COMPOSITION UNDERSTANDING WHOLE WHOLE CELL FERMENTATION CELLS OF BACILLUS SP. WITH PESTICIDE ACTIVIT

专利摘要:
composition comprising whole cell fermentation medium of bacillus sp. with pesticidal and growth-promoting activity, a method for modulating pest infestation and plant growth, and seed. the present invention relates to a bacillus strain, isolated f727 from bacillus sp. which produces metabolites with pesticidal activities. bioactive compositions and metabolites derived from cultures of bacillus sp. able to control pests; as well as processes for using the strain and its metabolites for pest control.
公开号:BR112015003123B1
申请号:R112015003123-4
申请日:2013-08-13
公开日:2021-04-13
发明作者:Ratnakar Asolkar；Ana Lucia Cordova-Kreylos；Christopher Mccort；Debora Wilk；Carly Todd；Hai Su；Pamela Marrone
申请人:Marrone Bio Innovations, Inc；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED-RELATED PATENT APPLICATIONS
[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 61 / 683,174 filed on August 14, 2012 and U.S. Patent Application No. 13 / 835,677, filed on March 15, 2013; whose disclosures are hereby incorporated as a reference in their entirety for all purposes. TECHNICAL FIELD
[0002] The present disclosure is in the field of biopesticides and pest control; in particular microbial pesticides and the microbial strains that produce them. BACKGROUND
[0003] Natural products are substances produced by microorganisms, plants and other organisms. Microbial natural products offer an abundant source of chemical diversity and there is a long history of using natural products for pharmaceutical purposes. Despite the emphasis on natural products for therapeutic agents for humans, when more than 50% are derived from natural products, only 11% of pesticides are derived from natural sources. However, natural product pesticides have the potential to play an important role in pest control on both conventional and organic farms.
[0004] Secondary metabolites produced by microorganisms (bacteria, actinomycetes and fungi) provide new chemical compounds that can be used alone or in combination with compounds known to efficiently control insect pests and to reduce the risk of developing resistance. There are several well-known examples of natural microbial products that are successful as insecticides for agriculture (Thompson et al., 2000; Arena et al., 1995; Krieg et al. 1983).
[0005] The development of a microbial pesticide begins with the isolation of a microorganism in a pure culture. This then proceeds with efficiency and spectrum verification using in vitro, in vivo or pilot scale tests in a greenhouse and in the field. At the same time, active compounds produced by the microorganism are isolated and identified. For the commercialization of a microbial pesticide, the micro-organism has to be produced economically by fermentation on an industrial scale and formulated with biologically compatible additives approved to increase efficiency and to maximize ease of application.
[0006] With the development of increasing resistance to chemical pesticides, the spectrum of available pesticides is decreasing. In addition, non-naturally occurring pesticides can have harmful environmental effects. Consequently, there is a need for new, naturally occurring pesticides for which plant pathogens have not developed resistance and which have minimal environmental effects. SUMMARY
[0007] A microbial strain, isolated F727 from Bacillus sp., Which has pesticidal activity, is disclosed here. This strain produces bioactive metabolites that are active in pest control and that promote plant growth. Methods for using Bacillus sp. 727 isolate are also disclosed. and its metabolites for pest control and to promote plant growth. In a particular embodiment, Bacillus sp. it can have at least one of the identification characteristics of NRRL B-50768.
[0008] In addition, Bacillus sp. may have a 16S rRNA gene sequence with at least 99% identity and particularly 99.5% identity to the consensus sequence shown in SEQ ID NO: 3 and which comprises a forward sequence that has at least 99% identity and particularly 99.5% identity to the sequence shown in SEQ ID NO: 1 and a reverse sequence that has at least 99% identity and particularly 99.5% identity to the sequence shown in SEQ ID NO: two.
[0009] A substantially pure culture or whole cell medium is additionally provided comprising said cell strain or fraction, extract, supernatant and / or substances or compounds derived from said strain or extract thereof.
[00010] A method for modulating infestation by pests in a plant is additionally provided, comprising the application to the plant and / or its seeds and / or to the substrate used for the growth of said plant of an amount of said isolate F727 of Bacillus sp. (and / or a culture, a cell fraction, an extract, a supernatant and / or substances or compounds derived from said strain or extract) that is efficient to modulate said pest infestation. In certain embodiments, the pest is a plant fungus such as, for example, Bremia, Botrytis, Sclerotinia, Sphaerotheca, Rhizoctonia, Colletotrichum, Fusarium, Verticillium, Phytophthora or Bipolaris. In additional embodiments, the pest is a bacterium such as, for example, Erwinia, Pseudomonas, Xanthomonas, Acidovorax or Clavibacter.
[00011] Methods are also provided to promote plant growth and / or seed germination, in which the methods comprise application to the plant and / or its seeds and / or the substrate used for the growth of said plant. an amount of said F727 isolate from Bacillus sp. (and / or a culture, a cell fraction, an extract, a supernatant and / or substances or compounds derived from said strain or extract) that is efficient to promote plant growth and / or seed germination.
[00012] In particular embodiments, said Bacillus produces a compound selected from the group consisting of: (a) compound "A" which (i) can be obtained from Bacillus sp., Particularly, isolated 727 from Bacillus sp. 727; (ii) has pesticidal activity; (iii) it has a molecular weight of approximately 10201060 and more particularly, 1044 which is determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); (iv) has 1H NMR values of δ 7.15, 6.72, 4.81, 4.70, 4.65, 4.40, 4.35, 4.25, 4.15, 3.85, 3.65, 3.50, 3.22, 2.85, 2.80, 2.65, 2.45, 2.35, 2.30, 2.20, 1.95, 1.55, 1, 31, 1.20 and 0.85; (v) has a retention time in High Pressure Liquid Chromatography (HPLC) of approximately 6-12 minutes, more specifically approximately 8 minutes and even more specifically approximately 8.31 min on a reverse phase HPLC C-18 column ( Phenomenex, Luna 5μ C18 (2) 100 A, 100 x 4.60 mm) using a water gradient solvent system: acetonitrile (CH3CN) (0-20 min; 90-0% aqueous CH3CN, 20-24 min ; 100% CH3CN, 24-27 min; 0-90% aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at a flow rate of 0.5 mL / min and UV detection at 210 nm; (vi) optionally contains 47 carbons, 72 hydrogens, 12 nitrogens and 15 oxygen; and (vii) is optionally a peptide and can comprise glutamine (1 unit), proline (1 unit), serine (1 unit), tyrosine (1 unit) and asparagine (3 units); (b) Compound "B" that (i) has pesticidal activity; (ii) it has a molecular weight of approximately 10301080 and more particularly, 1058 which is determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); (iii) has a retention time in High Pressure Liquid Chromatography (HPLC) of approximately 6-14 minutes, more specifically approximately 8 minutes and even more specifically approximately 8.67 min on a HPLC C-18 reverse phase column using a water gradient solvent system: acetonitrile (CH3CN) (0-20 min; 90 - 0% aqueous CH3CN, 2024 min; 100% CH3CN, 24-27 min; 0-90% aqueous CH3CN, 27- 30 min; 90% aqueous CH3CN) at a flow rate of 0.5 mL / min and UV detection at 210 nm; (iv) optionally comprises 48 carbons, 74 hydrogens, 12 nitrogens and 15 oxygen; and (v) is optionally a peptide and can comprise glutamine (1 unit), proline (1 unit), serine (1 unit), tyrosine (1 unit) and asparagine (3 units); and (c) Compound "C" that (i) has pesticidal activity; (ii) it has a molecular weight of approximately 10501120 and more particularly, 1072 which is determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); (iii) it has a retention time in High Pressure Liquid Chromatography (HPLC) of approximately 6-14 minutes, more specifically approximately 9 minutes and even more specifically approximately 9.19 min on an HPLC C-18 reverse phase column using a water gradient solvent system: acetonitrile (CH3CN) (0-20 min; 90 - 0% aqueous CH3CN, 2024 min; 100% CH3CN, 24-27 min; 0-90% aqueous CH3CN, 27- 30 min; 90% aqueous CH3CN) at a flow rate of 0.5 mL / min and UV detection at 210 nm; (iv) optionally contains 49 carbons, 76 hydrogens, 12 nitrogens and 15 oxygen; and (v) is optionally a peptide and can comprise glutamine (1 unit), proline (1 unit), serine (1 unit), tyrosine (1 unit) and asparagine (3 units).
[00013] A Bacillus strain that has the following characteristics is also provided here: (a) at least one of: (1) a nucleotide sequence that has at least 99.5% identity to a 16SrRRNA sequence presented in SEQ ID NO: 3; (2) a nucleotide sequence that has at least 95% identity to a recA sequence shown in SEQ ID NO: 10 and (3) a nucleotide sequence that has at least 90% identity to a sequence of inverse phoR shown in SEQ ID NO: 13; (b) produces one or more compounds that (i) have pesticidal activity; (ii) have a molecular weight of approximately 1020-1120 which is determined by Liquid Chromatography / Mass Spectroscopy (LC / MS) and (iii) have a retention time in High Pressure Liquid Chromatography (HPLC) of approximately 6-15 minutes on a C-18 HPLC reverse phase column using a water gradient solvent system: acetonitrile (CH3CN) (0-20 min; 90 - 0% aqueous CH3CN, 20-24 min; 100% CH3CN, 24-27 min; 0-90% aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at a flow rate of 0.5 mL / min and UV detection at 210 nm and (iv) are optionally peptides; (c) is resistant to kanamycin, chloramphenicol, ampicillin, penicillin, cefuroxime, piperacillin, tetracycline; and (d) it has alkaline phosphatase, esterase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activity.
[00014] A combination is also provided which comprises said F727 isolate from Bacillus sp., A substantially pure culture, cell fraction, extract, supernatant and substances, metabolites or compounds derived from said strain or extract thereof and at least one of ( a) a second substance that can be a chemical or biological pesticide and (b) at least one from a carrier, a diluent, a surfactant, an adjuvant. The combination can be a composition and can be coated on a seed.
[00015] A method for modulating infestation by pests in a plant is additionally provided, comprising the application in the plant and / or in the seeds of the same and / or in the substrate used for the growth of said plant of an amount of said efficient combination to modulate said pest infestation. In certain embodiments, the pest is a plant fungus such as, for example, Bremia, Botrytis, Sclerotinia, Sphaerotheca, Rhizoctonia, Colletotrichum, Fusarium, Verticillium, Phytophthora or Bipolaris. In additional embodiments, the pest is a bacterium such as, for example, Erwinia, Pseudomonas, Xanthomonas, Acidovorax or Clavibacter.
[00016] The use of a composition, optionally in combination with one or more second substances, is further provided to formulate a pesticidal composition, wherein the composition is selected from the group consisting of one or more of: (a) a substantially culture strain of Bacillus sp. F727 isolate, (b) a cell fraction of a culture of Bacillus sp. F727 isolate, (c) a supernatant obtained from a culture of Bacillus sp. F727 isolate, (d) a filtrate obtained starting from a culture of the F727 isolate from Bacillus sp., (e) an extract from either (a), (b), (c) or (d), (f) a metabolite produced by a culture of the F727 isolate from Bacillus sp., (G) compound A, (h) compound B and (i) compound C; and the second substance is selected from the group consisting of: (a) a pesticide, (b) an agent that promotes plant growth, (c) a carrier, (d) an adjuvant, (e) a surfactant, (f ) a fertilizer, and (g) an anti-phytopathogenic agent. BRIEF DESCRIPTION OF THE FIGURES
[00017] Figure 1 shows a schematic representation of the purification scheme for obtaining the compounds of the invention from the culture medium.
[00018] Figure 2 represents the ESI-LCMS chromatogram for compound "A".
[00019] Figure 3 represents the (+) ESIMS for compound "A".
[00020] Figure 4 represents the ESI-LCMS chromatogram for compound "B".
[00021] Figure 5 represents the (+) ESIMS for compound "B".
[00022] Figure 6 represents the ESI-LCMS chromatogram for compound "C".
[00023] Figure 7 represents the (+) ESIMS for compound "C"
[00024] "C".
[00025] Figure 8 represents the biological activity of VLC fraction 3 (F727F3 in the Figure) and Compound A (F727F3H11 in the Figure), Compound B (F727F3H14 in the Figure) and Compound C (F727F3H17 in the Figure) purified by HPLC against four fungal pathogens: Botrytis cinerea (Botrytis in the Figure), Sclerotinia homeocarpa (Sclerotina in the Figure), Rhizoctonia solani (Rhizoctonia in the Figure) and Bipolaris maydis (Bipolaris in the Figure).
[00026] Figure 9 shows the effect of the F727 supernatant on Botrytis cinerea in tomatoes. The plants were inoculated with B. cinerea spores at the concentrations indicated in the Figure and were treated with the supernatant from a fermentation of Bacillus sp. F727 (second bus from the left) or Switch® (third bus from the left). Controls included inoculated plants (leftmost bar) and untreated pesticide plants inoculated with two different concentrations of the fungus (fourth and fifth bars from the left).
