Pseudomonas strains and their metabolites that control plant diseases

Abstract

The present disclosure relates to novel bacterial strains, cell broths, and methods of using novel metabolites produced from the bacterial strains, identified as 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328, which are capable of inhibiting the growth of a variety of microbial species related to a variety of crop and fungal pathogens. The methods involve the use of novel, potent antibacterial metabolites produced from the bacterial strains corresponding to compounds having formulas (I), (II), and (III). TIFF2023546531000032.tif73166

Description

[Technical Field]

[0001] This application is a continuation-in-part application of International Patent Application PCT / US2020 / 54303, filed on October 5, 2020, entitled "PSEUDOMONAS STRAINS AND THEIR METABOLITES TO CONTROL PLANT DISEASES," claiming priority thereto, as well as priority to U.S. Provisional Application No. 17 / 063,540, filed on October 5, 2020, Argentine Patent Application No. P200102757, filed on October 5, 2020, and Taiwan Patent Application No. 109134454, filed on October 5, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] This invention relates to the field of biopesticides. In particular, it relates to seven novel strains of the genus Pseudomonas (Pseudomonas spp.), 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328, their cell broths, and novel metabolites produced from these bacterial strains, which can suppress the growth of various microbial species. The Pseudomonas strains 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328 are deposited with the American Type Culture Collection (ATCC) and have ATCC accession numbers PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802, respectively. [Background technology]

[0003] Plant diseases caused by pathogenic microorganisms are increasing exponentially and are costly to treat. Plant pathogens include fungi, bacteria, mycoplasmas, viruses, viroids, nematodes, or parasitic flowering plants. Currently, there are 14 common plant diseases caused by bacteria, including bacterial leaf spot, bacterial canker, and bacterial wilt. Burn disease (Erwinia amylovora), citrus gall disease [Xanthomonas axonopodis pv. citri (Xac)], bacterial leaf spot disease (BLS) [Xanthomonas campestris pv. vesicatoria (XV-16)], olive gall disease [Pseudomonas savastanoi pv. Savastanoi (Psv)], soft rot (Dickeya dadantii, Pectobacterium parmentieri, Pectobacterium atrosepticum) Pectobacterium carotovorum (also known as citrus crown gall) is a devastating plant disease. Nationwide, the cost of controlling this disease is estimated to exceed $100 million (Norelli et al., (2003)). In Florida alone, the cost of implementing eradication programs from 1995 to 2005, as well as compensation for commercial growers and homeowners of devastated residential citrus crops, is approaching $1 billion.

[0004] Apple blight is a devastating disease of pear-shaped fruits caused by infection with the Gram-negative bacterium Erwinia amylovola, affecting pears and apples in many parts of the world, including Europe, Germany, Austria, and Switzerland (Chen et al., (2009)). While apple blight rarely kills an entire orchard, the disease and its control still result in significant economic losses. In the Pacific Northwest and Northern California, small outbreaks have occurred annually since 1991, with large outbreaks occurring every 3-4 years in at least some areas. Pruning to remove infected parts of the plants can ruin the tree's shape and reduce future productivity, making even minor outbreaks costly. 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)).

[0005] Microbial natural products have provided a wealth of biological compounds for use as pesticides (Gwinn, (2018)). However, current methods for preventing bacterial plant diseases have limited effectiveness. The antibiotics streptomycin sulfate (FireWall, AgroSource, Inc.) and oxytetracycline hydrochloride (FireLine, AgroSource, Inc.) have been primarily used to combat E. amylovara when the risk of infection is high. Because these compounds are also used in human and animal health management, using the same antibiotics in crop agriculture is controversial (Stockwell, (2012)). The use of streptomycin sulfate is restricted due to concerns about antibiotic resistance (Vrancken et al., (2013)). Kasugamycin is another antibiotic being studied for use against burn disease. One drawback is that frequent administration of kasugamycin can lead to phytotoxicity, destroying plants (Adaskaveg et al., (2010)). Another drawback is that kasugamycin is expensive compared to other antibiotics. Therefore, kasugamycin needs to be used in combination with other antibiotics.

[0006] Over the past few decades, numerous non-antibiotic products have been developed, registered with the Environmental Protection Agency (EPA), approved by the National Organic Program (NOP), and marketed to orchard owners for burn disease control (Tianna et al., (2018)). Historically, in Europe, two products based on Bacillus subtilis have been registered for burn disease control: Serenade®, based on the QST 713 strain, and Biopro®, based on the BD 170 strain (Broggini et al., (2005)). Biologics based on spore-forming Bacillus species are advantageous for biological control because they can survive for extended periods (Haas et al., (2005)). The moderate success of two Bacillus-based biopharmaceuticals has been demonstrated in numerous field trials in the United States and Germany (Aldwinckle et al., (2002); Kunz et al., (2011); Laux et al., (2003)). This suggests that Bacillus species may be promising for controlling floral infections caused by E. amylovola. However, Bacillus species are only effective at low infection pressures. They are ineffective at moderate and high infection pressures. The results obtained for both biopharmaceuticals were inconsistent, with disease control rates fluctuating between 71% and 0% (Broggini et al., (2005)).

[0007] Future biological protection products must be able to effectively compete with E. amylovola on the one hand, and colonize the same microenvironment on different organs of the target plant on the other hand. Protective bacteria produce secondary metabolites that affect pathogens, compete for food and space, and prevent disease caused by E. amylovola in relation to the plant. In this regard, bacteria of the genus Pseudomonas fit the above biological protection factors (Haas et al., (2005)). Analysis of the species composition of colonizing bacteria in various plants has shown that fluorescent bacteria of the genus Pseudomonas are widely present.

[0008] In France, Pseudomonas species are the main components of populations inhabiting both healthy and diseased apple, pear, and hawthorn trees, and many of them have been found to be able to restrict the growth of E. amylovara in vitro (Paulin et al., (1978)). However, little information has been reported regarding the metabolites that have efficacy.

[0009] In California, Thomson et al. (1976) selected three fluorescent Pseudomonas species effective in protecting pear blossoms (Thomson et al., (1976)). In the mid-1980s, P. fluorescens strain A506, isolated from the leaves of California pear trees, showed characteristic activity to restrict the growth of E. amylovara and the ability to protect apples and pears from burn disease (Lindow et al., (1996)). The product BlightBan® A506, containing P. fluorescens, was developed and has been commercially available since 1996. Numerous experiments conducted in California, Oregon, and Washington have demonstrated the usefulness of this formulation in various apple and pear protection programs (Johnson, (2000)).

[0010] In the UK, two isolated strains of Pseudomonas fluorescein were used to protect hawthorn flowers and shoots (Wilson et al., (1992)).

[0011] In Italy and New Zealand, the suitability of two strains of the genus Pseudomonas, designated BO 3371 and BO G19, was investigated (Galasso et al., (2002)). Under greenhouse conditions, they are highly effective in protecting apple and pear flowers and shoots. For example, the relative protective effect of the BO3371 strain on pear shoots reaches 87% (Galasso et al., (2002)). However, the results obtained were not always consistent, which may be related to the sensitivity of flowers, which is linked to the length of time from flowering to the end of flowering.

[0012] In New Zealand, the fluorescent Pseudomonas strain IPV-BO G19 protected 79% of apple blossoms under field conditions. In another experimental orchard, when sprayed 24 hours before inoculating Braeburn apple blossoms with E. amylovara, fluorescent Pseudomonas strains IPV-BO G19 and IPV-BO 3371 suppressed the occurrence of burn disease by 78% and 58%, respectively (Biondi et al., (2006)).

[0013] In Spain, the strain P. fluorescein EPS62e significantly suppressed burn disease in field assays on apple blossoms, pear fruits, and pear blossoms. The improved adaptability and efficacy of P. fluorescein EPS62e against burn disease were achieved through a strategy combining nutrient enhancement and osmotic adaptation. Field treatment of pear blossoms with physiologically improved P. fluorescein EPS62e yielded a high efficacy rate of up to 90%, although results varied between studies (Cabrefiga et al., (2011); Mikicinski et al., (2020)).

[0014] In Poland, 47 colonies of bacteria that can mitigate the effects of burn disease on pear fruit were isolated from the foliage and soil of apple trees (Mikicinski et al., (2008)).

[0015] Metabolites produced by Gram-negative Pseudomonas species have been comprehensively outlined (Masschelein et al., (2017)). Pseudomonas metabolites are classified into types such as phenolic compounds, phenazine, and lipopeptides. The functions of Pseudomonas species and their metabolites include the following (Alsohim et al., (2014)): 1) hormone production or induction of systemic resistance; 2) many naturally occurring strains significantly improve plant growth (plant growth regulators, IAAs, viscosine); 3) production of siderophores and surfactants such as viscosine and viscosinamide, and antagonism can be conferred by antimicrobial compounds such as hydrogen cyanide, phenazine, pyrrolnitrin, or 2,4-diacylhologlucinol (DAPG). Our research has identified bacterial strains, and fermented products and novel metabolites have been produced from the bacteria. In particular, RejuAgro A and RejuAgro B show high efficacy against multiple pathogenic microorganisms, including bacteria and fungi that have not been previously reported.

