JP2023553190A - Pseudomonas strains and their metabolites that control plant diseases - Google Patents

Abstract

The present disclosure discloses that the present disclosure can suppress the growth of various microbial species for various crops. A novel bacterial strain, its cell broth, and methods of using novel metabolites produced from the bacterial strain. The method involves the use of novel and potent antimicrobial metabolites produced from strains corresponding to compounds having formulas (I), (II), and (III). TIFF2023553190000063.tif71162

Description

The present invention is in the field of biopesticides. In particular, the present invention is capable of inhibiting the growth of various microbial species, including Pseudomonas spp. and 0418-T328, their cell broths and novel metabolites produced from 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), Each ATCC accession number PTA -126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802.

Plant diseases caused by pathogenic microorganisms increase exponentially and are costly. Plant pathogenic organisms include fungi, bacteria, mycoplasmas, viruses, viroids, nematodes, or parasitic flowering plants. Currently, there are 14 types of common plant diseases caused by bacteria, including bacterial spot, bacterial blight, and bacterial wilt. fire blight (Erwinia amylovora), citrus carcinoma disease [Xanthomonas axonopodis pv. citri (Xac)], bacterial spot disease (BLS) [Xanthomonas campestris pathogen] vesicatoria (Xanthomonas campestris pv. vesicatoria) (XV-16)], olive carcinoma disease [Pseudomonas savastanoi pv. Dickeya dadantii , Pectobacterium parmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum, It is a devastating plant disease.In the United States, the cost of controlling fire blight is For citrus carcinoma, the cost of implementing an eradication program from 1995 to 2005 in Florida alone has been estimated to exceed $100 million (Norelli et al. (2003)). And compensation to homeowners whose residential citrus trees have been destroyed is approaching $1 billion.

Fire blight is a devastating disease of pome fruits caused by infection with the gram-negative bacterium Erwinia amylovora that affects pears and apples in many parts of the world, including Europe, Germany, Austria, and Switzerland (Chen (2009)). Although fire blight rarely kills entire orchards, this disease and its control still result in significant economic losses. The Pacific Northwest and northern California have experienced small outbreaks every year since 1991, and at least some areas have experienced large outbreaks every three to four years. Even minor disease outbreaks can be costly, as pruning to remove infected plant parts disrupts tree shape and reduces 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)).

Microbial natural products have provided a wealth of biological compounds 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.) are used to treat E. coli when the risk of infection is high. This product has been mainly used as a countermeasure against Amylobora. The use of these same antibiotics in crop agriculture is controversial, as these compounds are also used in human and animal health care (Stockwell, (2012)). Concerns about antibiotic resistance have limited the use of streptomycin sulfate (Vrancken et al. (2013)). Kasugamycin is also an antibiotic being studied for fire blight. One of the disadvantages is that frequent administration of kasugamycin leads to phytotoxicity that destroys plants (Adaskaveg et al. (2010)). Another disadvantage is the high price of kasugamycin compared to other antibiotics. Therefore, kasugamycin must be used in combination with other antibiotics.

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 products based on Bacillus subtilis have been registered for fire blight control in Europe: Serenade®, based on the QST 713 strain, and Serenade®, based on the BD 170 strain. Biopro® (Brogini et al. (2005)). Biologics based on sporulating Bacillus are advantageous for biological control due to their long-term viability (Haas et al. (2005)). The reasonable success of two Bacillus-based biologics has been demonstrated in a number of field trials in the United States and Germany (Aldwinckle et al. (2002); Kunz et al. (2011); Laux et al. (2003)). ). This means that E. Our results suggest that Bacillus species hold promise in the control of floral infections caused by Amylobora. However, Bacillus species are only effective when the infection pressure is low. It is not effective when the infection pressure is moderate and high. The results obtained were unstable for both biological products, with disease control rates varying between 71% and 0% (Brogini et al. (2005)).

Future biological protection products will, on the one hand, be based on E. It must be able to compete effectively with Amylobora and on the other hand colonize the same microenvironment on different organs of the target plant. Protective bacteria produce secondary metabolites that affect pathogens, compete for food and space, and protect E. Prevents the onset of disease caused by Amylobora. In this matter, bacteria of the genus Pseudomonas are compatible with the biological protection factors mentioned above (Haas et al. (2005)). Analysis of the species composition of colonizing bacteria of various plants showed that fluorescent bacteria of the genus Pseudomonas are widely present.

In France, the main constituents of populations on both healthy and diseased apple trees, pears and hawthorns are Pseudomonas species, many of which have been tested in vitro with E. It was found to exhibit the ability to limit the growth of Amylobora (Paulin et al. (1978)). However, little information on metabolites with efficacy has been reported.

In California, Thomson et al. (1976) selected three fluorescent Pseudomonas species that were effective in protecting pear flowers (Thomson et al. (1976)). In the mid-1980s, P. spp. was isolated from the leaves of a California pear tree. P. fluorescens strain A506 is an E. fluorescens strain A506. It has shown characteristic activity in restricting the growth of Amylobora and the ability to protect apples and pears from fire blight (Lindow et al. (1996)). P. A product containing fluorescence, BrightBan® A506, was developed and has been on the market since 1996. Numerous experiments conducted in California, Oregon, and Washington demonstrated the usefulness of this formulation in various protection programs for apples and pears (Johnson, (2000)).

In the UK, two isolates of Pseudomonas fluorescens have been used for the protection of hawthorn flowers and shoots (Wilson et al. (1992)).

In Italy and New Zealand, the suitability of two strains of the genus Pseudomonas with the codes BO 3371 and BO G19 was investigated (Galasso et al. (2002)). In greenhouse conditions they are very effective in protecting apple and pear flowers 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, and this may be related to flower sensitivity linked to the length of the period from anthesis to the end of anthesis.

In New Zealand, the fluorescent Pseudomonas sp. IPV-BO G19 strain protected 79% of apple flowers under field conditions. In another experimental orchard, 'Braeburn' apple blossoms have E. When applied 24 hours before inoculation with Amylobora, fluorescent Pseudomonas sp. IPV-BO G19 and IPV-BO 3371 inhibited fire blight outbreaks by 78% and 58%, respectively (Biondi et al. (2006)).

In Spain, strain EPS62e P. Fluorescence significantly suppressed fire blight in tests on apple flowers, pear fruits, and pear flowers in field assays. P. Increased adaptability and efficacy of F. EPS62e against fire blight was obtained by a combined strategy of nutritional fortification and osmotic adaptation. Physiologically improved P. Field treatments of P. fluorescens EPS62e on pear flowers produced efficacy as high as 90%, but results varied between trials (Cabrefiga et al. (2011); Mikicinski et al. (2020)).

In Poland, 47 colonies of bacteria were isolated from apple phyllosphere and soil that can reduce the impact of fire blight on pear fruits (Mikicinski et al. (2008)).

Metabolites produced by Gram-negative Pseudomonas species have been comprehensively reviewed (Maschelein et al. (2017)). The types of Pseudomonas metabolites are classified into phenolic compounds, phenazines, lipopeptides, and the like. Functions of Pseudomonas species and their metabolites include (Alsohim et al. (2014)): 1) produce hormones or induce systemic resistance; 2) many naturally occurring strains also (plant growth regulators, IAA, viscosin), 3) production of siderophores and surfactants such as viscosin and viscosinamide, as well as hydrogen cyanide, phenazine, pyrrolnitrine or 2,4-diacylhologlucinol ( Antagonistic effects can be imparted by antimicrobial compounds such as DAPG). In our research, bacterial strains have been identified and fermented products and novel metabolites have been produced from bacteria, and in particular, RejuAgro A and RejuAgro B have been shown to be effective against multiple pathogenic microorganisms, including hitherto unreported bacteria and fungi. Shows high efficacy against.

Norelli et al. (2003) Chen et al. (2009) Gwinn, (2018) Stockwell, (2012) Vranken et al. (2013) Adaskaveg et al. (2010) Tianna et al. (2018) Broggini et al. (2005) Haas et al. (2005) Aldwinckle et al. (2002) Kunz et al. (2011) Laux et al. (2003) Broggini et al. (2005) Paulin et al. (1978) Thomson et al. (1976) Lindow et al. (1996) Johnson, (2000) Wilson et al. (1992) Galasso et al. (2002) Biondi et al. (2006) Cabrefiga et al. (2011) Mikicinski et al. (2020) Mikicinski et al. (2008) Masschelein et al. (2017) Alsohim et al. (2014)

There is a need for new biopesticides derived from novel bacterial strains, cell broths, and novel metabolites produced from such strains that can inhibit the growth of various pathogenic bacteria that cause crop diseases. .

(Brief summary of the invention)
In a first aspect, a method of growing bacteria to promote production of protective metabolites is provided. The method includes alternative steps. One method provides the step of growing Pseudomonas bacteria in a liquid medium in a container to produce a bacterial ferment. The ratio of medium volume to container volume is between about 1:2 and 1:10, and the container is shaken at a speed between about 100 and 250 RPM. According to an alternative step, the method includes growing Pseudomonas bacteria in a liquid medium in a fermentor to produce a bacterial ferment. The fermentor air flow rate is between about 1 and 3 L/min. The concentration of dissolved oxygen is between 5 mg/L and 12 mg/L.

In a second aspect, an agricultural composition comprising a bacterial fermentation or a protected supernatant is provided. The agricultural composition is produced according to the method of the first aspect and any of the points disclosed in relation to the first aspect. In the first respect, the agricultural composition further comprises an adjuvant. In this respect, adjuvants are surfactants.

In a third aspect, a method of controlling bacterial crop diseases is provided. The method includes several steps. The first step comprises producing an agricultural composition comprising the bacterial fermentation or protected supernatant produced in accordance with the first aspect or any point thereof. The second step involves applying the agricultural composition to the crop to inhibit the growth of pathogenic microorganisms.

In a fourth aspect, a method of controlling bacterial crop diseases is provided. The method includes one step. The step includes applying to the crop an agronomic composition comprising between about 1.0×10 ⁵ and 1.0×10 ⁹ cfu/mL of Pseudomonas bacteria to inhibit growth of pathogenic microorganisms. .

In a fifth aspect, a method is provided for purifying a protected metabolite from a Pseudomonas bacterium. The method includes several steps. The first step comprises producing a bacterial fermentation or protected supernatant according to the method of the first aspect and in that respect. The second step involves extracting the bacterial fermentation or protected supernatant with a solvent mixture of similar polarity or properties. The third step is to elute the bacterial fermentation or protected supernatant with a mixture of hexane and ethyl acetate, or by eluting the bacterial fermentation or protected supernatant with a mixture of hexane and ethyl acetate. , including producing an eluate containing the protected metabolite.

