TWI890698B - Pseudomonas strains and their metabolites to control plant diseases - Google Patents

Abstract

The present disclosure concerns methods of using novel bacterial strains of 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328, the cell broth and novel metabolites produced from the bacterial strains, that can inhibit the growth of a variety of microbial species for a variety of crops. The methods include use of novel, potent antimicrobial metabolites produced from the strains corresponding to compounds having Formulas (I), (II), and (III): , , and

Description

Pseudomonas strains and their metabolites for controlling plant diseases

The present invention relates to the field of biological insecticides. Specifically, the invention relates to seven novel Pseudomonas spp. strains: 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328; and to cell broths and novel metabolites produced by these strains that inhibit the growth of various microbial species. Pseudomonas strains 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328 have been deposited with the American Type Culture Collection (ATCC) and have ATCC accession numbers PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802, respectively. In addition, Pseudomonas strains 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328 have been deposited with the Food Industry Research and Development Institute (FIRDI) and have FIRDI accession numbers BCRC911020, BCRC911021, BCRC911022, BCRC911023, BCRC911024, BCRC911025, and BCRC911026, respectively.

Plant diseases caused by pathogenic microorganisms are increasing exponentially and are costly. Plant pathogens include fungi, bacteria, molds, viruses, viroids, nematodes, or parasites of flowering plants. Currently, there are 14 common plant diseases caused by bacterial organisms, including bacterial spot, bacterial light, and bacterial wilt. Fire burn ( Erwinia amylovora ), citrus ulcer [Xanthomonas axonopodis pv. citri (Xac) ], bacterial leaf spot (BLS) [Xanthomonas campestris pv. vesicatora (XV-16)], olive knot [ Pseudomonas savastanoi pv . Savastanoi ( Psv )], and soft rot ( Dickeya dadantii , Pectobacterium parmentieri , Pectobacterium spp . atrosepticum , and Pectobacterium carotovorum are devastating plant diseases. Nationally, the cost of controlling fireburn has been estimated at over $100 million (Norelli et al. (2003)). For citrus scab, the cost of running an eradication program in Florida alone from 1995 to 2005, combined with compensation to commercial growers and homeowners whose residential citrus was damaged, approached $1 billion.

Fireburn is a devastating disease of pome fruit caused by infection with the Gram-negative bacterium Erwinia amylovora, which affects pears and apples in many parts of the world, including Europe, Germany, Australia, and Switzerland (Chen et al. (2009)). Although fireburn rarely wipes out entire orchards, the disease and its control still cause significant economic losses. In the Pacific Northwest and Northern California, small outbreaks have occurred annually since 1991, with major outbreaks occurring every three to four years in at least some areas. Even small outbreaks can be costly because pruning to remove infected plant parts results in tree damage and reduced future productivity. For example, a 10% incidence of rhizome dieback 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 biochemical compounds used as insecticides (Gwinn (2018)). However, current preventive measures for bacterial plant diseases have limited effectiveness. The antibiotics streptomycin sulfate (FireWall, AgroSource, Inc.) and terpenoid hydrochloride (FireLine, AgroSource, Inc.) are the primary products used against Erwinia amylovora when the risk of infection is high. Because these compounds are also used to manage human and animal health, their use in crop agriculture may be controversial (Stockwell (2012)). For streptomycin sulfate, concerns about antibiotic resistance have limited its use (Vrancken et al. (2013)). Another antibiotic under investigation for fire fever is kasugamycin. One drawback is that frequent doses of kasugamycin can lead to plant-damaging phytotoxic effects (Adaskaveg et al. (2010)). Another drawback is the high cost of kasugamycin compared to other antibiotics. Therefore, kasugamycin needs to be combined with various other antibiotics.

In recent decades, a number of non-antibiotic products have been developed, registered with the Environmental Protection Agency (EPA), approved by the National Organic Program (NOP), and marketed to fruit growers for the control of fireburn (Tianna et al., 2018). Historically, two products based on Bacillus subtilis have been registered in Europe for the control of fireburn: Serenade®, based on strain QST 713, and Biopro®, based on strain BD 170 (Broggini et al., 2005). Bioformulations based on spore-forming Bacillus bacteria offer the advantage of biocontrol due to their long-lasting viability (Haas et al., 2005). Two Bacillus-based bioformulations have demonstrated modest success in numerous field trials in the United States and Germany (Aldwinckle et al. (2002); Kunz et al. (2011); Laux et al. (2003)). This suggests the potential of Bacillus species in controlling bloom infections of Erwinia amylovora. However, Bacillus was only effective under low infection pressures. Bacillus was ineffective under moderate and high infection pressures. Results obtained with both biological products were inconsistent, ranging between 71% and 0% disease inhibition (Broggini et al. (2005)).

The desired bioprotective product must, on the one hand, effectively compete with E. amylovora and, on the other hand, be able to colonize different organs of the target plant within the same ecological niche. Protective bacteria produce secondary metabolites that affect the pathogen and compete for food and space, thereby preventing the pathogenesis of E. amylovora associated with plants. In this case, bacteria from the genus Pseudomonas have adapted to the bioprotective factors described above (Haas et al. (2005)). Analyses of the composition of species that colonize various plant species have revealed the widespread presence of fluorescent bacteria from the genus Pseudomonas.

In France, Pseudomonas species were found to predominate in the colonies of healthy and diseased apple, pear, and hawthorn trees, and many of these bacteria were shown to restrict the growth of Erwinia amylovora in vitro (Paulin et al. (1978)). However, little information on potent metabolites has been reported.

In California, Thomson et al. (1976) selected three fluorescent Pseudomonas strains that were effective in protecting pear blossoms (Thomson et al. (1976)). In the mid-1980s, the fluorescent Pseudomonas strain A506, isolated from pear trees in California, demonstrated unique activity in limiting the growth of Erwinia starch and protecting apple and pear trees from fire burn (Lindow et al. (1996)). A product containing fluorescent Pseudomonas was developed, BlightBan® A506, which has been commercially available since 1996. Numerous experiments conducted in California, Oregon, and Washington state have shown the preparation to be useful in various apple and pear preservation procedures (Johnson (2000)).

In the UK, two isolates of Pseudomonas fluorescens were used to protect flowers and branches of hawthorn trees (Wilson et al. (1992)).

In Italy and New Zealand, the suitability of two Pseudomonas strains, designated BO 3371 and BO G19, has been studied (Galasso et al. (2002)). Under greenhouse conditions, these bacteria are highly effective in protecting apple and pear blossoms and branches. For example, strain BO3371 achieved 87% relative protection of pear branches (Galasso et al. (2002)). However, the results obtained were not always consistent, which may be related to the ease with which flowers entangle branches, coupled with the length of time from flowering to the end of flowering.

In New Zealand, the fluorescent Pseudomonas strain IPV-BO G19 protected 79% of apple blossoms under field conditions. In another experimental garden, when sprayed 24 hours before inoculation of Braeburn apple blossoms with Erwinia amylovora, fluorescent Pseudomonas strains IPV-BO G19 and IPV-BO 3371 reduced fire burn by 78% and 58%, respectively (Biondi et al. (2006)).

In Spain, the fluorescent Pseudomonas strain EPS62e significantly limited fireburn in field tests on apple blossoms, pear fruit, and pear blossoms. A strategy combining nutritional enhancement with osmoadaptation was used to improve the adaptability and efficacy of Pseudomonas fluorescens EPS62e against fireburn. Field treatments of pear blossoms with the physiologically modified strain EPS62e resulted in efficacy rates as high as 90%, however, results were mixed depending on the test (Cabrefiga et al. (2011); Mikiciński et al. (2020)).

In Poland, 47 bacterial colonies capable of reducing the effects of fire burn on pear fruitlets were isolated from apple phyllosphere and soil (Mikiciński et al. (2008)).

Metabolites produced by Gram-negative Pseudomonas species have been comprehensively reviewed (Masschelein et al. (2017)). Pseudomonas metabolites can be categorized into phenolic compounds, phenotypes, lipopeptides, and others. 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 also significantly improve plant growth (plant growth regulators, IAA, mucin); 3) antagonism can conserve the production of siderochelatins and surfactants such as mucin and viscosinamide, as well as antimicrobial compounds such as hydrogen cyanide, phenotypes, pyrrolnitrin (pyrrolnitrin), or 2,4-diacetylphloroglucinol (DAPG). During the work, bacterial strains were identified, fermentation products produced by bacteria, and novel metabolites were identified. Specifically, RejuAgro A and RejuAgro B showed higher efficacy against a variety of pathogenic microorganisms, including previously unreported bacteria and fungi.

There is a need for new biopesticides derived from novel bacterial strains, cytosolic fluids and novel metabolites produced by such strains that can inhibit the growth of a variety of pathogens that cause crop diseases.

In a first aspect, a method for growing bacteria to enhance the production of protective metabolites is provided. The method includes alternate steps. In one method, a step is provided for growing Pseudomonas bacteria in a liquid culture medium in a container to produce a bacterial fermentation product. The ratio of the culture medium volume to the container volume is between approximately 1:2 and 1:10, and the container is vibrated at a rate of between approximately 100 and 250 RPM. According to an alternate step, the method includes a step for growing Pseudomonas bacteria in a liquid culture medium in a fermenter to produce a bacterial fermentation product. The air flow rate in the fermenter is between approximately 1 and 3 L/min. The dissolved oxygen concentration is between 5 mg/L and 12 mg/L.

In a second aspect, an agricultural composition comprising a bacterial fermentation product or a protective supernatant is provided. The agricultural composition is produced according to the method of the first aspect and any aspect disclosed therein. In the first aspect, the agricultural composition further comprises an adjuvant. In this regard, the adjuvant is a surfactant.

In a third aspect, a method for controlling bacterial crop diseases is provided. The method comprises several steps. The first step comprises producing an agricultural composition comprising a bacterial fermentation product or protective supernatant produced by the first aspect or any aspect thereof. The second step comprises applying the agricultural composition to crops to inhibit the growth of pathogenic microorganisms.

In a fourth aspect, a method for controlling bacterial crop diseases is provided. The method comprises applying an agricultural composition comprising between approximately 1.0 x 10 ⁵ and 1.0 x 10 ⁹ cfu of Pseudomonas bacteria per mL to a crop to inhibit the growth of pathogenic microorganisms.

In a fifth aspect, a method for purifying a protective metabolite from Pseudomonas bacteria is provided. The method comprises several steps. The first step comprises producing a bacterial fermentation product or protective supernatant using the method of the first aspect and aspects thereof. The second step comprises extracting the bacterial fermentation product or protective supernatant using a solvent mixture having similar polarity or characteristics. The third step comprises producing an eluate containing the protective metabolite by dissolving the bacterial fermentation product or protective supernatant using a mixture of hexane and ethyl acetate or by dissolving the bacterial fermentation product or protective supernatant using a mixture of hexane and ethyl acetate.

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

In a seventh aspect, a method for controlling bacterial crop diseases is provided. The method comprises several steps. The first step comprises producing an agricultural composition comprising protective metabolites from Pseudomonas bacteria purified by the method of the fifth aspect and any aspect thereof. The second step comprises applying the agricultural composition. Formulations of the protective supernatant or its metabolites can be in the form of solutions (SL), soluble powders (SP), soluble granules (SG), and encapsulated formulations. Alternatively, agricultural compositions of bacterial fermentation products and cells can be in the form of 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: (Formula (I)), (Formula (II)), and (Formula (III)).

The present invention relates to novel metabolites produced by seven Pseudomonas strains listed in this patent, such as 0617-T307, which exhibit antimicrobial activity against pathogenic microorganisms, including bacteria and fungi. The strains can be identified as Pseudomonas soli 0617-T307, a strain of the Pseudomonas putida group, based on 16S rRNA and other housekeeping gene sequences. The cytosol of these seven bacterial strains, such as 0617-T307, contains the novel, potent, six-membered, multicyclic natural product, designated RejuAgro A, as shown below, as well as the dimer RejuAgro B. RejuAgro A& RejuAgro B.

These compounds, their production methods, and their use in inhibiting plant microbial pathogens are described in more detail herein. Definitions

When introducing elements of aspects or embodiments of the present disclosure, the articles "a," "an," and "the" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Unless otherwise indicated, the term "or" refers to any one member of a particular list and also includes any combination of members of that list.

As intended herein, the terms "substantially," "approximately," and "about," and similar terms are intended to have a broad meaning consistent with common and accepted usage in the art to which this disclosure pertains. Those skilled in the art who review this disclosure will understand that these terms are intended to allow a description of certain features described and claimed without limiting the scope of such features to the precise numerical ranges provided. Accordingly, these terms should be interpreted as indicating that minor or insignificant modifications or variations of the described and claimed subject matter are considered to be within the scope of the present invention as described in the appended claims.

Biological control agents (or BCAs) are safe, durable, and cost-effective ways to manage pests such as pathogens, weeds, and insects. These agents are introduced into the environment to target specific pest species with the goal of reducing their populations or abundance.

Biologics are preparations of live microorganisms (bacteria and yeasts) that colonize a host. These microorganisms are primarily used to delay the accumulation of pathogens during the epiphytic phase (Tianna et al. (2018)).

“Biorational” is a term applied to biocides based on microorganisms. These biocides are typically produced by fermenting microbial strains. Many of these products have antibacterial and antifungal activity (Tianna et al. (2018)).

The U.S. Environmental Protection Agency (EPA) defines “biopesticides” as insecticides derived from natural materials and categorizes them as biochemical insecticides containing substances that control pests by non-toxic mechanisms, microbial insecticides composed of microorganisms that normally produce bioactive natural products (BNPs), or plant-embedded protective agents that have plant-derived activity due to the addition of genetically modified substances (Gwinn K.D. (2018)).

The compounds referred to as RejuAgro A, RejuAgro B and RejuAgro C correspond to the chemical compounds having the formulae (I), (II) and (III) shown below, respectively: (I) (II) and (III).

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

In a second aspect, an agricultural composition comprising a bacterial fermentation product or a protective supernatant is provided. The agricultural composition is produced according to the method of the first aspect and any aspect disclosed therein. In the first aspect, the agricultural composition further comprises an adjuvant. In this regard, the adjuvant is a surfactant.

In a third aspect, a method for controlling bacterial and fungal crop diseases is provided. The method comprises several steps. The first step comprises producing an agricultural composition comprising a bacterial fermentation product or protective supernatant produced by the first aspect or any aspect thereof. The second step comprises applying the agricultural composition to crops to inhibit the growth of pathogenic microorganisms.

In a first aspect, the crop disease is selected from the group consisting of tomato and pepper burn, citrus scab, olive knot, and soft rot. In a second aspect, the pathogenic microorganism is selected from the group consisting of: black leaf spot pathogen, gray mold, Erwinia starch (Ea), Xanthomonas amylovora var. citri (Xac), Pectobacterium spp., Pectobacterium rot, Pectobacterium carotovorum subsp. brasiliensis, Pectobacterium carotovorum subsp. carotovorum, Erwinia chrysanthemi, Pseudomonas sarsoni var. sarsoni (Psv), Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. syringae), Pseudomonas syringae pv. lachrymans, Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. vesicatoria, Xanthomonas arboricola pv. juglandis, Ralstonia solanacearum, Clavibacter michiganensis subsp. michiganensis, Phytophthora infestans, Apple Vent, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzae pv. Oryzicola), and Xanthomonas citri pv. citri. In a third aspect, the crop is selected from one or more of the following: banana, apple, pear, crabapple, citrus, potato, pumpkin, onion, rice, African violet, species of plants from the family Rutaceae, Solanaceae, Cucurbitaceae (including carrots, potatoes, tomatoes, eggplants, leafy greens, squash, and melons), peppers and bell peppers, olives, stone fruits, and pome fruits (including olives, peaches, and walnuts).

In a fourth aspect, a method for controlling bacterial crop diseases is provided. The method comprises applying an agricultural composition comprising between approximately 1.0 x 10 ⁵ and 1.0 x 10 ⁹ cfu of Pseudomonas bacteria per mL to a crop to inhibit the growth of pathogenic microorganisms.