[00027] Figure 10 shows the effect of the F727 supernatant on downy mildew on lettuce. The plants were treated with the supernatant of Bacillus sp. (F727); Ridomil or were not treated (UTC).
[00028] Figure 11 compares the effect of the F727 supernatant with Fenhexamid on Botrytis in tomatoes. Plants that were experimentally infected with B. cinerea were pre-sprayed once (F727 supernatant) or twice (F727 x 2 supernatant) with F727 supernatant, with water or with Fenhexamid (Elevate®) and severity of the disease was analyzed.
[00029] Figure 12 compares the effect of the F727 supernatant with Fenhexamid on Botrytis in peppers. The plants were sprayed with the supernatant from a fermentation with Bacillus sp. (F727), water, (UTC) or Fenhexamid (Elevate®). The sprayed plants were then experimentally infected with B. cinerea, grown and analyzed after 13 days in relation to the severity of the disease.
[00030] Figure 13 shows disease control measures in cucumbers infected with powdery mildew and treated with different F727 preparations. F727 cells were grown in three different media: SPY, SMP and TSB, as shown in the figure. Whole cell medium, cells (suspended in 10 mM MgSO4) and supernatant were obtained for each of these growth conditions. Water ("DI Water" in the figure) was used as a negative control. Blanks for SMP medium, SPY medium, TSB medium and 10 mM MgSO4 were also included.
[00031] Figure 14 shows disease control measures in tomato plants infected with Botrytis cinerea and treated with different F727 preparations. F727 cells were grown in three different media: SPY, SMP and TSB, as shown in the figure.
[00032] Medium with whole cells, cells (suspended in 10 mM MgSO4) and supernatant were obtained for each of these growth conditions. Water ("DI Water" in the figure) was used as a negative control. Blanks for SMP medium, SPY medium, TSB medium and 10 mM MgSO4 were also included.
[00033] Figure 15 shows disease control measures in cucumber plants infected with powdery mildew. Before inoculation with fungus spores, the plants were sprayed with water ("DI water" in the Figure, negative control), medium with whole cells from fermentation with isolate F727 (MBI-110 WCB) or one of a number of pesticides commercial (Double Nickel® (Certis, Bacillus amyloliquefaciens strain D747) Sonata® (Bacillus subtilus), Vacciplant®, Companion®, Serenade® (Bacillus pumilus) or Regalia® (Reynoutria sachalinensis) or a combination of Regalia® (Reynoutria sachalinensis) and F727 WCB).
[00034] Figure 16 shows disease control measures in tomato plants infected with Phytophthora infestans. Before inoculation with P. infestans, the plants were sprayed with water ("DI water" in the Figure, negative control), Regalia®, Double Nickel® (Certis, Bacillus amyloliquefaciens strain D747) or medium with whole cells from fermentation with the isolated F727 (MBI-110 WCB).
[00035] Figure 17 shows the effects of supernatants from fermentation with F727 and controls, on the growth of S. rolfsii mycelia in an in vitro assay. The effects of water (DI water), unfiltered F727 supernatant (unfiltered F727), filtered F727 supernatant (filtered F727) and Pristine® are shown. For each test and control material, two volumes were evaluated: in each pair of bars, the leftmost bar shows results using 25 μL and the rightmost bar shows results using 50 μL.
[00036] Figure 18 shows the effect of a soil bath with F727 WCB on the infection caused by lettuce downy mildew. UTC: untreated control lettuce plants infected with approximately 5x104 downy mildew spores; bath with F727: lettuce plants that underwent a soil bath with F727 WCB one hour before inoculation with approximately 5x104 of spores of downy mildew. The severity of the disease was measured as the percentage of leaf / cotyledon coverage with diseased tissue.
[00037] Figure 19 shows ESIMS / MS results for compound A.
[00038] Figure 20 shows a schematic diagram of the structure of Compound A. DETAILED DESCRIPTION
[00039] Although the compositions and methods disclosed here are susceptible to various modifications and alternative forms, examples of modalities will be described here in detail. It should be understood, however, that there is no intention to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives that fit within the spirit and scope of the invention that are defined by the attached claims.
[00040] When a range of values is provided, it is understood that each intermediate value, up to a tenth part of the lower limit unit, unless the context clearly determines the contrary, between the upper limit and the lower limit of such range and any other cited or intermediate value in such cited range, is included in it. Smaller ranges are also included. The upper and lower limits of these minor ranges are also included here, subject to any limits specifically excluded in the above range.
[00041] Unless otherwise stated, all technical and scientific terms used here have the same meaning as is commonly understood by a common expert in the art to which this invention belongs. While any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
[00042] It has to be mentioned that as used here and in the appended claims, the forms in the singular "um", "uma", "o" and "a" include references in the plural unless the context clearly determines otherwise .
[00043] As defined here, "derived from" means directly isolated or obtained from a particular source or alternatively that has identification characteristics of a substance or an organism isolated or obtained from a particular source. In the event that the "source" is an organism, "derived from" means that it can be isolated or obtained from the organism itself or the medium used to cultivate or grow said organism.
[00044] As defined here, the terms "culture in total medium" and "medium with whole cells" refer to a liquid culture that contains both the cells and the medium. If bacteria are grown on a plate, cells can be collected in water or other liquid, to provide a culture in total medium.
[00045] The term "supernatant" refers to the remaining liquid when cells that are grown in medium or collected in another liquid from an agar plate are removed by centrifugation, filtration, sedimentation or other means well known in the art.
[00046] As defined here, "filtrate" refers to the liquid from a culture in total medium that has been passed through a membrane.
[00047] As defined here, "extract" refers to the liquid substance removed from cells by a solvent (water, detergent, buffer) and separated from the cells by centrifugation, filtration or another method.
[00048] As defined herein, "metabolite" refers to a compound, a substance or a by-product of a fermentation of a microorganism or supernatant, filtrate or extract obtained from a microorganism that has pesticidal activity and particularly, bactericidal or fungicide. As defined herein, an "isolated compound" is essentially free of other compounds or substances, for example, at least approximately 20% pure, preferably at least approximately 40% pure, more preferably approximately 60% pure, even more preferably approximately 80% pure , more preferably approximately 90% pure and even more preferably approximately 95% pure, as determined by analytical methods, including, but not limited to chromatographic and electrophoretic methods. The terms "metabolite" and "compound" can be used interchangeably.
[00049] A "carrier" as defined here is an inert organic or inorganic material, with which the active ingredient is mixed or formulated to facilitate its application to a plant or other object that will be treated or to facilitate its storage, transport and / or manipulation.
[00050] The term "modular" as defined here is used to mean changing the amount of pest infestation or the spread rate of pest infestation.
[00051] The term "pest infestation", as defined here, is the presence of a pest in an amount that causes a harmful effect including a disease or infection in a host population or the emergence of an unwanted weed in a system of growth.
[00052] A "pesticide" as defined here, is a substance derived from a biological product or a chemical substance, which increases mortality or inhibits the growth rate of plant pests and includes, but is not limited to, nematocides, insecticides, fungicides for plants, bactericides for plants and antiviral agents for plants. Identification and Characterization of Bacillus sp. F727
[00053] Bacillus sp. has been identified as a new strain of Bacillus using a polyphasic approach that combines the determination of 16S rRNA sequence, fatty acid analysis, protein analysis by MALDI-TOF and characterization using various biochemical assays. See Examples 1-4, below.
[00054] The metabolites produced by fermentation of Bacillus sp. F727 were isolated and characterized. See Examples 5, 25 and 26 below. Certain of these metabolites demonstrated activity against fungal and bacterial pathogens both in vitro and in vivo. See Examples 6-17, 20, 22, 23 and 27-29 below. The effects that promote the growth of plants, starting from Bacillus sp. F727 and its metabolites have also been observed in a number of plants. See Examples 18 and 19 and 21, 24 below.
[00055] Thus Bacillus sp. F727 and / or its metabolites, can be used as natural products for the control of fungal and bacterial diseases in agriculture; and to promote plant growth. Production Methods
[00056] As previously mentioned, compounds or metabolites can be obtained, are obtainable or can be derived from an organism that has one or more identification characteristics of a F727 strain of Bacillus. The methods include cultivating these organisms and obtaining the compounds and / or compositions of the present invention by isolating these compounds from the culture of these organisms.
[00057] In particular, organisms are grown in nutrient media using methods known in the art. Organisms can be cultivated by shaking flask, small-scale or large-scale fermentation (including, but not limited to continuous, batch, fed batch or solid fermentation) in laboratory or industrial fermenters carried out in a suitable medium. and under conditions that allow cell growth. Cultivation can take place in a medium with suitable nutrients that includes sources of carbon and nitrogen and inorganic salts, using procedures known in the art. Suitable media are available from commercial sources or can be prepared according to published compositions.
[00058] After cultivation, a supernatant, a filtrate and / or an extract of or derived from said strain of Bacillus (for example, Bacillus sp. F727) can be used in the formulation of a pesticidal composition.
[00059] Alternatively, after cultivation, compounds and / or metabolites can be extracted, enriched and / or purified from the culture medium.
[00060] The extract can be fractionated by chromatography. Chromatographic fractions can be analyzed for toxic activity against, for example, fungi (for example, Botrytis, Sclerotinia, Rhizoctonia & Bipolaris) using methods known in the art. The fractionation can be repeated one or more times using the same or different chromatographic methods.
[00061] In one embodiment, a composition produced by the F727 strain comprises one or more compounds that (i) have pesticidal activity; (ii) have molecular weights between 1020-1120 which are determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); (iii) have retention times in High Pressure Liquid Chromatography (HPLC) between 6-15 minutes on a C-18 HPLC reverse phase column using a water gradient solvent system: acetonitrile (CH3CN) (0-20 min; 90 - 0% aqueous CH3CN, 2024 min; 100% CH3CN, 24-27 min; 0-90% aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at a flow rate of 0.5 mL / min and UV detection at 210 nm; and (iv) can optionally be obtained from a species of Bacillus. Compounds in one embodiment are peptides.
[00062] In a specific embodiment, the compound "A" (i) can be obtained starting from a species of Bacillus; (ii) it is toxic to a pest; (iii) it has a molecular weight of approximately 1020-1060 and more particularly, 1044 which is determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); (iv) has 1H NMR values of δ 7.15, 6.72, 4.81,4.70, 4.65, 4.40, 4.35, 4.25, 4.15, 3.85, 3.65, 3.50, 3.22, 2.85, 2.80, 2.65, 2.45, 2.35, 2.30, 2.20, 1.95, 1.55, 1, 31, 1.20, 0.85; and (v) has a retention time in High Pressure Liquid Chromatography (HPLC) of approximately 6-12 minutes, more specifically approximately 8 minutes and even more specifically approximately 8.31 min on a C-18 column in reverse HPLC phase (Phenomenex, Luna 5μ C18 (2) 100 A, 100 x 4.60 mm) using a water: acetonitrile (CH3CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH3CN, 20- 24 min; 100% CH3CN, 24-27 min; 0-90% aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at a flow rate of 0.5 mL / min and UV detection at 210 nm. In addition, Compound "A" reveals signals for 47 carbons, 72 hydrogens, 12 nitrogens and 15 oxygenates that are determined by 1H NMR, 13C NMR & MS analyzes. The 1H NMR spectrum exhibits characteristics of a typical peptide. Detailed analysis of Compound "A" by 1H NMR, 13C NMR, MS / MS and amino acid analyzes revealed the presence of glutamine (1 unit), proline (1 unit), serine (1 unit), tyrosine (1 unit) and asparagine (3 units).