[0016] Septoria tritici blotch, caused by Septoria tritici, is a problem found primarily in temperate regions worldwide. In the EU, it is one of the most important leaf diseases due to the high yield and value of grain production. Highly susceptible varieties can show yield reductions of more than 50% if not protected by fungicides. A major challenge to the chemical control of Septoria is disease resistance. Almost all populations of Septoria have developed resistance to strobilurin and triazole fungicides, which have been widely used for more than the past 20 years.

[0017] The fungal genus Colletotrichum includes numerous plant pathogenic species that infect a wide variety of hosts. Colletotrichum can cause significant damage to a wide range of fruit crops, including apples, peaches, grapes, and other berry crops (strawberries, blueberries, cranberries). In recent years, the main crops of interest regarding anthracnose damage are strawberries, stone fruits, and almonds. Disease outbreaks can be catastrophic under favorable conditions in the absence of control measures. Chemical fungicide resistance is a concern for growers. Resistance has been demonstrated to several classes of fungicides, including demethylation inhibitors, quinone-outside inhibitors, and methylbenzimidazole carbamates.

[0018] Fusarium head blight, caused by Fusarium graminearum, is a devastating disease of wheat and barley that produces mycotoxins that render grain unsaleable for livestock or human consumption. Under high-risk conditions, chemical fungicides must be used to prevent unacceptable levels of mycotoxin in crops. Some biological fungicides may also be used. The main advantage of using biological fungicides is a shorter pre-harvest interval, which allows for later application than chemical fungicides. Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum, is a widespread plant disease. Some important crops that are highly susceptible include tomatoes, sweet potatoes, melons, legumes, and bananas (Panama disease). The pathogen is spread by water spray, planting machines, and infected seeds. Fusarium species infect through lateral roots or root wounds and proliferate within cells until they reach the xylem. Historically, soil fumigation has been carried out at the beginning of the growing season as a means of eliminating Fusarium, but the removal of methyl bromide from the market has further limited the choice of fumigants.

[0019] Rice blast caused by Magnaporthe oryzae is the most severe disease that attacks rice. Rice blast can cause losses of up to 30% or more under severe conditions. The disease is most severe under warm and high humidity conditions, which are commonly found in rice cultivation areas. [PRIOR ART DOCUMENTS] [NON-PATENT DOCUMENTS]

[0020] [Non-Patent Document 1] Norelli et al., (2003) [Non-Patent Document 2] Chen et al., (2009) [Non-Patent Document 3] Gwinn, (2018) [Non-Patent Document 4] Stockwell, (2012) [Non-Patent Document 5] Vrancken et al., (2013) [Non-Patent Document 6] Adaskaveg et al., (2010) [Non-Patent Document 7] Tianna et al., (2018) [Non-Patent Document 8] Broggini et al., (2005) [Non-Patent Document 9] Haas et al., (2005) [Non-Patent Document 10] Aldwinckle et al., (2002) [Non-Patent Document 11] Kunz et al., (2011) [Non-Patent Document 12] Laux et al., (2003) [Non-Patent Document 13] Broggini et al., (2005) [Non-Patent Document 14] Paulin et al., (1978) [Non-Patent Document 15] Thomson et al., (1976) [Non-Patent Document 16] Lindow et al., (1996) [Non-Patent Document 17] Johnson, (2000) [Non-Patent Document 18] Wilson et al., (1992) [Non-Patent Document 19] Galasso et al., (2002) [Non-Patent Document 20] Biondi et al., (2006) [Non-Patent Document 21] Cabrefiga et al., (2011) [Non-Patent Document 22] Mikicinski et al., (2020) [Non-Patent Document 23] Mikicinski et al., (2008) [Non-Patent Document 24] Masschelein et al., (2017) [Non-Patent Document 25] Alsohim et al., (2014) [Overview of the Initiative] [Problems that the invention aims to solve]

[0021] There is a need for novel biopesticides derived from new strains, cell broths, and novel metabolites produced from such strains that can suppress the growth of various crop pathogens and fungal pathogens. [Means for solving the problem]

[0022] (Brief summary of the invention) In a first embodiment, a method for controlling bacterial crop diseases is provided. The method comprises several steps. The first step is formula (I):

[0023] [ka] This includes producing agricultural compositions containing [the specified substance].

[0024] The second step includes applying the above-mentioned agricultural composition to crops to suppress the growth of pathogenic microorganisms.

[0025] In a second embodiment, a method for controlling bacterial crop diseases is provided. The method comprises the step of applying an agricultural composition containing Pseudomonas bacteria at a concentration of about 1.0 × 10⁵ to 1.0 × 10⁹ cfu / mL to a crop to suppress the growth of pathogenic microorganisms.

[0026] In a third embodiment, a method for controlling fungal pathogens in bacterial crops is provided. The method comprises several steps. One step is formula (I)

[0027] [ka] The process includes producing an agricultural composition containing [a certain substance]. Another step involves applying the agricultural composition to a crop to suppress the growth of fungal pathogens.

[0028] In a fourth embodiment, a method for controlling bacterial crop diseases is provided. The method involves approximately 1.0 × 10⁻⁶ 5 ~1.0×10 9 The method includes the step of applying an agricultural composition containing Pseudomonas bacteria at a concentration of cfu / mL to crops to suppress the growth of fungal pathogens. [Brief explanation of the drawing]

[0029] [Figure 1] This figure shows an illustrative plot of the maximum likelihood phylogenetic tree of representative Pseudomonas lineages based on ligation alignment of 16S rDNA, gyrB, rpoB, and rpoD. Bootstrap support values ​​are shown below the four internal branches that received less than 100% support. Branches not shown represent 100% support. [Figure 2] This figure shows an example of isolation of strain 0617-T307 based on an assay of ethyl acetate extract. [Figure 3A]This figure shows an exemplary culture plot illustrating the distribution of RejuAgro A in cell broth, supernatant, and cells during shaking flask fermentation. [Figure 3B] This graph shows an exemplary plot regarding the production of RejuAgro A from cellular fermentation over time. [Figure 4] This figure shows exemplary agar plates demonstrating that V. inaequilis can be grown on PDA plates on day 14 using PDA alone (Plate A); 0.25% 0.01M PBS (Plate B); 0.8% DMSO (Plate C); or 1.6% DMSO (Plate D). [Figure 5] This figure shows an exemplary agar plate demonstrating that V. inaeculalis cannot grow on PDA plates containing four selected biological control bacteria (Plate A: 0617-T307; Plate B: 0118-T319; Plate C: 0318-T327; Plate D: 0418-T328) on day 14. [Figure 6] This figure shows exemplary agar plates demonstrating that V. inaecuris cannot grow on PDA plates containing 40-80 μg / mL of RejuAgro A on day 14 (Plate A: 10 μg / mL in PDA plate; Plate B: 20 ​​μg / mL in PDA plate; Plate C: 40 μg / mL in PDA plate; Plate D: 80 μg / mL in PDA plate). [Figure 7] This figure shows exemplary agar plates demonstrating that V. inaecuris can be grown on PDA plates containing 10-80 μg / mL of RejuAgro B on day 14 (Plate A: 10 μg / mL in PDA plate; Plate B: 20 ​​μg / mL in PDA plate; Plate C: 40 μg / mL in PDA plate; Plate D: 80 μg / mL in PDA plate). [Figure 8]This figure shows exemplary agar plates demonstrating that V. inaecuris can be grown on PDA plates containing 200-1000 μg / mL of copper sulfate on day 14 (Plate A: PDA plate containing 500 μg / mL CuSO4; Plate B: PDA plate containing 1000 μg / mL CuSO4). [Figure 9] This figure shows an exemplary volume-peak area curve of RejuAgro A analyzed by HPLC at a wavelength of 407 nm. [Figure 10] This figure shows exemplary data regarding the production of RejuAgro A from different bacterial strains. [Figure 11] This figure shows exemplary antifungal assays against Botrytis cinerea CA17. Panel A shows (1) 40 μL of 50 mg / mL nistatin and (2) 40 μL of DMSO; Panel B shows (1) M9 medium for 24 hours, (2) M8 medium for 24 hours, (3) M7 medium for 24 hours, and (4) M6 medium for 24 hours; Panel C shows (1) M9 medium for 12 hours, (2) M8 medium for 12 hours, (3) M7 medium for 12 hours, and (4) M6 medium for 12 hours. [Figure 12] This figure shows exemplary agar plates of M. fijiensis, demonstrating that growth was inhibited in the presence of 600 μg / mL RejuAgro A (Panel A), and that growth occurred in the presence of 60 μg / mL RejuAgro A (Panel B) or in the absence of RejuAgro A (Panel C). [Figure 13A] This graph shows the suppression of L. crescens BT1 growth in BM7 medium at 28°C after application of certain doses of RejuAgro A (RAA: 10 mg / L, 20 mg / L, and 40 mg / L). [Figure 13B] This graph shows the partial suppression of L. cressens proliferation after the application of RejuAgro A (RAA) at a dosage of 1 mg / L. [Figure 13C]This graph shows the suppression of L. cressens proliferation over 16 days after the application of RejuAgro A (RAA) at a dosage of 2.5 mg / L, which is comparable to the same concentrations of oxytetracycline (Oxy-Tet) and streptomycin (Str). [Modes for carrying out the invention]

[0030] This invention relates to novel metabolites produced by seven strains of the genus Pseudomonas, including 0617-T307 described in this patent, which exhibit antimicrobial activity against pathogenic microorganisms, including bacteria and fungi. Based on 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 broth of the seven bacterial strains, including 0617-T307, contains a novel, potent six-membered heterocyclic natural product named RejuAgro A, along with the dimer RejuAgro B, as shown below.