In a sixth aspect, an agricultural composition comprising a protected metabolite from a Pseudomonas bacterium is purified by the method of and in respect of the fifth aspect.

In a seventh aspect, a method of controlling bacterial crop diseases is provided. The method includes several steps. The first step comprises producing an agricultural composition comprising a protected metabolite derived from a Pseudomonas bacterium purified by the method of the fifth aspect or any point thereof. The second step is applying said agricultural composition, wherein the formulation of the protective supernatant or its metabolites can be a solution (SL), soluble powder (SP), soluble granule (SG) and encapsulated formulation. including. Additionally, agricultural compositions of bacterial ferments and cell preparations can be suspension concentrates (SC), wettable powders (WP), and water-dispersible granules (WG).

In an eighth aspect, the crystalline compound is selected from one of the following structures.

FIG. 3 shows an exemplary plot of a maximum likelihood phylogenetic tree of representative Pseudomonas strains based on concatenated alignments of 16S rDNA, gyrB, rpoB and rpoD. Bootstrap support values were displayed below the four internal branches that received less than 100% support. Those not shown represent 100% support. FIG. 3 shows an example of assay-based isolation of an ethyl acetate extract of strain 0617-T307. Exemplary culture plots showing the distribution of RejuAgro A in cell broth, supernatant and cells (panel A) and the time course of RejuAgro A production by cell fermentation (panel B) for the amount of RejuAgro A in shake flask fermentation. FIG. Exemplary culture plots showing the distribution of RejuAgro A in cell broth, supernatant and cells (panel A) and the time course of RejuAgro A production by cell fermentation (panel B) for the amount of RejuAgro A in shake flask fermentation. FIG. V. On day 14, V. inaequalis was treated with PDA alone without additives (Plate A); 0.25% 0.01M PBS (Plate B) or 0.8% DMSO (Plate C) or 1 FIG. 6 shows an exemplary agar plate showing the ability to grow on PDA plates with .6% DMSO (Plate D). V. Ina equalis contains four selected biocontrol bacteria (Plate A: 0617-T307; Plate B: 0118-T319; Plate C: 0318-T327; Plate D: 0418-T328) on day 14. FIG. 3 shows an exemplary agar plate showing inability to grow on PDA plates. V. FIG. 3 shows an exemplary agar plate showing that P. ina equalis is unable to grow on PDA plates containing 40-80 μg/mL RejuAgro A on day 14 (Plate A: 10 μg/mL in PDA plate; B: 20 μg/mL in PDA plates; Plate C: 40 μg/mL in PDA plates; Plate D: 80 μg/mL in PDA plates). V. FIG. 12 shows exemplary agar plates showing that P. ina equalis can grow on PDA plates containing 10-80 μg/mL RejuAgro B on day 14 (Plate A: 10 μg/mL in PDA plate; Plate B Plate C: 40 μg/mL in PDA plate; Plate D: 80 μg/mL in PDA plate). V. FIG. 3 shows exemplary agar plates showing that P. ina equalis can grow on PDA plates containing 200-1000 μg/mL copper sulfate on day 14 (Plate A: PDA plate containing 500 μg/mL _CuSO4) . ; Plate B: PDA plate containing 1000 μg/mL CuSO ₄ ). FIG. 3 shows an exemplary amount-peak area curve of RejuAgro A analyzed by HPLC at a wavelength of 407 nm. FIG. 2 shows exemplary data regarding the production of RejuAgro A from different bacterial strains. FIG. 3 shows an exemplary antifungal assay against Botrytis cinerea CA17, where panel A shows (1) 40 μL of 50 mg/mL nystatin, (2) 40 μL DMSO; panel B shows (1) 40 μL of 50 mg/mL nystatin; Panel C shows (1) M9 medium for 12 hours, (2) M8 medium for 24 hours, (3) M7 medium for 24 hours, and (4) M6 medium for 24 hours. ) M8 medium for 12 hours, (3) M7 medium for 12 hours, and (4) M6 medium for 12 hours. Growth was inhibited in the presence of 600 μg/mL RejuAgro A (panel A) and M. FIG. 2 shows an exemplary agar plate of M. fijiensis. FIG. 3 shows that the RejuAgro A molecule has a planar structure in which the S-Me group is rotated by 8.7° with respect to the heterocycle. This molecule shows significant π-conjugation cleavage at the C4-C5 bond (1.531 Å), which clearly has some orbital reason. The Me group attached to the sp ² carbon atom is rotationally disordered in two positions. N-H. ．． ．． FIG. 3 shows RejuAgro A molecules forming crystalline centrosymmetric H-bonded dimers due to O interactions. Furthermore, these dimers have a weaker C-H. ．． ．． A two-dimensional layer is formed along the [-3 0 1] plane through O interaction. FIG. 3 shows a RejuAgro B crystal with two symmetrically independent RejuAgro B molecules. Each molecule has a twisted structure, and the dihedral angles between the average planes of the connected heterocycles are 70.3° and 80.6°. In each heterocycle, significant π-conjugation cleavage is observed at the C(sp ² )-C(sp ² ) bond between two adjacent carbonyl groups (bond length is 1.534-1.539 Å). range), there is clearly some orbital reason for this. N-H. ．． ．． FIG. 3 shows RejuAgro B molecules forming crystalline centrosymmetric H-bonded dimers due to O interactions. These dimers are similar to other N-H. ．． ．． They are stacked and connected along the x direction by O interaction. Finally, the stack is the third type of N-H. ．． ．． Due to the O interaction, they are connected in a layered manner along the [0 1 1] direction. FIG. 2 is a diagram showing a RejuAgro C molecule having a π-conjugated planar shape in which the amide group is rotated by 42° from the plane of other atoms. FIG. 3 shows that RejuAgro C molecules in a crystal are stacked along the x-axis. This stack is N-H. ．． ．． They are connected in layers along the ab plane by O H bonds. This layer is connected in a three-dimensional network with solvated water molecules (3 molar equivalents) via multiple hydrogen bonds.

The present invention relates to novel metabolites produced by seven Pseudomonas strains, such as 0617-T307 described in this patent, that exhibit antibacterial activity against pathogenic microorganisms, including bacteria and fungi. From the sequences of 16S rRNA and other housekeeping genes, this strain was identified as Pseudomonas soli 0617-T307 of the Pseudomonas putida group. The cell broths of seven bacterial strains, such as 0617-T307, contain a novel and potent 6-membered heterocyclic natural product named RejuAgro A along with dimeric RejuAgro B, as shown below.

These compounds, their production methods and uses for the inhibition of plant microbial pathogens are disclosed in more detail herein.

DEFINITIONS When introducing elements of an aspect of the present disclosure or a particular embodiment, the articles "a,""an,""the," and "said" may mean that one or more of the elements are present. intended. The terms "comprising", "including" and "having" are intended to be inclusive, meaning that additional elements other than those listed may be present. do. The term "or", unless specified otherwise, means any one element of a particular list and also includes any combination of elements of that list.

As intended herein, the terms "substantially," "approximately," and "about" and similar terms have common and acceptable uses in the art to which the subject matter of this disclosure pertains. They are intended to have a broad meaning in conjunction. These terms are intended to enable description of the specific features described and claimed without limiting the scope of those features to the precise numerical ranges provided. , should be understood by one of ordinary skill in the art upon reviewing this disclosure. Accordingly, these terms are intended to mean that non-substantive or inconsequential modifications or variations of the subject matter described and claimed fall within the scope of the invention as set forth in the appended claims. should be interpreted as indicating that it is considered possible.

"Biological control agents (or BCAs)" are a safe, sustainable, and cost-effective way to manage pests such as pathogens, weeds, and insects. These drugs are introduced into the environment to target pest species and are intended to reduce the number and abundance of pests in the environment.

A "biological product" is a preparation of live microorganisms (bacteria, yeast) that colonize a host. These microorganisms are mainly applied to slow down the accumulation of pathogens during the epiphytic phase (Tianna et al. (2018)).

"Biorational" is a term applied to biopesticides based on microorganisms. These biopesticides are often made by fermenting microbial strains. Many of these products have both antibacterial and antifungal activity (Tianna et al. (2018)).

“Biopesticides” are defined by the U.S. Environmental Protection Agency (EPA) as pesticides derived from natural sources and typically produce biochemical pesticides, biologically active natural products (BNPs), including substances that control pests by non-hazardous mechanisms. It is classified as a microbial pesticide made up of microorganisms that have a chemical effect, and a plant-integrated protective agent that has activity produced by plants due to the addition of genetic material (Gwinn K.D. (2018)).

The compounds called RejuAgro A, RejuAgro B, and RejuAgro C correspond to compounds having the formulas (I), (II), and (III) illustrated below, respectively.

In a first aspect, a method of growing bacteria to promote production of protective metabolites is provided. The method includes alternative steps. One method provides the step of growing Pseudomonas bacteria in a liquid medium in a container to produce a bacterial ferment. The ratio of medium volume to container volume is between about 1:2 and 1:10, and the container is shaken at a speed between about 100 and 250 RPM. According to an alternative step, the method includes growing Pseudomonas bacteria in a liquid medium in a fermentor to produce a bacterial ferment. The fermentor air flow rate is between about 1 and 3 L/min. In one aspect, the method further includes separating the liquid medium from the bacteria after a period of time to produce a protected supernatant containing protected metabolites. In the second respect, the bacterium comprises a Pseudomonas strain selected from 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328. In the third point, the growth temperature is between about 10°C and 35°C. In the fourth point, the liquid medium is LB/YME medium for production of cells. In the fifth point, the liquid medium is YME medium for the production of RejuAgro A. In a sixth point, the ratio of medium volume to container volume is between about 1:5 and 1:10. In a seventh point, the ratio of medium volume to container volume is between about 1:7 and 1:9. In the eighth point, the ratio of medium volume to container volume is about 1:8. At the ninth point, the container is shaken at a speed of between about 200-250 RPM. At the tenth point, the container is shaken at a speed of between about 210-230 RPM. In the eleventh point, the fermentor air flow rate is between about 1.5-2.5 L/min and the concentration of dissolved oxygen is between 5 mg/L and 12 mg/L. At the twelfth point, the growth temperature is between about 10°C and 20°C. In the thirteenth point, the growth temperature is between about 15°C and 17°C. In the fourteenth point, the bacteria are grown for a period of 18 hours to 7 days. In the fifteenth point, the bacteria are grown for a period of seven days. In the sixteenth point, the bacteria are grown for a period of between one and two days.

