Pseudomonas strains and their metabolites for controlling plant diseases

Abstract

To provide new biopesticides derived from metabolites. The present invention relates to novel bacterial strains 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328, which are capable of inhibiting the growth of a variety of microbial species for a variety of crops, as well as methods of using the cell broths and novel metabolites produced from the bacterial strains. The methods involve the use of novel, potent antibacterial metabolites produced from the strains corresponding to compounds having formulas (I), (II), and (III). TIFF2026035671000063.tif71162

Description

[Technical Field]

[0001] The present invention is in the field of biopesticides. In particular, the present invention relates to seven novel strains of Pseudomonas spp., 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328, which are capable of inhibiting the growth of various microbial species, their cell broths, and novel metabolites produced by the bacterial strains. Pseudomonas strains 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328 have been deposited with the American Type Culture Collection (ATCC) and have ATCC accession numbers PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802, respectively. [Background technology]

[0002] Plant diseases caused by pathogenic microorganisms are exponentially increasing and costly. Plant pathogenic organisms include fungi, bacteria, mycoplasmas, viruses, viroids, nematodes, or parasitic flowering plants. Currently, there are 14 common plant diseases caused by bacteria, including bacterial spot, bacterial blight, and bacterial wilt. Fire blight (Erwinia amylovora), citrus canker [Xanthomonas axonopodis pv. citri (Xac)], bacterial leaf spot (BLS) [Xanthomonas campestris pv. vesicatoria (XV-16)], olive canker [Pseudomonas savastanoi pv. Savastanoi (Psv)], soft rot (Dickeya dadantii, Pectobacterium parmentieri, Pectobacterium atrosepticum) Atrosepticum and Pectobacterium carotovorum are devastating plant diseases. Nationally, fire blight control costs have been estimated at over $100 million (Norelli et al., 2003). For citrus canker, Florida alone is approaching $1 billion in eradication program costs from 1995 to 2005, as well as compensation to commercial growers and homeowners of destroyed residential citrus trees.

[0003] Fire blight, a devastating disease of pome fruit caused by infection with the gram-negative bacterium Erwinia amylovora, affects pears and apples in many parts of the world, including Europe, Germany, Austria, and Switzerland (Chen et al., 2009). While fire blight rarely destroys entire orchards, the disease and its control still result in significant economic losses. The Pacific Northwest and northern California have experienced small-scale outbreaks annually since 1991, with at least some areas experiencing large-scale outbreaks every 3–4 years. Even minor disease outbreaks can be costly, as pruning to remove infected plant parts can lead to poor tree shape and reduced future productivity. For example, a 10% incidence of rootstock blight in a 4-year-old apple orchard can result in losses of up to $3,500 per acre (Norelli et al., 2003).

[0004] Microbial natural products have provided a wealth of biological compounds for use as pesticides (Gwinn, 2018). However, current methods for preventing bacterial plant diseases have limited effectiveness. The antibiotics streptomycin sulfate (FireWall, AgroSource, Inc.) and oxytetracycline hydrochloride (FireLine, AgroSource, Inc.) have primarily been used to combat E. amylovora when the risk of infection is high. Because these compounds are also used in human and animal health management, the use of these same antibiotics in crop agriculture is controversial (Stockwell, 2012). Concerns about antibiotic resistance have limited the use of streptomycin sulfate (Vrancken et al., 2013). Kasugamycin is also an antibiotic being investigated for fire blight. One drawback is that frequent application of kasugamycin can lead to plant-destructive phytotoxicity (Adaskaveg et al., 2010). Another drawback is the high cost of kasugamycin compared to other antibiotics, so it must be used in combination with other antibiotics.

[0005] In recent decades, numerous non-antibiotic products have been developed, registered with the Environmental Protection Agency (EPA), approved by the National Organic Program (NOP), and sold to orchardists for fire blight control (Tianna et al., 2018). Historically, two Bacillus subtilis-based products have been registered for fire blight control in Europe: Serenade®, based on the QST 713 strain, and Biopro®, based on the BD 170 strain (Broggini et al., 2005). Biologics based on spore-forming Bacillus species are advantageous for biological control due to their long-term viability (Haas et al., 2005). Moderate success of two Bacillus-based biological products has been demonstrated in numerous field trials in the United States and Germany (Aldwinckle et al., 2002; Kunz et al., 2011; Laux et al., 2003). This suggests that Bacillus species hold promise in controlling E. amylovora flower infections. However, Bacillus species are only effective at low infection pressures; they are ineffective at moderate and high infection pressures. Results have been inconsistent for both biological products, with disease suppression rates ranging from 71% to 0% (Broggini et al., 2005).

[0006] Future bioprotection products must be able to effectively compete with E. amylovora and colonize the same microenvironment on different organs of the target plant. Protective bacteria produce secondary metabolites that affect the pathogen, compete for food and space, and prevent E. amylovora from causing disease in plant tissue. In this regard, Pseudomonas bacteria fit the above-mentioned bioprotective factors (Haas et al., 2005). Analysis of the species composition of colonizing bacteria on various plants showed that Pseudomonas fluorescent bacteria are widespread.

[0007] In France, Pseudomonas species were found to be major components of populations inhabiting both healthy and diseased apple, pear, and hawthorn trees, many of which exhibited the ability to limit the growth of E. amylovora in vitro (Paulin et al., 1978), but little information on the active metabolites has been reported.

[0008] In California, Thomson et al. (1976) screened three fluorescent Pseudomonas species for their effectiveness in protecting pear blossoms (Thomson et al., 1976). In the mid-1980s, P. fluorescens strain A506, isolated from California pear tree leaves, demonstrated distinctive activity in limiting the growth of E. amylovora and the ability to protect apples and pears from fire blight (Lindow et al., 1996). A P. fluorescens-containing product, BlightBan® A506, was developed and has been commercially available since 1996. Extensive experiments conducted in California, Oregon, and Washington have demonstrated the usefulness of this formulation in various apple and pear protection programs (Johnson, 2000).

[0009] In the UK, two isolates of Pseudomonas fluorescens were used to protect hawthorn flowers and shoots (Wilson et al., 1992).

[0010] In Italy and New Zealand, the suitability of two strains of the genus Pseudomonas, designated BO 3371 and BO G19, was investigated (Galasso et al., 2002). Under greenhouse conditions, they are highly effective in protecting apple and pear blossoms and shoots. For example, the relative protective effect of strain BO3371 on pear shoots reaches 87% (Galasso et al., 2002). However, the results obtained are not always consistent, which may be related to the susceptibility of flowers linked to the length of the period from flowering to the end of flowering.

[0011] In New Zealand, the fluorescent Pseudomonas spp. IPV-BO G19 strain protected 79% of apple blossoms under field conditions. In another experimental orchard, fluorescent Pseudomonas spp. IPV-BO G19 and IPV-BO 3371 suppressed fire blight incidence by 78% and 58%, respectively, when applied 24 hours before inoculation of 'Braeburn' apple blossoms with E. amylovora (Biondi et al., 2006).

[0012] In Spain, the strain EPS62e P. fluorescens significantly suppressed fire blight in field assays on apple blossoms, pear fruit, and pear blossoms. The improved adaptation and efficacy of P. fluorescens EPS62e against fire blight was achieved through a strategy combining nutritional enrichment and osmoadaptation. Field treatments of physiologically modified P. fluorescens EPS62e on pear blossoms produced efficacy as high as 90%, although results varied between trials (Cabrefiga et al., 2011; Mikicinski et al., 2020).

[0013] In Poland, 47 colonies of bacteria capable of reducing the effects of fire blight on pear fruit were isolated from apple phyllosphere and soil (Mikicinski et al., (2008)).

[0014] The metabolites produced by Gram-negative Pseudomonas species have been comprehensively reviewed (Masschelein et al., 2017). Pseudomonas metabolites are classified into phenolic compounds, phenazines, lipopeptides, and others. The functions of Pseudomonas species and their metabolites include (Alsohim et al., 2014): 1) producing hormones or inducing systemic resistance; 2) many naturally occurring strains also significantly improve plant growth (plant growth regulators, IAA, viscosin); 3) producing siderophores and surfactants such as viscosin and viscosinamide, and antagonizing antibacterial compounds such as hydrogen cyanide, phenazine, pyrrolnitrin, or 2,4-diacylhoroglucinol (DAPG). In our research, strains have been identified and fermentation products and novel metabolites have been produced from the bacteria, and in particular, RejuAgro A and RejuAgro B show high efficacy against multiple pathogenic microorganisms, including previously unreported bacteria and fungi. [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Norelli et al. (2003) [Non-patent document 2] Chen et al. (2009) [Non-patent document 3] Gwinn, (2018) [Non-patent document 4] Stockwell, (2012) [Non-patent document 5] Vrancken et al. (2013) [Non-patent document 6] Adaskaveg et al. (2010) [Non-Patent Document 7] Tianna et al. (2018) [Non-patent document 8] Broggini et al. (2005) [Non-Patent Document 9] Haas et al. (2005) [Non-Patent Document 10] Aldwinckle et al. (2002) [Non-Patent Document 11] Kunz et al. (2011) [Non-Patent Document 12] Laux et al. (2003) [Non-Patent Document 13] Broggini et al. (2005) [Non-Patent Document 14] Paulin et al. (1978) [Non-Patent Document 15] Thomson et al. (1976) [Non-Patent Document 16] Lindow et al. (1996) [Non-Patent Document 17] Johnson, (2000) [Non-Patent Document 18] Wilson et al. (1992) [Non-Patent Document 19] Galasso et al. (2002) [Non-Patent Document 20] Biondi et al. (2006) [Non-Patent Document 21] Cabrefiga et al. (2011) [Non-Patent Document 22] Mikicinski et al. (2020) [Non-Patent Document 23] Mikicinski et al. (2008) [Non-Patent Document 24] Masschelein et al. (2017) [Non-Patent Document 25] Alsohim et al. (2014) Summary of the Invention [Problem to be solved by the invention]

[0016] There is a need for new biopesticides derived from novel strains, cell broths, and novel metabolites produced from such strains that can inhibit the growth of a variety of pathogenic bacteria that cause crop diseases. [Means for solving the problem]

[0017] (Brief Summary of the Invention) In a first aspect, a method for growing bacteria to promote the production of protective metabolites is provided. The method includes alternative steps. In one method, a step of growing Pseudomonas bacteria in a liquid medium in a vessel to produce a bacterial fermentate is provided. The ratio of medium volume to vessel volume is between about 1:2 and 1:10, and the vessel is shaken at a speed between about 100 and 250 RPM. In an alternative step, the method includes growing Pseudomonas bacteria in a liquid medium 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 and 12 mg / L.

[0018] In a second aspect, an agricultural composition is provided comprising the bacterial fermentate or protected supernatant. The agricultural composition is produced according to the method of the first aspect and any of the aspects disclosed with respect to the first aspect. In the first aspect, the agricultural composition further comprises an adjuvant. In this aspect, the adjuvant is a surfactant.

[0019] In a third aspect, there is provided a method for controlling bacterial crop diseases. The method comprises several steps. The first step comprises producing an agricultural composition comprising the bacterial fermentate or protective supernatant produced according to the first aspect or any point thereof. The second step comprises applying the agricultural composition to a crop to inhibit the growth of pathogenic microorganisms.

[0020] In a fourth aspect, a method for controlling bacterial crop diseases is provided, the method comprising the step of: infecting a plant with a bacterial extract of about 1.0 x 10 microbial cells to inhibit the growth of pathogenic microorganisms; 5 ~1.0×10 9 The method includes applying to a crop an agricultural composition containing Pseudomonas bacteria between about cfu / mL.

[0021] In a fifth aspect, a method for purifying protected metabolites from Pseudomonas bacteria is provided. The method comprises several steps. The first step involves producing a bacterial fermentation product or a protected supernatant according to the method of the first aspect and its equivalents. The second step involves extracting the bacterial fermentation product or the protected supernatant with a solvent mixture having similar polarity or properties. The third step involves eluting the bacterial fermentation product or the protected supernatant with a mixture of hexane and ethyl acetate, or eluting the bacterial fermentation product or the protected supernatant with a mixture of hexane and ethyl acetate, to produce an eluate containing the protected metabolites.

[0022] In a sixth aspect, an agricultural composition comprising a protective metabolite from a Pseudomonas bacterium is purified by the method of the fifth aspect and points thereof.

