (see Colby, SR, "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations," Weeds 1967, 15, 20-22):

[0305] if

[0306] X represents the efficacy of active compound A when applied at a rate of m ppm (or g / ha).

[0307] Y represents the efficacy of active compound B when applied at a rate of n ppm (or g / ha).

[0308] Z represents the efficacy of active compound B when applied at a rate of r ppm (or g / ha).

[0309] E1 represents the efficacy of active compounds A and B when applied at application rates m and n ppm (or g / ha), respectively.

[0310] E2 represents the efficacy of active compounds A, B, and C when applied at application rates m, n, and r ppm (or g / ha), respectively.

[0311] For binary mixtures:

[0312]

[0313] For ternary mixtures:

[0314]

[0315] The percentage indicates the level of efficacy. % indicates efficacy compared to the control, while 100% efficacy indicates no disease was observed.

[0316] If the actual fungicidal activity exceeds the calculated value, the combined activity is superadditive, meaning a synergistic effect exists. In this case, the actual observed efficacy must be greater than the expected efficacy (E) calculated from the above formula.

[0317] Another method to demonstrate synergism is the Tammes method (see "Isoboles, A Graphic Representation of Synergism in Pesticides" in Neth. J. Plant Path., 1964, 70, 73-80).

[0318] Example 22. Selection of variant strains of Bacillus spp. strain NRRL B-50972

[0319] Under standard laboratory conditions, *Bacillus* strain NRRL B-50972 produced multiple colony morphologies on solid agar. Several colonies with different morphologies were identified and stored in glycerol at -80°C. Liquid culture media were inoculated with stock solutions from different colony phenotypes, and after several rounds of growth in liquid medium, the cultures were re-inoculated onto solid agar. From this, one isolate was identified as having a stable colony phenotype under the test conditions, yet still producing heat-resistant spores and exhibiting fusarium-killing chemical activity. Isolates with stable colony morphology are needed for further strain improvement (see Example 23). This isolate was deposited in NRRL on September 1, 2015, and assigned the following accession number: NRRL B-67129.

[0320] Example 23. Random mutagenesis to generate modified Bacillus-like mutants

[0321] Chemical mutagenesis

[0322] To generate a group of genetically distinct isolates of the genus *Bacillus* NRRL B-67129, the liquid growth culture of this strain was precipitated by centrifugation and resuspended in a buffer containing 1-methyl-3-nitro-1-nitroguanidine (NTG) to a final concentration of 400 μg / ml. As a control, a second sample without NTG was prepared. The sample was incubated at 30°C and 220 rpm for 1 hour. After 1 hour, the sample was precipitated by centrifugation, washed with NTG-free buffer, and finally resuspended in the same volume of fresh buffer. Aliquots of the undiluted culture were frozen as stock glycerol at -80°C. The samples were diluted and plated on agar plates to determine colony-forming units, and the percentage of inactivation was used as a reference for the degree of mutation for each genome. As described above, the improved isolates selected from the first round of screening were subjected to one or more subsequent rounds of NTG treatment, and further improvements in fusarium production were screened. The production of fusarium oxysporin is determined by relative amounts of several compounds, including fusarium oxysporin A (also known as LiF04a or "Fus A"), LiF08a, Paeniserine A1 and B1 (also known as "M868" or "868"), and Paeniprolixin A2 and B2 (also known as "M938" or "938").

[0323] High-throughput screening and isolate characterization

[0324] NTG-treated samples were diluted and plated on agar plates to obtain single colonies. Single colonies were inoculated into 96-well blocks containing seed culture medium and incubated with shaking at 30°C for 2 days. Subsequently, new 96-well blocks containing soybean-based production medium were inoculated and incubated with shaking at 30°C for 5 days. After 5 days, glycerol stock solutions were prepared from each sample in individual wells and stored at -80°C. Each sample was then subjected to chemical analysis for the four Fusarium oxysporin biomarkers identified above. In this preliminary screening, a single isolate was considered a hit if its "total Fusarium oxysporin value" (i.e., the sum of the four analyzed Fusarium oxysporin biomarkers relative to the mean wild-type value) was higher than the mean wild-type value + 3x (the standard deviation of the wild-type value). Eight replicates of each isolate were selected according to this criterion and analyzed as described above. Next, the confirmed isolates that excessively produced fusarium oxysporin were scaled up to 50 mL in 250 mL shake flasks, and spore formation, fusarium oxysporin production, and bioactivity were characterized. Preferred isolates were further scaled up in a bioreactor, and spore formation, viscosity, fusarium oxysporin production, and bioactivity were similarly characterized. Several mutant strains were obtained from the second round of NTG treatment and screening, and they were found to have good fusarium oxysporin biomarker production and bioactivity.

[0325] Example 24. Characterization of antibiotic susceptibility of Bacillus subtilis strain NRRL B-50972

[0326] Bacillus subtilis strain NRRL B-50972 was inoculated onto solid sLB agar medium supplemented with typical concentrations of antibiotics. Agar plates were incubated at 30°C, and growth was assessed at 24, 48, and 72 hours. The susceptibility of Bacillus subtilis strain NRRL B-50972 to each tested antibiotic is shown in Table 22.

[0327] Table 22. Antibiotic susceptibility of Bacillus subtilis strain NRRL B-50972

[0328]

[0329] Example 25. Characterization of spo0A in Bacillus subtilis strains NRRL B-50972 and NRRL B-67129

[0330] The genomes of *Bacillus* strains NRRL B-50972 and NRRL B-67129 were sequenced. Comparison of the two genome sequences identified characteristic differences in the spo0A gene between the two strains. Figure 15Sequence alignment showed that the difference between *Bacillus* strain NRRL B-50972 and *Bacillus* strain NRRL B-67129 lies in a single nucleotide near the 3' end of the *spo0A* gene. Identifying single nucleotide differences... Figure 15 The sequence number is indicated by a red arrow below the sequence. The nucleotide number relative to the first nucleotide of the spo0A gene is indicated above the sequence.

[0331] Alignment of Spo0A orthologs from endospore-forming bacteria revealed that nucleotide changes in the coding sequence of the Bacillus spp. strain NRRL B-67129 resulted in a single amino acid substitution in a conserved region (see [link to original text]). Figure 16 The Spo0A amino acid sequences from the following strains were compared with those from Bacillus species NRRL B-50972 and NRRL B-67129: Bacillus terrestris (NCBI reference sequence: WP_044647644.1), Bacillus polymyxa SQR-21 (GenBank: AHM66630.1), Bacillus subtilis subsp. 168 (NCBI reference sequence: NP_390302.1), Bacillus cereus E33L (GenBank: AJI26924.1), and Clostridium pasteurellii DSM 525 (GenBank: AAA18883.1). Figure 16 The arrows in the text indicate single amino acid substitutions in Spo0A from Bacillus spp. strain NRRL B-67129.

[0332] Example 26. Study on the structure-activity relationship of fusarium oxysporin, paeniserine, and paeniprolixin

[0333] The structure-activity relationship of several purified fusaric acid, paeniserine, and paeniprolixin was investigated using the in vitro assays described in Example 16. In the first experiment, the most common fusaric acid pairs were compared. Variations in these fusaric acid pairs occur at amino acid positions of rings / chains containing asparagine or glutamine (5). In this study, fusaric acid A was compared with fusaric acid B, and LiF08a was compared with LiF08b against the plant pathogen Alternaria solanacea (ALTESO). In both cases, the potency of the asparagine analog was more than twice that of its glutamine counterpart (see Example 16). Figure 17 ).

[0334] In another experiment, the cyclic and acyclic forms of fusarium were compared. It is unclear whether the acyclic forms of fusarium are precursors to the final compound or degradation products; however, they are widely present in the fermentation broth of *Bacillus* strain NRLRB-50972 and in common contaminants of fusarium purified from this broth. In an in vitro assay using the plant pathogen ALTESO, the antifungal activity of fusarium A was compared with that of LiF04c and LiF04d (acyclic analogs of fusarium A and B). The opening of the peptide ring at the ester bond had a significant effect. The acyclic analogs were inactive at the highest tested concentration (see [link to study]). Figure 17 This is important for structural information, and for demonstrating that these compounds (which typically constitute impurities in otherwise purified fusarium oxysporins) do not promote antifungal activity.

[0335] Amino acid substitutions at the amino acid positions (2) and (3) of the ring / chain were also investigated. The analogues Fusarium A, LiF05a, LiF06a, and LiF08a differed at these positions in either valine or isoleucine combinations. They were tested in an in vitro assay using the plant pathogen ALTESO. The two most potent analogues were Fusarium A (valine / valine) and LiF08a (isoleucine / isoleucine). The other two analogues (including mixtures of valine / isoleucine) were more than three times less potent (see [link to study]). Figure 17 ).

[0336] The differences in antifungal activity of the novel Paeniserine were also investigated. The differences in the amino acid positions of the ring / chain (1) and (4) were evaluated, also against ALTESO. Typical Fusarium oxysporins are confined to threonine at these positions, while Paeniserine is able to interchange between threonine and threonine. In this assay, Paeniserine showed antifungal activity similar to that of Fusarium oxysporin A (see [link to assay]). Figure 17 ).

[0337] The antifungal activity of Paeniprolixin (i.e., analogues with different side chain lengths) was also investigated in in vitro assays against the fungal pathogens ALTESO and Collla anthracnose. Typical Fusarium oxysporins contain a 15-guanidino-3-hydroxypentadecanoic acid side chain. Paeniprolixin has been shown to have two of the four additional methylene groups in its chain. Side chain length showed a significant effect on bioactivity and exhibited differences across different fungal pathogens. Against ALTESO, the unchanged length of GHPD was the most effective, with potency decreasing with each additional methylene group. Against Collla, the most effective length was GHPD+2CH2 (see [link to relevant documentation]). Figure 17 ).

[0338] Example 27. Synergistic antifungal activity of a mixture of Fusarium oxysporin A with Paeniserine A1 or Paeniprolixin C1

[0339] Using rezamidophos-based cell viability reagents An in vitro antifungal 96-well plate assay (see Example 18) was used to evaluate the antifungal activity of individual fusarium oxysporins, paeniserine, and paeniprolixin, as well as two-way combinations. The antifungal activity compared to the untreated control was calculated using the following equation:

[0340] Efficacy = (100 - relative growth of untreated control)

[0341] 100% efficacy means no fungal growth compared to the untreated control, and 0% efficacy means no inhibition of fungal growth compared to the untreated control.