In a first aspect, the Pseudomonas bacteria are strains of Pseudomonas selected from the group consisting of 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328. In a second aspect, the composition comprises between about 5.0 x 10 ⁷ and 2.0 x 10 ⁸ cfu of Pseudomonas bacteria per mL. In a third aspect, the crop disease is selected from the group consisting of black leaf spot, gray mold, fire burn, citrus scab, soft rot, olive scab, bacterial spot of tomato, bacterial scab or rice blast (stone fruit and pome fruit), angular spot of cucurbits, bacterial spot of peach, bacterial spot of tomato, black rot of walnut, bacterial wilt, tomato scab, potato late blight, apple scab, bacterial leaf blight, and bacterial leaf streak. In a fourth aspect, the pathogenic microorganism is selected from the group consisting of: black leaf spot fungus , gray mold, Erwinia starch-soluble ( Ea ), Xanthomonas amylovora var. citri ( Xac ) , Pectinobacterium tuberosum, Pectinobacterium rotum, Pectinobacterium brasiliensis, Pectinobacterium carrotsii, Erwinia chrysanthemi, Pseudomonas sarsii var. sarsii ( Psv ), Pseudomonas syringae var. tomato, Pseudomonas syringae var. syringae, Pseudomonas syringae var. cucumber, Xanthomonas campestris var. plum, Xanthomonas campestris var. scabra, Xanthomonas arborescens var. juglans regia, Ralstonia solanacearum, Cladosporium michiganense, Phytophthora spp., Venturia spp., Xanthomonas oryzae var. oryzae var . oryzae, and Xanthomonas citri var . In a fifth aspect, the crop is selected from one or more of the following: bananas, apples, pears, crabapples, citrus, potatoes, pumpkins, onions, rice, African violets, species of plants from the family Rutaceae, Solanaceae, Cucurbitaceae (including carrots, potatoes, tomatoes, eggplants, leafy greens, squashes, and melons), peppers and bell peppers, olives, stone fruits, and pome fruits (including olives, peaches, and walnuts).

In a fifth aspect, a method for purifying a protective metabolite from Pseudomonas bacteria is provided. The method comprises several steps. The first step comprises producing a bacterial fermentation product or a protective supernatant by the method of the first aspect and aspects thereof. The second step comprises extracting the bacterial fermentation product or the protective supernatant by extraction with ethyl acetate. The third step comprises producing an eluate containing the protective metabolite by solubilizing the bacterial fermentation product or the protective supernatant using a mixture of hexane and ethyl acetate, such as, for example, a mixture of 50% hexane and 50% ethyl acetate, or by solubilizing the ethyl acetate extract using a mixture of hexane and ethyl acetate, such as, for example, a mixture of 25% hexane and 75% ethyl acetate.

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

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

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

In a first aspect, the crop disease is selected from the group consisting of tomato and pepper fire burn, citrus scab, olive knot, and soft rot. In a second aspect, the pathogenic microorganism is selected from the group consisting of banana black leaf spot fungus , gray mold, Erwinia starch ( Ea ) (especially a streptomycin-resistant strain of Erwinia starch) , Xanthomonas amylovora var. citri ( Xac ) , potato pectin, black rot pectin, carrot pectin, carrot pectin, carrot pectin, Erwinia chrysanthemi, Pseudomonas sarcoma var. sarcoma ( Psv ), Pseudomonas syringae var. tomato, Pseudomonas syringae var. syringae, Pseudomonas syringae var. cucumber, Xanthomonas campestris var. plum, Xanthomonas campestris var. scabra, Xanthomonas arborescens var. juglans var., Ralstonia solanacearum, Cladosporium michiganense, Phytophthora spp., Venturia spp., Xanthomonas oryzae var. oryzae , and Xanthomonas citri var. citri . In a third aspect, the pathogenic Erwinia amylovora is streptomycin-resistant Erwinia amylovora. In a fourth aspect, the crop is selected from one or more of the following: banana, apple, pear, crabapple, citrus, potato, pumpkin, onion, rice, African violet, species of plants from the family Rutaceae, Solanaceae, and Cucurbitaceae (including carrots, potatoes, tomatoes, eggplants, leafy greens, pumpkins, and melons), peppers and green bell peppers, olives, and stone and pome fruits (including olives, peaches, and walnuts). In a fifth aspect, the pathogen is Flavobacterium columnare #2 or Flavobacterium columnare MS-FC-4. In a sixth aspect, the pathogen is Escherichia coli O157:H7.

In an eighth aspect, the crystalline compound is selected from one of the following structures: Formula (I), Formula (II), and Formula (III).

In a first aspect, the crystalline compound has the following structure: Formula (I), wherein the crystalline compound comprises at least one physical property selected from Tables 13-22.

In a second aspect, the crystalline compound is the following structure: Formula (II), wherein the crystalline compound comprises at least one physical property selected from Tables 23-29.

In a third aspect, the crystalline compound has the following structure: Formula (III), wherein the crystalline compound comprises at least one physical property selected from Tables 30-37.

Bacterial strains Pseudomonas aestivus 0617-T307, Pseudomonas aestivus 0917-T305, Pseudomonas aestivus 0917-T306, Pseudomonas aestivus 0917-T307, Pseudomonas mosselii 0118-T319, Pseudomonas mosselii 0318-T327, and Pseudomonas mosselii 0418-T328 were submitted to the American Type Culture Collection (ATCC®), PO Box 1549, Manassas, VA 20110 USA, on June 25, 2020. ("ATCC Patent Depository") and were granted unofficial ATCC patent numbers PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802, respectively. After viability testing, the ATCC Patent Depository assigned the following accession numbers to the deposited bacterial strains, effective June 25, 2020: Pseudomonas aeruginosa 0617-T307 (Accession No. PTA-126796), Pseudomonas aeruginosa 0917-T305 (Accession No. PTA-126797), Pseudomonas aeruginosa 0917-T306 (Accession No. PTA-126798), Pseudomonas aeruginosa 0917-T307 (Accession No. PTA-126799), Pseudomonas morganii 0118-T319 (Accession No. PTA-126800), Pseudomonas morganii 0318-T327 (Accession No. PTA-126801), and Pseudomonas morganii 0418-T328 (Accession No. PTA-126802). In addition, the bacterial strains Pseudomonas aeruginosa 0617-T307, Pseudomonas aeruginosa 0917-T305, Pseudomonas aeruginosa 0917-T306, Pseudomonas aeruginosa 0917-T307 , Pseudomonas morganii 0118-T319, Pseudomonas morganii 0318-T327, and Pseudomonas morganii 0418-T328 were deposited with the Food Industry Development Institute (FIRDI) in Taiwan. After viability testing, FIRDI assigned the following registration numbers to these bacteria, effective September 18, 2020: Pseudomonas aeruginosa 0617-T307 (registration number BCRC 911020), Pseudomonas aeruginosa 0917-T305 (registration number BCRC 911021), soil Pseudomonas aeruginosa 0917-T306 (Accession No. BCRC 911022), soil Pseudomonas aeruginosa 0917-T307 (Accession No. BCRC 911023); effective from September 17, 2020: Pseudomonas morganii 0118-T319 (Accession No. BCRC 911024), Pseudomonas morganii 0318-T327 (Accession No. BCRC 911025), and Pseudomonas morganii 0418-T328 (Accession No. BCRC 911026) Examples Example 1. Identification and characterization of strain 0617-T307.

Partial sequences from 16S rDNA, gyrB , rpoB , and rpoD were analyzed. These four genes are recommended markers for multiple sequence analysis (MLSA) in Pseudomonas species (Peix et al. (2018)).

For species identification, a BLASTN run was performed against the NCBI non-redundant nucleotide database using these four sequences. Based on the results, strain 0617-T307 is closely related to Pseudomonas species within the Pseudomonas fluorescens group. The "MLSA species occurrence" and "Genome list of type strains from the genus Pseudomonas" (Peix et al. (2018); see Figure 2 and Table 2 in Peix et al. (2018)) guidelines for the selection of taxonomic units were used (Figure 1). Based on this information, genomes were obtained from GenBank. All species within the Pseudomonas fluorescens group for which high-quality genome assemblies were available were included. Because 0617-T307 shares the highest rpoD sequence similarity (i.e., the gene with the highest discriminant function for Pseudomonas species nomenclature) with soil Pseudomonas, all four available genomes of soil Pseudomonas (including the type strain LMG 27941 ^T ) were included in the sample. For other species within the fluorescent Pseudomonas lineage, one species was selected as a representative of each group. Pseudomonas aeruginosa ( P. aeruginosa group; P. aeruginosa strains) was included as an outgroup of fixed trees.

Four MLSA genes were extracted from the sampled genomes. Each gene was aligned individually, and all four nucleotide alignments were concatenated for phylogenetic analysis. The concatenated alignment contained 9,912 aligned nucleotide positions. Maximum likelihood inference was performed using PhyML (Guindon et al. (2003)). Bootstrapping was performed using 1,000 replicates.

Based on polytomous speciation (Figure 1), 0617-T307 and all four soil Pseudomonas strains with available genomic sequences formed a monophyletic clade with 100% self-enlistment support. This result provides strong support for the assignment of 0617-T307 to the soil Pseudomonas strain reportedly isolated from a soil sample in Sierra Nevada National Park, Spain (Pascual et al. (2014)).

In addition, based on the guidance provided by García-Valdés and Lalucat for the nomenclature of Pseudomonas species (García-Valdés et al. (2016)), additional support for assigning 0617-T307 to soil Pseudomonas is included: (a) 16S rDNA > 98.7-99% identity. Compared with the type strain of soil Pseudomonas, 0617-T307 shares 99.2% sequence identity. Compared with the sister species of insect-borne Pseudomonas ( P. entomophila ), 0617-T307 shares 99.5% sequence identity. Note that 16S rDNA is known to lack sufficient discriminatory power for species identification in Pseudomonas (García-Valdés et al. (2016); Peix et al. (2018)); (b) rpoD gene > 95-96% identity. Compared to a type strain of soil Pseudomonas, 0617-T307 shares 96.5% sequence identity. Compared to a sister species of insect-borne Pseudomonas, 0617-T307 shares only 89.1% sequence identity; and (c) MLSA > 97% identity. Compared to a type strain of soil Pseudomonas, 0617-T307 shares 98.0% sequence identity. Compared to a sister species of insect-borne Pseudomonas, 0617-T307 shares only 95.1% sequence identity. Example 2. Preparation, isolation, and characterization of RejuAgro A and RejuAgro B from an ethyl acetate extract of the cell fluid of strain 0617-T307.

RejuAgro A and B preparations were obtained by ethyl acetate extraction of cell broth from fermentation jars, followed by chromatographic separation and purification. Briefly, a stock of Pseudomonas sp. 0617-T307 was streaked onto LB plates (tryptophan, 10 g/L; yeast extract, 5 g/L; NaCl, 10 g/L; agar, 15 g/L; water) and grown in a 28°C incubator for 24 h. To prepare seed culture, a single 0617-T307 colony was inoculated into a 2.0-L flask containing 500 mL of autoclaved YME medium (yeast extract, 4 g/L; glucose, 4 g/L; and malt extract, 10 g/L) and grown at 28°C with shaking at 200 rpm for 24 hours. The seed culture was then inoculated into a 20-L NBS fermenter containing 12 L of autoclaved YME medium. Fermentation was carried out at 16°C for 1-7 days. Agitation and air flow rates were 200 rpm and 2 L/min, respectively.

After harvest, the bacterial culture was extracted four times with ethyl acetate. The ethyl acetate layer was separated and hydrated with sodium sulfate and dried by rotary evaporation at 35°C. This yielded 2.9 g of crude extract from a 12 L culture of strain 0617-T307.

The concentrated sample was dissolved in ethyl acetate and mixed with silica gel. The silica gel was packaged as an injection column (φ3.0 x 20 cm) and mounted atop a Universal column (4.8 x 18.5 cm) in a rapid chromatography system (Yamazen AI-580) equipped with a UV detector. After loading, the sample was eluted with 280 mL of each of the following solvents 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 at UV 254 nm and fractions were collected in time mode at 20 mL/tube. In total, 114 fractions or tubes were generated by flash chromatography.

The resulting fractions were used for subsequent plate assays. One mL of each fraction was pipetted into a 1.5 mL test tube and vacuum-dried using an Eppendorf vacuum concentrator. The dried sample was dissolved in 50 µL of DMSO, of which 2 µL was used for the plate assay. Briefly, Erwinia amylovora 273 was streaked onto an LB plate and grown in a 28°C incubator. A single colony obtained 24 hours later was inoculated into 5 mL of LB medium and allowed to grow overnight in a shaker at 200 rpm at 28°C. The bacteria were diluted 1:100 with sterile water, and 225 µL was plated onto a 50% LB plate (tryptin, 5.0 g/L; yeast extract, 2.5 g/L; NaCl, 5.0 g/L; agar, 15 g/L). After drying in a biosafety cabinet for 10 minutes, the DMSO solution of each fraction was dispensed onto a pre-marked section of the culture dish and allowed to dry for an additional 10 minutes. DMSO and kasugamycin were used as negative and positive controls, respectively, in the assay. The plates were then incubated in a 28°C incubator and examined for zones of inhibition one day later.

An in vitro plate assay of 114 fractions showed that two fractions inhibited the growth of E. amylovora 273. Notably, fractions 38-40 (abbreviated as T3840 or Flash-RejuAgro A), eluted with 50% hexane/50% ethyl acetate, had a relatively large clearance zone, which is considered 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.

Preparative HPLC (Prep-HPLC) purification of fractions 3840 and 5054 yielded 15 mg of a yellow compound, RejuAgro A (Rt 17.5), and 103.3 mg of a dark green compound, RejuAgro B, respectively. RejuAgro A is soluble in both methanol and chloroform. RejuAgro B (Rt 10.5) is poorly soluble in methanol or chloroform, but is highly soluble in dimethyl sulfoxide (DMSO), resulting in a dark green color. The structures of these two compounds were elucidated by high-resolution mass spectrometry (HR-MS), infrared (IR), ultraviolet (UV), 1D and 2D nuclear magnetic resonance (NMR), and X-ray crystallography. It has been shown that the two compounds are structurally similar, with compound RejuAgro A containing seven types of carbon groups (three types of carbonyls, two types of tertiary carbons, and two types of methyl carbons), but RejuAgro B lacks one type of methyl group, as shown below: RejuAgro A& RejuAgro B.

RejuAgro A crystals were further obtained by slowly evaporating the chloroform solution at room temperature. These crystals were identified as orange tablets. Data were collected at 100 K using Cu (Kα) radiation using an Oxford SuperNova diffractometer. The molecule has a planar structure in which the S-Me (methyl) group rotates only 8.7° relative to the ring. The molecule has a significant break in π-conjugation at the C4-C5 bond (1.531 Å), apparently due to some orbital reasons. The Me group attached to the ^sp2 carbon atom is rotationally disordered in two positions. The molecule in the crystal forms a centrosymmetric H-bonded dimer via NH…O interactions. Furthermore, these dimers form a two-dimensional layer along the [-3 0 1] plane via weak C—H…O interactions. RejuAgro A molecules represent a six-membered heterocyclic ring [-NH-C(=O)-C(-SMe)=C(-Me)-C(=O)-C(=O)-]. Crystals of RejuAgro B were identified as a triclinic unit cell. The structure of RejuAgro B consists of two symmetrical, independent molecules. Each molecule has a helical structure, with dihedral angles between the average planes of the connected heterocyclic rings being 70.3° and 80.6°. Each dicyclic ring exhibits significant π-coherence at the C( ^sp2 )-C( ^sp2 ) bonds (bond lengths in the range of 1.534-1.539 Å) between two adjacent carbonyl groups, apparently due to orbital reasons. The molecules in the crystal form centrosymmetric H-bonded dimers via NH…O interactions. These dimers are connected in the stack along the x-direction by additional NH…O interactions. Finally, the stacks are connected to the layers along the [0 1 1] direction by a third NH…O interaction. When a solution of RejuAgro B was used for crystal growth, two crystals were obtained and were named RejuAgro B and RejuAgro C. The crystals of RejuAgro B and RejuAgro C have very similar molecular masses (see Example 20).

The crystal structure information of RejuAgro A, RejuAgro B, and RejuAgro C is presented in Example 20, the contents of which form part of this application and are incorporated by reference in their entirety.

