Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
  • A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
  • A01N63/27—Pseudomonas
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
  • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/90—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having more than three double bonds between ring members or between ring members and non-ring members
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/04—Ortho-condensed systems
  • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
  • C07D491/048—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being five-membered
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/38—Pseudomonas

Definitions

• This invention is in the field of biopesticides.
• the invention pertains to seven novel strains of Pseudomonas spp, 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328, the cell broth and novel metabolites produced from the bacterial strain that can inhibit the growth of a variety of microbial species.
• the Pseudomonas strains of 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318- T327, and 0418-T328 have been deposited in the American Type Culture Collection (ATCC) and have ATCC accession number PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802, respectively.
• ATCC American Type Culture Collection
• Plant diseases caused by pathogenic microorganisms are exponentially increasing and cost-consuming.
• the plant pathogenic organisms include fungus, bacterium, mycoplasma, virus, viroid, nematode, or parasitic flowering plant.
• fungus fungus
• bacterium mycoplasma
• virus virus
• viroid virus
• nematode nematode
• parasitic flowering plant Currently, there are 14 common plant diseases caused by bacterial organisms including bacterial spot, bacterial light and bacterial wilt etc.
• Fire blight Erwinia amylovora
• citrus cankers ⁇ Xanthomonas axonopodis pv. citri (Xac) ⁇ , bacterial leaf spot (BLS) ⁇ Xanthomonas campestris pv.
• Fire blight is a devastating disease of pome fruit resulting from the infection of a gram-negative bacterium Erwinia amylovora which impacts pear and apple in many parts of the world such as Europe, Germany, Austria and Switzerland (Chen et al. (2009)). While fire blight rarely kills an entire orchard, the disease and its control still cause significant economic losses. In the Pacific Northwest and northern California, there have been minor outbreaks annually since 1991 with at least some areas experiencing major outbreaks every 3 to 4 years. Even minor disease outbreaks can be costly as pruning to remove infected plant parts leads to disfigured trees with reduced future productivity. For example, a 10% incidence of rootstock blight in a 4-year old apple orchard can result in losses up to $3,500 per acre (Norelli et al. (2003)).
• Microbial natural products have provided rich amounts of biological compounds as pesticides (Gwinn (2016)).
• current prevention methods for the bacterial plant diseases have limited effectiveness.
• the antibiotics streptomycin sulfate (FireWall, AgroSource, Inc.) and oxytetracycline hydrochloride (FireLine, AgroSource, Inc.) have been the primary products used to combat A. amylovora when infection risk is high. Because these compounds are also used in the management of human and animal health, use of these same antibiotics in crop agriculture can be controversial (Stockwell (2012)). For streptomycin sulfate, the concerns over antibiotic resistance has limited its use (Vrancken et al. (2013)).
• kasugamycin Another antibiotic that is being researched against fire blight is kasugamycin.
• One disadvantage is the frequent dosages of kasugamycin lead to phytotoxic effects that destroy the plant (Adaskaveg et al. (2010)).
• the other disadvantage is the high cost of kasugamycin in comparison to other antibiotics. So, kasugamycin needs to be paired with an assortment of other antibiotics.
• Pseudomonas species have been comprehensively reviewed (Masschelein et al. (2017)).
• the types of Pseudomonas metabolites can be classified as phenolic compounds, phenazine, lipopeptides, etc.
• Function of Pseudomonas species and their metabolites include the following (Alsohim et al.
• a method of growing bacteria to enhance production of protective metabolites includes alternative steps.
• a step of growing Pseudomonas bacteria in liquid media in a vessel to produce a bacterial fermentate is provided.
• the ratio of media volume to vessel volume is between about 1 :2 and 1 : 10 and the vessel is shaken at a rate between about 100 and 250 RPM.
• the method includes growing Pseudomonas bacteria in liquid media in a fermenter to produce a bacterial fermentate.
• the air flow rate of the fermenter is between about 1 and 3 L/min.
• the concentration of dissolved oxygen is between 5 mg/L to 12 mg/L.
• an agricultural composition comprising the bacterial fermentate or the protective supernatant.
• the agricultural composition is produced according to the method of the first aspect and any of the respects disclosed with regard to the first aspect.
• the agricultural composition further includes adjuvants.
• the adjuvant is a surfactant.
• a method of controlling bacterial crop diseases includes several steps.
• a first step includes producing an agricultural composition comprising the bacterial fermentate or the protective supernatant produced by the first aspect or any of respects thereof.
• a second step includes applying said agricultural composition to crops to inhibit the growth of pathogenic microorganisms.
• a method of controlling bacterial crop diseases includes one step.
• a step includes applying an agricultural composition comprising between about 1.0 x 10 5 and 1.0 x 10 9 cfu per mL Pseudomonas bacteria to crops to inhibit the growth of pathogenic microorganisms.
• a method of purifying protective metabolites from Pseudomonas bacteria includes several steps.
• a first step includes producing a bacterial fermentate or protective supernatant by the method of the first aspect and the respects thereof.
• a second step includes extracting the bacterial fermentate or protective supernatant by solvent mixtures with similar polarities or characters.
• a third step includes producing an eluate containing protective metabolites by eluting the bacterial fermentate or protective supernatant using a mixture of hexane and ethyl acetate or by eluting the bacterial fermentate or protective supernatant using mixture of hexane and ethyl acetate.
• an agricultural composition comprising protective metabolites from Pseudomonas bacteria purified by the method of the fifth aspect and respects thereof.
• a method of controlling bacterial crop diseases includes several steps.
• a first step includes producing an agricultural composition comprising protective metabolites from Pseudomonas bacteria purified by the method of the fifth aspect or any of the respects thereof.
• a second step includes applying said agricultural composition that the formulation of the protective supernatant or its metabolites can be solution (SL), soluble powder (SP), soluble granules (SG) and encapsulated formulation.
• the agricultural composition of the formulation of bacteria fementate and cells can be suspension concentrate (SC), wettable powder (WP), and water dispersible granule (WG).
• a crystalline compound selected from one of the following structures:
• FIG. 1 illustrates exemplary plot of the maximum likelihood phylogeny of representative Pseudomonas lineages based on a concatenated alignment of 16S rDNA, gyrB, rpoB and rpoD.