[0031] [ka]

[0032] These compounds, methods for their production, and their applications for inhibiting plant microbial pathogens are disclosed in more detail herein.

[0033] definition When introducing elements of the aspects of this disclosure or particular embodiments, the articles “a,” “an,” “the,” and “said” are intended to mean that one or more of the elements exist. The terms “comprising,” “including,” and “having” are intended to be comprehensive and mean that additional elements other than those listed may exist. The term “or” means any one of the elements of a particular list, including any combination of the elements of that list, unless otherwise specified.

[0034] As intended herein, the terms “substantially,” “almost,” and “about,” and similar terms, are intended to have a broad meaning in accordance with common and acceptable usage in the art relating to the subject matter of this disclosure. It should be understood by those skilled in the art considering this disclosure that these terms are intended to enable the description of specific features described and claimed without limiting the scope of those features to the numerical range provided. Accordingly, these terms should be interpreted as indicating that a non-substantial or non-material modification or alteration of the subject matter described and claimed may fall within the scope of the invention as set forth in the appended claims.

[0035] Biological control agents (BCAs) are safe, sustainable, and cost-effective methods for managing pests such as pathogens, weeds, and insects. These agents are introduced into the environment targeting pest species, with the aim of reducing the population or abundance of pests in the environment.

[0036] "Biological agents" are preparations of live microorganisms (bacteria, yeast) that colonize a host. These microorganisms are primarily used to slow the accumulation of pathogens during the inoculation phase (Tianna et al., (2018)).

[0037] "Biorational" is a term applied to biopesticides based on microorganisms. These biopesticides are often produced by fermenting microbial strains. Many of these products possess both antimicrobial and antifungal activity (Tianna et al., (2018)).

[0038] "Biopesticides" are defined by the U.S. Environmental Protection Agency (EPA) as pesticides derived from natural materials and are classified into biochemical pesticides containing substances that control harmful organisms through harmless mechanisms, microbial pesticides consisting of microorganisms that normally produce bioactive natural products (BNPs), and plant-integrated protective agents that have the activity produced by plants due to the addition of genetic material (Gwinn KD (2018)).

[0039] The compounds called RejuAgro A, RejuAgro B, and RejuAgro C correspond to compounds having formulas (I), (II), and (III) as exemplified below, respectively.

[0040] [ka]

[0041] In a first embodiment, a method for controlling bacterial crop diseases is provided. The method comprises several steps. The first step is formula (I):

[0042] [ka] This includes producing agricultural compositions containing [the specified substance].

[0043] The second step includes applying the above-mentioned agricultural composition to crops to suppress the growth of pathogenic microorganisms.

[0044] In one respect, the method includes diseases of crops selected from the group consisting of black sigatoka, gray mold, burn, citrus gall, soft rot, olive gall, tomato bacterial leaf spot, bacterial gall or blast (stone fruit and pear fruit), cucurbit horn spot, peach bacterial spot, tomato bacterial spot, walnut canker, bacterial wilt, tomato gall, potato leaf blight, apple red mold, bacterial leaf blight, citrus greening disease, potato zebra chip disease, and bacterial streaks. In the second point, the method involves Mycosphaerella fijiensis, Botrytis cinerea, Erwinia amylovora (Ea) (especially streptomycin-resistant E. amylovora strains), Xanthomonas axonopodis pv. citri (Xac), Pectobacterium parmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. brasiliensis, and Pectobacterium carotovorum subsp. carotovorum. Carotovorum subsp.carotovorum), Dickeya dadantii, Pseudomonas savastanoi pv.savastanoi (Psv), Pseudomonas syringae pv.tomato, Pseudomonas syringae pv.syringae, Pseudomonas syringae pv.lachrymans, Xanthomonas campestris pv.Xanthomonas campestris pv. vesicatoria, Xanthomonas arboricola pv. juglandis, Ralstonia solanacearum, Clavibacter michiganensis subsp. michiganensis, Phytophthora infestans, Venturia inaequalis, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzae The method includes pathogenic microorganisms selected from the group consisting of Xanthomonas citri pv. citri (pv. oryzicola) and Xanthomonas citri pv. citri. In a third point, the method includes crops selected from the group consisting of plant species of the Brassicaceae, Solanaceae, and Cucurbitaceae families, such as bananas, apples, pears, crabapples, citrus fruits, potatoes, pumpkins, onions, rice, African violets; carrots, potatoes, tomatoes, eggplants, leafy vegetables, squash, and gourds; pepper and green pepper; olives; and stony and pome-fruit plants, including olives, peaches, and walnuts.

[0045] In a second embodiment, a method for controlling bacterial crop diseases is provided. The method involves approximately 1.0 × 10 5 ~1.0×10 9 The method includes the step of applying an agricultural composition containing Pseudomonas bacteria at a concentration of cfu / mL to crops to suppress the growth of pathogenic microorganisms.

[0046] In the first point, the method involves 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 mosselii The method includes Pseudomonas bacteria selected from the group consisting of 0418-T328 (accession number PTA-126802). In the second point, the method is approximately 5.0 × 10 7 ~2.0×10 8The method comprises an agricultural composition of Pseudomonas bacteria in a concentration of cfu / mL. In third points, the method includes diseases of crops selected from the group consisting of black sigatoka, gray mold, burn, citrus gall, soft rot, olive gall, tomato bacterial leaf spot, bacterial gall or blast (stone fruit and pear fruit), cucurbit horn spot, peach bacterial spot, tomato bacterial spot, walnut canker, bacterial wilt, tomato gall, potato leaf blight, apple red mold, bacterial leaf blight, citrus greening disease, potato zebra chip disease, and bacterial streaks. In the fourth point, the method involves Mycosferella phiensis, Botrytis cinerea, Erwinia amylovola (Ea) (especially streptomycin-resistant E. amylovola strains), Xanthomonas axonopodis pathogenic form citri (Xac), Pectobacterium palmentieri, Pectobacterium atrosepticum, Pectobacterium carotoborum subspecies brassiliensis, Pectobacterium carotoborum subspecies carotoborum, Dikeyer dadanthii, Pseudomonas sabastanoi pathogenic form sabastanoi (Psv), Pseudomonas syringae pathogenic form tomato, Pseudomonas syringae pathogenic form syringae, Pseudomonas syringae pathogenic form lacrimans, and Xanthomonas campestris. This group includes pathogenic microorganisms selected from the group consisting of pathogenic forms of Xanthomonas pruni, Xanthomonas campestris, pathogenic forms of Vesicatoria, Xanthomonas arboricola, pathogenic forms of Jugrandis, Ralstonia solanacearum, Clavibacter machinansis subspecies machinansis, Phytophthora infestans, Venturia inaeculis, Xanthomonas oryzae pathogenic form oryzae, Xanthomonas oryzae pathogenic form origicola, and Xanthomonas citri pathogenic form citri. In the fifth point, the method includes crops from the group consisting of bananas, apples, pears, crabapples, citrus fruits, potatoes, pumpkins, onions, rice, African violets; Brassicaceae, Solanaceae, and Cucurbitaceae plant species such as carrots, potatoes, tomatoes, eggplants, leafy vegetables, squash, and gourds, pepper and green pepper, olives; and stony and pome-fruit plants including olives, peaches, and walnuts.

[0047] In a third embodiment, a method for controlling fungal pathogens in bacterial crops is provided. The method comprises several steps. One step is formula (I)

[0048] [ka] The process includes producing an agricultural composition containing [a certain substance]. Another step involves applying the agricultural composition to a crop to suppress the growth of fungal pathogens.

[0049] In the first point, the method includes a fungal pathogen, which is selected from the group consisting of Septoria tritisi, Colletotrichum dematium, Fusarium oxysporum f.sp. Melonis, and Magnaporte oryzae. In the second point, the crop is selected from the group consisting of apples, peaches, grapes, berry crops, wheat, barley, tomatoes, sweet potatoes, melons, legumes, bananas, and rice.

[0050] In a fourth embodiment, a method for controlling bacterial crop diseases is provided. The method involves approximately 1.0 × 10⁻⁶ 5 ~1.0×10 9 The method includes the step of applying an agricultural composition containing Pseudomonas bacteria at a concentration of cfu / mL to crops to suppress the growth of fungal pathogens.

[0051] In a first aspect, the method comprises a Pseudomonas bacterium selected from the group consisting of 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 mosselii 0418-T328 (accession number PTA-126802). In a second aspect, the method comprises about 5.0 × 10 7 ~2.0 × 10 8 cfu / mL of Pseudomonas bacteria in a composition. In a third aspect, the method comprises a fungal pathogen selected from the group consisting of Zymoseptoria tritici, Colletotrichum dematium, Fusarium oxysporum f. sp. melonis and Magnaporthe oryzae. In a fourth aspect, the method comprises a crop selected from the group consisting of apple, peach, grape, berry crops, wheat, barley, tomato, sweet potato, melon, legumes, banana and rice.