The molecular formula of RejuAgro A is _{C₁₈H₁₈NO₃S} , and _its molecular weight is _185.2004 . This is consistent with the observed molecular species _[ M+H] at m / z 186.2177 (theoretical value 186.2083) in the HR-MS data. _The molecular formula of RejuAgro B is _{C₁₂H₈N₂O₆S} , and _its molecular weight is 276.2017. This is consistent with the observed molecular species [MH] at m / z 275.0278 (theoretical value 275.1960) in the HR-MS data. As of August 4, 2020, a CCDC structural database search indicates that no crystal structures exist for RejuAgro A, RejuAgro B, or RejuAgro C. Searches of other chemical databases such as SciFinder, Reaxys, and Google Patents and Patent-Related Databases indicated that there were no analogs of RejuAgro A or RejuAgro C, except for one reference to RejuAgro B found in SciFinder and Reaxys (Knackmuss et al. (1968)). Example 3. In vitro antimicrobial activity of RejuAgro A and RejuAgro B from strain 0617-T307

MIC values for RejuAgro A and RejuAgro B were determined for five bacterial types: wild-type Gram-negative plant pathogens, Streptomycin-resistant Erwinia amylovora, fish pathogens, Gram-positive and Gram-negative human pathogens, and the RejuAgro A producer (strain 0617-T307). Antimicrobial testing was performed according to the CLSI Antimicrobial Susceptibility Testing (AST) protocol. Briefly, a stock solution of each test bacterial strain was streaked onto LB (Luria-Bertani) plates (trypticum, 10 g/L; yeast extract, 5 g/L; sodium, 10 g/L; agar, 15 g/L). For special cultures, NA (nutrient broth + agar) plates (beef extract, 3 g/L; yeast extract, 1 g/L; aggrecan, 5 g/L; sucrose, 10 g/L; and agar, 15 g/L) were used for Xa c. SHIEH (tryptin, 5 g/L; yeast extract, 0.5 g/L; sodium acetate, 0.01 g/L; BaCl ₂ (H ₂ O) ₂ , 0.01 g/L; K ₂ HPO ₄ , 0.1 g/L; KH ₂ PO ₄ , 0.05 g/L; MgSO ₄ ∙7H ₂ O, 0.3 g/L; CaCl ₂ ∙2H ₂ O, 0.0067 g/L; FeSO ₄ ∙7H ₂ O, 0.001 g/L; NaHCO ₃ , 0.05 g/L; agar, 10 g/L) and TYES (tryptin, 4 g/L; yeast extract, 0.4 g/L; MgSO 4 , 0.5 g/L; CaCl ₂ 0.5 g/L; pH to 7.2, agar, 15 g/L) for F. columnaris strains MS-FC-4 and #2, respectively. A single colony from each plate was picked and inoculated into the corresponding liquid culture medium and grown overnight. The culture was diluted to an OD ₅₉₀ of 0.01 in LB or the corresponding medium and plated at 200 µL/well in a 96-well plate. Compounds RejuAgro A and RejuAgro B, as well as streptomycin, were diluted and 4 µL of each concentration was added to each well to produce the following final concentrations: 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.

Test results showed that RejuAgro A, rather than RejuAgro B, was the most active metabolite of strain 0617-T307. RejuAgro A was particularly effective against all tested bacteria, with MIC values ranging from 5 to 40 µg/mL, compared to its activity against Gram-positive MRSA (MIC > 40 µg/mL) and Gram-negative E. coli O157:H7 (MIC = 40 µg/mL), a major foodborne and waterborne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans. RejuAgro A exhibits antimicrobial activity equivalent to that of streptomycin against the strains Erwinia starch 1189 , Xanthomonas aegypti var. citri, Pseudomonas sarsnerii var. sarsnerii, Pectinobacterium tuberosum UPP163936, Pectinobacterium carotenoides var. brasiliensis var. brasiliensis var. brasiliensis var. wpp14945, and Erwinia chrysanthemi 3937, demonstrating an MIC value of 5 µg/mL against Erwinia starch and MIC values of 20-40 µg/mL against other soft pathogenic bacteria. Xanthomonas species are highly sensitive to streptomycin, with a MIC of 0.16 µg/mL, which is lower than the MIC of RejuAgro A, 5 µg/mL. The MIC of RejuAgro A for Pseudomonas sarsii var. sarsii is 40 µg/mL. The MIC of RejuAgro A for Xanthomonas arborescens var. juglans 219 is 6.25 µg/mL. The MICs of RejuAgro A for Ralstonia solanacearum K60 and Pss4 are 3.13 and 6.25 µg/mL, respectively. The MICs of RejuAgro A for Corynebacterium michiganensis strains NCPPB382, Cmm 0317, and Cmm 0690 are 6.25, 1.56, and 12.5 µg/mL, respectively. The MIC value of RejuAgro A against Ralstonia solanacearum K60 and Pss4 was 40 μg/mL.

RejuAgro A was also tested against other strains of E. amylovora, including a viral E. amylovora strain and three streptomycin-resistant E. amylovora strains. RejuAgro A demonstrated comparable efficacy to streptomycin against E. amylovora 110 (MIC 5 µg/mL). However, RejuAgro A was more effective than streptomycin against E. amylovora 1189. The MIC values for RejuAgro A and streptomycin against E. amylovora 1189 were 5 µg/mL and 10 µg/mL, respectively. Furthermore, RejuAgro A was more effective against streptomycin-resistant E. amylovora CA11, DM1, and 898, with a MIC value (10 µg/mL) lower than the MIC for streptomycin (>40 µg/mL) observed for RejuAgro A. These results indicate that RejuAgro A is the most potent compound tested against E. amylovora and represents a potential candidate for replacing streptomycin. There was no cross-resistance to RejuAgro A among streptomycin-resistant strains.

Regarding its activity against Flavobacterium spp., the MIC of RejuAgro A against Flavobacterium spp. strains MS-FC-4 and #2 (which cause columnar disease in wild and cultured fish) was 5 µg/mL. This value is higher than the MIC of streptomycin (0.31 µg/mL and 1.25 µg/mL for strains #2 and MS-FC-4, respectively).

The effect of RejuAgro A on strain 0617-T307 was tested. The results showed that the MIC value of RejuAgro A against Pseudomonas aeruginosa 0617-T307 (the producer of RejuAgro A) was greater than 40 µg/mL in the tested LB medium, indicating that strain 0617-T307 can exist and be resistant to at least 40 µg/mL of RejuAgro A produced by itself.

RejuAgro A was tested together with streptomycin against tomato pathogens (Pseudomonas syringae var. tomato PT30, Pseudomonas syringae var. syringae var. 7046, and Pseudomonas syringae var. cucumber 1188-1) and other citrus ulcer pathogens ( Xanthomonas campestris var. plum and Xanthomonas campestris var. XV-16). The MIC value of RejuAgro A against P. syringae was 40 µg/mL, while the MIC value of streptomycin ranged from 2.5 to 5 µg/mL. For X. campestris, the MIC values of RejuAgro A were 2.5 µg/mL or 40 µg/mL, which were lower than the MIC values of streptomycin, which had MIC values of 20 µg/mL or greater than 40 µg/mL. These values indicate that the Xanthomonas campestris pathogen is more sensitive to RejuAgro A than to streptomycin when compared to the tomato pathogen caused by Pseudomonas.

RejuAgro A demonstrated efficacy against all tested fungal pathogens (Table 1). RejuAgro A was tested against Phytophthora spp., Venturia spp., and Xanthomonas aeruginosa. RejuAgro A demonstrated 100% inhibition against both Phytophthora spp. and Venturia spp. at concentrations of 40 µg/mL, 80 µg/mL, and 600 µg/mL (Table 1).

Table 1. Summary of antimicrobial effects of RejuAgro A strains (related diseases) MIC (µg/mL) RejuAgro A Streptomycin Erwinia starch 1189 (Apple/Apricot Burn) 5 20 Erwinia starch ^110a (apple/apricot burn) 5 5 Erwinia starch ^CA11b (apple/apricot burn) 10 >40 Erwinia starch ^DM1b (Apple/Apricot Burn) 10 >40 Erwinia starch ^898c (Apple/Apricot Burn) 10 >40 Xanthomonas aeruginosa var. citri - Miami XC2002-00010 (Citrus ulcer disease) 5 0.16 Xanthomonas aeruginosa var. citri N40-SO5 (citrus ulcer disease) 5 0.16 Xylene penicillin-resistant Staphylococcus aureus USA300 (skin infections, sepsis) >40 10 Potato pectinobacterium UPP163 936 (causes soft rot in various crops) 40 40 Black rot fungus 942 (causes soft rot in various crops) 20 20 Carrot fungus Brasiliensis 944 (causes soft rot in various crops) 40 40 Carrot pectin fungus Carrot species wpp14 945 (causes soft rot in various crops) 40 40 Erwinia chrysanthemi 3937 (causes soft rot in various crops) 40 20 Pseudomonas sarcoma var. sarcoma (olive knot) 40 0.31 E. coli O157:H7 (foodborne illness) 40 20 Flavobacterium columnaris #2 (Columnaris fish disease) 5 0.31 Flavobacterium columnaris MS-FC-4 (columnar fish disease) 5 1.25 Soil Pseudomonas aeruginosa 0617-T307 (RejuAgro A producer) >40 >40 Pseudomonas syringae var. tomato PT30 (tomato bacterial spot) 40 2.5 Pseudomonas syringae var. syringae 7046 (bacterial Ba scab or rice blast (drupe and pome)) 20 2.5 Pseudomonas syringae var. cucumber 1188-1 (angular leaf spot of cucurbits) 10 5 Xanthomonas campestris var. prunus (Peach Leaf Spot) 40 >40 Xanthomonas campestris var. XV-16 (tomato bacterial spot disease) 2.5 20 Xanthomonas arborescens var. juglans 219 (walnut black rot) 6.25 0.39 Ralstonia solanacearum K60 (bacterial wilt) 3.13 12.5 Ralstonia solanacearum Pss4 (bacterial wilt) 6.25 12.5 Cladosporium michiganensis strain NCPPB382 (tomato ulcer) 6.25 12.5 Cladosporium michiganense Cmm 0317 (tomato ulcer) 1.56 3.12 Cladosporium michiganense Cmm 0690 (tomato ulcer) 12.5 12.5 Phytophthora solani Pi 1306 (potato late blight) 40 NA Phytophthora solani Pi 88069 (potato late blight) 40 NA Apple scab (Apple scab) 80 NA ^e Black leaf spot pathogen 10CR-25 (Banana Black Leaf Spot) 600 NA ^a Ea110 is a viral strain used in field trials in Michigan; ^b CA11 and DM1 are streptomycin-resistant strains containing Tn5393 with a transposon on the plasmid pEa34 and can be grown in medium containing 100 μg/mL streptomycin; ^c Ea898 is a spontaneously streptomycin-resistant strain with a mutation in the chromosomal rpsL gene and can be grown in medium containing 2000 μg/mL streptomycin; ^d Copper-resistant bacteria; ^e A positive control copper solution at 1000 μg/mL inhibited growth by 61%. Example 4. Production and stability of RejuAgro A from strain 0617-T307 in shake flask fermentation

The 0617-T307 fermentation used to produce and prepare RejuAgro A can be obtained by two methods: shake flask fermentation and fermentation jar fermentation. The fermentation jar fermentation is described in Example 2. In this example, the shake flask fermentation can be obtained as follows. The bacterium Pseudomonas genus 0617-T307 stock solution is streaked onto YME agar plates (yeast extract, 4 g/L; glucose, 4 g/L, and malt extract, 10 g/L; agar, 15 g/L) and grown in a 28°C incubator for 24 hours. A seed culture medium is prepared by growing a single 0617-T307 colony in a 250 mL flask containing 50 mL of sterile YME liquid medium at 16°C and 220 rpm for 24 hours. The seed culture was then inoculated into a 4-L flask containing 0.5 L of sterile YME medium at a 4% concentration ( v / v ). After inoculation (2%, v / v ) into eight 4-L flasks each containing 2 L of YME medium, the bacteria were grown at 16°C in a shaker at 200-220 rpm for 1-7 days.

RejuAgro A concentrations were determined by LC-MS analysis based on a developed standard curve. Two methods were used to prepare samples for LC-MS analysis. One method involved extracting the cell suspension with ethyl acetate (1 mL:1 mL, vortexing for 1 min), centrifuging the ethyl acetate layer, and vacuum-drying it to obtain an ethyl acetate extract. The dried ethyl acetate extract was dissolved in 40 µL of methanol, and 2 µL of the methanol solution was used for LC-MS analysis. The other method involved centrifuging the cell suspension to obtain a supernatant, which was then mixed with an equal volume of methanol to prepare a 50% methanol solution. 10 µL of this solution was injected into the LC-MS. The second approach was used because RejuAgro A production was an extracellular secretion process, as evidenced by the observation of a major amount of RejuAgro A in the supernatant rather than inside the cells (Figure 3, Panel A).

During the 7-day fermentation, total RejuAgro A production reached a peak concentration on the first day and then began to decrease over time (Figure 3, Panel B). Further detailed studies of RejuAgro A production and cell concentration were conducted every 6 hours during shake flask fermentation. These results showed that the RejuAgro A concentration (total RejuAgro A) reached a maximum of 13.8 mg/L at 18 hours, and the bacterial cell concentration reached a maximum of 2 x 10 ¹¹ CFU/mL at 12 hours, indicating that RejuAgro A production is a cell growth-related process.

The volume of culture medium in 4-L shake flasks affected the production of RejuAgro A. In 4-L flasks with YME medium, RejuAgro A production was only observed for the 500 mL volume and not for the 1.0 L or 1.5 L volumes. This observation suggests that RejuAgro A production occurs preferentially under highly aerated conditions.

The type of culture medium and the culture temperature affect the production of RejuAgro A. LB medium and YME medium were tested in parallel at 16°C or 28°C. RejuAgro A production was observed in YME medium at 16°C, but not in LB medium. Regarding colony-forming units, strain 0617-T307 grew well in LB medium at 16°C and 28°C, and in YME medium at 28°C. These results indicate that the production of RejuAgro A is culture medium-specific and temperature-dependent. The activity of the product from 0617-T307 was monitored by plate assay against Erwinia amylovora, which was consistent with the production of RejuAgro A.

To verify the suitability of the production conditions for RejuAgro A, ten other Pseudomonas strains were tested in parallel with Pseudomonas strain 0617-T307 under the same conditions. Based on analysis of housekeeping genes, strains 0917-T305, 0917-T306, and 0917-T307 were identified as Pseudomonas aeruginosa, while strains 0118-T319, 0318-T327, and 0418-T328 were identified as Pseudomonas morganii. Type strains of Pseudomonas aeruginosa and Pseudomonas morganii have been reported (Daboussi et al. (2002); Pascual et al. (2014)).

This showed that strain 0617-T307 and its closely related strains could produce RejuAgro A in YME at 28°C and 220 rpm. This result demonstrates that the method is specific for strain 0617-T307 and its closely related strains to produce RejuAgro A (Table 2). In 40-hour cultures of 0617-T307 grown in YME medium on a shaker at 16°C and 220 rpm, RejuAgro A was present and stable in cultures at room temperature for at least four weeks, as determined by LCMS.

Table 2. Summary of the RejuAgro A production capacity of selected Pseudomonas strains cultured in medium YME at 16°C, 220 rpm for 18 hours. strain coding Popular categories The creation of RejuAgro A 0617-T307 Soil Pseudomonas yes 0617-T318 Protection against Pseudomonas protegens no 0817-T317 Protection against Pseudomonas no 0717-T327 Pseudomonas koreensis no 0717-T314 Korean Pseudomonas no 0917-T305 Soil Pseudomonas yes 0917-T306 Soil Pseudomonas yes 0917-T307 Soil Pseudomonas yes 0118-T319 Pseudomonas morganii yes 0318-T327 Pseudomonas morganii yes 0418-T328 Pseudomonas morganii yes Example 5. Antimicrobial activity against the cell effluent of strain 0617-T307 and Erwinia amylovora.

Two assays were used to test the antimicrobial activity of 0617-T307 cell suspensions and metabolites. One assay was a plate diffusion assay and the other was a microplate assay. LB plates were used to test the antimicrobial activity of RejuAgro A-containing fractions and cell suspensions against E. amylovora (Table 3). Cell suspensions containing live 0617-T307 cells and suspensions containing 2 mg/mL RejuAgro A showed antimicrobial activity against E. amylovora. However, no zone of inhibition was observed when ^Serenade® was applied.

Table 3. Activity of 0617-T307 cells and RejuAgro A against Erwinia amylovora in LB plates Sample Concentration (mg/mL) Diameter of inhibition zone (cm) RejuAgro A 2 1.1 0617-T307 cells ND ^a 0.8 RejuAgro B 2 0 Serenade Initial solution 0 Streptomycin 2 2.4 Kasugamycin 1.3 DMSO 0 ^aThe concentration of bacterial cells has not yet been determined.

To identify a biological control formulation consisting of 0617-T307 cells and the active ingredient RejuAgro A, the following experiment was conducted. The supernatant (abbreviated as "supernatant") of a 40-hour cell culture of 0617-T307 containing RejuAgro A was used for antimicrobial testing against its producer, 0617-T307. This assay demonstrated that strain 0617-T307 was able to grow in LB medium, but not in YME medium, in a 2x dilution of the supernatant. Further investigation revealed that the inhibitory effect of the supernatant was due to a lower pH. Controlling the pH to 6.5-6.8 established a "yes" answer to questions 1 and 2.

Bioactive fractions (crude extract, 100 µg/mL; flash-RejuAgro A, 20 µg/mL; HPLC-RejuAgro A, 10 µg/mL) were tested against strain 0617-T307, Ea , and Xac . The bioactive fractions did not inhibit the growth of strain 0617-T307, indicating that RejuAgro A can be mixed with 0617-T307 cells for the preparation of a biocontrol agent. Bioactive fractions containing RejuAgro A showed inhibitory effects against Ea and Xac , with flash-RejuAgro A and HPLC-RejuAgro A, in particular, nearly eliminating the growth of Ea and Xac under the tested conditions. This suggests that RejuAgro A solutions can be used for biocontrol of fire burn and citrus scab at concentrations of 10-20 µg/mL. Example 6. Identification and characterization of bioactive metabolites from the ethyl acetate extract of the acidified supernatant (pH 2.0) of strain 0617-T307.