[00063] In another particular modality, a compound produced by the F727 strain is a "B" compound that (i) has pesticidal activity; (ii) it has a molecular weight of approximately 1030-1080 and more particularly, 1058 which is determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); and (iii) it has a retention time in High Pressure Liquid Chromatography (HPLC) of approximately 6-14 minutes, more specifically approximately 8 minutes and even more specifically approximately 8.67 min in an HPLC C-18 reverse phase column using a water gradient solvent system: acetonitrile (CH3CN) (0-20 min; 90 - 0% aqueous CH3CN, 20-24 min; 100% CH3CN, 24-27 min; 0-90% aqueous CH3CN , 27-30 min; 90% aqueous CH3CN) at a flow rate of 0.5 mL / min and UV detection at 210 nm. Data from the 1H and 13C NMR spectra, along with MS data, reveal signals for 48 carbons, 74 hydrogens, 12 nitrogens and 15 oxygen. The 1H NMR spectrum exhibits characteristics of a typical peptide. Detailed analysis of Compound "B" by 1H NMR, 13C NMR, MS / MS and amino acid analyzes revealed the presence of glutamine (1 unit), proline (1 unit), serine (1 unit), tyrosine (1 unit) and asparagine (3 units).
[00064] In yet another preferred embodiment, a compound produced by the F727 strain is a "C" compound that (i) has pesticidal activity; (ii) it has a molecular weight of approximately 10501120 and more particularly, 1072 which is determined by Liquid Chromatography / Mass Spectroscopy (LC / MS); and (iii) it has a retention time in High Pressure Liquid Chromatography (HPLC) of approximately 6-14 minutes, more specifically approximately 9 minutes and even more specifically approximately 9.19 min on a reverse phase HPLC C-18 column using a water gradient solvent system: acetonitrile (CH3CN) (0-20 min; 90 - 0% aqueous CH3CN, 20-24 min; 100% CH3CN, 24-27 min; 0-90% aqueous CH3CN , 27-30 min; 90% aqueous CH3CN) at a flow rate of 0.5 mL / min and UV detection at 210 nm. The data from the 1H and 13C NMR spectra, along with the MS data, reveal signals for 49 carbons, 76 hydrogens, 12 nitrogens and 15 oxygen. The 1H NMR spectrum exhibits characteristics of a typical peptide. Detailed analysis of Compound "C" by 1H NMR, 13C NMR, MS / MS and amino acid analyzes revealed the presence of glutamine (1 unit), proline (1 unit), serine (1 unit), tyrosine (1 unit) and asparagine (3 units). Compositions
[00065] The compositions may comprise whole media cultures, whole cell media, liquid cultures or suspensions of or derived from a Bacillus strain, specifically a Bacillus strain that has at least one of the identification characteristics of the F727 isolate from Bacillus sp ., as well as supernatants, filtrates or extracts obtained from said Bacillus sp. The compositions can also comprise one or more metabolites or isolated compounds derived from the F727 isolate from Bacillus sp., Which in particular have bactericidal, fungicidal activity and / or which promotes plant growth.
[00066] The compositions presented above can be formulated in any way.
[00067] Examples of formulations include, but are not limited to, concentrates that can be emulsified (EC), powders that can be moistened (WP), soluble liquids (SL), aerosols, ultra low volume concentrated solutions (ULV), soluble powders (SP), microencapsulated, granules dispersed in water, flow agents (FL), microemulsions (ME), nanoemulsions (NE) etc. In any formulation described here, the percentage of the active ingredient is within a range of 0.01% to 99.99%.
[00068] The compositions can be in the form of a liquid, a gel or a solid. A solid composition can be prepared by suspending a solid carrier in a solution of active ingredient (s) and drying the suspension under mild conditions, such as evaporation at room temperature or vacuum evaporation at 65 ° C or below .
[00069] A composition can comprise gel-encapsulated active ingredient (s). Such gel-encapsulated materials can be prepared by mixing a gel-forming agent (for example, gelatin, cellulose or lignin) with a culture or cell suspension of the F727 strain of Bacillus sp. live or inactivated or with a cell-free filtrate or a cell fraction from a culture or a suspension of the F727 strain of Bacillus sp. or with a culture, cell or cell fraction spray-dried or frozen by the F727 strain of Bacillus sp .; or with a solution of pesticidal compounds used in the method of the invention; and inducing gel formation of the agent.
[00070] The composition may additionally comprise a surfactant that will be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients and improvement of fluidity or rust inhibition. In a particular embodiment, the surfactant is a non-phytotoxic non-ionic surfactant that preferably belongs to EPA List 4B. In another particular modality, the non-ionic surfactant is polyoxyethylene monolaurate (20). The concentration of surfactant (s) can vary between 0.1-35% of the total formulation, a preferred range is 5-25%. The choice of dispersing and emulsifying agents, such as non-ionic, anionic, amphoteric and cationic dispersing agents and emulsifiers and the amount employed, are determined by the nature of the composition and the ability of the agent to facilitate the dispersion of the compositions.
[00071] The compositions presented above can be combined with another agent, microorganism and / or pesticide (for example, nematocide, bactericide, fungicide, acaricide, insecticide). Microorganisms include, but are not limited to Bacillus sp. (for example, Bacillus firmus, Bacillus thuringiensis, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis), Paecilomyces sp. (P. lilacinus), Pasteuria sp. (P. penetrans), Chromobacterium sp., Pseudomonas sp., Brevabacillus sp., Lecanicillium sp., Ampelomyces sp., Pseudozyma sp., Streptomyces sp (S. bikiniensis, S. costaricanus, S. avermitilis), Burkholderia sp., Trichoderma sp., Gliocladium sp., Avermectin, Myrothecium sp., Paecilomyces sp. , Hirsutella rhossiliensis, Pochonia chlamydosporia, Pleurotus ostreatus, Omphalotus olearius, Lampteromyces japonicas, Brevudimonas sp., Muscodor sp.
[00072] The agent may be a natural oil or oily product that has nematocidal, fungicidal, bactericidal and / or insecticidal activity (eg paraffinic oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil , citrus oil, rosemary oil, pyrethrum, citrus oil (including, but not limited to lime, orange and lemon oils); rosemary oil, English pepper, bergamot, common eucalyptus, chamomile, citronella, common jasmine , common juniper, common lavender, common myrrh, mint, freesia, gray santolina, herbaceous hyssop, holy basil, incense tree, jasmine, lavender, marigold, mint, peppermint, marigold, green mint, tree ylang-ylang, saponins).
[00073] In addition, the pesticide may be an isolated site antifungal agent that may include, but is not limited to benzimidazole, a demethylation inhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholine, hydroxypyrimidine , anilinopyrimidine, phosphorothiolate, external quinone inhibitor, quinoline, dicarboximide, carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzene; a demethylation inhibitor selected from the group consisting of imidazole, piperazine, pyrimidine and triazole (for example, bitertanol, myclobutanil, penconazole, propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole, tibaconazole, tb) of external quinone (for example, strobilurin). Strobilurin may include, but is not limited to, azoxystrobin, kresoxim-methoxy or trifloxystrobin. In yet another preferred embodiment, the antifungal agent is a quinone, for example, quinoxyphen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether). The antifungal agent can also be derived from a Reynoutria extract.
[00074] The fungicide can also be a multi-site non-inorganic chemical fungicide selected from the group consisting of chloronitrile, quinoxaline, sulfamide, phosphonate, phosphite, dithiocarbamate, chloralkyls, phenylpyridin-amine and cyanoacetamide oxime.
[00075] As previously mentioned, the composition can also comprise a nematocide. The nematocide can include, but is not limited to, chemical agents such as organophosphates, carbamates and fumigation agents and microbial products such as avermectin, Myrothecium sp. Biome (Bacillus firmus), Pasteuria spp., Paecilomyces and organic products such as saponins and vegetable oils.
[00076] The compositions can be applied using methods known in the art.
[00077] Specifically, these compositions are applied to and around plants or parts of plants. Plants are to be understood as meaning in the present context all plants and plant populations such as desired and unwanted wild plants or cultivation plants (including naturally occurring cultivation plants). Cultivation plants can be plants that can be obtained through conventional plant breeding and optimization methods or through biotechnological and genetic engineering methods or through combinations of these methods, including transgenic plants and plant cultivars that can be protected or not protected by the rights of plant growers. Plant parts should be understood to mean all plant parts and organs above and below ground, such as bud, leaf, flower and root, examples that can be mentioned being leaves, needles, peduncles, stems, flowers, fruiting bodies, fruits, seeds, roots, tubers and rhizomes. Plant parts also include harvested material and material for vegetative and productive propagation, for example, seedlings, tubers, rhizomes, branches and seeds.
[00078] The treatment of plants and parts of plants with the compositions presented above can be carried out directly or allowing the compositions to act on the neighborhood, habitat or storage space of a plant, for example, by immersion, spraying, evaporation, nebulization, dispersion, surface painting or injection.
[00079] The compositions disclosed here can also be applied to the soil using methods known in the art. These include, but are not limited to (a) drip irrigation or "chemigation"; (b) incorporation into the soil; (c) soaking the soil; (d) seed treatment and waterlogging; and (e) immersion of the bare root. Seed Treatments
[00080] Seed treatments include applying a composition that is disclosed here, optionally in combination with other bioactive, antagonistic or symbiotic agents on the surface of a seed prior to sowing. Toxins, proteins and / or pesticidal compounds disclosed here can be applied to seeds in the form of dry powders, powders in suspension or sprayed on the seed before planting.
[00081] The compositions disclosed here can be formulated for seed treatments in any of the following ways: dry powder, powder that can be suspended in water, liquid solution, concentrate or emulsion that can be drained, emulsion, microcapsules, gel or granules that can be dispersed in water.
[00082] In the case of a dry powder, the active ingredient is formulated similarly to a powder that can be moistened, but with the addition of an adhesive agent, such as mineral oil, instead of a wetting agent. For example, one kg of purified talcum powder (sterilized for 12 h), 15 g of calcium carbonate and 10 g of carboxymethyl cellulose are mixed under aseptic conditions following the method described by Nandakumar et al. (2001). The active ingredient (s) is / are mixed in a ratio of 1: 2.5 (suspension to dry mix) and the product is dried in the shade to reduce the moisture content for 20-35%.
[00083] In embodiments in which the compositions disclosed herein are applied to a seed, a composition may be applied in the form of one or more coatings prior to planting the seed using one or more seed coating agents including, but not limited to, ethylene glycol, polyethylene glycol, chitosan, carboxymethyl chitosan, peat slime, resins and waxes. The compositions can also be applied to seeds in combination with, for example, chemical fungicides or bactericides with a single-site, multi-site or unknown mode of action using methods known in the art.
[00084] In additional modalities, the disclosed compositions can be applied to the seeds through imbibition of the seeds or in the form of a powdered inoculum.
[00085] The seeds can be conventional seeds or they can be a genetically modified seed such as Liberty Link seeds (Bayer CropScience), Roundup Ready (Monsanto) or another herbicide resistant seed and / or seeds engineered to be resistant to insects or seeds that are "pyrimaded" with herbicide and insect resistance genes. Promotion of Plant Growth
[00086] Plant-bacteria interactions in the rhizosphere are important determinants of soil fertility and plant health. Free-living bacteria that are beneficial for plant growth are known as plant growth-promoting rhizobacteria (PGPR). Plant growth promoters generally work in one of three ways: by synthesizing plant growth regulators, by facilitating the uptake of nutrients from the soil and / or by preventing plant disease. Therefore, the effects of PGPRs can be both direct and indirect. The promotion of indirect plant growth may involve an antagonistic effect against phytopathogenic agents. This can be achieved, for example, through the production of siderophores, the synthesis of antibiotics and the production of HCN and / or enzymes that degrade the cell wall. The effects of promoting direct plant growth are achieved through the regulation of phytohormones (which help in the development of the plant and roots and in the protection against stress) and the solubilization of mineral phosphates and other nutrients.
[00087] The compositions disclosed here, in particular, the F727 isolate from Bacillus sp. and / or a supernatant, a filtrate, an extract, a compound, a metabolite or a cell fraction obtained from a culture of Bacillus sp. F727, can be used to modulate or more particularly to promote the growth of plants, for example, cultivation plants such as fruit (for example, strawberry), vegetable (for example, tomato, pumpkin, pepper, eggplant) plants , vegetables or grains (for example, soy, wheat, rice, corn), tree, flower, ornamental plants, thickets (for example, cotton, roses), peat (for example, annual ryegrass, Bermuda grass, buffalo grass, colonial grass, creeping grass, dicondra, hard fescue, Kentucky grass, Pennisetum clandestinum, perennial ryegrass, red fescue, Poa trivialis, Paspalum vaginatum, Saint Augustine grass, tall fescue, zoysia, etc.), bulb plants (for example, onion, garlic) or vine (for example, wine grape). The compositions can also be used to modulate the germination of seed (s) in plant (s).