• the bootstrap support values were labeled below the four internal branches that received ⁇ 100% support. Those not labeled represent 100% support.
• FIG. 2 illustrates an example of assay-guided isolation of ethyl acetate extract of strain 0617-T307.
• FIG. 3 depicts exemplary culture plots showing the amount of RejuAgro A in a shaking flask fermentation in which the distribution of RejuAgro A in the cell broth, supernatant and cells (panel A) and the production of RejuAgro A from cell fermentation over time (panel B).
• FIG. 4 depicts exemplary agar plates showing V. inaequalis can grow on PDA plates with PDA alone without additives (plate A); with 0.25% 0.01M PBS (plate B) or 0.8% DMSO (plate C) or 1.6% DMSO (plate D) on day 14.
• FIG. 5 depicts exemplary agar plates showing V. inaequalis cannot grow on PDA plates containing the selected four biocontrol bacteria (plate A: 0617-T307; plate B: 0118- T319; plate C: 0318-T327; plate D: 0418-T328) on day 14.
• FIG. 6 depicts exemplary agar plates showing V. inaequalis cannot grow on PDA plates containing 40-80 ⁇ g/mL RejuAgro A on day 14 (plate A: 10 ⁇ g/mL in PDA plate; plate B: 20 ⁇ g/mL in PDA plate; plate C: 40 ⁇ g/mL in PDA plate; plate D: 80 ⁇ g/mL in PDA plate).
• FIG. 7 depicts exemplary agar plates showing V. inaequalis can grow on PDA plates containing 10-80 ⁇ g/mL RejuAgro B on day 14 (plate A: 10 ⁇ g/mL in PDA plate; plate B: 20 ⁇ g/mL in PDA plate; plate C: 40 ⁇ g/mL in PDA plate; plate D: 80 ⁇ g/mL in PDA plate).
• FIG. 8 depicts exemplary agar plates showing V. inaequalis can grow on PDA plates containing 200-1000 ⁇ g/mL copper sulfate on day 14 (plate A: PDA plate with 500 ⁇ g/mL CuSO 4 ; plate B: PDA plate with 1000 ⁇ g/mL CuSO 4 ).
• FIG. 9 depicts an exemplary amount-peak area curve of RejuAgro A analyzed by HPLC at the wavelength of 407 nm.
• FIG. 10 depicts exemplary data on RejuAgro A production from different bacterial strains.
• FIG. 11 depicts an exemplary antifungal assay against Botrytis cinerea CA17, wherein panel A depicts (1) 40 ⁇ L Nystatin at 50 mg/mL, (2) 40 ⁇ L DMSO; panel B depicts (1) M9 medium for 24 h, (2) M8 medium for 24 h, (3) M7 medium for 24 h, (4) M6 medium for 24 h; panel C depicts (1) M9 medium for 12 h, (2) M8 medium for 12 h, (3) M7 medium for 12 h, and (4) M6 medium for 12 h.
• FIG. 12 depicts an exemplary agar plates of M. fijiensis showing inhibitory growth in the presence of RejuAgro A at 600 ⁇ g/mL (panel A) but growth in the presence of RejuAgro A at 60 ⁇ g/mL (panel B) or without RejuAgro A (panel C).
• FIG. 13A depicts the RejuAgro A molecule as having a planar structure - with S-Me group rotated only by 8.7° relative to the heterocycle. There is a notable break of ⁇ - conjugation in the molecule at C4-C5 bond (1.531 A) - apparently, because of some orbital reasons. The Me-group connected to sp 2 carbon atom is rotationally disordered over 2 positions.
• FIG. 13B depicts the RejuAgro A molecule in crystal form centrosymmetric H - bonded dimers through N-H...O interactions. Further, these dimers form 2-dimensional layers along [-3 0 1] plane via weaker C-H...0 interactions.
• FIG. 14A depicts the RejuAgro B crystal with two symmetrically independent RejuAgro B molecules. Each molecule has a twisted structure - with dihedral angle between mean planes of the linked heterocycles of 70. 3 and 80.6°. There is a notable break of ⁇ - conjugation in each heterocycle at C(sp 2 )-C(sp 2 ) bond between two adjacent carbonyl groups (the bond lengths are in the 1.534-1.539 A range) - apparently, because of some orbital reasons.
• FIG. 14B depicts RejuAgro B molecules in crystal form centrosymmetric 14- bonded dimers through N-H...0 interactions. These dimers are linked in stacks along x direction by other N-H...O interactions. Finally, the stacks are linked by third kind of N- H...0 interactions into layers along [011],
• FIG. 15A depicts RejuAgro C molecule having a planar pi-conjugated shape with amide group rotated out of the plane of the rest of the atoms by 42°.
• FIG. 15B depicts the RejuAgro C molecules in crystal are stacked along x-axis.
• the stacks are connected into layers along ab plane through H-bonds N-H. . .0.
• the layers are connected through multiple hydrogen bonds with solvate water molecules (3 mol. eq.) into a 3 -dimensional network.
• the present invention relates to a novel metabolite produced by seven Pseudomonas strains listed in this patent, such as 0617-T307, that exhibits antimicrobial activity against pathogenic microorganisms, including bacteria and fungi. From the 16S rRNA and other housekeeping gene sequences, the strain was identified as Pseudomonas soli 0617-T307 in the Pseudomonas putida group.
• the cell broth of the 7 bacterial strains, such as 0617-T307 contains a novel, potent 6-membered heterocycle natural product which is designated as RejuAgro A, along with a dimer RejuAgro B, as depicted below:
• Biocontrol agents are a way of managing pests, such as pathogens, weeds and insects, safely, sustainably, and cost-effectively. These agents are introduced into the environment to target a pest species, with the aim of reducing the pest's population or abundance in the environment.
• Bios are preparations of living microorganisms (bacteria and yeasts) that produce colonies on the hosts. These microorganisms are applied mainly to slow the pathogen buildup during the epiphytic phase (Tianna et al. (2016)).
• Biorational is a term applied to microbe-based biopesticides. These biopesticides are often made by fermenting microbial strains. Most of these products have both anti-bacterial and anti-fungal activity (Tianna et al. (2016)).
