NOVEL PAENIBACILLUS STRAINS, ANTIFUNGAL COMPOUNDS, AND METHODS FOR THEIR USE

Abstract

To provide anti-fungal compounds and methods of treating plants to control plant diseases.SOLUTION: The present invention relates to a composition comprising a biologically pure culture of a fungicidal strain of Paenibacillus sp. having a variant fusaricidin synthetase lacking a functional adenylation domain in the third module. The present invention also provides a composition comprising a biologically pure culture of a fungicidal Paenibacillus sp. strain or a cell-free extract thereof comprising at least one Paeniserine and at least one Paeniprolixin. Also provided are isolated compounds and methods of treating a plant to control a plant disease with the disclosed compositions and compounds.SELECTED DRAWING: Figure 14

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 138,765, filed March 26, 2015. No. 62 / 232,205, filed September 24, 2015. The contents of both patent applications are incorporated herein by reference in their entirety. is incorporated herein by reference.

[0002] Reference to an electronically submitted sequence listing The official copy of the sequence listing is 68 kilobytes long and was created on March 21, 2016. The file "BCS159002WO_ST25.txt" with the same size as the ASCII submitted electronically via EFS-Web as a sequence listing of the mat and contemporaneously herewith. The sequence listing contained in this ASCII formatted document is incorporated herein by reference in its entirety. No. 6,119,523, filed on Dec. 1, 2007, which is incorporated herein by reference in its entirety.

[0003] The present invention relates to bacterial strains and their ability to control plant diseases. In particular, the present invention relates to: Paenibacillus species have relatively high levels of broad-spectrum antifungal activity. The target is a strain of C. cillus sp. [Background technology]

[0004] Fungicides are used in crop protection; as food, feed, and cosmetic preservatives; and in human and veterinary medicine. It has myriad uses, including use as a therapeutic agent for both developed and developing countries. In the region, reduced crop yields, foodborne diseases and fungal infections in both humans and animals are a problem. This is the topic.

[0005] Synthetic insecticides or fungicides are often non-specific and therefore may not react with other naturally occurring They can act on non-target organisms, including beneficial organisms that are synergistic. Adult insecticides or fungicides can also be toxic and non-biodegradable. Consumers worldwide are increasingly using them in their food products, especially in their food. are increasingly aware of potential environmental and health issues associated with residual chemicals in Therefore, consumer demands to reduce the use or at least the amount of chemical (i.e., synthetic) pesticides are increasing. Therefore, it is important to maintain a high level of food chain protection while still allowing effective pest control. You need to manage requirements.

[0006] A further problem that arises with the use of synthetic insecticides or fungicides is the Repeated and exclusive application of pesticides often leads to the selection of resistant pathogens. Usually, such strains are also cross-resistant to other active ingredients with the same mode of action. Effective control of the pathogen with the active compound is then no longer possible. However, active ingredients with new mechanisms of action are difficult and expensive to develop.

[0007] Plant diseases are becoming more prevalent due to the risk of resistance development in pathogen populations and environmental and human health concerns. There is growing interest in identifying alternatives to synthetic insecticides and fungicides for managing biological The use of biological control agents is also an option.

[0008] Nonribosomal peptides such as fusaricidins are well recognized for their antibacterial properties. They are known to be effective against non-ribosomal steroids and are used in the field of crop protection. Peptides also have potential uses in biopharmaceuticals and other biotechnology applications. Fusaricidin can be isolated from Paenibacillus species and contains 15-guanidino It has a ring structure containing six amino acid residues in addition to 3-hydroxypentadecanoic acid. Isolated from Paenibacillus polymyxa The fusaricidins tested were LI-F03, LI-F04, LI-F05, LI-F07 and and LI-F08 (Kurusu K, Ohba K, Arai T and Fu kushima K., J. Antibiotics, 40:1506-1514,19 87), additional fusaricidins A, B, C, and D have been reported (Kajimura Y and Kaneda M., J. Antibiotics,49:129-13 5,1996;Kajimura Y and Kaneda M.,J.Antibi otics,50:220-228,1997).

[0009] Specific fusaricidins are found in Fusarium oxysporum orum), Aspergillus niger, Aspergillus Aspergillus oryzae and Penicillium tomi It has fungicidal activity against plant pathogenic fungi such as Penicillium thomii. Some fusaricidins are also known to have Against Gram-positive bacteria, including Staphylococcus aureus It has fungicidal activity (Kajimura Y and Kaneda M., J. Anti biotics,49:129-135,1996;Kajimura Y and K aneda M., J. Antibiotics, 50:220-228, 1997). Additionally, certain fusaricidins inhibit the growth of Leptosphaeria, which causes black root rot of canola. Antifungal activity against Leptosphaeria maculans (Beatty PH and Jensen SE., C an. J. Microbiol., 48:159-169, 2002). Fusaricidination The compound was further characterized and found to provide broad-spectrum antifungal activity at relatively low application rates. There is a need to identify strains of Paenibacillus species that produce these fusaricidins.

[0010] Fusaricidin and other antifungal metabolites can be obtained by fermentation of Paenibacillus species. However, many Paenibacillus species strains produce antibiotics known as polymyxins. Polymyxins are selectively toxic to Gram-negative bacteria and are effective against human diseases. When administered to humans, they may have neurotoxic or nephrotoxic effects. The global issue of relative toxicity of remyxins requires careful use and administration of these antibiotics. For this reason, Paenibacillus species strains developed for agricultural use express relatively high levels of fusaricidin and no detectable polymyxins. Such strains pose little or no risk to researchers and consumers. Furthermore, a Paenibacillus species strain exhibiting broad-spectrum activity was identified. The effectiveness of existing fungicides, especially those that are less susceptible to the development of fungal resistance, needs to be assessed. Improvement is highly desirable. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Kurusu K, Ohba K, Arai T and Fukushima K., J. Antibiotics, 40:1506-1514, 1987 [Non-patent document 2] Kajimura Y and Kaneda M., J. Antibiotics, 49:129-135, 1996 [Non-patent document 3] Kajimura Y and Kaneda M., J. Antibiotics, 50:220-228, 1997 [Non-patent document 4] Beatty PH and Jensen SE.,Can.J.Microbiol.,48:159-169,2002 Summary of the Invention [Means for solving the problem]

[0012] The present invention relates to a method for detecting adenylation domains in the third module (FusA-A3). Biological purity of fungicidal Paenibacillus sp. strains containing mutant fusaricidin synthetase. and a composition comprising the culture, wherein the lack of functional FusA-A3 is a wild-type fusaricidin. The amino acid residues were compared with the synthesis of fusaricidin by Paenibacillus species strains containing fusaricidin. (3) inhibits the synthesis of fusaricidins containing tyrosine or phenylalanine. In certain embodiments, the mutant fusaricidin synthetase is a substrate for FusA-A3. At least one, at least two, at least three, or a few of the amino acid residues that determine specificity At least four, at least five, at least six, at least seven, at least eight, In other embodiments, the amino acid residues are at least 9 or 10 deleted. sp235, Ala236, Ser239, Thr278, Leu299, Ala301 , Ala / Gly322, Val330, Cys331, Lys517, and these combinations thereof.

[0013] In one embodiment, the amino acid residues are 3203, 3204, 3205, 3206, 3207, 3208, 3209, 3210, 3211, 3212, 3213, 3214, 3215, 3216, 3217, 3218, 3219, 3220, 3221, 32 07, 3246, 3267, 3269, 3290, 3298, 3299 and / or 3 In another embodiment, the mutant fusaricidin synthetase is located at position 486. In sA-A3, Asp235, Ala236, Ser239, Thr278, and Leu 299, Ala301, Ala / Gly322, Val330, and Cys331 deletions In some embodiments, the mutant fusaricidin synthetase comprises a deletion of SEQ ID NO: Includes 10.

[0014] The present invention also provides biologically pure cultures or at least fungicidal Paenibacillus species strains. Another species of Paeniserine and at least one species of Paeniprol and compositions comprising these cell-free extracts containing Paeniprolixin. do.

[0015] In certain embodiments, the at least one paenicelin is paenicelin A1, paenicelin B1, paenicelin C2, paenicelin D3, paenicelin E4, paenicelin F5, paenicelin G6, paenicelin H7, paenicelin H8, paenicelin H9, paenicelin H10, paenicelin H11, paenicelin H12, paenicelin H13, paenicelin H14, paenicelin Paenicelin A2, Paenicelin A3, Paenicelin A4, Paenicelin B1, Paenicelin Paenicerin B2, Paenicerin B3, Paenicerin B4, Paenicerin C1, Paenicerin It is selected from the group consisting of nicerin C2 and paenicerin C3.

[0016] In other embodiments, the at least one paeniprolixin is paeniprolixin A 1. Paeniproxin A2, Paeniproxin B1, Paeniproxin B2 , Paeniproxin C1, Paeniproxin D1, Paeniproxin E1, Paeniproxin E2, Paeniproxin F1, Paeniproxin F2, Paeniproxin It is selected from the group consisting of eniprolixin G1 and paeniprolixin G2.

[0017] In certain embodiments, the composition comprises Fusaricidin A, LiF08a, Paenicelin A1, Paenitellin B1, Paeniprolixin A2 and Paeniprolixin B2 Includes:

[0018] In some embodiments, the composition comprises LiF03a, LiF03b, LiF03c , LiF03d, LiF07a, LiF07b, LiF07c and / or LiF07 In another embodiment, the composition comprises a synergistically effective amount of paenicelin A1 , Paeniprolixin B1, Paeniprolixin A2 and Paeniprolixin B2 nothing.

[0019] In one aspect, the present invention provides a method for the preparation of a Paenibacillus sp. strain comprising administering to a subject a Paenibacillus sp. strain selected from the group consisting of Paenibacillus sp. NRRL B -50972 strain, Paenibacillus species NRRL B-67129 strain, or these fungicidal The composition is a Paenibacillus sp. NRRL B-509 mutant. 72, Paenibacillus species NRRL B-67129, or their fungicidal mutants It may contain fermentation products of different strains.

[0020] In some embodiments, the fungicidal mutant strain is Paenibacillus sp. NRRL B It has a genome sequence with greater than 90% sequence identity to 50972. In an embodiment, the fungicidal mutant strain is Paenibacillus sp. NRRL B-50972. Equal or greater fungicidal activity and / or fusaricidin, paenicelin and / or or the level of paeniprolixin. does not contain polymyxin.

[0021] In some embodiments, the fermentation product is a liquid formulation. A liquid formulation may be a suspension concentrate or In one embodiment, the composition contains at least about 1×10 4 C In another embodiment, the composition comprises about 1% to about 25% of a strain / mL liquid formulation of FU. Contains fermentation solids.

[0022] In other embodiments, the present invention provides a method for producing a fusaricidin comprising: a) at least one fusaricidin; b) at least one The compound contains at least one species of paenitelin or at least one species of paeniprolixin in a synergistically effective amount. In one embodiment, the paenicelin is selected from the group consisting of paenicelin A1, paenicelin A2, paenicelin A3, paenicelin B4, paenicelin C5, paenicelin D6, paenicelin E7, paenicelin F8, paenicelin F9, paenicelin F10, paenicelin F11, paenicelin F12, paenicelin F13, paenicelin F14, paenicelin F15, Paenicelin A3, Paenicelin A4, Paenicelin B1, Paenicelin B2, Paenicelin Paenicelin B3, Paenicelin B4, Paenicelin C1, Paenicelin C2 and In another embodiment, the compound is at least one of paeniproloxamine C1 and paeniproloxamine C2. Paeniproxin A1, Paeniproxin A2, Paeniproxin B 1, Paeniproxin B2, Paeniproxin C1, Paeniproxin D1 , Paeniproxin E1, Paeniproxin E2, Paeniproxin F1, Paeniprolixin F2, Paeniprolixin G1 and Paeniprolixin G2 At least one of the following is true.

[0023] In particular, in one embodiment, at least one fusaricidin and at least one paenibacillus The synergistic ratio with serine or at least one paeniprolixin is 1:1000-1:100 0:1, preferably 1:500 to 500:1, more preferably 1:250 to 250 In another embodiment, the range of at least one fusaricin and at least The synergistic weight ratio with one paenicelin or at least one paeniprolixin is 1: in the range of 100 to 100:1, preferably in the range of 1:100 to 10:1 or even 1:5 In one embodiment, the fusaricidin is fusaricidin A. In another embodiment, the paenicelin is paenicelin A1. Paeniprolixin is Paeniprolixin C1.

[0024] In another aspect, the present invention provides a compound of structure (I): [ka]

[0025] (In the formula, R 1 and R 2 are each independently -CH(CH3)2 or -CH(CH3)CH2 CH3, R 3 is -CH2C(O)NH2 or -(CH2)2C(O)NH2, n is an integer between 13 and 20. and salts, hydrates, solvates, polymorphs, optical isomers thereof, It relates to geometric isomers, enantiomers, diastereomers, acyclic analogues, and mixtures.

[0026] In some embodiments, R 3is —CH2C(O)NH2. In another embodiment, Hey, R 3 is —(CH)C(O)NH. In one embodiment, R 1 -CH( In another embodiment, R 1 is -CH(CH3)CH2CH3. In an embodiment, R 2 is —CH(CH). In yet another embodiment, R 2 Ha-C H(CH3)CH2CH3.

[0027] In yet another aspect, the present invention provides a compound of structure (II): [ka]

[0028] (In the formula, R 1 is -CH2OH or -CH(OH)CH3 、 R 2 is —CH2C(O)NH2 or —(CH2)2C(O)NH2, and to R 3 is H or CH3, However, R 1 is -CHOH, and R 2 is -CH2C(O)NH2, R 3 is H) and salts, hydrates, solvates, polymorphs, optical isomers, and polymorphic forms thereof. What about isomers, enantiomers, diastereomers, acyclic analogs, and mixtures?

[0029] In some embodiments, R 3 is CH3. In another embodiment, R 3 is H In one embodiment, R 1 is —CHOH. In another embodiment, R1 Ha-CH (OH)CH3. In one embodiment, R 2 is -CH2C(O)NH2. In another embodiment, R 2 is -(CH2)2C(O)NH2.

[0030] In one embodiment, the present invention provides the isolated compounds and agriculturally acceptable The present invention relates to a composition comprising a carrier.

[0031] In certain embodiments, the present invention provides a compound of structure (I) in an amount of at least 0.001 m g / mL, at least 0.01 mg / mL, or at least 0.1 mg / mL In another embodiment, the present invention is directed to a solution comprising a compound of structure (II) at least 0.001 mg / mL, at least 0.01 mg / mL, or at least 0. In certain embodiments, the disclosed solutions are suitable for use in agricultural applications. The composition further comprises a suitable carrier.

[0032] In yet another embodiment, the present invention is a method for treating plants to control disease. and administering an effective amount of the composition disclosed herein to the plant, plant part, and / or plant locus. In certain embodiments, the composition comprises applying to a plant a Paenibacillus species. NRRL B-50972, Paenibacillus species NRRL B-67129, or In another embodiment, the method further comprises administering the composition to the leaves. In yet other embodiments, the composition comprises applying to plant parts of about 1 x 10 10 ~Approx. 1×10 12 Colony forming units (CFU) / hectare of Paenibacillus species NRRL B-50972, Paenibacillus species NRRL B-67129, or their sterilizing agents In one embodiment, the composition is applied in an amount of about 0.5 kg to about 5 kg. applied at g of fermentation solids / ha.

[0033] In some embodiments, the plant disease is caused by a fungus. In one embodiment, the plant disease is mold or rust. In another embodiment, the rust is wheat leaf rust, barley leaf rust, or downy mildew. leaf rust, rye leaf rust, red rust, crown rust and black rust. do.

[0034] In some embodiments, the fungus is Alternaria alternata (Alter naria alternata, Alternaria solani (Alternaria solani), Botrytis cinerea, Kore Colletotrichum lagenarium, Fusarium culmorum, Phaeophaea Phaeosphaeria nodorum, Zymoseptoria trichi Zymoseptoria tritici, Phytophthora cryptogaea Phytophthora cryptogea), Phytophthora infestans (Ph ytophthora infestans, Pythium ultimum m ultimum), Magnaporthe oryzae ), Thanatephorus cucumeris , Ustilago segetum var. avenae venae), Uromyces appendiculatus latus and Puccinia triticina is selected from the group consisting of:

[0035] In other embodiments, the plant disease is caused by a bacterium. The bacterium is Xanthomonas campestris s), Pseudomonas syringae and From the group consisting of Erwinia carotovora be selected.