[00088] The compositions described herein or the formulated product, can be used alone or in combination with one or more other components that are described below, such as growth promoting agents and / or antiphytopathogenic agents in a tank mixture or in a program (sequential application called rotation) with a predetermined order and application interval during the growing season. When used in combination with the products mentioned above, in a concentration lower than that recommended on the product label, the combined effectiveness of the two or more products (of which one is the said composition disclosed here) is, in certain modalities, greater than the sum of each effect of the individual component. Consequently, the effect is intensified by synergy between these two (or more) products and the risk for the development of pesticide resistance among pathogenic plant strains is reduced.
[00089] The composition can be applied by immersing the root in the transplant, specifically by treating a fruit or vegetable with the composition by immersing the roots of the fruit or vegetable in a suspension of said composition (approximately 0.25 to approximately 1.5% and more particularly approximately 0.5% to approximately 1.0% by volume) before transplanting the fruit or vegetable into the soil.
[00090] Alternatively, the composition can be applied by drip or another irrigation system. Specifically, the composition can be injected into a drip irrigation system. In a particular embodiment, the composition is applied in a concentration of 1x108 colony-forming units (CFU) / mL in a volume of approximately 11 to approximately 4 quarts per acre.
[00091] In yet another modality, the composition can be added in the form of an application to the plow. Specifically, the composition can be added in the form of a spray into the plow at planting using calibrated nozzles to provide a final output of 2-6 gallons / acre. The nozzles are placed in the plow opener over the planter so that the application of pesticide and the drop of seed into the plow are simultaneous.
[00092] Mixtures of the disclosed compositions with, for example, a solid or liquid adjuvant are prepared in a known manner. For example, mixtures can be prepared by mixing homogeneously and / or by grinding the active ingredients with diluents such as solvents, solid carriers and, where appropriate, surfactant compounds. The compositions can also contain additional ingredients such as stabilizers, viscosity regulators, binding agents, adjuvants as well as fertilizers or other active ingredients for the purpose of obtaining special effects. Combinations with Plant Growth Promoting Agents
[00093] The compositions disclosed here can also be used in combination with other growth-promoting agents such as synthetic or organic fertilizers (for example, di-ammonium phosphate, in granular or liquid form), composting, seaweed extracts, hormones plant growth agents such as IAA (indole acetic acid) used in a treatment with rooting hormone for transplants alone or in combination with plant growth regulators such as IBA (indole butyric acid) and NAA (naphthalene acetic acid) and microorganisms that promote growth, such as, for example, PPFM (pink pigmented optional methylotrophs), Bacillus spp., Pseudomonads, Rhizobia and Trichoderma. Anti-Phytopathogenic Agents
[00094] The compositions disclosed here can also be used in combination with other anti-phytopathogenic agents, such as plant extracts, biopesticides, inorganic crop protection agents (such as copper), surfactants (such as ramnolipids; Gandhi and others , 2007) or natural oils such as paraffinic oil and tea tree oil that have chemical pesticidal or fungicidal or bactericidal properties with a single-site, multi-site or unknown mode of action. As defined here, an "anti-phytopathogenic agent" is an agent that modulates the growth of a plant pathogen, particularly a pathogen that causes soil-borne disease on a plant or alternatively prevents infection of a plant by a pathogen of plants. A plant pathogen includes, but is not limited to, a fungus, bacteria, actinomycete or virus.
[00095] As mentioned earlier, the anti-phytopathogenic agent may be a single-site antifungal agent that may include, but is not limited to benzimidazole, a demethylation inhibitor (DMI) (eg, imidazole, piperazine, pyrimidine, triazole) , morpholine, hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, external quinone inhibitor, quinoline, dicarboximide, carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, benzylamine, xylamine, acoxyamine, polyoxin, acylamine, xylamine, acylamine. In a more particular embodiment, the antifungal agent is a demethylation inhibitor selected from the group consisting of imidazole, piperazine, pyrimidine and triazole (e.g., bitertanol, myclobutanil, penconazole, propiconazole, triadimefon, bromuconazole, cyproconazole, hexiconazole, hexiconazole, hexiconazole, hexiconazole, hexiconazole, hexiconazole, hexiconazole, hexiconazole, hexiconazole, hexiconazole, hexonazole, hexonazole, hexonazole, hexonazole. , tebuconazole, tetraconazole). In a more particular embodiment, the antifungal agent is miclobutanil. In yet another preferred embodiment, the antifungal agent is an external quinone inhibitor (for example, strobilurin). Strobilurin may include, but is not limited to, azoxystrobin, kresoxim-methyl or trifloxystrobin. In yet another preferred embodiment, the antifungal agent is a quinone, for example, quinoxyphen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether).
[00096] Still in an additional modality, the fungicide is a non-inorganic chemical fungicide of several sites selected from the group consisting of chloronitrile, quinoxaline, sulfamide, phosphonate, phosphite, dithiocarbamate, chloralkyl, phenylpyridine-amine and cyanoacetamide oxime.
[00097] Still in an additional modality, the anti-phytopathogenic agent can be streptomycin, tetracycline, oxytetracycline, copper or kasugamycin. EXAMPLES Example 1: Isolation and characterization of isolate F727 from Bacillus sp. by 16S rRNA, recA and phoR sequences
[00098] The F727 strain of Bacillus sp. was isolated from a soil sample collected in Jonesville, CA, using traditional plate dilution methods. The isolate was identified as a Bacillus sp. through PCR amplification and sequencing of 16S rRNA, recA and phoR genes using universal bacterial primers. Cerritos et al. (2008) Int. J. Sys. Evol. Microbiol. 58: 919923; Guo et al. (2012) Can. J. Microbiol. 58: 1295-1305.
[00099] Growth from a 24-hour potato dextrose plate was scraped with a sterile loop and resuspended in DNA extraction buffer. The DNA was extracted using the MoBio Ultra Clean Microbial DNA extraction kit. The DNA extract was checked for quality / quantity through the electrophoresis of a 5 μL aliquot in a 1% agarose gel. RRNA sequences
[000100] The PCR reactions for the amplification of the 16S rRNA gene were assembled by combining 2 μL of the clean DNA extract with 25 μL of GoTaq Green Mastermix, 1.5 μL of forward primer (FD1 primer, 5'- AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 4) and 1.5 μL of reverse primer (primer RD1, 5'- AAGGAGGTGATCCAGCC-3' (SEQ ID NO: 5)). The reaction volume was adjusted to 50 μL with water free of sterilized nucleasse The amplification reaction was conducted using a thermocycler under the following conditions: 10 minutes at 95 ° C (initial denaturation), 30 cycles of 45 seconds at 94 ° C, 45 seconds at 55 ° C and 2 minutes at 72 ° C, followed by 5 minutes at 72 ° C (final extension) and a final hold temperature of 10 ° C.
[000101] The size, quality and quantity of the amplification product were evaluated by electrophoresis of a 5 μL aliquot on a 1% agarose gel and comparing the product band with a mass scale.
[000102] The primers, nucleotides, enzyme and excess template were removed from the PCR product using the MoBio PCR clean up Kit. The cleaned PCR product was subjected to direct sequencing using the primers described above.
[000103] The forward and reverse sequences were aligned using the BioEdit software and a consensus sequence of 1459 bp was created. Following 16S FD1 of F727: TATACATGCAAGTCGAGCGGACAGATGGGAGCTTGTCCCTGATGT TAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAG ACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTT TGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTT ACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC TCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGC CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGC AGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACG CCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTA GGGAAGAACAAGTGCCGTTCGAATAGGGCGGCACCTTGACGGTA CCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGT AATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGG GCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCA ACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGA GGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGT GGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGA CGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCC TGGTAGTCCACGCCGTAACGATGAGTGCTAAGTGTTAGGGGGTTT CCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGG GGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGC CCGCACAAGCGGTGGAGCATGTGGTTT AATTCGAAGCAACGCNAGAACCTTACCANGTCTTG ACATCCTCTG ACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACNN NGGNGCATGGNNGTCGTCAGCTCGTGTCGTGAGATGTTGGGTAA GTCCCGCACNAGCGCAACCCNTTGATCTTANTTGCCAGCATTCAN TTGGNNNNNNNNNNNNNACTGCCNNNACNANCCGNNNAAGGNNN GGGNATNACGTNNANNNATNCNNGCCCNNNNTGACNNNNNNCAC NCCNNNNNNNNNNANNGNNNNNNAANNANNGGGNCNNNNNGNN NNNNAAANNNCNNNCNCNNNN GNGNN (SEQ ID NO: 1) of the 16S sequence RD1 F727: TCATCTGTCCCACCTTCGGCGGCTGGCTCCATAAAGGTTACCTCA CCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGT GTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCG ATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGA TCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTT TCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGG TCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGG TTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCA ACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATC TCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGC CCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAA GACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCT CCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCT TGCGACCGTACTCCCCAGGCGGAGTGCTTTAATGCGTTAGCTGCA GCAC TAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTT ACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCCACG CTTTCGCTCCCTCAGCGTCAGTTACAGACC CAGAGAGTCGCCTTCGCCCCACTGGTGTTCCTCCACATCCTCTAC GCATTTCACCCGGCTACAACGTGGAATTCCACTCTCCTCTTCTGC ACTCAAGTTTCCCCAGTTTCCAATGACCCCTCCCCGGTTGAGCCC GGGGGCTTTCACATCAGACTTAAAGAAACCCGCCTGCGAGCCCTT TACGCCCAATAATTCCGGACACGCTTGGCCACCTACGTATTACCG CGCTTGCTTGGCACGTTAGTAGCCGTGGCTTTTCTGGTTAGTTAA CCGTCAGTGCCGCCTATTCGGAACGGTACTTGTTCTTCCCTACAC AGAGCTTTACGATCGAAACTCATCACCTCCACGCGCGTGCTCGTC AGAACTTTCGTCATGCGAAGATCCTACTGCTGCCTCCGTAGGGTT GGCGTTTCTCTCAGTCCAGTGGCCATACGTCAGTAGCTACCCATC GTGCCTAGTGAGCGTTACCTCACCCACCTAGGC (SEQ ID NO: 2) consensus sequence of 16S F727: TATACATGCAAGTCGAGCGGACAGATGGGAGCTTGTCCCTGATGT TAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAG ACTGGGATAACTCCGGGAAACCGG GGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAAACATAAAA GGTGGCTTCGG CTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAG GTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGG TGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGG GAGGCAGCAGTAGGGAATCTTCCGCAA TGGACGAAAGTCTGACG GAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTC TGTTGTTAGGGAAGAACAAGTGCCGTTCGAATAGGGCGGCACCTT GACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAG CCGCGGTAATACGTAGGTGGCCAAGCGTTGTCCGGAATTATTGG GCGTAAAGGGCTCGCAGGCGGGTTTCTTTAAGTCTGATGTGAAAG CCCCCGGGCTCAACCGGGGAGGGGTCATTGGAAACTGGGGAAAC TTGAGTGCAGAAGA GGAGAGTGGAATTCCACGTTGTAGCCGGGTGAAATGCGTAGAGG ATGTGGAGGAACACCAGTGGGGCGAAGGCGACTCTCTGGGTCTG TAACTGACGCTGAGGGAGCGAAAGCGTGGGGGAGCGAACAGGAT TAGATACCCTGGTAGTCCACGCCGTAAAACGATGAGTGC TAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCAT TAAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAA AGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTA ATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACA TCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGA GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGT TGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCA GCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACA AACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTAT GACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAG CGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTC GGATCGCAGTCTGCAACTCGACTGCGTGAA GCTGGAA TCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCG GGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACC CGAAGTCGGTGAGGTAACCTTTATGGAGCCT
[000104] The consensus sequence of the 16S rRNA gene of the F727 strain was compared with those available sequences representative of the bacterial domain using BLAST. The combination with the closest species was with Bacillus sp. (accession number GU250449.1), with 99% similarity. No single 16S sequence in publicly available databases exhibited 100% similarity to the F727 strain.
[000105] Additionally, the consensus sequence was analyzed using the EzTaxon-e server (eztaxon-e.ezbiocloud.net/; Kim et al., 2012) based on the 16S rRNA sequence data. The closest combinations (shown in Table 1) included strains of types for several species of the genus Bacillus that cannot be differentiated based solely on the 16S rRNA sequences. Table 1