[0052] Biological Deposit Information One of the inventors, Dr. Ching-Hong Yang (Resident of 10120 North Sheridan Drive, Mequon, Wisconsin 53902, United States of America) is as evidenced by the “Indication of Deposited Microorganisms” (PCT / RO / 134) (submitted in this application) under PCT Rule 13-2. deposited the bacterial strains Pseudomonas soli 0617-T307, Pseudomonas soli 0917-T305, Pseudomonas soli 0917-T306, Pseudomonas soli 0917-T307, Pseudomonas mosselii 0118-T319, Pseudomonas mosselii 0318-T327, and Pseudomonas mosselii 0418-T328 with the American Type Culture Collection (ATCC®), P.O. Box 1549, Manassas, VA 20110 USA (the "ATCC Patent Depository") on June 25, 2020 Ta.Following the survival rate tests, the ATCC Patent Depository assigned the following accession numbers to these deposited bacterial strains, effective from June 25, 2020: 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 mosseri 0118-T319 (accession number PTA-126800), Pseudomonas mosseri 0318-T327 (accession number PTA-126801) and Pseudomonas mosseri 0418-T328 (accession number PTA-126802). Dr. Yang gave the applicant permission to include the disclosure of this biological deposit in this application. Furthermore, it grants unconditional and irrevocable consent to make it available to the public as of the filing date. . [Examples]

[0053] [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 multilocust sequence analysis (MLSA) of the genus Pseudomonas (Peix et al., (2018)).

[0054] For species attribution, BLASTN was performed against the NCBI non-redundant nucleotide database using these four sequences. Based on the results, strain 0617-T307 is closely related to a Pseudomonas species belonging to the P. putida group of the P. fluorescein lineage. The "MLSA phylogenetic tree" and the "list of genomes from standard strains of Pseudomonas species" (see Figure 2 and Table 2 of Peix et al., (2018); Peix et al., (2018)) were used as guides for classification sampling (Figure 1). Based on this information, genomes were obtained from GenBank. All species of the P. putida group with high-quality genome assemblies were included. Since 0617-T307 has the highest rpoD (i.e., the gene with the highest resolution for assigning Pseudomonas species) sequence similarity to P. soli, all four available genomes of P. soli were included in the sampling (standard strain LMG 27941 of P. soli). T (including). For other species in the P. fluorescein lineage, one species was selected as a representative of each group. P. elginosa (P. elginosa group, P. elginosa lineage) was included as an outgroup to form the root of the tree.

[0055] 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 alignments contained 9,912 sequenced nucleotide sites. Maximum likelihood estimation was performed using PhyML (Guindon et al., (2003)). Bootstrap support was evaluated by 1,000 copies.

[0056] Based on a polydentate molecular phylogenetic tree (Figure 1), 0617-T307 and four P. soli strains for which genome sequences are available form a monophyletic clade with 100% bootstrap support. This result provides strong support for assigning 0617-T307 to P. soli (Pascual et al., (2014)), a standard strain reported to have been isolated from soil samples in Sierra Nevada National Park, Spain.

[0057] Furthermore, based on the guidelines for Pseudomonas species assignment provided by Garcia-Valdes and Lalucat (Garcia-Valdes et al., (2016)), additional support for assigning 0617-T307 to P. soli included: (a) 16S rDNA > 98.7-99% identical. Compared to standard strains of P. soli, 0617-T307 had 99.2% sequence identity. Compared to its sister species, P. entomophila, 0617-T307 had 99.5% sequence identity. It should be noted that rDNA is known to lack sufficient resolution for species identification in the genus Pseudomonas (Garcia-Valdes et al., (2016); Peix et al., (2018)); (b) rpoD gene > 95-96% identical. Compared to the standard strain of P. soli, 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. soli, 0617-T307 had 98.0% sequence identity. Compared to its sister species, P. entomophila, 0617-T307 had only 95.1% sequence identity.

[0058] [Example 2] Preparation, isolation, and characterization of RejuAgro A and RejuAgro B derived from ethyl acetate extract of cell broth of strain 0617-T307. RejuAgro A and B can be prepared by ethyl acetate extraction of cell broth from fermenter fermentation, followed by isolation and purification by chromatography. Briefly, the stock bacterium Pseudomonas species 0617-T307 was streaked onto LB plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, water) and grown in an incubator at 28°C for 24 hours. For the preparation of the seed medium, a single colony of 0617-T307 was inoculated into a 2.0 L flask containing 500 mL of autoclaved YME medium (4 g / L yeast extract, 4 g / L glucose, 10 g / L malt extract) and grown at 28°C with a shaking rate of 200 rpm for 24 hours. The seed medium was then inoculated into a 20 L NBS fermenter containing 12 L of autoclaved YME medium. Fermentation was carried out at 16°C for 1 to 7 days. The stirring speed was 200 rpm and the airflow rate was 2 L / min.

[0059] After collection, the bacterial culture was extracted four times with ethyl acetate. The ethyl acetate layer was separated, dehydrated 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.

[0060] The concentrated sample was dissolved in ethyl acetate, mixed with silica gel, and packed into an injection column (φ3.0 × 20 cm), which was then loaded onto a silica gel universal column (4.8 × 18.5 cm) of a flash chromatography system (Yamazen AI-580) equipped with a UV detector. After loading the sample, it was eluted with 280 mL of each solvent in the following order of increasing polarity: 100% hexane, 75% hexane / 25% ethyl acetate, 50% hexane / 50% ethyl acetate, 25% hexane / 75% ethyl acetate, 100% ethyl acetate, 50% ethyl acetate / 50% acetone, 100% acetone, and 100% methanol. The sample was eluted at a flow rate of 20 mL / min. The eluate was monitored with UV 254 nm, and fractions were collected in time mode at 20 mL / test tube. A total of 114 fractions or test tubes were obtained from the flash chromatography.

[0061] The generated fractions were then applied to the plate assay. 1 mL of each fraction was taken into a 1.5 mL test tube and vacuum-dried using an Eppendorf vacuum concentrator. The dried samples were dissolved in 50 μL of DMSO, of which 2 μL was used for the plate assay. Briefly, Erwinia amylovola 273 was streaked onto LB plates and grown in a 28°C incubator. After 24 hours, the resulting single colonies were inoculated into 5 mL of LB medium and grown overnight at 28°C with a 200 rpm shaker. The cells were diluted 1:100 with sterile water, and 225 μL of this was plated onto a 50% LB plate (5.0 g / L tryptone, 2.5 g / L yeast extract, 5.0 g / L NaCl, 15 g / L agar). After drying in a biosafety cabinet for 10 minutes, the DMSO solution of each fraction was distributed to the pre-labeled portion of the petri dish and dried for another 10 minutes. DMSO and kasugamycin were used as negative and positive controls, respectively, for the assay. The plates were then incubated at 28°C, and a zone of inhibition was observed after 1 day.

[0062] In a 114-fraction in vitro plate assay, two fractions were shown to inhibit the growth of E. amylovara 273. Notably, fractions 38–40 (abbreviated as T3840 or Flash-RejuAgro A) were eluted with 50% hexane / 50% ethyl acetate and had a relatively large clearance zone that may be promising for further testing. Other bioactive compounds in this assay were found in fractions 50–52 (coded as T5052). These fractions were eluted with 25% hexane / 75% ethyl acetate.

[0063] Purification of fractions 3840 and 5054 by preparative HPLC (Prep-HPLC) yielded 15 mg of the yellow-stained compound RejuAgro A (Rt 17.5) and 103.3 mg of the dark green-stained compound RejuAgro B. RejuAgro A is soluble in methanol and chloroform. RejuAgro B (Rt 10.5) is not sufficiently soluble in methanol and chloroform, but is very soluble in dimethyl sulfoxide (DMSO) and exhibits 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. The results showed that the two compounds are structurally similar, with RejuAgro A having seven types of carbon groups (three carbonyl groups, two tertiary carbons, and two methyl carbons), while RejuAgro B lacks one methyl group, as shown below:

[0064] [ka]

[0065] [Example 3] In vitro antimicrobial activity of RejuAgro A and RejuAgro B derived from strain 0617-T307 The MIC values ​​of RejuAgro A and RejuAgro B were determined for five types of bacteria: wild-type Gram-negative plant pathogenic bacteria, streptomycin-resistant E. amylovola, fish pathogenic bacteria, Gram-positive and Gram-negative human pathogenic bacteria, and the RejuAgro A-producing strain (0617-T307). Antimicrobial assays were performed according to CLSI antimicrobial susceptibility testing (AST) standards. Briefly, stock solutions of each tested bacterium were streaked onto LB (Luria-Bertani) plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium salt, 15 g / L agar). For special cultures, 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, 10g / L agar) and TYES (4g / L tryptone, 0.4g / L yeast extract, 0.5g / L MgSO4, 0.5g / L CaCl2, pH 7.2, 15g / L agar) were used against Flavobacterium columnale strains MS-FC-4 and #2, respectively. Subsequently, a single colony was picked from the plate and inoculated into the corresponding liquid medium for overnight growth. This culture was then sterilized in LB or the corresponding medium. 590The compounds were diluted to 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, resulting in 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) were used as controls.