[0036] The present invention also relates to the use of the disclosed compositions to control plant pathogenic organisms in useful plants. In certain embodiments, the plant pathogenic organism is Alternaria alternata (A Alternaria alternata, Alternaria solani (Alterna ria solani), Botrytis cinerea ), Colletotrichum lagenarium (Colletotrichum lagenari um), Fusarium culmorum, Phaeophyceae Phaeosphaeria nodorum, Zymoseptoria Zymoseptoria tritici, Phytophthora cryptogae Phytophthora cryptogea, Phytophthora infestans Phytophthora infestans, Pythium ultimum thium ultimum), Magnaporthe oryzae yzae), Thanatephorus cucume ris), Ustilago segetum var. avenae (Ustilago segetum v ar.avenae), Uromyces appen diculatus) and Puccinia tritic In another embodiment, the plant pathogenic organism is selected from the group consisting of Xanthomonas or Xanthomonas campestris, Pseudomonas Pseudomonas syringae and Erwinia mosquitoes and Erwinia carotovora.

[0037] In yet another embodiment, the useful plants are apples, bananas, citrus fruits, kiwis, melons , peaches, pears, pineapples, pome fruits, pomegranates, cabbage, cauliflower, cucumber, The plant is selected from the group consisting of corn, tomato, potato, wheat, rice and soybean. [Brief explanation of the drawings]

[0038] [Figure 1] Figure 1 shows the plant fungicidal activity of whole broths of Paenibacillus species strains against tomato late blight (PHYTIN), gray mold (BOTRCI), and wheat leaf rust (PUCCRT). [Figure 2]In vitro antifungal activity of fusaricidin extracts from the whole broth of Paenibacillus species strains against Alternaria alternata (ALTEAL), Botrytis cinerea (BOTRCI), Fusarium culmorum (FUSACU), Phaeophaea nodrum (LEPTNO), Zymoseptoria trisiti (SEPPTR), Phytophthora cryptogaea (PHYTCR), Phytophthora infestans (PHYTIN), Pythium ultimum (PYTHUL), Magneporthe oryzae (PYRIOR), Tanatephorus cucumeris (RHIZSO), Ustilago segetum var. avenae (USTIAV), and Uromyces appendiculatus (UROMAP) was demonstrated. [Figure 3] The cleavage of the ring structure of LiF04a (also known as fusaricidin A) to produce the acyclic analog, LiF04c, is shown. The acyclic analogs of each of fusaricidin and fusaricidin-like compounds arise in a similar manner. [Figure 4A] Figure 1 shows a diagram outlining the structures of known fusaricidins, with conserved amino acids identified at positions (1), (4), and (6), and amino acid changes indicated as AA (amino acid). The 15-guanidino-3-hydroxypentadecanoic acid (GHPD) tail 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 the arrow pointing to the "O." [Figure 4B] 1 shows an HPLC / MS TOF chromatogram of a cell extract of Paenibacillus species in which known fusaricidins have been identified. [Figure 4C] 1 shows known fusaricidins detectable in cell extracts from Paenibacillus sp. strain NRRL B-50972 and / or strains derived therefrom. [Figure 5A] A diagram outlining the structure of paenicelin is shown. This class of compounds is similar to fusaricidin, except that one or both of the conserved threonines at positions (1) and (4) are replaced with serine. [Figure 5B] 1 shows HPLC / MS TOF chromatograms of cell extracts from Paenibacillus sp. strain NRRL B-50972 and / or strains derived therefrom in which paenicelin was identified. [Figure 5C] 1 shows the paenicelin detectable in cell extracts from Paenibacillus sp. strain NRRL B-50972 and / or strains derived therefrom. m / z values ​​and retention times (RT) are shown for all detected compounds. [Figure 6A] The chemical structure of paenicelin A1 derived from the UPLC / MS Triple TOF spectrum shown in Figure 6B is shown. [Figure 6B] 1 shows the UPLC / MS Triple TOF spectrum of paenicelin A1. [Figure 7A] The chemical structure of paenicelin B1 derived from the UPLC / MS Triple TOF spectrum shown in Figure 7B is shown. [Figure 7B] 1 shows the UPLC / MS Triple TOF spectrum of paenicelin B1. [Figure 8A] A diagram outlining the structure of paeniprolixin is shown. This class of compounds is similar to fusaricidin, except that the length of the GHPD tail is extended from -(CH2)12- to -(CH2)14- or -(CH2)16-. [Figure 8B] 1 shows HPLC / MS TOF chromatograms of cell extracts from Paenibacillus sp. strain NRRL B-50972 and / or strains derived therefrom in which paeniprolixin was identified. [Figure 8C] 1 shows paeniprolixin detectable in cell extracts from Paenibacillus sp. strain NRRL B-50972 and / or strains derived therefrom. m / z values ​​and retention times (RT) are shown for all detected compounds. [Figure 9A] The chemical structure of paeniprolixin C1 derived from the UPLC / MS Triple TOF spectrum shown in Figure 9B is shown. [Figure 9B]1 shows the UPLC / MS Triple TOF spectrum of paeniprolixin C1. [Figure 10A] The chemical structure of paeniprolixin D1 derived from the UPLC / MS Triple TOF spectrum shown in Figure 10B is shown. [Figure 10B] 1 shows the UPLC / MS Triple TOF spectrum of paeniprolixin D1. [Figure 11] Kirby-Bauer antibiotic disk diffusion assay in which fusaricidins A and B ("AB"), paenicellins A1 and B1 ("868"), paeniprolixins A2 and B2 ("938"), or a combination of 868 and 938 were applied to Colletotrichum lagenarium (COLLLA) spores spread over an agar plate. The diameter of each disk, with a zone of inhibition of fungal growth, is shown in millimeters. [Figure 12] Figure 12A shows the chemical structure of fusaricidin A and a simplified depiction of this structure. Figures 12B-12E show simplified depictions of the combinations of fusaricidin, paenicellin, and / or paeniprolixin produced by Paenibacillus sp. NRRL B-50972 and strains derived therefrom. These combinations produce synergistic antifungal effects and are responsible for the relatively high potency and broad-spectrum antifungal activity observed with Paenibacillus sp. NRRL B-50972 and strains derived therefrom. [Figure 13]The following Paenibacillus spp. strains were tested: Paenibacillus peoriae A (SEQ ID NO: 1); Paenibacillus polymyxa A (SEQ ID NO: 2); Paenibacillus polymyxa PKB1 (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); Paenibacillus spp. NRRL Figure 1 shows a multiple sequence alignment of segments of the FusA fusaricidin synthetase expressed by the B-50972 strain (SEQ ID NO: 10); and the Paenibacillus sp. A strain (SEQ ID NO: 11). Amino acid residues that determine substrate specificity are identified with black outlines (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 NOs: 1-5 and 11, and at positions 3204, 3205, 3208, 3247, 3268, 3270, 3291, 3299, 3300, and 3487 in SEQ ID NOs: 6-9. [Figure 14]The fusaricidin gene cluster in Paenibacillus sp. strain NRRL B-50972 and Paenibacillus sp. strain A ("strain A"). Arrows represent individual genes within the cluster (i.e., fusG is represented by the arrow "G," fusF is represented by the arrow "F," etc.). The largest arrow represents the fusA fusaricidin synthetase gene, with the following abbreviations and symbols: A = adenylation domain (substrate recognition and activation); C = condensation domain (peptide bond formation); E = epimerization domain (substrate racemization); TE = thioesterase domain (product release); oval without 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. While strain A has a typical fusaricidin gene cluster, the Paenibacillus sp. NRRL B-50972 fusaricidin gene cluster lacks a functional A domain in module 3. As a result, fusaricidin produced by Paenibacillus sp. NRRL B-50972 lacks tyrosine and phenylalanine at position (3) and incorporates only valine or isoleucine. [Figure 15] 1 shows a sequence alignment of the spoOA gene in Paenibacillus sp. strain NRRL B-50972 (SEQ ID NO: 12) and Paenibacillus sp. strain NRRL B-67129 (SEQ ID NO: 13). [Figure 16]1 shows a sequence alignment of Spo0A orthologs from endospore-forming bacteria showing that nucleotide changes in the Paenibacillus sp. strain NRRL B-67129 coding sequence result in a single amino acid substitution in a conserved region. The aligned Spo0A orthologous sequences are: Paenibacillus terrae Spo0A (SEQ ID NO: 14), Paenibacillus sp. NRRL B-50972 Spo0A (SEQ ID NO: 15), Paenibacillus sp. NRRL B-67129 Spo0A (SEQ ID NO: 16), Paenibacillus polymyxa Spo0A (SEQ ID NO: 17), Bacillus subtilis Spo0A (SEQ ID NO: 18), Bacillus cereus Spo0A (SEQ ID NO: 19), and Clostridium pasteurianum Spo0A (SEQ ID NO: 20). [Figure 17] Figure 1 shows the 80% minimum inhibitory concentration (MIC80) values ​​of several fusaricidins, paenicellin, and paeniprolixin with the fungal pathogens Alternaria solani (ALTESO) and Colletotrichum lagenarium (COLLLA). DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention relates to Paenibacillus sp. NRRL B-50972 strain or a cytocidal compound derived therefrom. The fungal mutant (strain) Paenibacillus sp. NRRL B-50972 is , was found to have broad-spectrum activity against plant pathogens.

[0040] All of the microorganisms and specific strains described herein are naturally isolated unless otherwise specified. isolated and in shake flask cultures, or to maximize the production of bioactive metabolites, e.g. To achieve this, artificial conditions are created using scaled-up manufacturing processes such as bioreactors. Growth under these conditions results in the "acclimation" of the strain. Such "adapted" strains are not exposed to the selective pressures found in the natural environment, but rather to artificial selective pressures. They differ from their counterparts found in nature in that they are cultivated as homogeneous populations exposed to become.

[0041] As used herein, the term "isolated" refers to a culture enriched in whole broth or fermentation products. or concentrated, or partially or substantially derived from whole broth or fermentation product Refers to a purified compound.

[0042] In one embodiment, a mutant strain of Paenibacillus sp. NRRL B-50972 is provided. The term "mutant" refers to a strain derived from Paenibacillus sp. NRRL B-50972. In one embodiment, the mutant refers to a genetic variant induced in Paenibacillus sp. Possess one or more or all of the distinguishing (functional) characteristics of strain RRL B-50972. In certain instances, the mutant or its fermentation product may be (as a distinguishing functional characteristic) a fungus, an oomycete, and / or bacteria at least as similar as Paenibacillus species NRRL B-50972 strain These mutants are similar to Paenibacillus sp. strain NRRL B-50972. have greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% sequence identity The mutant may be a gene mutant having a genomic sequence that is The cells of the L B-50972 strain were treated with chemicals or irradiation, or or spontaneous mutants (Fu) from cell populations of Paenibacillus sp. NRRL B-50972 strain. by selecting mutants (e.g., phage-resistant or antibiotic-resistant mutants) or by methods known to those skilled in the art. It can be obtained by other means well known in the art.

[0043] Paenibacillus sp. NRRL B-50972 strain and its mutants have been shown to inhibit a wide range of plant growth. In one embodiment, the strain is active against cucumber anthracnose, cucumber Fungi such as powdery mildew, wheat leaf rust, barley powdery mildew and botrytis; Oomycetes such as late blight, cucumber downy mildew and brassica downy mildew; and / or spores Pseudomonas, Xanthomonas and It has activity against bacteria such as cerebrospinal fluid (CSF) and Erwinia.

[0044] In certain embodiments, the Paenibacillus species strain has a sequence which is at least 75% identical to SEQ ID NO: 10. identity, at least 80% sequence identity, at least 90% sequence identity, at least 9 5% sequence identity, at least 96% sequence identity, at least 97% sequence identity, DNA sequences that show at least 98% sequence identity, or at least 99% sequence identity include.

[0045] In certain embodiments, the present invention provides a method for the production of fusaricidin, paenicelin, and / or paenicelin. The present invention relates to a fermentation product containing a Paenibacillus species strain that produces prolixin. The 15-guanidino-3-hydroxypentadecanoic acid (GHPD) tail and these A family of depsipeptides with linear counterparts. Specific conserved properties of fusaricidins The GHPD tail and three of the six amino acids in the sequence, namely (1) threonine, (4) threonine, and (6) alanine.

[0046] It was originally published in the mid-1970s by Nakajima et al. (J. Antibiot. 1972, 2 5,243-247) but has not been characterized, and In the late 1980s, Kurusu et al. (J. Antibiot., 1987, 40, 15 Fusaricidin was first described by the researchers in the mid-1990s and 2000s. In the early 1990s, Kajimura et al. (J. Antibiot., 1996, 49, 12 9-135;J.Antibiot.,1997 50,220-228), Kurod a 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) During this period of intense research, these compounds were renamed several times depending on the author (Fusarisiji LiF04a, Gatavalin, or KT-6291A is also known as There are many publications on this topic, but from the same group of 24 known fusaricidins The selected compounds are described each time.

[0047] After a period of silence on this topic, Vater et al. (J. Am. Soc. Mass Spectrom.,2015,26,1130-1141) is a method for mass spectrometry We describe the structural elucidation of fusaricidin by α-hydroxybenzoates and some analogues of the family. Vater et al. found that 7 amino acids (i.e., (4) threonine residues in the peptide sequence) identified a novel class of fusaricidin-like compounds with an extra alanine linked to As used herein, the term "acyclic analog" refers to a fusaricidin or fusaricidin-like compound. corresponding to compounds (e.g., paenicelin or paeniprolixin) but lacking an ester bond. refers to compounds that give rise to linear structures.

[0048] The amino acid chain of fusaricidin is synthesized by nonribosomal peptide synthetases (NRPSs). Multi-domain NRPSs can contain up to 15,000 amino acids. It consists of 100 amino acids and is therefore considered to be one of the longest proteins in nature ( Schwarzer et al.,(2003)Nonribosomal Pept ides:From Genes to Products.Nat.Prod.Rep NRPS incorporation is essential for the translation of 21 proteins by ribosomes. This promiscuity is not limited to the standard amino acids of the ribosomal peptides, but accounts for the large structural diversity of nonribosomal peptides. contributes to the activity and biological activity of the plant (Li and Jensen, (2008)). Nonribosomal biosynthesis of fusaricidin by Lactobacillus polymyxa PKB1 is dependent on the d-amino acid This involves direct activation of the enzyme (Chem. Biol. 15, 118-127).

[0049] In P. polymyxa E68, the fusaricidin biosynthetic gene cluster ( fusGFEDC BA) has been characterized and is the largest coding DNA sequence (CDS) in the cluster, NRP It was observed that the S coding sequence encodes a six-module peptide (Choi et al. al.,Identification and Functional Analysis is of the Fusaricidin Biosynthetic Gene of Paenibacillus polymyxa E681.Biochem.B iophys.Res.Commun.365,89-95;Li and Jense n,Identification and Functional Analysis of the Fusaricidin Biosynthetic Gene of Paenibacillus polymyxa E681.Biochem.Bio phys.Res.Commun.365,89-95;Li et al.,(201 3).Promoter Analysis and Transcription R egulation of fus Gene Cluster Responsibl e for Fusaricidin Synthesis of Paenibaci llus polymyxa SQR-21.Appl.Microbiol.Biot Echnol.97,9479-9489). The biosynthetic cluster controls the biosynthesis of the lipid moiety. It contains other CDSs that carry the transporter genes but does not contain any transporter genes (Li and Jens en,(2008).Nonribosomal Biosynthesis of F usaricidins by Paenibacillus polymyxa PK B1 Involves Direct Activation of a d-ami no acid. Chem. Biol. 15, 118-127). In P. polymyxa, The promoter of the fus operon was identified and has been shown to be a regulator of sporulation. It has been shown that the transcriptional repressor AbrB is bound by this gene. This suggests that fusaricidin is synthesized during sporulation and regulates the secondary metabolism of microorganisms throughout their life cycle. It is interesting to note that the α-glucan is regulated by the α-glucan chain reaction (Li et al., (2013). omoter Analysis and Transcription Regula tion of fus Gene Cluster Responsible for Fusaricidin Synthesis of Paenibacillus polymyxa SQR-21.Appl.Microbiol.Biotechno l.97,9479-9489).