A stock of Pseudomonas sp. 0617-T307 was inoculated onto LB agar plates (trypticum, 10 g/L; yeast extract, 5 g/L; NaCl, 10 g/L; agar, 15 g/L; water) and grown in a 28°C incubator for 24 hours. To prepare seed culture, a single 0617-T307 colony was inoculated into 500 mL of autoclaved YME medium (yeast extract, 4 g/L; glucose, 4 g/L; and malt extract, 10 g/L) and grown at 28°C with shaking at 150 rpm for 24 hours. The seed culture was then inoculated into eight 4-L flasks, each containing 2 L of autoclaved YME medium. Fermentation was carried out at 16°C in a shaker with a shaking rate of 150 rpm for 7 days.

After 7 days of growth, the bacterial culture was centrifuged at 4000 rpm for 15 minutes to obtain the supernatant. The pH of the supernatant was then adjusted to 2.0 by adding 6N HCl. The acidified supernatant was then extracted with ethyl acetate. This yielded 3.0 g of crude extract from a 14 L culture of strain 0617-T307.

The concentrated sample was dissolved in acetone and mixed with silica gel. The silica gel was loaded onto a silica gel column (φ3.0 x 20 cm) in a rapid chromatography system (Yamazen AI-580) equipped with a UV detector. After loading, the sample was eluted with 280 mL of each of the following solvents 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 at UV 254 nm and fractions were collected in time mode at 20 mL/tube. In total, 114 fractions or tubes were generated by flash chromatography.

The resulting fractions were used for subsequent plate assays. One mL of each fraction was pipetted into a 1.5 mL test tube and vacuum-dried using an Eppendorf vacuum concentrator. The dried sample was dissolved in 50 µL of DMSO, of which 2 µL was used for the plate assay. Briefly, Erwinia amylovora 273 was inoculated onto a 50% LB medium (trypticum, 5.0 g/L; yeast extract, 2.5 g/L; NaCl, 5.0 g/L) plate, and a single colony was inoculated into 5 mL of LB medium. The bacteria were diluted 1:100 with sterile water, and 225 µL of this was plated onto a 50% LB plate. After drying in a biosafety cabinet for 10 minutes, the DMSO solution of each fraction was dispensed onto a pre-labeled section of a culture dish and allowed to dry for an additional 10 minutes. DMSO and kasugamycin were used as negative and positive controls, respectively, in the assay. The plates were then incubated in a 28°C incubator and examined for zones of inhibition one day later.

In vitro assays of 114 active fractions revealed that three bioactive fractions (T3234, T5058, and T7882) inhibited the growth of E. amylovora 273. Fractions 3234 and 5258 exhibited relatively small clearance zones. Fraction 3234 was eluted with 50% hexane/50% ethyl acetate. Fraction 5058 was eluted with 25% hexane/75% ethyl acetate. For the negative control, DMSO exhibited no inhibition zone, and the positive control, kasugamycin, showed no inhibition zone. Another active fraction, T7882, was eluted with acetone/ethyl acetate (50%/50%). This fraction also inhibited the growth of E. amylovora.

Another HPLC separation and purification guided by activity against Erwinia amylovora resulted in the identification of 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 tryptophan-derived natural products, and their structures, but not their biological activities, were reported in the Scifinder database (Loots et al. (2015)). 0617_T307_7882_Rt18 was predicted to be a difuryl derivative previously reported by Osipov et al. (1978). The natural products are described below: and Example 7. Identification of other metabolites from strain 0617-T307 using LCMS/MS and spectral library searching.

Crude extracts from unadjusted and pH-adjusted cell fluids (pH 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 from Phenomenex C18 column, 100A, 50 x 2.1 mm Kinetex, 12 min gradient. 5-95% ACN on a Bruker Maxis Impact II. Data were acquired on a Bruker MaXis Impact II UHR-QqTOF (Ultra-High Resolution Qq-Time-Of-Flight) mass spectrometer. Collision-induced dissociation (CID) fragmentation of the eight most abundant ions in the spectrum was used, followed by a full MS scan followed by tandem MS (MS/MS). The scan rate was 3 Hz.

Spectral library searches were then performed based on bioinformatics and molecular networking analyses to identify new and known compounds. Sample MS/MS spectra were searched against the following spectral libraries: 1) GNPS Community Library; 2) FDA Library; PhytoChemical Library; 3) NIH Clinical Collection; 4) NIH Natural Products Library; 5) Pharmacologically Active NIH Small Molecule Depository; 6) Faulkner Legacy Library; 7) Insecticides; 8) Dereplicator Identified MS/MS Peptide Natural Products; 9) PNNL Lipids; 10) Massbank; 11) Massbank EU; 12) MoNA; 13) ReSpect – Phytochemicals; and 14) HMDB.

MS/MS spectra were searched in the above library and compared to deviations from the reference spectrum. Matching parameters were identical. These results can be explored to identify structural analogs of known compounds. An MS/MS molecular network was generated with a minimum cluster size of 2, a minimum edge of 0.7 cosine, and 6 minimum matching peaks. As an example, a new molecular species at m / z 303.16 was identified as corresponding to the new compound 0617-T307_5058_Rt25.0 from the active fraction. Several known compounds were identified from the crude extract, including indole-3-carboxylic acid, plant growth promoting factor, and xantholysin A. It has been reported that 1) the broad antifungal activity of Pseudomonas aeruginosa BW11M1 is primarily dependent on xantholysin production; and 2) xantholysin is required for swarming and contributes to biofilm formation (Li et al. (2013)). Indeed, high concentrations of xantholysin A were observed in strains 0617-T307, 0418-T328, and 0318-T327 cultured at 28°C. Therefore, in addition to the bioactive compound RejuAgro A, xantholysin A is another metabolite contributing to the antimicrobial activity of the biocontrol bacterium 0617-T307 and its closely related species 0318-T3027 and 0418-T328. Example 8. Greenhouse and field infection assays of RejuAgro A-producing strain 0617-T307 and some closely related species.

To evaluate the biocontrol activity of strain 0617-T307 against Erwinia amylovora, infection assays were conducted on crabapple trees in greenhouses at the University of Wisconsin-Milwaukee. Biocontrol agents (0617-T307, 0717-T327, and 0617-T318) containing 1.0 x ¹⁰⁸ cfu/mL were sprayed onto flowers (80% to fully opened) in multiple tree plots. Briefly, strain 0617-T307 was grown overnight in 26 mL glass tubes containing 5 mL of LB medium. Cells were then inoculated (1:100) into the LB medium and grown on a shaker at 28°C and 200 rpm for 14-18 hours. Cells were harvested and resuspended in 10x water to achieve a concentration of ¹⁰⁸ CFU/mL. The resuspended solution can be used in greenhouse and field assays to control fireburn. Control flowers were sprayed with distilled water. All flowers were then inoculated by spraying 1.0 x ¹⁰⁶ cfu/mL of the Erwinia amylovora strain 273. Three treatments with 0617-T307 were performed on September 7, October 9, and October 19, 2018. Referring to Table 4, all spray treatments with 0617-T307 (Pseudomonas aeruginosa) provided 100% control of blossom blight symptoms on crabapple flowers, compared to 0% control with distilled water. This suggests that 0617-T307 is a promising biocontrol agent for fire burn caused by Erwinia amylovora. Two other Pseudomonas species, 0717-T327 (Pseudomonas koreana) and 0617-T318 (Pseudomonas spp.), achieved lower control rates of 16.7% and 25%, respectively. Overall, of the three Pseudomonas species tested, only 0617-T307 demonstrated good control efficiency against crabapple fire burn. No phytotoxicity was observed.

Table 4. Summary of greenhouse infection tests September 7, 2018 October 9, 2018 October 19, 2018 biological control agents Processed flowers# Infected Flower# Control rate (%) Processed flowers# Infected Flower# Control rate (%) Processed flowers# Infected Flower# Control rate (%) Comparison 8 8 0 16 16 0 18 18 0 0617-T307 3 0 100 9 0 100 10 0 100 0717-T327 6 5 16.7 0617-T318 4 3 25

For the field test, biocontrol bacteria producing RejuAgro A (0617-T307, 0118-T319, 0318-T327, 0418-T328; see Table 2) were applied to apple tree flowers in the orchard (apple flowers were 40% and 70% open) at a concentration of 5 x 10 ⁸ CFU/mL on May 6, 2019. The bacterial pathogen Erwinia amylovora Ea110 was inoculated at a concentration of 5 x 10 ⁶ CFU/mL on May 7 (90% open). The percentages of diseased blossoms in the water control, streptomycin, 0617-T307, 0118-T319, 0318-T327, and 0418-T328 were 32.9%, 13.3%, 16.8%, 18.5%, 16.7%, and 11.8%, respectively. Compared to streptomycin, the biocontrol bacteria producing RejuAgro A showed similar or better efficacy in controlling apple burn in orchards. Example 9. Antifungal activity of RejuAgro A and B and their producers against Venturia odorifera.

Venturia osmoticus, the fungus that causes apple scab, was maintained on PDA agar in the dark at room temperature (approximately 24°C). Conidia and mycelial suspensions were harvested from the PDA (potato dextrose agar) in 0.01 M PBS. Ten microliters of the conidia and mycelial suspensions were then dropped onto plates containing biocontrol bacteria, RejuAgro A, or a modified RejuAgro A. Control plates were PDA plates without biocontrol bacteria or RejuAgro A or B. The plates were incubated in the dark at room temperature, and the diameter of each Venturia osmoticus colony was examined after 7 days.

When compared to the control (Figure 4), the four selected biocontrol bacteria, 0617-T307, 0118-T319, 0318-T327, and 0418-T328, inhibited the growth of Venturia spp. on PDA plates (Figure 5). RejuAgro A inhibited the growth of Venturia spp. on PDA plates at concentrations of 40-80 µg/mL (Figure 6). However, no inhibitory effect of RejuAgro B on the growth of Venturia spp. was observed at concentrations of 10-80 µg/mL (Figure 7). Finally, no inhibition of Venturia spp. was observed on PDA plates containing 200-1000 µg/mL copper sulfate (Figure 8). Example 10. Production of RejuAgro A by Pseudomonas species.

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

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 cultures were prepared by growing the bacteria in YME medium at 16°C and 220 rpm for 24 hours. HPLC analysis showed that all seven strains produced RejuAgro A (Figure 10). Example 11. Preparation and Greenhouse Assay of RejuAgro A.

RejuAgro A formulation (solution, SL; see Table 5). Before application to flowers, 10 μg/mL was mixed in a jar with 1% polyethylene glycol (PEG) 4000 as a safener. Subsequent testing showed that 0.03% polyvinyl alcohol (PVA) as a safener achieved better protection for flowers. The surfactant Alligare 90 can be added to enhance efficacy (Table 6).

Table 5. RejuAgro-A 1% SL ^a formulation Components Ratio (%, w / w ) Grams Notice RejuAgro-A 1 5 Active ingredient Alligare 90 (poly(alkyl EO/PO), etc.) 10 50 Wetters and diffusers Ethylene glycol/propylene glycol 5 25 antifreeze PVA 30 150 safener water Measure (add to 100%) 270 Carrier Total 100 500 ^a 1% solution (SL) of RejuAgro A formulation.

To evaluate the biocontrol activity of RejuAgro A against E. amylovora, greenhouse infection assays were conducted on crabapple trees at the University of Wisconsin-Milwaukee. Ten μg/mL was supplemented with 1% polyethylene glycol (PEG) 4000 or 1% PEG 4000 (negative control) was applied to flowers of fully opened trees 3 hours before and 24 hours after inoculation. Approximately 10 ^⁸ CFU/mL of E. amylovora 110 strain suspended in water was used as inoculum. Infection rates were calculated approximately 6 days after inoculation. The experiments were conducted from January 24 to January 31, 2020. RejuAgro A effectively inhibited blossom blight (Table 6).

Table 6. Flower blight test of RejuAgro A with 1% PEG 4000 handle infection rate 1% PEG4000 (negative control) 50.8% RejuAgro A 10μg/mL with 1% PEG4000 12.3% Streptomycin 200 μg/mL 5.7% Example 12. Antifungal activity of 0617-T307 cell fluid against Botrytis cinerea CA17.

Seeds of strain 0617-T307 were prepared by growing bacterial cells in YME medium at 28°C and 180 rpm for 24 hours. A 4% aliquot (2 mL to 50 mL) was then inoculated into a 250 mL flask containing 50 mL of M8 (IAA medium), M9 (CN medium), M7 (PRN medium), or M6 (DAPG medium) medium and grown at 28°C and 180 rpm for 48 hours. 0.5 mL aliquots of cell suspension were collected at 12 and 24 hours and stored in a -20°C freezer. For antifungal testing, the cell suspension was thawed and 5 μL was applied to sample wells on PDA (potato dextrose agar) plates at equal radii from the center of the plate inoculated with Botrytis cinerea (Figure 11). This showed that the cell suspension had antifungal activity against Botrytis cinerea CA17 on PDA (potato dextrose agar) plates. Example 13. Antimicrobial activity of the crude extract, RejuAgro A, and RejuAgro B against plant pathogenic bacteria.

The metabolites of bacteria 0917-T305, 0318-T327, and 0418-T328 showed good efficacy against Ralstonia solanacearum, Coryneformis michiganensis, and Xanthomonas arborescens var. juglone (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 from 0917-T305, 0318-T327, and 0418-T328 were prepared at 5 mg/mL and tested against three different plant pathogens: Ralstonia solanacearum, Coryneformis michiganensis, and Xanthomonas arborescens var. juglans. In the agar plate diffusion assay, metabolites of bacteria 0917-T305, 0318-T327, and 0418-T328 grown in YME at both 16°C and 28°C showed relatively good efficacy against the tested Ralstonia solanacearum, Coryneformis michiganensis, and Xanthomonas arborescens var. juglans. (Table 7). This indicates that, along with RejuAgro A, the other metabolites also have good efficacy against Ralstonia solanacearum, Coryneformis michiganensis, and Xanthomonas arborescens var. juglans. RejuAgro B also showed good efficacy against Ralstonia solanacearum (Table 7).

Table 7. Effects of crude bacterial extracts on selected pathogenic bacteria in plate assays Concentration (mg/mL) Bacterial strains and compounds Culture medium and temperature Xanthomonas arborescens var. juglans Ralstonia solanacearum Cladosporium michiganensis Xaj219 Xaj417 K60 Pss Cmm382 Cmm0317 Cmm0690 5 0917-T305 crude extract YME 16℃ 0 ^a 0.2 0.5 0.5 0.5 0.5 0.5 5 0318-T327 crude extract YME 28 ℃ 0.2 0.4 0.5 1.0 0.7 0.7 0.8 5 0418-T328 crude extract YME 28 ℃ 0.2 0.4 0.5 1.0 0.7 0.8 0.8 5 0318-T327 crude extract YME 16℃ 0.1 0.4 0.7 0.6 0.5 0.3 0.4 5 0418-T328 crude extract YME 16℃ 0.2 0.4 0.8 0.9 0.5 0.5 0.5 5 Vancomycin 3.0 3.0 3.0 0.1 Streptomycin 1.3 1.9 1.5 1.5 2 RejuAgro A 0.5 1.0 0.4 0.5 2 RejuAgro B 0.0 0.0 0.4 0.4 ^a Diameter of the inhibition zone (cm) Example 14. Antimicrobial effects of Rt 18.9, Rt 22.9, and Rt 25.0.

A stock of Pseudomonas sp. 0617-T307 was inoculated onto LB agar plates (trypticum, 10 g/L; yeast extract, 5 g/L; NaCl, 10 g/L; agar, 15 g/L; water) and grown in a 28°C incubator for 24 h. Fermentation and crude extract preparation were performed as described in Example 6.

HPLC separation and purification of ethyl acetate extracts of acidified cell fluid of Pseudomonas sp. 0617-T307 identified two antimicrobial compounds (Rt22.9 and Rt25.0) from acute fraction T5058 and one antimicrobial compound (Rt18.9) from acute fraction T7882. The antimicrobial activity was tested against the bacterial strains listed in Table 8. Two µL of DMSO, Rt18.9, Rt22.9 or Rt25.0 were spotted onto agar plates where different bacterial strains were grown, and the inhibition area was further examined (Table 8).