[0342] Tables 23 and 24 clearly show that the observed activity of the active compound combinations of the present invention is greater than the calculated activity, i.e., there is a synergistic effect.

[0343] Table 23. Antifungal activity of Fusarium oxysporum, Paeniserine A1 alone, and Fusarium oxysporum + Paeniserine A1 against Alternaria solanacea.

[0344]

[0345] *Observation = Observed activity

[0346] **Calculation = Activity calculated using the Colby formula**

[0347] Table 24. Antifungal activity of Fusarium oxysporum (Fusarium oxysporum) against Alternaria solanaceus alone, Paeniprolixin alone, and Fusarium oxysporum (Fusarium oxysporum) + Paeniprolixin C1.

[0348] *Observation = Observed activity

[0349] **Calculation = Activity calculated using the Colby formula**

[0350] 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 pertains. All publications, patents, and patent publications cited are incorporated herein by reference in their entirety for all purposes.

[0351] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and materials described, as they are capable of variation. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims.

[0352] Those skilled in the art, using only conventional experimental methods, will understand or be able to identify many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims. sequence list <110> Bayer Crop Science LLC <120> Novel Bacillus strains, antifungal compounds, and their methods of use <130> BCS159002 WO <150> US 62 / 138,765 <151> 2015-03-26 <150> US 62 / 232,205 <151> 2015-09-24 <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 550 <212> PRT <213> Paenibacillus peoriae <400> 1 Asn Ala Leu Val Tyr Asp Pro Val Thr Ile Glu Gln Ile Lys Gly His 1 5 10 15 Leu Val His Leu Met Glu Gln Ile Val Glu Asn Pro Ala Ile Ser Val 20 25 30 Asp Ala Leu Glu Leu Val Thr Pro Gln Glu Arg Glu Gln Ile Leu Asn 35 40 45 Val Trp Gly Asn Thr Lys Gly Ile Tyr Glu His Cys Asn Thr Phe His 50 55 60 Gly Leu Leu Glu Glu Gln Ala Gly Arg Thr Pro Asp Ala Thr Ala Ile 65 70 75 80 Trp Phe Glu Asp Glu Ser Leu Thr Tyr Ala Glu Leu Asn Ala Lys Ala 85 90 95 Asn Gly Leu Ala Arg Arg Leu Arg Thr Gln Gly Ile Lys Thr Gly Asp 100 105 110 Leu Val Gly Leu Ile Ala Glu Arg Ser Leu Glu Met Ile Val Gly Ile 115 120 125 Tyr Gly Ile Met Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu 130 135 140 Tyr Pro Lys Glu Arg Ile Ser Tyr Met Leu Glu Asp Ser Gly Ala Lys 145 150 155 160 Leu Ile Leu Thr Gln Ala His Phe Leu Glu His Leu Gly Trp Thr Glu 165 170 175 Asn Val Leu Leu Leu Asp Glu Ser Ser Thr Tyr Asp Ala Asp Thr Ser 180 185 190 Asn Leu Glu Asp Thr Ala Gly Pro Asp Asp Leu Ala Tyr Val Ile Tyr 195 200 205 Thr Ser Gly Thr Thr Gly Gln Pro Lys Gly Val Leu Val Glu His Arg 210 215 220 Gly Leu Pro Asn Leu Ser Asp Val Tyr Gly Ala His Phe Glu Val Thr 225 230 235 240 Pro Gln Asp Arg Ile Val Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser 245 250 255 Val Ser Glu Ile Leu Thr Ala Leu Ser His Gly Gly Val Leu Cys Ile 260 265 270 Pro Ser Thr Glu Asp Ile Leu Asp His Ala Leu Phe Glu Gln Phe Met 275 280 285 Asn Asp Lys Gly Val Thr Val Ala Thr Leu Pro Pro Ala Tyr Ala Ile 290 295 300 His Leu Asp Pro Glu Arg Leu Pro Thr Leu Arg Cys Leu Leu Thr Ala 305 310 315 320 Gly Ser Ala Ala Ser Val Glu Leu Ile Glu Glu Trp Arg Lys His Val 325 330 335 Arg Tyr Ser Asn Gly Tyr Gly Pro Thr Glu Asp Ser Val Cys Thr Thr 340 345 350 Ile Trp Ser Val Pro Asp Ser Glu Glu Ala Thr Asp Ile Val Ser Ile 355 360 365 Gly Arg Pro Ile Ala Asn His Ser Val Tyr Ile Leu Asp Asp His Phe 370 375 380 Arg Leu Gln Pro Val Gly Val Ala Gly Glu Leu Cys Ile Ser Ser Ile 385 390 395 400 Gly Leu Ala Arg Gly Tyr His Asn Gln Pro Glu Leu Met Asp Glu Lys 405 410 415 Phe Val Asp Asn Pro Phe Ala Pro Gly Glu Arg Met Tyr Arg Thr Gly 420 425 430 Asp Leu Val Arg Trp Leu Pro Asn Gly Thr Ile Glu Tyr Leu Gly Arg 435 440 445 Ile Asp His Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu 450 455 460 Val Glu Ala Gln Met Leu Arg Val Pro Ser Val Gln Glu Val Val Ala 465 470 475 480 Met Ala Ala Glu Gly Glu Asp Gly Tyr Lys Asp Leu Val Ala Tyr Phe 485 490 495 Val Ala Ala Gln Lys Leu Glu Val Ser Glu Leu Arg Ala Val Leu Ser 500 505 510 Glu Met Leu Pro Gly Tyr Met Ile Pro Ser Arg Phe Ile Gln Leu Glu 515 520 525 Asp Met Pro Leu Thr Ser Asn Gly Lys Ile Asp Arg Lys Ala Leu Gln 530 535 540 Gly Glu Arg Gly Trp Ala 545 550 <210> 2 <211> 550 <212> PRT <213> Paenibacillus polymyxa <400> 2 Asn Ala Leu Val Tyr Asp Gln Val Thr Ile Gly Gln Ile Lys Gly His 1 5 10 15 Leu Val His Leu Met Glu Gln Ile Val Glu Asn Pro Ala Ile Ser Val 20 25 30 Asp Ala Leu Glu Leu Val Thr Pro Gln Glu Arg Glu Gln Ile Leu Asn 35 40 45 Val Trp Gly Asn Met Lys Gly Ile Tyr Glu His Cys Asn Thr Phe His 50 55 60 Gly Leu Leu Glu Glu Gln Ala Gly Arg Thr Pro Asp Ala Thr Ala Ile 65 70 75 80 Trp Phe Glu Asp Glu Ser Leu Thr Tyr Ala Glu Leu Asn Ala Lys Ala 85 90 95 Asn Gly Leu Ala Arg Arg Leu Arg Thr Gln Gly Ile Lys Thr Gly Asp 100 105 110 Leu Val Gly Leu Ile Ala Glu Arg Ser Leu Glu Met Ile Val Gly Ile 115 120 125 Tyr Gly Ile Met Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu 130 135 140 Tyr Pro Lys Glu Arg Ile Ser Tyr Met Leu Glu Asp Ser Gly Ala Lys 145 150 155 160 Leu Ile Leu Thr Gln Ala His Leu Leu Glu His Leu Gly Trp Thr Glu 165 170 175 Asn Val Leu Leu Leu Asp Glu Ser Ser Thr Tyr Asp Ala Asp Thr Ser 180 185 190 Asn Leu Glu Asp Thr Ala Gly Pro Asp Asp Leu Ala Tyr Val Ile Tyr 195 200 205 Thr Ser Gly Thr Thr Gly Gln Pro Lys Gly Val Leu Val Glu His Arg 210 215 220 Gly Leu Pro Asn Leu Ser Asn Val Tyr Gly Ala His Phe Glu Val Thr 225 230 235 240 Pro Gln Asp Arg Ile Val Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser 245 250 255 Val Ser Glu Ile Leu Thr Ala Leu Ser His Gly Gly Val Leu Cys Ile 260 265 270 Pro Ser Thr Glu Asp Ile Leu Asp His Ala Leu Phe Glu Gln Phe Met 275 280 285 Asn Asp Lys Gly Ile Thr Val Ala Thr Leu Pro Pro Ala Tyr Ala Ile 290 295 300 His Leu Asp Pro Glu Arg Leu Pro Thr Leu Arg Cys Leu Leu Thr Ala 305 310 315 320 Gly Ser Ala Ala Ser Val Glu Leu Ile Glu Glu Trp Arg Lys His Val 325 330 335 Arg Tyr Ser Asn Gly Tyr Gly Pro Thr Glu Asp Ser Val Cys Thr Thr 340 345 350 Ile Trp Ser Val Pro Asp Ser Glu Glu Ala Thr Asp Ile Val Ser Ile 355 360 365 Gly Arg Pro Ile Ala Asn His Ser Val Tyr Ile Leu Asp Asp His Phe 370 375 380 Arg Leu Gln Pro Val Gly Val Ala Gly Glu Leu Cys Ile Ser Ser Ile 385 390 395 400 Gly Leu Ala Arg Gly Tyr His Asn Arg Pro Glu Leu Met Asp Glu Lys 405 410 415 Phe Val Asp Asn Pro Phe Ala Pro Gly Glu Arg Met Tyr Arg Thr Gly 420 425 430 Asp Leu Val Arg Trp Leu Pro Asn Gly Thr Ile Glu Tyr Leu Gly Arg 435 440 445 Ile Asp His Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu 450 455 460 Val Glu Ala Gln Met Leu Arg Val Pro Ser Val Gln Glu Val Val Ala 465 470 475 480 Met Ala Val Glu Gly Asp Asp Gly Tyr Lys Asp Leu Val Ala Tyr Phe 485 490 495 Val Ala Ala Gln Lys Leu Glu Val Ser Glu Leu Arg Ala Val Leu Ser 500 505 510 Glu Ile Leu Pro Gly Tyr Met Ile