[0023] In a seventh aspect, a method for controlling bacterial crop diseases is provided. The method comprises several steps. The first step involves producing an agricultural composition containing a protective metabolite from Pseudomonas bacteria purified by the method of the fifth aspect or any point thereof. The second step involves applying the agricultural composition, where the formulation of the protective supernatant or its metabolites can be a solution (SL), a soluble powder (SP), a soluble granule (SG), or an encapsulated formulation. Furthermore, agricultural compositions of bacterial ferment and cell formulations can be suspension concentrates (SC), wettable powders (WP), and water-dispersible granules (WG).

[0024] In an eighth embodiment, the crystalline compound is selected from one of the following structures:

[0025] [ka] [Brief explanation of the drawings]

[0026] [Figure 1]Figure 1 shows an exemplary plot of a maximum likelihood phylogenetic tree of representative Pseudomonas lineages based on a concatenated alignment of 16S rDNA, gyrB, rpoB, and rpoD. Bootstrap support values ​​are displayed under the four internal branches that received less than 100% support. No display represents 100% support. [Figure 2] FIG. 1 shows an example of assay-based isolation of an ethyl acetate extract of strain 0617-T307. [Figure 3A] FIG. 1 shows exemplary culture plots for the amount of RejuAgro A in shake flask fermentation, showing the distribution of RejuAgro A in the cell broth, supernatant, and cells (Panel A) and the time course of RejuAgro A production by cell fermentation (Panel B). [Figure 3B] FIG. 1 shows exemplary culture plots for the amount of RejuAgro A in shake flask fermentation, showing the distribution of RejuAgro A in the cell broth, supernatant, and cells (Panel A) and the time course of RejuAgro A production by cell fermentation (Panel B). [Figure 4] FIG. 1 shows exemplary agar plates showing that V. inaequalis can grow on PDA plates with no additives alone (Plate A); 0.25% 0.01M PBS (Plate B) or 0.8% DMSO (Plate C) or 1.6% DMSO (Plate D) at day 14. [Figure 5] Figure 1 shows exemplary agar plates demonstrating that V. inaeculis is unable to grow on PDA plates containing four selected biocontrol bacteria (Plate A: 0617-T307; Plate B: 0118-T319; Plate C: 0318-T327; Plate D: 0418-T328) at day 14. [Figure 6]Figure 1 shows exemplary agar plates demonstrating that V. inaeculis is unable to grow on PDA plates containing 40-80 μg / mL of RejuAgro A at day 14 (Plate A: 10 μg / mL in PDA plate; Plate B: 20 ​​μg / mL in PDA plate; Plate C: 40 μg / mL in PDA plate; Plate D: 80 μg / mL in PDA plate). [Figure 7] Figure 1 shows exemplary agar plates demonstrating that V. inaeculis can grow on PDA plates containing 10-80 μg / mL of RejuAgro B at day 14 (Plate A: 10 μg / mL in PDA plate; Plate B: 20 ​​μg / mL in PDA plate; Plate C: 40 μg / mL in PDA plate; Plate D: 80 μg / mL in PDA plate). [Figure 8] Figure 1 shows exemplary agar plates demonstrating that V. inaeculis can grow on PDA plates containing 200–1000 μg / mL copper sulfate at day 14 (Plate A: PDA plate containing 500 μg / mL CuSO; Plate B: PDA plate containing 1000 μg / mL CuSO). [Figure 9] FIG. 1 shows an exemplary quantity-peak area curve of RejuAgro A analyzed by HPLC at a wavelength of 407 nm. [Figure 10] FIG. 1 shows exemplary data on the production of RejuAgro A from different bacterial strains. [Figure 11] FIG. 1 shows an exemplary antifungal assay against Botrytis cinerea CA17. Panel A shows (1) 40 μL of 50 mg / mL nystatin, (2) 40 μL DMSO; Panel B shows (1) M9 medium for 24 hours, (2) M8 medium for 24 hours, (3) M7 medium for 24 hours, and (4) M6 medium for 24 hours; Panel C shows (1) M9 medium for 12 hours, (2) M8 medium for 12 hours, (3) M7 medium for 12 hours, and (4) M6 medium for 12 hours. [Figure 12]FIG. 1 shows an exemplary agar plate of M. fijiensis showing growth inhibition in the presence of 600 μg / mL RejuAgro A (Panel A) and growth in the presence of 60 μg / mL RejuAgro A (Panel B) or without RejuAgro A (Panel C). [Figure 13A] The RejuAgro A molecule is shown here, with the S-Me group rotated 8.7° relative to the heterocycle, resulting in a planar geometry. This molecule exhibits significant π-conjugation disruption at the C4-C5 bond (1.531 Å), which clearly has some orbital reason. The Me group attached to the sp2 carbon atom is rotationally disordered in two positions. [Figure 13B] Figure 1 shows RejuAgro A molecules forming centrosymmetric H-bonded dimers in the crystal form via NH...O interactions. Furthermore, these dimers form two-dimensional layers along the [-3 0 1] plane via weaker CH...O interactions. [Figure 14A] This is a diagram of a RejuAgro B crystal with two symmetrically independent RejuAgro B molecules. Each molecule has a twisted structure, with the average interplanar dihedral angles of the linked heterocycles being 70.3° and 80.6°. Each heterocycle exhibits significant π-conjugation breaks at the C(sp2)-C(sp2) bond between the two adjacent carbonyl groups (bond lengths in the 1.534-1.539 Å range), which clearly have some orbital reason. [Figure 14B] This figure shows RejuAgro B molecules forming centrosymmetric H-bonded dimers in crystalline form through NH...O interactions. These dimers are linked in stacks along the x direction by other NH...O interactions. Finally, the stacks are linked in layers along the 0011 direction by a third type of NH...O interaction. [Figure 15A] FIG. 1 shows the RejuAgro C molecule with a π-conjugated planar geometry in which the amide group is rotated 42° out of the plane of the other atoms. [Figure 15B]RejuAgro C molecules in the crystal stack along the x-axis. The stacks are connected in layers along the ab plane by NH...O H-bonds. The layers are connected to a 3D network with solvated water molecules (3 molar equivalents) through multiple hydrogen bonds. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates to novel metabolites produced by seven Pseudomonas strains, including 0617-T307, described in this patent, that exhibit antibacterial activity against pathogenic microorganisms, including bacteria and fungi. Based on 16S rRNA and other housekeeping gene sequences, the strain was identified as Pseudomonas soli 0617-T307, a member of the Pseudomonas putida group. Cell broths from the seven bacterial strains, including 0617-T307, contain novel, potent six-membered heterocyclic natural products, designated RejuAgro A, along with the dimeric RejuAgro B, as shown below.

[0028] [ka]

[0029] These compounds, their methods of production, and their use for inhibiting plant microbial pathogens are disclosed in more detail herein.

[0030] definition When introducing elements of aspects of the disclosure or specific embodiments, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. The term "or," unless otherwise specified, means any one element of a particular list and also includes any combination of elements of that list.

[0031] As intended herein, the terms "substantially," "approximately," and "about" and similar terms are intended to have broad meanings consistent with common and accepted usage in the art to which the subject matter of this disclosure pertains. These terms should be understood by those of skill in the art reviewing this disclosure to enable description of the particular features described and claimed without limiting the scope of those features to the precise numerical ranges provided. Accordingly, these terms should be interpreted to indicate that insubstantial or insignificant modifications or variations of the subject matter described and claimed are considered to be within the scope of the invention as set forth in the appended claims.

[0032] "Biological control agents (or BCAs)" are a safe, sustainable, and cost-effective way to manage pests such as pathogens, weeds, and insects. These agents are introduced into the environment to target pest species, with the goal of reducing the population or abundance of pests in the environment.

[0033] "Biological preparations" are preparations of live microorganisms (bacteria, yeasts) that colonize the host. These microorganisms are primarily applied to slow the accumulation of pathogens during the epiphytic stage (Tianna et al., (2018)).

[0034] "Biorational" is a term applied to microbial-based biological pesticides. These biopesticides are often produced by fermenting microbial strains. Many of these products have both antibacterial and antifungal activity (Tianna et al., 2018).

[0035] "Biopeticides" are defined by the US Environmental Protection Agency (EPA) as pesticides derived from natural sources and are classified into biochemical pesticides, which contain substances that control pests through harmless mechanisms; microbial pesticides, which are typically composed of microorganisms that produce biologically active natural products (BNPs); and plant-incorporated protectants, whose activity is produced by plants due to the addition of genetic material (Gwinn KD (2018)).

[0036] The compounds designated RejuAgro A, RejuAgro B, and RejuAgro C correspond to compounds having formulas (I), (II), and (III), respectively, as illustrated below.

[0037] [ka]

[0038] In a first aspect, a method for growing bacteria to promote production of protective metabolites is provided. The method includes alternative steps. In one method, a step of growing Pseudomonas bacteria in a liquid medium in a vessel to produce a bacterial fermentate is provided. The medium volume to vessel volume ratio is between about 1:2 and 1:10, and the vessel is shaken at a speed between about 100 and 250 RPM. In an alternative step, the method includes growing Pseudomonas bacteria in a liquid medium in a fermenter to produce a bacterial fermentate. The airflow rate of the fermenter is between about 1 and 3 L / min. In one aspect, the method further includes a step of separating the liquid medium from the bacteria after a period of time to produce a protective supernatant containing the protective metabolites. In a second aspect, the bacterium comprises a Pseudomonas strain selected from 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328. In a third aspect, the growth temperature is between about 10°C and 35°C. In a fourth aspect, the liquid medium is LB / YME medium for cell production. In a fifth aspect, the liquid medium is YME medium for RejuAgro A production. In a sixth aspect, the ratio of medium volume to vessel volume is between about 1:5 and 1:10. In a seventh aspect, the ratio of medium volume to vessel volume is between about 1:7 and 1:9. In an eighth aspect, the ratio of medium volume to vessel volume is about 1:8. At point 9, the vessel is shaken at a speed between about 200-250 RPM. At point 10, the vessel is shaken at a speed between about 210-230 RPM. At point 11, the airflow rate of the fermentor is between about 1.5-2.5 L / min, and the dissolved oxygen concentration is between 5 mg / L and 12 mg / L. At point 12, the growth temperature is between about 10°C and 20°C. At point 13, the growth temperature is between about 15°C and 17°C. At point 14, the bacteria are grown for a period of 18 hours to 7 days. At point 15, the bacteria are grown for a period of 7 days. At point 16, the bacteria are grown for a period of 1 day to 2 days.

[0039] In a second aspect, an agricultural composition is provided comprising the bacterial fermentate or protected supernatant. The agricultural composition is produced according to the method of the first aspect and any of the aspects disclosed with respect to the first aspect. In the first aspect, the agricultural composition further comprises an adjuvant. In this aspect, the adjuvant is a surfactant.

[0040] In a third aspect, there is provided a method for controlling bacterial and fungal crop diseases. The method comprises several steps. The first step comprises producing an agricultural composition comprising the bacterial fermentate or protective supernatant produced in accordance with the first aspect or any point thereof. The second step comprises applying the agricultural composition to a crop to inhibit the growth of pathogenic microorganisms.

[0041] In a first respect, the crop disease is selected from the group consisting of fire blight, citrus canker, olive canker, and soft rot of tomato and pepper. In the second respect, the pathogenic microorganisms are Mycospherella figiensis, Botrytis cinerea, Erwinia amylovora (Ea), Xanthomonas axonopodis pv. citri (Xac), Pectobacterium parmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp. carotovorum, Dickeja dadantii, Pseudomonas savastanoi pv. savastanoi (Psv), Pseudomonas syringae pv. tomato (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.michiganensis, Phytophthora infestans, Venturia inaequalis, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Xanthomonas citri pv. citri. In a third respect, the crop is selected from one or more of bananas, apples, pears, crabapples, citrus fruits, potatoes, pumpkins, onions, rice, African violets; plant species of the families Cruciferae, Solanaceae, and Cucurbitaceae, such as carrots, potatoes, tomatoes, eggplants, leafy vegetables, squash, and gourds; peppers and green peppers; olives; stone fruits and pome fruits, including olives, peaches, and walnuts.