[0066] The assay results showed that the most active metabolite of the 0617-T307 strain was RejuAgro A, not RejuAgro B. Compared to its effects on the Gram-positive bacterium MRSA (MIC > 40 μg / mL) and the Gram-negative bacterium E. korrai O157:H7 (a foodborne and waterborne important pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans) (MIC = 40 μg / mL), RejuAgro A is particularly efficient against test bacteria with MIC values ​​of 5–40 μg / mL. The antibacterial activity of RejuAgro A was equivalent to that of streptomycin against Erwinia amylovola 1189, Xanthomonas axonopodis pathogenic form citri, Pseudomonas savastanoi pathogenic form savastanoi, Pectobacterium palmentieri UPP163 936, Pectobacterium carotoborum subspecies brassiliensis 944, Pectobacterium carotoborum subspecies carotoborum wpp14 945, and Dikeyer dadantii 3937 strains. The MIC value for E. amylovola was 5 μg / mL, while for other less pathogenic bacteria it was 20-40 μg / mL. Xanthomonas bacteria were highly susceptible to streptomycin, with an MIC value of 0.16 μg / mL, which is lower than the MIC value of 5 μg / mL for RejuAgro A. The MIC value of RejuAgro A against Pseudomonas savastanoi pathogen is 40 μg / mL. The MIC value of RejuAgro A against Xanthomonas arboricola pathogen jugrandis 219 is 6.25 μg / mL. The MIC values ​​of RejuAgro A against Ralstonia soranacearum K60 and Pss4 are 3.13 and 6.25 μg / mL, respectively. The MIC values ​​of RejuAgro A against Clavibacter machinansis subspecies NCPPB382, Cmm0317, and Cmm0690 are 6.25, 1.56, and 12.5 μg / mL, respectively. The MIC value of RejuAgro A against Ralstonia soranacearum K60 and Pss4 is 40 μg / mL.

[0067] Furthermore, RejuAgro A was tested against other E. amylovola strains, including one pathogenic strain and three streptomycin-resistant strains. Against E. amylovola 110, RejuAgro A showed comparable efficacy to streptomycin (MIC value 5 μg / mL). However, RejuAgro A was more effective than streptomycin against E. amylovola 1189. The MIC values ​​for RejuAgro A and streptomycin against E. amylovola 1189 were 5 μg / mL and 10 μg / mL, respectively. Moreover, a lower MIC value (10 μg / mL) was observed for RejuAgro A than that of streptomycin (over 40 μg / mL), making it more effective against streptomycin-resistant E. amylovola CA11, DM1, and 898. These results suggest that RejuAgro A is the most potent compound in the tests against E. amylovara and could be a viable alternative to streptomycin. There was no indication of cross-resistance to RejuAgro A in streptomycin-resistant strains.

[0068] Against Flavobacterium, the causative agent of columnaris disease in fish, RejuAgro A had an MIC value of 5 μg / mL against Flavobacterium columnare MS-FC-4 strain and strain #2 (which causes columnaris disease in wild and farmed fish), 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).

[0069] The effect of RejuAgro A on the 0617-T307 strain was investigated. The MIC value of RejuAgro A for Pseudomonas soli 0617-T307 (a RejuAgro A-producing strain) was shown to be greater than 40 μg / mL in the tested LB medium, which means that the 0617-T307 strain is able to survive and tolerate at least the 40 μg / mL of RejuAgro A it produces itself.

[0070] RejuAgro A was tested together with streptomycin against tomato pathogens (P. syringae pathogenic tomato PT30, P. syringae pathogenic syringae 7046, P. syringae pathogenic lacrimans 1188-1) and other citrus crown gall pathogens (Xanthomonas campestris pathogenic purni, Xanthomonas campestris pathogenic vesicatoria XV-16). The MIC value of RejuAgro A against P. syringae was 40 μg / mL, and the MIC value of streptomycin was 2.5–5 μg / mL. Against X. campestris species, the MIC value of RejuAgro A was 2.5 μg / mL or 40 μg / mL, which is lower compared to the MIC value of streptomycin, which is 20 μg / mL or greater than 40 μg / mL. These findings indicate that, compared to tomato pathogens caused by the Pseudomonas genus, Xanthomonas campestris is more susceptible to RejuAgro A than streptomycin.

[0071] RejuAgro A was effective against all pathogenic fungi tested (Table 1). RejuAgro A was tested against Phytophthora infestans, Venturia inaeculais, and Mycosferella fijiensis. RejuAgro A showed 100% inhibition against P. infestans and V. inaeculais at 40 μg / mL, 80 μg / mL, and 600 μg / mL (Table 1).

[0072] [Table 1] TIFF0007914116000009.tif212166

[0073] [Example 4] Production and stability of RejuAgro A from strain 0617-T307 in shaking flask fermentation Fermentation of 0617-T307 used in the production and preparation of RejuAgro A can be obtained by two approaches: shaking flask fermentation and fermenter fermentation. Fermenter fermentation is described in Example 2. In this example, flask fermentation can be obtained as follows: The stock bacterium, Pseudomonas species 0617-T307, was streaked onto YME agar medium (4 g / L yeast extract, 4 g / L glucose, 10 g / L malt extract, 15 g / L agar) and grown in a 28°C incubator for 24 hours. A seed medium was prepared by growing a single colony of 0617-T307 in a 250 mL flask containing 50 mL of sterile YME liquid medium at 16°C and 220 rpm for 24 hours. The seed medium was then inoculated into a 4 L flask containing 0.5 L of sterile YME medium at a ratio of 4% (v / v). Following inoculation (2%, v / v) into eight 4L flasks containing 2L of YME medium, the bacteria were grown at 16°C for 1–7 days in a shaker at 200–220 rpm.

[0074] The concentration of RejuAgro A was obtained by LC-MS analysis according to the developed standard curve. Two methods were used to prepare the samples for LC-MS analysis. One approach was to extract the cell broth with ethyl acetate (1 mL:1 mL, vortex for 1 minute), and obtain an 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. The other method was to centrifuge the cell broth to obtain the supernatant, then mix the supernatant with an equal volume of methanol to make a 50% methanol solution, and inject 10 μL of this solution into the LC-MS. The second method was adopted because it was confirmed that RejuAgro A is produced extracellularly and that it is present in greater quantities in the supernatant rather than in the cells (Figure 3A).

[0075] During the 7-day fermentation period, the total production of RejuAgro A reached its peak concentration on the first day and then began to decrease with increasing time (Figure 3B). Furthermore, detailed examinations of RejuAgro A production and cell concentration were conducted every 6 hours during shaking flask fermentation. The concentration of RejuAgro A (total amount of RejuAgro A) reached a maximum of 13.8 mg / L at 18 hours, and the bacterial cell concentration reached a maximum of 2 × 10⁻⁶ at 12 hours. 11 The CFU / mL level was reached, indicating that RejuAgro A production is a process associated with cell proliferation.

[0076] The volume of culture medium in a 4L shaking flask affects the production of RejuAgro A. In a 4L flask using YME medium, RejuAgro A production was observed only at a volume size of 500mL, and not at volume sizes of 1.0L or 1.5L. This observation suggests that RejuAgro A production prefers to be present under high aeration conditions.

[0077] The type of culture medium and culture temperature affect the production of RejuAgro A. LB medium was tested at 16°C or 28°C in parallel with YME medium. RejuAgro A production was confirmed in YME medium at 16°C but not in LB medium. Regarding colony-forming units, the 0617-T307 strain grew well in both LB medium at 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 of the product derived from 0617-T307 was monitored by a plate assay against E. amylovora, which was consistent with the production of RejuAgro A.

[0078] To confirm the applicability of the production conditions for RejuAgro A, ten other strains of the Pseudomonas genus were tested under the same conditions as the Pseudomonas strain 0617-T307. Based on housekeeping gene analysis, 0917-T305, 0917-T306, and 0917-T307 were identified as Pseudomonas soli, while 0118-T319, 0318-T327, and 0418-T328 were identified as Pseudomonas mosseri. Type strains of both Pseudomonas soli and Pseudomonas mosseri have been reported (Daboussi et al., (2002); Pascual et al., (2014)).

[0079] The 0617-T307 strain and its phylogenetically related species were shown to be able to produce RejuAgro A in YME at 28°C and 220 rpm. This result suggests that this method has specificity for producing RejuAgro A in the 0617-T307 strain and some of its related species (Table 2). When LCMS was performed on a 40-hour culture obtained by culturing 0617-T307 in YME medium at 16°C and 220 rpm with a shaker, RejuAgro A was found to be stable and present at room temperature for at least 4 weeks.

[0080] [Table 2]

[0081] [Example 5] Antimicrobial activity of cell broth of strain 0617-T307 against 0617-T307 and E. amylovora.

[0082] Two assays were used for the antimicrobial testing of cell broth and metabolites of 0617-T307: one a plate diffusion assay and the other a microplate assay. LB plates were used for the plate diffusion assay of the antimicrobial activity of RejuAgro A-containing fractions and cell broth against E. amylovora (Table 3). Both cell broth containing live 0617-T307 cells and a suspension containing 2 mg / mL of RejuAgro A showed antimicrobial activity against E. amylovora. However, no zone of inhibition was observed when Serenade® was applied.

[0083] [Table 3]

[0084] To find a biological control method consisting of both 0617-T307 cells and the active ingredient RejuAgro A, the following experiments were conducted. For an antimicrobial assay against 0617-T307, the RejuAgro A-producing strain, the supernatant (abbreviated as "supernatant") of cell broth cultured for 40 hours with 0617-T307 containing RejuAgro A was used. It was shown that the 0617-T307 strain could grow in LB medium, not YME medium, at a 2-fold dilution of the supernatant. Further studies showed that the inhibitory effect of the supernatant was due to its low pH value. Subsequently, by controlling the pH to 6.5-6.8, questions 1 and 2 could be answered "yes".