Pro Ser Arg Phe Ile Gln Leu Glu 515 520 525 Asp Met Pro Leu Thr Ser Asn Gly Lys Ile Asp Arg Lys Ala Leu Gln 530 535 540 Gly Glu Arg Gly Trp Ala 545 550 <210> 3 <211> 550 <212> PRT <213> Paenibacillus polymyxa <400> 3 Asn Ala Leu Val Tyr Asp Pro Val Thr Ile Glu Gln Ile Lys Gly His 1 5 10 15 Leu Val His Leu Met Glu Gln Ile Val Glu Asn Pro Ala Ile Ser Val 20 25 30 Asp Ala Leu Glu Leu Val Thr Pro Gln Glu Arg Glu Gln Ile Leu Asn 35 40 45 Val Trp Gly Asn Thr Lys Gly Ile Tyr Glu His Cys Asn Thr Phe His 50 55 60 Gly Leu Leu Glu Glu Gln Ala Gly Arg Thr Pro Asp Ala Thr Ala Ile 65 70 75 80 Trp Phe Glu Asp Glu Ser Leu Thr Tyr Ala Glu Leu Asn Ala Lys Ala 85 90 95 Asn Gly Leu Ala Arg Arg Leu Arg Thr Gln Gly Ile Lys Thr Gly Asp 100 105 110 Leu Val Gly Leu Ile Ala Glu Arg Ser Leu Glu Met Ile Val Gly Ile 115 120 125 Tyr Gly Ile Met Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu 130 135 140 Tyr Pro Lys Glu Arg Ile Ser Tyr Met Leu Glu Asp Ser Gly Ala Lys 145 150 155 160 Leu Ile Leu Thr Gln Ala His Leu Leu Glu His Leu Gly Trp Thr Glu 165 170 175 Asn Val Leu Leu Leu Asp Glu Ser Ser Thr Tyr Asp Ala Asp Thr Ser 180 185 190 Asn Leu Glu Asp Thr Ala Gly Pro Asp Asp Leu Ala Tyr Val Ile Tyr 195 200 205 Thr Ser Gly Thr Thr Gly Gln Pro Lys Gly Val Leu Val Glu His Arg 210 215 220 Gly Leu Pro Asn Leu Ser Asp Val Tyr Gly Thr His Phe Glu Val Thr 225 230 235 240 Pro Gln Asp Arg Ile Val Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser 245 250 255 Val Ser Glu Ile Leu Thr Ala Leu Ser His Gly Gly Val Leu Cys Ile 260 265 270 Pro Ser Thr Gln Asp Ile Leu Asp His Ala Leu Phe Glu Gln Phe Met 275 280 285 Asn Asp Lys Gly Ile Thr Val Ala Thr Leu Pro Pro Ala Tyr Ala Ile 290 295 300 His Leu Asp Pro Glu Arg Leu Pro Thr Leu Arg Cys Leu Leu Thr Ala 305 310 315 320 Gly Ser Ala Ala Ser Val Glu Leu Ile Glu Glu Trp Arg Lys His Val 325 330 335 Arg Tyr Ser Asn Gly Tyr Gly Pro Thr Glu Asp Ser Val Cys Thr Thr 340 345 350 Ile Trp Ser Val Pro Asp Ser Glu Glu Ala Thr Asp Ile Val Ser Ile 355 360 365 Gly Arg Pro Ile Ala Asn His Ser Val Tyr Ile Leu Asp Asp His Phe 370 375 380 Arg Leu Gln Pro Val Gly Val Ala Gly Glu Leu Cys Ile Ser Ser Ile 385 390 395 400 Gly Leu Ala Arg Gly Tyr His Asn Arg Pro Glu Leu Met Asp Glu Lys 405 410 415 Phe Val Asp Asn Pro Phe Ala Pro Gly Glu Arg Met Tyr Arg Thr Gly 420 425 430 Asp Leu Val Arg Trp Leu Pro Asn Gly Thr Ile Glu Tyr Leu Gly Arg 435 440 445 Ile Asp His Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu 450 455 460 Val Glu Ala Gln Met Leu Arg Val Pro Ser Val Gln Glu Val Val Ala 465 470 475 480 Met Ala Val Glu Gly Asp Asp Gly Tyr Lys Asp Leu Val Ala Tyr Phe 485 490 495 Val Ala Ala Gln Lys Leu Glu Val Ser Glu Leu Arg Ala Val Leu Ser 500 505 510 Glu Met Leu Pro Gly Tyr Met Ile Pro Ser Arg Phe Ile Gln Leu Glu 515 520 525 Asp Met Pro Leu Thr Ser Asn Gly Lys Ile Asp Arg Lys Ala Leu Lys 530 535 540 Gly Glu Arg Gly Trp Ala 545 550 <210> 4 <211> 550 <212> PRT <213> Paenibacillus polymyxa <400> 4 Asn Ala Leu Val Tyr Asp Pro Val Thr Ile Gly Gln Ile Lys Gly His 1 5 10 15 Leu Val His Leu Met Glu Gln Ile Val Glu Asn Pro Ala Ile Ser Val 20 25 30 Asp Ala Leu Glu Leu Ile Thr Pro Gln Glu Arg Glu Gln Ile Leu Asn 35 40 45 Val Trp Gly Asn Thr Lys Ala Ile Tyr Glu His Tyr Asn Thr Phe His 50 55 60 Gly Leu Leu Glu Glu Gln Ala Gly Arg Thr Pro Asp Ala Ala Ala Ile 65 70 75 80 Trp Phe Glu Asp Glu Ser Leu Thr Tyr Ala Glu Leu Asn Ala Lys Ala 85 90 95 Asn Gly Leu Ala Arg Arg Leu Arg Thr Gln Gly Ile Lys Thr Gly Asp 100 105 110 Leu Val Gly Leu Ile Ala Glu Arg Ser Leu Glu Met Ile Val Gly Ile 115 120 125 Tyr Gly Ile Met Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu 130 135 140 Tyr Pro Gln Glu Arg Ile Ser Tyr Met Leu Glu Asp Ser Gly Ala Lys 145 150 155 160 Leu Ile Leu Thr Gln Ala His Leu Leu Glu His Leu Gly Trp Thr Glu 165 170 175 Asn Val Leu Leu Leu Asp Glu Ser Ser Thr Tyr Asp Ala Asp Thr Ser 180 185 190 Asn Leu Glu Asp Thr Ala Gly Pro Asp Asp Leu Ala Tyr Val Ile Tyr 195 200 205 Thr Ser Gly Thr Thr Gly Gln Pro Lys Gly Val Leu Val Glu His Arg 210 215 220 Gly Leu Pro Asn Leu Ser Asp Val Tyr Gly Ala His Phe Glu Val Thr 225 230 235 240 Pro Gln Asp Arg Ile Val Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser 245 250 255 Val Ser Glu Ile Leu Thr Ala Leu Ser His Gly Gly Val Leu Cys Ile 260 265 270 Pro Ser Ala Gln Asp Ile Leu Asp His Ala Leu Phe Glu Gln Phe Met 275 280 285 Asn Asp Lys Gly Ile Thr Val Ala Thr Leu Pro Pro Ala Tyr Ala Ile 290 295 300 His Leu Asp Pro Glu Arg Leu Pro Thr Leu Arg Cys Leu Leu Thr Ala 305 310 315 320 Gly Ser Thr Ala Ser Ile Glu Leu Ile Glu Glu Trp Arg Lys His Val 325 330 335 Arg Tyr Ser Asn Gly Tyr Gly Pro Thr Glu Asp Ser Val Cys Thr Thr 340 345 350 Ile Trp Ser Val Pro Asp Ser Glu Glu Ala Thr Asn Ile Val Ser Ile 355 360 365 Gly Arg Pro Ile Ala Asn His Ser Val Tyr Ile Leu Asp Asp His Phe 370 375 380 Arg Leu Gln Pro Val Gly Val Ala Gly Glu Leu Cys Ile Ser Ser Ile 385 390 395 400 Gly Leu Ala Arg Gly Tyr His Asn Arg Pro Glu Leu Met Asp Glu Lys 405 410 415 Phe Val Asp Asn Pro Phe Ala Pro Gly Glu Arg Met Tyr Arg Thr Gly 420 425 430 Asp Leu Val Arg Trp Leu Pro Asn Gly Asn Ile Glu Tyr Leu Gly Arg 435 440 445 Ile Asp His Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu 450 455 460 Val Glu Ala Gln Met Leu Arg Val Pro Ser Val Gln Glu Val Val Ala 465 470 475 480 Met Ala Val Glu Gly Asp Asp Gly Tyr Lys Asp Leu Val Ala Tyr Phe 485 490 495 Val Ala Ala Gln Lys Leu Glu Val Ser Glu Leu Arg Ala Val Leu Ser 500 505 510 Glu Met Leu Pro Gly Tyr Met Ile Pro Ser Arg Phe Ile Gln Leu Glu 515 520 525 Asp Met Leu Leu Thr Ser Asn Gly Lys Ile Asp Arg Lys Ala Leu Gln 530 535 540 Gly Glu Arg Gly Trp Ala 545 550 <210> 5 <211> 550 <212> PRT <213> Paenibacillus polymyxa <400> 5 Asn Ala Leu Val Tyr Asp Gln Val Thr Ile Glu Gln Ile Lys Glu His 1 5 10 15 Leu Val His Leu Met Glu Gln Ile Ile Glu Asn Pro Ala Thr Ser Val 20 25 30 Asp Ala Leu Glu Leu Val Thr Pro Gln Glu Arg Glu Gln Ile Leu Asn 35 40 45 Val Trp Gly Asn Thr Asn Val Cys Tyr Glu His Asn Ser Thr Phe His 50 55 60 Gly Leu Leu Glu Glu Gln Ala Gly Arg Thr Pro Asp Ala Thr Ala Leu 65 70 75 80 Leu Phe Gly Asp Glu Met Leu Thr Tyr Ala Glu Leu Asn Ala Lys Ala 85 90 95 Asn Gly Leu Ala Arg Arg Leu Arg Thr Gln Gly Ile Lys Thr Gly Asp 100 105 110 Leu Val Gly Leu Ile Ala Glu Arg Ser Leu Glu Met Ile Val Gly Ile 115 120 125 Tyr Gly Ile Met Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu 130 135 140 Tyr Pro Lys Glu Arg Ile Ser Tyr Met Leu Glu Asp Ser Gly Ala Lys 145 150 155 160 Leu Ile Leu Thr Gln Ala His Leu Leu Glu His Leu Gly Trp Thr Glu 165 170 175 Ser Val Leu Leu Leu Asp Glu Ser Ser Thr Tyr Asp Ala Asp Thr Ser 180 185 190 Lys Leu Glu Asp Thr Ala Gly Pro Asp Asp Leu Ala Tyr Val Ile Tyr 195 200 205 Thr Ser Gly Thr Thr Gly Gln Pro Lys Gly Val Leu Val Glu His Arg 210 215 220 Gly Leu Pro Asn Leu Ser Asp Val Tyr Gly Ala His Phe Glu Val Thr 225 230 235 240 Pro Gln Asp Arg Ile Val Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser 245 250 255 