[0042] In a fourth aspect, a method for controlling bacterial crop diseases is provided, the method comprising the step of: infecting a plant with a bacterial extract of about 1.0 x 10 microbial cells to inhibit the growth of pathogenic microorganisms; 5 ~1.0×10 9 The method includes applying to a crop an agricultural composition containing Pseudomonas bacteria between about cfu / mL.

[0043] In a first respect, the Pseudomonas bacterium is a Pseudomonas strain selected from 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328. In a second respect, the composition comprises about 5.0 x 10 7 ~2.0×10 8In a third aspect, the crop disease is selected from the group consisting of black sigatoka, gray mold, fire blight, citrus canker, soft rot, olive canker, tomato bacterial leaf spot, bacterial canker or blast (stone fruit and pome fruit), cucurbit angular spot, peach bacterial spot, tomato bacterial spot, walnut canker, bacterial wilt, tomato canker, potato leaf blight, apple scab, bacterial leaf blight, and bacterial streak. In the fourth point, the pathogenic microorganisms are Mycospherella fijiensis, Botrytis cinerea, Erwinia amylovora (Ea), Xanthomonas axonopodis pathotype citri (Xac), Pectobacterium parmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp. carotovorum, Dickeyia dadantii, Pseudomonas savastanoi pathotype savastanoi (Psv), Pseudomonas syringae pathotype tomato, Pseudomonas syringae pathotype syringae, Pseudomonas syringae pathotype lachrymans, Xanthomonas campestris pathotype prunii, Xanthomonas campestris and Xanthomonas citri pathotype citri. In a fifth aspect, the crop is selected from the group consisting of banana, apple, pear, crabapple, citrus fruits, potato, pumpkin, onion, rice, African violet; plant species of the Brassicaceae, Solanaceae, and Cucurbitaceae families such as carrot, potato, tomato, eggplant, leafy vegetables, squash, and cucurbits; pepper and green pepper; olive; and stone fruit and pome-shaped fruits including olive, peach, and walnut.

[0044] In a fifth aspect, a method for purifying protected metabolites from Pseudomonas bacteria is provided. The method comprises several steps. The first step comprises producing a bacterial fermentate or protected supernatant according to the method of the first aspect and in accordance therewith. The second step comprises extracting the bacterial fermentate or protected supernatant by ethyl acetate extraction. The third step comprises producing an eluate containing the protected metabolites by eluting the bacterial fermentate or protected supernatant with a mixture of hexane and ethyl acetate, e.g., a mixture of 50% hexane and 50% ethyl acetate, or by eluting the ethyl acetate extract with a mixture of hexane and ethyl acetate, e.g., a mixture of 25% hexane and 75% ethyl acetate.

[0045] In a first aspect, the Pseudomonas bacterium is a Pseudomonas strain selected from 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328.

[0046] In a sixth aspect, an agricultural composition comprising a protective metabolite from a Pseudomonas bacterium is purified by the method of the fifth aspect and points thereof.

[0047] In a seventh aspect, there is provided a method for controlling bacterial crop diseases. The method comprises several steps. The first step comprises producing an agricultural composition comprising protective metabolites derived from Pseudomonas bacteria purified by the method of the fifth aspect or any point thereof. The second step comprises applying the agricultural composition to crops to inhibit the growth of pathogenic microorganisms.

[0048] In a first respect, the crop disease is selected from the group consisting of fire blight, citrus canker, olive canker, soft rot, tomato and pepper. In the second respect, the pathogenic microorganisms are Mycospherella fijiensis, Botrytis cinerea, Erwinia amylovora (Ea) (especially streptomycin-resistant E. amylovora strains), Xanthomonas axonopodis pathotype citri (Xac), Pectobacterium parmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp. carotovorum, Dickeyer dadantii, Pseudomonas savastanoi pathotype savastanoi (Psv), Pseudomonas syringae pathotype tomato, Pseudomonas syringae pathotype syringae, Pseudomonas syringae pathotype lachrymans, and Xanthomonas campestris. In a third respect, the pathogenic E. amylovora is selected from the group consisting of streptomycin-resistant E. amylovora, Xanthomonas campestris pathotype vesicatoria, Xanthomonas arboricola pathotype juglandis, Ralstonia solanacearum, Clavibacter michiganensis subsp. michiganensis, Phytophthora infestans, Venturia inaeculis, Xanthomonas oryzae pathotype oryzae, Xanthomonas oryzae pathotype oryzicola, and Xanthomonas citri pathotype citri. In a fourth aspect, the crop is selected from one or more of bananas, apples, pears, crabapples, citrus fruits, potatoes, pumpkins, onions, rice, African violets; plant species of the Brassicaceae, Solanaceae, and Cucurbitaceae families, such as carrots, potatoes, tomatoes, eggplants, leafy vegetables, squash, and gourds; peppers and green peppers; olives; and stone fruits and pome fruits, including olives, peaches, and walnuts. In a fifth aspect, the pathogenic bacterium is Flavobacterium columnare #2 or Flavobacterium columnare MS-FC-4. In a sixth aspect, the pathogenic bacterium is E. coli O157:H7.

[0049] In an eighth embodiment, the crystalline compound is selected from one of the following structures:

[0050] [ka]

[0051] In a first respect, the crystalline compound has the structure:

[0052] [ka] The crystalline compound has at least one physical property selected from Tables 13-22.

[0053] In a second respect, the crystalline compound has the structure:

[0054] [ka] The crystalline compound has at least one physical property selected from Tables 23-29.

[0055] In a third respect, the crystalline compound has the structure:

[0056] [ka] The crystalline compound has at least one physical property selected from Tables 30-37.

[0057] Biological deposit information One of the present inventors, Dr. Ching-Hong Yang (resident of 10120 North Sheridan Drive, Mequon, Wisconsin 53902, USA), has obtained the bacterial strains Pseudomonas sori 0617-T307, Pseudomonas sori 0917-T305, Pseudomonas sori 0917-T306, Pseudomonas sori 0917-T307, Pseudomonas mosserii 0118-T319, Pseudomonas mosserii 0318-T327, and Pseudomonas mosserii 0418-T328, as evidenced by the "Indication Relating to the Deposited Microorganisms" under PCT Rule 13bis (PCT / RO / 134) (filed herein), from the American Type Culture Collection (ATCC®), P.O. Box 1000, San Jose, CA 95210. 1549, Manassas, VA 20110 USA (the "ATCC Patent Depository"), filed on June 25, 2020. After viability testing, the ATCC Patent Depository has assigned the following accession numbers to these deposited bacterial strains, effective from June 25, 2020: Pseudomonas sori 0617-T307 (accession number PTA-126796), Pseudomonas sori 0917-T305 (accession number PTA-126797), Pseudomonas sori 0917-T306 (accession number PTA-126798), Pseudomonas sori 0917-T307 (accession number PTA-126799), Pseudomonas mosserii 0118-T319 (accession number PTA-126800), Pseudomonas mosserii 0318-T327 (Accession No. PTA-126801) and Pseudomonas mosserii 0418-T328 (Accession No. PTA-126802). Dr. Yang has given the applicant his unconditional and irrevocable consent to the inclusion in this application of the disclosure of this biological deposit, which will be made available to the public as of the filing date. [Example]

[0058] Example 1 Identification and Characterization of Strain 0617-T307

[0059] Partial sequences of 16S rDNA, gyrB, rpoB, and rpoD were analyzed. These four genes are recommended markers for multilocus sequence analysis (MLSA) of Pseudomonas (Peix et al., (2018)).

[0060] For species assignment, these four sequences were used to perform BLASTN against the NCBI nonredundant nucleotide database. Based on the results, strain 0617-T307 is closely related to Pseudomonas species belonging to the P. putida group of P. fluorescens lineage. The MLSA phylogenetic tree and the genome list derived from type strains of Pseudomonas species (see Figure 2 and Table 2 in Peix et al., 2018; Peix et al., 2018) were used as a guide for taxonomic sampling (Figure 1). Based on this information, genomes were retrieved from GenBank. All species in the P. putida group with high-quality genome assemblies were included. Because 0617-T307 shares the highest rpoD sequence similarity (i.e., the gene with the highest resolution for Pseudomonas species assignment) with P. soli, all four available genomes of P. soli were included in the sampling (P. soli type strain LMG 27941). T For other species in the P. fluorescens lineage, one species was selected as a representative for each group. P. aeruginosa (P. aeruginosa group, P. aeruginosa lineage) was included as an outgroup to create the root of the tree.

[0061] Four genes for MLSA were extracted from the sampled genomes. After each gene was sequenced individually, all four nucleotide alignments were concatenated for phylogenetic analysis. The concatenated alignment contained 9,912 aligned nucleotide sites. Maximum likelihood estimation was performed using PhyML (Guindon et al., 2003). Bootstrap support was assessed with 1,000 replicates.

[0062] Based on the multilocus molecular phylogenetic tree (Figure 1), 0617-T307 and the four P. sori strains for which genome sequences are available form a monophyletic clade with 100% bootstrap support. This result provided strong support for assigning 0617-T307 to P. sori, the type strain reportedly isolated from soil samples in the Sierra Nevada National Park, Spain (Pascual et al., 2014).

[0063] Furthermore, based on the guidelines for Pseudomonas species assignment provided by Garcia-Valdes and Lalucat (Garcia-Valdes et al., 2016), additional support for assigning 0617-T307 to P. sori included the following: (a) 16S rDNA >98.7–99% identity. Compared to the reference strain of P. sori, 0617-T307 had 99.2% sequence identity. Compared to the sister species P. entomophila, 0617-T307 had 99.5% sequence identity. Note that rDNA is known to lack sufficient resolution for species identification in the genus Pseudomonas (Garcia-Valdes et al., 2016; Peix et al., 2018); (b) rpoD gene >95–96% identity. Compared to the type strain of P. sori, 0617-T307 had 96.5% sequence identity. Compared to the sister species P. entomophila, 0617-T307 had only 89.1% sequence identity; and (c) MLSA >97% identity. Compared to the type strain of P. sori, 0617-T307 had 98.0% sequence identity. Compared to the sister species P. entomophila, 0617-T307 had only 95.1% sequence identity.

[0064] Example 2 Preparation, isolation, and characterization of RejuAgro A and RejuAgro B from ethyl acetate extract of cell broth of strain 0617-T307

[0065] RejuAgro A and B preparations were obtained by ethyl acetate extraction of the cell broth from the fermenter fermentation, followed by isolation and purification by chromatography. Briefly, the stock bacteria, Pseudomonas sp. 0617-T307, was streaked onto an LB plate (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, water) and grown for 24 hours in an incubator at 28°C. To prepare the seed medium, a single colony of 0617-T307 was inoculated into a 2.0 L flask containing 500 mL of autoclaved YME medium (4 g / L yeast extract, 4 g / L glucose, 10 g / L malt extract) and grown for 24 hours at 28°C with a shaking speed of 200 rpm. The seed medium was then inoculated into a 20 L NBS fermenter containing 12 L of autoclaved YME medium. Fermentation was carried out for 1 to 7 days at 16°C, with an agitation speed of 200 rpm and an airflow rate of 2 L / min.

[0066] After harvesting, the bacterial culture was extracted four times with ethyl acetate. The ethyl acetate layer was separated, dehydrated using sodium sulfate, and then dried by rotary evaporation at 35°C. As a result, 2.9 g of crude extract was obtained from a 12 L culture of the 0617-T307 strain.

[0067] The concentrated sample was dissolved in ethyl acetate, mixed with silica gel, and packed into an injection column (φ3.0 × 20 cm) and loaded onto a silica gel universal column (4.8 × 18.5 cm) in a flash chromatography system (Yamazen AI-580) equipped with a UV detector. After loading, the sample was eluted with 280 mL of each of the following solvents in order of 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 eluate was monitored by UV at 254 nm, and fractions were collected in a timed mode of 20 mL per tube. A total of 114 fractions or tubes were obtained from the flash chromatography.