• Biopesticides is defined by The US Environmental Protection Agency (EP A) to be pesticides derived from natural materials and categorizes them as either biochemical pesticides, containing substances that control pests by nontoxic mechanisms, microbial pesticides, consisting of microorganisms that typically produce bioactive natural products (BNPs), or plant-incorporated-protectants with activity produced by plants because of added genetic materials Gwinn K.D. (2018)).
• RejuAgro A corresponds to chemical compounds having the formulas (I), (II) and (III), respectively, as illustrated below:
• a method of growing bacteria to enhance production of protective metabolites includes alternative steps.
• a step of growing Pseudomonas bacteria in liquid media in a vessel to produce a bacterial fermentate is provided.
• the ratio of media volume to vessel volume is between about 1 :2 and 1 : 10 and the vessel is shaken at a rate between about 100 and 250 RPM.
• the method includes growing Pseudomonas bacteria in liquid media in a fermenter to produce a bacterial fermentate.
• the air flow rate of the fermenter is between about 1 and 3 L/min.
• the method further includes the step of separating the liquid media from the bacteria after a period of time to produce a protective supernatant comprising the protective metabolites.
• the bacteria include a Pseudomonas strain selected from 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328.
• the growing temperature is between about 10 degrees C and 35 degrees C.
• the liquid media is LB/YME media for the production of cells.
• the liquid media is YME media for the production of RejuAgro A.
• the ratio of media volume to vessel volume is between about 1 :5 and 1 :10. In a seventh respect, the ratio of media volume to vessel volume is between about 1 :7 and 1 :9. In an eighth respect, the ratio of media volume to vessel volume is about 1 :8.
• the vessel is shaken at a rate between about 200 and 250 RPM. In a tenth respect, the vessel is shaken at a rate between about 210 and 230 RPM. In an eleventh respect, the air flow rate of the fermenter is between about 1.5 and 2.5 L/min and the concentration of dissolved oxygen is between 5 mg/L to 12 mg/L. In a twelfth respect, the growing temperature is between about 10 degrees C and 20 degrees C.
• the growing temperature is between about 15 degrees C and 17 degrees C.
• the bacteria are grown for a period of at 18 h to 7 days.
• the bacteria are grown for a period of seven days.
• the bacteria are grown for a period between one and two days.
• an agricultural composition comprising the bacterial fermentate or the protective supernatant.
• the agricultural composition is produced according to the method of the first aspect and any of the respects disclosed with regard to the first aspect.
• the agricultural composition further includes a adjuvant.
• the adjuvant is a surfactant.
• a method of controlling bacterial and fungal crop diseases includes several steps.
• a first step includes producing an agricultural composition comprising the bacterial fermentate or the protective supernatant produced by the first aspect or any of respects thereof.
• a second step includes applying said agricultural composition to crops to inhibit the growth of pathogenic microorganisms.
• the crop diseases are selected from the group consisting of fire blight, citrus cankers, olive knot, and soft rot, tomatoes and pepper.
• the pathogenic microorganism is selected from the group consisting of Mycosphaerella fijiensis, Botrytis cinereal, Erwinia amylovora (Ea), Xanthomonas axonopodis pv. citri (Xac), P ectobacterium parmentieri, P ectobacterium atrosepticum, P ectobacterium carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp.
• Psv Pseudomonas savastanoi pv. savastanoi
• Psv Pseudomonas syringae pv. tomato
• Pseudomonas syringae pv syringae Pseudomonas syringae pv. lachrymans
• Xanthomonas campestris pv. pruni Xanthomonas campestris pv. vesicatoria
• Xanthomonas arboricola pv. juglandis Ralstonia solanacearum
• the crop is selected from one or more of Bananas, apples, pears, crabapples, citrus, potatoes, pumpkins, onions, rice, African violets, plant species of Cruciferae, Solanaceae, Cucurbitaceae including carrots, potatoes, tomatoes, eggplants, leafy greens, squashes and cucurbits, peppers and green peppers, olive, stone and pome fruit plants including olives, peaches, walnuts.
• a method of controlling bacterial crop diseases includes one step.
• a step includes applying an agricultural composition comprising between about 1.0 x 10 5 and 1.0 x 10 9 cfu per mL Pseudomonas bacteria to crops to inhibit the growth of pathogenic microorganisms.
• the Pseudomonas bacteria is a Pseudomonas strain selected from 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328.
• the composition comprises between about 5.0 x 10 7 and 2.0 x 10 8 cfu per mL Pseudomonas bacteria.
• the crop disease is selected from the group consisting of Black sigatoka, Grey mould, Fire blight, Citrus canker, soft rot, Olive knot, Tomato bacterial speck, Bacterial canker or blast (stone and pome fruits), Angular Leaf Spot of Cucurbits, Bacterial Spot of Peach, Tomato bacterial spot, walnut blight, bacterial wilt, Tomato canker, Potato late blight, apple scab, bacterial leaf blight, and bacterial leaf streak.
• the pathogenic microorganism is selected from the group consisting of Mycosphaerella fijiensis, Botrytis cinereal, Erwinia amylovora (Ea), Xanthomonas axonopodis pv. citri (Xac), P ectobacterium parmentieri, P ectobacterium atrosepticum, P ectobacterium carotovorum subsp. brasiliensis, P ectobacterium carotovorum subsp. carotovorum, Dickeya dadantii, Pseudomonas savastanoi pv.
• Psv Pseudomonas syringae pv. tomato, Pseudomonas syringae pv syringae, Pseudomonas syringae pv. lachrymans, Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. vesicatoria, Xanthomonas arboricola pv. juglandis, Ralstonia solanacearum, Clavibacter michiganensis subsp.
• Psv Pseudomonas syringae pv. tomato, Pseudomonas syringae pv syringae, Pseudomonas syringae pv. lachrymans, Xanthomonas campestris pv. pruni, Xanthomonas camp
• the crop is selected from one or more of Bananas, apples, pears, crabapples, citrus, potatoes, pumpkins, onions, rice, African violets, plant species of Cruciferae, Solanaceae, Cucurbitaceae including carrots, potatoes, tomatoes, eggplants, leafy greens, squashes and cucurbits, peppers and green peppers, olive, stone and pome fruit plants including olives, peaches, walnuts.