Val Ser Glu Ile Leu Thr Ala Leu Ser His Gly Gly Val Leu Cys Ile 260 265 270 Pro Ser Thr Gln Asp Ile Leu Asp His Val Leu Phe Glu Gln Phe Met 275 280 285 Asn Asp Lys Gly Ile Thr Val Ala Thr Leu Pro Pro Ala Tyr Ala Ile 290 295 300 His Leu Asp Pro Glu Arg Leu Pro Thr Leu Arg Cys Leu Leu Thr Ala 305 310 315 320 Gly Ser Ala Ala Ser Val Glu Leu Ile Glu Glu Trp Arg Lys His Val 325 330 335 Arg Tyr Ser Asn Gly Tyr Gly Pro Thr Glu Asp Ser Val Cys Thr Thr 340 345 350 Ile Trp Ser Val Pro Asp Ser Glu Gly Ala Thr Asp Ile Val Ser Ile 355 360 365 Gly Arg Pro Ile Ala Asn His Ser Val Tyr Ile Leu Asp Asp His Phe 370 375 380 Arg Leu Gln Pro Val Gly Val Ala Gly Glu Leu Cys Ile Ser Gly Ile 385 390 395 400 Gly Leu Ala Arg Gly Tyr His Asn Gln Pro Glu Leu Met Asp Glu Lys 405 410 415 Phe Val Asp Asn Pro Phe Ala Pro Gly Glu Arg Met Tyr Arg Thr Gly 420 425 430 Asp Leu Val Arg Trp Leu Pro Asn Gly Thr Ile Glu Tyr Leu Gly Arg 435 440 445 Ile Asp His Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu 450 455 460 Val Glu Ala Gln Ile Leu Arg Val Pro Ser Val Gln Glu Val Val Ala 465 470 475 480 Met Ala Val Glu Gly Asp Asp Gly Tyr Lys Asp Leu Val Ala Tyr Phe 485 490 495 Val Ala Ala Gln Lys Leu Glu Val Ser Glu Leu Arg Ala Val Leu Ser 500 505 510 Glu Met Leu Pro Gly Tyr Met Ile Pro Ser Arg Phe Ile Gln Leu Asp 515 520 525 Asp Met Pro Leu Thr Ser Asn Gly Lys Val Asp Arg Lys Ala Leu Gln 530 535 540 Gly Glu Arg Gly Trp Ala 545 550 <210> 6 <211> 550 <212> PRT <213> Paenibacillus polymyxa <400> 6 Asn Ala Leu Val Tyr Asp Pro Ala Thr Ile Glu Gln Ile Lys Gly His 1 5 10 15 Leu Phe His Leu Met Glu Gln Ile Val Glu Asn Pro Ala Ile Ser Val 20 25 30 Asp Ala Leu Glu Leu Val Thr Pro Gln Glu Arg Glu Gln Ile Leu Ser 35 40 45 Val Trp Gly Glu Thr Glu Ala Ser Ser Lys His Arg Thr Thr Phe His 50 55 60 Gly Leu Leu Glu Glu Gln Ala Ala Arg Thr Pro Asp Ala Thr Ala Ile 65 70 75 80 Leu Phe Glu Asn Glu Met Leu Thr Tyr Ala Glu Leu Asn Ala Lys Ala 85 90 95 Asn Gly Leu Ala Arg Arg Leu Arg Ala Glu Gly Ile Lys Thr Gly Asp 100 105 110 Leu Val Gly Leu Leu Val Glu Arg Ser Thr Asp Met Ile Val Gly Met 115 120 125 Tyr Gly Ile Met Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu 130 135 140 Tyr Pro Lys Glu Arg Ile Asn Tyr Met Leu Glu Asp Ser Gly Thr Lys 145 150 155 160 Met Ile Leu Thr Gln Ala His Leu Leu Glu His Ile Gly Trp Met Gly 165 170 175 Asn Val Leu Leu Leu Glu Glu Pro Ser Thr Tyr Asp Ala Asp Glu Ser 180 185 190 Asn Leu Lys Asp Thr Ala Asp Ser Asp Asp Leu Ala Tyr Val Ile Tyr 195 200 205 Thr Ser Gly Thr Thr Gly Gln Pro Lys Gly Val Leu Val Glu His Arg 210 215 220 Gly Leu Arg Asn Leu Ser Asp Val Tyr Arg Gly Leu Phe Glu Val Thr 225 230 235 240 Pro Gln Asp Arg Ile Val Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser 245 250 255 Val Ser Glu Ile Ile Thr Ala Leu Ser His Gly Ala Thr Leu Cys Ile 260 265 270 Pro Ser Thr Gln Asp Ile Leu Asp His Ala Leu Phe Glu Gln Phe Met 275 280 285 Asn Ser Lys Ala Ile Thr Ile Ala Thr Leu Pro Pro Ala Tyr Ile Ile 290 295 300 His Leu Glu Pro Glu Arg Leu Pro Ala Leu Arg Cys Leu Leu Thr Ala 305 310 315 320 Gly Ser Ala Thr Ser Val Glu Leu Ile Glu Lys Trp Arg Lys His Val 325 330 335 Gln Tyr Phe Asn Gly Tyr Gly Pro Thr Glu Asp Ser Val Cys Thr Thr 340 345 350 Met Trp Thr Val Pro Asp Ser Glu Glu Thr Met Glu Arg Val Ser Ile 355 360 365 Gly Gln Pro Ile Ala Asn His Arg Val Tyr Ile Leu Asp Asp His Phe 370 375 380 Arg Val Leu Pro Val Gly Val Ala Gly Glu Leu Cys Ile Ser Gly Ile 385 390 395 400 Gly Leu Ala Arg Gly Tyr His Asn Gln Pro Ala Leu Met Asp Glu Lys 405 410 415 Phe Val Asp Asn Pro Phe Thr Pro Gly Glu Arg Met Tyr Arg Thr Gly 420 425 430 Asp Leu Val Arg Trp Leu Pro Asp Gly Thr Ile Glu Tyr Leu Gly Arg 435 440 445 Ile Asp His Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu 450 455 460 Val Glu Ala His Met Leu Arg Val Pro Phe Val Gln Glu Val Val Ala 465 470 475 480 Leu Ala Val Glu Ser Glu Asp Gly Tyr Lys Asp Leu Val Ala Tyr Phe 485 490 495 Val Ala Ala Gln Lys Leu Glu Val Ser Glu Leu Arg Ala Val Leu Ser 500 505 510 Glu Met Leu Pro Gly Tyr Met Ile Pro Ser Arg Phe Val Gln Leu Glu 515 520 525 Asp Met Pro Leu Thr Ser Asn Gly Lys Ile Asp Arg Lys Ala Leu Gln 530 535 540 Gly Glu Gln Gly Trp Ala 545 550 <210> 7 <211> 550 <212> PRT <213> Paenibacillus polymyxa <400> 7 Asn Ala Leu Val Tyr Asp Pro Ser Thr Ile Glu Gln Ile Lys Gly His 1 5 10 15 Leu Phe His Leu Met Glu Gln Ile Val Glu Asn Pro Ala Ile Ser Val 20 25 30 Asp Ala Leu Glu Leu Val Thr Pro Gln Glu Arg Glu Gln Ile Leu Asn 35 40 45 Val Trp Gly Glu Thr Glu Ala Ser Ser Lys His Arg Thr Thr Phe His 50 55 60 Gly Leu Leu Glu Glu Gln Ala Ala Arg Thr Pro Asp Ala Thr Ala Ile 65 70 75 80 Leu Phe Glu Asn Glu Met Leu Thr Tyr Ala Glu Leu Asn Ala Lys Ala 85 90 95 Asn Gly Leu Ala Arg Arg Leu Arg Ala Glu Gly Ile Lys Thr Gly Asp 100 105 110 Leu Val Gly Leu Leu Val Glu Arg Ser Thr Asp Met Ile Val Gly Met 115 120 125 Tyr Gly Ile Met Lys Ala Gly Gly Ala Tyr Val Pro Met Asp Pro Glu 130 135 140 Tyr Pro Lys Glu Arg Ile Asn Tyr Met Leu Glu Asp Ser Gly Thr Lys 145 150 155 160 Met Ile Leu Ala Gln Ala His Leu Leu Glu His Ile Asp Trp Met Gly 165 170 175 Asn Val Leu Leu Leu Glu Glu Pro Ser Thr Tyr Asp Ala Asp Glu Ser 180 185 190 Asn Leu Lys Asp Thr Ala Asn Ser Asp Asp Leu Ala Tyr Val Ile Tyr 195 200 205 Thr Ser Gly Thr Thr Gly Gln Pro Lys Gly Val Leu Val Glu His Arg 210 215 220 Gly Leu Arg Asn Leu Ser Asp Val Tyr Arg Gly Leu Phe Glu Val Thr 225 230 235 240 Pro Gln Asp Arg Ile Val Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser 245 250 255 Val Ser Glu Ile Ile Thr Ala Leu Ser His Gly Ala Thr Leu Cys Ile 260 265 270 Pro Ser Thr Gln Asp Ile Leu Asp His Ala Leu Phe Glu Gln Phe Met 275 280 285 Asn Ser Lys Ala Ile Thr Ile Ala Thr Leu Pro Pro Ala Tyr Ile Ile 290 295 300 His Leu Glu Pro Glu Arg Leu Pro Ala Leu Arg Cys Leu Leu Thr Ala 305 310 315 320 Gly Ser Ala Thr Ser Val Glu Leu Ile Glu Lys Trp Arg Lys His Val 325 330 335 Gln Tyr Phe Asn Gly Tyr Gly Pro Thr Glu Asp Ser Val Cys Thr Thr 340 345 350 Met Trp Thr Val Pro Asp Ser Glu Glu Thr Met Glu Arg Val Ser Ile 355 360 365 Gly Gln Pro Ile Ala Asn His Arg Val Tyr Ile Leu Asp Asp His Phe 370 375 380 Arg Val Leu Pro Val Gly Val Ala Gly Glu Leu Cys Ile Ser Gly Ile 385 390 395 400 Gly Leu Ala Arg Gly Tyr His Asn Gln Pro Ala Leu Met Asp Glu Lys 405 410 415 Phe Val Asp Asn Pro Phe Thr Pro Gly Glu Arg Met Tyr Arg Thr Gly 420 425 430 Asp Leu Val Arg Trp Leu Pro Asp Gly Thr Ile Glu Tyr Leu Gly Arg 435 440 445 Ile Asp His Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu 450 455 460 Val Glu Ala His Met Leu Arg Val Pro Phe Val Gln Glu Val Val Ala 465 470 475 480 Leu Ala Val Glu Ser Glu Asp Gly Tyr Lys Asp Leu Val Ala Tyr Phe 485 490 495 Val Ala Ala Gln Lys Leu Glu Val Ser Glu Leu Arg Ala Val Leu Ser 500 505 510 Glu Met Leu Pro Gly Tyr Met Ile Pro Ser Arg Phe Val Gln Leu Glu 515 520 525 Asp Met Pro Leu Thr Ser Asn Gly Lys Ile Asp Arg Lys Ala Leu Gln 530 535 540 Gly Glu Gln Gly Trp Ala 545 550 <210> 8 <211> 550 <212> PRT <213> Paenibacillus