[0068] The resulting fractions were then applied to the plate assay. 1 mL of each fraction was collected in a 1.5 mL test tube and vacuum-dried using an Eppendorf vacuum concentrator. The dried sample was dissolved in 50 μL of DMSO, and 2 μL of the solution was used for the plate assay. Briefly, Erwinia amylovora 273 was streaked onto an LB plate and grown in an incubator at 28°C. After 24 hours, a single colony was inoculated into 5 mL of LB medium and grown overnight at 28°C in a shaker at 200 rpm. The cells were diluted 1:100 with sterile water, and 225 μL of the diluted solution was plated onto a 50% LB plate (5.0 g / L tryptone, 2.5 g / L yeast extract, 5.0 g / L NaCl, 15 g / L agar). After drying for 10 minutes in a biosafety cabinet, the DMSO solution of each fraction was dispensed into pre-labeled sections of a Petri dish and allowed to dry for another 10 minutes. DMSO and kasugamycin were used as negative and positive controls, respectively, during the assay. The plates were then incubated in an incubator at 28°C, and inhibition zones were confirmed after one day.

[0069] In vitro plate assays of 114 fractions showed that two fractions inhibited the growth of E. amylovorus 273. Notably, fractions / tubes 38-40 (abbreviated as T3840 or Flash-RejuAgro A) were eluted with 50% hexane / 50% ethyl acetate and had a relatively large clearance zone that may be promising for further testing. Other bioactive compounds in this assay were found in fractions 50-52 (coded as T5052). These fractions were eluted with 25% hexane / 75% ethyl acetate.

[0070] Fractions 3840 and 5054 were purified by preparative HPLC (Prep-HPLC), yielding 15 mg of the yellow-colored compound RejuAgro A (Rt 17.5) and 103.3 mg of the dark green-colored compound RejuAgro B, respectively. RejuAgro A is soluble in methanol and chloroform. RejuAgro B (Rt 10.5) is poorly soluble in methanol and chloroform, but is highly soluble in dimethyl sulfoxide (DMSO), exhibiting a dark green color. The structures of these two compounds were investigated using high-resolution mass spectrometry (HR-MS), infrared (IR), ultraviolet (UV), one- and two-dimensional nuclear magnetic resonance (NMR), and X-ray crystallography. The results showed that the two compounds are structurally similar. RejuAgro A contains seven carbon groups (three carbonyl groups, two tertiary carbons, and two methyl carbons), while RejuAgro B lacks one methyl group, as shown below:

[0071] [ka]

[0072] Furthermore, crystals of RejuAgro A were obtained by slowly evaporating the chloroform solution at room temperature. The crystals were identified as orange platelets. The data set was collected at 100 K on an Oxford SuperNova diffractometer using Cu(Kα) radiation. The molecule has a planar structure, with the S-Me (methyl) group rotated by 8.7° relative to the heterocycle. The molecule exhibits significant π-conjugation breakage at the C4-C5 bond (1.531 Å), which is apparently due to some orbital reason. sp 2 The Me group attached to the carbon atom of is rotationally disordered at two positions. The molecules in the crystal form centrosymmetric H-bonded dimers through NH...O interactions. Furthermore, these dimers form two-dimensional layers along the [-3 0 1] plane through weaker CH...O interactions. RejuAgro A molecules exhibit a six-membered heterocycle [-NH-C(=O)-C(-SMe)=C(-Me)-C(=O)-C(=O)-]. The crystal of RejuAgro B was identified as a triclinic crystal. The structure of RejuAgro B contains two symmetrically independent molecules. Each molecule has a twisted structure, with dihedral angles between the average planes of the connected heterocycles of 70.3° and 80.6°. Each heterocycle also contains a C(sp) bond between two adjacent carbonyl groups. 2 )-C(sp 2 There is significant π-conjugation disruption at the ) bond (bond lengths ranging from 1.534 to 1.539 Å), which clearly has some orbital reason. The molecules in the crystal form centrosymmetric H-bonded dimers through NH...O interactions. These dimers are linked in stacks along the x direction by other NH...O interactions. Finally, the stacks are linked in layers along the

[0011] direction by a third type of NH...O interaction. When RejuAgro B solution was used for crystal growth, two crystals were obtained, designated RejuAgro B and RejuAgro C. Both RejuAgro B and RejuAgro C crystals have very similar molecular weights (see Example 20).

[0073] Crystal structure information for RejuAgro A, RejuAgro B, and RejuAgro C is provided in Example 20, the contents of which are incorporated by reference in their entirety and form part of this application.

[0074] The molecular formula of RejuAgro A is C7H7NO3S and the molecular weight is 185.2004. This is consistent with the observed [M+H] molecular species m / z of 186.2177 (theoretical value 186.2083) in the HR-MS data. The molecular formula of RejuAgro B is C 12 The molecular mass is H8N2O6S, with a molecular mass of 276.2017. This is consistent with the molecular mass of [MH] observed in the HR-MS data, with a molecular mass of 275.0278 (theoretical value: 275.1960). A CCDC structural database search as of August 4, 2020, suggests that no crystal structures exist for RejuAgro A, RejuAgro B, or RejuAgro C. Searches of other chemical databases, including SciFinder, Reaxys, and Google Patent and Related Databases, indicate that no analogues of RejuAgro A or RejuAgro C exist, except for one reference (Knackmuss et al., (1968)) found for RejuAgro B in SciFinder and Reaxys.

[0075] Example 3: In vitro antibacterial activity of RejuAgro A and RejuAgro B derived from strain 0617-T307

[0076] MIC values ​​of RejuAgro A and RejuAgro B were determined against five bacterial species: wild-type Gram-negative plant pathogenic bacteria, streptomycin-resistant E. amylovora, fish disease-causing bacteria, Gram-positive and Gram-negative human pathogenic bacteria, and the producer of RejuAgro A (strain 0617-T307). Antimicrobial assays were performed according to CLSI antimicrobial susceptibility testing (AST) standards. Briefly, stock solutions of each tested bacterium were streaked onto Luria-Bertani (LB) plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium salt, 15 g / L agar). For specific cultures, nutrient broth and agar (NA) plates (3 g / L beef extract, 1 g / L yeast extract, 5 g / L polypeptone, 10 g / L sucrose, 15 g / L agar) were used for Xacin. SHIEH (5 g / L tryptone, 0.5 g / L yeast extract, 0.01 g / L sodium acetate, 0.01 g / L BaCl2(H2O)2, 0.1 g / L K2HPO4, 0.05 g / L KH2PO4, 0.3 g / L MgSO4 7H2O, 0.0067 g / L CaCl2 2H2O, 0.001 g / L FeSO4 7H2O, 0.05 g / L NaHCO3, 10 g / L agar) and TYES (4 g / L tryptone, 0.4 g / L yeast extract, 0.5 g / L MgSO4, 0.5 g / L CaCl2, pH 7.2, 15 g / L agar) were used for Flavobacterium columnarum strains MS-FC-4 and #2, respectively. Single colonies were then picked from the plates and inoculated into the corresponding liquid medium for overnight growth. This culture reached OD in LB or the corresponding medium. 590The compounds were diluted to a pH of 0.01 and dispensed at 200 μL / well into a 96-well plate. The compounds RejuAgro A and RejuAgro B and streptomycin were diluted and 4 μL of each concentration was added to each well to achieve final concentrations of 40 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.3125 μg / mL, 0.15625 μg / mL, and 0.078 μg / mL. Vehicle water (for streptomycin) or DMSO (for RejuAgro A and RejuAgro B) served as controls.

[0077] The assay results showed that the most active metabolite of strain 0617-T307 was RejuAgro A, not RejuAgro B. Compared to its effectiveness against the Gram-positive bacterium MRSA (MIC > 40 μg / mL) and the Gram-negative bacterium Escherichia coli O157:H7 (a food- and water-borne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans) (MIC = 40 μg / mL), RejuAgro A was particularly effective against test bacteria with MIC values ​​between 5 and 40 μg / mL. The antibacterial activity of RejuAgro A was comparable to that of streptomycin against Erwinia amylovora 1189, Xanthomonas axonopodis pathovar citri, Pseudomonas savastanoi pathovar savastanoi, Pectobacterium parmentieri UPP163 936, Pectobacterium carotovorum subsp. brasiliensis 944, Pectobacterium carotovorum subsp. carotovorum wpp14 945, and Dickeja dadantii 3937. The MIC values ​​for E. amylovora were 5 μg / mL and for other weakly pathogenic bacteria were 20-40 μg / mL. Xanthomonas bacteria were highly susceptible to streptomycin, with an MIC of 0.16 μg / mL, lower than the MIC of 5 μg / mL for RejuAgro A. The MIC value of RejuAgro A against Pseudomonas savastanoi pathovar savastanoi is 40 μg / mL. The MIC value of RejuAgro A against Xanthomonas arboricola pathovar juglandis 219 is 6.25 μg / mL. The MIC values ​​of RejuAgro A against Ralstonia solanacearum K60 and Pss4 are 3.13 and 6.25 μg / mL, respectively. The MIC values ​​of RejuAgro A against Clavibacter michiganensis subsp. michiganensis NCPPB382, Cmm0317, and Cmm0690 are 6.25, 1.56, and 12.5 μg / mL, respectively. The MIC value of RejuAgro A against Ralstonia solanacearum K60 and Pss4 is 40 μg / mL.

[0078] RejuAgro A was also tested against other E. amylovora strains, including one virulent strain and three streptomycin-resistant strains. Against E. amylovora 110, RejuAgro A was as effective as streptomycin (MIC 5 μg / mL). However, RejuAgro A was more effective than streptomycin against E. amylovora 1189. The MICs of RejuAgro A and streptomycin against E. amylovora 1189 were 5 μg / mL and 10 μg / mL, respectively. Furthermore, RejuAgro A was more effective against streptomycin-resistant E. amylovora CA11, DM1, and 898, as a lower MIC (10 μg / mL) was observed for RejuAgro A compared with streptomycin (>40 μg / mL). These results suggest that RejuAgro A was the most potent compound tested against E. amylovora and could be a potential alternative to streptomycin. There was no indication of cross-resistance to RejuAgro A in streptomycin-resistant strains.

[0079] Against Flavobacterium, the causative bacterium of columnar disease in fish, RejuAgro A had an MIC value of 5 μg / mL against Flavobacterium columnare strains MS-FC-4 and #2 (which cause columnar disease in wild and farmed fish), which was higher than the MIC values ​​of streptomycin (0.31 μg / mL and 1.25 μg / mL against #2 and MS-FC-4 strains, respectively).

[0080] The effect of RejuAgro A on the 0617-T307 strain was examined. The MIC value of RejuAgro A against Pseudomonas sori 0617-T307 (a RejuAgro A-producing strain) was shown to be greater than 40 μg / mL in the tested LB medium, which means that the 0617-T307 strain survives and is resistant to at least 40 μg / mL of RejuAgro A that it produces.

[0081] RejuAgro A was tested with streptomycin against tomato pathogens (P. syringae pathovar tomato PT30, P. syringae pathovar syringae 7046, and P. syringae pathovar lachrymans 1188-1) and other citrus canker pathogens (Xanthomonas campestris pathovar prunii and Xanthomonas campestris pathovar vesicatoria XV-16). The MIC of RejuAgro A against P. syringae was 40 μg / mL, while the MIC of streptomycin ranged from 2.5 to 5 μg / mL. Against X. campestris, the MIC of RejuAgro A was 2.5 μg / mL or 40 μg / mL, which is lower than the MIC of streptomycin, which was 20 μg / mL or >40 μg / mL. These results indicate that the Xanthomonas campestris pathogen is more sensitive to RejuAgro A than to streptomycin when compared with the Pseudomonas-caused tomato pathogens.

[0082] RejuAgro A was effective against all pathogenic fungi tested (Table 1). RejuAgro A was tested against Phytophthora infestans, Venturia inaeculis, and Mycospherella fisiensis. RejuAgro A showed 100% inhibition against P. infestans and V. inaeculis at 40 μg / mL, 80 μg / mL, and 600 μg / mL (Table 1).

[0083] [Table 1] TIFF2026035671000011.tif212167

[0084] Example 4: Production and stability of RejuAgro A from strain 0617-T307 in shake flask fermentation

[0085] Fermentation of 0617-T307 for use in the production and preparation of RejuAgro A can be achieved by two approaches: shake flask fermentation and fermentor fermentation. Fermentor fermentation is described in Example 2. In this example, flask fermentation can be achieved as follows: Stock bacteria, Pseudomonas sp. 0617-T307, was streaked onto YME agar medium (4 g / L yeast extract, 4 g / L glucose, 10 g / L malt extract, 15 g / L agar) and grown for 24 hours in an incubator at 28°C. Seed medium was prepared by growing a single colony of 0617-T307 in a 250 mL flask containing 50 mL of sterile YME liquid medium at 16°C and 220 rpm for 24 hours. The seed medium was then inoculated at a 4% ratio (v / v) into a 4 L flask containing 0.5 L of sterile YME medium. Following inoculation (2%, v / v) into eight 4-L flasks containing 2 L of YME medium, bacteria were grown for 1–7 days on a shaker at 16°C and 200–220 rpm.