Table 8. Antimicrobial effects of Rt 18.9, Rt 22.9, and Rt 25.0 Strain (related disease) ^a DMSO Rt18.9 (5 mg/mL) Rt22.9 (10 mg/mL) Rt25.0 (5 mg/mL) Culture medium ^b Cladosporium michiganense Cmm 0317 (tomato ulcer) no yes yes yes LB Pseudomonas syringae var. cucumber 1188-1 (angular leaf spot of cucurbits) no yes yes yes LB Xanthomonas aeruginosa var. citri N40-SO5 (citrus ulcer disease) no yes no yes NA Erwinia starch 1189 (Apple/Apricot Burn) no yes no no LB Carrot fungus Brasiliensis 944 (causes soft rot in various crops) no yes no yes LB Ralstonia solanacearum K60 (bacterial wilt) no yes yes yes LB Xanthomonas arborescens var. juglans var. 417 (walnut black rot) no yes no no NA Pseudomonas syringae var. tomato PT30 (tomato bacterial spot) no yes yes yes LB Black rot fungus 942 (causes soft rot in various crops) no yes no no LB Potato pectinobacterium UPP163 936 (causes soft rot in various crops) no yes no no LB Pseudomonas sarcoma var. sarcoma (Olive Knot) 01-26 no yes yes yes LB Pseudomonas syringae var. syringae var. 7046 (bacterial Ba scab or rice blast (drupe and pome)) no yes yes yes LB Xanthomonas arborescens var. juglans 219 (walnut black rot) no yes no yes NA Xanthomonas aeruginosa var. citri - Miami XC2002-00010 (Citrus ulcer disease) no no no yes NA ^a Examine the area of inhibition between 2 and 5 days after spotting with DMSO, Rt18.9, Rt22.9, or Rt25.0. ^b. Agar plates used for bacterial growth consisted of either LB medium (10.0 g/L trypsin, 5.0 g/L yeast extract, 10.0 g/L sodium salt, 15.0 g/L agar, and tap water to a final volume of 1.0 L) or NA medium (3.0 g/L beef extract, 1.0 g/L yeast extract, 5.0 g/L peptone, 10.0 g/L sucrose, and 15 g/L agar, and tap water to a final volume of 1.0 L). Table 14.2 Examples of medium compositions for LB and NA agar plates. 15. Antimicrobial activity of RejuAgro A against banana black leaf spot pathogens

The antimicrobial activity of RejuAgro A against X. chinensis was examined by adding HPLC-purified RejuAgro A to PDA agar medium at final concentrations of 60 and 600 µg/mL, respectively. 480 µL of either 0.5 mg/mL or 5 mg/mL RejuAgro A was added to 3.52 mL of PDA in wells of a 6-well plate, yielding final RejuAgro A concentrations of 60 (Figure 12, middle 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 and 3.52 mL of PDA were used as a control treatment (Figure 12, right well (panel C). After the agar solidified, an agar disc containing X. oryzae was placed on the center of the agar surface. Complete inhibition of X. oryzae was observed in the 600 µg/mL RejuAgro A treatment two weeks after inoculation (Figure 12). Example 16. Antimicrobial Effect of RejuAgro A against Xanthomonas oryzae var. oryzae (Xon507)

The antimicrobial activity of RejuAgro A against Xanthomonas oryzae var. oryzae (Xon507) was examined. A bacterial suspension of Xanthomonas oryzae var. oryzae (Xon507) ( _OD600 = 0.3) was sprayed onto PSG agar plates. Paper discs containing 50 µL of HPLC-purified aqueous RejuAgro A at concentrations of 5.5 µg/mL, 11.1 µg/mL, 22.1 µg/mL, 33.2 µg/mL, 55.4 µg/mL, and 110.7 µg/mL were placed on the agar plates. The zone of inhibition was measured 44 hours after the discs were placed on the agar plates. Inhibition was observed at all concentrations of RejuAgro A suspensions used to soak paper discs (Table 9).

Table 9. Antimicrobial effect of RejuAgro A against Xanthomonas oryzae var. oryzae (Xon507). RejuAgro A concentration Water contrast 5.5 µg/mL 11.1 µg/mL 22.1 µg/mL 33.2 µg/mL 55.4 µg/mL 110.7 µg/mL Inhibition zone (cm) 0 0.27±0.06 0.5±0.1 0.73±0.15 0.83±0.15 0.93±0.06 1.33±0.12 Example 17. Antimicrobial Effect of RejuAgro A against Xanthomonas citri var. aurantii (XW19)

The antimicrobial activity of RejuAgro A against Xanthomonas citriodora var. citriodora (XW19) was examined. A bacterial suspension of Xanthomonas citriodora var. citriodora (XW19) (OD ₆₀₀ = 0.3) was sprayed onto PSG agar plates. Paper discs containing 50 µL of HPLC-purified aqueous RejuAgro A at concentrations of 5.5 µg/mL, 11.1 µg/mL, 22.1 µg/mL, 33.2 µg/mL, 55.4 µg/mL, and 110.7 µg/mL were placed on the agar plates. The zone of inhibition was measured 44 hours after the discs were placed on the agar plates. Inhibition was observed at concentrations of 55.37 µg/mL and 110.74 µg/mL of RejuAgro A (Table 10).

Table 10. Antimicrobial activity of RejuAgro A against Xanthomonas citri var. aurantii (XW19). RejuAgro A concentration Water contrast 5.5 µg/mL 11.1 µg/mL 22.1 µg/mL 33.2 µg/mL 55.4 µg/mL 110.7 µg/mL Inhibition zone (cm) 0 0 0 0 0 0.23±0.06 0.27±0.12 Example 18. Composition of the culture medium used in the examples.

Table 11 includes exemplary media compositions used in the Examples.

Table 11. Culture medium composition. No. Medium name Composition g/L pH at 25°C References M1 YME Yeast extract 4.0 g NA (Hamamoto, H. et al. (2015)) Malt extract 10 g glucose 4.0 g tap water 1.0 L M6 DAPG medium Malt extract 15.0 g NA (Gnanamanickam, Samuel S. (2008)) water M7 PRN culture medium glycerin 30.0 g NA (Gnanamanickam, Samuel S. (2008)) K ₂ HPO ₄ 3.0 g NaCl 5.0 g MgSO ₄ ∙7H ₂ O 0.5 g D-Tryptophan 0.61 g M8 IAA medium D-glucose 5.0 g NA (Gnanamanickam, Samuel S. (2008)) Tyrosinase 25.0 g MgSO ₄ ∙7H ₂ O 0.3 g K ₂ HPO ₄ 1.7 g NaH ₂ PO ₄ 2.0 g M9 CN Tyrosinase 10.0 g NA (Gavrish, E. et al. (2008)) Nutritious Meat Soup 10.0 g Example 19. Bacterial strains, natural products, and references cited therein.

The bacterial strains and natural products described in this application and presented in the appended claims are well known in the microbiology literature. Table 12 below lists these references for each cited bacterial strain and natural product disclosed herein, the contents of which are incorporated herein by reference in their entirety.

Table 12. Bacterial strains, natural products, and references cited as evidence of their availability. bacterial strains References 0617-T307, 0917-T305, 0917-T306, and 0917-T307 Pascual, J., García-López, M., Carmona, C., Sousa, T. da S., de Pedro, N., Cautain, B., Martín, J., Vicente, F., Reyes, F., Bills, GF, & Genilloud, O. (2014). Pseudomonas soli sp. nov., a novel producer of xantholysin congeners. Syst Appl Microbiol , 37: 412–416. 0118-T319, 0318-T327, and 0418-T328 Dabboussi, F., Hamze, M., Singer, E., Geoffroy, V., Meyer, J., & Izard, D. (2002). Pseudomonas mosselii sp. nov., a novel species. Int J Syst Bacteriol , 52: 363–376. natural products References RejuAgro B Knackmuss, H., Medizinische, M., & Chemie, I. (1968). Methyl-substituted 2,3,6-trihydroxypyridines and their oxidation products. Eur. J. Inorg. Chem. 2689: 2679–2689. Rt22.9 and Rt25.0 Loots, D. T., Erasmus, E., & Mienie, L. J. (2005). Identification of 19 new metabolites induced by abnormal amino acid conjugation in isovaleric acidemia. Clin Chem , 51: 1510–1512. Rt18.9 Osipov, AM, Metlova, LP, Baranova, N. V, & Rudakov, ES (1978). New derivatives of difuryl: 2,2'-difuryl-5,5'-dicarbinol and 2,2'-difuryl-5,5'-dicarboxylic acid. Ukrainskii Khimicheskii Zhurnal (Russian edition), 44: 398. Example 20. Crystal structure information of RejuAgro A, RejuAgro B, and RejuAgro C. A. Crystal structure information of RejuAgro A

Single crystals of RejuAgro A (C ₇ H ₇ NO ₃ S) were obtained by slow evaporation of a chloroform solution of RejuAgro A. Orange tablets were obtained. Suitable crystals were selected and mounted on a SuperNova, Dual, Cu at home/near, Atlas diffractometer. The crystals were held at 100.05(10) K during data collection. The structure was solved using Olex2 (Dolomanov et al. (2009)) using the ShelXS structure solution program using direct methods (Sheldrick (2008)) and refined using least-squares minimization using the ShelXL refinement package (Sheldrick, GM (2015)).

The data set was collected at 100 K using 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) = 3.429 mm ^-1 , D calc = 1.576 g/cm ³ , 13936 measured reflections (10.522° ≤ 2Θ ≤ 140.8°), 1496 unique ( R _int = 0.0220, _RΣ = 0.0083), which was used in all calculations. _The final R1 was 0.0253 (I > 2σ(I)) and wR2 _was 0.0702 (all data).

The detailed model description was created using Olex2, compiled based on OlexSys svn.r3508 from May 29, 2018. The number of restraints is 0, and the number of limits is unknown. Details: 1. Fixed Uiso: 1.2x at all C(H,H,H,H,H,H) groups; 1.5x at 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, refined into the rotating group: C6(H6A,H6B,H6C,H6D,H6E,H6F); b Ideal Me, refined into the rotating group: C7(H7A,H7B,H7C).

Referring to Figure 13A, the RejuAgro A molecule has a planar structure, with the S-Me group rotated only 8.7° relative to the ring. The molecule exhibits a significant break in π-conjugation at the C4-C5 bond (1.531 Å), apparently due to orbital factors. The Me group attached to the ^sp2 carbon atom is rotationally disordered in two positions.

Referring to Figure 13B , RejuAgro A molecules in the crystal form centrosymmetric H-bonded dimers via N-H…O interactions. Furthermore, these dimers form two-dimensional layers along the [-3 0 1] plane via weaker C-H…O interactions.

The chemical structure of RejuAgro A is shown below: (Formula (I))

Additional crystal structure data for the RejuAgro A molecule are presented in Tables 13-21.

Table 13. Crystalline data and structural refinement of RejuAgro A Identification code RejuAgro A Experiential C ₇ H ₇ NO ₃ S Formula 185.20 Temperature/K 100.05(10) Crystal system Monoclinic Space Group P2 ₁ /n a/Å 5.30391(6) b/Å 13.97822(13) c/Å 10.74471(13) α/° 90 β/° 101.5883(12) γ/° 90 Volume/Å ³ 780.367(15) Z 4 ρ _calculated g/cm ³ 1.576 μ/mm ^-1 3.429 F(000) 384.0 Crystal size/mm ³ 0.874 × 0.274 × 0.118 Radiation CuKα (λ = 1.54184) Data collection 2Θ range/° 10.522 to 140.8 Index range -6 ≤ h ≤ 6, -17 ≤ k ≤ 17, -13 ≤ l ≤ 12 Collected reflections 13936 Independent reflex 1496 [R _int = 0.0220, R _Σ = 0.0083] Data/Constraints/Parameters 1496/0/117 Regarding the goodness of fit of ^F2 1.067 Final R index [I＞=2σ (I)] _R1 = 0.0253, _wR2 = 0.0700 Final R Index [All Data] _R1 = 0.0254, _wR2 = 0.0702 Maximum difference: peak/hole/e Å ^-3 0.33/-0.29

Table 14. Partial atomic coordinates (×10 ⁴ ) and equivalent isotropic displacement parameters (Å ² ×10 ³ ) of RejuAgro A. U _eq is defined as 1/3 of the trace of the orthogonal U _IJ tensor. atom X Y z U(eq) S1 6015.2(6) 2181.9(2) 7360.1(3) 14.21(13) O1 2838.4(18) 479.2(7) 5956.1(10) 21.0(2) O2 -1864.8(19) 3816.9(7) 4920.5(10) 20.3(2) O3 -4128.5(19) 2125.4(7) 3998.6(10) 19.1(2) N1 -625(2) 1317.3(8) 5012.2(11) 14.8(2) C1 1795(2) 1257.7(9) 5770.8(12) 14.1(3) C2 3003(2) 2163.2(9) 6326.8(12) 12.9(3) C3 1866(3) 3025.4(10) 6015.7(12) 14.5(3) C4 -699(3) 3073.5(10) 5196.4(12) 14.7(3) C5 -2010(3) 2140.2(9) 4672.1(13) 14.9(3) C6 3049(3) 3965.2(10) 6489.7(14) 19.0(3) C7 7080(3) 978.0(10) 7802.9(13) 17.9(3)

Table 15. Anisotropic displacement parameters (Å ² ×10 ³ ) of RejuAgro A. The anisotropic displacement factor exponent is expressed as -2π ² [h ² a* ² U ₁₁ +2hka*b*U ₁₂ +…]. atom U ₁₁ U ₂₂ U ₃₃ U ₂₃ U ₁₃ U ₁₂ S1 12.06(19) 13.72(19) 14.8(2) -0.65(11) -2.16(13) -1.00(10) O1 18.6(5) 11.9(5) 27.2(5) -1.4(4) -7.8(4) 1.5(4) O2 20.2(5) 15.0(5) 24.3(5) 2.8(4) 1.0(4) 4.3(4) O3 13.9(5) 22.2(5) 18.4(5) 0.8(4) -3.7(4) 2.2(4) N1 13.8(6) 11.8(5) 16.4(6) -1.3(4) -2.8(4) -1.4(4) C1 13.7(6) 14.9(7) 12.8(6) 1.0(5) 0.3(5) 0.2(5) C2 11.8(6) 15.1(7) 11.3(6) -0.5(5) 1.1(5) -0.6(5) C3 15.4(6) 14.5(6) 13.4(6) -0.4(5) 2.6(5) -0.6(5) C4 16.1(6) 14.9(6) 13.4(6) 1.7(5) 3.3(5) 2.3(5) C5 14.9(6) 16.3(7) 13.1(6) 1.4(5) 1.9(5) 1.3(5) C6 20.7(7) 12.3(6) 21.9(7) -0.9(5) -0.8(5) 0.7(5) C7 15.9(6) 16.6(6) 18.3(7) 2.0(5) -3.1(5) 2.0(5)

Table 16. Bond lengths of RejuAgro A. atom atom Length/Å atom atom Length/Å S1 C2 1.7529(13) N1 C5 1.3742(17) S1 C7 1.8082(14) C1 C2 1.4886(17) O1 C1 1.2190(16) C2 C3 1.3593(18) O2 C4 1.2150(17) C3 C4 1.4658(18) O3 C5 1.2083(18) C3 C6 1.5002(18) N1 C1 1.3778(17) C4 C5 1.5314(18)

Table 17. Key features of RejuAgro A. atom atom atom angle atom atom atom angle C2 S1 C7 110.46(6) C2 C3 C6 123.92(12) C5 N1 C1 126.34(12) C4 C3 C6 116.00(12) O1 C1 N1 119.28(12) O2 C4 C3 123.44(12) O1 C1 C2 123.28(12) O2 C4 C5 117.88(12) N1 C1 C2 117.44(11) C3 C4 C5 118.68(11) C1 C2 S1 122.08(9) O3 C5 N1 121.87(12) C3 C2 S1 116.47(10) O3 C5 C4 122.26(12) C3 C2 C1 121.41(12) N1 C5 C4 115.87(12) C2 C3 C4 120.06(12)

Table 18. Hydrogen bonds of RejuAgro A. D H A d(DH)/Å d(HA)/Å d(DA)/Å DHA/° N1 H1 O1 ¹ 0.845(18) 2.032(19) 2.8768(15) 178.8(17) C7 H7C O2 ² 0.98 2.58 3.5549(16) 175.3 ¹ -X,-Y,1-Z; ² 3/2+X,1/2-Y,1/2+Z

Table 19. Torsion angle of RejuAgro A. A B C D angle A B C D angle S1 C2 C3 C4 -177.63(9) C1 C2 C3 C6 -176.67(12) S1 C2 C3 C6 0.76(18) C2 C3 C4 O2 177.16(13) O1 C1 C2 S1 -2.63(19) C2 C3 C4 C5 -1.90(19) O1 C1 C2 C3 174.66(13) C3 C4 C5 O3 179.49(12) O2 C4 C5 O3 0.4(2) C3 C4 C5 N1 -0.66(18) O2 C4 C5 N1 -179.77(11) C5 N1 C1 O1 -177.26(13) N1 C1 C2 S1 177.32(9) C5 N1 C1 C2 2.78(19) N1 C1 C2 C3 -5.39(19) C6 C3 C4 O2 -1.36(19) C1 N1 C5 O3 179.92(13) C6 C3 C4 C5 179.58(11) C1 N1 C5 C4 0.07(19) C7 S1 C2 C1 -8.68(13) C1 C2 C3 C4 4.9(2) C7 S1 C2 C3 173.90(10)

Table 20. Hydrogen atomic coordinates (Å×10 ⁴ ) and equivalent isotropic displacement parameters (Å ² ×10 ³ ) of RejuAgro A. atom X y z U(eq) H1 -1260(30) 789(13) 4721(17) 20(4) H6A 2408.52 4468.05 5871.25 twenty three H6B 4925.78 3921.96 6602.43 twenty three H6C 2587.06 4119.49 7305.04 twenty three H6D 4205.71 3871.61 7314.56 twenty three H6E 1688.46 4417.71 6583.38 twenty three H6F 4027.18 4220.17 5880.78 twenty three H7A 7306.25 625.34 7044.62 27 H7B 5790.31 654.5 8190.27 27 H7C 8721.96 1001.76 8413.78 27

Table 21. Market share of RejuAgro A. atom Market share atom Market share atom Market share H6A 0.485(15) H6B 0.515(15) H6C 0.515(15) H6D 0.485(15) H6E 0.515(15) H6F 0.485(15) B. Crystal structure information of RejuAgro B.