polymyxa <400> 8 Asn Ala Leu Val Tyr Asp Pro Ala Thr Ile Glu Gln Ile Lys Gly His 1 5 10 15 Leu Phe His Leu Met Glu Gln Ile Val Glu Asn Pro Ala Ile Ser Val 20 25 30 Asp Ala Leu Glu Leu Val Thr Pro Gln Glu Arg Glu Gln Ile Leu Asn 35 40 45 Val Trp Gly Asp Thr Gly Ala Ser Ser Lys His Arg Thr Thr Phe His 50 55 60 Gly Leu Leu Glu Glu Gln Ala Gly Arg Thr Pro Asp Ala Thr Ala Ile 65 70 75 80 Val Phe Glu Asn Glu Val Leu Thr Tyr Ala Glu Leu Asn Ala Lys Ala 85 90 95 Asn Gly Leu Ala Arg Arg Leu Arg Ala Glu Gly Ile Lys Thr Gly Asp 100 105 110 Leu Val Gly Leu Ile Val Glu Arg Ser Thr Asp Met Ile Val Gly Met 115 120 125 Tyr Gly Ile Met Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu 130 135 140 Tyr Pro Lys Glu Arg Ile Asn Tyr Met Leu Glu Asp Ser Gly Thr Lys 145 150 155 160 Met Ile Leu Ala Gln Ala His Leu Leu Glu His Arg Gly Trp Thr Gly 165 170 175 Asn Val Leu Leu Leu Asp Glu Pro Ser Thr Tyr Asp Ala Asp Thr Ser 180 185 190 Asn Leu Lys Asp Thr Ala Asp Pro Asp Asp Leu Ala Tyr Val Ile Tyr 195 200 205 Thr Ser Gly Thr Thr Gly Arg Pro Lys Gly Val Leu Val Glu His Arg 210 215 220 Gly Leu Gln Asn Leu Ser Asp Val Tyr Arg Gly Leu Phe Glu Val Thr 225 230 235 240 Pro Gln Asp Arg Ile Val Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser 245 250 255 Val Ser Glu Ile Leu Thr Thr Leu Ser His Gly Ala Thr Leu Cys Ile 260 265 270 Pro Ser Thr Gln Glu Ile Leu Asp His Ala Leu Phe Glu Gln Phe Met 275 280 285 Asn Asp Lys Gly Ile Thr Val Ala Thr Leu Pro Pro Ala Tyr Ala Ile 290 295 300 His Leu Glu Pro Glu Arg Leu Pro Thr Leu Arg Cys Leu Leu Thr Ala 305 310 315 320 Gly Ser Ala Thr Ser Val Glu Leu Ile Glu Lys Trp Arg Lys His Val 325 330 335 Gln Tyr Phe Asn Ala Tyr Gly Pro Thr Glu Asp Ser Val Cys Thr Thr 340 345 350 Ile Trp Asn Ala Gln Asn Ser Glu Glu Thr Val Gly Ile Val Ser Ile 355 360 365 Gly Gln Pro Ile Ala Asn His Arg Val Tyr Ile Leu Asp Glu His Phe 370 375 380 Arg Leu Leu Pro Val Gly Val Ala Gly Glu Leu Cys Ile Ser Gly Ile 385 390 395 400 Gly Leu Ala Arg Gly Tyr His Asn Arg Pro Glu Leu Met Asp Glu Lys 405 410 415 Phe Val Asp Asn Pro Tyr Ala Pro Gly Glu Arg Met Tyr Arg Thr Gly 420 425 430 Asp Leu Val Arg Trp Leu Ser Asn Gly Thr Ile Glu Tyr Leu Gly Arg 435 440 445 Ile Asp His Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu 450 455 460 Val Glu Ala His Met Leu Arg Val Pro Ser Val Gln Glu Val Val Val 465 470 475 480 Leu Ala Val Glu Ser Asp Asp Gly Tyr Lys Asp Leu Val Ala Tyr Phe 485 490 495 Val Ala Ala Gln Lys Leu Glu Val Ser Glu Leu Arg Ala Asp Leu Ser 500 505 510 Glu Met Leu Pro Gly Tyr Met Ile Pro Ser Arg Phe Ile Gln Leu Glu 515 520 525 Asp Met Pro Leu Thr Ser Asn Gly Lys Ile Asp Arg Lys Ala Leu Gln 530 535 540 Gly Glu Arg Gly Trp Ala 545 550 <210> 9 <211> 550 <212> PRT <213> Paenibacillus polymyxa <400> 9 Asn Ala Leu Val Tyr Asp Pro Ala Thr Ile Glu Gln Ile Lys Gly His 1 5 10 15 Leu Phe His Leu Met Glu Gln Ile Val Glu Asn Pro Ala Ile Ser Val 20 25 30 Asp Ala Leu Glu Leu Val Thr Pro Gln Glu Arg Glu Gln Ile Leu Asn 35 40 45 Val Trp Gly Asp Thr Gly Ala Ser Ser Lys His Arg Thr Thr Phe His 50 55 60 Gly Leu Leu Glu Glu Gln Ala Gly Arg Thr Pro Asp Ala Thr Ala Ile 65 70 75 80 Val Phe Glu Asn Glu Val Leu Thr Tyr Ala Glu Leu Asn Ala Lys Ala 85 90 95 Asn Gly Leu Ala Arg Arg Leu Arg Ala Glu Gly Ile Lys Thr Gly Asp 100 105 110 Leu Val Gly Leu Ile Val Glu Arg Ser Thr Asp Met Ile Val Gly Met 115 120 125 Tyr Gly Ile Met Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu 130 135 140 Tyr Pro Lys Glu Arg Ile Asn Tyr Met Leu Glu Asp Ser Gly Thr Lys 145 150 155 160 Met Ile Leu Ala Gln Ala His Leu Leu Glu His Arg Gly Trp Thr Gly 165 170 175 Asn Val Leu Leu Leu Asp Glu Pro Ser Thr Tyr Asp Ala Asp Thr Ser 180 185 190 Asn Leu Lys Asp Thr Ala Asp Pro Asp Asp Leu Ala Tyr Val Ile Tyr 195 200 205 Thr Ser Gly Thr Thr Gly Arg Pro Lys Gly Val Leu Val Glu His Arg 210 215 220 Gly Leu Gln Asn Leu Ser Asp Val Tyr Arg Gly Leu Phe Glu Val Thr 225 230 235 240 Pro Gln Asp Arg Ile Val Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser 245 250 255 Val Ser Glu Ile Leu Thr Thr Leu Ser His Gly Ala Thr Leu Cys Ile 260 265 270 Pro Ser Thr Gln Glu Ile Leu Asp His Ala Leu Phe Glu Gln Phe Met 275 280 285 Asn Asp Lys Gly Ile Thr Val Ala Thr Leu Pro Pro Ala Tyr Ala Ile 290 295 300 His Leu Glu Pro Glu Arg Leu Pro Thr Leu Arg Cys Leu Leu Thr Ala 305 310 315 320 Gly Ser Ala Thr Ser Val Glu Leu Ile Glu Lys Trp Arg Lys His Val 325 330 335 Gln Tyr Phe Asn Ala Tyr Gly Pro Thr Glu Asp Ser Val Cys Thr Thr 340 345 350 Ile Trp Asn Ala Gln Asn Ser Glu Glu Thr Val Gly Ile Val Ser Ile 355 360 365 Gly Gln Pro Ile Ala Asn His Arg Val Tyr Ile Leu Asp Glu His Phe 370 375 380 Arg Leu Leu Pro Val Gly Val Ala Gly Glu Leu Cys Ile Ser Gly Ile 385 390 395 400 Gly Leu Ala Arg Gly Tyr His Asn Arg Pro Glu Leu Met Asp Glu Lys 405 410 415 Phe Val Asp Asn Pro Tyr Ala Pro Gly Glu Arg Met Tyr Arg Thr Gly 420 425 430 Asp Leu Val Arg Trp Leu Ser Asn Gly Thr Ile Glu Tyr Leu Gly Arg 435 440 445 Ile Asp His Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu 450 455 460 Val Glu Ala His Met Leu Arg Val Pro Ser Val Gln Glu Val Val Val 465 470 475 480 Leu Ala Val Glu Ser Asp Asp Gly Tyr Lys Asp Leu Val Ala Tyr Phe 485 490 495 Val Ala Ala Gln Lys Leu Glu Val Ser Glu Leu Arg Ala Asp Leu Ser 500 505 510 Glu Met Leu Pro Gly Tyr Met Ile Pro Ser Arg Phe Ile Gln Leu Glu 515 520 525 Asp Met Pro Leu Thr Ser Asn Gly Lys Ile Asp Arg Lys Ala Leu Gln 530 535 540 Gly Glu Arg Gly Trp Ala 545 550 <210> 10 <211> 250 <212> PRT <213> Paenibacillus sp. <400> 10 Asn Thr Leu Val Tyr Asp Ser Ser Asn Ile Glu Arg Ile Arg Gly His 1 5 10 15 Leu Val His Leu Met Glu Gln Ile Val Lys Asn Pro Gly Ile Ser Val 20 25 30 Asp Ala Leu Glu Leu Val Thr Pro Gln Glu Arg Asp His Ile Leu Asn 35 40 45 Ile Trp Lys Asp Ile Ala Val Pro Tyr Glu His Tyr Ala Glu Leu His 50 55 60 Ala Gln Ala Gln Thr Ala Pro Ile Gly Gln Pro Asn Val Tyr Ile Val 65 70 75 80 Asp Asp His Phe Arg Leu Leu Pro Val Gly Val Ala Gly Glu Leu Cys 85 90 95 Ile Ala Gly Val Gly Phe Thr Arg Glu His His Asn His Pro Glu Leu 100 105 110 Thr Asp Glu Lys Phe Val Asp Asn Pro Phe Ala Pro Gly Glu Arg Met 115 120 125 Tyr Arg Thr Gly Asp Leu Ala Arg Trp Leu Pro Asp Gly Thr Ile Gln 130 135 140 Tyr Leu Gly Arg Val Asp His Gln Val Lys Ile Arg Gly Tyr Arg Val 145 150 155 160 Glu Leu Ser Glu Val Glu Ala Gln Met Leu Lys Val Gln Ser Val Gln 165 170 175 Asp Val Val Val Met Ala Val Glu Gly Asp Asp Gly His Lys Asp Leu 180 185 190 Phe Ala Tyr Phe Val Ala Asp Gln Thr Ile Glu Ile Ser Glu Leu Arg 195 200 205 Ala Val Leu Ser Glu Leu Leu Pro Val Tyr Met Ile Pro Ser His Phe 210 215 220 Val Gln Leu Glu Asn Pro Leu Leu Thr Pro Ser Gly Lys Ile Asp Arg 225 230 235 240 Lys Ala Leu Gln Gly Glu Arg Gly Trp Ala 245 250 <210> 11 <211> 550 <212> PRT <213> Paenibacillus sp. <400> 11 Asn Ala Leu Val Tyr Asp Gln Val Thr Ile Glu Gln Ile Lys Gly His 1 5 10 15 Leu Val His Leu Met Glu Gln Ile Val Glu Asn Pro Ala Ile Ser Val 20 25 30 Asp Ala Leu Glu Leu Val Thr Pro Gln Glu Arg