In a second aspect, an agricultural composition comprising a bacterial fermentation or a protected supernatant is provided. The agricultural composition is produced according to the method of the first aspect and any of the points disclosed in relation to the first aspect. In the first respect, the agricultural composition further comprises an adjuvant. In this respect, adjuvants are surface-active substances.

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

In the first respect, the crop disease is selected from the group consisting of fire blight, citrus carcinoma, olive carcinoma, and soft rot, tomato and pepper. In the second respect, the pathogenic microorganisms include Mycospherella fisiensis, Botrytis cinerea, Erwinia amylovora (Ea), Xanthomonas axonopodis pv. citri (Xac), Pectobacterium palmentieri , Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp. orum subsp. carotovorum), Dikeya dadantii, Pseudomonas savastanoi pathotypes savastanoi (Psv), Pseudomonas syringae pv. tomato, Pseudomonas syringae pv syringae, Pseudomonas syringae pv. lacrimans (Pseudomonas syringae pv. lachrymans), Xanthomonas campest Squirrel Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. vesicatoria, Xanthomonas arboricola pv. s arboricola pv. juglandis), Ralstonia solanacearum , Clavibacter michiganensis subsp. michiganensis, Phytophthora infestans, Venturia inaeq ualis), Xanthomonas oryzae pv. oryzae), Xanthomonas oryzae pv. oryzicola, and Xanthomonas citri pv. citri. In the third respect, crops include cruciferous plants such as bananas, apples, pears, crabapples, citrus fruits, potatoes, pumpkins, onions, rice, African violets; carrots, potatoes, tomatoes, eggplants, leafy vegetables, squash and cucurbits; Cruciferae, Solanaceae, Cucurbitaceae, peppers and green peppers, olives; stone fruit and pome fruit plants, including olives, peaches, and walnuts.

In a fourth aspect, a method of controlling bacterial crop diseases is provided. The method includes one step. The step includes applying to the crop an agronomic composition comprising between about 1.0×10 ⁵ and 1.0×10 ⁹ cfu/mL of Pseudomonas bacteria to inhibit growth of pathogenic microorganisms. .

In the 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 point, the composition comprises between about 5.0×10 ⁷ and 2.0×10 ⁸ cfu/mL of Pseudomonas bacteria. In the third point, the crop disease is black sigatoka, botrytis blight, fire blight, citrus carcinoma disease, soft rot, olive carcinoma disease, tomato leaf spot bacterial disease, bacterial carcinoma disease or potato blight. diseases (stone fruits and pome fruits), horn spot of cucurbits, bacterial spot of peaches, bacterial spot of tomato, walnut blight, bacterial wilt, tomato carcinoma, potato leaf blight, apple red selected from the group consisting of mildew, leaf blight, and bacterial streak disease. In the fourth point, the pathogenic microorganism is Mycospherella fisiensis, Botrytis cinerea, Erwinia amylovora (Ea), Xanthomonas axonopodis citri (Xac), Pectobacterium palmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum subspecies brasiliensis, Pectobacterium carotovorum subspecies carotovorum, Dikeya dadantii, Pseudomonas savastanoi Pathotype savastanoi (Psv), Pseudomonas syringae Pathotype tomato, Pseudomonas syringae Pathotype syringae, Pseudomonas savastanoi Syringae Pathotype lacrimans, Xanthomonas campestris Pathotype Pruni, Xanthomonas campestris Pathotype Vesicatoria, Xanthomonas arboricola Pathotype Jugrandis, Ralstonia solanacearum, Clavibacter michiganensis Subspecies michiganensis, Phytophthora infestans, selected from the group consisting of Venturia inaequalis, Xanthomonas oryzae, Pathotype oryzae, Xanthomonas oryzae, Pathotype orydicola, and Xanthomonas citri, Pathotype citri. In the fifth point, the crops include bananas, apples, pears, crabapples, citrus fruits, potatoes, pumpkins, onions, rice, African violets; cruciferous crops such as carrots, potatoes, tomatoes, eggplants, leafy vegetables, squash and cucurbits; selected from one or more of plant species of Solanaceae, Cucurbitaceae, pepper and green pepper, olive; stone fruit and pome fruit plants including olive, peach, walnut.

In a fifth aspect, a method is provided for purifying a protected metabolite from a Pseudomonas bacterium. The method includes several steps. The first step comprises producing a bacterial fermentation or protected supernatant according to the method of the first aspect and in that respect. The second step involves extracting the bacterial fermentation or protected supernatant by ethyl acetate extraction. The third step is by eluting the bacterial fermentation or protected supernatant with a mixture of hexane and ethyl acetate, such as a mixture of 50% hexane and 50% ethyl acetate, or by elution of the bacterial fermentation or protected supernatant, such as a mixture of 25% hexane and 75% acetic acid. eluting the ethyl acetate extract with a mixture of hexane and ethyl acetate, such as a mixture of ethyl acetate, to produce an eluate containing the protected metabolite.

In the 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 sixth aspect, an agricultural composition comprising a protected metabolite from a Pseudomonas bacterium is purified by the method of and in respect of the fifth aspect.

In a seventh aspect, a method of controlling bacterial crop diseases is provided. The method includes several steps. The first step comprises producing an agricultural composition comprising a protected metabolite derived from a Pseudomonas bacterium purified by the method of the fifth aspect or any point thereof. The second step involves applying the agricultural composition to the crop to inhibit the growth of pathogenic microorganisms.

In the first respect, the crop disease is selected from the group consisting of fire blight, citrus carcinoma, olive carcinoma, soft rot, tomato and pepper. In the second respect, pathogenic microorganisms include Mycospherella fisiensis, Botrytis cinerea, Erwinia amylovora (Ea) (particularly streptomycin-resistant E. amylovora strains), Xanthomonas axonopodis pathovar citri (Xac), Pectobacterium pallidum. Mentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum subspecies brasiliensis, Pectobacterium carotovorum subspecies carotovorum, Dikeya dadantii, Pseudomonas savastanoi (Psv), Pseudomonas syringae pathogenic type tomato, Pseudomonas syringae Pathotype syringae, Pseudomonas syringae Pathotype lacrimans, Xanthomonas campestris Pathotype purni, Xanthomonas campestris Pathotype Vesicatoria, Xanthomonas arboricola Pathotype jugrandis, Ralstonia solanacearum, Clavibacter michiganensis species Michiganensis, Phytophthora infestans, Venturia inaecualis, Xanthomonas oryzae, Xanthomonas oryzae, Xanthomonas orygicola, and Xanthomonas citri, Xanthomonas oryzae. In the third point, pathogenic E. Amylovora is a streptomycin-resistant E. It is Amylovora. In the fourth respect, the crops are cruciferous, such as bananas, apples, pears, crabapples, citrus fruits, potatoes, pumpkins, onions, rice, African violets; carrots, potatoes, tomatoes, eggplants, leafy vegetables, squash and cucurbits; , Solanaceae, Cucurbitaceae, peppers and green peppers, olives; stone fruit and pome fruit plants, including olives, peaches, and walnuts. In the fifth point, the pathogenic bacteria are Flavobacterium columnare #2, Flavobacterium columnare MS-FC-4. In the sixth point, the pathogenic bacterium is E. E. coli O157:H7.

In an eighth aspect, the crystalline compound is selected from one of the following structures.

In the first point, the crystalline compound has the following structure:

結晶性化合物は、表１３～２２から選択される少なくとも１つの物性を含む。

The crystalline compound includes at least one physical property selected from Tables 13-22.

In the second point, the crystalline compound has the following structure:

結晶性化合物は、表２３～２９から選択される少なくとも１つの物性を含む。

The crystalline compound includes at least one physical property selected from Tables 23-29.

In the third point, the crystalline compound has the following structure:

結晶性化合物は、表３０～３７から選択される少なくとも１つの物性を含む。

The crystalline compound includes at least one physical property selected from Tables 30-37.

Biological deposits bacterial stocks Monas Soli 0617 -T307, Subu Monas Soli 0917 -T305, Subu Monas Soli 0917 -T306, Subu Demonas Soli 0917 -T307, Subu Demonas Mosceri 0118 -T319, Subud Monas Monas 0318 -T327, and Sched Monas Mosseri 0418-T328 is available from the American Type Culture Collection (ATCC (registered trademark)), P.M. O. Box 1549, Manassas, VA 20110 USA (the "ATCC Patent Depositary") on June 25, 2020, and unofficial ATCC Patent Numbers PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA, respectively. -126800, PTA-126801, and PTA-126802. After viability testing, the ATCC Patent depository has assigned these deposited bacterial strains the following accession number, effective 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 mosselii 0118-T319 ( Accession number PTA-126800), Pseudomonas mosselii 0318-T327 (Accession number PTA-126801), and Pseudomonas mosseri 0418-T328 (Accession number PTA-126802).

[Example 1] Identification and characterization of strain 0617-T307

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)).

For species assignment, these four sequences were used to run BLASTN against the NCBI non-redundant nucleotide database. Based on the results, strain 0617-T307 is P. Fluorescens strain P. It is closely related to Pseudomonas species belonging to the group Putida. The “MLSA phylogenetic tree” and the “genome list derived from standard strains of Pseudomonas species” (Peix et al., (2018); see Figure 2 and Table 2 of Peix et al., (2018)) were used as a guide for taxonomic sampling (Fig. 1). Based on this information, the genome was obtained from GenBank. P. with a high-quality genome assembly. All species of the Putida group were included. 0617-T307 is P. Because it has the highest rpoD (i.e., the gene with the highest resolution for Pseudomonas species assignment) sequence similarity to P. All four available genomes of P. soli were included in the sampling (including the standard strain of P. soli LMG 27941 ^T ). P. For other species of the fluorescens lineage, one species was selected as a representative of each group. P. P. aeruginosa (P. aeruginosa group, P. aeruginosa lineage) was included as an outgroup to create the root of the tree.

Four genes for MLSA were extracted from the sampled genome. After each gene was sequenced individually, all four nucleotide alignments were linked together for phylogenetic analysis. The linked alignment includes 9,912 aligned nucleotide sites. Maximum likelihood estimation was performed using PhyML (Guindon et al. (2003)). Bootstrap support was estimated by 1,000 replicates.