[0085] The bioactive fractions (crude extract, 100 μg / mL; flash-RejuAgro A, 20 μg / mL; HPLC-RejuAgro A, 10 μg / mL) were tested against strains 0617-T307, Ea, and Xac. The bioactive fractions failed to inhibit the growth of strain 0617-T307, indicating that RejuAgro A can be mixed with 0617-T307 cells for the preparation of a biological control agent. The bioactive fractions containing RejuAgro A showed inhibitory effects against Ea and Xac, with flash-RejuAgro A and HPLC-RejuAgro A particularly halting the growth of Ea and Xac under test conditions. This demonstrates that RejuAgro A solutions can be used for the biological control of scalding and citrus crown gall at concentrations of 10-20 μg / mL.

[0086] [Example 6] Identification and characterization of bioactive metabolites derived from ethyl acetate extract of the acidified supernatant (pH 2.0) of the 0617-T307 strain.

[0087] The stock bacterium Pseudomonas species 0617-T307 was inoculated onto LB agar plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, water) and grown in a 28°C incubator for 24 hours. For the preparation of the seed medium, a single colony of 0617-T307 was inoculated into 500 mL of autoclaved YME medium (4 g / L yeast extract, 4 g / L glucose, 10 g / L malt extract) and grown at 28°C with a shaking rate of 150 rpm for 24 hours. Subsequently, the seed medium was inoculated into eight 4 L flasks, each containing 2 L of autoclaved YME medium. Fermentation was carried out for 7 days at 16°C with a shaking rate of 150 rpm.

[0088] After 7 days of growth, the supernatant was obtained by centrifugation of the bacterial culture at 4000 rpm for 15 minutes. The pH of the supernatant was then adjusted to 2.0 by adding 6N HCl. The acidified supernatant was then subjected to ethyl acetate extraction. As a result, 3.0 g of crude extract was obtained from a 14 L culture of strain 0617-T307.

[0089] 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 sample, it was eluted with 280 mL of each solvent in the following order of increasing polarity: 100% hexane, 75% hexane / 25% ethyl acetate, 50% hexane / 50% ethyl acetate, 25% hexane / 75% ethyl acetate, 100% ethyl acetate, 50% ethyl acetate / 50% acetone, 100% acetone, and 100% methanol. The sample was eluted at a flow rate of 20 mL / min. The eluate was monitored with UV 254 nm, and fractions were collected in time mode at 20 mL / test tube. A total of 114 fractions or test tubes were obtained from the flash chromatography.

[0090] The generated fractions were then applied to the plate assay. 1 mL of each fraction was taken into a 1.5 mL test tube and vacuum-dried using an Eppendorf vacuum concentrator. The dried samples were dissolved in 50 μL of DMSO, of which 2 μL was used for the plate assay. Briefly, Erwinia amylovola 273 was inoculated into a 50% LB (5.0 g / L tryptone, 2.5 g / L yeast extract, 5.0 g / L NaCl) plate, and single colonies were inoculated into 5 mL of LB medium. The bacteria were diluted 1:100 with sterile water, and 225 μL of this was plated into the 50% LB plate. After drying for 10 minutes in a biosafety cabinet, the DMSO solution of each fraction was distributed into pre-labeled portions of petri dishes and dried for another 10 minutes. DMSO and kasugamycin were used as negative and positive controls, respectively, in the assay. Next, the plates are incubated in a 28°C incubator, and a zone of inhibition is observed after one day.

[0091] In in vitro plate assays of 114 flash fractions, three bioactive fractions (T3234, T5058, and T7882) were shown to inhibit the growth of E. amylovara 273. Fractions 3234 and 5258 showed relatively small clearance zones. Fraction 3234 was eluted by 50% hexane / 50% ethyl acetate. Fraction 5058 was eluted by 25% hexane / 75% ethyl acetate. In the negative control, DMSO did not show a zone of inhibition, while the positive control kasugamycin showed a zone of inhibition. Another flash fraction, T7882, was eluted by acetone / ethyl acetate (50% / 50%), which similarly inhibited the growth of E. amylovara.

[0092] Furthermore, isolation and purification by HPLC based on anti-E. amylovara activity identified two antimicrobial compounds (Rt22.9 and Rt25.0) from T5058 (see compound formulas 0617_T307_5058_Rt22.9 and 0617_T307_5058_Rt25.0) and one antimicrobial compound (Rt18.9) from T7882 (see compound formula 0617_T307_7882_Rt18.9). T307_5058_Rt22.9 and T307_5058_Rt25.0 are natural products derived from tryptophan, and their structures have been reported in the SciFinder database, but their biological activity has not been reported (Loots et al., (2015)). 0617_T307_7882_Rt18 was predicted to be a derivative of the previously reported difryl (Osipov et al., (1978)). These natural products are listed below:

[0093] [ka]

[0094] [Example 7] Identification of other metabolites derived from strain 0617-T307 using LCMSMS and spectral library search.

[0095] Crude extracts of un-pH-adjusted and pH-adjusted cell broth (pH of the cell broth was adjusted to 2.0 with 6N HCl) were concentrated and resuspended in 250 μL of 100% MeOH containing an internal standard (m / z 311.08) for LC-MS / MS analysis. LC injection volume: 5 μL; LC column: 1.7 μM C18, 100A, 50 × 2.1 mm Kinetex C18 column from Phenomenex, 12 min gradient. 5–95% ACN on Bruker Maxis Impact II. Data were acquired by Bruker Maxis Impact II, UHR-QqTOF (Ultra High Resolution Qq-Time-Of-Flight) mass spectrometer. Following each full MS scan, tandem MS (MS / MS) was performed using collision-activated dissociation (CID) fragmentation of the eight most abundant ions in the spectrum. Scan rate was 3 Hz.

[0096] Next, based on bioinformatics analysis and molecular network analysis, a precise spectral library search was performed with the aim of identifying novel 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 Products Library, 5) Pharamacologically Active NIH Small Molecule Repository, 6) Faulkner Legacy Library, 7) Pesticides, 8) Dereplicator Identified MS / MS Peptidic Natural Products, 9) PNNL Lipids, 10) Massbank, 11) Massbank EU, 12) MoNA, 13) ReSpect-Phytochemicals, and 14) HMDB.

[0097] The MS / MS spectra of the samples were searched in the above library and aligned with the reference spectrum offset. The matching parameters were the same. These results can be explored to identify structural analogs of known compounds. The MS / MS molecular network was generated with a minimum cluster size of 2, a minimum edge of 0.7 cosines, and a minimum number of matching peaks of 6. As an example, a new molecular species m / z 303.16 was identified as corresponding to a new compound from the active fraction 0617-T307_5058_Rt25.0. Some known compounds were confirmed from the crude extract, which contains indole-3-carboxylic acid and xantholysin A, which are plant growth promoters. 1) The broad antifungal activity of P. putida BW11M1 is mainly dependent on xantholysin production, and 2) xantholysin is required for swarming and contributes to biofilm formation, as reported (Li et al., (2013)). In fact, higher concentrations of xantholidine A were observed when 0617-T307, 0418-T328, and 0318-T327 were cultured at 28°C. In other words, apart from the bioactive compound RejuAgro A, xantholidine A is a metabolite that contributes to the antimicrobial activity of the biologically controlled bacterium 0617-T307 and its close relatives 0318-T3027 and 0418-T328.

[0098] [Example 8] Greenhouse and field infection assays of strain 0617-T307 and several closely related species that produce RejuAgro A.

[0099] To evaluate the bioregulatory activity of 0617-T307 against Erwinia amylovola, an infection assay was performed using crabapple trees in a greenhouse at the University of Wisconsin-Milwaukee. 1.0 × 10 8Biocontrol agents containing cfu / mL (0617-T307, 0717-T327, 0617-T318) were sprayed onto flowers (80% to full bloom) in multiple small plots. Briefly, strain 0617-T307 was grown overnight in a 26 mL glass tube containing 5 mL of LB medium, and then the cells were inoculated into LB medium (1:100) and grown for 14-18 hours at 28°C with a 200 rpm shaker. The cells were collected and 10 8 The solution was resuspended in 10 times the amount of water until the concentration was CFU / mL. This resuspended solution can be used in field assays for burn disease control in greenhouses and outdoors. Control flowers were sprayed with distilled water. Then, all flowers were sprayed with 1.0 × 10⁶ solution. 6 Inoculation was performed by spraying with the E. amylovola strain Erwinia amylovola 273 at cfu / mL. Treatment with 0617-T307 was carried out three times on September 7, October 9, and October 19, 2018. Referring to Table 4, all spray treatments with 0617-T307 (Pseudomonas soli) resulted in 100% control of flower blight symptoms in crabapple flowers compared to 0% control with distilled water, suggesting that 0617-T307 is a promising biocontroller against E. amylovola-induced blight. The control rates for two other Pseudomonas species, 0717-T327 (Pseudomonas couriensis) and 0617-T318 (Pseudomonas protegens), were lower at 16.7% and 25%, respectively. In conclusion, of the three Pseudomonas species tested, only 0617-T307 showed good control efficacy against crabapple burn disease. No plant toxicity was observed.