RecA strings
[000106] The PCR reactions for the amplification of the recA gene were assembled by combining 2 μL of the clean DNA extract with 25 μL of GoTaq Green Mastermix, 1.5 μL of forward primer (recAf, 5'-GATCGTCARGCAGSCYTWGAT - 3 ', SEQ ID NO: 6) and 1.5 μL of reverse primer (recAr, 5'-TTWCCRACCATAACSCCRAC-3', SEQ ID NO: 7). The reaction volume was adjusted to 50 μL using sterile nuclease-free water. The amplification reaction was conducted in a thermocycler under the following conditions: 5 minutes at 95 ° C (initial denaturation), 30 cycles of 30 seconds at 95 ° C, 30 seconds at 45 ° C and 1 minute at 72 ° C , followed by 5 minutes at 72 ° C (final extension) and a final hold temperature of 4 ° C.
[000107] The size, quality and quantity of the PCR product were evaluated by electrophoresis of a 5 μL aliquot on a 1% agarose gel and comparing the product band with a mass scale.
[000108] The primers, nucleotides, enzyme and excess template were removed from the PCR product using the MoBio PCR clean up Kit. The cleaned PCR product was subjected to direct sequencing using the primers described above.
[000109] The forward and reverse sequences were aligned using the BioEdit software and a 505 bp consensus sequence was created. Sequence of recA Forward F727: AACATTCGGCAAGGTTCCATCATGAAACTCGGGGAAAAGACGGAT ACAAGAATTTCAACAGTTCCGAGCGGTTCCCTTGCACTTGATACC GCTCTCGGAATAGGCGGATACCCGCGCGGACGGATTATTGAAGT ATACGGACCTGAAAGCTCAGGTAAAACGACTGTAGCGCTTCATGC GATTGCTGAAGTTCAGGAGAAAGGCGGACAAGCCGCATTTATTGA TGCTGAGCATGCCCTTGACCCTGTTTACGCGCAAAAGCTCGGTGT AAATATTGAGGAGCTGCTGCTTTCTCAGCCTGATACGGGAGAGCA GGCGCTTGAGATTGCCGAAGCGCTGGTACGAAGCGGAGCCGTCG ATATCGTAGTTGTCGACTCTGTTGCGGCGCTTGTCCCGAAAGCTG AAATCGAAGGAGACATGGGGGATTCCCACGTCGGTTTGCAGGCC CGTTTGATGTCTCAAGCGCTCCGTAAGCTTTCCGGTGCCATCAAT AAATCTAAAACAATCGCAATCTTTATTAACCAAATTCGTGAAAAAGT CGGCGTTAGGGTCGGAAAAAA (SEQ ID NO: 8) Reverse Sequence of recA F727: GTATAAGATTGCGATTGTTTTAGATTTATTGATGGCACCGGAAAGC TTACGGAGCGCTTGAGACATCAAACGGGCCTGCAAACCGACGTG GGAATCCCCCATGTCTCCTTCGATTTCAGCTTTCGGGACAAGCGC CGCAACAGAGTCGACAACTACGATATCGACGGCTCCGCTTCGTAC CAGCGCTTCGGCAATCTCAAGCGCCTGCTCTCCCGTATCAGGCTG AGAAAGCAGCAGCTCCTCAATATTTACACCGAGCTTTTGCGCGTA AACAGGGTCAAGGGCATGCTCAGCATCAATAA ATGCGGCTTGTCCGCCTTTCTCCTGAACT TCAGCAATCGCATGAA GCGCTACAGTCGTTTTACCTGAGCTTTCAGGTCCGTATACTTCAAT AATCCGTCCGCGCGGGTATCCGCCTATTCCGAGAGCGGTATCAA GTGCAAGGGAACCGCTCGGAACTGTTGAAATTCTTGTATCCGTCT TTTCCCCGAGTTTCATGATGGAACCTTTGCCGAATTGTTTTTCTATT TGCTTAAGAGCCATATCWAAGRCTGWA WTRAMRATCAA (SEQ ID NO: 9) Consensus Sequence of recA F727: AAGGTTCCATCATGAAACTCGGGGAAAAGACGGATACAAGAATTT CAACAGTTCCGAGCGGTTCCCTTGCACTTGATACCGCTCTCGGAA TAGGCGGATACCCGCGCGGACGGATTATTGAAGTATACGGACCT GAAAGCTCAGGTAAAACGACTGTAGCGCTTCATGCGATTGCTGAA GTTCAGGAGAAAGGCGGACAAGCCGCATTTATTGATGCTGAGCAT GCCCTTGACCCTGTTTACGCGCAAAAGCTCGGTGTAAATATTGAG GAGCTGCTGCTTTCTCAGCCTGATACGGGAGAGCAGGCGCTTGA GATTGCCGAAGCGCTGGTACGAAGCGGAGCCGTCGATATCGTAG TTGTCGACTCTGTTGCGGCGCTTGTCCCGAAAGCTGAAATCGAAG GAGACATGGGGGATTCCCACGTCGGTTTGCAGGCCCGTTTGATG TCTCAAGCGCTCCGTAAGCTTTCCGGTGCCATCAATAAATCTAAAA CAATCGCAATCTT (SEQ ID NO: 10)
[000110] The consensus sequence of the recA gene of the F727 strain (SEQ ID NO: 10) was compared with representative bacterial sequences using BLAST. The closest species combination was with the complete genome of Bacillus amyloliquefaciens (accession number CP002927.1), with 92% similarity. PhoR strings
[000111] The PCR reactions for the amplification of the phoR gene were assembled by combining 2 μL of the clean DNA extract with 25 μL of GoTaq Green Mastermix, 1.5 μL of forward primer (phoR- f: 5'- TTYARYTCATGRGAVACATT-3 ', SEQ ID NO: 11) and 1.5 μL of reverse primer (phoR-r: 5'-GGNTAYAAANARGAGGAGCC-3', SEQ ID NO: 12). The reaction volume was adjusted to 50 μL using sterile nuclease-free water. The amplification reaction was conducted in a thermocycler under the following conditions: 5 minutes at 95 ° C (initial denaturation), 35 cycles of 45 seconds at 95 ° C, 45 seconds at 48 ° C and 1 minute at 72 ° C , followed by 10 minutes at 72 ° C (final extension) and a final hold temperature of 4 ° C.
[000112] The size, quality and quantity of the PCR product were evaluated by electrophoresis of a 5 μL aliquot on a 1% agarose gel and comparing the product band with a mass scale.
[000113] The primers, nucleotides, enzyme and excess template were removed from the PCR product using the MoBio PCR clean up Kit. The cleaned PCR product was subjected to direct sequencing using the primers described above.
[000114] A 998 nucleotide phoR sequence was obtained using the reverse primer described above. Following Phor Inverse F727: TCGTTGTCTGTATCATATTGGTTTTCAGTGTTCTCGGCCTTTTCTTG CAGCAGCTCATTTCTTCATCCGCCAAGGAAAGAACGGAGGGACAG CTTGAAAAGGAAGCCGCATACATAGCCGGACTCCTTGACGCCGG CCAAGTAAACAATAAAAGAAACGAAACGGTCATTAA AGATGCCAGCCGTACATTAGATATCGACGTGTCCGTATTAAATGAA AAAGGCCGCGGTTTATATCACTCAGGCAGACGCGCTGATGACTCG GCTATAAAGGAATTCGTCTCCCGTAATAAAAATGCGGCGGCGATT CAGAACGGAGAGAAAGTATGGCATGGAACGGCCCTTAAAAACGC CGCCGGCCAAACGGCGGGATATGTGCTCGTTTCCTCGCGGATCG ATAAAGGTTCGAATATAACAGGGGAAATGTGGGGCATGCTGGCTG CAAGCCTTTGTACTGCTTTTATTATTATCGTTTTCTTCTATACGAAT ATGACCTCCCGTTACAAAAGGTCAAT CGACTCCGCGACAAAAGTGGCCACTGAGCTGTCTAAGGGGAACT ATGACGCCCGCTCCTACGGCGGGTACGCAAGACGCTCAGACCGT CTCGGGCGCGCTATGAACAGCCTCGCTGTGGATTTGATGGAAATG ACGAGAACGCAGGATATGCAGCGCGACCGCCTGCTGACCGTCAT CGAAAATATCGGATCAGGTTTGATTTTAATAGACGGGAGAGGCTTT ATTAATCTCGTGAACAGGTCGTATACGAAGCAGTTCCATACAAATC CTGAACGTCTGCTTCGGCGTCTCTACCATGACGCATTTGAGCATG AGGAAATCATTCGGCTGGTCGAAGACATCT TTATGACAGAAACGAAGAAACGCCAGCTGCTCACGCTTCCCATCA AAATCGAACGGCGCTATTTTGAGGTTGACGGCGTCCCGATT ATGG GCCCTGACGATGAATGGAAAAGGCATTGTTCTCGTGTTTCATGAT ATGAC (SEQ ID NO: 13)
[000115] The phoR inverse sequence was compared to representative bacterial sequences using BLAST. The closest species combination was with the complete genome of several strains of Bacillus amyloliquefaciens with only 83% similarity. Example 2: Fatty Acid Composition of Isolate F727
[000116] A fatty acid profile of the F727 isolate was obtained from MIDI Labs, Inc (Newark, DE), according to commercial standards. The results are shown in Table 2. The comparison of its fatty acid profile with the RTSBA6 6.10 fatty acid database showed that the F727 isolate had a similarity index of 0.885 with Bacillus subtilis.Table 2
Example 3: Characterization of isolate F727 by the protein profile by MALDI-TOF
[000117] A protein fingerprint by MALDI-TOF mass spectroscopy of isolate F727 was performed at MIDI Labs, Inc. (Newark, DE). Isolate F727 exhibited a protein profile by MALDI-TOF different from that of any other microorganism present in the mass spectra database. Some similarities with protein profiles of Bacillus vallismortis, Bacillus mojaviensis and Bacillus subtilis were observed; however, none of the similarity scores were high enough to be indicative of even a generic combination. Example 4: Biochemical Characterization of Bacillus sp. Gram stain
[000118] Gram staining is a method of differentiating bacteria based on the physical properties of the cell wall, primarily of the peptidoglycan composition. Gram-positive bacterial isolates have a thick peptidoglycan layer resulting in a violet / blue color; while Gram-negative bacterial isolates have a thinner layer of peptidoglycan on the cell wall, resulting in a red / pink color. Microscopic inspection of the F727 isolate after staining with Gram revealed violet cells, indicating that the F727 isolate from Bacillus sp. it is a Gram-positive bacterium. Urease activity
[000119] The urease test is used to detect the activity of the enzyme urease, which catalyzes the conversion of urea to ammonia and bicarbonate. The urea medium contains urea and the phenol red pH indicator. The indicator turns yellow in an acidic environment and pink in an alkaline environment. If the enzymatic activity of urease is present, the urea in the medium is degraded to produce ammonia and the medium turns pink, indicating a positive test.
[000120] After inoculation with isolate F727, the urea medium changed its color from red to yellow, indicating a negative test for urease activity. Thus, isolate F727 from Bacillus sp. created an acidic environment, indicative of the absence of urease activity. Catalase activity
[000121] The catalase test is used to detect the activity of the enzyme, catalase.
[000122] Catalase decomposes hydrogen peroxide into oxygen and water. Organisms that have catalase activity produce gas bubbles when treated with hydrogen peroxide. The bubbles formed within seconds of applying the reagent to a culture of Bacillus sp. F727 isolate, indicating that this organism has catalase activity. Oxidase activity
[000123] The oxidase test is used to detect the presence of cytochrome c oxidase activity. Bacteria that contain cytochrome c as part of their respiratory chain are positive for the oxidase and make the reagent violet. Conversely, bacteria that are negative for oxidase do not oxidize the reagent, leaving it colorless. Isolate F727 from Bacillus sp. turned the reagent violet, demonstrating that it had oxidase activity. TSI Agar
[000124] Triple sugar and iron agar (TSI) is used to determine a microorganism's ability to ferment glucose, lactose and / or sucrose, as well as the ability of enteric bacteria to produce hydrogen sulfide. The medium contains the phenol red pH indicator as well as ferrous sulfate, which reacts with hydrogen sulfide to produce a black precipitate. When isolate F727 was tested, the tilted medium remained red while the top changed from red to yellow and no black color was observed. These results indicate that the isolate F727 does not produce hydrogen sulfide and ferment only glucose, not lactose or sucrose. Antibiotic susceptibility
[000125] The antibiotic susceptibility of Bacillus sp. was tested using antibiotic discs over Muller-Hinton medium. A handle filled with F727 was resuspended in 1 mL of sterile deionized water and 100 μL of this suspension was streaked onto a plate with Mueller-Hinton agar. After absorbing the striatum into the agar, the pre-loaded antibiotic discs were placed on the plate and the plate was incubated at 25 ° C for 48 hours. The results are shown in Table 3.