Based on the multilocus molecular phylogenetic tree (Figure 1), 0617-T307 and the four P. elegans for which genome sequences are available. The Soli strains form a monophyletic clade with 100% bootstrap support. This result indicates that 0617-T307 is a standard strain of P. spp. that has been reported to be isolated from soil samples in Sierra Nevada National Park, Spain. provided strong support for assigning it to Soli (Pascual et al., (2014)).

Additionally, 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 is provided below. (a) 16S rDNA>98.7-99% identical. Compared to the standard strain of P. sori, 0617-T307 had 99.2% sequence identity. When compared to P. entomophila, 0617-T307 had 99.5% sequence identity. 16S rDNA is known to lack sufficient resolution for species identification in the genus Pseudomonas. Note that (Garcia-Valdes et al., (2016); Peix et al., (2018)); (b) rpoD gene >95-96% identical. 0617- T307 had 96.5% sequence identity. Compared to its sister species P. entomophila, 0617-T307 had only 89.1% sequence identity; and (c ) MLSA>97% identical. Compared to the standard strain of P. sori, 0617-T307 had 98.0% sequence identity. Compared to its sister species, P. entomophila, 0617-T307 had 98.0% sequence identity. T307 had only 95.1% sequence identity.

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

The preparation of RejuAgro A and B can be obtained by ethyl acetate extraction of the cell broth of the fermentor fermentation followed by chromatographic isolation and purification. Briefly, stock bacteria Pseudomonas sp. 0617-T307 were streaked onto LB plates (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L agar, water) and incubated at 28°C. The cells were grown for 24 hours in an incubator. For seed medium preparation, 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); Grow for 24 hours at 28°C with a shaking speed of 200 rpm. A 20 L NBS fermentor containing 12 L of autoclaved YME medium was then inoculated with the seed medium. Fermentation proceeded for 1-7 days at 16°C. The stirring speed was 200 rpm, and the air flow rate was 2 L/min.

After collection, the bacterial culture was extracted four times with ethyl acetate. The ethyl acetate layer was separated, dried with 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 strain 0617-T307.

The concentrated sample was dissolved in ethyl acetate, mixed with silica gel and packed as an injection column (φ 3.0 × 20 cm), and placed in a silica gel universal column (4) of a flash chromatography system (Yamazen AI-580) equipped with a UV detector. .8 x 18.5 cm). After loading the samples, the samples were tested 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. Samples were eluted at a flow rate of 20 mL/min. The eluate was monitored by UV 254 nm and fractions were collected by time mode at 20 mL/tube. A total of 114 fractions or test tubes were obtained from flash chromatography.

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

In an in vitro plate assay of 114 fractions, E. Two fractions were shown to inhibit the proliferation of Amylobora 273. Notably, fraction/tube 38-40 (which was abbreviated as T3840 or Flash-RejuAgro A) was eluted with 50% hexane/50% ethyl acetate, showing a relatively large clearance that may be promising for further testing. It had a zone. The other bioactive compound in this assay was in fractions 50-52 (which was coded as T5052). These fractions were eluted with 25% hexane/75% ethyl acetate.

As a result of purifying fractions 3840 and 5054 by preparative HPLC (Prep-HPLC), 15 mg of a yellow colored compound RejuAgro A (Rt17.5) and 103.3 mg of a dark green colored compound RejuAgro B were discovered, respectively. RejuAgro A can be dissolved in methanol and chloroform. RejuAgro B (Rt10.5) is not well soluble in methanol and chloroform, but is very well soluble in dimethyl sulfoxide (DMSO) and has a dark green color. The structures of these two compounds were investigated by high-resolution mass spectrometry (HR-MS), infrared (IR), ultraviolet (UV), one-dimensional and two-dimensional nuclear magnetic resonance (NMR), and X-ray crystallography. As a result, the two compounds are structurally similar, and the compound RejuAgro A has seven types of carbon groups (three types of carbonyl groups, two types of tertiary carbons, and two types of methyl carbons), whereas RejuAgro A has seven types of carbon groups (three types of carbonyl groups, two types of tertiary carbons, and two types of methyl carbons). B lacks one methyl group as shown below:

Furthermore, crystals of RejuAgro A were obtained by slowly evaporating the chloroform solution at room temperature. The crystals were identified as orange plate-like pieces. Data sets were collected at 100K on an Oxford SuperNova diffractometer using Cu (Kα) radiation. This molecule has a planar structure, with the S-Me (methyl) group rotated by 8.7° relative to the heterocycle. This molecule shows significant π-conjugation cleavage at the C4-C5 bond (1.531 Å), which clearly has some orbital reason. The Me group attached to the sp ² carbon atom is rotationally disordered in two positions. The molecules in the crystal are NH. ．． ．． A centrosymmetric H-bond dimer is formed by O interaction. Furthermore, these dimers have a weaker C-H. ．． ．． A two-dimensional layer is formed along the [-3 0 1] plane through O interaction. The RejuAgro A molecule exhibits a 6-membered heterocycle [-NH-C(=O)-C(-SMe)=C(-Me)-C(=O)-C(=O)-]. The crystals of RejuAgro B were identified as triclinic. The structure of RejuAgro B includes two symmetrically independent molecules. Each molecule has a twisted structure, and the dihedral angles between the average planes of the connected heterocycles are 70.3° and 80.6°. In each heterocycle, significant π-conjugation cleavage is observed at the C(sp ² )-C(sp ² ) bond between two adjacent carbonyl groups (bond length is 1.534-1.539 Å). range), there is clearly some orbital reason for this. The molecules in the crystal are NH. ．． ．． A centrosymmetric H-bond dimer is formed by O interaction. These dimers are similar to other N-H. ．． ．． They are stacked and connected along the x direction by O interaction. Finally, the stack is the third type of N-H. ．． ．． Due to the O interaction, they are connected in a layered manner along the [0 1 1] direction. When RejuAgro B solution was used for crystal growth, two crystals were obtained and named RejuAgro B and RejuAgro C. Both RejuAgro B and RejuAgro C crystals have very similar molecular weights (see Example 20).

Crystal structure information for RejuAgro A, RejuAgro B, and RejuAgro C is provided in Example 20, the contents of which constitute a part of this application and are incorporated by reference in its entirety.

The molecular formula of RejuAgro A is C ₇ H ₇ NO ₃ S, and the molecular weight is 185.2004. This agrees with the molecular species m/z of [M+H] observed in HR-MS data, m/z 186.2177 (theoretical value 186.2083). The molecular formula of RejuAgro B is C ₁₂ H ₈ N ₂ O ₆ S, and the molecular weight is 276.2017. This agrees with the molecular species m/z of [MH] observed in HR-MS data, m/z 275.0278 (theoretical value 275.1960). CCDC structure database search as of August 4, 2020 suggests that crystal structures for RejuAgro A, RejuAgro B, and RejuAgro C do not exist. Other chemical databases such as SciFinder, Reaxys, Google Patent and Patent Related Database Searches showed no similarity to RejuAgro A or RejuAgro C except for one publication (Knackmuss et al., (1968)) for RejuAgro B from SciFinder and Reaxys. The body was shown not to exist.

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

A wild-type Gram-negative phytopathogenic bacterium, streptomycin-resistant E. The MIC values of RejuAgro A and RejuAgro B were determined for five types of bacteria: Amylobora, 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 fungus tested were streaked onto LB (Luria-Bertani) plates (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium salts, 15 g/L agar). For special culture, NA (nutrient broth + agar) 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 Xac. SHIEH (5g/L tryptone, 0.5g/L yeast extract, 0.01g/L sodium acetate, 0.01g/L _BaCl2 ( _H2O ) ₂ , _0.1g /L _K2HPO4 , 0.05g /L _KH2PO4 , _{0.3g /} L _MgSO4.7H2O , 0.0067g/L _CaCl2.2H2O , _0.001g /L _FeSO4.7H2O , _0.05g /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 CaCl ₂ , pH 7.2, 15 g/L agar), respectively. It was used against Bacterium columunale strains MS-FC-4 and #2. A single colony was then picked from the plate and inoculated into the corresponding liquid medium and grown overnight. This culture was diluted with LB or the corresponding medium to OD ₅₉₀ =0.01 and distributed in 96-well plates at 200 μL/well. Compounds RejuAgro A and RejuAgro B and streptomycin were diluted and 4 μL of each concentration was added to each well, with 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) was used as a control.

The assay results showed that the most active metabolite of strain 0617-T307 was RejuAgro A rather than RejuAgro B. Gram-positive bacteria MRSA (MIC>40 μg/mL) and Gram-negative bacteria E. RejuAgro A has a MIC value of 40 μg/mL when compared to its efficacy against K. coli O157:H7 (an important food- and water-borne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans) (MIC = 40 μg/mL). It is particularly efficient against test bacteria of 5-40 μg/mL. The antibacterial activity of RejuAgro A was shown to be against Erwinia amylovora 1189, Xanthomonas axonopodis pathotype citri, Pseudomonas savastanoi pathotype sabastanoi, Pectobacterium palmentieri UPP163 936, Pectobacterium carotovorum subsp. brasiliensis 944, Pectobacterium E. carotovorum subspecies carotovorum wpp14 945 and D. dadantii 3937 strains are equivalent to streptomycin; The MIC value for Amylobora was 5 μg/mL and for other weakly pathogenic bacteria 20-40 μg/mL. Xanthomonas bacteria are highly susceptible to Streptomyces with a MIC value of 0.16 μg/mL, which is lower than the MIC value for RejuAgro A of 5 μg/mL. The MIC value of RejuAgro A against Pseudomonas savastanoi pathotype is 40 μg/mL. The MIC value of RejuAgro A against Xanthomonas arboricola pathotype Jugrandis 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 for Clavibacter michiganensis subspecies 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.

In addition, RejuAgro A is a pathogenic E. Amylobora 1 strain and streptomycin-resistant E. Other E. coli including 3 strains of Amylobora. It was also tested against Amylobora strains. E. RejuAgro A showed the same effect as streptomycin against Amylobora 110 (MIC value 5 μg/mL). However, RejuAgro A is E. It is more effective than streptomycin against Amylovora 1189. E. The MIC values of RejuAgro A and streptomycin against Amylobora 1189 are 5 μg/mL and 10 μg/mL, respectively. In addition, a lower MIC value (10 μg/mL) than that of streptomycin (>40 μg/mL) was observed for RejuAgro A, indicating that streptomycin-resistant E. It is more effective against Amylobora CA11, DM1 and 898. These results show that RejuAgro A is superior to E. It was the most potent compound tested against Amylovora, suggesting it may be a potential alternative to streptomycin. There is no sign of cross-resistance to RejuAgro A in streptomycin-resistant strains.