[0086] The concentration of RejuAgro A was obtained by LC-MS analysis according to the developed standard curve. Two methods were used to prepare samples for LC-MS analysis. One approach involved extracting the cell broth with ethyl acetate (1 mL:1 mL, vortexing for 1 min) and obtaining an ethyl acetate extract by centrifugation and vacuum drying the ethyl acetate layer. The dried ethyl acetate extract was dissolved in 40 μL of methanol, and 2 μL of the methanol solution was used for LC-MS analysis. The other method involved centrifuging the cell broth to obtain the supernatant, then mixing the supernatant with an equal volume of methanol to make a 50% methanol solution, and injecting 10 μL of this solution into the LC-MS. Because RejuAgro A production was confirmed to be extracellularly secreted and the supernatant contained a large amount of RejuAgro A, rather than intracellularly, we adopted the second method (Figure 3, Panel A).

[0087] During the 7-day fermentation, the total production of RejuAgro A reached a peak concentration on the first day and then began to decrease with increasing time (Figure 3, Panel B). Furthermore, detailed investigations of RejuAgro A production and cell concentration were performed every 6 hours in shake flask fermentation. The RejuAgro A concentration (total RejuAgro A) reached a maximum of 13.8 mg / L at 18 hours, and the bacterial cell concentration reached a maximum of 2 × 10 cells at 12 hours. 11 CFU / mL, indicating that the production of RejuAgro A is a production process accompanied by cell proliferation.

[0088] The volume of medium in a 4 L shake flask affects the production of RejuAgro A. In 4 L flasks using YME medium, RejuAgro A production was observed only in the 500 mL volume, but not in the 1.0 L or 1.5 L volumes. This observation indicates that RejuAgro A production prefers a highly aerated environment.

[0089] The type of medium and incubation temperature affect the production of RejuAgro A. LB medium was tested at 16°C or 28°C in parallel with YME medium. RejuAgro A production was confirmed in YME medium at 16°C but not in LB medium. Regarding colony-forming units, strain 0617-T307 grew well in LB medium at both 16°C and 28°C, and in YME medium at 28°C. These results suggest that RejuAgro A production is medium-specific and temperature-dependent. Activity against the 0617-T307-derived product was monitored by plate assay against E. amylovora, which is consistent with RejuAgro A production.

[0090] To confirm the applicability of the RejuAgro A production conditions, 10 other Pseudomonas strains were tested in parallel with Pseudomonas strain 0617-T307 under the same conditions. Based on housekeeping gene analysis, strains 0917-T305, 0917-T306, and 0917-T307 were identified as Pseudomonas sori, while strains 0118-T319, 0318-T327, and 0418-T328 were identified as Pseudomonas mosserii. Type strains of both Pseudomonas sori and Pseudomonas mosserii have been reported (Daboussi et al., 2002; Pascual et al., 2014).

[0091] Strain 0617-T307 and its phylogenetically related species were shown to be able to produce RejuAgro A in YME at 28°C and 220 rpm. This result suggests that this method has the specificity to produce RejuAgro A for strain 0617-T307 and some of its related species (Table 2). LCMS testing of a 40-hour culture obtained by culturing 0617-T307 in YME medium at 16°C in a shaker at 220 rpm demonstrated that RejuAgro A was present and stable at room temperature for at least 4 weeks.

[0092] [Table 2]

[0093] Example 5: Antibacterial activity of cell broth of strain 0617-T307 against 0617-T307 and E. amylovora.

[0094] Two assays were used to test the antibacterial activity of 0617-T307 cell broth and metabolites: a plate diffusion assay and a microplate assay. LB plates were used for the plate diffusion assay of the antibacterial activity of RejuAgro A-containing fractions and cell broth against E. amylovora (Table 3). Both the cell broth containing viable cells of 0617-T307 and a suspension containing 2 mg / mL of RejuAgro A demonstrated antibacterial activity against E. amylovora. However, no inhibition zones were observed when Serenade® was applied.

[0095] [Table 3]

[0096] To identify a biological control method consisting of both 0617-T307 cells and the active ingredient RejuAgro A, the following experiments were conducted. For antibacterial assays against 0617-T307, the producer of RejuAgro A, the supernatant of a 40-hour culture of 0617-T307 containing RejuAgro A (abbreviated as "supernatant") was used. It was shown that the 0617-T307 strain could grow in LB medium but not in YME medium at a two-fold dilution of the supernatant. Further studies indicated that the inhibitory effect of the supernatant was due to its low pH value. By subsequently controlling the pH to 6.5-6.8, questions 1 and 2 could be answered "yes."

[0097] Bioactive fractions (crude extract, 100 μg / mL; flash-RejuAgro A, 20 μg / mL; HPLC-RejuAgro A, 10 μg / mL) were tested against strains 0617-T307, Ea, and Xac. The bioactive fractions failed to inhibit the growth of strain 0617-T307, indicating that RejuAgro A can be mixed with 0617-T307 cells to prepare a biocontrol agent. Bioactive fractions, including RejuAgro A, exhibited inhibitory effects against Ea and Xac. In particular, flash-RejuAgro A and HPLC-RejuAgro A nearly stopped the growth of Ea and Xac under the test conditions. This demonstrates that RejuAgro A solutions at 10–20 μg / mL can be used for the biological control of fire blight and citrus canker.

[0098] Example 6 Identification and characterization of bioactive metabolites from the ethyl acetate extract of acidified supernatant (pH 2.0) of strain 0617-T307.

[0099] Stock bacteria, Pseudomonas sp. 0617-T307, were inoculated onto LB agar (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, water) plates and grown for 24 hours in an incubator at 28°C. To prepare the seed medium, a single colony of 0617-T307 was inoculated into 500 mL of autoclaved YME medium (4 g / L yeast extract, 4 g / L glucose, 10 g / L malt extract) and grown for 24 hours at 28°C with a shaking speed of 150 rpm. Eight 4-L flasks, each containing 2 L of autoclaved YME medium, were then inoculated with the seed medium. Fermentation proceeded for 7 days at 16°C with a shaking speed of 150 rpm.

[0100] After 7 days of growth, the supernatant was obtained by centrifuging the bacterial culture at 4000 rpm for 15 minutes. The pH of the supernatant was then adjusted to 2.0 by adding 6N HCl. The acidified supernatant was then subjected to ethyl acetate extraction. As a result, 3.0 g of crude extract was obtained from a 14 L culture of the 0617-T307 strain.

[0101] The concentrated sample was dissolved in acetone, mixed with silica gel, and loaded onto a silica gel column (φ3.0 × 20 cm) in a flash chromatography system (Yamazen AI-580) equipped with a UV detector. After loading, the sample was eluted with 280 mL of each of the following solvents in order of 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 eluate was monitored by UV at 254 nm, and fractions were collected in a timed mode at 20 mL / tube. A total of 114 fractions or tubes were obtained from the flash chromatography.

[0102] The resulting fractions were then applied to the plate assay. 1 mL of each fraction was collected in a 1.5 mL test tube and vacuum-dried using an Eppendorf vacuum concentrator. The dried sample was dissolved in 50 μL of DMSO, and 2 μL of this was used for the plate assay. Briefly, Erwinia amylovora 273 was inoculated onto a 50% LB (5.0 g / L tryptone, 2.5 g / L yeast extract, 5.0 g / L NaCl) plate, and a single colony was inoculated into 5 mL of LB medium. The bacteria were diluted 1:100 with sterile water, and 225 μL of this was plated onto a 50% LB plate. After drying for 10 minutes in a biosafety cabinet, the DMSO solution of each fraction was dispensed into a pre-labeled portion of a Petri dish and allowed to dry for another 10 minutes. DMSO and kasugamycin were used as negative and positive controls, respectively, during the assay. The plates are then incubated in an incubator at 28°C and the inhibition zones are checked after one day.

[0103] In vitro plate assays of 114 flush fractions revealed three bioactive fractions (T3234, T5058, and T7882) that inhibited the growth of E. amylovorus 273. Fractions 3234 and 5258 exhibited relatively small zones of clearance. Fraction 3234 was eluted with 50% hexane / 50% ethyl acetate. Fraction 5058 was eluted with 25% hexane / 75% ethyl acetate. As a negative control, DMSO showed no zone of inhibition, while the positive control, kasugamycin, did. Another flush fraction, T7882, was eluted with acetone / ethyl acetate (50% / 50%). This similarly inhibited the growth of E. amylovorus.

[0104] Further isolation and purification by HPLC based on anti-E. amylovora activity identified two antibacterial compounds (Rt22.9 and Rt25.0) from T5058 (see compound formulas 0617_T307_5058_Rt22.9 and 0617_T307_5058_Rt25.0) and one antibacterial compound (Rt18.9) from T7882 (see compound formula 0617_T307_7882_Rt18.9). T307_5058_Rt22.9 and T307_5058_Rt25.0 are tryptophan-derived natural products whose structures are reported in the Scifinder database, but whose biological activity has not been reported (Loots et al., 2015). 0617_T307_7882_Rt18 was predicted to be a derivative of difuryl, which has been previously reported (Osipov et al., (1978)). These natural products are shown below:

[0105] [ka]

[0106] Example 7: Identification of other metabolites from strain 0617-T307 using LCMSMS and spectral library searching.

[0107] Crude extracts of unadjusted and pH-adjusted cell broth (pH of the cell broth was adjusted to 2.0 with 6N HCl) were concentrated and resuspended in 250 μL of 100% MeOH containing an internal standard (m / z 311.08) for LC-MS / MS analysis. LC injection volume: 5 μL; LC column: 1.7 μM C18, 100A, 50 x 2.1 mm Kinetex C18 column from Phenomenex, 12 min gradient. 5–95% ACN was measured on a Bruker Maxis Impact II. Data were acquired using a Bruker Maxis Impact II UHR-QqTOF (Ultra-High Resolution Qq-Time-Of-Flight) mass spectrometer. Each full MS scan was followed by tandem MS (MS / MS) analysis using collision-activated dissociation (CID) fragmentation of the eight most abundant ions in the spectrum. The scan rate was 3 Hz.

[0108] Based on bioinformatics and molecular network analysis, accurate spectral library searches were then performed to identify novel and known compounds. MS / MS spectra of samples were searched against the following spectral libraries: 1) GNPS Community Library, 2) FDA Library, PhytoChemical Library, 3) NIH Clinical Collections, 4) NIH Natural Products Library, 5) Pharmacologically Active NIH Small Molecule Repository, 6) Faulkner Legacy Library, 7) Pesticides, 8) Dereplicator-Identified MS / MS Peptidic Natural Products, 9) PNNL Lipids, 10) Massbank, 11) Massbank EU, 12) MoNA, 13) ReSpect-Phytochemicals, and 14) HMDB.

[0109] The MS / MS spectra of the samples were searched against the above libraries and aligned to the reference spectrum with offsets. Matching parameters were the same. These results can be explored to identify structural analogs of known compounds. MS / MS molecular networks were generated with a minimum cluster size of 2, a minimum edge cosine of 0.7, and a minimum number of matching peaks of 6. As an example, a new molecular species at m / z 303.16 was identified as corresponding to a new compound from the active fraction 0617-T307_5058_Rt25.0. Some known compounds were confirmed in the crude extract, which contains the plant growth promoters indole-3-carboxylic acid and xantholysin A. It has been reported that 1) the broad antifungal activity of P. putida BW11M1 is primarily dependent on xantholysin production, and 2) xantholysin is required for swarming and contributes to biofilm formation (Li et al., 2013). Indeed, higher concentrations of xanthridine A were observed when 0617-T307, 0418-T328, and 0318-T327 were cultured at 28°C. Thus, with the exception of the bioactive compound RejuAgro A, xanthridine A is the metabolite that contributes to the antibacterial activity of the biocontrol bacterium 0617-T307 and its related species 0318-T3027 and 0418-T328.