[0100] [Table 4]

[0101] In field assays, on May 5th and 6th, 2019 (when apple blossoms were 40% and 70% in bloom), the number of biocontrolling bacteria producing RejuAgro A (0617-T307, 0118-T319, 0318-T327, 0418-T328; see Table 2) was 5 × 10⁶. 8The bacteria was applied to the flowers of apple trees in an orchard at a concentration of CFU / mL. The bacterial pathogen E. amylovola Ea110 was found on May 7th (90% flowering) in 5 × 10 6 Inoculation was performed at a concentration of CFU / mL. The diseased flower cluster rates for water control, streptomycin, 0617-T307, 0118-T319, 0318-T327, and 0418-T328 were 32.9%, 13.3%, 16.8%, 18.5%, 16.7%, and 11.8%, respectively. Compared to streptomycin, the biocontrol bacteria producing RejuAgro A are equally or more effective in controlling apple orchard burn disease.

[0102] [Example 9] Antifungal activity of RejuAgro A and B and their producing fungi against Venturia inaeculis.

[0103] The fungus Venturia inaeculalis, which causes apple red mold, was maintained on PDA agar at room temperature (approximately 24°C) in the dark. A mixed suspension of conidia and hyphae (in 0.01 M PBS) was collected from the PDA (potato dextrose agar). 10 μL of the conidia and hyphae suspension was dropped onto plates containing biological control bacteria, RejuAgro A, or modified RejuAgro A. The control was a PDA plate without the addition of biological control bacteria, RejuAgro A, or B. The petri dishes were incubated at room temperature in the dark, and the diameter of each V. inaeculalis colony was confirmed after 7 days.

[0104] Compared to the control (Figure 4), the four selected biological control bacterial strains 0617-T307, 0118-T319, 0318-T327, and 0418-T328 were able to suppress the growth of V. inaeculais on PDA plates (Figure 5). RejuAgro A could suppress the growth of V. inaeculais on PDA plates at 40–80 μg / mL (Figure 6). However, no inhibitory effect of RejuAgro B on the growth of V. inaeculais was observed at 10–80 μg / mL on PDA plates (Figure 7). Finally, no suppression of V. inaeculais was observed on PDA plates containing 200–1000 μg / mL of copper sulfate (Figure 8).

[0105] [Example 10] Production of RejuAgro A by Pseudomonas species The amount of RejuAgro A was analyzed by HPLC-MS on the broth after fermentation of a 4L flask containing 500mL of YME medium at 16°C and 220rpm with shaking for 24 hours. A volume-peak area curve was created to investigate the relationship between the HPLC peak area and the amount of RejuAgro A (Figure 9). Analytical method: 1) 25mL of cell broth was extracted with 25mL of ethyl acetate. 2) 5mL of the ethyl acetate extract was dried and dissolved in 0.1mL of methanol. 3) 4μL was injected into HPLC-MS.

[0106] Seven bacterial strains (0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328) were evaluated for RejuAgro A production. Seed media were prepared by growing the bacteria in YME medium at 16°C and 220 rpm for 24 hours. HPLC analysis showed that all seven bacterial strains produced RejuAgro A (Figure 10). [Example 11] Formulation and greenhouse assay of RejuAgro A.

[0107] Formulation of RejuAgro A (solution, SL; see Table 5). Before application to flowers, 10 μg / mL was tank-mixed with 1% polyethylene glycol (PEG) 4000 as a toxicity mitigator. Subsequent tests showed that using 0.03% polyvinyl alcohol (PVA) as a toxicity mitigator resulted in superior flower protection. Additionally, the surfactant Alligare90 can be added for even greater efficacy (Table 5).

[0108] [Table 5]

[0109] To evaluate the bioregulatory activity of RejuAgro A against Erwinia amylovola, a greenhouse infection assay was conducted using crabapple trees at the University of Wisconsin-Milwaukee. A solution of 10 μg / mL of RejuAgro A supplemented with 1% polyethylene glycol (PEG) 4000, or 1% PEG 4000 (negative control), was applied to fully bloomed tree flowers 3 hours before inoculation and 24 hours after inoculation. For inoculation, approximately 100 units of E. amylovola strain 110 suspended in water were used. 8 CFU / mL was used. Infection rates were calculated around 6 days after inoculation. RejuAgro A can effectively suppress flower blight (Table 6).

[0110] [Table 6]

[0111] [Example 12] Antifungal activity of 0617-T307 cell broth against Botrytis cinerea CA17 The inoculum of strain 0617-T307 was prepared by growing it in YME medium at 28°C and 180 rpm for 24 hours. Then, 4% (2 mL to 50 mL) was inoculated into 250 mL flasks containing 50 mL of M8 (IAA medium), M9 (CN medium), M7 (PRN medium), or M6 (DAPG medium), and grown at 28°C and 180 rpm for 48 hours. 0.5 mL of cell broth was collected at 12 and 24 hours and stored in a -20°C freezer. For the antifungal assay, the cell broth was thawed and 5 μL was spread onto the sample wells of a PDA (potato dextrose agar) plate at equal radial distances from the center inoculated with Botrytis cinerea (Figure 11). The cell broth was shown to have antifungal activity against Botrytis cinerea CA17 on the PDA (potato dextrose agar) plate.

[0112] [Example 13] Antimicrobial activity of crude extracts, RejuAgro A, and RejuAgro B against plant pathogenic bacteria.

[0113] Metabolites of bacteria 0917-T305, 0318-T327, and 0418-T328 showed excellent efficacy against Ralstonia solanacearum, C. machinansis subspecies machinansis, and Xanthomonas arboricola pathogenic form 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 tested by plate diffusion assay against three plant pathogens: Ralstonia solanacearum, C. machinansis subspecies machinansis, and Xanthomonas arboricola pathogenic form jugrandis. In agar plate diffusion assays, metabolites of bacteria 0917-T305, 0318-T327, and 0418-T328 grown in YME at 16°C and 28°C showed relatively good efficacy against Ralstonia solanacearum, C. machinansis subspecies machinansis, and Xanthomonas arboricola pathogenic form jugrandis (Table 7). This indicates that, along with RejuAgro A, other metabolites also have excellent efficacy against Ralstonia solanacearum, Clavibacter machinansis subspecies machinansis, and Xanthomonas arboricola pathogenic form jugrandis. RejuAgro B showed good efficacy against Ralstonia solanacearum (Table 7).

[0114] [Table 7]

[0115] [Example 14] Antibacterial effect of Rt18.9, Rt22.9, and Rt25.0.

[0116] The stock bacterium, Pseudomonas species 0617-T307, was inoculated onto LB agar plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, water) and grown in a 28°C incubator for 24 hours. Fermentation and preparation of the crude extract were carried out in the same manner as described in Example 6.

[0117] HPLC isolation and purification of ethyl acetate extracts from acidified cell broth of Pseudomonas species 0617-T307 identified two antimicrobial compounds (Rt22.9 and Rt25.0) from flash fraction T5058 and one antimicrobial compound (Rt18.9) from flash fraction T7882. These were tested for antimicrobial activity against the bacterial strains shown in Table 8. 2 μL of DMSO, Rt18.9, Rt22.9, or Rt25.0 were spotted onto agar plates grown with different bacterial strains, and the inhibition zones were further tested (Table 8).

[0118] [Table 8] TIFF0007914116000018.tif129164

[0119] [Example 15] Antimicrobial effect of RejuAgro A against Mycosferella fijiensis The antimicrobial effect of RejuAgro A against Mycosferella fijiensis was investigated by adding RejuAgro A at final concentrations of 60 μg / mL and 600 μg / mL, respectively, to PDA agar plates, after purification by HPLC. 480 μL of 0.5 mg / mL or 5 mg / mL RejuAgro A was added to 3.52 mL of PDA in a well of a 6-well plate to achieve final concentrations of 60 μg / mL (Figure 12, center well (Panel A)) and 600 μg / mL (Figure 12, left well (Panel B)), respectively. The plate was gently shaken to dissolve the compound. 480 μL of water containing 3.52 mL of PDA was used as a control (Figure 12, right well (Panel C)). After the agar solidified, the agar plate on which M. fijiensis was grown was placed in the center of the agar surface. Complete suppression of M. phiensis proliferation was observed 2 weeks after inoculation when treated with a concentration of RejuAgro A at 600 μg / mL (Figure 12).

[0120] [Example 16] Antimicrobial effect of RejuAgro A against Xanthomonas oryzae pathogenic form (Xon507) The antibacterial effect of RejuAgro A against the pathogenic form of Xanthomonas oryzae (Xon507) was investigated. X. oryzae pathogenic form (Xon507) bacterial suspension (OD 600 A solution (=0.3) was sprayed onto a PSG agar plate. Paper discs loaded with aqueous RejuAgro A purified by HPLC 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, each with a loading volume of 50 μL, were placed on the agar plate, and the zone of inhibition was measured 44 hours after the paper discs were placed. Inhibition was observed at all concentrations of the RejuAgro A suspension on the paper discs (Table 9).