[0050] Allelic diversity is typically thought to be involved in the generation of chemical diversity. However, an interesting feature of the fus cluster is the incorporation of amino acids (Tyr, Val). Various fusaricidins with different nucleotides (Ile, allo-Ile, Phe) are expressed in a single fusA It can be produced by alleles, and the underlying mechanism involves amino acid recognition. The NRPS A domain, which plays a key role in the NRPS synthesis, relaxes substrate specificity (Han et al., (2012).Site-Directed Modification of the Adenylation Domain of the Fusaricidin N onribosomal Peptide Synthetase for Enhan ced Production of Fusaricidin Analogs.Bi otechnol.Lett.34,1327-1334;Mousa et al., (2015) Biodiversity of Genes Encoding Ant i-Microbial Traits within Plant Associat ed Microbes, Front Plant Sci.2015;6:231).

[0051] The structure of the A domain responsible for substrate recognition and activation in the fusA gene was determined by X-ray crystallography. The ten amino acid residues (Asp2) that determine the substrate specificity were determined from GrsA using 35,Ala236,Trp239,Thr278,Ile299,Ala301,Al a322, Ile330, Cys331 and Lys517) have been identified (Cha llis et al.,(2000)Predictive,Structure-B ased Model of Amino Acid Recognition by Nonribosomal Peptide Synthetase Adenylat ion Domains.Chem Biol 7:211-224;Stachelh aus et al.,(1999)The Specificity Conferrer ing Code of Adenylation Domains in Nonri bosomal Peptide Synthetases.Chem Biol 6: 493-505). These 10 signature residues are responsible for their functions within the substrate binding site. They can be divided into three subgroups based on their function: Asp235 and Lys5 17 interacts with the carboxyl and amino termini of the substrate, respectively, and sequence analysis revealed Their positions in the A domains of NRPSs were found to be invariant. 236, Ala301, and Ile330 are specific for amino acid substrates with aliphatic side chains. Moderately variable within the A domain: Trp239, Thr278, Ile299, A Ia322 and Cys331 are highly positionally variable, contributing to the discrimination and selection of different substrates. It is believed to be important in edictive,Structure-Based Model of Amino Acid Recognition by Nonribosomal Peptide Synthetase Adenylation Domains.Chem Bio l 7:211-224;Stachelhaus et al.,(1999)The Specificity Conferring Code of Adenylat ion Domains in Nonribosomal Peptide Synt hetases. Chem Biol 6:493-505). Ile299 is a substrate specific It was the most positionally variable of all the sequences that conferred isomerism (Stachelhau s et al.,(1999)The Specificity Conferrin g Code of Adenylation Domains in Nonribo somal Peptide Synthetases.Chem Biol 6:49 3-505).

[0052] The 10 amino acid residues that determine the substrate specificity of fusaricidin synthetase are listed in Table 1. The adenylation domain (A domain) of each of the six modules of the synthetase is shown in , the first module FusA-A1, the second module FusA-A2, the third module FusA-A3, These 10 amino acid residues are shown in Figure 13. The same sequence was also found in the multiple sequence alignment of FusA from various Paenibacillus species shown in Fig. It is set forth. [Table 1]

[0053] In certain embodiments, the fungicidal Paenibacillus species strain has substrate specificity in FusA-A3. At least one, at least two, at least three, or at least two amino acid residues that determine isomerism At least four, at least five, at least six, at least seven, at least eight, expressing mutant fusaricidin synthetase containing at least nine or all ten deletions In another embodiment, the fungicidal Paenibacillus species strain has an Asp in FusA-A3. 235, Ala236, Ser239, Thr278, Leu299, Ala301, A Ia / Gly322, Val330, Cys331, Lys517 and combinations thereof a fusarizid strain having a deletion of at least one amino acid residue selected from the group consisting of: It expresses phosphodiesterase.

[0054] The deletions in FusA-A3 disclosed herein are fusarizidin or fusaricidin A tether for incorporating specific amino acids into amino acid position (3) of the peptide ring in peptide-like compounds. It affects the activity of lysidine synthetase. For example, Paenibacillus species NRRL B- Strain 50972 contains a deletion in FusA-A3, resulting in a tyrosine amino acid at amino acid position (3). or inability to produce fusaricidin compounds having phenylalanine amino acids Without wishing to be bound by any theory, the deletion in FusA-A3 may be involved in metabolic away from classical fusaricidin biosynthesis, paenicelins and paeniprolixins This can shift the biosynthesis of fusaricidin-like compounds such as

[0055] In a particular embodiment, the present invention provides a functional adenylation domain (F A mutant fusaricidin synthetase lacking fusaricidin A-A3) and at least one species of Paenibacillus Fungicidal Paenibacillus Further Comprising Serine and At Least One Paeniprolixin In certain embodiments, compositions comprising a biologically pure culture of a species strain are directed to the composition. Both paenicelin and at least one paeniprolixin are isolated in the composition. Or concentrated.

[0056] In some embodiments, the isolated compound or paeniprolixin is is [ka] TIFF0007802733000005.tif211148TIFF0007802733000006.tif217149TIFF0007802733000007.tif217147TIFF0007802733000008.tif208148

[0057] In some embodiments, the isolated compound or paenicelin is: Ru, [ka] TIFF0007802733000010.tif185151TIFF0007802733000011.tif200158

[0058] In another aspect, the present invention identifies fungicidal Paenibacillus species strains and / or The present invention relates to a method for producing the corresponding fermentation product, which method comprises the step of: to characterize the enzymes and assay the fungicidal activity of Paenibacillus species strains In certain embodiments, the FusA-A3 gene is sequenced in a FusA-A3 strain. are primers based on one or more sequences shown in Figure 13 (i.e., SEQ ID NOs: 1 to 11). In some embodiments, prior to screening, The cells are grown and cells with one or more of the following characteristics are selected: wild-type Fusarizi A reference Paenibacillus species strain containing a fusin synthetase (i.e., expressing functional FusA-A3) The amino acid residue (3) was tyrosine or phenylalanine-containing fusaricidins (e.g., LiF03a, LiF03b, Li F03c, LiF03d, LiF07a, LiF07b, LiF07c, and / or Reduced or undetectable levels of LiF07d); and / or wild-type fusaricidin Reference Paenibacillus species strain containing the fusase (i.e., expressing functional FusA-A3) Paenicelins (e.g., paenicelin A1 and / or Paeniselin B1) and / or increased levels of Paeniprolixin.

[0059] In one aspect, the present invention provides a method for producing a fermentation product having broad-spectrum antifungal activity. A method for culturing a Paenibacillus species strain having a mutant fusaricidin synthetase, and allowing the bacteria to sporulate.

[0060] In another embodiment, the present invention provides a fungicidal paste having broad spectrum antifungal activity. A method for identifying a Paenibacillus species strain, comprising: a) detecting FusA-A in a Paenibacillus species strain; b) sequencing the mutant fusaricidin synthetase; To assay the fungicidal activity of Paenibacillus species strains possessing lysidine synthetase; and c) the Paenibacillus species strain contains a mutant fusatericidin synthetase and a wild-type fusatericidin synthetase. showed increased fungicidal activity compared to reference Paenibacillus species strains containing salicidin synthetase. In this case, it is advisable to select a fungicidal Paenibacillus species strain with broad-spectrum antifungal activity. and a method for producing a Paenibacillus strain. Quantifying phosphorus and / or paeniprolixin, and determining whether Paenibacillus sp. strains , increased levels compared to a reference Paenibacillus species strain containing wild-type fusaricidin synthetase If the compound produces paenicelin and / or paeniprolixin, it may have a broad-spectrum anti-inflammatory effect. and selecting a Paenibacillus species strain having fungicidal activity. The method comprises culturing a fungicidal Paenibacillus species strain to produce a fungicidal fermentation product. and further including.

[0061] In one embodiment, the present invention provides a fungicidal paenibacterium having broad spectrum antifungal activity. 1. A method for producing an antifungal fermentation comprising a Bacillus species strain, comprising: a) FusA-A3 in Paenibacillus species strains was sequenced to identify mutant fusaricidins b) characterizing the fusaricidin synthetase; c) assaying the fungicidal activity of Paenibacillus sp. strains; and reference Paenibacillus sp. containing wild-type fusaricidin synthetase. fungicides with broad-spectrum antifungal activity if they show increased fungicidal activity compared to strains d) selecting a fungicidal Paenibacillus sp. strain; and d) culturing a fungicidal Paenibacillus sp. strain. and producing a fungicidal fermentation product.

[0062] In some embodiments, the mutant fusaricidin synthetase is FusA-A3. At least one, at least two, or at least two amino acid residues that determine substrate specificity in at least three, at least four, at least five, at least six, at least seven, at least In other embodiments, the mutant form comprises at least eight, at least nine, or all ten deletions. Fusaricidin synthetase converts Asp235, Ala236, Ser239, Thr278, Leu299, Ala301, Ala / Gly322, V Selected from the group consisting of al330, Cys331, Lys517 and combinations thereof The amino acid sequence of the present invention includes a deletion of at least one amino acid residue.

[0063] The present invention also relates to Paenibacillus sp. NRRL B-50972 strain or a mutant thereof. Or the cell-free preparation or metabolites may be extracted from leaves, stems, flowers, fruits, roots, seeds, etc. By administering to the plant or plant part, or when the plant or plant part is growing, The present invention also includes a method for treating plants by applying the composition to soil, for example, to control plant diseases. Includes.

[0064] In the method according to the present invention, Paenibacillus species NRRL B-50972 strain or The compositions containing the fungicidal mutants may be used in any type of fungicide used to grow plants. Any of the plants grown in a medium (e.g., soil, vermiculite, shredded cardboard, and water) It can be applied to any plant or any part of any plant, or to plants such as orchids and staghorn ferns. The composition can be applied to plants or plant parts that grow in the air. spraying, atomizing, vaporizing , scattering, dusting, watering , squirting, sprinkling, pouring It can be applied by ring or fumigation. As mentioned above, agriculture, horticulture, forestry, plantations, orchards, nurseries, organically grown Application is made at any desired location where the plants of interest are located, such as in cultivated crops, turfgrass, and urban environments. It is possible.

[0065] The compositions of the present invention comprise Paenibacillus sp. strain NRRL B-50972 or a derivative thereof. The fungicidal mutants (strains) to be cultured were grown in the media and other methods described in the examples below. and culturing the cells by methods well known in the art, including using Conventional large-scale microbial cultivation processes include submerged fermentation, solid-state fermentation, or liquid surface culture. Towards the end of the fermentation, as nutrients are depleted, the cells transition from the vegetative phase to the spore form. The transition to adulthood begins, and the end products of fermentation are primarily spores, metabolic products, and residual fermentation medium. Sporulation is part of the natural life cycle of Paenibacillus and generally occurs when nutrient limitation is present. Fermentation is initiated by cells in response to the bacteria, resulting in the acquisition of high levels of colony-forming units and the production of spores. The fermentation process is designed to promote the formation of bacterial cells, spores, and progeny in the culture medium. The product can be used directly or purified by other methods such as centrifugation, tangential flow filtration, depth filtration, and evaporation. Concentration can be achieved by conventional industrial methods.

[0066] The compositions of the present invention comprise a fermentation product. In some embodiments, the concentrated fermentation broth , for example, by washing via a diafiltration process to remove residual fermentation broth and metabolic products. The term "broth concentrate" as used herein refers to a broth concentrate obtained by conventional industrial methods as described above. It refers to whole broth (fermentation broth) that has been concentrated by fermentation but remains in liquid form. The term "fermentation solids" as used herein refers to the solid material remaining after the fermentation broth has been dried. The term "fermentation product" as used herein refers to whole broth, broth concentrate, and / or fermentation solids. The composition of the present invention comprises a fermentation product.

[0067] Fermentation broth or broth concentrate can be dried by spray drying, freeze drying, tray drying, fluidized bed drying, The effectiveness of the addition of a carrier may be achieved using conventional drying processes or methods such as ram drying, or evaporation. It can be dried regardless of whether it is used or not.

[0068] The resulting dried product may be further processed, such as by milling or granulation, to produce a particular particle size or The physical format can be achieved by adding the carrier described below after drying. It is also possible to do so.

[0069] Cell-free preparations of the fermentation broth of the strains of the present invention can be prepared by extraction, centrifugation and / or The soluble fraction can be obtained by any means known in the art, such as by filtration or by other suitable means. Therefore, so-called cell-free preparations may not be devoid of cells, but rather may be prepared by removing cells. Depending on the technique used (e.g., centrifugation speed), the cells may be nearly cell-free or essentially It will be appreciated that the resulting cell-free preparation may be dried and / or The fermentation broth may be formulated with ingredients to aid in application to the plant or plant growth medium. The concentration methods and drying techniques described above for cell-free preparations are also applicable to cell-free preparations.

[0070] In one embodiment, the fermentation product is at least about 1 x 10 4 Colony forming units (CFU) ) microorganisms (e.g., Paenibacillus species NRRL B-50972 strain or its fungicidal In another embodiment, the fermentation product comprises at least about 1 x10 5 Colony forming units (CFU) of microorganisms (e.g., Paenibacillus species NRRL B In another embodiment, the fungicidal mutant strain (strain 1-50972 or a fungicidal mutant thereof) is present in a broth containing 100 mL of the fungicidal mutant strain 1-50972. The fermentation product is at least about 1 x 10 6 CFU of microorganisms (e.g., Paenibacillus strain NRRL B-50972 or its fungicidal mutants) / mL broth. In yet another embodiment, the fermentation product is at least about 1 x 10 7 CFU of microorganisms (e.g. For example, Paenibacillus species NRRL B-50972 or its fungicidal mutants) In another embodiment, the fermentation product comprises at least about 1 x 10 mL broth. 8 CF U microorganisms (e.g., Paenibacillus species NRRL B-50972 strain or its fungicidal In another embodiment, the fermentation product comprises at least about 1 x10 9 CFU of microorganisms (e.g., Paenibacillus species NRRL B-50972 or In another embodiment, the fermentation product comprises: At least about 1 x 10 10 CFU of microorganisms (e.g., Paenibacillus species NRRL B- 50972 or a fungicidal mutant thereof) / mL broth. The fermentation product is at least about 1 x 10 11 CFU of microorganisms (e.g., Paenibacillus species NRRL B-50972 strain or its fungicidal mutant strain) / mL broth.

[0071] The compositions of the present invention may be prepared in a variety of ways, depending on their specific physical and / or chemical properties. or use forms prepared therefrom, such as aerosols, capsule suspensions, , cold-fogging concentrate, warm fogging concentrate (warm-fogging concentrate), encapsulated granules, granules, seeds Flowable concentrates for treatment, ready-to-use solutions, dusts, emulsions, oil-in-water emulsions, water-in-oil Emulsifiable concentrates, granules, microgranules, oil-dispersible powders, oil-miscible flowable concentrates, oil-miscible liquids, gases (under pressure), gas generators, foams, pastes, insecticide-coated seeds, suspension concentrates, oil dispersions Suspoemulsion concentrates, soluble concentrates, suspensions, wet powders, soluble powders, dusts and and granules, water-soluble and water-dispersible granules or tablets, water-soluble and water-dispersible for seed treatment Active powders, wettable powders, natural and synthetic products impregnated with active ingredients, and seeds Microencapsulation in polymeric substances and coating materials for ULV cooling It can be used in aerosol and warm aerosol formulations.

[0072] In some embodiments, the compositions of the present invention are liquid formulations. Suitable examples include suspension concentrates and oil dispersions. The composition is a solid formulation. Non-limiting examples of liquid formulations include lyophilized powders and spray formulations. Dry powders are included.

[0073] The compositions of the present invention may be modified and / or processed to improve efficacy, stability and usability. , cells, cell-free preparations or metabolites for ease of packaging and end-use application A formulation inert added to a composition comprising Such formulation inactive ingredients and components may be added individually or in combination. In some embodiments, the composition may include a suitable carrier, stabilizer, nutrient, or physical property modifier. In the present invention, the carrier may be a liquid material such as water, oil, or other organic or inorganic solvent; Inorganic, polymeric or polymer composites derived from biological or chemical synthesis In some embodiments, the carrier is a seed or any solid material. It is a binder or adhesive that promotes adhesion of the composition to plant parts such as roots. ylor,AG,et al.,“Concepts and Technolog ies of Selected Seed Treatments”, Annu.Re See, e.g., J. Phytopathol. 28:321-339 (1990). The agents may include anti-caking agents, antioxidants, desiccants, protectants or preservatives. , sugars, polysaccharides, oils, proteins, amino acids, fatty acids and phosphates, etc. Physical property modifiers include bulking agents, humectants, thickeners, p H regulator, rheology modifier, dispersant, adjuvant, surfactant, antifreeze or colorant In some embodiments, cells produced by fermentation, cellular The cell preparation or metabolite-containing composition may be optionally administered with or without water as a diluent. In some embodiments, the formulation can be used directly without further formulation preparation. Inactive ingredients are added after concentrating the fermentation broth and during and / or after drying. can be.