Glu Leu Ile Leu Asp 35 40 45 Val Trp Gly Asn Thr Lys Val Ser Tyr Glu His Cys Asn Thr Phe His 50 55 60 Gly Leu Leu Glu Glu Gln Ala Gly Arg Thr Pro Glu Ala Thr Ala Ile 65 70 75 80 Val Phe Glu Asp Glu Met Leu Thr Tyr Ala Glu Leu Asn Ala Lys Ala 85 90 95 Asn Gly Leu Ala Arg Lys Leu Arg Asn Gln Gly Ile Gln Thr Gly Asp 100 105 110 Leu Val Gly Leu Ile Ala Asp Arg Ser Ser Glu Met Ile Val Gly Ile 115 120 125 Tyr Gly Ile Met Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu 130 135 140 Tyr Pro Lys Glu Arg Ile Ser Tyr Met Leu Glu Asp Ser Gly Ala Lys 145 150 155 160 Leu Val Leu Thr Gln Ala Arg Leu Leu Glu His Leu Gly Trp Thr Glu 165 170 175 Asn Val Leu Leu Leu Asp Glu Pro Ser Thr Tyr Asp Ala Asp Thr Ser 180 185 190 Asn Leu Lys Asp Thr Val Gly Pro Asp Asn Leu Ala Tyr Val Ile Tyr 195 200 205 Thr Ser Gly Thr Thr Gly Gln Pro Lys Gly Val Leu Val Glu His Arg 210 215 220 Gly Leu Gln Asn Leu Ser Asp Val Tyr Gly Thr Tyr Phe Glu Val Thr 225 230 235 240 Pro Gln Asp Arg Ile Val Gln Phe Ala Ser Leu Ser Phe Asp Ala Ser 245 250 255 Val Ser Glu Val Leu Thr Ala Leu Ser His Gly Ala Ala Leu Cys Ile 260 265 270 Pro Ser Thr Gln Asp Ile Leu Asp Tyr Ala Leu Phe Glu Gln Phe Ile 275 280 285 Asn Asp Lys Gly Ile Thr Ile Ala Thr Leu Pro Pro Ala Tyr Ala Ile 290 295 300 His Leu Glu Pro Glu Arg Leu Pro Ala Leu Arg Cys Leu Leu Thr Ala 305 310 315 320 Gly Ser Ala Ala Ser Val Glu Leu Ile Glu Lys Trp Arg Lys His Val 325 330 335 Arg Tyr Ser Asn Gly Tyr Gly Pro Thr Glu Asp Ser Ile Cys Thr Thr 340 345 350 Ile Trp Ser Val Pro Asp Ser Glu Glu Thr Leu Glu Thr Val Ser Ile 355 360 365 Gly Arg Pro Ile Ala Asn His Ser Val Tyr Val Leu Asp Glu His Leu 370 375 380 Arg Leu Gln Pro Val Gly Val Val Gly Glu Leu Cys Ile Ser Gly Ile 385 390 395 400 Gly Leu Ala Arg Gly Tyr His Asn Arg Pro Ala Leu Met Asp Glu Lys 405 410 415 Phe Val Glu Asn Pro Phe Thr Pro Gly Glu Arg Met Tyr Arg Thr Gly 420 425 430 Asp Leu Val Arg Trp Leu Pro Asn Gly Thr Ile Glu Tyr Val Gly Arg 435 440 445 Ile Asp His Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Leu Gly Glu 450 455 460 Val Glu Ala Gln Met Leu Arg Val Gln Ser Val Gln Glu Val Val Ala 465 470 475 480 Met Ala Val Glu Gly Asp Asp Gly Gln Lys Asp Leu Val Ala Tyr Phe 485 490 495 Val Ala Ala Arg Glu Leu Glu Val Ser Glu Leu Gln Thr Val Leu Ser 500 505 510 Glu Met Leu Pro Gly Tyr Met Ile Pro Ser Arg Phe Ile Gln Leu Glu 515 520 525 Asp Met Pro Leu Thr Ser Asn Gly Lys Ile Asn Arg Lys Ala Leu Gln 530 535 540 Gly Glu Arg Gly Trp Ala 545 550 <210> 12 <211> 804 <212> DNA <213> Paenibacillus sp. <400> 12 ttgcaaaaaa ttgaggtatt gttggctgat gacaaccggg aatttacgaa tctgcttgcc 60 gaatatattt ccgatcagga gcatatggaa gttacaggaa tcgcctataa tggtgaagaa 120 gtgctccaac acatcgcaga atcccgcaac gtacctgatg tacttatttt agatattatc 180 atgcctcatc tggatggtct cggcgtattg gagcgcttga gagaaatgaa cctgtctcca 240 cagccgaaaa tcattatgct gactgcattc ggtcaagaaa atattacgca aagagccgta 300 cagctcgggg catcttatta tattttgaag ccgtttgaca tggaagtgct tcccaaccgt 360 gttcgtcaat tggtgggacc acaattagtc agcagcagtc cggtgacggt ttcttccatg 420 cggtctaatg tggtgccaat cggcaaaacg aaaaacctgg atgccagtat tacggccatt 480 atccatgaaa tcggtgtgcc agctcatatt aagggctatc aatatttacg cgaagccatt 540 actatcgtgt acaataatat cgaaattttg ggtgccatca ccaaaacatt atatcccgca 600 atcgccgaaa aatttaaaac gacggcatcc cgcgtggaac gcgccattcg tcatgccatc 660 gaggtagcat ggacacgtgg caacatcgac agcatctctc atctgttcgg ctacaccatt 720 aatatctcca aatccaagcc gaccaactca gagtttattg cgatggtagc tgacaagctt 780 cggattgaga ataaggtgtc ctga 804 <210> 13 <211> 804 <212> DNA <213> Paenibacillus sp. <400> 13 ttgcaaaaaa ttgaggtatt gttggctgat gacaaccggg aatttacgaa tctgcttgcc 60 gaatatattt ccgatcagga gcatatggaa gttacaggaa tcgcctataa tggtgaagaa 120 gtgctccaac acatcgcaga atcccgcaac gtacctgatg tacttatttt agatattatc 180 atgcctcatc tggatggtct cggcgtattg gagcgcttga gagaaatgaa cctgtctcca 240 cagccgaaaa tcattatgct gactgcattc ggtcaagaaa atattacgca aagagccgta 300 cagctcgggg catcttatta tattttgaag ccgtttgaca tggaagtgct tcccaaccgt 360 gttcgtcaat tggtgggacc acaattagtc agcagcagtc cggtgacggt ttcttccatg 420 cggtctaatg tggtgccaat cggcaaaacg aaaaacctgg atgccagtat tacggccatt 480 atccatgaaa tcggtgtgcc agctcatatt aagggctatc aatatttacg cgaagccatt 540 actatcgtgt acaataatat cgaaattttg ggtgccatca ccaaaacatt atatcccgca 600 atcgccgaaa aattaaaac gacggcatcc cgcgtggaac gcgccattcg tcatgccatc 660 gaggtagcat ggacacgtgg caacatcgac agcatctctc atctgttcgg ctacaccatt 720 aatatctcca aatccaagcc gaccaactca gagtttattg cgatggtagt tgacaagctt 780 cggattgaga ataaggtgtc ctga 804 <210> 14 <211> 267 <212> PRT <213> Paenibacillus terrae <400> 14 Met Gln Lys Ile Glu Val Leu Leu Ala Asp Asp Asn Arg Glu Phe Thr 1 5 10 15 Asn Leu Leu Ala Glu Tyr Ile Ser Asp Gln Glu Asp Met Glu Val Thr 20 25 30 Gly Ile Ala Tyr Asn Gly Glu Glu Val Leu Gln His Ile Ala Glu Ser 35 40 45 Arg Asn Val Pro Asp Val Leu Ile Leu Asp Ile Ile Met Pro His Leu 50 55 60 Asp Gly Leu Gly Val Leu Glu Arg Leu Arg Glu Met Asn Leu Ser Pro 65 70 75 80 Gln Pro Lys Ile Ile Met Leu Thr Ala Phe Gly Gln Glu Asn Ile Thr 85 90 95 Gln Arg Ala Val Gln Leu Gly Ala Ser Tyr Tyr Ile Leu Lys Pro Phe 100 105 110 Asp Met Glu Val Leu Ala Asn Arg Val Arg Gln Leu Val Gly Pro Gln 115 120 125 Leu Val Ser Ser Ser Pro Leu Thr Val Ser Ser Met Arg Ser Asn Val 130 135 140 Val Pro Met Gly Lys Thr Lys Asn Leu Asp Ala Ser Ile Thr Ala Ile 145 150 155 160 Ile His Glu Ile Gly Val Pro Ala His Ile Lys Gly Tyr Gln Tyr Leu 165 170 175 Arg Glu Ala Ile Thr Met Val Tyr Asn Asn Ile Glu Ile Leu Gly Ala 180 185 190 Ile Thr Lys Thr Leu Tyr Pro Ala Ile Ala Glu Lys Phe Lys Thr Thr 195 200 205 Ala Ser Arg Val Glu Arg Ala Ile Arg His Ala Ile Glu Val Ala Trp 210 215 220 Thr Arg Gly Asn Ile Asp Ser Ile Ser His Leu Phe Gly Tyr Thr Ile 225 230 235 240 Asn Ile Ser Lys Ser Lys Pro Thr Asn Ser Glu Phe Ile Ala Met Val 245 250 255 Ala Asp Lys Leu Arg Ile Glu Asn Lys Val Ser 260 265 <210> 15 <211> 267 <212> PRT <213> Paenibacillus sp. <400> 15 Leu Gln Lys Ile Glu Val Leu Leu Ala Asp Asp Asn Arg Glu Phe Thr 1 5 10 15 Asn Leu Leu Ala Glu Tyr Ile Ser Asp Gln Glu Asp Met Glu Val Thr 20 25 30 Gly Ile Ala Tyr Asn Gly Glu Glu Val Leu Gln His Ile Ala Glu Ser 35 40 45 Arg Asn Val Pro Asp Val Leu Ile Leu Asp Ile Ile Met Pro His Leu 50 55 60 Asp Gly Leu Gly Val Leu Glu Arg Leu Arg Glu Met Asn Leu Ser Pro 65 70 75 80 Gln Pro Lys Ile Ile Met Leu Thr Ala Phe Gly Gln Glu Asn Ile Thr 85 90 95 Gln Arg Ala Val Gln Leu Gly Ala Ser Tyr Tyr Ile Leu Lys Pro Phe 100 105 110 Asp Met Glu Val Leu Ala Asn Arg Val Arg Gln Leu Val Gly Pro Gln 115 120 125 Leo Val Ser Ser Ser Pro Val Thr Val Ser Ser Met Arg Ser Asn Val 130 135 140 Val Pro Met Gly Lys Thr Lys Asn Leu Asp Ala Ser Ile Thr Ala Ile 145 150 155 160 Ile His Glu Ile Gly Val Pro Ala His Ile Lys Gly Tyr Gln Tyr Leu 165 170 175 Arg Glu Ala Ile Thr Met Val Tyr Asn Asn Ile Glu Ile Leu Gly Ala 180 185 190 Ile Thr Lys Thr Leu Tyr Pro Ala Ile Ala Glu Lys Phe Lys Thr Thr 195 200 205 Ala Ser Arg Val Glu Arg Ala Ile Arg His Ala Ile Glu