• a method of purifying protective metabolites from Pseudomonas bacteria includes several steps.
• a first step includes producing a bacterial fermentate or protective supernatant by the method of the first aspect and the respects thereof.
• a second step includes extracting the bacterial fermentate or protective supernatant by ethyl acetate extraction.
• a third step includes producing an eluate containing protective metabolites by eluting the bacterial fermentate or protective supernatant using a mixture of hexane and ethyl acetate, such as, for example, a mixture of 50% hexane and 50% ethyl acetate, or by eluting the ethyl acetate extracts using a mixture of hexane and ethyl acetate, such as, for example, a mixture of 25% hexane and 75% ethyl acetate.
• the Pseudomonas bacteria is a Pseudomonas strain selected from 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328.
• an agricultural composition comprising protective metabolites from Pseudomonas bacteria purified by the method of the fifth aspect and respects thereof.
• a method of controlling bacterial crop diseases is provided. The method includes several steps.
• a first step includes producing an agricultural composition comprising protective metabolites from Pseudomonas bacteria purified by the method of the fifth aspect or any of the respects thereof.
• a second step includes applying said agricultural composition to crops to inhibit the growth of pathogenic microorganisms.
• the crop disease is selected from the group consisting of fire blight, citrus cankers, olive knot, soft rot, tomatoes and peppers.
• the pathogenic microorganism is selected from the group consisting of Mycosphaerella fijiensis, Botrytis cinereal, Erwinia amylovor a (Ea) (especially the streptomycin-resistant A. amylovora strains), Xanthomonas axonopodis pv. citri (Xac), P ectobacterium parmentieri, P ectobacterium atrosepticum, P ectobacterium carotovorum subsp.
• Psv Pseudomonas syringae pv. tomato, Pseudomonas syringae pv syringae, Pseudomonas syringae pv. lachrymans, Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. vesicatoria, Xanthomonas arboricola pv.
• the pathogenic E. amylovora is the streptomycin-resistant A. amylovora.
• the crop is selected from one or more of Bananas, apples, pears, crabapples, citrus, potatoes, pumpkins, onions, rice, African violets, plant species of Cruciferae, Solanaceae, Cucurbitaceae including carrots, potatoes, tomatoes, eggplants, leafy greens, squashes and cucurbits, peppers and green peppers, olive, stone and pome fruit plants including olives, peaches, walnuts.
• the pathogenic bacterium is Flavobacterium columnare #2, Flavobacterium columnare MS-FC- 4.
• the pathogenic bacterium is E. coli O157:H7.
• a crystalline compound selected from one of the following structures:
• the crystalline compound is the following structure:
• Formula (I), wherein the crystalline compound includes at least one physical property is selected from
• the crystalline compound is the following structure:
• Formula (II), wherein the crystalline compound includes at least one physical property is selected from
• the crystalline compound is the following structure: 3 H 2 O Formula (III), wherein the crystalline compound includes at least one physical property is selected from
• the bacterial strains Pseudomonas soli 0617-T307, Pseudomonas soli 0917-T305, Pseudomonas soli 0917-T306, Pseudomonas soli 0917-T307, Pseudomonas mosselii 0118- T319, Pseudomonas mosselii 0318-T327, and Pseudomonas mosselii 0418-T328 were submitted to the American Type Culture Collection (ATCC®), P.O.
• ATCC Patent Depository Box 1549, Manassas, VA 20110 USA (“ATCC Patent Depository”) on June 25, 2020 and were accorded unofficial ATCC patent numbers PTA- 126796, PTA- 126797, PTA- 126798, PTA- 126799, PTA- 126800, PTA-126801, and PTA-126802, respectively. Following viability testing, the ATCC Patent Depository accorded these deposited bacterial strains the following Accession numbers, effective June 25, 2020: Pseudomonas soli 0617-T307 (Accession No. PTA- 126796), Pseudomonas soli 0917-T305 (Accession No.
• PTA-126797 Pseudomonas soli 0917-T306 (Accession No. PTA-126798), Pseudomonas soli 0917-T307 (Accession No. PTA-126799), Pseudomonas mosselii 0118-T319 (Accession No. PTA-126800), Pseudomonas mosselii 0318-T327 (Accession No. PTA-126801), and Pseudomonas mosselii 0418-T328 (Accession No. PTA-126802).
• Example 1 Identification and characterization of strain 0617-T307.
• strain 0617-T307 is closely related to Pseudomonas species in the P. putida group within the P. fluorescens lineage.
• the “MLSA phylogeny” and “list of genomes from the type strains of Pseudomonas spp.” of (Peix et al. (2016); see Fig. 2 and Table 2 in Peix et al. (2016)) were used as the guide for taxon sampling (FIG. 1). Based on this information, the genomes were obtained from GenBank. All species in the P.
• the four genes for MLSA were extracted from the genomes sampled. Each gene was aligned individually, then all four nucleotide alignments were concatenated for phylogenetic analysis. The concatenated alignment contains 9,912 aligned nucleotide sites. The maximum likelihood inference was performed using PhyML (Guindon et al. (2003)). The bootstrap support was assessed by 1,000 replicates.
• the preparation of RejuAgro A and B can be obtained by ethyl acetate extraction of the cell broth from the fermenter fermentation, followed by the chromatographic isolation and purification. Briefly, the stock bacterium Pseudomonas sp. 0617-T307 was streaked onto LB plate (Tryptone, 10 g/L; Yeast extract, 5 g/L; NaC1, 10 g/L; agar, 15 g/L; water) and grew in a 28°C incubator for 24 h.
• single colony of 0617-T307 was inoculated into a 2.0 L flask containing 500 mL autoclaved YME media (yeast extract, 4 g/L; glucose 4 g/L and malt extract 10 g/L) and grow at 28°C for 24 h in a shaking speed of 200 rpm. Then the seed media was inoculated into a 20 L NBS fermenter containing 12 L autoclaved YME media. The fermentation was proceeded at 16°C for 1-7 days. The agitation speed and the airflow rate were 200 rpm and 2 L/min, respectively.
• the concentrated sample was dissolved in ethyl acetate and mixed with silica gel, which was packed as an injection column ( ⁇ 33.0 X 20 cm) and mounted atop a silica gel Universal Column (4.8 x 18.5 cm) on a flash chromatography system (Yamazen AI-580) equipped with an UV detector.