Regarding Flavobacterium, which is the causative agent of columnaris disease in fish, RejuAgro A has an MIC value of 5 μg against Flavobacterium columnarie MS-FC-4 strain and strain #2 (which causes columnaris disease in wild and farmed fish). /mL, which was higher than the MIC value of streptomycin (0.31 μg/mL and 1.25 μg/mL for strain #2 and MS-FC-4, respectively).

The effect of RejuAgro A on the 0617-T307 strain was verified. The MIC value of RejuAgro A against Pseudomonas sori 0617-T307 (RejuAgro A-producing strain) was shown to be greater than 40 μg/mL in the tested LB medium, indicating that the 0617-T307 strain at least contained 40 μg/mL produced by itself. It means survival and resistance to RejuAgro A.

RejuAgro A is a tomato pathogen (P. syringae tomato PT30, P. syringae 7046, P. syringae lacrimans 1188-1) and other citrus carcinoma disease pathogens (Xanthomonas campestris tomato). Pruni, Xanthomonas campestris (P. vesicatoria XV-16) was tested with streptomycin. P. The MIC value of RejuAgro A against S. syringae was 40 μg/mL, and the MIC value of streptomycin was 2.5 to 5 μg/mL. X. For Campestris species, the MIC value of RejuAgro A is 2.5 μg/mL or 40 μg/mL, which is small compared to the MIC value of streptomycin of 20 μg/mL or over 40 μg/mL. These results indicate that the pathogen of Xanthomonas campestris is more sensitive to RejuAgro A than to streptomycin when compared to the pathogen of tomato caused by the genus Pseudomonas.

RejuAgro A was effective against all pathogenic fungi tested (Table 1). RejuAgro A was tested against Phytophthora infestans, Venturia inaequalis, and Mycospherella fisiensis. RejuAgro A has P. Infestans and V. It showed 100% inhibition against Ina equalis (Table 1).

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

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

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 is to extract the cell broth with ethyl acetate (1 mL:1 mL, vortex 1 min) and obtain the ethyl acetate extract by centrifugation and vacuum drying of 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. Another method is to centrifuge the cell broth to obtain a supernatant, then mix the supernatant with an equal volume of methanol to make a 50% methanol solution, and inject 10 μL of the solution into the LC-MS. be. Since RejuAgro A is produced by extracellular secretion and it was confirmed that a large amount of RejuAgro A was contained in the supernatant rather than inside the cells, the second method was adopted (Figure 3, Panel A).

During the 7-day fermentation, the total production of RejuAgro A reached a peak concentration on the first day and then started to decrease with increasing time (Fig. 3, panel B). Furthermore, in the shake flask fermentation, detailed studies were conducted on the production amount and cell concentration of RejuAgro A every 6 hours. The concentration of RejuAgro A (total amount of RejuAgro A) reached a maximum value of 13.8 mg/L in 18 hours, and the bacterial cell concentration reached a maximum value of 2 × 10 ¹¹ CFU/mL in 12 hours, which indicates that the production of RejuAgro A was shown to be a production process associated with cell proliferation.

The volume of medium in the 4L shake flask influences the production of RejuAgro A. In a 4L flask using YME medium, production of RejuAgro A was confirmed only in the volume size of 500 mL, and not in the volume sizes of 1.0 L and 1.5 L. This observation indicates that RejuAgro A production prefers to exist in high aeration conditions.

The type of medium and culture temperature affect the production of RejuAgro A. LB medium was tested in parallel with YME medium at 16°C or 28°C. Production of RejuAgro A was confirmed in YME medium at 16°C, but not in LB medium. For 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 the production of RejuAgro A is medium specific and temperature dependent. Activity against products derived from E. 0617-T307 is This is consistent with RejuAgro A production, as monitored by plate assay for Amylobora.

In order to confirm the applicability of the production conditions of RejuAgro A, in parallel with the Pseudomonas 0617-T307 strain, 10 other Pseudomonas strains were also tested under the same conditions. According to housekeeping gene analysis, 0917-T305, 0917-T306, and 0917-T307 were identified as Pseudomonas sori, and 0118-T319, 0318-T327, and 0418-T328 were identified as Pseudomonas mosselii. . Both type strains of Pseudomonas sori and Pseudomonas mosselii have been reported (Daboussi et al. (2002); Pascual et al. (2014)).

It was shown that strain 0617-T307 and its closely related species can produce RejuAgro A at 28° C. and 220 rpm YME. This result suggests that this method has specificity for producing RejuAgro A for strain 0617-T307 and some of its related species (Table 2). RejuAgro A is present and stable at room temperature for at least 4 weeks as tested by LCMS on a 40 hour culture obtained by culturing 0617-T307 in YME medium at 16° C. on a shaker at 220 rpm.

Example 5 Cell broth of strains 0617-T307 and E. Antibacterial activity against Amylobora.

Two assays were used for antimicrobial testing of cell broth and metabolites of 0617-T307. One is a plate diffusion assay and the other is a microplate assay. E. LB plates were used for plate diffusion assays of the antibacterial activity of RejuAgro A-containing fractions and cell broth against Amylobora (Table 3). Both the cell broth containing live cells of E. 0617-T307 and the suspension containing 2 mg/mL RejuAgro A were derived from E. 0617-T307. It showed antibacterial activity against Amylobora. However, no inhibition circle was observed when applying Serenade®.

In order to find a biological control method consisting of both 0617-T307 cells and the active ingredient RejuAgro A, the following experiments were conducted. For the antibacterial assay against 0617-T307, the producer of RejuAgro A, the supernatant (abbreviated as "supernatant") of cell broth cultured for 40 hours of 0617-T307 containing RejuAgro A was used. It was shown that the 0617-T307 strain could be grown in LB medium, but not in YME medium, by diluting the supernatant two-fold. Further studies showed that the inhibitory effect of the supernatant was due to the low pH value. By then controlling the pH to 6.5 to 6.8, questions 1 and 2 can be answered with "yes".

The bioactive fraction (crude extract, 100 μg/mL; flash-RejuAgro A, 20 μg/mL; HPLC-RejuAgro A, 10 μg/mL) was tested against strains 0617-T307, Ea and Xac. It was shown that the bioactive fraction was not able to inhibit the growth of strain 0617-T307, indicating that RejuAgro A can be mixed with 0617-T307 cells to prepare a biocontrol agent. The bioactive fraction containing RejuAgro A showed an inhibitory effect on Ea and Xac, especially flash-RejuAgro A and HPLC-RejuAgro A almost stopped the proliferation of Ea and Xac under the tested conditions. This demonstrates that RejuAgro A solution can be used for biocontrol of fire blight and citrus carcinoma disease at 10-20 μg/mL.

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

Stock bacteria Pseudomonas sp. 0617-T307 was 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 in an incubator at 28°C for 24 hours. I let it happen. For seed medium preparation, 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 shaken at 150 rpm at 28 °C. Grow at speed for 24 hours. The seed medium was then inoculated into eight 4L flasks each containing 2L of autoclaved YME medium. Fermentation proceeded for 7 days in a shaker at 16° C. and a shaking speed of 150 rpm.

After 7 days of growth, the supernatant was obtained by centrifuging the bacterial culture at 4000 rpm for 15 min. 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 strain 0617-T307.

The concentrated sample was dissolved in acetone, mixed with silica gel, and loaded onto a silica gel column (φ3.0×20 cm) of a flash chromatography system (Yamazen AI-580) equipped with a UV detector. After loading the samples, the samples were tested 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. Samples were eluted at a flow rate of 20 mL/min. The eluate was monitored by UV 254 nm and fractions were collected by time mode at 20 mL/tube. A total of 114 fractions or test tubes were obtained from flash chromatography.

The generated fractions were applied to subsequent plate assays. 1 mL of each fraction was collected in a 1.5 mL test tube and vacuum dried in an Eppendorf vacuum concentrator. The dried sample was dissolved in 50 μL of DMSO, of which 2 μL was used for plate assay. Briefly, Erwinia amylovora 273 was inoculated onto 50% LB (5.0 g/L tryptone, 2.5 g/L yeast extract, 5.0 g/L NaCl) plates, and single colonies were transferred to 5 mL LB medium. I will be vaccinated. Bacteria were to be diluted 1:100 in sterile water, of which 225 μL was plated on 50% LB plates. After drying in a biosafety cabinet for 10 minutes, the DMSO solution of each fraction was dispensed onto a pre-labeled area of the Petri dish and allowed to dry for an additional 10 minutes. Along with the assay, DMSO and kasugamycin were used as negative and positive controls, respectively. The plates are then incubated in a 28° C. incubator and inhibition zones are confirmed after one day.

In an in vitro plate assay of 114 flash fractions, E. Three bioactive fractions (T3234, T5058, T7882) were shown to inhibit the growth of Amylobora 273. Fractions 3234 and 5258 showed relatively small clearance zones. Fraction 3234 is eluted with 50% hexane/50% ethyl acetate. Fraction 5058 was eluted with 25% hexane/75% ethyl acetate. As a negative control, DMSO did not show a circle of inhibition, and a positive control, kasugamycin, showed a circle of inhibition. Another flash fraction, T7882, was eluted with acetone/ethyl acetate (50%/50%). This is E. The proliferation of Amylobora activity was similarly inhibited.

Furthermore, anti-E. Isolation and purification by HPLC based on Amylobora activity resulted in two antibacterial compounds (Rt22.9 and Rt25.0) from T5058 (see compound formulas, 0617_T307_5058_Rt22.9 and 0617_T307_5058_Rt25.0), one antibacterial compound from T7882 The compound (Rt18.9) (see compound formula 0617_T307_7882_Rt18.9) was identified. T307_5058_Rt22.9 and T307_5058_Rt25.0 are tryptophan-derived natural products whose structures have been reported in the Scifinder database but no biological activity (Loots et al. (2015)). 0617_T307_7882_Rt18 was predicted to be a previously reported derivative of difuryl (Osipov et al. (1978)). These natural products are shown below:

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

Crude extracts of non-pH adjusted cell broth and pH adjusted cell broth (pH of cell broth was adjusted to 2.0 with 6N HCl) were concentrated and added to 250 μL 100% MeOH containing internal standard (m/z 311.08). and used 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 with Bruker Maxis Impact II. Data were acquired by 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) using collisionally activated dissociation (CID) fragmentation of the eight most abundant ions in the spectrum. The scan rate was 3Hz.