[0074] All plants and plant parts can be treated according to the invention. Plants include both desirable and undesirable wild or crop plants (including naturally occurring crop plants). Crop plants are understood to mean all plants and plant populations, including those that are traditionally cultivated. by means of advanced breeding and optimization methods, or by means of biotechnology and recombinant methods, or and the plants obtained by the combination of these methods, and are called transgenic plants and plants. Plant parts include plant species that may or may not be protected by breeder's rights. It means all the aerial and underground parts and organs of a plant, such as shoots, leaves, flowers and roots. Examples are leaves, needles, rachises, stems, flowers, fruiting bodies, fruits and seeds. Plant parts include the roots, tubers and rhizomes. Plant parts also include crop material and vegetative growth. Propagation material and generative propagation material include, for example, cuttings, tubers, rootstocks, scions and seeds.

[0075] As already mentioned above, all plants and their parts can be treated according to the invention. In a preferred embodiment, wild-type strains or hybridized or protozoal strains can be used. Plant species and plant cultivars obtained by traditional biological breeding methods such as plastogenesis, In a further preferred embodiment, traditional methods are used where appropriate. Transgenic organisms obtained by recombinant methods in combination with other methods (genetically modified organisms) Plants and plant cultivars as well as their parts are treated. The terms "plant" or "plant part" are defined herein above. Plants of the plant cultivars which are currently in use or are in use are particularly preferably treated according to the invention. Plant varieties are developed either through traditional breeding, mutagenesis or recombinant DNA techniques. These are understood to mean plants with new traits bred by cultivars, species, It takes the form of a biotype and a genotype.

[0076] The treatment of plants and plant parts with the compositions according to the invention can be carried out by direct or conventional treatment methods, For example, immersion, spraying, atomization, misting, evaporation, dusting, fogging, scattering, foaming, coating, diffusing, injection by acting on the environment, habitat or storage space by irrigation, drip irrigation, etc. In the case of propagation material, particularly in the case of seeds, further methods of dry seed treatment, wet seed treatment, coatings, such as by one or more coatings, by methods, slurry processing methods, encrustation Furthermore, the application of the active substance by the ultra-low volume method is also possible. Alternatively, it is possible to inject the active substance preparation or the active substance itself into the soil.

[0077] A preferred direct treatment of plants is a foliar application treatment, i.e., applying a composition according to the invention to the leaves. The frequency and rate of application can be adapted to the infection pressure of the pathogen in question.

[0078] In the case of systemically active compounds, the compositions according to the invention reach the plant via the root system. In this case, the treatment of the plant is carried out by applying the composition according to the invention to the environment of the plant. This can be done, for example, by irrigation, by incorporating it into the soil or into a nutrient solution, i.e. by positioning the plant. impregnating a substrate (e.g., soil or a hydroponic system) with a liquid form of a composition according to the present invention; or soil application, i.e., the composition according to the invention can be applied in solid form (e.g. This can be done by incorporating the fertilizer (in the form of granules) into the plant. This involves applying the composition according to the invention in a solid application form (for example in the form of granules) to a flooded rice field. This can also be done by weighing.

[0079] Preferred plants are useful plants, ornamental plants, turf plants, and plants used as ornamentals in public and domestic settings. The forest trees are commonly used trees and plants from the group of forest trees. Includes wood for the production of cellulose, paper and products made from parts of trees.

[0080] The term "useful plants" as used in this context means plants grown for the purpose of obtaining food, feed, fuel, and refers to crop plants used as plants for industrial purposes.

[0081] Useful plants that can be treated and / or improved with the compositions and methods of the present invention include: Examples of plants that can be mentioned include: grasses, grapes, cereals, e.g. corn. wheat, barley, rye, oats, rice, corn and millet / sorghum; beets fruits, such as pome fruits, stone fruits and soft fruits, e.g., sugar beets and fodder beets; apples, pears, plums, peaches, almonds, cherries and berries, e.g., strawberries Berries, raspberries, blackberries, etc.; legumes, e.g., beans, lentils, peas and soybeans; oilseeds, e.g., rapeseed, mustard, poppy, olive, sunflower, coco Nuts, castor oil plants, cocoa and peanuts; Cucurbits, e.g. pumpkin / squash cucumbers and melons; fiber plants such as cotton, flax, hemp and jute; citrus fruits , e.g., oranges, lemons, grapefruits and tangerines; vegetables, e.g., Spinach, lettuce, asparagus, cabbage seeds, carrots, onions, tomatoes, potatoes potatoes and peppers; Lauraceae, e.g., avocado, cinnamon, camphor, or tobacco , nuts, coffee, eggplant, sugarcane, tea, pepper, grapes, hops, banana, latte Other plants, such as ornamental plants, flowers, shrubs, deciduous trees and conifers. The list is not exhaustive.

[0082] The following plants are particularly suitable target crops for applying the compositions and methods of the present invention: Considered: cotton, eggplant, grass, pome fruits, stone fruits, soft fruits, corn, wheat, barley cereals, pears, beans, soybeans, rapeseed, tomatoes, peas Mango, melon, cabbage, potato and apple.

[0083] Examples of trees that can be improved according to the methods of the present invention are Abies sp. .), Eucalyptus species (Eucalyptus sp.), Picea species (Picea sp.) .), pine species (Pinus sp.), horse chestnut species (Aesculus sp.), Platanus sp., Tilia sp. , Acer sp., Tsuga sp., and Aconite sp. Fraxinus sp.), Rowan species (Sorbus sp.), Birch species (Betula sp.), Hawthorn species (Crataegus sp.), Ulmus species ( Ulmus sp.), Quercus sp., Fagus sp. sp.), Salix sp., Populus sp. .)

[0084] Preferred trees that can be improved according to the method of the present invention are those of the tree species Aesculus (Ae sculus) Origin: A. hippocastanum, A. A. pariflora, A. carnea; tree species From the genus Platanus: P. aceriflora ), P. occidentalis, P. racemois cemosa); from the tree species Picea: P. abies ;From the genus Pinus: P. radiata, P. ponder P. ponderosa, P. contorta, P. si P. sylvestre, P. elliottii ), P. montecola, P. albicaulis aulis, P.resinosa, P.palustris lustris), P. taeda, P. flexi lis), P. jeffregi, P. baksiana ana), P. strobus; tree species Eucalyptus tus) Origin: E. grandis, E. globulus bulus), E.camadentis, E.nitens itens), E. obliqua, E. regnans ans), and can be obtained from E. pilularus.

[0085] Particularly preferred trees that can be improved according to the method of the present invention are those of the tree species Pinus spp. (Pi nus) origin: P. radiota, P. ponderosa, P. contorta, P. cibeles, P. Strobus; from the tree species Eucalyptus: E. grandis, E. globulus, and E. camadentis.

[0086] Very particularly preferred trees which can be improved according to the method of the invention are horse chestnuts. These are the Japanese zelkova, Platanaceae, linden, and maple.

[0087] The present invention may also be applied to any turfgrass, including cool-season and warm-season turfgrass. Examples of terrestrial turfgrasses include Kentucky bluegrass (Poa pratensis L. pratensis L.), Poa trivialis L. (Poa a trivialis L.), Canada bluegrass (Poa compressa L. (P Poa compressa L.), Annual bluegrass (Poa annua L. annua L.), Upland bluegrass (Poa glaucantha gauginosa ( Poa glaucantha Gaudin), Wood bluegrass (Poa nemora) Squirrel L. (Poa nemoralis L.)) and bulbous bluegrass (Poa Bluegrass species (Poa spp. (P) oa spp.); Creeping bentgrass (Agrostis palustris Hud s.(Agrostis palustris Huds.)), Colonial Bentgra Sibth.(Agrostis tenuis Sibth th.), velvet bentgrass (Agrostis canina L. canina L.), South German mixed bentgrass (Agrostis Agrostis tenius Sibth., Agros Agrostis canina L. and Agrostis Agrostis palustris Huds. Agrostis spp., which includes the genus Agrostis, and the common bean grass (Agroste Bentgrass (Agrostis alba L.) and other bentgrasses (Agrostis Agrostis spp.); Red fescue (Festuca rubra L.) L. spp. rubra)), creeping fescue (Festuca rubra L. (F estuca rubra L.), Chewing's fescue (Festuca rubra L.) ·Commutata Gaud.(Festuca rubra commutata Gaud. .)), sheep fescue (Festuca ovina L. .)), hard fescue (Festuca longifolia Thuill.(Festuca longifolia Thuill.), Hair fescue (Festuca capillata) Lam. (Festuca capillata Lam.)), Tall fescue (Festuca Festuca arundinacea Schreb. Schreb.)) and meadow fescue (Festuca elanor L. (Festuc Festuca species such as fescue (Festuca spp.) spp.)); Lolium multiflorum (Lam.) um Lam.), ryegrass (Lolium perenne L. L.)) and Italian ryegrass (Lolium multiflorum Lam. (Loli Lolium species (Lolium spp.) ium spp.)); and fairway wheatgrass (Agropyron cristatum (L.) Gae rtn.(Agropyron cristatum(L.)Gaertn.)) Steady wheatgrass (Agropyron decertorum (Fisch.) Schult) (Agropyron desertorum (Fisch.) Schult.) and and Western wheatgrass (Agropyron sumithii Rydb.) n smithii Rydb.) and other wheatgrass (Agropyllum species ... opyron spp.)).

[0088] Another example of a cool-season turfgrass is beachgrass (Ammophila breviligulata Fern. (Ammophila breviligulata Fern.)), Smooth Brome Grass (Bromus inermis Leyss. s.), Timothy grass (Phleum pratens L. e L.), Sandcuttail (Phleum subulata L. ulatum L.), Orchard grass (Dactylis glomerata L. glomerata L.), Puccinia distans (Puccinia distans) Puccinellia distans(L.) Parl. ) and comb grass (Cynosurus cristatus L. (Cynosurus crista tus L.)) and other cattails.

[0089] An example of a warm-season turfgrass is Cynodon spp. LC Rich. spp.LCRich), Zoysia Willd. spp.Willd.), dog grass (Stenotafulum secundum Wald. Kunt) (Stenotaphrum secundatum Walt Kuntze)), Centipede grass (Eremochlora ophiuroides Munro) chloa ophiuroides Munro Hack.)), carpet grass Axonopus affinis Chase )), Bahiagrass (Paspalum notatum flug) um Flugge), Kikuyu grass (Pennisetum clandestinum Hochst .ex Chiov.(Pennisetum clandestinum Hochs t.ex Chiov.), Buffalo grass (Buctodactyloides (Nut t.)Engelm.(Buchloe dactyloids(Nutt.)Enge lm.), Medakasedge (Bouteroa gracilis (HBK) Lag.ex G Liphitos (Bouteloua gracilis (HBK) Lag.ex G riffiths), Seashore Paspalum (Paspalum vaginatum Swarts ( Paspalum vaginatum Swartz) and the Japanese bush cricket (Bow Bouteloua curtipenzura (Michx.Torr.)(Bouteloua curtipenzura) ipendula (Michx. Torr.))). Generally, cool-season turfgrasses are Particularly preferred are bluegrass, bentgrass and conker grass. Grass, fescue and ryegrass are particularly preferred. Bentgrass is particularly preferred.

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

[0091] The present invention also relates to a method for treating plant pathogenic fungi, plant pathogenic bacteria and / or fungi comprising administering to said fungi, bacteria and / or fungi the composition of the present invention. The present invention relates to a method for controlling undesirable microorganisms, characterized in that the method is applied to the habitat of the microorganisms. do.

[0092] Fungicides can be used in crop protection for the control of plant pathogenic fungi. In particular, Plasmodiophoromycetes, Peronosporum Peronosporomycetes (synonym: Oomycetes), Chytridiomycota (C hytridiomycetes), zygomycetes (Zygomycetes), ascomycetes ( Ascomycetes), Basidiomycetes and Deuteromycetes Members of the Deuteromycetes (synonym: Fungi imperfecti) Outstanding efficacy against a broad spectrum of plant pathogenic fungi, including soil-borne pathogens Some fungicides are systemically active and can be applied as foliar or seed dressings. They can also be used in plant protection as soil fungicides. Suitable for fighting fungi that infest wood or plant roots.

[0093] The fungicide is a fungicide for the Pseudomonadaceae, Ericaceae (Rh izobiaceae, Enterobacteriaceae, Coli Corynebacteriaceae and Streptomycetaceae can be used in crop protection to control Streptomycetaceae .