Val Ala Trp 210 215 220 Thr Arg Gly Asn Ile Asp Ser Ile Ser His Leu Phe Gly Tyr Thr Ile 225 230 235 240 Asn Ile Ser Lys Ser Lys Pro Thr Asn Ser Glu Phe Ile Ala Met Val 245 250 255 Ala Asp Lys Leu Arg Ile Glu Asn Lys Val Ser 260 265 <210> 16 <211> 267 <212> PRT <213> Paenibacillus sp. <400> 16 Leu Gln Lys Ile Glu Val Leu Leu Ala Asp Asp Asn Arg Glu Phe Thr 1 5 10 15 Asn Leu Leu Ala Glu Tyr Ile Ser Asp Gln Glu Asp Met Glu Val Thr 20 25 30 Gly Ile Ala Tyr Asn Gly Glu Glu Val Leu Gln His Ile Ala Glu Ser 35 40 45 Arg Asn Val Pro Asp Val Leu Ile Leu Asp Ile Ile Met Pro His Leu 50 55 60 Asp Gly Leu Gly Val Leu Glu Arg Leu Arg Glu Met Asn Leu Ser Pro 65 70 75 80 Gln Pro Lys Ile Ile Met Leu Thr Ala Phe Gly Gln Glu Asn Ile Thr 85 90 95 Gln Arg Ala Val Gln Leu Gly Ala Ser Tyr Tyr Ile Leu Lys Pro Phe 100 105 110 Asp Met Glu Val Leu Ala Asn Arg Val Arg Gln Leu Val Gly Pro Gln 115 120 125 Leo Val Ser Ser Ser Pro Val Thr Val Ser Ser Met Arg Ser Asn Val 130 135 140 Val Pro Met Gly Lys Thr Lys Asn Leu Asp Ala Ser Ile Thr Ala Ile 145 150 155 160 Ile His Glu Ile Gly Val Pro Ala His Ile Lys Gly Tyr Gln Tyr Leu 165 170 175 Arg Glu Ala Ile Thr Met Val Tyr Asn Asn Ile Glu Ile Leu Gly Ala 180 185 190 Ile Thr Lys Thr Leu Tyr Pro Ala Ile Ala Glu Lys Phe Lys Thr Thr 195 200 205 Ala Ser Arg Val Glu Arg Ala Ile Arg His Ala Ile Glu Val Ala Trp 210 215 220 Thr Arg Gly Asn Ile Asp Ser Ile Ser His Leu Phe Gly Tyr Thr Ile 225 230 235 240 Asn Ile Ser Lys Ser Lys Pro Thr Asn Ser Glu Phe Ile Ala Met Val 245 250 255 Val Asp Lys Leu Arg Ile Glu Asn Lys Val Ser 260 265 <210> 17 <211> 267 <212> PRT <213> Paenibacillus polymyxa <400> 17 Met Gln Lys Ile Glu Val Leu Leu Ala Asp Asp Asn Arg Glu Phe Thr 1 5 10 15 Asn Leu Leu Ala Glu Tyr Ile Ser Asp Gln Glu Asp Met Glu Val Thr 20 25 30 Gly Ile Ala Tyr Asn Gly Glu Glu Val Leu Gln Arg Ile Ala Glu Ser 35 40 45 Arg Asn Val Pro Asp Val Leu Ile Leu Asp Ile Ile Met Pro His Leu 50 55 60 Asp Gly Leu Gly Val Leu Glu Arg Leu Arg Glu Met Asn Leu Thr Pro 65 70 75 80 Gln Pro Lys Ile Ile Met Leu Thr Ala Phe Gly Gln Glu Asn Ile Thr 85 90 95 Gln Arg Ala Val Gln Leu Gly Ala Ser Tyr Tyr Ile Leu Lys Pro Phe 100 105 110 Asp Met Glu Val Leu Ala Asn Arg Val Arg Gln Leu Val Gly Pro Gln 115 120 125 Leu Val Ser Ser Ser Pro Val Thr Val Ser Ser Met Arg Ser Asn Val 130 135 140 Val Pro Met Gly Lys Thr Lys Asn Leu Asp Ala Ser Ile Thr Ala Ile 145 150 155 160 Ile His Glu Ile Gly Val Pro Ala His Ile Lys Gly Tyr Gln Tyr Leu 165 170 175 Arg Glu Ala Ile Thr Met Val Tyr Asn Asn Ile Glu Ile Leu Gly Ala 180 185 190 Ile Thr Lys Thr Leu Tyr Pro Ala Ile Ala Glu Lys Phe Lys Thr Thr 195 200 205 Ala Ser Arg Val Glu Arg Ala Ile Arg His Ala Ile Glu Val Ala Trp 210 215 220 Thr Arg Gly Asn Ile Asp Ser Ile Ser His Leu Phe Gly Tyr Thr Ile 225 230 235 240 Asn Ile Ser Lys Ser Lys Pro Thr Asn Ser Glu Phe Ile Ala Met Val 245 250 255 Val Asp Lys Leu Arg Ile Glu Asn Lys Val Ser 260 265 <210> 18 <211> 267 <212> PRT <213> Bacillus subtilis <400> 18 Met Glu Lys Ile Lys Val Cys Val Ala Asp Asp Asn Arg Glu Leu Val 1 5 10 15 Ser Leu Leu Ser Glu Tyr Ile Glu Gly Gln Glu Asp Met Glu Val Ile 20 25 30 Gly Val Ala Tyr Asn Gly Gln Glu Cys Leu Ser Leu Phe Lys Glu Lys 35 40 45 Asp Pro Asp Val Leu Val Leu Asp Ile Ile Met Pro His Leu Asp Gly 50 55 60 Leu Ala Val Leu Glu Arg Leu Arg Glu Ser Asp Leu Lys Lys Gln Pro 65 70 75 80 Asn Val Ile Met Leu Thr Ala Phe Gly Gln Glu Asp Val Thr Lys Lys 85 90 95 Ala Val Asp Leu Gly Ala Ser Tyr Phe Ile Leu Lys Pro Phe Asp Met 100 105 110 Glu Asn Leu Val Gly His Ile Arg Gln Val Ser Gly Asn Ala Ser Ser 115 120 125 Val Thr His Arg Ala Pro Ser Ser Gln Ser Ser Ile Ile Arg Ser Ser 130 135 140 Gln Pro Glu Pro Lys Lys Lys Asn Leu Asp Ala Ser Ile Thr Ser Ile 145 150 155 160 Ile His Glu Ile Gly Val Pro Ala His Ile Lys Gly Tyr Leu Tyr Leu 165 170 175 Arg Glu Ala Ile Ser Met Val Tyr Asn Asp Ile Glu Leu Leu Gly Ser 180 185 190 Ile Thr Lys Val Leu Tyr Pro Asp Ile Ala Lys Lys Phe Asn Thr Thr 195 200 205 Ala Ser Arg Val Glu Arg Ala Ile Arg His Ala Ile Glu Val Ala Trp 210 215 220 Ser Arg Gly Asn Ile Asp Ser Ile Ser Ser Leu Phe Gly Tyr Thr Val 225 230 235 240 Ser Met Thr Lys Ala Lys Pro Thr Asn Ser Glu Phe Ile Ala Met Val 245 250 255 Ala Asp Lys Leu Arg Leu Glu His Lys Ala Ser 260 265 <210> 19 <211> 264 <212> PRT <213> Bacillus cereus <400> 19 Met Glu Lys Ile Lys Val Cys Leu Val Asp Asp Asn Lys Glu Leu Val 1 5 10 15 Ser Met Leu Glu Ser Tyr Val Ala Ala Gln Asp Asp Met Glu Val Ile 20 25 30 Gly Thr Ala Tyr Asn Gly Gln Glu Cys Leu Asn Leu Leu Lys Asp Lys 35 40 45 Gln Pro Asp Val Leu Val Leu Asp Ile Ile Met Pro His Leu Asp Gly 50 55 60 Leu Ala Val Leu Glu Lys Met Arg His Ile Glu Arg Leu Arg Gln Pro 65 70 75 80 Ser Val Ile Met Leu Thr Ala Phe Gly Gln Glu Asp Val Thr Lys Lys 85 90 95 Ala Val Asp Leu Gly Ala Ser Tyr Phe Ile Leu Lys Pro Phe Asp Met 100 105 110 Glu Asn Leu Thr Ser His Ile Arg Gln Val Ser Gly Lys Ala Asn Ala 115 120 125 Thr Ile Lys Arg Pro Leu Pro Ser Phe Arg Ser Ala Thr Thr Val Asp 130 135 140 Gly Lys Pro Lys Asn Leu Asp Ala Ser Ile Thr Ser Ile Ile His Glu 145 150 155 160 Ile Gly Val Pro Ala His Ile Lys Gly Tyr Met Tyr Leu Arg Glu Ala 165 170 175 Ile Ser Met Val Tyr Asn Asp Ile Glu Leu Leu Gly Ser Ile Thr Lys 180 185 190 Val Leu Tyr Pro Asp Ile Ala Lys Lys Tyr Asn Thr Thr Ala Ser Arg 195 200 205 Val Glu Arg Ala Ile Arg His Ala Ile Glu Val Ala Trp Ser Arg Gly 210 215 220 Asn Ile Asp Ser Ile Ser Ser Leu Phe Gly Tyr Thr Val Ser Met Ser 225 230 235 240 Lys Ala Lys Pro Thr Asn Ser Glu Phe Ile Ala Met Val Ala Asp Lys 245 250 255 Leu Arg Leu Glu His Lys Ala Ser 260 <210> 20 <211> 273 <212> PRT <213> Clostridium pasteurianum <400> 20 Met Glu Tyr Ser Lys Ile Ser Val Leu Ile Ala Asp Asp Asn Lys Glu 1 5 10 15 Phe Cys Asn Ile Leu Asn Asp Tyr Leu Leu Asn Gln Ser Asp Ile Val 20 25 30 Val Val Gly Ile Ala Lys Asp Gly Ile Glu Ala Leu Lys Leu Ile Glu 35 40 45 Glu Lys Lys Pro Asp Leu Val Ile Leu Asp Ile Ile Met Pro Asn Met 50 55 60 Asp Gly Leu Val Val Leu Glu Lys Leu Ala Asn Ile Asn Ile Asp Pro 65 70 75 80 Val Pro Asn Val Ile Val Leu Ser Ala Val Gly Gln Asp Lys Ile Thr 85 90 95 Gln Arg Ala Ile Thr Leu Gly Ala Asp Tyr Tyr Val Val Lys Pro Phe 100 105 110 Asp Met Asp Val Phe Thr Lys Arg Ile Arg Gln Met Phe Asn Asn Thr 115 120 125 Ile Leu Asp Ser Glu Thr Lys Lys Thr Met Pro Ile Ser Glu Lys Ala 130 135 140 Ala Asp Val Lys Ile Ser Gln Ser Val Pro Leu Asp Leu Glu Asp Glu 145 150 155 160 Ser Ile Ile His Glu Ile Gly Val Pro Ala His Ile Lys Gly Tyr Met 165 170 175 Tyr Leu Arg Glu Ala Ile Asn Met Val Val Asp Asn Ile Glu Leu Leu 180 185 190 Ser Ala Val Thr Lys Glu Leu Tyr Pro Ser Ile Ala Lys Lys Tyr Asn 195 200 205 Thr Thr Ala Ser Arg Val Glu Arg Ala Ile Arg His Ala Ile Glu Val 210 215 220 Ala Trp Ser Arg Gly Gln Val Asp Thr Ile Asn Lys Leu Phe Gly Tyr 225 230 235 240 Thr Ile His Asn Gly Lys Gly Lys Pro Thr Asn Ser Glu Phe Ile Ala 245 250 255 Met Ile Ala Asp Lys Leu Arg Leu Lys Asn Lys Val Lys Asn Val Ala 260 265 270 Gln