[0110] Example 8 Greenhouse and field infection assays of RejuAgro A-producing strain 0617-T307 and some of its relatives.

[0111] To evaluate the biocontrol activity of 0617-T307 against Erwinia amylovora, an infection assay was performed on crabapple trees in the greenhouse at the University of Wisconsin-Milwaukee. 8Biocontrol agents (0617-T307, 0717-T327, and 0617-T318) containing cfu / mL were sprayed on flowers (80% to full bloom) in multiple plots. Briefly, strain 0617-T307 was grown overnight in a 26 mL glass tube containing 5 mL of LB medium, and the cells were then inoculated (1:100) into LB medium and grown at 28°C and 200 rpm on a shaker for 14 to 18 hours. Cells were harvested and stored at 10 °C. 8 The flowers were resuspended in 10x water to a concentration of 1.0 x 10 CFU / mL. This resuspension can be used in greenhouse and field assays for fire blight control. Control flowers were sprayed with distilled water. All flowers were then sprayed with 1.0 x 10 6 Crabapple flowers were inoculated by spraying with E. amylovora strain Erwinia amylovora 273 at 1000 cfu / mL. Treatments with 0617-T307 were performed three times: September 7, October 9, and October 19, 2018. Table 4 shows that all spray treatments of 0617-T307 (Pseudomonas sori) resulted in 100% control of blossom blight symptoms compared with 0% control with distilled water, suggesting that 0617-T307 is a promising biocontrol agent for E. amylovora fire blight. The other two Pseudomonas species, 0717-T327 (Pseudomonas coriensis) and 0617-T318 (Pseudomonas protegens), had lower control rates of 16.7% and 25%, respectively. In conclusion, of the three Pseudomonas species tested in this study, only 0617-T307 showed good control of fire blight on crabapple, without any phytotoxicity.

[0112] [Table 4]

[0113] In field assays, on May 5 and May 6, 2019 (at 40% and 70% bloom of apple blossoms), the RejuAgro A-producing biocontrol bacteria (0617-T307, 0118-T319, 0318-T327, and 0418-T328; see Table 2) reached 5 × 10 8The bacterial pathogen E. amylovora Ea110 was applied to the flowers of apple trees in the orchard at a concentration of 5 × 10 CFU / mL on May 7 (90% flowering). 6 The inoculation concentrations were 100 CFU / mL. The diseased inflorescence rates for the water control, streptomycin, 0617-T307, 0118-T319, 0318-T327, and 0418-T328 were 32.9%, 13.3%, 16.8%, 18.5%, 16.7%, and 11.8%, respectively. Compared with streptomycin, the biocontrol bacteria producing RejuAgro A were equally or more effective in controlling fire blight in apple orchards.

[0114] [Example 9] Antifungal activity of RejuAgro A and B and their producing fungi against Venturia inaeculis.

[0115] The fungus Venturia inaeculis, which causes apple rot, was maintained on PDA agar medium in the dark at room temperature (approximately 24°C). A mixed suspension of conidia and mycelia (in 0.01M PBS) was harvested from the PDA (potato dextrose agar). 10 μL of the conidia and mycelia suspension was dropped onto a plate containing biocontrol bacteria, RejuAgro A, or RejuAgro A-amended plates. Control plates were PDA plates without the addition of biocontrol bacteria, RejuAgro A or B. The dishes were incubated in the dark at room temperature, and the diameter of each colony of V. inaeculis was measured after 7 days.

[0116] When compared with the control (Figure 4), the four selected biocontrol bacterial strains 0617-T307, 0118-T319, 0318-T327, and 0418-T328 were able to suppress the growth of V. inaeculis on PDA plates (Figure 5). RejuAgro A was able to suppress the growth of V. inaeculis on PDA plates at 40–80 μg / mL (Figure 6). However, no inhibitory effect of RejuAgro B on the growth of V. inaeculis was observed on PDA plates at 10–80 μg / mL (Figure 7). Finally, no inhibition of V. inaeculis was observed on PDA plates containing 200–1000 μg / mL copper sulfate (Figure 8).

[0117] Example 10: Production of RejuAgro A by Pseudomonas sp.

[0118] The amount of RejuAgro A was analyzed by HPLC-MS in the broth after 24 hours of fermentation in a 4 L flask containing 500 mL of YME medium at 16 °C and 220 rpm. A quantity-peak area curve was constructed to examine the relationship between the HPLC peak area and the amount of RejuAgro A (Figure 9). Analysis method: 1) 25 mL of cell broth was extracted with 25 mL of ethyl acetate. 2) 5 mL of the ethyl acetate extract was dried and dissolved in 0.1 mL of methanol. 3) 4 μL was injected into the HPLC-MS.

[0119] Seven bacteria (0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328) were evaluated for RejuAgro A production, and seed medium was prepared by growing the bacteria in YME medium at 16°C and 220 rpm for 24 hours. HPLC analysis showed that all seven bacteria produced RejuAgro A (Figure 10).

[0120] Example 11. RejuAgro A formulation and greenhouse assay.

[0121] RejuAgro A was formulated (solution, SL; see Table 5). 10 μg / mL was tank-mixed with 1% polyethylene glycol (PEG) 4000 as a safener before application to flowers. Subsequent testing showed that the use of 0.03% polyvinyl alcohol (PVA) as a safener provided better flower protection. Additionally, the surfactant Alligare 90 could be added for even greater efficacy (Table 6).

[0122] [Table 5]

[0123] To evaluate the biocontrol activity of RejuAgro A against Erwinia amylovora, a greenhouse infection assay was conducted at the University of Wisconsin-Milwaukee using crabapple trees. A 10 μg / mL solution containing 1% polyethylene glycol (PEG) 4000 or 1% PEG 4000 (negative control) was applied to fully bloomed trees 3 hours before and 24 hours after inoculation. Approximately 10 of E. amylovora 110 strains suspended in water were inoculated. 8 CFU / mL was used. The infection rate was calculated around day 6 after inoculation. The experiment was conducted for one week from January 24 to January 31, 2020. RejuAgro A can effectively suppress flower blight (Table 6).

[0124] [Table 6]

[0125] Example 12: Antifungal activity of 0617-T307 cell broth against Botrytis cinerea CA17

[0126] An inoculum of strain 0617-T307 was prepared by growing it in YME medium at 28°C and 180 rpm for 24 hours. Then, 4% (2 mL to 50 mL) of the inoculum was inoculated into a 250 mL flask containing 50 mL of M8 (IAA medium), M9 (CN medium), M7 (PRN medium), or M6 (DAPG medium) and grown at 28°C and 180 rpm for 48 hours. 0.5 mL of cell broth was collected at 12 and 24 hours and stored in a -20°C freezer. For antifungal assays, the cell broth was thawed and 5 μL was spread onto sample wells of a PDA (potato dextrose agar) plate at equal radial distances from the center of the inoculated Botrytis cinerea (Figure 11). The cell broth was shown to have antifungal activity against Botrytis cinerea CA17 on PDA (potato dextrose agar) plates.

[0127] Example 13: Antibacterial activity of crude extracts, RejuAgro A and RejuAgro B against plant pathogenic bacteria.

[0128] Metabolites from bacteria 0917-T305, 0318-T327, and 0418-T328 showed excellent activity against R. solanacearum, C. michiganensis subsp. michiganensis, and X. arboricola pathovar juglandis (Table 7). Bacteria 0917-T305, 0318-T327, and 0418-T328 were grown in YME medium at 16°C and 28°C, respectively. Natural product extracts of 0917-T305, 0318-T327, and 0418-T328 were prepared at 5 mg / mL and tested against three plant pathogens: Ralstonia solanacearum, Clavibacter michiganensis subsp. michiganensis, and Xanthomonas arboricola pathovar juglandis, in a plate diffusion assay. In agar plate diffusion assays, metabolites from bacteria 0917-T305, 0318-T327, and 0418-T328 grown in YME at 16°C and 28°C showed relatively good activity against the tested R. solanacearum, C. michiganensis subsp. michiganensis, and X. arboricola pathov. juglandis (Table 7). This indicates that RejuAgro A, along with other metabolites, also has excellent activity against R. solanacearum, Clavibacter michiganensis subsp. michiganensis, and Xanthomonas arboricola pathov. juglandis. RejuAgro B also shows good activity against R. solanacearum (Table 7).

[0129] [Table 7]

[0130] [Example 14] Antibacterial effects of Rt18.9, Rt22.9, and Rt25.0.

[0131] Stock bacteria, Pseudomonas sp. 0617-T307, were inoculated onto LB agar (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, water) plates and grown for 24 hours in a 28°C incubator. Fermentation and crude extract preparation were performed as described in Example 6.

[0132] HPLC separation and purification of the ethyl acetate extract of the acidified cell broth of Pseudomonas sp. 0617-T307 identified two antibacterial compounds (Rt 22.9 and Rt 25.0) from flash fraction T5058 and one antibacterial compound (Rt 18.9) from flash fraction T7882, which were tested for antibacterial activity against the bacterial strains shown in Table 8. Two microliters of DMSO, Rt 18.9, Rt 22.9, or Rt 25.0 were each spotted onto agar plates on which different bacterial strains were grown, and the inhibition zones were further examined (Table 8).

[0133] [Table 8] TIFF2026035671000020.tif102162

[0134] [Example 15] Antibacterial effect of RejuAgro A against Mycosphaerella fisiensis

[0135] The antibacterial effect of RejuAgro A against Mycosphaerella fisiensis was investigated by adding HPLC-purified RejuAgro A to PDA agar plates at final concentrations of 60 μg / mL and 600 μg / mL, respectively. 480 μL of 0.5 mg / mL or 5 mg / mL RejuAgro A was added to 3.52 mL of PDA in a 6-well plate to achieve final RejuAgro A concentrations of 60 μg / mL (Figure 12, center well (Panel A)) and 600 μg / mL (Figure 12, left well (Panel B)), respectively. The plate was gently shaken to dissolve the compound. 480 μL of water containing 3.52 mL of PDA was used as a control treatment (Figure 12, right well (Panel C)). After the agar solidified, an agar strip containing M. fisiensis was placed on the center of the agar surface. Complete inhibition of M. fisiensis growth was observed 2 weeks after inoculation when treated with RejuAgro A at a concentration of 600 μg / mL (FIG. 12).

[0136] [Example 16] Antibacterial effect of RejuAgro A against Xanthomonas oryzae pathotype oryzae (Xon507)

[0137] The antibacterial effect of RejuAgro A against Xanthomonas oryzae pathovar oryzae (Xon507) was investigated. 600 Aqueous solutions of RejuAgro A (p = 0.3) were sprayed onto PSG agar plates. Paper discs loaded with HPLC-purified aqueous RejuAgro A at concentrations of 5.5 μg / mL, 11.1 μg / mL, 22.1 μg / mL, 33.2 μg / mL, 55.4 μg / mL, and 110.7 μg / mL in a 50 μL loading volume were placed on the agar plates, and inhibition zones were measured 44 hours after disc placement. Inhibition was observed for all concentrations of paper discs soaked with RejuAgro A suspension (Table 9).

[0138] [Table 9]

[0139] [Example 17] Antibacterial effect of RejuAgro A against Xanthomonas citri pv. citri citrange (XW19)

[0140] The antibacterial effect of RejuAgro A against Xanthomonas citri pathovar citri citranjae (XW19) was investigated. The bacterial suspension (OD ) of X. citri pathovar citri citranjae (XW19) was 600Aqueous solutions of 5.5 μg / mL, 11.1 μg / mL, 22.1 μg / mL, 33.2 μg / mL, 55.4 μg / mL, and 110.7 μg / mL of HPLC-purified RejuAgro A were sprayed onto PSG agar plates. Paper discs loaded with 50 μL of aqueous RejuAgro A at concentrations of 5.5 μg / mL, 11.1 μg / mL, 22.1 μg / mL, 33.2 μg / mL, 55.4 μg / mL, and 110.7 μg / mL were placed on the agar plates, and inhibition zones were measured 44 hours after the discs were placed on the agar plates. Inhibition was observed at RejuAgro A concentrations of 55.37 μg / mL and 110.74 μg / mL (Table 10).

[0141] [Table 10]

[0142] [Example 18] Medium culture composition used in the example

[0143] Table 11 contains exemplary media compositions used in the examples.

[0144] [Table 11]

[0145] Example 19: Bacterial strains, natural products, and references cited therein.