+++ indicates highly susceptible (without growth); ++ indicates moderately susceptible (reduced growth); - indicates no susceptibility ZYM API strip
[000126] The API ZYM strip (BioMerrieux) provides a method for testing various enzymatic activities of a microorganism. The test was performed according to the manufacturer's instructions and a summary of the data is shown in Table 4.
API 20 NE strip
[000127] The API ® 20 NE strip consists of 20 microtubes that contain dehydrated substrates. Conventional tests were inoculated with a suspension of Bacillus sp. that reconstitutes the medium. The metabolism produced changes in the color in the microtubes. The assimilation tests were inoculated with a minimal medium and the isolate F727 from Bacillus sp. grows if the bacteria is able to use the substrate.
[000128] The reactions were analyzed according to the manufacturer's reading table and the results are summarized in Table 5.


Example 5: Isolation and Characterization of compounds A, B & C Purification procedure
[000129] The following procedure (described in Figure 1) was used for the purification of compounds extracted from a cell culture of the F727 isolate from Bacillus sp ..
[000130] The culture medium from a 1 L fermentation of Bacillus sp. the growth medium was extracted with Amberlite XAD-7 resin (Asolkar et al., 2006) by stirring the cell suspension with the resin at 155 rpm for two hours at room temperature. The resin and cell mass were collected by filtration through cotton gauze and washed with deionized water to remove salts. The resin, cell mass and cotton gauze were then soaked for 2 hours in acetone, after which the acetone was filtered and vacuum dried using a rotary evaporator to provide a crude extract.
[000131] The crude extract was subjected to liquid chromatography with vacuum in C18 in reverse phase (VLC, H2O / CH3OH; gradient from 80:20 to 0: 100%) to provide 6 fractions. These fractions were concentrated to dryness using a rotary evaporator and the resulting dry residues were checked for biological activity using an agar disk assay. See Example 16 below. This assay identified VLC Fraction 3 with C-18 as having fungicidal activity.
[000132] Active fraction 3 was subjected to reverse phase HPLC (Spectra System P4000, Thermo Scientific) to provide pure compounds, which were then verified in the biological tests mentioned above to locate / identify the active compounds.
[000133] The active fraction 3 was further purified on an HPLC C-18 column (Fenomenex, Luna 10u C18 (2) 100 A, 250 x 30) using a water gradient solvent system: acetonitrile (containing 0.01 % TFA) (0-10 min; 70% aqueous CH3CN, 10-20 min; 70-45% aqueous CH3CN, 20-40 min; 45 - 30% aqueous CH3CN, 40-60 min; 30-0 % CH3CN, 60-65 min; 100% CH3CN, 65-70 min; 0 - 30% aqueous CH3CN) at a flow rate of 8 mL / min with UV detection at 210 nm. Three purified compounds were obtained: Compound A (F727F3H11), which has a retention time of 35.95 min, Compound B (F727F3H14), which has a retention time of 37.26 min and Compound C (F727F3H17), which has a retention time of 38.11 min. Mass spectroscopy
[000134] Mass spectroscopy analysis of compounds A, B and C was performed on a Thermo Finnigan LCQ Deca XP Plus electrospray (ESI) instrument using both positive and negative ionization modes in a full scan mode (m / z 1001500 Da) in an LCQ DECA XPplus Mass Spectrometer (Thermo Electron Corp., San Jose, CA). A Thermo high performance liquid chromatography (HPLC) instrument equipped with a Finnigan Surveyor PDA plus detector, self-sampling plus, MS pump and a 4.6 mm x 100 mm Luna C18 5 μm column (Phenomenex) was used. The solvent system consisted of water (solvent A) and acetonitrile (solvent B). The mobile phase starts at 10% solvent B and is linearly increased to 100% solvent B over 20 min and then maintained in 100% solvent B for 4 min and finally returned to 10% solvent B over 3 min and maintained at 10% B for 3 min. The flow rate was 0.5 ml / min. The injection volume was 10 μL and the samples were kept at room temperature in an autosampler.
[000135] The compounds were analyzed by LC-MS using LC and reverse phase chromatography. The mass spectroscopy analysis of the present compounds was carried out under the following conditions: the nitrogen gas flow was fixed at 30 and 15 arb for the coating and aux / sweep gas flow, respectively. Electrospray ionization was carried out with a spray voltage set at 5000 V and a capillary voltage set at 35.0 V. The capillary temperature was set at 400 ° C. The data were analyzed using the Xcalibur software.
[000136] The molecular weight of Compound A (F727F3H11) was determined to be 1044, based on a molecular ion peak at 1043.84 (M - H) in the negative ionization mode (Figure 2). This determination was supported by the ionization pattern in ESIMS in the positive mode, which exhibited a peak at 1045.48 (M + H) and a pseudomolecular ion peak at 1067.55 (M + Na) (Figure 3).
[000137] The molecular weight of Compound B (F727F3H14) was determined to be 1058, based on a molecular ion peak at 1057.83 (M - H) in the negative ionization mode (Figure 4). This determination was supported by the ionization pattern in ESIMS in the positive mode that exhibited a peak at 1059.56 (M + H) and a pseudomolecular ion peak at 1081.63 (M + Na) (Figure 5).
[000138] The molecular weight of Compound C (F727F3H17) was determined to be 1072, based on a molecular ion peak at 1071.85 (M - H) in the negative ionization mode (Figure 6). This determination was supported by the ionization pattern in the positive ESEVIS that exhibited a peak at 1073.57 (M + H) and a pseudomolecular ion peak at 1095.62 (M + Na) (Figure 7). Plug analysis method for antifungal test of fraction and pure compounds
[000139] Purified fractions and compounds were tested for antifungal activity as follows. A filter disk was placed in each quadrant of a medium-sized Petri dish (total of four disks). Each disc was placed 2 cm from the center of the plate. 15 μL of column fraction or purified compost (20 mg / mL) was dispensed on the surface of each of the two disks opposite each other. Ethanol was dispensed over the other two disks as a control. After the filter discs were loaded, small plugs (ca. 1 x 1 cm) of fungi were placed in the center of the Petri dish. The fungal pathogens used were Bipolaris maydis, Botrytis cinerea, Sclerotinia homeocarpa and Rhizoctonia solani. The plates were incubated at 25 ° C and, after 48 hours, the zone of inhibition around each filter disc was measured. The results are shown in Figure 8 for fraction 3 of VLC and compounds A, B and C; and indicate that all three compounds have significant fungicidal activity. Amino acid analysis of compounds A, B & C
[000140] Compound A (F727F3H11, 0.05 mg) was hydrolyzed by hydrolysis in liquid phase (6N HCl, 1% Phenol, 110 ° C, 24 h, under vacuum). After cooling, the reaction mixture was dried and the hydrolyzed product was dissolved in the Norleu dilution buffer to a volume of 1.0 mL. A 50 μL aliquot of this sample was loaded onto an ion exchange column for analysis.
[000141] For standards and calibration, a standardized amino acid solution for the protein hydrolyzate in the Na-based Hitachi 8800 (Sigma, A-9906) was used to determine response factors and thus calibrate the Hitachi 8800 analyzer for all amino acids. Each injection contained norleukin as an internal standard, to allow the results to be corrected for variations in sample volume and chromatography variables. The system used Na Pickering buffers, Pierce Sequanal grade HCl (hydrolysis), a Transgenomic Ion-Exchange column and an optimized method developed by the Molecular Structure Facility (MSF), UC Davis. The individual amino acids present in each sample were reported. The amino acids present in compound A were found to be glutamine (1 unit), proline (1 unit), serine (1 unit), tyrosine (1 unit) and asparagine (3 units).
[000142] The amino acid compositions of compounds B and C were analyzed in a similar way. It was observed that compounds B and C have the same amino acids, in the same proportion, as compound A. Example 6: Effect of isolate F727 from Bacillus sp. on Botrytis in tomato plants
[000143] Tomato plants (Solatium lycopersicum) var. Roma were treated with supernatant from fermentation with F727. Each plant was sprayed with approximately 3 mL of cell-free fermentation supernatant. The plants were allowed to dry and they were inoculated with 2 ml of a spore suspension of Botrytis cinerea in a concentration of 6.67 x 107 spores / ml. One control plant was sprayed with deionized water, two negative control plants were sprayed with spores only (in concentrations of 1x107 spores / mL and 6.67x107 spores / mL) and a positive control plant was sprayed with SWITCH® 65.2 WG ( Cypronidil and Fludioxonil, marketed by Bayer Crop Sciences, Inc.) at a rate of 14oz / 100gal / acre. The treatments were carried out in triplicates. The plants were placed in a transparent plastic container in a growth chamber with lights and constant temperature control. The disease classification was performed 8 days after treatment. Plants were evaluated for disease severity by visual assessment of a symptomatic leaf area of the disease and a disease classification was obtained. See, for example, WC James (1971) "A Manual of Assessment Keys in Plant Diseases." American Phytopathological Society. ISBN 978-0-89054081-7. The results, shown in Figure 9, show that the severity of the disease was reduced from 65% (untreated infected control) to 40% in infected plants treated with F727 supernatant.
[000144] In additional experiments, two-fold, four-fold and ten-fold dilutions of F727 WCB were tested. Using WCB from two separate fermentations, the reduction in disease severity was observed in all three dilutions. Example 7: Effect of Bacillus sp. F727 on downy downy mildew on lettuce
[000145] Lettuce plants (Lactuca sativa) var. Celtuce, were planted at a density of four seedlings per pot. Each vessel was sprayed with 2 ml of F727 fermentation supernatant.
[000146] The plants were allowed to dry and were then inoculated with 2 ml of a suspension of spores of Bremia lactuca (downy mildew) (1x105 spores / ml). The treatments were carried out in 5 replicates. The treated plants were incubated in sealed trays with a plastic cover, at 15 ° C in a growth chamber with a 12-hour photoperiod. 10 days after treatment, the severity of the disease was assessed as described in Example 6.
[000147] The results are shown in Figure 10. The average severity of the disease in the untreated control was 54.47%, while plants treated with F727 exhibited a disease severity of only 14.64%. The severity of the disease in plants treated with F727 was comparable to that obtained after treatment of the plants with the chemical control RIDOMIL® GOLD EC (4% w / w metalaxyl-M and 64% w / w mancozeb) (150 ppm ai) (Syngenta), which provided 11.67% severity.
[000148] In additional experiments, two-fold, four-fold and ten-fold dilutions of F727 WCB were tested. Using WCB from two separate fermentations, the reduction in disease severity was observed in all three dilutions. Example 8: Comparison of the effect of Bacillus sp. F727 with ELEVATE
[000149] Tomato plants (Solatium lycopersicum) var. Roma were treated with 3 mL of F727 fermentation supernatant and allowed to dry. The plants were then inoculated with approximately 2 ml of Botrytis cinerea spore suspension (2.8 x 10 7 spores / ml). A subset of the infected plants was sprayed a second time with F727, after 2 hours or after the first treatment had dried. The inoculated plants were also treated with water (negative control) and ELEVATE® 50 WDG (Fenhexamid, Bayer Crop Science, Inc.) as a positive control.
[000150] Treatments were performed in replicates of 4. Nine days after treatment, the severity of the disease was assessed as described in Example 6. The results are shown in Figure 11. The severity of the disease for the water control was 38 , 3%, while the positive control (ELEVATE® 50 WDG, Fenhexamid, Bayer Crop Science, Inc.) reduced the severity of the disease to 13.33%. Plants treated with 1x and 2x F727 supernatant had a disease severity of 8.3 and 5% respectively. Example 9: Effect of the F727 supernatant on B. cinerea infection in peppers
[000151] Pepper plants (Capscicum annuum) var. Serrano were sprayed with approximately 2 mL of F727 supernatant and allowed to dry. The plants were then inoculated with 2 ml of a spore suspension of Botrytis cinerea (2.7 x 10 7 spores / ml). The plants were treated in triplicate. Thirteen days after treatment, disease severity was assessed and compared with an untreated control (sprayed with water) and a positive control (sprayed with Elevate® 50 WDG (Fenhexamid, Bayer Crop Science, Inc.)) applied to the rate of label.
[000152] The results, shown in Figure 12, indicate that disease control in plants infected with Botrytis cinerea that had been treated with F727 was comparable to that obtained by treating the plants with Elevate®. Example 10: Evaluation of medium with whole cells, supernatant and cells with F727 produced by fermentation in three media against powdery mildew in the cucumber.
[000153] F727 cells were fermented in three different growth media (SPY, SMP and TSB). Two-week-old cucumber plants (Cucumis sativus) var. SMR58 were sprayed with approximately 3 mL of whole cell medium with F727, F727 supernatant or F727 cells obtained from each of these three fermentations. The cells were pelleted and then resuspended in 10 mM magnesium sulfate for spraying. Four replicates per treatment were produced. The plants were allowed to dry for two hours before being sprayed with approximately 2 ml of a powdery mildew spore suspension at a concentration of 3.0 x 10 5 spores / ml, which had been prepared from an infected plant. The plants were then incubated in a growth chamber until the disease developed and the severity of the disease was assessed as described in Example 6.
[000154] The results, expressed as the percentage of disease control, are shown in Figure 13. The antifungal activity in the cucumber was observed with whole cells, medium with whole cells and cell supernatants obtained from all three media. Example 11: Evaluation of the efficacy of medium with whole cells, supernatant and cells with F727 produced by fermentation in three media against Botrytis cinerea in tomatoes.
[000155] Two-week-old tomato plants (Solatium lycopersicum) var. Stupice were sprayed with approximately 2 ml of F727 whole cell medium, F727 supernatant and F727 cells, each prepared starting from three separate fermentations in different growth media (SPY, SMP and TSB). The cells were pelleted and resuspended in 10 mM magnesium sulfate for spraying. Three replicates per treatment were produced. The plants were allowed to dry for an hour, then placed under humid conditions to open the stomata. Approximately 1 mL of a spore suspension of Botrytis cinerea prepared from a 10-day old agar plate grown at 1.0x107 spores / mL in medium with 2% Sabouraud maltose was sprayed onto each plant. The plants were incubated in a growth chamber with a 12 hour photoperiod until the disease developed and the severity of the disease was assessed as described in Example 6.
[000156] The results, expressed as the percentage of disease control, are shown in Figure 14. The antifungal activity in tomatoes was observed with whole cells, medium with whole cells and cell supernatants obtained from all three media. Example 12: Evaluation of the efficacy of F727 whole cell medium, commercial Bacillus-based products and F727 whole cell medium mixed with Regalia® against powdery mildew in cucumber.
[000157] Two-week-old cucumber plants (Cucumis sativus) var. SMR58 were sprayed on the first true leaf with approximately 3 mL of: Regalia® 5% (Renoutria sachalinensis, Marrone Bio Innovation, Inc., Davis, CA) at 1: 2000, Regalia® at 5% at 1: 200, medium with cells whole with F727, medium with whole cells with F727 + 5% Regalia® at 1: 2000, Serenade® (Bayer Crop Science, Inc.) at 1: 200, Sonata® (Bayer Crop Science, Inc.) at 1: 200 , Vacciplant® (Laboratoires Goêmar SA) at 40 μL / 50 mL, Companion® (Growth Products, Ltd.) at 1: 200 and Double Nickel 55® (Certis USA, LLC) at 0.06 g / 50 mL. Four replicates per treatment were prepared. The plants were allowed to dry for two hours before approximately 2 mL of a powdery mildew spore suspension at a concentration of 3.0 x 10 5 spores / mL was sprayed onto each plant. The plants were incubated in a growth chamber until the disease developed.
[000158] The results, shown in Figure 15, indicate that the medium with whole cells from a fermentation with the isolate F727 from Bacillus sp. (identified as MBI-110 WCB in the Figure) is more effective against powdery mildew than many existing fungicides. The results for Regalia® with and without F727 supernatant are shown in Table 6. Colby's synergy coefficient indicates that there is synergy between medium with whole cells with F727 and Regalia® 5% at 1: 2000.
Example 13: Evaluation of the efficacy of whole cell medium with F727, Regalia® and Double Nickel 55® against Phytophthora infestans in tomato
[000159] Tomato plants (Solatium lycopersicum) var. Stupice in a stage of two true leaves were sprayed with 2 mL of medium with whole cells with F727, Regalia® at 1: 200 (Marrone Bio Innovations, Inc.) and Double Nickel 55® at 0.06 g / 50mL (Certis USA , LLC). The plants were allowed to dry before being sprayed to cover with a spore solution of Phytophthora infestans, in a concentration of 104 spores / mL, prepared from infected tomato leaves. The plants were incubated in a growth chamber at 20 ° C under artificial light. Three days after treatment, the plants were evaluated for disease severity as described in Example 6.
[000160] The results, shown in Figure 16, indicate that the medium with whole cells from a fermentation with the isolate F727 from Bacillus sp. (identified as MBI-110 WCB in the Figure) provides robust protection against infection caused by P. infestans from tomatoes. Example 14: Evaluation of Sclewtium rolfsii F727 control in vitro
[000161] Isolate F727 was fermented in liquid medium and the supernatant was removed by centrifugation. Half of the supernatant was filtered through a 0.2 μm filter. Isolated sclerotia of Sclewtium rolfsii were placed in the centers of 10 cm Petri dishes and four 0.5 cm diameter discs per plate were placed at a distance of 2 cm from the fungus and at equal distances from each other. The F727 supernatant, filtered F727 supernatant and Pristine® (BASF) at 0.5 mL / L were added to the disks in 12.5 μL aliquots, until two opposing disks contained a total of 25 μL and the other two contained 50 μL. Two plates per treatment were prepared. The plates were incubated at 25 ° C for three days.
[000162] The percentage of inhibition for each test substance was determined by measuring the growth of the mycelium from the sclerotia to the farthest edge of the colony towards each disk. The results, shown in Figure 17, indicate that filtered F727 supernatants when unfiltered were effective in inhibiting the growth of S. rolfsii in the disc assay. The unfiltered supernatant was consistently more efficient than the filtrate and its effectiveness was comparable to that of the commercial standard. Example 15: Evaluation of F727 control of Rhizoctonia solani in soybeans
[000163] Sterilized barley grains were inoculated with Rhizoctonia solani and incubated for 1-2 weeks. The grains were dried, mixed and mixed with sand in a one to one ratio to generate an R. solani inoculum. The soil was vigorously mixed with this inoculum to a volume of 500 ml of soil per pot, watered with 100 ml of water and incubated in a growth chamber for 24 hours. Whole cell medium with isolate F727 was prepared at a concentration of 100%, 50% and 25%. The soil was then soaked with 40 mL of each dilution of the medium and nine soybean seeds were planted in each pot. For each replica, there were three vessels and there were three replicates per treatment. The plants were incubated in a growth chamber for 14 days. Germination, plant height, fresh shoot weight and fresh root weight were evaluated.
[000164] Sprout support measures (Table 7), emergence (Table 8), average sprout weight (Table 9) and average sprout height (Table 10) were determined. The results indicate that the medium with whole cells with F727 increased the emergence, the weight of the shoots and the height of the shoots in soybean plants infected with R. solani.