[0094] Non-limiting examples of fungal disease pathogens that can be treated according to the present invention include: Included: Diseases caused by powdery mildew pathogens, e.g., Blumeria species ia species), such as Blumeria graminis (Blumeria graminis) inis); Podosphaera species, e.g. Podosphaera leucotricha; Spa Sphaerotheca species, e.g. Sphaerotheca fu Liginea (Sphaerotheca fuliginea); Unc. inula species, such as Uncinula necator cator); Diseases caused by rust pathogens, e.g., Gymnosporangium species (Gy Gymnosporangium species), e.g. Gymnosporangium rust Gymnosporangium sabinae; Hemi leia species, such as Hemileia wastatrix (Hemileia vastatrix); Phakopsora species, e.g. For example, Phakopsora pachyrhizi and Phakopsora meibomiae; Puckinia genus Puccinia species, such as Puccinia recondita nia recondite), P. triticina, P. gra minis (P. graminis) or P. striiformis (P. striiform Uromyces species, e.g., Uromyces a pendicularis (Uromyces appendiculatus); Diseases caused by pathogens from the group of oomycetes, e.g., Albugo species go species, such as Algubo candida Bremia species, such as Bremia lactucae ( Bremia lactucae); Peronospora sp. ecies), such as Peronospora pisi or P. brassicae; Phytophthora species ra species), such as Phytophthora infestans ora infestans; Plasmopara species (Plasmopara spec ies), e.g. Plasmopara viticola ;Pseudoperonospora species , for example, Pseudoperonospora hum uli) or Pseudoperonospora cubensis (Pseudoperonospor a cubensis); Pythium species, e.g. Pythium ultimum; Leaf spot and leaf wilt caused by, for example, Alternaria species (A Alternaria species, such as Alternaria solani (Alternaria aria solani); Cercospora species ), e.g., Cercospora beticola; Cladiosporium species, e.g. Cladiosporium Cladiosporium cucumerinum Cochliobolus species, e.g. Cochlio Cochliobolus sativus (conidial morphology: Drechslera, synonym: Helminthosporium sporium), Cochliobolus mi yabeanus); Colletotrichum species es), for example Colletotrichum lindemtanium (Colletotrichum l indemuthanium; Cycloconium spe cies), e.g. Cycloconium oleaginum (Cycloconium olea ginum); Diaporthe species, e.g. Diaporthe Diaporthe citri; Elsinoe species e species), such as Elsinoe fawc ettii); Gloeosporium species , for example, Gloeosporium laeticolor (Gloeosporium laeticolor) Glomerella species, e.g., Glomerella Glomerella cingulata; Guignardia Guignardia species, e.g. Guignardia bidwelli (Guignardia bidwelli); Leptosphaeria species (Leptos phaeria species, such as Leptosphaeria macranthus (Lept osphaeria maculans), Leptosphaeria nodrum (Lepto sphaeria nodorum; Magnaporthe s pecies, such as Magnaporthe grise a); Marssonia species, e.g. Marssonia ko Marssonia coronaria; Microdochium species odochium species), such as Microdochium nivale chium nivale);Mycosphaerella s pecies, such as Mycosphaerella graminicola (Mycosphaerella graminicola, M. arachidicola and and M. fijiensis; Phaeosphaeria species osphaeria species, such as Phaeophaeria nodrum (Pha eosphaeria nodorum; Pyrenophora species species, such as Pyrenophora tere s), Pyrenophora tritici repentis (Pyrenophora tritici i repentis); Ramularia species, e.g. For example, Ramularia collo-cygni, Ramularia Ramularia areola; Rhynchosporium species (Rh Rhynchosporium species), such as Rhynchosporium secharis (R hynchosporium secalis; Septoria species species, such as Septoria apii, Septoria Septoria lycopersii; Typhula species hula species, such as Typhula incarnata (Typhula inca rnata); Venturia species, e.g., Venturia Venturia inaequalis; Root and stem diseases caused by, for example, Corsicium species ticium species), e.g. Corticium graminearum (Cortic Fusarium graminearum; Fusarium species s), such as Fusarium oxysporum; Gaeumannomyces species, e.g., Gaeuman Gaeumannomyces graminis (Gaeumannomyces graminis); Rhizoctonia species, such as Rhizoctonia solani ( Rhizoctonia solani; e.g., Sarocladium oryzae (Saroc Sarocladium disease caused by Sclerophyll aeruginosa; e.g., Sclerophyll aeruginosa Sclerotinia caused by Sclerotium oryzae Tapesia species, e.g. Tapesia aquafu Tapesia acuformis; Thielaviopsis species Thiel aviopsis species, such as Thiel aviopsis bacicolae aviopsis basicola); ear and panicle diseases (including corncobs) caused by, for example: ), Alternaria species, e.g., Alternaria spp.; Aspergillus spp. Aspergillus species, e.g., Aspergillus flavus s flavus); Cladosporium species s), e.g., Cladosporium cladosporioides (Cladosporium c Ladosporioides; Claviceps speci es), e.g., Claviceps purpurea; Fusarium species, e.g. Fusarium culmorum (Fusarium culmorum); Gibberella sp ecies), e.g. Gibberella zeae; Monographella species, e.g., Monographella nivalis (Monographella nivalis); Septoria species (Sept oria species, such as Septoria nodrum orum); Diseases caused by smut fungi, such as Sphaceloteca species (S phacelotheca species), such as Sphacelotheca leiliana (S phacelotheca reiliana);Tilletia spp. pecies), e.g., Tilletia caries, T. controversa (T. controversa); Urocystis species is species), such as Urocystis occulta cculta); Ustilago species, e.g. Ustilago Ustilago nuda, U. nuda tritici tritici); Fruit rot caused by, for example, Aspergillus species illus species, e.g., Aspergillus flavus us flavus); Botrytis species, e.g. Botrytis cinerea; Penicillium spp. Penicillium species), such as Penicillium expansum (P enicillium expansum and P. purpurogenum (P. purpu rogenum); Sclerotinia species, For example, Sclerotinia sclerotiorum (Sclerotinia scleroti orum); Verticilium species, e.g. Verticilium alboatrum ; Seed and soil-borne decay, mould, wilt, rot caused by, for example: Blight and seedling damping-off, Alternaria species , such as Alternaria brassicicola Aphanomyces species, e.g., Aphanomyces Produced by Aphanomyces euteiches Ascochyta species, e.g. Ascochyta Caused by Ascochyta lentis; Aspergillus Aspergillus species, e.g., Aspergillus flavus Caused by Aspergillus flavus; Cladosporium Cladosporium species, e.g. Cladosporium herbicides Caused by Cladosporium herbarum; Cochliobolus species, e.g. Cochliobolus Cochliobolus sativus, (conidial morphology: Drexlera (Drechslera), Bipolaris (Synonym: Helminthospoli) Caused by Helminthosporium; Colletotrichum sp. Colletotrichum species, such as Colletotrichum cocco Caused by Colletotrichum coccodes; Fusarium species, such as Fusarium culmorum (F caused by Gibberella species (Gibberella spp.) rella species), such as Gibberella zeae ae) caused by Macrophomina spe cies), such as Macrophomina phaseolina (Macrophomina phae caused by Monographel species la species), such as Monographell caused by Penicillium nivalis; Penicilliu m species), such as Penicillium expansum expansum; Phoma species s), e.g. caused by Phoma lingam; Phomopsis species, e.g. Phomopsis soy Caused by Phomopsis sojae; Phytophthora species (P Phytophthora species), e.g., Phytophthora cactorum (Phy tophthora cactorum); Pyrenophora species, such as Pyrenophora graminea (Py renophora graminea); Pyricularia species (P yricularia species), e.g., Pyricularia oryzae laria oryzae; Pythium spp. pecies), such as Pythium ultimum Caused by Rhizoctonia species, For example, the disease caused by Rhizoctonia solani Rhizopus species, e.g. Rhizopus oryzae (Rhizopus oryzae); Sclerotium species (Sc lerotium species), e.g., Sclerotium rolfsii (Scler caused by Septoria rolfsii; Septoria species a species), such as Septoria nodoru m); Typhula species, e.g. It is caused by Typhula incarnata Verticillium species, e.g. For example, Verticillium dahliae caused by; For example, Nectria species, such as Nectria galli Galls and tumors caused by Nectria galligena Witches' broom disease; For example, Monilinia species, such as Monilinia Wilt caused by laxa (Monilinia laxa); For example, Exobasidium species, e.g. If it is Exobasidium vexans Leaf blister or leaf curl disease caused by; Taphrina species, e.g. Taphrina deformans Taphrina deformans; Decaying diseases of woody plants caused by, for example, Paemoniella Chlamydospora (Phaemoniella clamydospora), Paeoac Lemonium aleophilum (Phaeoacremonium aleophilum) and Fomitiporia mediterranea Esca disease caused by Eutypa lata Eutypa dyeback caused by Caused by Ganoderma boninense Reishi disease; e.g., Rigidoporus lignosus ) caused by rigidoporus disease; For example, Botrytis species, e.g., Botrytis Flower and seed damage caused by Botrytis cinerea Disease; tuber diseases caused by, for example, Rhizoctonia spp. tonia species, such as Rhizoctonia solani solani); Helminthosporium species (Helminthosporium s pecies, such as Helminthosporium solani (Helminthosporium um solani); For example, Plasmodiophora species ), for example, Plamodiophora brassicae (Plamodiophora bra Root galls caused by Bacillus ssicae; Diseases caused by bacterial pathogens, such as Xanthomonas spp. (Xanthomonas species), e.g., Xanthomonas campestris Xanthomonas campestris pv.oryz ae); Pseudomonas species, e.g. Pseudomonas syringae passover lachrymans (Pseudomonas syri ngae pv.lachrymans);Erwinia spe. cies, e.g., Erwinia amylovora ).

[0095] The following soybean diseases can be controlled with priority: Fungal diseases of leaves, stems, pods and seeds caused by, for example: Damage, Alternaria leaf spot disease (Alternaria species atlanticus tenuissima (Alter naria spec.atrans tenuissima), anthracnose (Colletotrichum Cam gloeosporoides dematius var. toruncatum (Colletotrich um gloeosporoides dematium var.truncatum )), brown spot (Septoria glycines), purple Leaf spot disease (Cercospora kikuchii), coreane Choanephora leaf blight (Choanephora infundibrifera Trispora (Choanephora ora infundibulifera trispora) (synonym)), Dacturio Dactuliophora glycine (Dactuliophora glycine) ines), downy mildew (Peronospora mans hurica), Drexlera blight (Drechslera grissini a glycini), leaf spot (Cercospora soina jina), freckle disease (Leptosphaerina trifolii erulina trifolii), Brown Rot (Phyllosticta sojaecola (Ph yllosticta sojaecola), black spot disease (Phomopsis sojae (P homopsis sojae), powdery mildew (Microsphaera diffusa (Mic rosphaera diffusa), Pyrenochaeta leaf spot (Pyrenochaeta grisea) Ness (Pyrenochaeta glycines), leaf rot (Rhizoctonia sora Rhizoctonia solani), rust (Phakopsora pachyrhizi (P hakopsora pachyrhizi, Phakopsora meibomiae (Phak opsora meibomiae), black spot disease (Sphaceloma glycines celoma glycines), Stemphylium leaf blight (Stemphylium borealis) Triosum (Stemphylium botryosum), brown ring disease (Corynes) Pora cassiicola (Corynespora cassiicola).

[0096] Fungal diseases of the roots and stem bases, such as black root rot (Castor's root rot), caused by Calonectria crotalariae , charcoal rot (Macrophomina phaseol ina), Fusarium head blight (Fusarium oxysporum rum), Fusarium orthoceras, Fusarium Fusarium semitectum, Fusarium Fusarium equiseti), Mycoleptodyscus root rot (Mu Mycoleptodiscus terrestris is)), root rot (Neocosmospora basinfecta vasinfecta), black spot disease (Diaporthe passerorum phaseolorum), stem rot (Diaporthe phaseolorum var. kauri) Mullet (Diaporthe phaseolorum var. caulivora) , stem blight (Phytophthora megasperma ma), leaf fall disease (Phialophora gregata a)), rhizome rot (Pythium aphanid ermatum), Pythium irregulare , Pythium debaryanum, Pythium milli Pythium myriotylum, Pythium ultimum hium ultimum), Rhizoctonia root rot (Rhizoctonia solani (Rhiz octonia solani), Sclerotinia sclerotiorum (Sc lerotinia sclerotiorum), Sclerotinia Southern Brite Disease (Sclerotinia rolfsii), Chie Thielaviopsis root rot (Thielaviopsis basicola) basicola)).

[0097] The fungicidal compositions of the present invention can be used for the curative or defensive / preventive control of plant pathogenic fungi. Therefore, the present invention also relates to a method for producing a plant comprising the steps of: The present invention provides a method for controlling plant pathogenic fungi by applying the composition of the present invention to the soil in which the plants grow. and therapeutic and protective methods for preventing and treating the disease.

[0098] The composition is well tolerated by plants at the concentrations required to control plant diseases. In fact, it allows the treatment of the above-ground parts of the plant, the propagation stock and seeds, and the soil.

[0099] According to the present invention, cultivars and plant varieties (plant varieties or plant breeders' rights) Treat all plants and plant parts, including vegetative and non-vegetative plants (whether or not they are protectable). Cultivars and plant varieties can be derived from doubled haploids, protoplast fusions, random and directed mutagenesis, the use of molecular or genetic markers, or biotechnology and and genetic engineering, assisted or complemented by one or more biotechnological methods. The plant may be obtained by conventional propagation and breeding methods capable of producing the desired plant.

[0100] In certain embodiments, the compositions of the present invention comprise about 1×10 8 ~Approx. 1×10 14 colony formation fungicidal Paenibacillus species NRRL B-50972 strain or In another embodiment, the compositions of the present invention are applied at about 1 x10 9 ~Approx. 1×10 13 Colony forming units (CFU) of fungicidal Paenibacillus sp. NR RL B-50972 or its fungicidal mutants per hectare. In another embodiment, the compositions of the present invention comprise about 1×10 10 ~Approx. 1×10 12 colony formation fungicidal Paenibacillus species NRRL B-50972 strain or applied at fungal mutants / hectare.

[0101] In some embodiments, the compositions of the present invention are used to prepare fermented solids containing about 0.1 kg to about 10 kg of fermented solids. In another embodiment, the compositions of the present invention are applied at about 0.25 min / hectare. In yet another embodiment, the fermentation solids are applied at a rate of from about 7.5 kg to about 7.5 kg of fermentation solids per hectare. The compositions of the present invention are applied at about 0.5 kg to about 5 kg of fermentation solids per hectare. The compositions of the present invention are applied at about 1 kg or about 2 kg of fermentation solids per hectare. It is also possible.

[0102] The compositions of the present invention have favorable thermotoxicity and are environmentally friendly when well tolerated by plants. It is well tolerated, protects plants and plant organs, improves yield and the quality of harvested material. The compositions of the present invention are preferably used as crop protection compositions. They can be used against normally sensitive and resistant species and They are active against all or some stages of development.

[0103] Plants that can be treated according to the invention include the following main crops: corn Shiitake mushrooms, soybeans, alfalfa, cotton, sunflowers, Brassica napus (Brassica napus) pus) (e.g. canola, rapeseed), Brassica rapa ), B. juncea (e.g., rapeseed, mustard) and Brassica Brassica (Brassica carinata) a) Oilseeds, Arecaceae species (oil palm, coconut), rice , wheat, sugar beet, sugarcane, oats, rye, barley, millet and sorghum wheat, triticale, flax, nuts, grapes and vines and various plant taxa, e.g. For example, various fruits and vegetables from Rosaceae sp. (e.g. For example, pome fruits such as apples and pears, as well as apricots, cherries, almonds, Stone fruits such as plums and peaches, as well as strawberries, raspberries, redcurrants and and berry fruits such as blackcurrants and gooseberries), Libesioidae species (Ribesioidae sp.), Juglandacea sp. e sp.), Betulaceae sp., Anacardia Anacardiaceae sp., Fagaceae sp. ae sp.), Moraceae sp., Oleaceae sp. leaceae sp.) (e.g. olive trees), Actinidaceae sp. nidaceae sp.), Lauraceae sp. (e.g. (e.g. avocado, cinnamon, camphor), Musaceae sp. ( For example, banana trees and banana plantations, Rubiaceae species eae sp.) (e.g., coffee), Theaceae sp. (e.g. tea), Sterculiceae sp., Lutacea Rutaceae species (e.g., lemons, oranges, mandarins, and grapes) grapefruit; Solanaceae species (e.g., tomato) potato, pepper, chili pepper, eggplant, tobacco), Liliaceae species (Lili aceae sp.), Compositae sp. (e.g., Lettuce, artichokes and chicories, e.g. root chicory, endive and common chicory coli), Umbelliferae sp. (e.g., ginseng parsley, celery and celery root), Cucurbitaceae species eae sp.) (e.g., cucumber, gherkin, pumpkin, watermelon, kaede and melon), Alliaceae sp. (e.g., chives, leek and onion), Cruciferae sp. (white cabbage) Vegetables, red cabbage, broccoli, cauliflower, Brussels sprouts, bok choy, kohlrabi , radish, horseradish, cress and Chinese cabbage), Leguminosae species (Leg uminosae sp.) (e.g., peanuts, peas, lentils and beans, For example, kidney beans and fava beans, Chenopodiaceae species ae sp.) (e.g., Swiss chard, fodder beet, spinach, beetroot Linaceae sp. (e.g. hemp), Cannabaceae ea sp.) (e.g., cannabis), Malvaceae sp. (e.g., octopus) La, cocoa), Papaveraceae (e.g. poppy), asparagus Asparagaceae (e.g., asparagus); and turfgrass, lawns, Useful and ornamental plants in gardens and forests, including grasses, and Stevia rebaudiana (St evia rebaudiana); and in either case, the genetic modification of these plants Type.

[0104] In certain embodiments, the fermentation product further comprises formulation ingredients. The formulation ingredients include humectants, thickeners, and the like. bulking agents, solvents, autogenous accelerators, emulsifiers, dispersing agents, anti-frost agents, thickeners, and / or adjuvants In one embodiment, the formulation component is a wetting agent. The fermentation product is a freeze-dried or spray-dried powder.

[0105] The compositions of the present invention may be used to improve recovery, efficacy or physical properties and / or to enhance To aid in processing, packaging, and administration, formulation ingredients may be added to the compositions of the present invention. Such formulation ingredients may be added individually or in combination.