[0146] The bacterial strains and natural products described in this application and claimed in the appended claims are well known in the microbiology literature, which is set forth below in Table 12 for each of the cited bacterial strains and natural products disclosed herein, the contents of which are incorporated herein by reference in their entirety.

[0147] [Table 12]

[0148] Example 20 Crystal structure information for RejuAgro A, RejuAgro B, and RejuAgro C.

[0149] A. Crystal structure information of RejuAgro A Single crystals of RejuAgro A (C7H7NO3S) were obtained by slow evaporation of a chloroform solution of RejuAgro A. Orange platelets were obtained. Suitable crystals were selected and mounted on SuperNova, Dua, Cu Home / Nearby, and Atlas diffractometers. Crystals were maintained at 100.05(10) K during data collection. Using Olex2 (Dolomanov et al., 2009), the structure was solved by the ShelXS structure solver using direct methods (Sheldrick, 2008) and refined with the ShelXL refinement package (Sheldrick, GM, 2015) using least-squares minimization.

[0150] The data set was collected at 100 K on an Oxford SuperNova diffractometer using Cu(Kα) radiation.

[0151] Crystal data for RejuAgro A (C7H7NO3S) (M = 185.20 g / mol): monoclinic, space group P21 / n (no. 14), a = 5.30391(6) Å, b = 13.97822(13) Å, c = 10.74471(13) Å, β = 101.5883(12)°, V = 780.367(15) Å 3 , Z=4, T=100.05(10)K, μ(CuKα)=3.429mm -1 , Dcalc=1.576g / cm 3 , 13936 reflections measured (10.522°≦2Θ≦140.8°), 1496 independent (R int =0.0220, R sigma = 0.0083) were used in all calculations. The final R1 was 0.0253 (I > 2σ(I)) and wR2 was 0.0702 (all data).

[0152] The refined model was written in Olex2 and compiled to svn.r3508 on May 29, 2018, at OlexSys. Number of restraints: 0, Number of constraints: unknown. Details: 1. Fixed Uiso: 1.2x: All C(H,H,H,H,H,H) groups; 1.5x: All C(H,H,H) groups; 2. Other: Sof(H6A) = Sof(H6D) = Sof(H6F) = 1-FVAR(1); Sof(H6B) = Sof(H6C) = Sof(H6E) = FVAR(1); 3. a. Disordered Me: C6 (H6A, H6B, H6C, H6D, H6E, H6F) refined as rotators; b. Idealized Me: C7 (H7A, H7B, H7C) refined as rotators.

[0153] Referring to Figure 13A, the RejuAgro A molecule has a planar structure with the S-Me group rotated by 8.7° relative to the heterocycle. This molecule exhibits significant π-conjugation disruption at the C4-C5 bond (1.531 Å), which is apparently due to some orbital reason. 2 The Me group attached to the carbon atom of is rotationally disordered in two positions.

[0154] Referring to Figure 13B, RejuAgro A molecules in the crystal form centrosymmetric H-bonded dimers through NH...O interactions. These dimers then form two-dimensional layers along the [-3 0 1] plane through weaker CH...O interactions.

[0155] The chemical structure of RejuAgro A is shown below:

[0156] [ka]

[0157] Additional crystallographic data for the RejuAgro A molecule is presented in Tables 13-21.

[0158] [Table 13]

[0159] [Table 14]

[0160] [Table 15]

[0161] [Table 16]

[0162] [Table 17]

[0163] [Table 18]

[0164] [Table 19]

[0165] [Table 20]

[0166] [Table 21]

[0167] B. Crystal structure information of RejuAgro B RejuAgro B(C 12Single crystals of H8N2O6 were obtained by slow evaporation of a methanol solution of RejuAgro B. Orange pyramids were obtained. A suitable crystal was selected and mounted on a SuperNova, Dual, Cu Home / Nearby, Atlas diffractometer. The crystal was kept at 100.05(10) K during data collection. Using Olex2 (Dolomanov et al., (2009)), the structure was solved by the ShelXS structure solver using direct methods (Sheldrick (2008)) and refined with the ShelXL refinement package (Sheldrick (2015)) using least-squares minimization.

[0168] The data set was collected at 100 K on an Oxford SuperNova diffractometer using Cu(Kα) radiation.

[0169] RejuAgro B(C 12 Crystal data of H8N2O6) (M=276.20g / mol): triclinic, space group P-1 (no. 2), a=7.0528(3)Å, b=11.7911(5) Å, c=14.6888(6)Å, α=72.249(4)°, β=79.265(3)°, γ=86.633(3)°, V=1143.02(8)Å 3 , Z=4, T=100.05(10)K, μ(CuKα)=1.139mm -1 , Dcalc=1.605g / cm 3 , 15292 reflections measured (7.872°≦2Θ≦141.144°), 4304 independent (R int =0.0258, R sigma = 0.0234) were used in all calculations. The final R1 was 0.0419 (I > 2σ(I)) and wR2 was 0.1124 (all data).

[0170] The refined model description was created in Olex2 and compiled to svn.r3508 on May 29, 2018, at OlexSys. Number of restraints: 0, number of constraints: unknown. Details are as follows: 1. Fixed Uiso: 1.2x: all N(H) groups, 1.5x: all C(H,H,H) groups. 2.a Aromatic / amide H refined in mounted coordinates: N1(H1), N2(H2), N1A(H1A), N2A(H2A); 2.b. Idealized Me refined as rotatable groups: C6(H6A, H6B, H6C), C12(H12A, H12B, H12C), C6A(H6AC, H6AA, H6AB), C12A(H12D, H12E, H12F).

[0171] Referring to Figure 14A, RejuAgro B crystals contain two symmetrically independent RejuAgro B molecules. Each molecule has a twisted structure, with dihedral angles between the average planes of the linked heterocycles of 70.3° and 80.6°. Each heterocycle also contains a C(sp) bond between two adjacent carbonyl groups. 2 )-C(sp 2 ) bond (bond lengths in the 1.534–1.539 Å range), there is clearly some orbital reason for this.

[0172] Referring to Figure 14B, RejuAgro B molecules in the crystal form centrosymmetric H-bonded dimers through NH...O interactions. These dimers are linked in stacks along the x direction by other NH...O interactions. Finally, the stacks are linked in layers along the

[0011] direction by a third type of NH...O interaction.

[0173] The chemical structure of RejuAgro B is shown below:

[0174] [ka]

[0175] Additional crystallographic data for the RejuAgro B molecule is presented in Tables 22-29.

[0176] [Table 22]

[0177] [Table 23]

[0178] [Table 24]

[0179] [Table 25]

[0180] [Table 26]

[0181] [Table 27]

[0182] [Table 28]

[0183] [Table 29]

[0184] C. Crystal structure information of RejuAgro C RejuAgro C(C 10 H 16Single crystals of N2O7 were obtained by slow evaporation of methanol solutions of RejuAgro B and RejuAgro C. Colorless needles were obtained with RejuAgro B. Suitable crystals were selected and mounted on a SuperNova, Dual, Cu Home / Nearby, Atlas diffractometer. The crystals were kept at 100.05(10) K during data collection. The structure was solved using Olex2 (Dolomanov et al., 2009) with the olex2.solve structure solving program (Bourhis et al., 2015) using charge flipping and refined with the ShelXL refinement package (Sheldrick, 2015) using least-squares minimization.

[0185] The data set was collected at 100 K on an Oxford SuperNova diffractometer using Cu(Kα) radiation.

[0186] RejuAgro C(C 10 H 16 Crystal data of N2O7) (M=276.25g / mol): triclinic, space group P-1 (no. 2), a=7.0334(4)Å, b=10.2354(7)Å , c=10.4693(7)Å, α=116.426(7)°, β=104.722(5)°, γ=97.680(5)°, V=625.72(8)Å 3 , Z=2, T=100.00(10)K, μ(CuKα)=1.081mm -1 , Dcalc=1.466g / cm 3 , 7480 reflections measured (10.068°≦2Θ≦140.528°), 2353 independent (R int =0.0405, R sigma = 0.0373) were used in all calculations. The final R1 was 0.0504 (I > 2σ(I)) and wR2 was 0.1388 (all data).

[0187] The refined model description was created in Olex2 and compiled to svn.r3508 of OlexSys on May 29, 2018. Number of restraints - 0, number of constraints - unknown. Details: 1. Fixed Uiso: 1.5 times: all C(H,H,H) groups, 2. Idealized Me refined as a rotatable group: C9(H9A,H9B,H9C),C10(H10A,H10B,H10C)

[0188] Referring to Figure 15A, the RejuAgro C molecule has a π-conjugated planar geometry in which the amide group is rotated 42° out of the plane of the other atoms.

[0189] Referring to Figure 15B, RejuAgro C molecules in the crystal stack along the x-axis. The stacks are connected in layers along the ab plane by NH...O H-bonds. The layers are connected to solvate water molecules (3 molar equivalents) through multiple hydrogen bonds into a three-dimensional network.

[0190] The chemical structure of RejuAgro C is shown below:

[0191] [ka]

[0192] Additional crystallographic data for the RejuAgro C molecule is presented in Tables 30-37.

[0193] [Table 30]

[0194] [Table 31]

[0195] [Table 32]

[0196] [Table 33]

[0197] [Table 34]

[0198] [Table 35]

[0199] [Table 36]

[0200] [Table 37]

[0201] [Table 38] TIFF2026035671000054.tif229166TIFF2026035671000055.tif230167TIFF2026035671000056.tif229165TIFF2026035671000057.tif44162

[0202] Incorporation by Reference All documents, publications, patents, patent applications and related materials cited herein are incorporated by reference as if fully set forth herein.

Claims

1. 1. A method for growing bacteria to enhance production of protective metabolites, comprising: i. growing Pseudomonas bacteria in a liquid medium in a vessel to produce a bacterial fermentate, wherein the ratio of medium volume to vessel volume is between about 1:2 and 1:10, and the vessel is shaken at a speed between about 100 and 250 RPM; or ii. Growing Pseudomonas bacteria in a liquid medium in a fermentor to produce a bacterial fermentate, wherein the airflow rate of the fermentor is between about 1-3 L / min. A method comprising:

2. 10. The method of claim 1, further comprising the step of separating the liquid medium from the bacteria after a period of time to produce a protective supernatant containing the protective metabolites.

3. The bacteria include Pseudomonas soli 0617-T307 (accession number PTA-126796), Pseudomonas soli 0917-T305 (accession number PTA-126797), Pseudomonas soli 0917-T306 (accession number PTA-126798), Pseudomonas soli 0917-T307 (accession number PTA-126799), Pseudomonas mosselii 0118-T319 (accession number PTA-126800), Pseudomonas mosselii 3. The method according to claim 1, wherein the bacterial strain is selected from the group consisting of Pseudomonas mosserii 0318-T327 (accession number PTA-126801) and Pseudomonas mosserii 0418-T328 (accession number PTA-126802).

4. The method of any one of claims 1 to 3, wherein the growth temperature is between about 10°C and 35°C.

5. The method according to any one of claims 1 to 4, wherein the liquid medium is LB medium for cell production.

6. The liquid medium comprises a compound represented by formula (I) and formula (II) 【Chemistry 1】 The method according to any one of claims 1 to 4, wherein the medium is a YME medium for the production of

7. The method according to any one of claims 1 to 6, wherein either a shaker or a fermenter can be used.

8. The method according to any one of claims 1 to 6, wherein when a shaker is used, the ratio of medium volume to container volume is between about 1:5 and 1:

10.

9. The method according to any one of claims 1 to 6, wherein when a shaker is used, the ratio of medium volume to container volume is between about 1:7 and 1:

9.

10. The method according to any one of claims 1 to 6, wherein when a shaker is used, the ratio of medium volume to container volume is about 1:

8.

11. The method of any one of claims 1 to 10, wherein the container is shaken at a speed of between about 200 and 250 RPM.

12. The method of any one of claims 1 to 10, wherein the container is shaken at a speed of between about 210 and 230 RPM.

13. The method according to any one of claims 1 to 6, wherein, when a fermenter is used, the air flow rate of the fermenter is between about 1.5 and 2.5 L / min, or the oxygen concentration is between 5 mg / L and 12 mg / L.