Claims

1. A composition comprising a biopure culture of a fungicide Bacillus species strain, wherein the Bacillus species strain is Bacillus species strain NRRL B-50972 or Bacillus species strain NRRL B-67129.

2. The composition of claim 1, comprising the fermentation products of Bacillus subtilis strains NRRL B-50972 and NRRL B-67129.

3. The composition of claim 2, wherein the fermentation product does not contain polymyxin.

4. The composition of claim 3, wherein the fermentation product is a liquid preparation.

5. The composition of claim 4, wherein the liquid formulation is a suspension concentrate or an oil dispersion.

6. The composition of claim 4 or 5, comprising at least 1 × 10 4 CFU strain / ml liquid formulation.

7. The composition of claim 6, comprising 1% to 25% fermentation solids.

8. A method of treating plants to prevent and control diseases, wherein the method comprises applying an effective amount of the composition of any one of claims 1 to 7 to the plant and / or the location of the plant; The disease described therein is caused by fungi or bacteria; The fungus mentioned is selected from Alternaria alternifolia (Alternaria alternifolia) Alternaria alternata ), Solanine Alternaria ( Alternaria solani Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinerea Anthracnose ( ) Colletotrichum lagenarium ), Fusarium moniliforme ( Fusarium culmorum ), clematis spp. ( Phaeosphaeria nodorum ), Gramineae ( Zymoseptoria tritici ), Phytophthora infestans ( Phytophthora cryptogea ), pathogenic fungus ( Phytophthora infestans ), ultimate humic acid ( Pythium ultimum Rice blast fungus ( Magnaporthe oryzae Rice sheath blight pathogen ( Thanatephorus cucumeris ), Ustilago maydis var. oatum ( Ustilago segetum var. avenae ), Coriolis rubrum ( Uromyces appendiculatus ) and wheat leaf rust ( Puccinia triticina ); The bacteria mentioned are selected from Xanthomonas campestris (Xanthomonas campestris). Xanthomonas campestris ), Pseudomonas syringae ( Pseudomonas syringae ) and Erwinia carotene ( Erwinia carotovora ).

9. The method of claim 8, wherein the method comprises applying an effective amount of the composition of any one of claims 1 to 7 to a portion of the plant.

10. The method of claim 8 or 9, wherein the composition is a fermentation product of Bacillus subtilis strain NRRL B-50972 or Bacillus subtilis strain NRRL B-67129.

11. The method of claim 8 or 9, wherein the method comprises applying the composition to the leaf plant portion.

12. The method of claim 8 or 9, wherein the composition is prepared at a concentration of 1 × 10⁻⁶ per hectare. 10 Up to 1×10 12 Use Bacillus species NRRL B-50972 and Bacillus species NRRL B-67129 with colony forming units (CFU) for application.

13. The method of claim 8 or 9, wherein the composition is applied at a rate of 0.5 kg to 5 kg of fermentation solids per hectare.

14. A method of treating a plant to prevent or control a disease, wherein the method comprises applying an effective amount of the composition of any one of claims 1 to 7 to the plant and / or the location of the plant; wherein the disease is mildew or rust; The mold mentioned above refers to cucumber powdery mildew or grape powdery mildew. The rust disease mentioned is wheat leaf rust.

15. The method of claim 14, wherein the method comprises applying an effective amount of the composition of any one of claims 1 to 7 to a portion of the plant.

16. The method of any one of claims 8, 9, 14 or 15, wherein the composition has residual activity, such that it controls fungi for at least 5 days after application to the plant, a portion of the plant and / or a location of the plant.

17. The method of claim 16, wherein the composition has residual activity, such that it controls fungi for at least 10 days after application to the plant, a portion of the plant, and / or a location of the plant.

18. The method of claim 17, wherein the composition has residual activity, such that it controls fungi for at least 15 days after application to the plant, a portion of the plant, and / or a location of the plant.

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