[0121] [Table 9]

[0122] [Example 17] Antimicrobial effect of RejuAgro A against Xanthomonas citri pv. citri citrange (XW19) pathogenic form. The antibacterial effect of RejuAgro A against Xanthomonas citri pathogenic form citri citrande (XW19) was investigated. (OD) 600 A solution (=0.3) was sprayed onto a PSG agar plate. Paper discs loaded with aqueous RejuAgro A purified by HPLC 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, each in a 50 μL loading volume, were placed on the agar plate, and the zone of inhibition was measured 44 hours after the paper discs were placed on the agar plate. Inhibition was observed at RejuAgro A concentrations of 55.37 μg / mL and 110.74 μg / mL (Table 10).

[0123] [Table 10]

[0124] [Example 18] Use of RejuAgro A and other Solanaceae hosts to suppress citrus greening disease and potato zebra tip disease.

[0125] Yellow dragon disease (HLB), also known as citrus greening, is one of the most devastating diseases affecting citrus fruits. HLB is believed to originate in Asia, and in the United States, it was first detected in Florida in 2005. Since 2005, HLB has spread to citrus-producing areas in Florida, reducing citrus yields by 75% while more than doubling production costs. In 2008, HLB was detected in Louisiana, and in 2009, the disease was detected in Georgia and South Carolina. In 2012, HLB was detected in residential areas of Texas and California (Hu & Wright (2019)). The disease is caused by Candidatus Liberibacter asiaticus, a bacterial pathogen that cannot be cultured in pure media. Liberibacter crescens is the only species of this genus that can be grown in sterile media and is used as a model for studying other unculturable Liberibacter pathogens, such as Ca. Liberibacter americanus and Ca. Liberibacter africanus, which cause citrus greening; and Ca. Liberibacter solanacearum, which causes potato zebra tip (ZC) disease and attacks tomatoes and other plants in the Solanaceae family as well as plants in the Apiaceae or Umbelliferae families (Sena-Velez et al. (2019)).

[0126] The HLB pathogen resides in the phloem of plants, and its spread requires the citrus psyllid, a pathogenic insect. While efforts have been made to control this disease, their effectiveness has been limited and unsustainable. Current methods for preventing infection and maintaining the productivity of HLB-infected trees include insecticidal control of the pathogen, antimicrobial treatments, and nutritional supplements. The antibiotics oxytetracycline and streptomycin are the only viable options for controlling this disease, but these antibiotics may lead to antibiotic resistance in human pathogens and disruption of the citrus tree ecosystem. Insect control through insecticide spraying also poses a potential threat to human health and non-target insects such as pollinators. Recent advances in the use of antimicrobial peptides to treat HLB are promising (Huang et al. (2021)), but they remain experimental, and the costs of large-scale application to the vascular tissue of citrus trees may be high.

[0127] The inventors have previously isolated the bacterial species Pseudomonas soli 0617-T307 from a soil sample in Wisconsin. This bacterial strain produces an active compound, RejuAgro A (RAA), which exhibits inhibitory effects against a variety of plant bacterial pathogens, including Erwinia amylovola, a causative agent of burn disease, and Xanthomonas axonopodis, a causative agent of citrus crown gall disease. The inventors successfully purified this compound, which has a molecular weight of 185.2 g, significantly lower than that of oxytetracycline (MW: 460.4 g) and streptomycin (MW: 581.6 g). Preliminary test results show that RAA can successfully inhibit the growth of Liberibacter cressens BT-1. The potency and minimum inhibitory concentration (MIC) are comparable to those of oxytetracycline and streptomycin, and are low at 2.5 mg / L over 16 days of inhibition (Figure 13). The target of RAA is plant bacterial diseases that cause economic damage to crops and fruits. Since RAA is a novel natural compound that has not been applied in humans or animals, the risk of enhancing antibiotic resistance is considered to be much lower than that of other conventional antibiotics. In addition, its smaller molecular size makes it easier for HLB to reach the vascular tissue of citrus trees where it inhabits. RAA can be used to control HLB, ZC, and many other Solanaceae host diseases caused by Candidatus with significantly lower environmental impact.

[0128] [Example 19] Use of RejuAgro A to suppress plant fungal pathogens The in vivo antimicrobial activity of HPLC-purified RejuAgro A was evaluated. Various concentrations of RejuAgro A (5, 10, 15, 25, 50, and 100 μg / mL) were added to 6-well plates containing agar. DMSO was used as a negative control, and specific antimicrobial compounds were also used as negative controls. Fungal cultures grown in Petri plates were cut into small pieces and transferred to the center of each well of the assay plate. Fungal growth was observed after 6 days. For Septoria tritisi and Coletotrichum dematium, the growth medium was potato dextrose agar (PDA), and the positive control was nystatin (50 μg / mL). For Fusarium oxysporum differentiated form melonis and Magnaporte oryzae, the growth medium used was PDA, and the positive control was cycloheximide (50 μg / mL). The antimicrobial effects of the fungal pathogens Septoria tritisi, Coletotricum dematium, Fusarium oxysporum, differentiated forms Melonis and Magnaporte oryzae, as measured by minimum inhibitory concentrations (MICs), were 100, 50, 100, and 75 μg / mL, respectively (Table 11). This study demonstrates that RejuAgro A has inhibitory activity against Septoria leaf blight caused by Septoria tritisi in wheat and other cereals. RejuAgro A shows inhibitory activity against Coletotricum species fungi that infect plant hosts, including apples, peaches, grapes, berry crops (e.g., strawberries, blueberries, cranberries) and other plant crops that are susceptible to Coletotricum infection. In addition, RejuAgro A exhibits inhibitory activity against Fusarium species, which cause Fusarium head blight in wheat and barley, as well as Fusarium wilt in tomatoes, sweet potatoes, melons, legumes, bananas (Panama disease), and other crops susceptible to Fusarium infection. RejuAgro A also exhibits inhibitory activity against Magnaporte oryzae, which causes rice rot.

[0129] [Table 11]

[0130] [Example 20] Culture medium composition used in the example Table 12 includes exemplary culture medium compositions used in the examples.

[0131] [Table 12]

[0132] [Example 21] Bacterial strains, natural products, and the literature cited therein.

[0133] The bacterial strains and natural products described in this application and the attached claims are well known in the microbiological literature. These documents are listed in Table 13 below for each of the cited bacterial strains and natural products disclosed herein, and their contents are incorporated herein by reference in their entirety.

[0134] [Table 13]

[0135] References

[0136] [Table 14] TIFF0007914116000025.tif229164TIFF0007914116000026.tif229164TIFF0007914116000027.tif237167TIFF0007914116000028.tif133163

[0137] Built-in by reference All documents, publications, patents, patent applications, and related materials cited herein are incorporated by reference as if they were fully contained herein.

Claims

1. A method for controlling bacterial crop diseases caused by pathogenic microorganisms, i. 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 mosselii From the group consisting of 0418-T328 (accession number PTA-126802), a bacterial strain of formula (I) 【Chemistry 1】 A step of producing an agricultural composition containing a compound represented by; and ii. A step of applying the agricultural composition to crops to suppress the growth of the pathogenic microorganisms. A method that includes this.

2. The method according to claim 1, wherein the crop disease is selected from the group consisting of black sigatoka, gray mold, burn, citrus gall, soft rot, olive gall, tomato bacterial leaf spot, bacterial gall or blast, cucurbit horn spot, peach bacterial spot, tomato bacterial spot, walnut canker, bacterial wilt, tomato gall, potato leaf blight, apple red mold, bacterial leaf blight, citrus greening disease, potato zebra chip disease, and bacterial stripe blight.

3. The pathogenic microorganisms include Mycosphaerella fijiensis, Botrytis cinerea, Erwinia amylovora, Xanthomonas axonopodis pv. citri, Pectobacterium parmentieri, Pectobacterium atrosepticum, and Pectobacterium carotoborum subspecies brassiliensis. Carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp. carotovorum, Dickeya dadantii, Pseudomonas savastanoi pv. savastanoi, Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. syringae Pseudomonas syringae pv. lachrymans, Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. vesicatoria, Xanthomonas arboricola pv. juglandis, Ralstonia solanacearum, Clavibacter machinanensis subspecies *Clavibacter miciganensis*The method according to claim 1, selected from the group consisting of subsp. michiganensis, Phytophthora infestans, Venturia inaequalis, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Xanthomonas citri pv. citri.

4. The method according to claim 1, wherein the crop is selected from the group consisting of bananas, apples, pears, crabapples, citrus fruits, potatoes, pumpkins, onions, rice, African violets, plants of the Brassicaceae family, plants of the Solanaceae family, plants of the Cucurbitaceae family, stony fruits, and pomelofruits.

5. A method for controlling fungal pathogens in crops, i. 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 mosseri 0118-T319 (Accession number PTA-126800), Pseudomonas mosseri 0318-T327 (Accession number PTA-126801) and Pseudomonas mosseri From the group consisting of 0418-T328 (accession number PTA-126802), a bacterial strain of formula (I) 【Chemistry 2】 A step of producing an agricultural composition containing a compound represented by; and ii. A step of applying the agricultural composition to the crop to suppress the growth of the fungal pathogen. A method that includes this.

6. The method according to claim 5, wherein the fungal pathogen is selected from the group consisting of Septoria tritici, Colletotrichum dematium, Fusarium oxysporum f.sp. Melonis, and Magnaporte oryzae.

7. The method according to claim 5, wherein the crop is selected from the group consisting of apples, peaches, grapes, berry crops, wheat, barley, tomatoes, sweet potatoes, melons, legumes, bananas, and rice.

Images