[0106] The formulation ingredients may be modified to improve efficacy, stability and physical properties, usability, and / or is a leading supplier of cells, cell-free preparations, and isolated proteins for ease of processing, packaging, and end-use applications. Such formulation ingredients can be added to compositions containing the compound and / or metabolite. does not contain any agriculturally acceptable carriers, inert ingredients, stabilizers, preservatives, nutrients or physical property modifiers. Modifiers may be included and may be added individually or in combination. In the present invention, the carrier may be a liquid material such as water, oil, or other organic or inorganic solvent; Inorganic, polymeric or polymer composites derived from biological or chemical synthesis In some embodiments, the formulation ingredients include solid materials such as leaves, seeds, a binder, adjuvant, or other agent that promotes adhesion of the composition to plant parts such as shoots or roots; For example, Taylor, AG, et al., "Concepts and Technologies of Selected Seed Treatm ents,”Annu.Rev.Phytopathol.,28:321-339(1 990). Stabilizers include anti-caking agents, antioxidants, anti-settling agents, defoamers, drying agents, etc. Nutrients may include sugars, polysaccharides, oils, proteins, amino acids, It can contain carbon, nitrogen, and phosphorus sources such as fatty acids and phosphates. Modifiers include bulking agents, wetting agents, thickeners, pH adjusters, rheology modifiers, dispersants, and additives. furants, surfactants, film formers, hydrotropes, builders, antifreeze or colorants In some embodiments, the present invention includes fermentation-produced cells, inorganic The compositions containing the cell preparation and / or metabolites may be prepared in water as a diluent without any other formulation preparation. In certain embodiments, the lyophilized powder can be used directly with or without water. A wetting agent or dispersing agent is added to the fermentation solid, such as a powder or spray-dried powder. , increasing the spreading and penetration properties, or dispersing agent when it is applied to a surface Increases the dispersibility and solubility of the active ingredient (once diluted). Exemplary wetting agents include: MULTIWET™ MO-70R (Croda Inc., E sulfosuccinates and derivatives such as (Blankton, NJ); BREAK-THRU siloxanes such as ATLOX™ (Evonik, Germany); ATLOX™ 489 nonionic compounds such as 4 (Croda Inc., Edison, NJ); TERWE T (registered trademark) 3001 (Huntsman International LLC, T Alkyl polyglucosides such as TERGIT (The Woodlands, Texas) OL(R) 15-S-15 (The Dow Chemical Company C12-C14 alcohol ethoxylates such as those from Midland, Michigan phosphoric acid, such as RHODAFAC® BG-510 (Rhodia, Inc.); esters; and EMULSOGEN™ LS (Clariant Corp. Alkyl ether carboxylates such as (North Carolina) Examples include:

[0107] Deposit information The samples of the Paenibacillus species strains of the present invention were collected from the National Agricultural Research Center (NCRC) in the United States. enter for Agricultural Utilization Resea rch, Agricultural Research Service, USDe part of Agriculture(NRRL), 1815 North University Street, Peoria, IL 61604, USA) It was deposited in the Agricultural Research Service Culture Collection and opened in Buda on August 28, 2014. It has been assigned the following accession number under the Plague Convention: NRRL B-50972.

[0108] Paenibacillus species NRRL B-50972 strain showing stable colony morphology Nibacillus species samples were collected from the National Center for Agricultural Research (NCAR). r Agricultural Utilization Research, Agri Cultural Research Service, USDepartment of Agriculture(NRRL), 1815 North Univers Agricultural Research Center, City Street, Peoria, IL 61604, USA Deposited in the Bisculture Collection and deposited under the Budapest Treaty on September 1, 2015. and has been assigned the following accession number: NRRL B-67129.

[0109] The Paenibacillus species strain was designated as a Paenibacillus species by the Director of the Patent and Trademark Office under 37 C.F.R. during the pendency of this patent application. § 1.14 and 35 U.S.C. § 122. The deposit has been made under conditions which ensure that access to the material will be secure. The right to use the invention is not a license to practice the invention in derogation of patent rights granted by government action. It should be understood that this does not constitute a license.

[0110] The following examples are provided solely for illustrative and non-limiting purposes of the present invention.

[0111] [Example] Example 1. Selection of Paenibacillus sp. NRRL B-50972 The genomes of several Paenibacillus species strains were sequenced. The genome data were analyzed to identify the It has the salicidin biosynthetic gene cluster, but not the polymyxin synthetase gene class. We identified a strain lacking the gene cluster (fusA) involved in fusaricidin biosynthesis. ) was previously identified and characterized as having a polymyxin synthetase gene cluster. For example, Li et al., "Nonribosomal Bios ynthesis of Fusaricidins by Paenibacillu s polymyxa PKB1 Involves Direct Activati on of a D-Amino Acid,”Chemistry&Biology, 15:118-127(2008);Li et al., “Promoter Ana lysis and transcription regulation of fu s Gene Cluster Responsible for Fusaricid in Synthesis of Paenibacillus polymyxa S QR-21,”Applied Microbiol Biotechnol,97:9 479-9489(2013);and Choi et al.,“Identifi cation of a Polymyxin Synthetase Gene Cl uster of Paenibacillus polymyxa and Hete rologous Expression of the Gene in Bacil lus subtilis,”Journal of Bacteriology,19 1(10):3350-3358(2009).

[0112] Strains identified in this analysis were further evaluated to confirm fusaricidin production. Each strain was cultured in a soy-based medium and the lipophilic fraction of the whole broth was extracted. Ross extracts were analyzed by high-performance liquid chromatography (HPLC) to determine the presence of fusaricidin A. The results were based on the HPLC profiles generated with a standard sample containing fusaricidin A. It was determined.

[0113] Example 2. Antifungal activity of whole broth of Paenibacillus sp. strains in planta Selected Paenibacillus species strains, including Paenibacillus species NRRL B-50972, The strains were grown in soy-based medium to produce whole broth cultures. Distilled water was added to each of the whole broths. were added individually to make a 10% final dilution.

[0114] The diluted whole broth is applied to the leaves of young plants and then used to treat tomato blight (PHYTIN), ash Exposure to fungal inoculum of Bacterial Blight (BOTRCI) or Wheat Leaf Rust (PUCCRT) An untreated control was included in each assay for comparison purposes. After 1 day, each plant was scored for percent control of the pathogen compared to untreated control plants. Each treatment was replicated three times using whole broth of each Paenibacillus species strain shown in Figure 1. The average percentage control was assessed.

[0115] Tested for antifungal activity against PHYTIN, BOTRCI and PUCCRT Among the 23 strains, Paenibacillus species NRRL B-50972 was the most effective against all three fungal pathogens. It was one of the few strains with a relatively high level of activity against the body.

[0116] Example 3. In vitro analysis of fusaricidin extract from Paenibacillus NRRL B-50972 strain Biological potency in Toro The total number of strains of several Paenibacillus species, including Paenibacillus species NRRL B-50972, Broth cultures were prepared using soy-based media. The lipophilic fraction containing fusaricidin was extracted from the culture medium. Three extracts containing various fusaricidins and antifungal metabolites were extracted from whole broth. Separate fractions (i.e., fraction 1, fraction 2, and fraction 3) were extracted from the original Paenibacillus sp. strain. Extracted from whole broth of Paenibacillus sp. strain NRRL B-50972 did not separate further.

[0117] Fusaricidin-containing fractions from each strain were tested against the following 12 fungal pathogens: Luternaria alternata (ALTEAL), Botrytis cinerea (BOTRC I), Fusarium culmorum (FUSACU), Phaeophaea nodrum (LE PTNO), Zymoseptoria trisiti (SEPPTR), Phytophthora cryptogea (PHYTCR), Phytophthora infestans (PHYTIN), Pythium urticae Timum (PYTHUL) Magneporthe oryzae (PYRIOR), Tanatephorus k cumeris (RHIZSO), Ustilago segetum var. avenae (Ustilago s egetum var. avenae) (USTIAV), and Uromyces appendici Inhibition of fungal cell growth by various fractions of UROMAP was investigated using a soy-based Growth was assessed in culture medium and compared to that of untreated controls. Eight doses of each fraction were administered at 0.005 ppm The 50% inhibition (ED) range was tested at concentrations ranging from 100 ppm to 100 ppm. 50 ) and 80% inhibition (ED80 ) are reported in the table of FIG.

[0118] The fusaricidin-containing fraction of Paenibacillus species NRRL B-50972 strain was significantly higher than that of other Paenibacillus species. A broad spectrum across 12 assays not observed with fractions derived from Bacillus species The fraction of Paenibacillus species NRRL B-50972 also showed antifungal activity. In this assay, the fractions were significantly higher than those observed with fractions from Paenibacillus species strains. showed significant activity (see Figure 2).

[0119] Example 4. In vitro prevention test of Phytophthora-infected tomatoes This plant pathogen laboratory assay demonstrated the development of Paenibacillus species NRRL B-50972. The yeast product was tested and showed relatively high antifungal activity in the previous screening assay. In order to prepare a suitable preparation of this compound, One part by weight of spray-dried powder of whole broth from each strain cultivated in bean medium was added to water and 0.1 parts by weight of the powder. parts of an emulsifier (alkylaryl polyglycol ether), and then diluted with water. to the desired concentration.

[0120] To test for preventive activity, young plants were sprayed with the compound preparations at the stated application rates. After the spray coating had dried, the plants were infested with Phytophthora infestans. The plants were then inoculated with an aqueous spore suspension of P. phthora infestans. It was placed in an incubation cabinet at 20° C. and 100% relative atmospheric humidity.

[0121] The test was evaluated 3 days after inoculation. 0% means efficacy equivalent to that of the untreated control. 100% efficacy means that no disease is observed. [Table 2]

[0122] Example 5. In vivo prognosis of Plasmopara-infected grapevines Prevention test This plant pathogen laboratory assay demonstrated the development of Paenibacillus species NRRL B-50972. The yeast product was tested and showed relatively high antifungal activity in the previous screening assay. In order to prepare a suitable formulation of this compound, One part by weight of the spray-dried powder prepared as described in Example 5 was mixed with water and 0.1 parts by weight of emulsifier ( alkylaryl polyglycol ether) and subsequently diluted with water to the desired concentration. did.

[0123] To test for preventive activity, young plants were sprayed with the compound preparations at the stated application rates. After the spray coating dried, Plasmopara vticola ( The plants were inoculated with an aqueous spore suspension of B. iticola and then incubated at approximately 20°C and relative atmospheric humidity The plants were then placed in a 100% incubation cabinet for 1 day. The plants were then placed in a greenhouse at 25°C and a relative atmospheric humidity of approximately 90% for 4 days. It was placed in an incubation cabinet for 1 day.

[0124] The test was evaluated 6 days after inoculation. 0% means efficacy equivalent to that of the untreated control, 1 An efficacy of 0.00% means that no disease is observed. [Table 3]

[0125] Example 6. In vivo prevention test against beans infected with Uromyces This plant pathogen laboratory assay demonstrated the development of Paenibacillus species NRRL B-50972. The yeast product was tested and showed relatively high antifungal activity in the previous screening assay. In order to prepare a suitable formulation of this compound, One part by weight of the spray-dried powder prepared as described in Example 5 was mixed with water and 0.1 parts by weight of emulsifier ( alkylaryl polyglycol ether) and subsequently diluted with water to the desired concentration. did.

[0126] To test for preventive activity, young plants were sprayed with the compound preparations at the stated application rates. After the spray coating had dried, the plants were inoculated with the causative agent of bean rust (Uromyces appendix). Inoculation with an aqueous spore suspension of Uromyces appendiculatus Incubate the seeds in an incubation cabinet at approximately 20°C and 100% relative humidity for 1 day. It was left for a while.

[0127] The plants were then placed in a greenhouse at approximately 21° C. and a relative atmospheric humidity of approximately 90%.

[0128] The test was evaluated 10 days after inoculation. 0% means efficacy equivalent to that of the untreated control. , 100% efficacy means that no disease is observed. [Table 4]

[0129] Example 7. Powdery mildew fungus (Sphaerotheca f Comparison of Paenibacillus strains in a field trial of zucchini infected with Paenibacillus uliginea Two field trials were conducted on zucchini artificially inoculated with Spaerotheca fuliginea. Five treatments were performed using whole broth injections from each Paenibacillus species strain cultivated in soy-based medium. The spray-dried powder was resuspended in water and applied at a rate of 1000 L / ha as outlined in Table 6. Plants were planted at 4-8 day intervals between July 15th and August 8th at the growth stages BBCH59 to BBCH72. The percent disease control shown in Table 5 was performed 10 days after the final application. is the result of the last evaluation, where disease symptoms were visually observed. 0% corresponds to the efficacy of the untreated control. 100% efficacy meant that no disease was observed. [Table 5] [Table 6]

[0130] The results in Table 4 demonstrate that the observed activity of Paenibacillus sp. strain NRRL B-50972 is The antifungal activity of the α-glucan derivatives tested in this field study was relatively high in the previous screening assay. This clearly demonstrates its superiority over other strains tested.

[0131] Example 8. Powdery mildew (Uncinula Necator) Comparison of Paenibacillus strains in field trials on infected grapevines. Two field trials with grapevines naturally infected with Uncinula nectar were conducted. Six treatments were performed by resuspending the spray dried powder described in Example 8 in water and applying 1000 L / ha. In quantity, between June 3rd and July 1st, at 5-7 day intervals at the growth stage of BBCH57-BBCH75 The percent disease control shown in Table 7 was measured at 100°C for 10 days and applied to the plants as shown in Table 8. The results are from the final evaluation of disease symptoms, which was conducted 15 days after application and was visually observed. means efficacy equivalent to that of an untreated control, and 100% efficacy means that no disease damage is observed. This meant that... [Table 7] [Table 8]

[0132] The results in Table 7 demonstrate that the observed activity of Paenibacillus sp. strain NRRL B-50972 is The antifungal activity of the α-glucan derivatives tested in this field study was relatively high in the previous screening assay. This clearly demonstrates its superiority over other strains tested.

[0133] Example 9. Summer blight (Alternaria solani) Comparison of Paenibacillus strains in field trials on infected tomatoes. Two field experiments were conducted using tomato plants artificially inoculated with Alternaria solani. Three treatments were performed by resuspending the spray-dried powder described in Example 8 in water and applying it at 1000 L / ha. The application rate was 6 to 10 days between June 26 and July 10 at the growth stages of BBCH51 to BBCH59. Plants were applied at 8 day intervals as shown in Table 10. Percent disease control was as shown in Table 9. The values ​​are the results of the final evaluation of disease symptoms by visual observation, which was carried out 8 days after the final application. 0% means efficacy equivalent to that of the untreated control, 100% efficacy means no disease observed. This meant that it was not possible. [Table 9] [Table 10]

[0134] The results in Table 9 demonstrate that the observed activity of Paenibacillus sp. strain NRRL B-50972 is The antifungal activity of the α-glucan derivatives tested in this field study was relatively high in the previous screening assay. This clearly demonstrates its superiority over other strains tested.

[0135] Example 10. Summer blight (Alternaria solani) Comparison of Paenibacillus strains in field trials on infected potatoes. One field trial was conducted using potato plants artificially inoculated with Alternaria solani. Five treatments were performed using the spray-dried powder described in Example 8 resuspended in water and applied at 500 L / ha. At the application rate of 4, the BBCH37 to BBCH55 growth stages were grown between June 26 and July 19. The plants were treated with the disease control agents shown in Table 11 at intervals of 1-8 days as shown in Table 12. The percentage is the result of the final evaluation of disease symptoms by visual observation, performed 6 days after the last application. 0% means efficacy equivalent to that of the untreated control, 100% efficacy means no disease observed. It meant not being noticed. [Table 11] [Table 12]

[0136] The results in Table 11 show that the observed activity of Paenibacillus sp. strain NRRL B-50972 was In this field study, It clearly demonstrates superiority compared to the other strains tested.

[0137] Example 11. Summer blight (Alternaria solani) Comparison of Paenibacillus strains in field trials on infected potatoes. One field trial was conducted using potato plants artificially inoculated with Alternaria solani. Three treatments were performed using the spray dried powder described in Example 8 resuspended in water and applied at 500 L / ha. At the growth stages of BBCH37 to BBCH51 between July 24th and August 5th, Plants were treated at 6-day intervals as shown in Table 14. Percent disease control was reported in Table 13. The results are the results of the final evaluation of disease symptoms by visual observation, which was carried out 6 days after the last application. 0% means efficacy equivalent to that of the untreated control, 100% efficacy means no disease observed. This meant that it would not be possible. [Table 13] [Table 14]

[0138] The results in Table 13 demonstrate that the observed activity of Paenibacillus sp. strain NRRL B-50972 was In this field study, It clearly demonstrates superiority compared to the other strains tested.