Single crystals of RejuAgro B (C ₁₂ H ₈ N ₂ O ₆ ) were obtained by slow evaporation of a methanolic solution of RejuAgro B. Orange pyramids were obtained. Suitable crystals were selected and mounted on a SuperNova, Dual, Cu at home/near, Atlas diffractometer. The crystals were held at 100.05(10) K during data collection. The structure was solved using Olex2 (Dolomanov et al. (2009)) with the ShelXS structure solution program using direct methods (Sheldrick (2008)) and refined using least-squares minimization with the ShelXL refinement package (Sheldrick (2015)).

The data set was collected at 100 K using 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 , D calc = 1.605 g/cm ³ , 15292 measured reflections (7.872° ≤ 2Θ ≤ 141.144°), 4304 unique values ( R _int = 0.0258, R _Σ = 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).

Use Olex2 to create a detailed model description, compiled based on OlexSys 2018.05.29 svn.r3508. Number of constraints - 0, number of limits - unknown. Details are as follows: 1. Fixed floppy disk: 1.2x at all N(H) groups; 1.5x at all C(H,H,H) groups; 2.a Aromatic/amide H, refined by sliding coordinates: N1(H1), N2(H2), N1A(H1A), N2A(H2A); 2.b Ideal Me, refined by position rotation groups: C6(H6A,H6B,H6C), C12(H12A,H12B,H12C), C6A(H6AC,H6AA,H6AB), C12A(H12D,H12E,H12F).

Referring to Figure 14A, a RejuAgro B crystal contains two symmetrical, independent RejuAgro B molecules. Each molecule has a helical structure, with dihedral angles of 70.3° and 80.6° between ^the average planes of the connected heterocyclic rings. The π-cohesion between two adjacent carbonyl groups in each ^heterocyclic ring is significantly broken (bond lengths range from 1.534 to 1.539 Å), apparently due to some orbital reasons.

Referring to Figure 14B , RejuAgro B molecules in the crystal form centrosymmetric H-bonded dimers via N-H…O interactions. These dimers are connected in the stack along the x-direction via additional N-H…O interactions. Finally, the stack is connected to the layer along the [011] direction via a third N-H…O interaction.

The chemical structure of RejuAgro B is shown below: (Formula (II))

Additional crystal structure data for the RejuAgro B molecule are presented in Tables 22-29.

Table 22. Crystallographic data and structural refinement of RejuAgro B. Identification code RejuAgro B Experiential C ₁₂ H ₈ N ₂ O ₆ Formula 276.20 Temperature/K 100.05(10) Crystal system Triclinic Space Group P-1 a/Å 7.0528(3) b/Å 11.7911(5) c/Å 14.6888(6) α/° 72.249(4) β/° 79.265(3) γ/° 86.633(3) Volume/Å ³ 1143.02(8) Z 4 ρ _calculated g/cm ³ 1.605 μ/mm ^-1 1.139 F(000) 568.0 Crystal size/mm ³ 0.3 × 0.22 × 0.2 Radiation CuKα (λ = 1.54184) Data collection 2Θ range/° 7.872 to 141.144 Index range -8 ≤ h ≤ 8, -12 ≤ k ≤ 14, -17 ≤ l ≤ 17 Collected reflections 15292 Independent reflex 4304 [R _int = 0.0258, R _Σ = 0.0234] Data/Constraints/Parameters 4304/0/365 Regarding the goodness of fit of ^F2 1.044 Final R index [I＞=2σ (I)] R ₁ = 0.0419, wR ₂ = 0.1043 Final R Index [All Data] _R1 = 0.0517, _wR2 = 0.1124 Maximum difference: peak/hole/e Å ^-3 0.31/-0.25

Table 23. Partial atomic coordinates (×10 ⁴ ) and equivalent isotropic displacement parameters (Å ² ×10 ³ ) of RejuAgro B. U _eq is defined as 1/3 of the trace of the orthogonal U _IJ tensor. atom x y z U(eq) O1 2961(2) 7383.5(12) 2001.1(11) 31.9(3) O2 -1826(2) 9293.7(13) 432.4(11) 35.5(4) O3 -3044(2) 7134.9(13) 421.6(10) 34.1(3) O4 182(2) 5943.5(11) 3820.2(9) 24.6(3) O5 3410(2) 2456.2(11) 4624.7(9) 27.0(3) O6 4494(2) 2636.3(12) 2701.7(10) 31.2(3) N1 593(2) 8361.8(14) 1206.5(12) 25.6(4) N2 1809(2) 4204.9(13) 4225.8(11) 22.5(3) C1 1436(3) 5154.4(16) 2526.1(12) 19.3(4) C2 1083(3) 5152.6(16) 3557.2(13) 20.0(4) C3 2860(3) 3281.2(16) 4009.9(13) 21.6(4) C4 3360(3) 3353.0(16) 2929.1(13) 22.3(4) C5 2479(3) 4308.1(16) 2217.5(13) 21.0(4) C6 2868(3) 4264.1(17) 1193.1(13) 27.0(4) C7 537(3) 6175.3(16) 1875.4(12) 19.6(4) C8 1477(3) 7336.7(16) 1708.5(13) 22.7(4) C9 -1015(3) 8384.0(17) 814.7(13) 25.8(4) C10 -1777(3) 7166.6(18) 869.1(13) 24.7(4) C11 -992(3) 6069.0(16) 1488.2(13) 22.5(4) C12 -2025(3) 4934.9(19) 1648.0(16) 33.7(5) O1A 3249(2) 2542.2(12) 7353.0(10) 29.1(3) O2A 6867(2) 4284.9(12) 4433.3(10) 33.3(3) O3A 8705(2) 2165.5(13) 4475.5(11) 36.7(4) O4A 7213(2) 37.3(12) 8198.9(10) 30.6(3) O5A 2666(2) -2784.5(13) 9706.4(10) 36.6(4) O6A 580(2) -1850.3(14) 8272.4(12) 42.8(4) N1A 5218(2) 3432.4(14) 5950.6(11) 24.6(4) N2A 4869(2) -1309.3(14) 9003.5(11) 26.3(4) C1A 4443(3) 197.8(16) 7467.5(13) 21.3(4) C2A 5632(3) -339.4(16) 8237.7(13) 23.9(4) C3A 3220(3) -1884.4(17) 9063.4(14) 27.5(4) C4A 2026(3) -1349.2(18) 8259.8(15) 27.9(4) C5A 2747(3) -250.9(17) 7474.9(14) 24.8(4) C6A 1500(3) 238(2) 6728.1(15) 33.8(5) C7A 5314(3) 1250.6(16) 6665.8(13) 20.2(4) C8A 4511(3) 2434.0(16) 6711.8(13) 21.9(4) C9A 6479(3) 3408.2(17) 5139.6(14) 25.0(4) C10A 7408(3) 2197.3(17) 5129.5(14) 25.4(4) C11A 6631(3) 1122.0(16) 5922.8(13) 23.0(4) C12A 7343(3) -57.7(18) 5817.8(15) 30.6(5)

Table 24. Anisotropic displacement parameters (Å ² ×10 ³ ) of RejuAgro B. The anisotropic displacement factor exponent takes the following form: -2π ² [h ² a* ² U ₁₁ +2hka*b*U ₁₂ +…]. atom U ₁₁ U ₂₂ U ₃₃ U ₂₃ U ₁₃ U ₁₂ O1 33.5(8) 24.6(7) 37.9(8) -5.9(6) -12.5(6) -2.0(6) O2 38.2(8) 25.5(8) 32.6(8) 3.8(6) -4.8(6) 7.4(6) O3 39.2(8) 37.3(8) 28.2(7) -9.7(6) -15.1(6) 8.0(7) O4 34.1(7) 21.0(7) 17.2(6) -5.5(5) -3.2(5) 6.3(6) O5 40.0(8) 19.7(7) 21.1(7) -3.3(5) -11.5(6) 5.2(6) O6 41.1(8) 23.2(7) 25.3(7) -5.6(6) -2.6(6) 10.7(6) N1 30.1(9) 15.4(8) 28.0(9) -2.9(6) -3.1(7) 0.5(6) N2 34.8(9) 19.1(8) 12.8(7) -4.1(6) -5.5(6) 5.0(7) C1 23.4(9) 17.5(9) 15.2(8) -2.4(7) -3.1(7) -1.0(7) C2 23.7(9) 17.6(9) 17.2(9) -3.0(7) -3.7(7) -0.5(7) C3 26.6(10) 17.2(9) 20.6(9) -4.0(7) -5.6(7) -1.3(7) C4 28.7(10) 16.2(9) 19.8(9) -3.5(7) -2.9(7) 1.3(7) C5 24.8(9) 18.4(9) 17.6(9) -2.8(7) -3.0(7) 0.1(7) C6 37.7(11) 23.1(10) 19.0(9) -6.5(8) -4.0(8) 7.2(8) C7 25.6(9) 18.4(9) 12.8(8) -4.1(7) -0.3(7) 3.2(7) C8 28.1(10) 18.4(9) 19.3(9) -3.6(7) -2.9(7) 2.8(7) C9 30.7(10) 23.1(10) 17.2(9) -0.2(8) 0.7(7) 4.0(8) C10 28.7(10) 29.8(10) 14.7(9) -6.4(8) -3.6(7) 5.9(8) C11 27.1(10) 21.4(9) 17.4(9) -5.2(7) -2.1(7) 3.4(8) C12 36.9(12) 28.6(11) 37.8(12) -7.7(9) -15.5(9) -1.4(9) O1A 32.5(8) 26.5(7) 25.4(7) -7.3(6) -0.5(6) 6.2(6) O2A 40.9(8) 21.2(7) 29.3(8) 0.0(6) 2.0(6) -1.0(6) O3A 39.6(9) 29.7(8) 33.2(8) -7.5(6) 9.6(7) -1.9(6) O4A 32.5(8) 28.8(8) 30.1(8) -5.3(6) -10.7(6) 0.1(6) O5A 46.2(9) 28.2(8) 25.3(8) 2.1(6) 1.6(6) -0.1(7) O6A 36.0(9) 34.1(9) 49.6(10) 1.6(7) -7.1(7) -7.7(7) N1A 31.8(9) 16.0(8) 24.2(8) -5.0(6) -2.8(7) 2.3(6) N2A 34.6(9) 22.7(8) 17.8(8) -1.0(6) -6.1(7) 6.9(7) C1A 27.1(10) 17.8(9) 16.7(9) -4.0(7) -1.4(7) 4.1(7) C2A 30.6(10) 19.3(9) 20.4(9) -4.8(7) -3.9(8) 4.6(8) C3A 32.7(11) 23.7(10) 21.9(10) -6.4(8) 3.9(8) 3.7(8) C4A 27.6(10) 25.1(10) 27.8(10) -6.4(8) 0.4(8) 1.3(8) C5A 28.8(10) 22.0(10) 21.0(9) -4.6(8) -1.3(8) 0.6(8) C6A 30.4(11) 39.5(12) 28.2(11) -3.0(9) -7.5(8) -5.5(9) C7A 22.8(9) 18.2(9) 19.1(9) -2.7(7) -7.0(7) 0.0(7) C8A 25.6(9) 20.7(9) 18.5(9) -3.5(7) -6.1(7) 1.4(7) C9A 26.2(10) 24.4(10) 22.8(10) -4.9(8) -3.2(8) -2.2(8) C10A 26.5(10) 24.8(10) 23.7(10) -7.3(8) -0.9(8) -2.1(8) C11A 25.5(9) 20.3(9) 22.7(9) -5.1(7) -5.4(7) -0.1(7) C12A 33.3(11) 25.6(10) 30.4(11) -9.9(9) 3.0(8) -0.3(8)

Table 25. Bond lengths of RejuAgro B. atom atom Length/Å atom atom Length/Å O1 C8 1.213(2) O1A C8A 1.202(2) O2 C9 1.214(2) O2A C9A 1.222(2) O3 C10 1.212(2) O3A C10A 1.205(2) O4 C2 1.217(2) O4A C2A 1.209(2) O5 C3 1.210(2) O5A C3A 1.213(2) O6 C4 1.208(2) O6A C4A 1.203(3) N1 C8 1.390(2) N1A C8A 1.394(2) N1 C9 1.360(3) N1A C9A 1.354(2) N2 C2 1.388(2) N2A C2A 1.388(2) N2 C3 1.366(2) N2A C3A 1.356(3) C1 C2 1.488(2) C1A C2A 1.496(3) C1 C5 1.344(3) C1A C5A 1.333(3) C1 C7 1.483(2) C1A C7A 1.495(2) C3 C4 1.538(3) C3A C4A 1.534(3) C4 C5 1.480(3) C4A C5A 1.488(3) C5 C6 1.495(3) C5A C6A 1.491(3) C7 C8 1.489(3) C7A C8A 1.491(3) C7 C11 1.338(3) C7A C11A 1.335(3) C9 C10 1.537(3) C9A C10A 1.539(3) C10 C11 1.481(3) C10A C11A 1.486(3) C11 C12 1.494(3) C11A C12A 1.493(3)

Table 26. Key features of RejuAgro B. atom atom atom angle atom atom atom angle C9 N1 C8 125.07(16) C9A N1A C8A 125.23(16) C3 N2 C2 125.15(15) C3A N2A C2A 125.18(17) C5 C1 C2 122.84(16) C5A C1A C2A 122.52(17) C5 C1 C7 123.43(16) C5A C1A C7A 121.91(17) C7 C1 C2 113.73(15) C7A C1A C2A 115.53(16) O4 C2 N2 120.22(16) O4A C2A N2A 120.41(17) O4 C2 C1 122.04(16) O4A C2A C1A 122.03(17) N2 C2 C1 117.74(15) N2A C2A C1A 117.55(17) O5 C3 N2 122.60(17) O5A C3A N2A 123.6(2) O5 C3 C4 121.02(17) O5A C3A C4A 119.50(19) N2 C3 C4 116.37(15) N2A C3A C4A 116.89(17) O6 C4 C3 117.92(16) O6A C4A C3A 118.93(18) O6 C4 C5 123.19(17) O6A C4A C5A 122.62(19) C5 C4 C3 118.87(16) C5A C4A C3A 118.42(17) C1 C5 C4 118.55(16) C1A C5A C4A 119.10(18) C1 C5 C6 125.01(17) C1A C5A C6A 125.12(18) C4 C5 C6 116.43(16) C4A C5A C6A 115.74(17) C1 C7 C8 113.48(16) C8A C7A C1A 115.80(16) C11 C7 C1 123.39(16) C11A C7A C1A 121.49(17) C11 C7 C8 123.12(17) C11A C7A C8A 122.54(16) O1 C8 N1 121.37(17) O1A C8A N1A 120.00(17) O1 C8 C7 121.04(17) O1A C8A C7A 122.46(17) N1 C8 C7 117.59(17) N1A C8A C7A 117.48(16) O2 C9 N1 123.67(19) O2A C9A N1A 123.12(18) O2 C9 C10 120.36(19) O2A C9A C10A 120.17(17) N1 C9 C10 115.97(16) N1A C9A C10A 116.70(16) O3 C10 C9 118.81(17) O3A C10A C9A 118.60(17) O3 C10 C11 121.85(19) O3A C10A C11A 123.38(18) C11 C10 C9 119.32(17) C11A C10A C9A 118.00(16) C7 C11 C10 117.85(17) C7A C11A C10A 119.31(17) C7 C11 C12 125.55(17) C7A C11A C12A 123.69(17) C10 C11 C12 116.58(17) C10A C11A C12A 116.97(17)