Table 8
Table 9

Table 10
Example 16: Evaluation of F727 control of bacterial plant pathogens in vitro
[000165] One ml of sterile water was inoculated with a loop of each of the bacterial plant pathogens, Erwinia amylovora, Pseudomonas syringae, Bacillus cereus, Erwinia carotovora, Xanthomonas campestris, Xanthomonas arboricola or Clavibacter michiganensis subsp. michiganensis, from plates with potato dextrose agar (PDA) grown. The bacteria were resuspended and 100 μL of the resuspension of the pathogen was streaked on a plate with PDA agar and allowed to be absorbed inside the plate for 10-15 minutes. Sterile filter discs were applied to the agar and loaded with 20 μL of VLC fractions of F727 (10 mg / mL in methanol) or combinations of VLC fractions. The fractions were obtained from a fermentation of Bacillus sp. in V8 medium as described in Example 5, above.
[000166] The plates were incubated 24-48 hours and then inspected for the appearance of an inhibition zone around the filter disc, indicating susceptibility of the pathogen to the F727 fraction. The results are shown in Table 11.Table 11: Susceptibility of bacterial phytopathogenic agents to F727 fractions
Identification: +++ very susceptible, ++ susceptible, - resistant
[000167] The inhibition of the largest number of bacterial species was observed with Fraction 3. Consequently, this fraction was subjected to additional fractionation, as described in Example 5, above. In addition, Fraction 5 exhibited antibacterial activity against Bacillus cereus, Erwinia carotovora and Clavibacter. Example 17: Evaluation of supernatant and crude extract of F727, prepared in different media, to control the germination of fungal pathogens in vitro
[000168] Isolate F727 was fermented in 12 different liquid media. Samples of supernatant and crude extract were tested in a dose-response spore germination assay. In a 48-well plate, 100 μL of supernatant sample or crude extract was combined with 200 μL of agar with potato dextrose (PDA) 1.5x and allowed to solidify. Spore suspensions from fungal plant pathogens were prepared at the concentrations shown in Table 12 and 50 μL of spore suspension was dispensed on the surface of the PDA / treatment mixture. The plates were incubated at 25 ° C for 3-5 days and a visual assessment of fungal spore germination was conducted.
[000169] The results are shown in Table 13. All tested supernatants inhibited the germination of all pathogens at the lowest concentration tested (3.13%). The activity of the crude extracts varied depending on the medium used to grow the F727 cells (a 0.4% dilution of crude extract was the lowest concentration tested). This indicated that the activity of F727 extracts may depend on the medium in which the cells are grown and, thus, the optimal activity against a particular pathogen can be adjusted based on the medium in which the cells are grown. For example, the M24 medium can be used to grow cells whose extracts will target Fusarium. Table 12
Table 13: Minimum inhibitory concentration of crude F727 extract from different media necessary to prevent the germination of fungal spores
Example 18: Evaluation of the growth characteristics of F727 plants in vitro
[000170] The F727 isolate was evaluated for plant growth characteristics in vitro, including the ability to solubilize phosphate, the production of ACC-deaminase, the production of indole-3-acetic acid (IAA), the production of siderophores (CAS agar) and the ability to grow with methanol as the only carbon source (AMS agar). Phosphate solubilization was evaluated on bromophenol-phosphate blue agar, ACC-deaminase activity was evaluated on agar plates with ACC as the only carbon and nitrogen source, siderophores production was evaluated on CAS agar and methylotrophy was evaluated on an agar with mineral salts corrected with methanol as the only carbon source. As shown in Table 14, Isolate F727 was positive in the test for all these plant growth promotion characteristics. Table 14
Identification: +++: very strong positive response, ++: positive response, +: weak positive response Example 19: Analysis of the vigor of seeds treated with F727
[000171] Corn, soy, wheat, rice, sorghum and tomato seeds had their surfaces sterilized for six minutes with bleach at 1% and washed with sterile water five times. The seeds were submerged for 24 hours in the F727 suspension prepared in 10 mM magnesium sulfate. The seeds were dried for 30 minutes for Experiment # 1 and overnight for Experiment # 2. The seeds were allowed to germinate on moistened paper towels for several days and the total fresh weight of the seeds was measured.
[000172] The results are shown in Table 15 and indicate that the cells of Bacillus sp. promoted the growth of corn, soybeans, sorghum and tomatoes.Table 1
# var. Kandy Korn * var. Trucker's Favorite Yellow, Boone County White and Silver King. Example 20: Antifungal activity against Aspergillus niger
[000173] Whole cell medium (WCB) from fermentation of Bacillus sp. was evaluated for its inhibitory effect on the pathogen Aspergillus niger postharvest, in a fruit immersion assay. The WCB was used undiluted and diluted in sterile water to concentrations of 5%, 20% and 50%.
[000174] Sterilized green grapes were immersed for five seconds in each concentration of WCB, then placed on a shelf in a container. Once the fruit was dry, each container received 24 sprays of A. niger inoculum adjusted to 3 x 103 spore / mL. 100 mL of deionized water was added to each box under the shelf to increase humidity and the boxes were sealed and incubated at room temperature. Two containers, each containing 5 grapes per treatment, were included in the experiment. A water treatment was used as a negative control and the commercial product Switch was used as a positive control.
[000175] The disease percentage of each fruit was determined by observing the mycelium cover on each grape. The results are shown in Table 16 and indicate that F727 WCB exhibits significant anti-Aspergillus activity. Table 16. Percentage of A. niger disease in grapes after immersion treatment with F727 WCB after 12 days of growth.
Example 21: Promotion of growth in maize
[000176] Corn seeds were planted in the soil mixture for planting in pots, in pots with a diameter of 4 inches, at a density of 10 seeds per pot. The seeded pots were soaked at the time of planting and a week later with medium with whole cells with F727. The pots were incubated in a greenhouse. A total of 10 plants per pot and 9 pots per treatment were evaluated.
[000177] Total fresh weight was recorded after 2 weeks' growth. Control plants (soaked with water) had an average fresh weight of 21.48 + 4.02 g, while plants that were soaked with F727 WCB had an average fresh weight of 26.5 + 3.55 g. These differences are statistically significant, which are determined by Minitab ANOVA Tukey's. These results provide additional evidence for the activity of promoting the growth of F727 WCB. Example 22: Control of downy mildew Bremia lactucae on lettuce by soaking the soil with whole cell medium with F727
[000178] Spores of Downy Mildew (Bremia lactucae) were obtained by cutting leaves containing spores of infected plants grown in culture boxes and shaking the leaves in 50 mL of deionized water. The liquid was then filtered through a 100 μm nylon screen. The number of spores in the filtered liquid was counted on a hemocytometer and the liquid was adjusted with deionized water to contain 5x104 spores / mL.
[000179] For the preparation of whole cell medium (WCB), the F727 strain of Bacillus was grown in SPY medium for 24-72 hours at 25 ° C.
[000180] Lettuce (c.v. Celtuce) was planted in 2.5-inch pots at a density of six plants per pot. The plants were grown in a growth chamber at 16 ° C with a 12 hour photoperiod. After 10 days of growth, the soil was soaked with 20 mL of F727 WCB.
[000181] One hour after soaking, approximately 1 mL of the downy mildew spore suspension described above was sprayed onto each pot of lettuce plants. During the first 48 hours after inoculation, the pots were incubated in covered plastic trays. After that, they were incubated for 8 days at 16 ° C with a 12 hour photoperiod.
[000182] For each cotyledon, the severity of the disease was classified as the percentage of the leaf surface that was diseased and the average severity was determined for each pot of plants. The results for treated (drenched soil) and untreated control (UTC) plants were compared using a t-test and are shown in Table 17 and Figure 18. The results show a statistically significant reduction (p = 0.0036) of severity of the disease caused by Downy Mildew on lettuce plants that received soil soaked with F727 whole cell medium.Table 17: Effect of soil soaked with F727 WCB on lettuce infection caused by downy mildew (Two-sample t-test assuming equivalent variances)
Example 23: Synergy between whole cell half with F727 and Bacillus amyloliquefaciens in the control of Sphaerotheca fuliginea Powdery Mildew in the cucumber
[000183] An inoculum of Sphaerotheca fuliginea was prepared by washing infected cucumber leaves with deionized water and filtering what was washed through two layers of cotton gauze.
[000184] Cucumber plants in the true two-leaf stage were sprayed with 2 ml of treatment (see below) and allowed to dry for three hours, after which the plants were inoculated by brushing the leaves with 2 ml of S inoculum. soot at a concentration of 2.5x105 conidia / mL.
[000185] The treatments were as follows (with three replicates per treatment): 1. Water (negative control) 2. Whole cell medium with F727 (total concentration) 3. Whole cell medium with F727 (50% concentration) 4. Whole cell medium with F727 (total concentration) + Double Nickel 55 (3 lbs. / Acre) 5. Whole cell medium with F727 (50% concentration) + Double Nickel 55 (3 lbs. / Acre) 6. Double Nickel 55 (3 lbs./acre)
[000186] For the preparation of medium with whole cells, F727 was grown in SPY medium for 5 days. Double Nickel 55 is a preparation of commercial B. amyloliquefaciens (Certis USA, Columbia, MD), which has broad spectrum fungicidal activity, including activity against Powdery Mildew.
[000187] After inoculation, the plants were incubated in a growth chamber at a temperature of approximately 25 ° C for ten days, during which time they were evaluated visually in relation to the severity of the disease, which was analyzed as the percentage surface of the leaf that was sick. The results, shown in Table 18, were analyzed using the Minitab 16 statistical software (Minitab, State College, PA) and the possible synergy was assessed by calculating Colby's synergy coefficient. Colby (1967) "Calculating synergistic and antagonistic responses of herbicide combinations." Weeds 15: 20-22.Table 18: Effect of whole cell medium with F727 and mixtures with Double Nickel 55, in the control of powdery mildew in cucumber.
* Data for disease severity are presented as means and standard deviations. The means that do not have the same letter are significantly different (p <0.05) using Fisher's LSD test. # Ee is the Efficacy expected from Colby's formula (Colby, 1967): Ee = A + B-AB / 100, where A and B are the efficacies of the individual products; There is a synergy if the effectiveness of the combination (% of control, E) is greater than Ee (E / Ee> 1.0).
[000188] The results in Table 18 indicate that the whole cell medium with F727 has a strongly synergistic effect with Double Nickel in the control of Powdery Mildew, with Colby synergy coefficients of 5.6 and 1.8, respectively, for medium with whole cells at full concentration and half the concentration. Example 24: Promotion of plant growth using F727 whole cell medium talc formulations