14. The method of any one of claims 1 to 13, wherein the growth temperature is between about 10°C and 20°C.

15. The method of any one of claims 1 to 13, wherein the growth temperature is between about 15°C and 17°C.

16. The method of any preceding claim, wherein the bacteria are grown for a period of between 18 hours and 7 days.

17. The method of any preceding claim, wherein the bacteria are grown for a period of at least 7 days.

18. The method of any preceding claim, wherein the bacteria are grown for a period of between 1 and 2 days.

19. An agricultural composition comprising a bacterial fermentate or a protective supernatant produced by the method of any one of claims 1 to 18.

20. 20. The agricultural composition of claim 19, wherein the formulation of the protected supernatant or its metabolites is selected from a solution (SL), a soluble powder (SP), a soluble granule (SG) and an encapsulated formulation.

21. 20. The agricultural composition of claim 19, wherein the bacterial ferment and cell preparation is selected from suspension concentrates (SC), wettable powders (WP), and water dispersible granules (WG).

22. 20. The agricultural composition of claim 19, further comprising an adjuvant.

23. 23. The agricultural composition of claim 22, wherein the adjuvant is a surfactant.

24. 20. The agricultural composition of claim 19, wherein the adjuvant is the selected active ingredient, water and a polar solvent.

25. 24. The agricultural composition of claim 23, wherein the surface-active substance is selected from wetting agents and spreading agents.

26. 26. The agricultural composition of claim 25, wherein the surfactant is a wetting agent compatible with alkyl polyethylene oxide or polypropylene oxide.

27. 1. A method for controlling bacterial crop diseases comprising: i. Producing an agricultural composition comprising a bacterial fermentate or protective supernatant produced according to any one of claims 1 to 18, or an agricultural composition according to any one of claims 19 to 27; and ii. applying the agricultural composition to crops to inhibit the growth of pathogenic microorganisms; A method comprising:

28. 28. The method of claim 27, wherein the crop disease is selected from the group consisting of black sigatoka, gray mold, fire blight, citrus canker, soft rot, olive canker, tomato bacterial leaf spot, bacterial canker or rice blast (stone fruit and pome fruit), cucurbit angular spot, peach bacterial spot, tomato bacterial spot, walnut canker, bacterial wilt, tomato canker, potato leaf blight, apple scab, bacterial leaf blight, and bacterial streak.

29. The pathogenic microorganisms include Mycosphaerella fijiensis, Botrytis cinerea, Erwinia amylovora (Ea) (especially streptomycin-resistant E. amylovora strains), Xanthomonas axonopodis pv. citri (Xac), Pectobacterium parmenttieri, Pectobacterium atrosepticum, and Pectobacterium carotovorum. Pectobacterium carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp. carotovorum, Dickeya dadantii, Pseudomonas savastanoi pv. savastanoi (Psv), Pseudomonas syringae pv. tomato, Pseudomonas syringae Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. lachrymans, Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. vesicatoria, Xanthomonas arboricola pv. juglandis, Xanthomonas arboricola pv. juglandis, Xanthomonas campestris pv. puruni, Xanthomonas campestris pv. vesicatoria, Xanthomonas arboricola pv. juglandis, Xanthomonas arboricola pv. juglandis, Xanthomonas arboricola pv. juglandis, Xanthomonas arboricola pv. juglandis, Xanthomonas arboricola pv. juglandis, Xanthomonas arboricola pv. juglandis, Xanthomonas arboricola pv. juglandis, Xanthomonas arboricola pv. puruni, Xanthomonas campestris pv. vesicatoria ... campestris pv. vesicatoria, Xanthomonas arboricola pv. juglandis, Xanthomonas arboricola pv. juglandis, Xanthomonas arboricola pv. juglandis, Xanthomonas pv. juglandis), Ralstonia solanacearum, Clavibacter michiganensis subsp. michiganensis29. The method of claim 27 or 28, wherein the pathogen is selected from the group consisting of: Phytophthora infestans, Venturia inaequalis, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Xanthomonas citri pv. citri.

30. 30. The method of any one of claims 27 to 29, wherein the crop is selected from the group consisting of banana; apple; pear; crabapple; citrus fruits; potato; pumpkin; onion; rice; African violet; plant species of the families Cruciferae, Solanaceae and Cucurbitaceae such as carrot, potato, tomato, eggplant, leafy vegetables, squash and gourds; pepper and green pepper; olive; stone fruit and pome-like fruit plants including olive, peach and walnut.

31. A method for controlling fish diseases, wherein the pathogenic microorganism is Flavobacterium columnare #2 or Flavobacterium columnare MS-FC-4, and the method is a bacterial immobilization material having any shape, composition or structure, such as a feed additive, an aquarium cleaner, or a gardening block enriched with the bacterial strain.

32. A method for controlling disease caused by a human pathogenic bacterium, wherein the bacterium is E. coli O157:H7.

33. 1. A method for controlling bacterial crop diseases comprising: Approximately 1.0×10 ５ ~1.0 x 10 ９ cfu / mL of Pseudomonas bacteria is applied to crops to suppress the growth of pathogenic microorganisms. A method comprising:

34. The Pseudomonas bacteria include Pseudomonas sori 0617-T307 (accession number PTA-126796), Pseudomonas sori 0917-T305 (accession number PTA-126797), Pseudomonas sori 0917-T306 (accession number PTA-126798), Pseudomonas sori 0917-T307 (accession number PTA-126799), Pseudomonas mosserii 0118-T319 (accession number PTA-126800), Pseudomonas mosserii 0318-T327 (accession number PTA-126801), and Pseudomonas mosserii 34. The method of claim 33, wherein the vector is selected from the group consisting of 0418-T328 (Accession No. PTA-126802).

35. The composition is about 5.0 x 10 ７ ~2.0 x 10 ８ 35. The method of claim 33 or 34, comprising Pseudomonas bacteria between 100 and 1500 cfu / mL.

36. The method according to any one of claims 33 to 35, wherein the crop disease is selected from the group consisting of black sigatoka, gray mold, fire blight, citrus canker, soft rot, olive canker, tomato bacterial leaf spot, bacterial canker or rice blast (stone fruit and pome fruit), cucurbit angular spot, peach bacterial spot, tomato bacterial spot, walnut canker, bacterial wilt, tomato canker, potato leaf blight, apple scab, bacterial leaf blight, and bacterial streak.

37. Pathogenic microorganisms include Mycospherella fijiensis, Botrytis cinerea, Erwinia amylovora (Ea) (especially streptomycin-resistant E. amylovora strains), Xanthomonas axonopodis pathotype citri, Pectobacterium parmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp. carotovorum, Dickeyer dadantii, Pseudomonas savastanoi pathotype savastanoi (Psv), Pseudomonas syringae pathotype tomato, Pseudomonas syringae pathotype syringae, Pseudomonas syringae pathotype lachrymans, and Xanthomonas campestris. The method of any one of claims 33 to 36, wherein the pathogen is selected from the group consisting of: Xanthomonas campestris pathotype vesicatoria, Xanthomonas arboricola pathotype juglandis, Ralstonia solanacearum, Clavibacter michiganensis subsp. michiganensis, Phytophthora infestans, Venturia inaeculis, Xanthomonas oryzae pathotype oryzae, Xanthomonas oryzae pathotype oryzicola, and Xanthomonas citri pathotype citri.

38. 38. The method of any one of claims 33 to 37, wherein the crop is selected from the group consisting of banana; apple; pear; crabapple; citrus fruits; potato; pumpkin; onion; rice; African violet; plant species of the families Brassicaceae, Solanaceae and Cucurbitaceae such as carrot, potato, tomato, eggplant, leafy vegetables, squash and cucurbits; pepper and green pepper; olive; stone fruit and pome fruit plants including olive, peach and walnut.

39. 1. A method for purifying a protected metabolite from a Pseudomonas bacterium, comprising: i. Producing a bacterial fermentate or protective supernatant by the method of any one of claims 1 to 17, or a preparation thereof produced by any one of claims 18 to 20; ii. Extracting the bacterial fermentate or protective supernatant by ethyl acetate extraction; and iii. Eluting the bacterial fermentate or the protected supernatant with 50% hexane and 50% ethyl acetate, or eluting the bacterial fermentate or the protected supernatant with 25% hexane and 75% ethyl acetate to produce an eluate containing the protected metabolites; A method comprising:

40. The Pseudomonas bacteria include Pseudomonas sori 0617-T307 (accession number PTA-126796), Pseudomonas sori 0917-T305 (accession number PTA-126797), Pseudomonas sori 0917-T306 (accession number PTA-126798), Pseudomonas sori 0917-T307 (accession number PTA-126799), Pseudomonas mosserii 0118-T319 (accession number PTA-126800), Pseudomonas mosserii 0318-T327 (accession number PTA-126801), and Pseudomonas mosserii 40. The method of claim 39, wherein the vector is selected from the group consisting of 0418-T328 (Accession No. PTA-126802).

41. 1. A method for controlling bacterial crop diseases comprising: i. Producing an agricultural composition comprising a protective metabolite from a Pseudomonas bacterium purified by the method of any one of claims 33 and 34, and ii. applying the agricultural composition to crops to inhibit the growth of pathogenic microorganisms. A method comprising:

42. 42. The method of claim 41, wherein the crop disease is selected from the group consisting of black sigatoka, gray mold, fire blight, citrus canker, soft rot, olive canker, tomato bacterial leaf spot, bacterial canker or blast (stone fruit and pome fruit), cucurbit angular spot, peach bacterial spot, tomato bacterial spot, walnut canker, bacterial wilt, tomato canker, potato leaf blight, apple scab, bacterial leaf blight, and bacterial streak.

43. Pathogenic microorganisms include Mycospherella fijiensis, Botrytis cinerea, Erwinia amylovora (Ea) (especially streptomycin-resistant E. amylovora strains), Xanthomonas axonopodis pathotype citri (Xac), Pectobacterium parmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp. carotovorum, Dickeyer dadantii, Pseudomonas savastanoi pathotype savastanoi (Psv), Pseudomonas syringae pathotype tomato, Pseudomonas syringae pathotype syringae, Pseudomonas syringae pathotype lachrymans, and Xanthomonas campestris.

43. The method of claim 41 or 42, wherein the pathogen is selected from the group consisting of: Xanthomonas campestris pathotype vesicatoria, Xanthomonas arboricola pathotype juglandis, Ralstonia solanacearum, Clavibacter michiganensis subsp. michiganensis, Phytophthora infestans, Venturia inaeculis, Xanthomonas oryzae pathotype oryzae, Xanthomonas oryzae pathotype oryzicola, and Xanthomonas citri pathotype citri.

44. 42. The method of claim 41, wherein the pathogenic microorganism is a pathogenic E. amylovora that is streptomycin-resistant E. amylovora.

45. 45. The method of any one of claims 41 to 44, wherein the crop is selected from the group consisting of bananas, apples, pears, crab apples, citrus fruits, potatoes, tomatoes, eggplants, leafy vegetables, squash and gourds, peppers and green peppers, olives; stone fruits and pome fruits including olives, peaches and walnuts.

46. 42. The method of claim 41, wherein the pathogenic microorganism is selected from Flavobacterium columnarium #2 and Flavobacterium columnarium MS-FC-4.

47. 42. The method of claim 41, wherein the pathogenic microorganism is E. coli O157:H7.

48. Approximately 1.0×10 ５ ~1.0 x 10 ９ 42. The method of claim 41, comprising applying to a crop an agricultural composition comprising Pseudomonas bacteria between 100 and 1500 cfu / mL to inhibit the growth of pathogenic microorganisms.

49. The agricultural composition is about 5.0 x 10 ７ ~2.0 x 10 ８ 49. The method of claim 48, comprising between about 100 and about 1000 cfu / mL of Pseudomonas bacteria.

50. The following structure: 【Chemistry 2】 A crystalline compound selected from one of:

51. The crystalline compound has the following structure: 【Transformation 3】 51. The crystalline compound of claim 50, wherein the crystalline compound comprises at least one physical property selected from Tables 13-22.

52. The crystalline compound has the following structure: 【Chemistry 4】 51. The crystalline compound of claim 50, wherein the crystalline compound comprises at least one physical property selected from Tables 23-29.

53. The crystalline compound has the following structure: 【Transformation 5】 60. The crystalline compound of claim 59, wherein the crystalline compound comprises at least one physical property selected from Tables 30-37.

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