• the sample was eluted by the 280 mL of each of the following solvents in order with an increasing polarity, 100% hexane, 75% hexane/25% ethyl acetate, 50% hexane/50% ethyl acetate, 25% hexane/75% ethyl acetate, 100% ethyl acetate, 50% ethyl acetate/50% acetone, 100% acetone, and 100% methanol.
• the sample was eluted at a flow rate of 20 mL/min.
• the elute was monitored at UV 254 nm, and fractions were collected by a time mode at 20 mL/tubes. Totally, there are 114 fractions or tubes generated from the flash chromatography.
• the generated fractions were applied for the subsequent plate assays.
• One mL of each fraction was picked up into a 1.5 mL test tube and vacuum dried by an Eppendorf vacuum concentrator.
• the dried sample was dissolved in 50 ⁇ L DMSO, of which 2 ⁇ L was used in the plate assay.
• Erwinia amylovora 273 was streaked onto LB plate to grow at 28°C incubator and single colony obtained after 24 h was inoculated into 5 mL LB media to allow an overnight growth at 28°C shaker at 200 rpm.
• the bacteria were diluted 1 : 100 in sterile water, of which 225 ⁇ L was plated onto 50% LB plate (Tryptone, 5.0 g/L; Yeast extract, 2.5 g/L; NaC1, 5.0 g/L, Agar, 15 g/L). After dried in the biosafety cabinet for 10 mins, the DMSO solution of each fraction was then distributed to its pre-labeled section of the petri dish and allowed to dry for another 10 min. Along with the assay, DMSO and Kasugamycin were used as negative and positive controls, respectively. The plates were then incubated at 28°C incubator and the inhibitory zone was checked one day later.
• Preparative HPLC Preparative HPLC purification of the fraction 3840 and 5054 lead to the discovery of 15 mg yellow colored compound RejuAgro A (Rtl7.5) and 103.3 mg darkgreen colored compound RejuAgro B, respectively.
• RejuAgro A can be dissolved in methanol and chloroform.
• RejuAgro B (Rtl0.5) does not dissolve well in methanol or chloroform, but it can be dissolved very well in dimethyl sulfoxide (DMSO) in a dark-green color.
• DMSO dimethyl sulfoxide
• the structures of the two compounds have been investigated by High resolution mass spectrometry (HR-MS), infrared (IR), Ultraviolet (UV), ID and 2D Nuclear magnetic resonance (NMR) as well as X-ray crystal structure analysis.
• the compound RejuAgro A contains 7 types of carbon groups (three types carbonyl, two types tertiary carbons, two types of methyl carbons), but the RejuAgro B lack one type of methyl group, as shown below:
• the crystal of RejuAgro B was identified as triclinic.
• the structure of RejuAgro B contains two symmetrically independent molecules. Each molecule has a twisted structure - with dihedral angle between mean planes of the linked heterocycles of 70. 3 and 80.6°.
• the molecule formula of RejuAgro A is C7H7NO3S, and the Molecular Weight: 185.2004. This is in consistent to the observed molecule species of [M+H] at mlz 186.2177 (theoretical 186.2083) in HR-MS data.
• the molecule formula of RejuAgro B is C 12 H 8 N 2 O 6 S, and the Molecular Weight: 276.2017. This is in consistent to the observed molecule species of [M-H] at mlz 275.0278 (theoretical 275.1960) in HR-MS data.
• CCDC structural database search as of August 4, 2020 indicates there are no crystal structures of RejuAgro A, RejuAgro B and RejuAgro C.
• the MIC values of RejuAgro A and RejuAgro B were determined for five types of bacteria: wild type gram-negative plant pathogenetic bacteria, streptomycin-resistant E. amylovora, fish disease causing bacteria, gram-positive and gram-negative human pathogenetic bacteria, and the producer of RejuAgro A (strain 0617-T307).
• the antimicrobial assay was performed according to the CLSI Antimicrobial Susceptibility Testing (AST) Standards. Briefly, the stock solution of each of the tested bacteria was streaked onto LB (Luria-Bertani) plate (tryptone, 10 g/L; yeast extract, 5 g/L; sodium salt, 10 g/L; Agar, 15 g/L).
• NA Nutrient broth+Agar
• yeast extract 3 g/L
• polypeptone 5 g/L
• sucrose 10 g/L
• SHIEH tryptone, 5 g/L; yeast extract, 0.5 g/L; sodium acetate, 0.01 g/L; BaC1 2 (H 2 O)2, 0.01 g/L; K 2 HPO 4 , 0.1 g/L; KH 2 PO 4 , 0.05 g/L; MgSO 4 -7H 2 O, 0.3 g/L; CaC1 2 -2H 2 O, 0.0067 g/L; FeSO 4 -7H 2 O, 0.001 g/L; NaHCO 3 , 0.05 g/L; agar, 10 g/L) and TYES (tryptone 4 g/L; yeast extract 0.4 g/L; MgSO4, 0.5 g/L; CaC1 2 0.5 g/L; pH to 7.2, agar, 15 g/L) were used for Flavobacterium columnare strains MS-FC-4 and #2, respectively.
• the compound RejuAgro A and RejuAgro B and streptomycin was diluted and 4 ⁇ L of each concentration was added into each well to make a final concentration of 40 ⁇ g/mL, 20 ⁇ g/mL, 10 ⁇ g/mL, 5 ⁇ g/mL, 2.5 ⁇ g/mL, 1.25 ⁇ g/mL, 0.625 ⁇ g/mL, 0.3125 ⁇ g/mL, 0.15625 ⁇ g/mL, 0.078 ⁇ g/mL.
• the vehicle water (for streptomycin) or DMSO (for RejuAgro A and RejuAgro B) were used as control.
• RejuAgro A rather than RejuAgro B is the most active metabolite of strain 0617-T307.
