BACTERIAL COMPOSITIONS AND METHODS FOR TOMATO PROTECTION AGAINST CLAVIBACTER MICHIGANENSIS SUBSP. Michigan (CMM)

Description

1 / 56 Bacterial compositions and methods for protecting tomatoes against Clavibacter michiganensis subsp. michiganensis (CMM) CROSS-REFERENCE ON RELATED REQUEST

[001] None FIELD OF THE INVENTION

[002] The present invention relates to the field of agriculture, and more particularly to the prevention and control of bacterial canker (Clavibacter michiganensis pv. michiganensis) in tomatoes. BACKGROUND OF THE INVENTION

[003] Bacterial canker is the most destructive of the bacterial diseases affecting tomatoes. It is caused by the bacterium Clavibacter michiganensis subsp. michiganensis (Cmm). This microorganism is introduced into crops mainly through infected seeds or transplants and is capable of spreading rapidly, resulting in devastating losses. This disease is particularly difficult to control, not only because there is no cure, but also because Cmm can be difficult to eradicate once introduced into a greenhouse, garden, or field. Therefore, there is a significant need to reduce the economic and environmental costs associated with Cmm infections.

[004] The international PCT publication WO 2020 / 069438 describes the use of a bacterial culture containing Bacillus pumilus to protect tomatoes against Cmm. However, there is still a need for improved methods and compositions to control, suppress and / or prevent Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato plants.

[005] In particular, there is a need for methods in which the application of an effective amount of bacterial compositions containing Bacillus subtilis and Bacillus pumilus provides measurable benefits in the field. There is also a need for methods in which the Petition 870250086123, dated 09 / 23 / 2025, p. 15 / 87 2 / 56 effective bacterial compositions come into contact with the roots of a tomato plant before planting the plant in the soil.

[006] There is also a need for greater yield predictability and a reduction in the economic and environmental costs associated with the intensive use of currently available canker control agents. Therefore, it would be desirable to provide bacterial compositions and methods for protecting tomato plants against Cmm that can be combined with existing herbicides, insecticides, fungicides, bactericides and nutrients, including, but not limited to, copper-containing compounds.

[007] The present invention meets these and other needs, as will become evident from the review of the disclosure and description of the invention's features that follows. BRIEF SUMMARY OF THE INVENTION

[008] According to one aspect, the invention relates to a bacterial composition comprising an aqueous mixture of live Bacillus subtilis and Bacillus pumilus, wherein the composition is effective in controlling, suppressing and / or preventing an infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato plants.

[009] According to another aspect, the invention relates to a bacterial composition for use in the control, suppression and / or prevention of an infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato, wherein said composition comprises an aqueous mixture of live Bacillus subtilis and Bacillus pumilus.

[0010] According to another aspect, the invention relates to a method for controlling, suppressing and / or preventing an infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato, comprising: - the supply of a bacterial composition comprising at least one of a mixture of Bacillus subtilis and Baci- Petition 870250086123, dated 09 / 23 / 2025, p. 16 / 87 3 / 56 Hus pumilus, a culture medium previously inoculated with Bacillus subtilis and Bacillus pumilus, a cell-free extract of Bacillus subtilis and Bacillus pumilus and / or metabolites produced by Bacillus subtilis and Bacillus pumilus; and - the application of an effective quantity of said bacterial composition to at least one of the following: a tomato plant, a tomato root, a tomato leaf, a tomato seed, a tomato stem, and a tomato fruit.

[0011] In some forms, the method also includes the application of at least one of a herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

[0012] According to another aspect, the invention relates to a method for controlling, suppressing and / or preventing an infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato, comprising: - the supply of a bacterial composition comprising at least one of a mixture of Bacillus subtilis and Bacillus pumilus, a culture medium previously inoculated with Bacillus subtilis and Bacillus pumilus, a cell-free extract of Bacillus subtilis and Bacillus pumilus and / or metabolites produced by Bacillus subtilis and Bacillus pumilus; - the supply of at least one copper fungicide selected from the group consisting of copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate and mixtures thereof; and - the application of an effective quantity of said bacterial composition and said copper fungicide to at least one of the following: a tomato plant, a tomato root, a tomato leaf, a tomato seed, a tomato stem, and a tomato fruit. Petition 870250086123, dated 09 / 23 / 2025, page 17 / 87 4 / 56

[0013] According to another aspect, the invention relates to a kit for protecting tomatoes against Clavibacter michiganensis subsp. michiganensis (Cmm), comprising: (i) a first container comprising a bacterial composition as defined in this document; and (ii) a second container comprising at least one of a herbicide, an insecticide, a fungicide, a bactericide and a nutrient.

[0014] According to another aspect, the invention relates to the use of a bacterial composition as defined in this document to control, suppress and / or prevent infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato plants.