A precise spectral library search was then performed based on bioinformatics analysis and molecular network analysis with the aim of identifying new and known compounds. The MS/MS spectra of the samples were searched against the following spectral libraries: 1) GNPS Community Library, 2) FDA Library, PhytoChemical Library, 3) NIH Clinical Collections, 4) NIH Natural Pr 5) Pharmacologically Active NIH Small Molecule Repository, 6) Faulkner Legacy Library, 7) Pesticides, 8) Dereplicator Identified MS/MS Peptidic Natural Produ cts, 9) PNNL Lipids, 10) Massbank, 11) Massbank EU, 12) MoNA, 13) ReSpect-Phytochemicals, 14) HMDB.

The MS/MS spectra of the samples were searched in the library described above and aligned with an offset to the reference spectrum. Matching parameters were the same. These results can be explored to identify structural analogs of known compounds. MS/MS molecular networks were generated with minimum cluster size = 2, minimum edge 0.7 cosine, and minimum number of matching peaks 6. As an example, a new molecular species with m/z 303.16 was identified that corresponds to a new compound from active fraction 0617-T307_5058_Rt25.0. Some of the known compounds were confirmed from the crude extract, and the crude extract contains indole-3-carboxylic acid and xantholysin A, which are plant growth promoting factors. 1) P. It has been reported that the broad antifungal activity of P. putida BW11M1 mainly depends on xantholysine production; 2) xantholysine is required for swarming and contributes to biofilm formation (Li et al. (2013)). In fact, higher concentrations of xantholysine A were observed by culturing 0617-T307, 0418-T328, and 0318-T327 at 28°C. That is, except for the bioactive compound RejuAgro A, xantholysine A is a metabolite that contributes to the antibacterial activity of the biocontrol bacteria 0617-T307 and its relatives 0318-T3027 and 0418-T328.

Example 8 Greenhouse and field infection assays of RejuAgro A producing strain 0617-T307 and several of its relatives.

To evaluate the biocontrol activity of 0617-T307 against Erwinia amylovora, infection assays were performed using crabapple trees in a greenhouse at the University of Wisconsin-Milwaukee. Biocontrol agents (0617-T307, 0717-T327, 0617-T318) containing 1.0×10 ⁸ cfu/mL were applied to 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, then the cells were inoculated (1:100) into LB medium and incubated at 28°C on a shaker at 200 rpm for 14 to 30 minutes. Grow for 18 hours. Cells were harvested and resuspended in 10x water to 10 ⁸ CFU/mL. This resuspension can be used in field assays for fire blight control in greenhouses and fields. Control flowers were sprayed with distilled water. All flowers were then injected with 1.0 x 10 ⁶ cfu/mL of E. It was inoculated by spraying the Amylovora strain Erwinia amylovora 273. The treatment using 0617-T307 was carried out three times on September 7, October 9, and October 19, 2018. Referring to Table 4, all spray treatments of 0617-T307 (Pseudomonas sori) resulted in 100% control of flower blight symptoms against 0% control of distilled water on crabapple flowers; E. It was suggested that it could be a promising biological control agent for fire blight caused by Amylobora. Control rates for the other two Pseudomonas species, 0717-T327 (Pseudomonas coriensis) and 0617-T318 (Pseudomonas protegens), were lower at 16.7% and 25%, respectively. In conclusion, among the three Pseudomonas species tested, only 0617-T307 shows a good control effect on crabapple fire blight. No phytotoxicity was observed.

In field assays, biocontrol bacteria producing RejuAgro A (0617-T307, 0118-T319, 0318-T327 , 0418-T328; see Table 2) was applied to the flowers of apple trees in an orchard at a concentration of 5×10 ⁸ CFU/mL. Bacterial pathogen E. Amylobora Ea110 was inoculated on May 7th (90% flowering) at a concentration of 5×10 ⁶ CFU/mL. The diseased inflorescence rates for water control, streptomycin, 0617-T307, 0118-T319, 0318-T327, and 0418-T328 were 32.9%, 13.3%, 16.8%, 18.5%, and 16.7, respectively. %, 11.8%. Compared to streptomycin, the biocontrol bacteria producing RejuAgro A is equally or more effective in controlling fire blight in apple orchards.

[Example 9] Antifungal activity of RejuAgro A and B and their producing bacteria against Venturia inaequalis.

The fungus Venturia inaequalis, which causes apple head blight, was maintained on PDA agar in the dark at room temperature (approximately 24°C). A mixed suspension of conidia and mycelia (in 0.01 M PBS) was collected from PDA (potato dextrose agar). 10 μL of conidial and hyphal suspensions were dropped onto biological control bacteria, RejuAgro A, or RejuAgro A modified plates. Controls were PDA plates without the addition of biocontrol bacteria, RejuAgro A or B. Petri dishes were incubated at room temperature in the dark and after 7 days V. The diameter of each colony of Ina equalis was confirmed.

When compared to the control (Figure 4), the four selected biocontrol bacterial strains 0617-T307, 0118-T319, 0318-T327, and 0418-T328 were found to be more susceptible to V. The proliferation of Ina equalis can be suppressed (Fig. 5). RejuAgro A was added to V.I. on PDA plates at 40-80 μg/mL. The proliferation of Ina equalis can be suppressed (Figure 6). However, on PDA plates at 10-80 μg/mL, V. No inhibitory effect of RejuAgro B on the proliferation of Ina equalis was observed (FIG. 7). Finally, V. No inhibition of Ina equalis was observed (Figure 8).

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

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

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

Example 11 RejuAgro A formulation and greenhouse assay.

Formulation of RejuAgro A (solution, SL; see Table 5). Prior to application to flowers, 10 μg/mL was tank mixed with 1% polyethylene glycol (PEG) 4000 as a safener. Subsequent tests showed that using 0.03% polyvinyl alcohol (PVA) as a safener provided better flower protection. Additionally, Alligare 90, a surfactant, can be added to obtain a higher effect (Table 6).

To evaluate the biocontrol activity of RejuAgro A against Erwinia amylovora, a greenhouse infection assay using crabapple trees was conducted at the University of Wisconsin-Milwaukee. 10 μg/mL with 1% polyethylene glycol (PEG) 4000 or 1% PEG 4000 (negative control) was applied to fully bloomed flowers 3 hours before and 24 hours after inoculation. For inoculation, E. Approximately 10 ⁸ CFU/mL of Amylobora 110 strain was used. Infection rates were calculated around day 6 after inoculation. The experiment was conducted for one week from January 24th to January 31st, 2020. RejuAgro A can effectively suppress flower blight (Table 6).

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

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

[Example 13] Antibacterial activity of crude extracts, RejuAgro A and RejuAgro B against phytopathogenic bacteria.

The metabolites of bacteria 0917-T305, 0318-T327 and 0418-T328 are R. Solanacearum, C. michiganensis subspecies michiganensis and X. michiganensis. It showed excellent efficacy against Arboricola arboricola pathogenic type Jugrandis (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 combined with three plant pathogens, Ralstonia solanacearum, Clavibacter michiganensis subsp. michiganensis, and Xanthomonas arboricola. It was tested by plate diffusion assay against the pathogenic type S. jugrandis. In agar plate diffusion assays, metabolites of bacteria 0917-T305, 0318-T327, 0418-T328 grown in YME at 16°C and 28°C were compared to the tested R. Solanacearum, C. michiganensis subspecies michiganensis, X. It showed a relatively good effect against Arboricola pathotype Jugrandis (Table 7). This indicates that, along with RejuAgro A, other metabolites also have excellent effects against Ralstonia solanacearum, Clavibacter michiganiensis subspecies michiganensis, and Xanthomonas arboricola pathotype juglandis. RejuAgro B shows good efficacy against Ralstonia solanacearum (Table 7).

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

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. I let it happen. Fermentation and crude extract preparation were carried out similarly to that described in Example 6.

HPLC separation and purification of ethyl acetate extract of acidified cell broth of Pseudomonas sp. antibacterial compound (Rt18.9) was identified. These were tested for antibacterial activity against the bacterial strains shown in Table 8. 2 μL of DMSO, Rt18.9, Rt22.9 or Rt25.0 were each spotted on agar plates on which different bacterial strains were grown and the inhibition zones were further tested (Table 8).

[Example 15] Antibacterial effect of RejuAgro A against Mycospherella fisiensis

The antibacterial effect of RejuAgro A against Mycosphaerella fisiensis was examined by adding final concentrations of HPLC-purified RejuAgro A of 60 μg/mL and 600 μg/mL to PDA agar medium, respectively. 480 μL of 0.5 mg/mL or 5 mg/mL RejuAgro A was added to 3.52 mL of PDA in the wells of a 6-well plate, resulting in a final concentration of RejuAgro A of 60 μg/mL each (Figure 12, center well (panel). A)) and 600 μg/mL (Figure 12, left well (panel B)). 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 solidification of the agar, M. A piece of agar on which P. fisiensis was grown was placed in the center of the agar surface. M. A complete inhibition of the growth of S. physiensis was confirmed 2 weeks after inoculation when treated with RejuAgro A at a concentration of 600 μg/mL (FIG. 12).

[Example 16] Antibacterial effect of RejuAgro A against Xanthomonas oryzae pathogenic type oryzae (Xon507)

The antibacterial effect of RejuAgro A against Xanthomonas oryzae (Xon507) was investigated. X. A bacterial suspension ( _OD600 = 0.3) of S. oryzae (Xon507) was sprayed onto PSG agar plates. Aqueous RejuAgro A purified by HPLC was added to 50 μL 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, respectively. Paper disks loaded with the loading volume were placed on agar plates and the inhibition circle was measured 44 hours after placing the paper disks. Inhibition was observed at all concentrations of paper discs impregnated with RejuAgro A suspension (Table 9).

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

The antibacterial effect of RejuAgro A against Xanthomonas citri pathogenic type Citri citrangae (XW19) was investigated. X. A bacterial suspension (OD ₆₀₀ =0.3) of the pathotype Citri citrangea (XW19) was sprayed onto PSG agar plates. Aqueous RejuAgro A purified by HPLC was added to 50 μL 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, respectively. Paper disks loaded with the loading volume were placed on agar plates, and the inhibition circle was measured 44 hours after placing the paper disks on the agar plates. Inhibition was observed at concentrations of RejuAgro A of 55.37 μg/mL and 110.74 μg/mL (Table 10).

[Example 18] Medium culture composition used in the example

Table 11 includes exemplary media compositions used in the examples.

[Example 19] Bacterial strains, natural products, and literature cited therein.

The bacterial strains and natural products described in this application and set forth in the appended claims are well known in the microbiological literature. These documents are shown in Table 12 below for each of the cited bacterial strains and natural products disclosed herein, the contents of which are incorporated herein by reference in their entirety.