• MRS A gram-positive MRS A
• gram-negative E coli O157:H7 an important food- and waterborne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS) in humans
• HUS hemolytic-uremic syndrome
• the antimicrobial activity of RejuAgro A is equivalent to streptomycin regarding the strain Erwinia amylovora 1189, Xanthomonas axonopodis pv. citri. Pseudomonas savastanoi pv. savastanoi, Pectobacterium parmentieri UPP163 936, Pectobacterium carotovorum subsp brasillensis 944, Pectobacterium carotovorum subsp. carotovorum wpp14 945, Dickeya dadantii 3937, which showed a MIC value of 5 ⁇ g/mL for E.
• the Xanthomonas bacteria are very sensitive to streptomycin, with a MIC value of 0.16 ⁇ g/mL, this is lower than the MIC value 5 ⁇ g/mL for RejuAgro A.
• the MIC value of RejuAgro A for Pseudomonas savastanoi pv. savastanoi is 40 ⁇ g/mL.
• the MIC value of RejuAgro A for Xanthomonas arboricola pv. Juglandis 219 is 6.25 ⁇ g/mL.
• the MIC value of RejuAgro A for Ralstonia solanacearum K60 and Pss4 is 3.13 and 6.25 ⁇ g/mL respectively.
• the MIC value of RejuAgro A for Clavibacter michiganensis subsp. michiganensis NCPPB382, Cmm 0317, Cmm 0690 is 6.25, 1.56, and 12.5 ⁇ g/mL respectively.
• the MIC value of RejuAgro A for Ralstonia solanacearum K60 and Pss4 is 40 ⁇ g/mL.
• RejuAgro A was also tested against other E. amylovora strains that include one virulence E. amylovora and three streptomycin-resistant E. amylovora strains. RejuAgro A showed the same efficacy as streptomycin against E. amylovora 110 (MIC value 5 ⁇ g/mL). However, RejuAgro A is more efficient against E. amylovora 1189 than streptomycin. The MIC values of RejuAgro A and streptomycin to amylovora 1189 are 5 ⁇ g/mL and 10 ⁇ g/mL respectively. In addition, RejuAgro A is much more efficient against streptomycin- resistant E.
• RejuAgro A has MIC values 5 ⁇ g/mL for Flavobacterium columnare strains MS-FC-4 and #2 (cause columnaris disease in wild and cultured fish), which is higher than the MIC values of streptomycin (0.31 ⁇ g/mL and 1.25 ⁇ g/mL for strain #2 and MS-FC-4, respectively).
• RejuAgro A was tested along with streptomycin against tomato pathogens (P. syringae. pv. tomato PT30, P. syringae. pv syringae 7046, P. syringae. pv. lachrymans 1188- 1) and other citrus canker pathogens (Xanthomonas campestris pv. pruni. Xanthomonas campe stris pv. vesicatoria XV- 16).
• the MIC values of RejuAgro A against P. syringae are 40 ⁇ g/mL, while the streptomycin’s MIC values are 2.5-5 ⁇ g/mL.
• RejuAgro A MIC values are 2.5 ⁇ g/mL or 40 pg /mL, which is smaller than the MIC values of streptomycin, which are 20 ⁇ g/mL or larger than 40 ⁇ g/mL.
• RejuAgro A showed efficacy against all of tested pathogenic fungi (Table 1). RejuAgro A was tested against Phytophthora infestans, Venturia inaequalis and Mycosphaerella fijiensis. RejuAgro A showed 100% inhibition against P. infestans. and V. inaequalis. at 40 ⁇ g/mL, 80 ⁇ g/mL and 600 ⁇ g/mL (Table 1).
• Pectohacterium atrosepticum 942 (Produce soft rot in 20 20 multiple crops)
• Dickeya dadantii 3937 (Produce soft rot in multiple 40 20 crops)
• Flavobacterium columnare #2 (Fish columnaris disease) 0.31
• Pseudomonas soli 0617-T307 (RejuAgro A producer) >40 >40
• Pseudomonas syringae pv syringae 7046 (Bacterial 20 2.5 canker or blast (stone and pome fruits))
• NCPPB382 Tomato canker
• MIC ⁇ g/mL
• Mycosphaerella fijiensis 1 OCR-25 (black sigatoka of 600 NA Banana)
• a Eal 10 is the virulent strain used for the field trials in Michigan state
• Both CA11 and DM1 are streptomycin-resistant strains containing Tn5393 with the transposon on the acquired plasmid pEa34 and can grow in 100 pg/mL streptomycin containing media
• c Ea898 is a spontaneous streptomycin-resistant strain with a mutation in the chromosomal rpsL gene and can grow in the media containing 2000 pg/mL streptomycin
• d Copper resistant bacteria e Positive control Copper solution at 1000 pg/mL inhibits 61% of the growth.
• Example 4 Production and stability of RejuAgro A from strain 0617-T307 in a shakingflask fermentation.
• the fermentation of 0617-T307 for the production and preparation of RejuAgro A can be obtained by two approaches, the shaking-flask fermentation and fermenter fermentation.
• the fermenter fermentation was described in Example 2.
• the flask fermentation can be obtained as below.
• the stock bacterium Pseudomonas sp. 0617- T307 was streaked onto YME agar plate (yeast extract, 4 g/L; glucose 4 g/L and malt extract 10 g/L; agar, 15 g/L) and grew at 28°C incubator for 24 h.
• the seed media were made by growing single colony of 0617-T307 in a 250 mL flask containing 50 mL sterile YME liquid media at 16°C and 220 rpm for 24 h. Then the seed media were inoculated into 4 L flask containing 0.5 L sterile YME media at 4% ratio (v/v). Following the inoculation (2%, v/v) into eight 4-L flasks each containing 2 L YME media, the bacteria were grown at 16°C in a shaker at 200-220 rpm for 1-7 days.
• the RejuAgro A concentration was obtained by LC-MS analysis according to the developed standard curves.
• Two methods were used for the preparation of samples for LC- MS analysis.
• One approach is to extract the cell broth by ethyl acetate (1 mL: 1 mL, vortex for 1 min), and to obtain the ethyl acetate extracts by centrifugation and vacuum drying of the ethyl acetate layer.
• the dried ethyl acetate extracts were dissolved in 40 ⁇ L methanol and 2 ⁇ L methanol solution was used for LC-MS analysis.
• the other method is to obtain the supernatant by centrifuging the cell broth, then mix the supernatant with equal volume of methanol to make the 50% methanol solution, of which 10 ⁇ L solution was injected into LC- MS.