[0139] Example 12. Identification of fusA mutations in Paenibacillus sp. NRRL B-50972 fixed To further characterize the Paenibacillus sp. NRRL B-50972 strain, the FusA fragment was The genomic sequence of the fusA gene encoding salicidin synthetase was determined by standard sequencing. The amino acid sequence of Paenibacillus sp. NRRL B-5097 was determined and related amino acid sequences were identified. The amino acid sequences derived from FusA expressed by the two strains are described in the following publications: It was compared with that of several other Paenibacillus strains, including: Li S., et al. .,(2014).“Complete Genome Sequence of Pa enibacillus polymyxa SQR-21,a Plant Grow th-Promoting Rhizobacterium with Antifun gal Activity and Rhizosphere Colonization n Ability,”Genome Announc,2(2):HASH(0x74 3db288);Niu B.,et al.,(2011).“The Genome of the Plant Growth-Promoting Rhizobacter erium Paenibacillus polymyxa M-1 Contain s Nine Sites Dedicated to Nonribosomal S Synthesis of Lipopeptides and Polyketides ,”J.Bacteriol.193(20):5862-3;Ma M.,et al .,(2011)“Complete Genome Sequence of Pae nibacillus polymyxa SC2, A Strain of Plan t Growth-Promoting Rhizobacterium with B road-Spectrum Antimicrobial Activity,”J. Bacteriol.193(1):311-2;and Li and Jensen ,(2008).Nonribosomal Biosynthesis of Fus aricidins by Paenibacillus polymyxa PKB1 Involves Direct Activation of a d-amino Acid.Chem.Biol.15,118-127.

[0140] The alignment shown in Figure 13 is based on the results of Paenibacillus sp. NRRL B-50972 Expressed mutant FusA revealed significant deletions in fusaricidin synthetase The first deletion is from position 3009 of the corresponding sequence of Paenibacillus sp. strain A (SEQ ID NO: 11). The second deletion spans positions 3037 to 3040 of the corresponding sequence of Paenibacillus sp. strain A (SEQ ID NO: 1 1) ranging from positions 3047 to 3317. Both deletions are located in the FusA fusaricidin synthetase. The enzyme is inserted into the A domain of the third module (i.e., FusA-A3).

[0141] As explained above, each A domain contains 10 amino acids involved in substrate recognition and activation. These conserved amino acid residues are shown in Figure 1. The alignment is outlined in Figure 3. Paenibacillus species NRRL B-50972 The deletion identified in the mutant FusA fusaricidin synthetase expressed by , the last conserved amino acid residue (i.e., Lys517 at position 3486 of SEQ ID NO: 11) ) to remove everything except

[0142] These two deletions in the mutant FusA fusaricidin synthetase are referred to herein as The strain, designated Paenibacillus sp. NRRL B-67129, had a stable colony morphology. It is present in strains derived from Paenibacillus species NRRL B-50972, including mutants that exhibit the Because reversion to wild-type FusA-A3 is unlikely due to the extensive nature of the deletion, , Random mutant strains derived from Paenibacillus species NRRL B-50972 were mutant It appears likely that this deletion in FusA-A3 will generally be maintained.

[0143] Example 13. Paenibacillus sp. NRRL B-50972 strain and Paenibacillus sp. A Comparison of fusaricidin production among strains To determine the effect of mutant FusA-A3, we performed paenicelin and fusaricidin assays. The cells were cultured using the method described in Example 14, using Paenibacillus sp. NRRL B-50972 strain. (expressing mutant FusA-A3) and Paenibacillus sp. strain A (wild-type FusA-A The identity of each compound was determined by its unique retention time and mass. The relative signal intensities of each peak in the spectrum are shown in Table 15. In the absence of a standard, absolute quantification was not possible. However, similar amounts of each cell extract were used. injection and the relative amounts of the compounds can be estimated from the resulting signal intensities. [Table 15]

[0144] In the wild-type FusA fusaricidin synthetase, FusA-A3 is located at the amino acid position ( 3) Fusaricidin compounds containing L-Tyr, L-Phe, L-Val, L-Ile or L -allo-Ile (see Table 1). The mutant FusA-A3 in strain B-50972 does not express detectable fusaricidin C, fusaricin. The result was an extract that did not contain LiF07a, LiF07b, or LiF07b. Fusaricidin C and Fusaricidin D both have tyrosine at amino acid position (3), and LiF0 Both 7a and LiF07b have a phenylalanine at amino acid position (3). These experimental data are based on the results of the experiments using Paenibacillus sp. strain NRRL B-50972. Mutations in the FusA-A3 gene result in a tyrosine or phenylalanine at amino acid position (3). It has been shown that the compound inhibits the biosynthesis of fusaricidin, which has the formula (see Figure 14).

[0145] Therefore, Paenibacillus sp. NRRL B-50972 strain and Paenibacillus sp. N The mutants derived from RRL B-50972 lack tyrosine or phenylalanine at amino acid position (3). a detectable amount of fusaricidin or fusaricidin-like compound having fluoranine (e.g., Fusaricidins C and D or LiF07a and LiF07b) can be produced. Analysis of mutant FusA-A3 in Paenibacillus sp. NRRL B-50972 strain Analysis showed that this strain and its mutants contain either a tyrosine amino acid or a phenylalanine amino acid at amino acid position (3). Fusaricidin or fusaricidin analogues having a peptide ring containing a hydroxyalanine amino acid This indicates that the gene cannot be produced genetically.

[0146] Of the two types of paenicellin analyzed, one was detectable in Paenibacillus species A strains. The signal intensity was 100% in the Paenibacillus species NRRL B-50972 strain extract. This was less than half the corresponding signal intensity observed. Without being bound by any theory, , but the first nine conserved amino acids in FusA-A3 (i.e., A sp235, Ala236, Ser239, Thr278, Leu299, Ala301 , Ala / Gly322, Val330, and Cys331) Responsible for the recognition and activation of tyrosine and phenylalanine at position (3) of lysidine compounds Furthermore, the mutant F expressed by Paenibacillus sp. NRRL B-50972 strain usA-A3 converts metabolic intermediates from the production of specific fusaricidins to a broader range of fusaricidins. This can shift the biosynthesis of gin-like compounds (e.g., paenicelin) towards

[0147] Example 14. Paenibacillus sp. NRRL B-50972 strain and Paenibacillus sp. A Comparison of biological activity of strains Paenibacillus species NRRL B-50972 strain (expressing mutant FusA-A3) and and Paenibacillus sp. strain A (expressing wild-type FusA-A3) were grown in soy-based medium. The whole broth was then diluted with a mixture of water and organic solvent at 10%, 5%, and 2%. The diluted whole broth was applied to young plants and then Puccinia tristinae (PUCCRT), Botrytis cinerea (BOTRCI ), or exposed to an inoculum of Phytophthora infestans (PHYTIN). After several days of exposure to the pathogen inoculum, each plant was compared to an untreated control plant for pathogen control. Each treatment was evaluated in triplicate and the average percent control was calculated as The results were recorded (see Tables 16-18).

[0148] In each assay, Paenibacillus species NRRL B-50972 strain was These experimental data show that the mutant fusaricidin synthetase exhibited superior control over the species A strain. in the biosynthesis of fusaricidin and fusaricidin-like compounds These changes enhance the control of plant pathogens by Paenibacillus sp. NRRL B-50972. suggests that it will bring about [Table 16] [Table 17] [Table 18]

[0149] Example 15. Identification of fusaricidins in cell extracts of Paenibacillus species Paenibacillus species NRRL B-50972 strain and / or strains derived therefrom were grown in soybeans. The cells were grown in the base medium until they reached stationary phase, at which point the whole broth culture was harvested and The cell extract was generated by extraction with organic solvent.

[0150] High performance liquid chromatography / mass spectrometry time-of-flight (HPLC / MS TOF) was used. We developed a chromatographic method to isolate many fusaricidin-like molecules from cell extracts. Column: YMC™ Basic 4.6 x 250 mm, 5 μm; water (0.1% F A) and acetonitrile (0.1% formic acid (FA)); Gradient (% B): 0–9 min 28–3 0%; 9~14 minutes 30~33%; 14~34 minutes 33~50%; Wash A chromatogram from a cell extract in which known fusaricidins have been identified is shown in Figure 4B. The general structure of fusaricidins is shown in Figure 4A. Each cyclic fusaricidin has a corresponding acyclic analogue. Have a body.

[0151] All detectable fusaricidins in the cell extracts were identified by their retention times and m / z values. (See Figure 4C.) Interestingly, the amino acid at position (3) is a tyrosine. or phenylalanine, fusaricidins C and D and other fusaricidins It was not detected in the cell extracts.

[0152] Example 16. Characterization of Paenicelins in Paenibacillus species cell extracts The cells of Paenibacillus species NRRL B-50972 strain and / or strains derived therefrom To identify other compounds in the cell extract, ultra-high performance liquid chromatography / mass spectrometry was performed. Chromatography using ripple time-of-flight (UPLC / MS Triple TOF) A method was developed to fragment a large number of fusaricidin-like molecules. Column: ZORBAX (trademark) ) Eclipse Plus, 2.1 x 100 mm, 1.8 μm; water (0.1% FA) and acetonitrile (0.1% FA). Gradient (% B): 0–5 min, 10–95%; wash.

[0153] Using this method, the applicant has developed an AB SCIEX TRIPLE TOF (registered trademark) ) by examining the mass fragmentation patterns obtained from the mass spectrometer, as well as by examining published By comparing the spectra with those of the literature, we identified a novel paenicelin family of fusaricidins. The applicants have named this new family Paeniseri The representative UPLC / MS analysis of paenicelin A1 and paenicelin B1 was performed. The MS Triple TOF fragmentation patterns and corresponding chemical structures are shown in Figure 5 and Figure 6, respectively. and 6. A similar analysis was performed for each of the paenicellins detected in the cell extracts.

[0154] Paenicelin is an important derivative of the fusaricidin backbone with one or more serine substitutions. Historically, fusaricidins were named for their (1) stimulating activity against fusaricin, and (2) their ability to inhibit fusaricin. Contains three conserved amino acids: leonine, (4) threonine, and (6) alanine However, Paenicelin is thought to be a peptide sequence of (1) and (4). This shows a novel substitution in which one or both of the propylamine residues are replaced with serine. In the labeled paenicelin, the amino acids at positions (2) and (3) are both valine. The chromatogram in which the peak corresponding to paenicelin was identified is shown in Figure 5B.

[0155] Applicants also identified a family of serine-substituted fusaricidin-like compounds in cell extracts. , and were characterized based on their retention times and m / z values ​​(see Figure 5C). Although C4 was not detectable, the production of fusaricidin was based on the previously characterized structure of fusaricidin. It is reasonable to expect that, like fusaricidin, each cyclic paenicellin has a corresponding acyclic analogue.

[0156] Applicant characterized the paenicelin as having valine amino acids at residues (2) and (3). However, it should be noted that compounds with mutations at these positions may exist. These potential mutations are isoleucine, phenylalanine, and phenylalanine as residues (2) and (3). Fusaricidin / LiF analogs containing amino acids such as phenylalanine and tyrosine Furthermore, the tail of GHPD is shown above, but the length of the tail is similar to that of the It is highly likely that compounds with similar changes to the niprolixin family exist (see Example 17).

[0157] Example 17. Characterization of Paeniprolixin in Paenibacillus species cell extracts Paenibacillus sp. strain NRRL B-50972 and / or cell lines derived therefrom The extract was further analyzed by the chromatographic method described in Example 14. Introducing the new family of AB SCIEX TRIPLE TOF® mass spectrometers by examining the mass fragmentation patterns obtained from the The applicant characterized this new family, Paeniprolii, by comparing the Paeniprolixin C1 and Paeniprolixin C2 were named Paeniprolixin. Representative UPLC / MS Triple TOF fragmentation pattern of eniprolixin D1 The corresponding chemical structures are shown in Figures 8 and 9, respectively. This was performed on each paeniprolixin detected.

[0158] Paeniprolixin represents another important departure from the fusaricidin backbone in the aliphatic tail. Its name comes from the Latin prolix (meaning long), which is ascribed to the Niprolixin has a longer tail than fusaricidin. Historically, fusaricidin was used as a This is the latest information on the subject. Publications (e.g., Vater et al., J. Am. Soc. Mass Spec trom.,2015,26,1130-1141) also showed that this is consistent. The authors concluded that the discovery that the tail of GHPD is strictly conserved. In contrast to many other lipopeptides reported in the literature, the fatty acid moiety is "It is the main target for structural changes in proteins such as factin, italin, and fengycin." Applicant has identified a longer tail in cell extracts of Paenibacillus sp. NRRL B-50972. (i.e., 17-guanidino-3-hydroxyheptadecanoic acid or GHPD + 2CH 2 and 19-guanidino-3-hydroxynonadecanoic acid (GHPD + 4CH2) A family of salicidin-like compounds was identified (see Figure 8A). Unlike paenicelin, Paeniprolixin contains L-threonine at position (1) and D-allo-threonine at position (4). Maintain the conserved amino acid residues of onine.

[0159] Applicant has characterized paeniprolixin as containing valine or isopropyl at residues (2) and (3). There are compounds that have either amino acid of soleucine but have mutations at those positions. It is important to note that these potential mutations may be present in (2) and and (3) residues such as valine, isoleucine, or phenylalanine and tyrosine. It is believed that other combinations of amino acids are similar to fusaricidin / LiF analogs. Furthermore, hybrids with the above-mentioned paenicelin having longer tail lengths are also possible. There may be a combination.

[0160] The chromatogram in which the peak corresponding to paeniprolixin was identified is shown in Figure 8B. This family of fusaricidin-like compounds with longer GHPD tails also exhibits these retention times. The compounds were characterized based on their m / z values ​​(see Figure 8C). D1 and D2 were not detected, but were produced based on the previously characterized structure of fusaricidin. As in the case of fusaricidin, it is reasonable to expect that each cyclic paenipro Rixin has a corresponding acyclic analogue.

[0161] Example 18. Antifungal properties of paenicelin, paeniprolixin, and other fusaricidins Fungal Bioactivity Profile The samples shown in Table 19 were isolated from Paenibacillus sp. cells. The fermentation whole broth was centrifuged and The supernatant was removed. The resulting pellet was then extracted with methanol. The fractions were then separated using reversed-phase medium pressure liquid chromatography. Further purification was carried out using column chromatography. [Table 19]

[0162] In vitro antifungal 96-well plate assay demonstrates the efficacy of resazurin as an indicator of fungal growth. Using the original cell viability reagent PRESTOBLUE®. Starting with fungal spores. Therefore, this assay is used to evaluate the ability of ATP to inhibit fungal spore germination and / or fungal cell growth. The efficacy of the sample was measured. The assay was used to measure the efficacy of three agriculturally relevant fungal diseases: Alternaria alternata, Naria solani (ALTESO), Colletotrichum lagenarium (COLLLA) and and Botrytis cinerea (BOTRCI) were used.

[0163] All samples outlined in Table 19 have been shown to be active against agriculturally relevant fungal diseases. It has been proven that the 80% maximum values ​​in parts per million (ppm) for each sample in Table 20 Interestingly, certain compounds have been shown to inhibit the growth of certain pathogens. For example, the asparagine analogue in sample 3 The glutamine of the same compound as sample 4 is thought to be important in controlling ALTESO. The longer tailed analogs of sample 6 were more involved in controlling COLLLA. was the most potent inhibitor of COLLLA. This indicates that all are active in their own right. On the other hand, the combination of these chemicals determines the ultimate potency and spectrum of disease control in the final product. This suggests that it is important for [Table 20]

[0164] Example 19. Antibacterial physiology of paenicelin, paeniprolixin, and other fusaricidins Activity Profile The in vitro antibacterial 96-well plate assay uses absorbance as an indicator of bacterial growth. This assay measures the absorbance of untreated wells by comparing it to sample wells. The potency of a sample to inhibit bacterial growth is measured. The final dilution / concentration that inhibits bacterial growth is called the MIC. (minimum inhibitory concentration The assay is called the potency (on) and this value can be used to compare the potencies of different samples. , Xanthomonas campestris (XANTAV), Pseudomonas syringae (PS DMTM), and Erwinia carotovora (ERWICA), three agriculturally relevant bacteria The evaluation was carried out using sexual diseases.

[0165] The samples outlined in Table 19 were applied to the antibacterial assay to determine the MIC80 with each bacterial pathogen. The assay results are shown in Table 21. Samples 1 to 5 were used to treat agriculturally relevant bacterial diseases. Interestingly, certain compounds were found to be active against specific diseases. For example, paenicelin has the weak point of fusaricidin A. It complements fusaricidin A in that it can control PSDMTM, a fungus. On the other hand, fusaricidin A compensates for the weakness of paenicelin in controlling ERWICA. As with the fungal assays, this means that while all are valid in their own right, The combination of chemicals plays a key role in the final efficacy and spectrum of disease control of the final product. This suggests that it is important. [Table 21]

[0166] Example 20. Kirby-Bauer Antibiotic Synergy by Disk Diffusion Assay Suggestion To obtain an initial assessment of synergy between different classes of fusaricidin-like compounds, Bioassays were performed using the pathogen Colla. A classic Kirby-Bauer antibiotic disk diffusion assay was performed (Bauer, A .W.,et al.,1966 Am.J.Clin.Pathol.36:493- 496) Briefly, similar amounts of various seeds were added to petri dishes inoculated with a lawn of COLLLA spores. A blank sterile disk loaded with each sample was placed on the Petri dish. Activity was recorded as the diameter of the inhibition zone around the disk. The results are shown in Figure 1. Shown in 1.