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

A. Crystal structure information of RejuAgro A A single crystal of RejuAgro A (C ₇ H ₇ NO ₃ S) was obtained by slowly evaporating a chloroform solution of RejuAgro A. Orange plate-like pieces were obtained. A suitable crystal was selected and mounted on a SuperNova, Dua, Cu home/nearby, Atlas diffractometer. The crystal was held at 100.05 (10) K during data collection. Using Olex2 (Dolomanov et al. (2009)), structures were solved by the ShelXS structure solver using direct methods (Sheldrick, 2008) and with the ShelXL refinement package (Sheldrick, 2009) using least squares minimization. G.M. (2015)).

Data sets were collected at 100K on an Oxford SuperNova diffractometer using Cu (Kα) radiation.

Crystal data of RejuAgro A (C ₇ H ₇ NO ₃ S) (M = 185.20 g/mol): monoclinic, space group P2 ₁ /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 R ₁ was 0.0253 (I>2σ(I)) and wR ₂ was 0.0702 (all data).

The description of the refined model was created in Olex2 and published on May 29, 2018 in OlexSys svn. Compiled to r3508. Number of restraints - 0, number of restraints - unknown. Details: 1. Fixed Uiso: 1.2 times: all C (H, H, H, H, H, H) groups, 1.5 times: all C (H, H, H) groups, 2. Others: Sof(H6A)=Sof(H6D)=Sof(H6F)=1-FVAR(1); Sof(H6B)=Sof(H6C)=Sof(H6E)=FVAR(1);3. a Disordered Me:C6 refined as a rotating group (H6A, H6B, H6C, H6D, H6E, H6F); b. Idealized Me:C7 (H7A, H7B, H7C) refined as a rotating group.

Referring to FIG. 13A, the RejuAgro A molecule has a planar structure in which the S-Me group is rotated by 8.7° relative to the heterocycle. This molecule shows significant π-conjugation cleavage at the C4-C5 bond (1.531 Å), which clearly has some orbital reason. The Me group attached to the sp ² carbon atom is rotationally disordered in two positions.

Referring to FIG. 13B, RejuAgro A molecules in the crystal are NH. ．． ．． The O interaction forms a centrosymmetric H-bonded dimer. Furthermore, these dimers have a weaker C-H. ．． ．． A two-dimensional layer is formed along the [-3 0 1] plane through O interaction.

The chemical structure of RejuAgro A is shown below:

Additional crystallographic data for RejuAgro A molecules are shown in Tables 13-21.

B. Crystal structure information of RejuAgro B A single crystal of RejuAgro B (C ₁₂ H ₈ N ₂ O ₆ ) was obtained by slowly evaporating 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 held at 100.05 (10) K during data collection. Using Olex2 (Dolomanov et al. (2009)), structures were solved by the ShelXS structure solver using direct methods (Sheldrick (2008)) and with the ShelXL refinement package (Sheldrick (2008)) using least squares minimization. (2015)).

Data sets were collected at 100K on an Oxford SuperNova diffractometer using Cu (Kα) radiation.

Crystal data of RejuAgro B (C ₁₂ H ₈ N ₂ O ₆ ) (M=276.20 g/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) = 1.139 mm ^-1 , Dcalc = 1.605 g/cm ³ , 15292 reflections measured (7 .872°≦2Θ≦141.144°), 4304 independent (R _int =0.0258, R _sigma =0.0234), which were used in all calculations. The final R ₁ was 0.0419 (I>2σ(I)) and wR ₂ was 0.1124 (all data).

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

Referring to FIG. 14A, a RejuAgro B crystal contains two symmetrically independent RejuAgro B molecules. Each molecule has a twisted structure, and the dihedral angles between the average planes of the connected heterocycles are 70.3° and 80.6°. In each heterocycle, significant π-conjugation cleavage is observed at the C(sp ² )-C(sp ² ) bond between two adjacent carbonyl groups (bond length is 1.534-1.539 Å). range), there is clearly some orbital reason for this.

Referring to FIG. 14B, RejuAgro B molecules in the crystal are NH. ．． ．． The O interaction forms a centrosymmetric H-bonded dimer. These dimers are similar to other N-H. ．． ．． Finally, the stack is a third type of NH. ．． ．． Due to the O interaction, they are connected in a layered manner along the [0 1 1] direction.

The chemical structure of RejuAgro B is shown below:

Additional crystallographic data for RejuAgro B molecules are shown in Tables 22-29.

C. Crystal structure information of RejuAgro C Single crystals of RejuAgro C (C ₁₀ H ₁₆ N ₂ O ₇ ) were obtained by slowly evaporating methanol solutions of RejuAgro B and RejuAgro C. With RejuAgro B, colorless needles were obtained. A suitable crystal was selected and mounted on a SuperNova, Dual, Cu home/nearby, Atlas diffractometer. The crystal was held at 100.05 (10) K during data collection. Using Olex2 (Dolomanov et al. (2009)), the structure was modified using Charge Flipping. Solved with the solve structure solving program (Bourhis et al. (2015)) and refined with the ShelXL refinement package (Sheldrick (2015)) using least squares minimization.

Data sets were collected at 100K on an Oxford SuperNova diffractometer using Cu (Kα) radiation.

Crystal data of RejuAgro C (C ₁₀ H ₁₆ N ₂ O ₇ ) (M=276.25 g/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) = 1.081 mm ⁻¹ , Dcalc = 1.466 g/cm ³ , 7480 reflection measured (10 .068°≦2Θ≦140.528°), 2353 independent (R _int =0.0405, R _sigma =0.0373), which were used in all calculations. The final R ₁ was 0.0504 (I>2σ(I)) and wR ₂ was 0.1388 (all data).

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

Referring to FIG. 15A, the RejuAgro C molecule has a π-conjugated planar shape in which the amide group is rotated by 42° from the plane of other atoms.

Referring to FIG. 15B, RejuAgro C molecules in the crystal are stacked along the x-axis. This stack is N-H. ．． ．． They are connected in layers along the ab plane by O H bonds. This layer is connected in a three-dimensional network with solvated water molecules (3 molar equivalents) via multiple hydrogen bonds.

The chemical structure of RejuAgro C is shown below:

Additional crystallographic data for RejuAgro C molecules are shown in Tables 30-37.

INCORPORATION BY REFERENCE All documents, publications, patents, patent applications, and related materials cited herein are incorporated by reference as if fully set forth herein.

Biological deposit information
Dr. Ching-Hong Yang, one of the inventors, of 10120 North Sheridan Drive, Mequon, Wisconsin 53902, United States of America, is the author of the Bacterial strains Pseudomonas sori 0617-T307, Pseudomonas sori 0917-T305 , Pseudomonas sori 0917-T306, Pseudomonas sori 0917-T307, Pseudomonas mosselii 0118-T319, Pseudomonas mosselii 0318-T327, and Pseudomonas mosselii 0418-T328 were purchased from the American Type Culture Collection (ATCC®), P. mosselii. O. Box 1549, Manassas, VA 20110 USA (the ``ATCC Patent Depository'') on June 25, 2020 . After viability testing, the ATCC Patent depository has assigned these deposited bacterial strains the following accession number, effective 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 mosselii 0118-T319 ( Accession number PTA-126800), Pseudomonas mosselii 0318-T327 (Accession number PTA-126801), and Pseudomonas mosseri 0418-T328 (Accession number PTA-126802). Dr. Yang hereby unconditionally and irrevocably grants applicant permission to include the disclosure of this biological deposit in this application and to make it available to the public as of the filing date. giving consent.

Claims (53)

A method of growing bacteria to promote production of protective metabolites, the method comprising:
i. growing Pseudomonas bacteria in a liquid medium in a container to produce a bacterial ferment, the ratio of medium volume to container volume being between about 1:2 and 1:10; shaking the container at a speed between about 100 and 250 RPM;
or ii. A method of growing Pseudomonas bacteria in a liquid medium in a fermenter to produce a bacterial fermentation, the method comprising the steps of: growing Pseudomonas bacteria in a liquid medium in a fermenter, the air flow rate of the fermenter being between about 1 and 3 L/min. 2. The method of claim 1, further comprising separating the liquid medium from the bacteria after a period of time to produce a protected supernatant containing protected metabolites. The bacteria are 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 0318-T327 (accession number PTA-126801), and Pseudomonas・Mosserii 0418- 3. The method of claim 1 or 2, wherein the method is selected from the group consisting of T328 (accession number PTA-126802). A method according to any of claims 1 to 3, wherein the growth temperature is between about 10°C and 35°C. The method according to any one of claims 1 to 4, wherein the liquid medium is an LB medium for cell production. The liquid medium has formula (I) and formula (II)