Table 27. Hydrogen bonds of RejuAgro B. D H A d(DH)/Å d(HA)/Å d(DA)/Å DHA/° N1 H1 O2 ¹ 0.88 2.51 3.104(2) 125.1 N1 H1 O4A ² 0.88 2.17 2.928(2) 143.6 N2 H2 O4 ³ 0.88 2.09 2.909(2) 154.7 N1A H1A O2A ² 0.88 2.14 2.940(2) 150.8 N2A H2A O3 ⁴ 0.88 2.08 2.892(2) 153.8 ¹ -X,2-Y,-Z; ² 1-X,1-Y,1-Z; ³ -X,1-Y,1-Z; ⁴ 1+X,-1+Y,1+Z

Table 28. Torsion angle of RejuAgro B. A B C D angle A B C D angle O2 C9 C10 O3 9.1(3) O2A C9A C10A O3A 10.0(3) O2 C9 C10 C11 -169.03(18) O2A C9A C10A C11A -169.05(18) O3 C10 C11 C7 173.21(18) O3A C10A C11A C7A 173.9(2) O3 C10 C11 C12 -8.5(3) O3A C10A C11A C12A -8.1(3) O5 C3 C4 O6 -8.1(3) O5A C3A C4A O6A -1.6(3) O5 C3 C4 C5 173.42(17) O5A C3A C4A C5A -179.46(18) O6 C4 C5 C1 -171.34(18) O6A C4A C5A C1A -174.5(2) O6 C4 C5 C6 7.3(3) O6A C4A C5A C6A 3.5(3) N1 C9 C10 O3 -170.34(17) N1A C9A C10A O3A -171.13(19) N1 C9 C10 C11 11.5(2) N1A C9A C10A C11A 9.8(3) N2 C3 C4 O6 170.50(17) N2A C3A C4A O6A 178.04(19) N2 C3 C4 C5 -7.9(2) N2A C3A C4A C5A 0.2(3) C1 C7 C8 O1 7.7(3) C1A C7A C8A O1A 0.3(3) C1 C7 C8 N1 -172.31(16) C1A C7A C8A N1A -176.82(16) C1 C7 C11 C10 178.53(16) C1A C7A C11A C10A 178.16(17) C1 C7 C11 C12 0.4(3) C1A C7A C11A C12A 0.3(3) C2 N2 C3 O5 -177.67(18) C2A N2A C3A O5A 174.19(19) C2 N2 C3 C4 3.7(3) C2A N2A C3A C4A -5.5(3) C2 C1 C5 C4 -1.8(3) C2A C1A C5A C4A -1.9(3) C2 C1 C5 C6 179.67(17) C2A C1A C5A C6A -179.68(19) C2 C1 C7 C8 70.3(2) C2A C1A C7A C8A -103.24(19) C2 C1 C7 C11 -108.6(2) C2A C1A C7A C11A 81.3(2) C3 N2 C2 O4 -179.02(17) C3A N2A C2A O4A -172.19(18) C3 N2 C2 C1 1.4(3) C3A N2A C2A C1A 6.9(3) C3 C4 C5 C1 7.0(3) C3A C4A C5A C1A 3.3(3) C3 C4 C5 C6 -174.34(16) C3A C4A C5A C6A -178.77(18) C5 C1 C2 O4 177.87(18) C5A C1A C2A O4A 176.18(19) C5 C1 C2 N2 -2.6(3) C5A C1A C2A N2A -2.9(3) C5 C1 C7 C8 -109.5(2) C5A C1A C7A C8A 78.8(2) C5 C1 C7 C11 71.6(3) C5A C1A C7A C11A -96.6(2) C7 C1 C2 O4 -2.0(3) C7A C1A C2A O4A -1.8(3) C7 C1 C2 N2 177.59(16) C7A C1A C2A N2A 179.16(16) C7 C1 C5 C4 178.00(16) C7A C1A C5A C4A 175.89(17) C7 C1 C5 C6 -0.5(3) C7A C1A C5A C6A -1.9(3) C8 N1 C9 O2 175.24(18) C8A N1A C9A O2A 169.92(19) C8 N1 C9 C10 -5.4(3) C8A N1A C9A C10A -8.9(3) C8 C7 C11 C10 -0.3(3) C8A C7A C11A C10A 3.0(3) C8 C7 C11 C12 -178.45(18) C8A C7A C11A C12A -174.84(18) C9 N1 C8 O1 176.67(18) C9A N1A C8A O1A -172.43(18) C9 N1 C8 C7 -3.3(3) C9A N1A C8A C7A 4.8(3) C9 C10 C11 C7 -8.7(3) C9A C10A C11A C7A -7.1(3) C9 C10 C11 C12 169.60(17) C9A C10A C11A C12A 170.89(17) C11 C7 C8 O1 -173.40(18) C11A C7A C8A O1A 175.71(19) C11 C7 C8 N1 6.6(3) C11A C7A C8A N1A -1.4(3)

Table 29. Hydrogen atomic coordinates (Å×10 ⁴ ) and equivalent isotropic displacement parameters (Å ² ×10 ³ ) of RejuAgro B. atom x y z U(eq) H1 1115.22 9049.78 1137.02 31 H2 1572.72 4198.81 4837.04 27 H6A 4232.36 4076.12 1013.7 40 H6B 2564.31 5038.79 758.38 40 H6C 2062.97 3648.38 1135.45 40 H12A -1685.96 4670.99 1066.64 51 H12B -3421.18 5067.41 1777.57 51 H12C -1645.33 4322.49 2204.55 51 H1A 4812.5 4134.48 6001.06 30 H2A 5506.16 -1572.03 9488.94 32 H6AC 1266.79 -381.27 6443.7 51 H6AA 2147.29 916.19 6217.2 51 H6AB 266.47 500.23 7032.58 51 H12D 6459.43 -365.46 5507.86 46 H12E 7403.89 -616.99 6460.59 46 H12F 8633.15 35.4 5415.92 46 C. Crystal structure information of RejuAgro C.

Single crystals of RejuAgro C (C ₁₀ H ₁₆ N ₂ O ₇ ) were obtained by slow evaporation of a methanol solution of RejuAgro B and RejuAgro C. Colorless needles were obtained along with RejuAgro B. Suitable crystals were selected and mounted on a SuperNova, Dual, Cu at home/near, Atlas diffractometer. The crystals were kept at 100.05(10) K during data collection. The structure was solved using Olex2 (Dolomanov et al. (2009)) using the olex2.solve structure solution program (Bourhis et al. (2015)) with charge flipping and refined using the ShelXL refinement package (Sheldrick (2015)) using least squares minimization.

The data set was collected at 100 K using 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 , D calc = 1.466 g/cm ³ , 7480 measured reflections (10.068° ≤ 2Θ) ≤ 140.528°), 2353 unique values ( R _int = 0.0405, R _Σ = 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 detailed model description was created using Olex2, compiled with OlexSys svn.r3508 from May 29, 2018. The number of restraints is 0, and the number of restrictions is unknown. Details: 1. Fixed floppy disks; at 1.5 times the radius of all C(H,H,H) groups; 2. Ideal Me, defined as rotational groups: C9(H9A,H9B,H9C), C10(H10A,H10B,H10C).

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

Referring to Figure 15B , RejuAgro C molecules in the crystal are stacked along the x-axis. These stacks are connected to layers along the ab plane via H-bonds (N-H…O). These layers are connected to the 3D network via multiple hydrogen bonds with solvate water molecules (3 molar equivalents).

The chemical structure of RejuAgro C is shown below: (Formula (III))

Additional crystal structure data for the RejuAgro C molecule are presented in Tables 30-37.

Table 30. Crystallographic data and structural refinement of RejuAgro C. Identification code RejuAgro C Experiential C ₁₀ H ₁₆ N ₂ O ₇ Formula 276.25 Temperature/K 100.00(10) Crystal system Triclinic Space Group P-1 a/Å 7.0334(4) b/Å 10.2354(7) c/Å 10.4693(7) α/° 116.426(7) β/° 104.722(5) γ/° 97.680(5) Volume/Å ³ 625.72(8) Z 2 ρ _calculated g/cm ³ 1.466 μ/mm ^-1 1.081 F(000) 292.0 Crystal size/mm ³ 0.461 × 0.063 × 0.021 Radiation CuKα (λ = 1.54184) Data collection 2Θ range/° 10.068 to 140.528 Index range -8 ≤ h ≤ 6, -11 ≤ k ≤ 12, -12 ≤ l ≤ 12 Collected reflections 7480 Independent reflex 2353 [R _int = 0.0405, R _Σ = 0.0373] Data/Constraints/Parameters 2353/0/214 Regarding the goodness of fit of ^F2 1.047 Final R index [I＞=2σ (I)] _R1 = 0.0504, _wR2 = 0.1270 Final R Index [All Data] _R1 = 0.0622, _wR2 = 0.1388 Maximum difference: peak/hole/e Å ^-3 0.41/-0.27

Table 31. Partial atomic coordinates (×10 ⁴ ) and equivalent isotropic displacement parameters (Å ² ×10 ³ ) of RejuAgro C. U _eq is defined as 1/3 of the trace of the orthogonal U _IJ tensor. atom x y z U(eq) O1 999(2) 3453.7(16) 3219.0(16) 25.9(3) O1W 5100(2) 8599(2) 11132.3(18) 30.5(4) O2 5632(2) 8416.1(16) 8638.5(17) 28.9(4) O2W 1123(3) 6910(2) 10264(2) 38.6(4) O3 3742(2) 8669.8(16) 6246.1(17) 28.8(4) O3W -137(3) 3837.8(19) 7792.6(18) 32.3(4) O4 2491(2) 2183.1(18) 6934.3(18) 31.4(4) N1 2349(3) 6104.7(19) 4717(2) 24.2(4) N2 2874(3) 441(2) 4805(2) 34.3(5) C1 1129(3) 2223(2) 3453(3) 28.0(5) C2 2308(3) 2755(2) 4951(2) 25.9(5) C3 2976(3) 4407(2) 5702(2) 23.6(4) C4 4286(3) 5669(2) 7152(2) 23.7(4) C5 4459(3) 7096(2) 7326(2) 24.7(4) C6 3504(3) 7361(2) 6092(2) 24.9(4) C7 2121(3) 4717(2) 4574(2) 23.4(4) C8 2582(3) 1776(2) 5638(3) 26.6(5) C9 -4(4) 717(3) 2106(3) 32.4(5) C10 5482(3) 5460(3) 8426(2) 29.8(5)

Table 32. Anisotropic displacement parameters (Å ² ×10 ³ ) of RejuAgro C. The anisotropic displacement factor exponent takes the following form: -2π ² [h ² a* ² U ₁₁ +2hka*b*U ₁₂ +…]. atom U ₁₁ U ₂₂ U ₃₃ U ₂₃ U ₁₃ U ₁₂ O1 26.2(7) 28.8(8) 26.6(8) 15.3(6) 10.5(6) 12.1(6) O1W 33.2(8) 29.8(9) 28.5(8) 14.4(7) 10.2(7) 11.1(7) O2 33.7(8) 27.5(8) 26.0(8) 13.9(6) 10.5(6) 8.5(6) O2W 37.4(10) 43.6(10) 34.9(9) 18.6(8) 15.5(8) 10.1(8) O3 35.1(8) 28.0(8) 32.5(8) 19.1(7) 15.4(7) 15.1(6) O3W 34.3(8) 42.2(9) 31.9(9) 22.2(8) 16.0(7) 22.5(7) O4 36.4(8) 36.7(8) 35.0(8) 23.6(7) 18.6(7) 19.6(7) N1 26.1(9) 28.6(9) 26.8(9) 17.9(8) 12.0(7) 13.7(7) N2 45.3(12) 36.3(11) 38.6(11) 25.3(9) 23.0(9) 22.9(9) C1 27.0(10) 30.6(11) 36.2(11) 19.6(9) 18.2(9) 13.3(9) C2 24.5(10) 28.7(11) 32.3(11) 17.4(9) 15.0(9) 12.7(8) C3 23.1(9) 28.6(10) 28.4(10) 17.8(9) 13.2(8) 14.4(8) C4 22.7(10) 29.7(10) 25.8(10) 16.4(9) 11.7(8) 12.7(8) C5 22.9(10) 29.4(11) 26.0(10) 14.7(9) 11.8(8) 11.2(8) C6 25.6(10) 30.2(11) 30.7(11) 19.3(9) 16.3(9) 15.7(9) C7 24.1(10) 28.0(10) 26.2(10) 16.1(9) 13.2(8) 13.9(8) C8 23.9(10) 30.7(11) 33.0(11) 19.1(9) 13.5(9) 12.5(8) C9 34.4(12) 30.8(11) 33.5(12) 16.3(10) 13.2(10) 10.5(9) C10 31.7(11) 33.1(11) 28.1(11) 17.7(9) 8.8(9) 14.3(9)

Table 33. Bond lengths of RejuAgro C. atom atom Length/Å atom atom Length/Å O1 C1 1.396(3) C1 C9 1.472(3) O1 C7 1.353(3) C2 C3 1.455(3) O2 C5 1.367(3) C2 C8 1.478(3) O3 C6 1.258(3) C3 C4 1.431(3) O4 C8 1.252(3) C3 C7 1.371(3) N1 C6 1.365(3) C4 C5 1.373(3) N1 C7 1.343(3) C4 C10 1.505(3) N2 C8 1.332(3) C5 C6 1.453(3) C1 C2 1.379(3)

Table 34. Key features of RejuAgro C. atom atom atom angle atom atom atom angle C7 O1 C1 105.97(16) C5 C4 C10 120.77(19) C7 N1 C6 119.61(17) O2 C5 C4 124.31(18) O1 C1 C9 114.99(19) O2 C5 C6 112.35(18) C2 C1 O1 109.38(19) C4 C5 C6 123.24(19) C2 C1 C9 135.6(2) O3 C6 N1 120.46(18) C1 C2 C3 107.33(18) O3 C6 C5 122.8(2) C1 C2 C8 123.9(2) N1 C6 C5 116.74(18) C3 C2 C8 128.4(2) O1 C7 C3 113.39(18) C4 C3 C2 138.62(19) N1 C7 O1 120.70(17) C7 C3 C2 103.93(19) N1 C7 C3 125.9(2) C7 C3 C4 117.30(19) O4 C8 N2 122.2(2) C3 C4 C10 122.16(18) O4 C8 C2 120.68(18) C5 C4 C3 117.05(18) N2 C8 C2 117.1(2)

Table 35. Hydrogen bonds of RejuAgro C. D H A d(DH)/Å d(HA)/Å d(DA)/Å DHA/° O2 H2 O1W 0.94(3) 1.73(3) 2.659(2) 169(3) N2 H2A O3 ¹ 0.96(3) 1.92(3) 2.863(2) 168(3) N1 H1 O3W ² 0.97(4) 1.76(4) 2.723(2) 169(3) O2W H2WA O3W 0.86(4) 2.03(4) 2.866(3) 164(3) N2 H2B O3 ³ 0.94(3) 2.36(3) 3.057(3) 130(2) O1W H1WA O2 ⁴ 0.79(4) 2.46(3) 3.080(2) 136(3) O1W H1WA O3 ⁴ 0.79(4) 2.01(4) 2.727(2) 151(3) O1W H1WB O4 ⁵ 0.89(4) 1.88(4) 2.770(2) 175(3) O2W H2WB O1W 0.92(5) 1.87(5) 2.767(3) 165(4) O3W H3WA O4 0.85(3) 1.88(3) 2.724(2) 173(3) O3W H3WB O2W ⁶ 0.91(4) 1.77(4) 2.677(2) 175(3) ¹ +X,-1+Y,+Z; ² -X,1-Y,1-Z; ³ 1-X,1-Y,1-Z; ⁴ 1-X,2-Y,2-Z; ⁵ 1-X,1-Y,2-Z; ⁶ -X,1-Y,2-Z

Table 36. Torsion angle of RejuAgro C. A B C D angle A B C D angle O1 C1 C2 C3 -0.6(2) C4 C3 C7 O1 176.52(16) O1 C1 C2 C8 173.11(17) C4 C3 C7 N1 -2.2(3) O2 C5 C6 O3 -0.5(3) C4 C5 C6 O3 -176.94(18) O2 C5 C6 N1 178.06(16) C4 C5 C6 N1 1.6(3) C1 O1 C7 N1 178.17(16) C6 N1 C7 O1 -179.40(16) C1 O1 C7 C3 -0.6(2) C6 N1 C7 C3 -0.7(3) C1 C2 C3 C4 -174.7(2) C7 O1 C1 C2 0.8(2) C1 C2 C3 C7 0.2(2) C7 O1 C1 C9 179.80(16) C1 C2 C8 O4 -136.2(2) C7 N1 C6 O3 179.69(17) C1 C2 C8 N2 42.2(3) C7 N1 C6 C5 1.1(3) C2 C3 C4 C5 179.1(2) C7 C3 C4 C5 4.6(3) C2 C3 C4 C10 1.0(4) C7 C3 C4 C10 -173.53(18) C2 C3 C7 O1 0.3(2) C8 C2 C3 C4 11.9(4) C2 C3 C7 N1 -178.46(18) C8 C2 C3 C7 -173.13(19) C3 C2 C8 O4 36.2(3) C9 C1 C2 C3 -179.4(2) C3 C2 C8 N2 -145.5(2) C9 C1 C2 C8 -5.7(4) C3 C4 C5 O2 179.50(17) C10 C4 C5 O2 -2.3(3) C3 C4 C5 C6 -4.5(3) C10 C4 C5 C6 173.71(18)

Table 37. Hydrogen atomic coordinates (Å×10 ⁴ ) and equivalent isotropic displacement parameters (Å ² ×10 ³ ) of RejuAgro C. atom x y z U(eq) H9A -1263.03 784.93 1500.56 49 H9B -356.49 -12.41 2434.48 49 H9C 851.94 376.04 1484.45 49 H10A 4654.31 5449.72 9047.62 45 H10B 6747.93 6301.33 9062.81 45 H10C 5824.37 4494.45 7996.93 45 H2 5530(40) 8390(30) 9510(30) 39(7) H2A 2960(40) -250(30) 5200(30) 43(7) H1 1690(50) 6240(40) 3870(40) 60(9) H2WA 840(50) 6070(40) 9430(40) 53(9) H2B 3190(50) 290(30) 3940(40) 43(7) H1WA 5240(50) 9470(40) 11680(40) 49(9) H1WB 5840(60) 8290(40) 11710(40) 66(10) H2WB 2360(80) 7470(50) 10400(50) 91(14) H3WA 770(50) 3390(30) 7570(30) 46(8) H3WB -500(60) 3520(40) 8410(50) 67(11) Citation Incorporated by reference

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

without

Figure 1 shows an exemplary diagram of the maximum possible speciation of representative Pseudomonas anemias based on a concatenated alignment of 16S rDNA, gyrB , rpoB , and rpoD . Four internal branches with <100% support are labeled with their own support values. Unlabeled branches indicate 100% support.