[000189] A talc formulation was prepared by mixing a dry carrier made of talc (1 kg), carboxymethyl cellulose (10 g), calcium carbonate (15 g) and medium with whole cells with F727 at a ratio of 1 : 1 (w / v) dry carrier mix: WCB. The mixture was dried overnight and then subjected to fine grinding. This formulation was applied in two ways. In the first method, this was applied to the soil at planting, under the seed (field corn) in the form of an application to the plow. In the second method, the talc formulation was dissolved in water and used to activate the seeds throughout the night before planting. The water was used as a control. The plants were grown for two weeks in a greenhouse and weighed.
[000190] The results of these analyzes are shown in Tables 19 and 20. The application to the plow resulted in a 16% increase in the average fresh weight (Table 19), while the activation resulted in a 15% increase in the average fresh weight (Table 20). Thus, the F727 WCB has the activity of promoting growth. Table 19: Application of the F727 WCB talc formulation to the plow
Table 20: Seed activation with the talc formulation of F727 WCB
Example 25: Fractionation of whole cell medium with F727
[000191] The F727 strain was grown in one liter of a suitable fermentation medium (for example, SPM, SPY, TSB, V8) for 4872 hours at 25 ° C. One liter of medium with whole cells was extracted with Amberlite XAD-7 resin by stirring the cell suspension with the resin at 155 rpm for two hours at room temperature. The resin and cell mass were collected by filtration through cotton gauze and washed with deionized water to remove salts. The resin, cell mass and cotton gauze were then immersed for 2 hours in acetone, after which the acetone was filtered and dried under vacuum, using a rotary evaporator, to provide a crude extract.
[000192] The crude extract was fractionated by vacuum liquid chromatography with C-18 in reverse phase. In a previous fractionation scheme (Example 5 and Figure 1), pesticidal activity was observed in Fractions 3 (cut 40-60% methanol) and 4 (cut 60-80% methanol). To maximize the amount of activity in a single fraction, the VLC procedure with C-18 in reverse phase was changed, as shown in Table 21, to provide a cut of 50-80% methanol (Fraction 4). Compounds A, B and C were then purified from Fraction 4 by HPLC with C-18 as described in Example 5.Table 21: VLC fractions of F727 extract with Amberlite
Example 26: Structure of Compound A determined by MS / MS analysis
[000193] The PM 1044 compound from Fraction 4 (Compound A) was analyzed by ionization mass spectroscopy with electrospray / mass spectroscopy (ESEVIS / MS) to obtain the sequence of its constituent amino acids. The results are shown in Figure 19. Based on these results, Compound A was determined to have a cyclic peptide structure shown in Figure 20. Example 27: Bactericidal activity of VLC compounds and fractions
[000194] Erwinia carotovora, Pseudomonas syringae, Xanthomonas arboricola, Acidovorax avenae subsp. citrulli and Clavibacter michiganensis subsp. michiganensis were plated on potato dextrose agar. After the accumulation of sufficient biomass, a handle filled with each pathogen was removed from its plate and suspended in sterile water. 100 μL of each suspension was streaked onto a potato dextrose agar plate and allowed to absorb within the plate for 10-15 minutes.
[000195] Samples of fractions of VLC 4 and 5, as described in Example 25 and of compounds A (PM = 1044), B (PM = 1058) and C (PM = 1072) starting from the VLC Fraction 4 described in Example 25 were prepared at 5 mg / mL in methanol and 5 sets of two-fold serial dilutions of each sample were made in water (ie, concentrations between 0.15625 mg / mL and 5 mg / mL, in increments of two times have been tested). Sterile filter discs were applied to the plates with agar and the discs were loaded with 20 μL of each sample. The plates were incubated at 25 ° C for 3 days and then observed in relation to the growth inhibition zones around the filter discs, indicative of the susceptibility of the pathogen to the sample. If an inhibition were detected, the minimum sample concentration exhibiting inhibitory activity was determined. The results are shown in Table 21. Fractions 4 and 5 have inhibitory activity against Erwinia caratovora and Clavibacter michagensis, while Compounds A and B inhibit the growth of Acidovorax avenae.Table 21: Minimum sample concentration that inhibits agent growth pathogenic
Table 21: -continuation-
Example 28: Fungicidal activity of VLC compounds and fractions
[000196] Fraction samples of VLC 4 and 5, as described in Example 25 and of compounds A (PM = 1044), B (PM = 1058) and C (PM = 1072) from the VLC Fraction 4 described in Example 25 were prepared at 5 mg / ml in methanol. Serial dilutions of each sample were prepared on a 48-well plate. The highest concentration of each sample tested was 500 μg / mL (10% of the original concentration) and the samples were thereafter serially diluted in water in two-fold increments to a concentration of 3.9062 μg / mL. 100 μL of dilution sample was added to each well, followed by 200 μL of agar with potato dextrose (PDA) 1.5X and the mixture was then allowed to solidify for 10-15 minutes.
[000197] Colletotrichum cereale, Fusarium oxysporum, Botrytis cinerea and Verticillium dahliae were plated on PDA and incubated at room temperature until the growth of mycelium covered the entire plate. Sterile water was added to each plate and a sterile spatula was used to dislodge mycelia and spores. The suspension was passed through a filter to separate mycelia and spores. The spores were counted with a hemocytometer and adjusted, through dilution with water, to a concentration of 103104 spores / mL.
[000198] Phytophthora capsici was plated on PARP agar and grown at room temperature until sufficient mycelial growth was observed. Sterile water was added to the plate and a spatula was used to dislodge the spores. The plate was incubated in a Conviron chamber at 16 ° C for 1-2 hours until the sporangia released the zoospores. The spores were then collected in a tube, counted with a hemocytometer and the spore suspension was adjusted, with water, to a concentration of 103-104 spores / mL.
[000199] 50 μL of the spore solution from each pathogen was inoculated into each sample well. The plates were incubated at 25 ° C for 4-5 days and then observed for inhibition of spore germination in the wells, indicative of the susceptibility of the pathogen to the sample. If an inhibition were detected, the minimum concentration of the sample exhibiting inhibitory activity was determined. The results are shown in Table 22. Table 22: Minimum sample concentrations that inhibit spore germination


[000200] Compounds A, B and C and Fraction 4 inhibited spore germination of all fungal pathogens tested except P. capsici. Compound C and Fraction 4 exhibited the highest inhibitory activities. DEPOSIT OF BIOLOGICAL MATERIAL
[000201] The following biological material was deposited under the terms of the Budapest Treaty at the Agricultural Research Culture Collection (NRRL), 1815 N. University Street, Peoria, Illinois 61604 USA and received the following number:

[000202] The strain was deposited under conditions that guarantee that access to the crop will be available pending this patent application to one determined by the Commissioner of Patents and Trademarks as being entitled under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of the deposited strain. The deposit is available when required by foreign patent laws in countries where correspondents of this patent application or their progeny are deposited. However, it must be understood that the availability of a deposit does not constitute a license to practice the present invention by abolishing the patent rights granted by the Government's action.
[000203] The invention described and claimed here should not be limited in scope by the specific aspects disclosed here, since these aspects are intended as illustrations of various aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. In fact, various modifications of the invention in addition to those shown and described here will become apparent to those skilled in the art from the foregoing description. It is also intended that such modifications fall within the scope of the appended claims. In the event of a conflict, this disclosure including definitions will control. Several references are cited here, whose disclosures are incorporated as a reference in their entirety.

权利要求:
Claims (7)
[0001]
1. Composition, characterized by the fact that it comprises: (a) a whole cell medium collected from fermentation of a Bacillus F727 strain (NRRL Accession No. B-50768), in which the Bacillus F727 strain has pesticidal activity or activity promoting plant growth; (b) at least one of a carrier, a diluent, a surfactant or an adjuvant; and (c) a chemical or biological pesticide.
[0002]
2. Method for the modulation of fungal infestation in a plant, characterized by the fact that it comprises the application in the plant and / or in the seeds of the same and / or in the substrate used for the growth of said plant of an amount of the composition comprising a whole cell medium collected from strain fermentation of a Bacillus F727 (NRRL Accession No. B-50768) and optionally comprising a pesticide.
[0003]
3. Method according to claim 2, characterized by the fact that the fungus is selected from the group consisting of Botrytis, Sclerotinia, Rhizoctonia and Bipolaris.
[0004]
4. Method according to claim 2, characterized in that the additional pesticide consists of extract of Reynoutria sachalinensis (sanguinary).
[0005]
5. Method for modulating the growth of a plant and / or the germination of a seed characterized by the fact that it comprises the contact of said plant, its growth substrate and / or a seed of said plant with a quantity of the composition comprising a whole cell medium collected from fermentation of a Bacillus F727 strain (NRRL Accession No. B-50768), and optionally, one or more substances effective to modulate the growth of said plant and / or the germination of said seed.
[0006]
6. Method according to claim 5, characterized by the fact that one or more substances comprises a growth promoting agent, a surfactant, a carrier, an adjuvant and / or a fertilizer.
[0007]
7. Seed coated with a composition, characterized by the fact that said composition comprises a whole cell medium collected from fermentation of strain of a Bacillus F727 (NRRL Accession No. B-50768).

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法律状态:
2019-10-15| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-04-07| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-04-14| B15K| Others concerning applications: alteration of classification|Free format text: AS CLASSIFICACOES ANTERIORES ERAM: C12N 1/20 , A01N 63/02 Ipc: C12N 1/20 (2006.01), A01N 63/00 (2020.01), A01N 63 |

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2020-09-08| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-11-24| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2021-03-02| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-23| B09W| Decision of grant: rectification|Free format text: REFERENCIA: RPI 2617 DE 02.03.2021 - CODIGO 9.1 |

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2021-04-13| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/08/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261683174P| true| 2012-08-14|2012-08-14|

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US61/683,174|2012-08-14|

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US13/835,677|2013-03-15|

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US13/835,677|US9125419B2|2012-08-14|2013-03-15|Bacillus sp. strain with antifungal, antibacterial and growth promotion activity|

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PCT/US2013/054775|WO2014028521A1|2012-08-14|2013-08-13|Bacillus sp. strain with antifungal, antibacterial and growth promotion activity|

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