[0167] The results of this preliminary assay suggest that specific paeniselin and paeniprolixin administered together This suggests that there is a synergistic effect when Paenicelin A1 is applied separately. and B1 ("868") or paeniprolixin A2 and B2 ("938") , show a relatively small zone of inhibition in this assay. However, their combination ( "868 / 938") is a fusion protein consisting of 868, 938, or fusaricidins A and B ("AB"). The largest and most distinct zone of inhibition is shown in Figure 1. For 868 and 938 samples, approximately 0.1 mg of total material was applied to each sterile disk. The disk containing both the 868 and 938 samples is the total amount of material on the 868 / 938 disk. Each sample contained approximately 0.05 mg, so that the total weight was approximately 0.1 mg.

[0168] A limitation of this assay is that the fusaricidin compounds must be diffused into the agar to inhibit fungal growth. This early indicator of synergy is determined by the presence of Further evaluation was performed using an in vitro antifungal assay.

[0169] Example 21. In vitro antifungal studies to demonstrate synergistic effects of fusaricidin combinations Assay In addition to the fusaricidin combinations outlined in Example 17, in vitro antifungal activity in liquid media Assays were performed to detect the fusaricidin and / or fusaricidin-like compounds shown in FIG. To demonstrate the proposed synergistic effects resulting from the application of the combination, each of the groups shown in Figure 12 Each was evaluated individually, first to assess structural properties, and then combined to address synergistic effects. Evaluate both binary and ternary mixtures.

[0170] Although individual compounds may exhibit weaknesses in terms of fungicidal activity, combinations may exhibit similar activity. It will have more activity than the pure addition.

[0171] Fungicide synergy is when the fungicidal activity of a combination of active compounds is greater than that of the active compounds when applied individually. In any case, the total activity of the active compounds in the active compound mixture is always greater than the sum of the activities of the active compounds in the active compound mixture.

[0172] The expected activity for a given combination of two or three active compounds is as follows: (Colby, SR, "Calculating Synthetic rgistic and Antagonistic Responses of He rbicide Combinations, Weeds 1967,15,20-22 ).

[0173] X is the efficacy of active compound A when applied at a rate of m ppm (or g / ha). can be, Y is the efficacy of active compound B when applied at a rate of n ppm (or g / ha). the law of nature, Z is the efficacy of active compound B when applied at a rate of r ppm (or g / ha). the law of nature, E1: Active compounds A and B applied at rates of m and n ppm (or g / ha) is the resistance when E2 contains the active compounds A, B and C at application rates of m, n and r ppm (or g / ha) and In this case, for a binary mixture:

number

[0174] and for ternary mixtures:

number

[0175] is.

[0176] The degree of efficacy is expressed as a percentage, with 0% meaning efficacy equivalent to that of the control and 100% efficacy. Power means that no disease is observed.

[0177] If the actual fungicidal activity exceeds the calculated value, the activity of the combination is superadditive, i.e. In this case, the actual observed efficacy is greater than the predicted efficacy calculated from the above formula. It must be greater than the value of Efficacy (E).

[0178] A further method for demonstrating synergy is the Tammes method ("Isobo les,A Graphic Representation of Synergis m in Pesticides”in Neth.J.Plant Path.,19 64, 70, 73-80).

[0179] Example 22. Selection of mutant strains of Paenibacillus sp. NRRL B-50972 Under standard laboratory conditions, Paenibacillus species NRRL B-50972 strain is Produces multiple colony morphologies on crystalline media. Several morphologically distinct colonies were identified. The liquid cultures were cultured at −80°C as glycerol stocks. -phenotype-derived stocks were inoculated and grown in liquid medium for several rounds before solidification. From this, the strains with stable colony phenotypes under the tested conditions were selected. , one isolate that is still capable of producing heat-resistant spores and fusaricidin chemicals Isolates with stable colony morphology were preferred for further strain improvement. This isolate was deposited with the NRRL on September 1, 2015, and is currently under construction. It has been assigned the number NRRL B-67129.

[0180] Example 23. Random mutagenesis to generate improved Paenibacillus species mutants Chemical mutagenesis Creation of a pool of genetically diverse isolates of Paenibacillus sp. NRRL B-67129 To obtain the desired product, a liquid culture of the strain was pelleted by centrifugation and 1-methyl-3-dimethyl- in a buffer containing 1-nitroguanidine (NTG) at a final concentration of 400 μg / mL As a reference, a second sample without NTG was prepared. The samples were incubated at 30°C and The mixture was incubated at 220 rpm for 1 hour. After 1 hour, the samples were centrifuged. The pellet was washed with NTG-free buffer and finally resuspended in the same volume of fresh buffer. Aliquots of the undiluted culture were stored as glycerol stocks at -80°C. The samples were diluted and plated on agar plates to measure colony forming units. The mortality rate was determined as a measure of the mutation rate per genome. The improved isolates selected from the screening were subjected to one or more rounds of NTG as described above. The strains were then subjected to treatment and screened for further improvement in fusaricidin production. The production of fusaricidin A (also known as LiF04a or "FusA"); LiF08a; Paenicellin A1 and B1 (also known as "M868" or "868") known); Paeniprolixin A2 and B2 ("M938" or "938" The relative amounts of several compounds, including benzodiazepines (also known as benzodiazepines), were determined.

[0181] High-throughput screening and separation characteristics The NTG-treated samples were diluted and plated on agar plates to identify single colonies. A single colony was inoculated into a 96-well deep well block containing seed medium and incubated for 30 The soybean-based production medium was then incubated at 4°C for 2 days with shaking. Inoculate a new 96-well deep well block containing 100 ml of PBS and incubate at 30°C for 5 days without shaking. After 5 days, glycerol stocks were extracted from each sample in individual wells. The samples were prepared and stored at -80°C. The four fusaricidin biomarkers identified above were analyzed. In this primary screen, individual isolates were subjected to chemical analysis. fusarizidin values” (i.e., the ratio of the four analyzed fusarizidin values ​​to the average of the wild-type values). When the mean of the wild-type values ​​(sum of biomarkers) was three times the standard deviation of the wild-type values, Eight replicates of each isolate selected based on this criterion were analyzed as described above. Confirmed fusaricidin overproducers were then grown in 250 mL shaker flasks and analyzed. Scale up to 50 mL in a scopoeia and evaluate sporulation, fusaricidin production, and bioactivity. The prioritized isolates were further scaled up in bioreactors and characterized for their spore form. The fusaricin-producing cells were characterized for their growth, viscosity, fusaricin production, and biological activity. Several mutant strains were obtained from the second round of screening, and they showed excellent fusaricidin- It was found to have biomarker production and biological activity.

[0182] Example 24. Characterization of antibiotic susceptibility of Paenibacillus sp. strain NRRL B-50972 hair Paenibacillus species NRRL B-50972 strain was supplemented with typical concentrations of antibiotics. Solid sLB agar and sLB agar were inoculated. The agar plates were incubated at 30°C. The cells were incubated and growth was assessed after 24, 48 and 72 hours. The susceptibility of Paenibacillus sp. NRRL B-50972 to the tested antibiotics is shown in Table 22. Shown below. [Table 22]

[0183] Example 25. Paenibacillus sp. NRRL B-50972 strain and Paenibacillus sp. N Characterization of spo0A in strain RRL B-67129 Paenibacillus species NRRL B-50972 strain and Paenibacillus species NRRL B- The genome of strain 67129 was sequenced. Comparison of the two genome sequences revealed that the two strains We identified characteristic differences in the spoOA gene in the genus spoOA, as shown in the sequence alignment in Figure 15. NRRL B-50972 and Paenibacillus sp. NRR strain L B-67129 differs by one nucleotide towards the 3' end of the spo0A gene. Single nucleotide differences were identified and are indicated by red arrows below the sequences in Figure 15. The nucleotide number for the first nucleotide of the A gene is shown above the sequence.

[0184] Alignment of Spo0A orthologues from endospore-forming bacteria reveals that Paenibacillus sp. NR Nucleotide changes in the coding sequence of strain RL B-67129 were found in a single conserved region. It was shown that this resulted in the amino acid substitution of (see Figure 16). Spo0A amino acid sequence (NCBI reference sequence: WP_044647644.1), Nibacillus polymyxa SQR-21 (GenBank: AHM66630.1), wasp R. subtilis subsp. subtilis strain 168 (NCBI Reference Sequence nce:NP_390302.1), Bacillus cereus E33L (GenBank:A JI26924.1), and Clostridium pasteurianum DSM 525 (Ge nBank:AAA18883.1) was used as the Paenibacillus species NRRL B-50972 strain. and Spo0A amino acid sequence and amino acid sequence from Paenibacillus sp. NRRL B-67129 strain The arrow in Figure 16 indicates the sequence of the Paenibacillus sp. NRRL strain B-67129. Single amino acid substitutions in the original Spo0A are shown.

[0185] Example 26. Structure-activity relationship study with fusaricidin, paenicelin, and paeniprolixin Using the in vitro assay described in Example 16, several purified fusaricidins In the first experiment, the structure-activity relationship of paenicelin and paeniprolixin was investigated. The most common fusaricidin pairs were compared. These fusaricidin mutations were This occurs at amino acid position (5) of the ring / chain, which has either glutamine or glutamine. In a study, fusaricidin was used to treat the plant pathogen Alternaria solani (ALTESO). A was compared with fusaricidin B, and LiF08a was compared with LiF08b. In both studies, the asparagine analog was more than twice as potent as its glutamine counterpart ( See Figure 17).

[0186] In a separate experiment, the cyclic versus acyclic forms of fusaricidin were compared. It is unclear whether the formula forms are precursors or decomposition products of the final compounds, but they It is ubiquitously present in the fermentation broth of Paenibacillus species NRRL B-50972 strain, and It is a common contaminant in purified fusaricidin from broth. In vitro assays using the plant pathogen Alteso, LiF04c, and L The ester bond was compared with a mixture of iF04d (acyclic analogues of fusaricidins A and B). There is a significant effect of peptide ring opening in the presence of cyclic hydroxybenzoates. The acyclic analogue was inactive at the highest concentration tested. (See Figure 17.) This is important for structural information that would otherwise be difficult to purify. These compounds, which typically constitute contaminants in fusaricidins, may contribute to the antifungal activity. Demonstrate that the likelihood of not giving

[0187] Amino acid substitutions at amino acid positions (2) and (3) of the ring / chain were also investigated. The bodies fusaricidin A, LiF05a, LiF06a, and LiF08a are located at their positions These differ in that they have a combination of either valine or isoleucine. The two most potent analogs were tested in an in vitro assay using the plant pathogen Alteso. The body contains fusaricidin A (valine / valine) and LiF08a (isoleucine / isoleucine). The other two analogs, containing a valine / isoleucine mixture, were less than one-third (See Figure 17.)

[0188] The difference in antifungal activity between the novel paenicelin and ALTESO was also investigated. The differences in amino acid positions (1) and (4) of the ring / chain were evaluated. Lysidine is restricted to threonine at these positions, while paenicelin is restricted to threonine and Paenicellin is a phosphodiesterase inhibitor that inhibits fusaricidal activity in this assay. The antifungal activity of the compound was similar to that of phenanthrene A (see FIG. 17).

[0189] Against the fungal pathogens Alteso and Colletotrichum lagenarium (Colla) Using an in vitro assay, paeniprolixin (i.e., with different side chain lengths) The antifungal activity of the classical fusaricins (15-guanidino-3-hydroxybenzoates) was also investigated. Paeniprolixin contains a hydroxypentadecanoic acid side chain. It has four additional methyl groups in the chain. It has been shown that side chain length has a significant effect on biological activity. There was a difference between different fungal pathogens. was the most potent, decreasing with each additional methylene group. The most potent length was GHPD+2CH2 (see Figure 17).

[0190] Example 27. Fusaricidin A and Paenicelin A1 or Paeniprolixin C1 Synergistic antifungal activity of the mixture The resazurin-based cell viability reagent PRESTOBLUE® (see Example 18) In vitro antifungal 96-well plate assays using fusaricidin, phenobarbital Eniselin and paeniprolixin, alone or in a two-way combination, are antifungal The activity was evaluated. Antifungal activity was calculated in relation to the untreated control value using the following formula: Efficacy = (100 - relative proliferation to untreated control) 100% efficacy indicates no fungal growth compared to untreated controls, 0% efficacy indicates no fungal growth compared to untreated controls. This shows no inhibition of fungal growth compared to the control.

[0191] Tables 23 and 24 show the observed activity of the active compound combinations according to the invention, calculated The activity was greater than that observed in the control group, clearly indicating that a synergistic effect was present. [Table 23] [Table 24]

[0192] Unless otherwise defined, all technical and scientific terms used herein are defined by the present invention. The terms have the same meaning as commonly understood by a person skilled in the art to which they pertain. All publications, patents and patent applications cited therein are hereby incorporated by reference in their entirety for all purposes. incorporated herein.

[0193] The disclosed invention is not limited to the particular methodology, protocols, and materials described, and variations are possible. It is understood that various modifications may be made. Also, the terminology used herein may be used to describe specific embodiments. It is for illustrative purposes only and is not intended to limit the scope of the invention, the scope of which is set forth in the accompanying drawings. It is also understood that one is limited only by the scope of the claims.

[0194] Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein. Such equivalents are likely to be recognized or ascertainable using no more than routine experimentation. are intended to be encompassed by the following claims.

Claims

1. Structure (II) 【Chemistry 1】 (In the formula, (a) R １ Ha-CH ２ OH and R ２ Ha-CH ２ C(O)NH ２ and R ３ is H; (b) R １ Ha-CH ２ OH and R ２ Ha-(CH ２ ) ２ C(O)NH ２ and R ３ is H; (c) R １ Ha-CH ２ OH and R ２ Ha-(CH ２ ) ２ C(O)NH ２ and R ３ is CH ３ Is it; (d) R １ -CH(OH)CH ３ and R ２ Ha-CH ２ C(O)NH ２ and R ３ is H; (e) R １ -CH(OH)CH ３ and R ２ Ha-(CH ２ ) ２ C(O)NH ２ and R ３ is H) or a salt, hydrate, solvate, polymorph, optical isomer, geometric isomer, enantiomer, diastereomer, or acyclic analog thereof, wherein the acyclic analog results from cleavage of the ester bond in structure (II) to form a linear structure.

2. The compound having the structure (II) or an acyclic analog thereof is 【Chemistry 2】 【change】 【change】 or 【change】 2. The composition of claim 1, wherein:

3. 10. The composition of claim 1, which is a fermentation product of a Paenibacillus sp. strain.

4. 4. The composition of claim 3, further comprising a formulation ingredient selected from the group consisting of humectants, bulking agents, solvents, dispersants, anti-frost agents, thickeners and adjuvants.

5. The composition of claim 1 further comprising fusaricidin.

6. The composition of claim 5, wherein the fusaricidin is fusaricidin A.

7. The compound having the structure (II) 【Transformation 3】 The composition of claim 5 , wherein

8. 10. A method for treating plants to control diseases, comprising applying a composition according to claim 1 to the plant, to a part of the plant and / or to the locus of the plant.

9. 9. The method of claim 8, comprising applying the composition of claim 1 or 2 to leafy plant parts.

10. The disease is caused by Alternaria alternata, Alternaria solani, Botrytis cinerea, Colletotrichum lagenarium, Fusarium culmorum, Phaeosphaeria nodorum, Zymoseptoria tritici, Phytophthora cryptogea, Phytophthora infestans, or the like.

9. The method of claim 8, wherein the pest is caused by Pythium infestans, Pythium ultimum, Magnaporthe oryzae, Thanatephorus cucumeris, Ustilago segetum var. avenae, Uromyces appendiculatus, or Puccinia triticina.

11. 9. The method of claim 8, wherein the disease is caused by a bacterium selected from the group consisting of Xanthomonas campestris, Pseudomonas syringae, and Erwinia carotovora.

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