• the second method was used because RejuAgro A production is an extracellular secretion process, which was demonstrated by the observation of the major amount of RejuAgro A in the supernatants rather than inside of the cells (FIG. 3, panel A).
• Table 2 Summary of RejuAgro A producing capabilities for the selected Pseudomonas strains that were cultured in medium YME at 16 °C, 18 hours, 220 rpm.
• Example 5 Antimicrobial activity of cell broth of strain 0617-T307 against 0617-T307 and E. amylovora.
• DMSO 0 a The concentration of the bacterial cells was not determined.
• the bioactive fractions (crude extracts, 100 ⁇ g/mL; flash -RejuAgro A, 20 ⁇ g/mL; HPLC-RejuAgro A, 10 ⁇ g/mL) were tested against strains 0617-T307, Ea and Xac. It showed that the bioactive fractions were not able to inhibit the growth of strain 0617-T307, which demonstrates RejuAgro A can be mixed with 0617-T307 cells for the preparation of biocontrol agents.
• the bioactive fractions containing RejuAgro A showed inhibitory effects against Ea and Xac, especially the flash-RejuAgro A and HPLC-RejuAgro A, almost abolish the growth of Ea and Xac under the tested conditions. This demonstrates that the RejuAgro A solution can be used for the biocontrol of fire blight and citrus cankers at 10-20 ⁇ g/mL.
• Example 6 Identification and characterization of the bioactive metabolites from ethyl acetate extracts of the acidified supernatant (pH 2.0) of strain 0617-T307.
• the seed media was inoculated into eight 4-L flasks each containing 2 L autoclaved YME media.
• the fermentation was proceeded at 16°C in a shaker with a shaking speed of 150 rpm for 7 days.
• the supernatants were obtained by centrifuging bacterial culture at 4000 rpm for 15 min.
• the pH of the supernatant was then adjusted to 2.0 by adding 6N HC1.
• the acidified supernatants were then subjected to the ethyl acetate extraction. This resulted 3.0 g crude extract from 14 L culture of strain 0617-T307.
• the sample was eluted by the 280 mL of each of the following solvents in order with an increasing polarity, 100% hexane, 75% hexane/25% ethyl acetate, 50% hexane/50% ethyl acetate, 25% hexane/75% ethyl acetate, 100% ethyl acetate, 50% ethyl acetate 50% acetone, 100% acetone, and 100% methanol.
• the sample was eluted at a flow rate of 20 mL/min.
• the elute was monitored at UV 254 nm, and fractions were collected by a time mode at 20 mL/tubes. Totally, there are 114 fractions or tubes generated from the flash chromatography.
• the generated fractions were applied for the subsequent plate assays.
• One mL of each fractions was picked up into a 1.5 mL test tube and vacuum dried by an Eppendorf vacuum concentrator.
• the dried sample was dissolved in 50 ⁇ L DMSO, of which 2 ⁇ L was used in the plate assay.
• Erwinia amylovora 273 was inoculated into 50% LB (Tryptone, 5.0 g/L; Yeast extract, 2.5 g/L; NaC1, 5.0 g/L) plate and single colony will be inoculated into 5 mL LB media.
• the bacteria will be diluted 1 : 100 in sterile water, of which 225 ⁇ L was plated onto 50% LB plate.
• the DMSO solution of each fraction was then distributed to its pre-labeled section of the petri dish and allowed to dry for another 10 min.
• DMSO and Kasugamycin were used as negative and positive controls, respectively. The plates were then incubated at 28°C incubator and the inhibitory zone will be checked after one day.
• T307_5058_Rt22.9 and T307_5058_Rt25.0 are tryptophan derived natural products, and their structures were reported in Scifinder database but not the biological activities (Loots et al. (2015)).
• 0617_T307_7882_Rtl8 was predicted to be a derivative of difuryl that has been reported previously (Osipov et al. (1978)). These natural products are depicted below:
• Example 7 Identification of other metabolites from strain 0617-T307 using LCMSMS and spectral library search.
• 0617-T307_5058_Rt25.0 Some of the known compounds were identified from the crude extract, which includes the Indole-3 -carboxylic acid, a plant growth-promoting factor, and xantholysin A. It is reported that 1) the broad antifungal activity of P. putida BW11M1 is mainly dependent on Xantholysin production; 2) Xantholysin is required for swarming and contributes to biofilm formation (Li et al. (2013)). Indeed, the higher concentration of xantholysin A was observed by culturing 0617-T307, 0418-T328 and 0318-T327 at 28°C.
• Xantholysin A is another contribution metabolite for the antimicrobial activity of the biocontrol bacteria 0617-T307 and its closely related species 0318-T3027 and 0418-T328.
• Example 8 Greenhouse and field infection assays for strain 0617-T307 and some of its closely related species that produce RejuAgro A.
• the percentage of diseased flower clusters for water control, streptomycin, 0617-T307, 0118- T319, 0318-T327, and 0418-T328 are 32.9%, 13.3%, 16.8%, 18.5%, 16.7%, and 11.8% respectively.
• streptomycin the biocontrol bacteria that produce RejuAgro A have similar or better efficacy on controlling fire blight in the apple orchards.
• the fungus Venturia inaqualis that causes apple scab was maintained on PDA agar in the dark at room temperature ( ⁇ 24 °C).
• a mixture of conidia and mycelia suspension (in 0.01 M PBS) was harvested from PDA (Potato dextrose agar).
• Ten ⁇ L the conidia and mycelia suspension were dropped onto biocontrol bacteria, RejuAgro A, or RejuAgro A amended plates.
• the control was PDA plates without the addition of the biocontrol bacteria or RejuAgro A or B.
• the dishes were incubated at room temperature in the dark and the diameter of each colony of V. inaqualis was checked 7 days later.
• the seed of strain 0617-T307 was prepared by growing bacterial cells in YME media at 28°C and 180 rpm for 24 h. Then 4% (2 mL to 50 mL) was inoculated in to 250 mL flask containing 50 mL M8 (IAA medium) or M9 (CN medium) or M7 (PRN medium) or M6 (DAPG medium) medium and grew at 28°C and 180 rpm for 48 h. A volume of 0.5 mL of the cell broth was collected at 12 h and 24 h, and was stored in -20°C freezer.