The method according to any one of claims 1 to 4, which is a YME medium for the production of. A method according to any of claims 1 to 6, wherein either a shaker or a fermenter can be used. 7. A method according to any of claims 1 to 6, wherein when using a shaker, the ratio of medium volume to container volume is between about 1:5 and 1:10. 7. A method according to any of claims 1 to 6, wherein when using a shaker, the ratio of medium volume to container volume is between about 1:7 and 1:9. 7. A method according to any of claims 1 to 6, wherein when using a shaker, the ratio of medium volume to container volume is approximately 1:8. 11. A method according to any preceding claim, wherein the container is shaken at a speed of between about 200 and 250 RPM. 11. A method according to any preceding claim, wherein the container is shaken at a speed between about 210 and 230 RPM. When using a fermenter, the air flow rate of the fermenter is between about 1.5 and 2.5 L/min, or the oxygen concentration is between about 5 mg/L and 12 mg/L, claims 1-6. The method described in any of the above. 14. The method of any of claims 1-13, wherein the growth temperature is between about 10°C and 20°C. 14. The method of any of claims 1-13, wherein the growth temperature is between about 15°C and 17°C. A method according to any of claims 1 to 15, wherein the bacteria are grown for a period of 18 hours to 7 days. A method according to any of claims 1 to 15, wherein the bacteria are grown for a period of at least 7 days. 16. A method according to any of claims 1 to 15, wherein the bacteria are grown for a period of between 1 and 2 days. An agricultural composition comprising a bacterial fermentation product or a protected supernatant produced by the method according to any of claims 1 to 18. Agricultural composition according to claim 19, wherein the formulation of the protected supernatant or its metabolites is selected from solutions (SL), soluble powders (SP), soluble granules (SG) and encapsulated formulations. Agricultural composition according to claim 19, wherein the bacterial fermentate and cell formulation is selected from suspension concentrates (SC), wettable powders (WP) and water-dispersible granules (WG). 20. The agricultural composition of claim 19, further comprising an adjuvant. 23. Agricultural composition according to claim 22, wherein the adjuvant is a surfactant. 20. Agricultural composition according to claim 19, wherein the adjuvant is the selected active ingredient, water and a polar solvent. 24. Agricultural composition according to claim 23, wherein the surfactant is selected from wetting agents and spreading agents. 26. Agricultural composition according to claim 25, wherein the surfactant is a wetting agent compatible with alkyl polyethylene oxide or polypropylene oxide. 1. A method for controlling bacterial crop diseases, the method comprising:
i. producing an agricultural composition comprising the bacterial fermentation or protected 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;
method including. Crop diseases include black sigatoka, botrytis blight, fire blight, citrus carcinoma, soft rot, olive carcinoma, tomato leaf spot, bacterial carcinoma, or potato root disease (stone fruit and pear). fruit spot), Cucurbitaceae horn spot disease, peach bacterial spot disease, tomato bacterial spot disease, walnut body blight, bacterial wilt, tomato carcinoma disease, potato leaf blight, apple leaf blight, leaf blight 28. The method of claim 27, wherein the method is selected from the group consisting of: , and bacterial streak disease. Pathogenic microorganisms include Mycosphaerella fijiensis, Botrytis cinerea, Erwinia amylovora (Ea) (particularly streptomycin-resistant E. amylovora strains), and Xanthomonas axonopodis pathotype citri (Ea). onas axonopodis pv. citri) (Xac), Pectobacterium parmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum subspecies brasiliensis otovorum subsp. brasiliensis), Pectobacterium carotovorum subsp. Species Pectobacterium carotovorum subsp. carotovorum, Dickeya dadantii, Pseudomonas savastanoi, Pseudomonas savastanoi pv. savastanoi (Psv), Pseudomonas syringae pv.tomato, Pseudomonas syringae pv. Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. lachrymans, Xanthomonas campestris s campestris pv. pruni), Xanthomonas campestris pathotype vesicatoria (Xanthomonas campestris pv. vesicatoria), Xanthomonas arboricola pv. jugrandis, Ralstonia solanacea arum), Clavibacter michiganensis subsp. michiganensis), Phytophthora infestans, Venturia inaequalis, Xanthomonas oryzae pv.ory zae), Xanthomonas oryzae pv. oryzicola and 29. The method according to claim 27 or 28, wherein the method is selected from the group consisting of Xanthomonas citri pv. citri. Crops include bananas; apples; pears; crabapples; citrus fruits; potatoes; pumpkins; onions; rice; African violets; carrots, potatoes, tomatoes, eggplants, leafy vegetables, squash and cucurbits; Solanaceae), a plant species of the Cucurbitaceae family; peppers and green peppers; olives; stone fruit and pome fruit plants, including olives, peaches and walnuts. Method described. A method for controlling fish diseases, in which the pathogenic microorganisms are Flavobacterium columna #2 and Flavobacterium columna MS-FC-4, and feed additives, aquarium cleaners, and bacterial strains are used. A method of bacterial fixation material having any formation, configuration or structure, such as reinforced garden blocks. A method for controlling diseases caused by human pathogenic bacteria, the bacteria being E. A method in which E. coli O157:H7 (E. coli O157:H7) is used. 1. A method for controlling bacterial crop diseases, the method comprising:
A method comprising applying to a crop an agricultural composition comprising between about 1.0×10 ⁵ and 1.0×10 ⁹ cfu/mL of Pseudomonas bacteria to inhibit growth of pathogenic microorganisms. 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 mosselii 0118-T319 (accession number PTA-126800), Pseudomonas mosselii 0318-T327 (accession number PTA-126801), and Pseudomonas mosselii 0418-T328 (accession number PTA- 34. The method of claim 33, wherein the method is selected from the group consisting of: 126802). 35. The method of claim 33 or 34, wherein the composition comprises between about 5.0 x ¹⁰⁷ and 2.0 x ¹⁰⁸ cfu/mL of Pseudomonas bacteria. Crop diseases include black sigatoka, botrytis blight, fire blight, citrus carcinoma, soft rot, olive carcinoma, tomato leaf spot, bacterial carcinoma, or potato root disease (stone fruit and pear). fruit spot), Cucurbitaceae horn spot disease, peach bacterial spot disease, tomato bacterial spot disease, walnut body blight, bacterial wilt, tomato carcinoma disease, potato leaf blight, apple leaf blight, leaf blight , and bacterial streak disease. The pathogenic microorganisms include Mycospherella fisiensis, Botrytis cinerea, Erwinia amylovora (Ea) (especially streptomycin-resistant E. amylovora strains), Xanthomonas axonopodis pathotype citri, Pectobacterium palmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum subspecies brasiliensis, Pectobacterium carotovorum subspecies carotovorum, Dikeya dadantii, Pseudomonas savastanoi Pathotype savastanoi (Psv), Pseudomonas syringae Pathotype tomato, Pseudomonas syringae Pathotype syringae, Pseudomonas savastanoi Syringae Pathotype lacrimans, Xanthomonas campestris Pathotype Pruni, Xanthomonas campestris Pathotype Vesicatoria, Xanthomonas arboricola Pathotype Jugrandis, Ralstonia solanacearum, Clavibacter michiganensis Subspecies michiganensis, Phytophthora infestans, 37. The method according to any one of claims 33 to 36, wherein the method is selected from the group consisting of Venturia inaecualis, Xanthomonas oryzae, Pathotype oryzae, Xanthomonas oryzae, Pathotype orydicola, and Xanthomonas citri, Pathotype citri. Crops include bananas; apples; pears; crabapples; citrus fruits; potatoes; pumpkins; onions; rice; African violets; carrots, potatoes, tomatoes, eggplants, leafy vegetables, squash, and cucurbits; 38. The method according to any of claims 33 to 37, wherein the plant species are selected from the group consisting of: pepper and green pepper; olive; stone fruit and pome fruit plants including olive, peach, walnut. A method for purifying a protected metabolite derived from a Pseudomonas bacterium, the method comprising:
i. producing a bacterial fermentation or a protected supernatant by the method of any one of claims 1 to 17, or a formulation thereof produced according to any one of claims 18 to 20;
ii. Extracting the bacterial fermentation or protected supernatant by ethyl acetate extraction; and iii. Protection by eluting the bacterial fermentation or protected supernatant with 50% hexane and 50% ethyl acetate, or by eluting the bacterial fermentation or protected supernatant with 25% hexane and 75% ethyl acetate. producing an eluate containing the metabolite;
method including. 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 mosselii 0118-T319 (accession number PTA-126800), Pseudomonas mosselii 0318-T327 (accession number PTA-126801), and Pseudomonas mosselii 0418-T328 (accession number PTA- 40. The method of claim 39, wherein the method is selected from the group consisting of: 1. A method for controlling bacterial crop diseases, the method comprising:
i. producing an agricultural composition comprising a protected metabolite derived from a Pseudomonas bacterium purified by the method of any one of claims 33 and 34; and ii. A method comprising applying the agricultural composition to a crop to inhibit the growth of pathogenic microorganisms. Crop diseases include black sigatoka, botrytis blight, fire blight, citrus carcinoma, soft rot, olive carcinoma, tomato leaf spot, bacterial carcinoma, or potato root disease (stone fruit and pear). fruit spot), Cucurbitaceae horn spot disease, peach bacterial spot disease, tomato bacterial spot disease, walnut body blight, bacterial wilt, tomato carcinoma disease, potato leaf blight, apple leaf blight, leaf blight 42. The method of claim 41, wherein the method is selected from the group consisting of , and bacterial streak disease. The pathogenic microorganisms include Mycospherella fisiensis, Botrytis cinerea, Erwinia amylovora (Ea) (especially streptomycin-resistant E. amylovora strains), Xanthomonas axonopodis pathotype citri (Xac), Pectobacterium palmentieri, and Pectobacterium.・Atrosepticum, Pectobacterium carotovorum subspecies brasiliensis, Pectobacterium carotovorum subspecies carotovorum, Dikeya dadantii, Pseudomonas sabastanoi Pathotype savastanoi (Psv), Pseudomonas syringae Pathotype tomato, Pseudomonas syringae Pathotype syringae , Pseudomonas syringae Pathotype lacrimans, Xanthomonas campestris Pathotype purni, Xanthomonas campestris Pathotype Vesicatoria, Xanthomonas arboricola Pathotype Jugrandis, Ralstonia solanacearum, Clavibacter michiganensis Subspecies michiganensis, Phytophthora michiganensis 43. The method according to claim 41 or 42, wherein the method is selected from the group consisting of Xanthomonas infestans, Venturia inaequalis, Xanthomonas oryzae, Xanthomonas oryzae, Xanthomonas oryzae, and Xanthomonas citri. The pathogenic microorganism is streptomycin-resistant E. Pathogenic E. amylovora. 42. The method of claim 41, wherein the amylovora is Amylobora. The crops consist of stone fruit and pome fruit plants, including bananas, apples, pears, crab apples, citrus fruits, potatoes, tomatoes, eggplants, leafy vegetables, squash and cucurbits, peppers and green peppers, olives; olives, peaches, and walnuts. 45. A method according to any of claims 41 to 44, selected from the group. 42. The method of claim 41, wherein the pathogenic microorganism is selected from Flavobacterium collumnale #2 and Flavobacterium collumnale MS-FC-4. The pathogenic microorganism is E. 42. The method of claim 41, wherein Kollai O157:H7. Claim 41 comprising applying to a crop an agricultural composition comprising between about 1.0 x 10 ⁵ and 1.0 x 10 ⁹ cfu/mL of Pseudomonas bacteria to inhibit growth of pathogenic microorganisms. The method described in. 49. The method of claim 48, wherein the agricultural composition comprises between about 5.0 x ¹⁰⁷ and 2.0 x ¹⁰⁸ cfu/mL of Pseudomonas bacteria. Structure below:

A crystalline compound selected from one of: The crystalline compound has the following structure,

51. The crystalline compound according to claim 50, wherein the crystalline compound includes at least one physical property selected from Tables 13-22. The crystalline compound has the following structure,

51. The crystalline compound according to claim 50, wherein the crystalline compound includes at least one physical property selected from Tables 23-29. The crystalline compound has the following structure,

60. The crystalline compound according to claim 59, wherein the crystalline compound includes at least one physical property selected from Tables 30-37.

Images