FIG2 shows an example of assay-guided separation of ethyl acetate extracts of strain 0617-T307.

FIG. 3A depicts an exemplary culture profile showing the amount of RejuAgro A in a shake flask fermentation showing its distribution in the cell slurry, supernatant, and cells, and FIG. 3B depicts the production of RejuAgro A from cell fermentation over time.

Figure 4 depicts exemplary agar plates showing that V. inaequalis was able to grow on PDA plates with PDA alone without additives (Panel A), 0.25% 0.01 M PBS (Panel B), 0.8% DMSO (Panel C), or 1.6% DMSO (Panel D) at day 14.

Figure 5 depicts exemplary agar plates demonstrating the inability of V. apple to grow on PDA plates containing four selected biocontrol bacteria (Plate A: 0617-T307; Plate B: 0118-T319; Plate C: 0318-T327; Plate D: 0418-T328) on day 14.

Figure 6 depicts exemplary agar plates demonstrating the inability of V. apple to grow on PDA plates containing 40-80 µg/mL RejuAgro A on day 14 (Plate A: 10 µg/mL on PDA plate; Plate B: 20 µg/mL on PDA plate; Plate C: 40 µg/mL on PDA plate; Plate D: 80 µg/mL on PDA plate).

Figure 7 depicts exemplary agar plates showing the growth of V. apple on PDA plates containing 10-80 µg/mL RejuAgro B on day 14 (Plate A: 10 µg/mL on PDA plate; Plate B: 20 µg/mL on PDA plate; Plate C: 40 µg/mL on PDA plate; Plate D: 80 µg/mL on PDA plate).

Figure 8 depicts exemplary agar plates showing the ability of V. apple to grow on PDA plates containing 200-1000 µg/mL copper sulfate (Plate A: PDA plate with 500 µg/mL CuSO ₄ ; Plate B: PDA plate with 1000 µg/mL CuSO ₄ ) on day 14.

Figure 9 depicts an exemplary mass-peak area curve of RejuAgro A analyzed by HPLC at a wavelength of 407 nm.

Figure 10 depicts exemplary data for the production of RejuAgro A by different bacterial strains.

Figure 11 depicts an exemplary antifungal assay against Botrytis cinerea CA17, where Panel A depicts (1) 40 µL of nystatin at 50 mg/mL, (2) 40 µL of DMSO; Panel B depicts (1) M9 culture for 24 hours, (2) M8 culture for 24 hours, (3) M7 culture for 24 hours, and (4) M6 culture for 24 hours; Panel C depicts (1) M9 culture for 12 hours, (2) M8 culture for 12 hours, (3) M7 culture for 12 hours, and (4) M6 culture for 12 hours.

Figure 12 depicts exemplary agar plates of M. fijiensis showing inhibition of reproduction in the presence of 600 µg/mL RejuAgro A (Panel A) or in the presence of 60 µg/mL RejuAgro A (Panel B) or in the absence of RejuAgro A ( Panel C).

Figure 13A depicts the RejuAgro A molecule as a planar structure, in which the S-Me group is rotated only 8.7° relative to the ring. The molecule exhibits a significant break in π-conjugation at the C4-C5 bond (1.531 Å), apparently due to orbital factors. The Me group attached to the ^sp2 carbon atom is rotationally disordered in two positions.

Figure 13B depicts RejuAgro A molecules in a crystal forming centrosymmetric H-bonded dimers via N-H…O interactions. Furthermore, these dimers form two-dimensional layers along the [-3 0 1] plane via weaker C-H…O interactions.

Figure 14A depicts a crystal of RejuAgro B with two symmetrically separated RejuAgro B molecules. Each molecule has a helical structure with dihedral angles of 70.3° and 80.6° between ^the average planes of the connected heterocyclic rings. The π-conjugation between the two adjacent carbonyl groups in each ^heterocyclic ring is significantly broken (bond lengths range from 1.534 to 1.539 Å), apparently due to some orbital reason.

Figure 14B depicts a RejuAgro B molecule in crystal form as centrosymmetric H-bonded dimers connected by N-H…O interactions. The dimers are connected in the stack along the x-direction by additional N-H…O interactions. Finally, the stack is connected to the layer along [011] by a third N-H…O interaction.

Figure 15A depicts a RejuAgro C molecule with a planar π-conjugated shape, in which the amide group is rotated 42° from the plane of the remaining atoms.

Figure 15B depicts the stacking of RejuAgro C molecules along the x-axis in a crystal. The stacks are connected to layers along the ab plane via H-bonds (N-H…O). The layers are connected to the 3D network via multiple hydrogen bonds with solvate water molecules (3 molar equivalents).

Domestic deposit information (please note the depository in order of institution, date, and number) 1. Pseudomonas soli 0617-T307: Food Industry Development Research Institute, September 4, 2020, R.O.C., BCRC 911020 2. Pseudomonas soli 0917-T305: Food Industry Development Research Institute, September 4, 2020, R.O.C., BCRC 911021 3. Pseudomonas soli 0917-T306: Food Industry Development Research Institute, September 4, 2020, R.O.C., BCRC 911022 4. Pseudomonas soli 0917-T307: Food Industry Development Research Institute, September 4, 2020, R.O.C., BCRC 911023 5. Pseudomonas mosselii 0118-T319: Food Industry Development Research Institute, September 4, 2020, R.O.C., BCRC 911024 6. Pseudomonas mosselii 0318-T327: Food Industry Development Research Institute, September 4, 2020, R.O.C., BCRC 911025 7. Pseudomonas mosselii 0418-T328: Food Industry Development Research Institute, September 4, 2020, R.O.C., BCRC 911026 International Deposit Information (Please indicate the deposited country, institution, date, and number in order) 1. Pseudomonas soli 0617-T307: American Type Culture Collection (ATCC®), June 25, 2020, PTA-126796 2. Pseudomonas soli 0917-T305: American Type Culture Collection (ATCC®), June 25, 2020, PTA-126797 3. Pseudomonas soli 0917-T306: American Type Culture Collection (ATCC®), June 25, 2020, PTA-126798 4. Pseudomonas soli 0917-T307: American Type Culture Collection (ATCC®), June 25, 2020, PTA-126799 5. Pseudomonas mosselii 0118-T319: American Type Culture Collection (ATCC®), June 25, 2020, PTA-126800 6. Pseudomonas mosselii 0318-T327: American Type Culture Collection (ATCC®), June 25, 2020, PTA-126801 7. Pseudomonas mosselii 0418-T328: American Type Culture Collection (ATCC®), June 25, 2020, PTA-126802

Claims (35)

A method for growing bacteria to enhance production of protective metabolites, the method comprising the steps of: i. growing Pseudomonas bacteria in a liquid culture medium in a container to produce a bacterial fermentation product, wherein the ratio of the volume of the culture medium to the volume of the container is between about 1:2 and 1:10, and wherein the container is agitated at a rate between about 100 and 250 RPM, or ii. growing Pseudomonas bacteria in a liquid culture medium in a fermenter to produce a bacterial fermentation product, wherein the air flow rate of the fermenter is between about 1 and 3 L/min, wherein the bacteria are selected from the group consisting of: Pseudomonas soli 0617-T307 (Accession No. PTA-126796, BCRC 911020), Pseudomonas aeruginosa 0917-T305 (Accession No. PTA-126797, BCRC 911021), Pseudomonas aeruginosa 0917-T306 (Accession No. PTA-126798, BCRC 911022), Pseudomonas aeruginosa 0917-T307 (Accession No. PTA-126799, BCRC 911023), Pseudomonas mosselii 0118-T319 (Accession No. PTA-126800, BCRC 911024), Pseudomonas mosselii 0318-T327 (Accession No. PTA-126801, BCRC 911025), and Pseudomonas mosselii 0418-T328 (Accession No. PTA-126802, BCRC 911026), wherein the liquid culture medium is selected from LB medium and YME medium, and wherein the Pseudomonas bacteria are grown at a growth temperature of about 16°C to about 28°C. The method of claim 1, further comprising the step of separating the liquid culture medium from the bacteria after a period of time to produce a protective supernatant containing the protective metabolites. The method of claim 1 or 2, wherein the liquid culture medium is LB medium for producing cells. The method of claim 1 or 2, wherein the liquid culture medium is a YME culture medium for producing formula (I) and formula (II): (I)and (II). The method according to claim 1 or 2, wherein the method can be carried out using a shaker or a fermentation tank. The method of claim 5, wherein when a shaker is used, the ratio of the volume of the culture medium to the volume of the container is between about 1:5 and 1:10. The method of claim 6, wherein when a shaker is used, the ratio of the volume of the culture medium to the volume of the container is between about 1:7 and 1:9. The method of claim 7, wherein when a shaker is used, the ratio of the volume of the culture medium to the volume of the container is about 1:8. The method of claim 1 or 2, wherein the container is vibrated at a rate between about 200 and 250 RPM. The method of claim 9, wherein the container is vibrated at a rate between about 210 and 230 RPM. The method of claim 1 or 2, wherein a fermenter is used, wherein the air flow rate of the fermenter is between about 1.5 and 2.5 L/min or the oxygen concentration is between 5 mg/L and 12 mg/L. The method of claim 1 or 2, wherein the growth temperature is about 16°C. The method of claim 1 or 2, wherein the growth temperature is about 28°C. The method of claim 1 or 2, wherein the bacteria are grown for a period of time ranging from 18 hours to 7 days. The method of claim 1 or 2, wherein the bacteria are grown for a period of at least seven days. The method of claim 16, wherein the bacteria are grown for a period of time between one and two days. An agricultural composition comprising a bacterial fermentation product or a protective supernatant produced by the method of any one of claims 1 to 16. The agricultural composition of claim 17, wherein the formulation of the protective supernatant or its metabolites is selected from a solution (SL), a soluble powder (SP), a soluble granule (SG) and a packaged formulation. The agricultural composition of claim 17, wherein the bacterial fermentation product and cell preparation is selected from a suspension concentrate (SC), a wettable powder (WP) and a water-dispersible granule (WG). The agricultural composition of claim 17, further comprising an adjuvant. The agricultural composition of claim 20, wherein the adjuvant is a surfactant. The agricultural composition of claim 20, wherein the adjuvant is a selected active ingredient, water and a polar solvent. The agricultural composition of claim 21, wherein the surfactant is selected from a wetting agent and a diffusing agent. The agricultural composition of claim 23, wherein the surfactant is a wetting agent, and the wetting agent includes alkyl polyethylene oxide or polypropylene oxide. A method for purifying a protective metabolite from a Pseudomonas bacterium, the method comprising the following steps: i. producing a bacterial fermentation product or protective supernatant produced by the method of any one of claims 1 to 16, or a formulation thereof; ii. extracting the bacterial fermentation product or protective supernatant by extraction with ethyl acetate; and iii. producing a solubilized product containing the protective metabolite by dissolving the bacterial fermentation product or protective supernatant using 50% hexane and 50% ethyl acetate or by dissolving the bacterial fermentation product or protective supernatant using 25% hexane and 75% ethyl acetate, wherein the Pseudomonas bacterium is selected from the group consisting of: Pseudomonas aeruginosa 0617-T307 (Accession No. PTA-126796, BCRC 911020), Pseudomonas aeruginosa 0917-T305 (Accession No. PTA-126797, BCRC 911021), Pseudomonas aeruginosa 0917-T306 (Accession No. PTA-126798, BCRC 911022), Pseudomonas aeruginosa 0917-T307 (Accession No. PTA-126799, BCRC 911023), Pseudomonas morganii 0118-T319 (Accession No. PTA-126800, BCRC 911024), Pseudomonas morganii 0318-T327 (Accession No. PTA-126801, BCRC 911025), and Pseudomonas morganii 0418-T328 (Accession No. PTA-126802, BCRC 911026). A method for controlling bacterial crop diseases, comprising the steps of: i. producing an agricultural composition comprising a bacterial fermentation product or protective supernatant produced by the method of any one of claims 1 to 16, or an agricultural composition of any one of claims 17 to 24, or a protective metabolite from Pseudomonas bacteria purified by the method of claim 25, and ii. applying the agricultural composition to a crop to inhibit the growth of a pathogenic microorganism. The method of claim 26, wherein the crop disease is selected from the group consisting of black leaf spot, gray mold, fire burn, citrus scab, soft rot, olive scab, tomato bacterial spot, bacterial scab or rice blast (stone fruit and pome fruit), cucurbit angular spot, peach bacterial spot, tomato bacterial spot, walnut black rot, bacterial wilt, tomato scab, potato late blight, apple scab, bacterial leaf blight, and bacterial leaf streak. The method of claim 26, wherein the pathogenic microorganism is selected from the group consisting of: banana black leaf spot fungus , gray mold, Erwinia starch ( Ea ) (especially a streptomycin-resistant strain of Erwinia starch) , Xanthomonas amylovora var. citri ( Xac ) , potato pectin rod, black rot pectin rod, carrot pectin rod brasiliensis, carrot pectin rod carrot, Erwinia chrysanthemum, Pseudomonas sarcoma var. sarcoma ( Psv ), Pseudomonas syringae var. tomato, Pseudomonas syringae var. syringae, Pseudomonas syringae var. cucumber, Xanthomonas campestris var. plum, Xanthomonas campestris var. scabra, Xanthomonas arborescens var. juglans regia, Ralstonia solanacearum, Cladosporium michiganense, Phytophthora spp., Venturia spp., Xanthomonas oryzae var. oryzae var . oryzae, and Xanthomonas citri var . The method of claim 26, wherein the pathogenic microorganism is pathogenic Erwinia amylovora, and the pathogenic Erwinia amylovora is streptomycin-resistant Erwinia amylovora. The method of claim 26, wherein the crop is selected from the group consisting of bananas, apples, pears, crabapples, citrus, potatoes, tomatoes, eggplants, leafy greens, pumpkins and melons, peppers and green peppers, olives, stone fruits and pome fruits, including olives, peaches, and walnuts. The method of claim 26, wherein the pathogenic microorganism is selected from Flavobacterium columnaris #2 and Flavobacterium columnaris MS-FC-4. The method of claim 26, wherein the pathogenic microorganism is Escherichia coli O157:H7. The method of claim 26, comprising applying the agricultural composition comprising between about 1.0 x 10 ⁵ and 1.0 x 10 ⁹ cfu of Pseudomonas bacteria per mL to a crop to inhibit the growth of pathogenic microorganisms. The method of claim 33, wherein the agricultural composition comprises between about 5.0 x 10 ⁷ and 2.0 x 10 ⁸ cfu of Pseudomonas bacteria per mL. A crystalline compound selected from one of the following structures: Formula (I), Formula (II), and Formula (III).