• the cell broth was thawed and 5 ⁇ L was applied onto the sample wells on a PDA (potato dextrose agar) plate with equal radius distance to the central that is inoculated with the Botrytis cinerea (FIG. 11). It showed that the cell broth has antifungal activity against Botrytis cinerea CAI 7 on PDA (Potato Dextrose Agar) plates.
• 0617-T307 identified two antimicrobial compounds (Rt22.9 and Rt25.0) from flash fraction T5058 and one antimicrobial compound (Rtl8.9) from flash fraction T7882. They were tested for their antimicrobial activities on bacterial strains listed in Table 8. Two ⁇ L of DMSO, Rtl8.9, Rt22.9 or Rt25.0 were spotted on agar plates respectively grown with different bacterial strains and the inhibitory zone was further examined (Table 8).
• Xanthomonas axonopodis pv. No No No Yes NA cz/zv-Miami XC2002-00010 (Citrus canker) inhibitory zone was examined between 2 to 5 days after spotted with DMSO, Rtl8.9, Rt22.9 or Rt25.0.
• Agar medium plate used for growing the bacteria was either LB Medium (10.0 g/L Tryptone, 5.0 g/L Yeast extract, 10.0 g/L Sodium salt, 15.0 g/L Agar and tap water to final volume 1.0 L) or NA Medium (3.0 g/L Beef extract, 1.0 g/L Yeast extract, 5.0 g/L Polypeptone, 10.0 g/L Sucrose and 15 g/L Agar and tap water to final volume of 1.0 L) Table 14.2 Medium composition of LB and NA agar plates
• the paper discs loaded with 50 ⁇ L loading volume of the HPLC purified aqueous RejuAgro A at the concentrations of 5.5 ⁇ g/mL, 11.1 ⁇ g/mL, 22.1 ⁇ g/mL, 33.2 ⁇ g/mL, 55.4 ⁇ g/mL, 110.7 ⁇ g/mL respectively, were put on the agar plates and the inhibition zone was measured 44 hours after placing the paper discs on the agar plates. An inhibition was observed at all concentrations of the paper discs soaked with RejuAgro A suspension (Table 9).
• the paper discs loaded with 50 ⁇ L loading volume of the HPLC purified aqueous RejuAgro A at the concentrations of 5.5 ⁇ g/mL, 11.1 ⁇ g/mL, 22.1 ⁇ g/mL, 33.2 ⁇ g/mL, 55.4 ⁇ g/mL, 110.7 ⁇ g/mL respectively, were put on the agar plates and the inhibition zone was measured 44 hours after placing the paper discs on the agar plates. An inhibition was observed at the concentrations of 55.37 ⁇ g/mL and 110.74 ⁇ g/mL of RejuAgro A (Table 10).
• Example 18 Media culture compositions used in the Examples.
• Table 11 includes exemplary media compositions used in the Examples.
• Example 19 Bacterial strains, natural products, and references cited to same.
• Example 20 Crystal structure information of RejuAgro A, RejuAgro B and RejuAgro C.
• the RejuAgro A molecule has a planar structure - with S- Me group rotated only by 8.7° relative to the heterocycle. There is a notable break of 71- conjugation in the molecule at C4-C5 bond (1.531 A) - apparently, because of some orbital reasons.
• the Me-group connected to sp 2 carbon atom is rotationally disordered over 2 positions.
• the RejuAgro A molecule in crystal form centrosymmetric H-bonded dimers through N-H. . .0 interactions. Further, these dimers form 2-dimensional layers along [-3 0 1] plane via weaker C-H. . .0 interactions.
• the Anisotropic displacement factor exponent takes the form:
• N1H1 O1 1 0.845(18) 2.032(19) 2.8768(15) 178.8(17)
• H6A 0.485(15) H6B 0.515(15) H6C 0.515(15)
• the RejuAgro B crystal contains two symmetrically independent RejuAgro B molecules. Each molecule has a twisted structure - with dihedral angle between mean planes of the linked heterocycles of 70.3 and 80.6°. There is a notable break of ⁇ -conjugation in each heterocycle at C(sp 2 )-C(sp 2 ) bond between two adjacent carbonyl groups (the bond lengths are in the 1.534-1.539 A range) - apparently, because of some orbital reasons.
• the RejuAgro B molecules in crystal form centrosymmetric H-bonded dimers through N-H. . .0 interactions. These dimers are linked in stacks along x direction by other N-H...O interactions, Finally, the stacks are linked by third kind of N-H...O interactions into layers along [011],
• Tables 22-29 [0160] Table 22. Crystal data and structure refinement for RejuAgro B.
• the Anisotropic displacement factor exponent takes the form: - 2 ⁇ 2 [h 2 a*U 11 i+2hka*b*U12+...].
• N2AH2AO3 4 0.88 2.08 2.892(2) 153.8 1 -X,2-Y -Z; 2 1-X,1-Y, 1-Z; 3 -X,l-Y,l-Z; 4 1+X ,-1+Y, 1+Z
• the RejuAgro C molecule has a planar pi-conjugated shape with amide group rotated out of the plane of the rest of the atoms by 42°.
• the RejuAgro C molecules in crystal are stacked along x-axis.
• the stacks are connected into layers along ab plane through H-bonds N-H. . .0.
• the layers are connected through multiple hydrogen bonds with solvate water molecules (3 mol. eq.) into a 3-dimensional network.
• solvate water molecules (3 mol. eq.) into a 3-dimensional network.
• Table 30 Crystal data and structure refinement for RejuAgro C.
• the Anisotropic displacement factor exponent takes the form: - ⁇ 2 [h 2 a* 2 U 11 +2hka*b*U 12 +...].
• N2 45.3(12) 36.3(11) 38.6(11) 25.3(9) 23.0(9) 22.9(9) C1 27.0(10) 30.6(11) 36.2(11) 19.6(9) 18.2(9) 13.3(9)
• Lysocin e is a new antibiotic that targets menaquinone in the bacterial membrane. Nat Chem Biol 11 : 127-133.
• the antimicrobial compound xantholysin defines a new group of Pseudomonas cyclic lipopeptides. PLoS One 8:e62946.

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