[0015] According to another aspect, the invention relates to the combined use of: (i) a bacterial composition comprising a mixture of live Bacillus subtilis and Bacillus pumilus; and (ii) at least one of a herbicide, an insecticide, a fungicide, a bactericide and a nutrient, to control, suppress and / or prevent infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato plants.

[0016] Aspects, advantages and additional features of the present invention will become more apparent after reading the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention. DETAILED DESCRIPTION OF THE MODALITIES

[0017] The following description provides examples of how the invention can be put into practice. It is understood that other examples of how the invention can be carried out without departing from the scope of the invention described. Unless otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by a person skilled in the art to which the invention pertains. Petition 870250086123, dated 09 / 23 / 2025, p. 18 / 87 5 / 56 Overview

[0018] The invention aims to meet an important need in the prevention and control of bacterial canker in tomato plants.

[0019] When used in this document, Clavibacter michiganensis subsp. Michiganensis and Cmm) are used interchangeably to refer to the Gram-positive, aerobic, plant pathogenic bacterium that causes wilt and canker in tomato plants.

[0020] As described below, the present invention proposes new compositions and methods for controlling, suppressing and / or preventing Cmm infections in tomato plants. Bacterial compositions

[0021] One aspect of the invention relates to bacterial compositions effective against Cmm. The present invention is particularly based on the combined use of Bacillus subtilis and Bacillus pumilus. The compositions according to the present invention preferably comprise a mixture of live Bacillus subtilis and Bacillus pumilus. In certain embodiments, the invention also encompasses any culture medium previously inoculated with Bacillus subtilis and Bacillus pumilus, cell-free extracts of Bacillus subtilis and Bacillus pumilus, and / or metabolites produced by Bacillus subtilis and Bacillus pumilus.

[0022] When used in this document, the reference to a “culture medium previously inoculated with Bacillus subtilis and Bacillus pumilus” means a medium previously inoculated with both bacteria, together or separately. Therefore, this term covers mixtures of two or more pooled culture media, previously inoculated with one bacterium and / or both bacteria.

[0023] When used in this document, the reference to a “cell-free extract of Bacillus subtilis and Bacillus pumilus” refers to a previously inoculated culture medium, as defined above, from which the bacteria have been removed (e.g., by filtration, centrifugation) Petition 870250086123, dated 09 / 23 / 2025, p. 19 / 87 6 / 56 gação, etc.). In some embodiments, the cell-free extract is a filtered fraction and / or a cell-free supernatant obtained from the culture of Bacillus subtilis and / or Bacillus pumilus.

[0024] When used in this document, reference to metabolites produced by Bacillus subtilis and Bacillus pumilus refers to a mixture comprising peptides, proteins, small molecules, lipids, etc., that have been produced by bacteria (synthesis, expression, extraction, secretion, etc.), separately or in combination.

[0025] In some embodiments, Bacillus pumilus consists of the Bacillus pumilus strain deposited in the American Type Culture Collection (ATCC) on September 26, 2018, and with deposit number PTA-125304. In some embodiments, Bacillus pumilus consists of the NES-CAP-1 strain (GenBank Accession Number MF079281.1).

[0026] In some embodiments, Bacillus subtilis consists of the Bacillus subtilis strain deposited at ATCC® on September 26, 2018, and with deposit number PTA 125303.

[0027] In some embodiments, Bacillus subtilis consists of the Bacillus subtilis strain deposited at ATCC® on September 26, 2018, and with deposit number PTA 125302.

[0028] In some embodiments, the composition comprises live Bacillus subtilis and live Bacillus pumilus in a 1:1 ratio. In other embodiments, this ratio is 2:1. In other embodiments, this ratio is 1:2.

[0029] In some embodiments, the composition comprises between 1 x 109 and 1 x 106 Bacillus pumilus bacteria / ml. In some embodiments, the composition comprises at least 1 x 109 Bacillus pumilus bacteria / ml, or at least 1 x 108 Bacillus pumilus bacteria / ml, or at least 1 x 107 Bacillus pumilus bacteria / ml, or at least 1 x 106 Bacillus pumilus bacteria / ml.

[0030] In some modalities, the composition comprises between Petition 870250086123, dated 09 / 23 / 2025, p. 20 / 87 7 / 56 x 109e 1 x 106 Bacillus subtilis bacteria / ml. In some embodiments, the composition comprises at least 1 x 109 Bacillus subtilis bacteria / ml, or at least 1 x 108 Bacillus subtilis bacteria / ml, or at least 1 x 107 Bacillus subtilis bacteria / ml, or at least 1 x 106 Bacillus subtilis bacteria / ml.

[0031] It is also possible to include additional bacteria, culture media, cell-free extracts and / or metabolites in the composition of the invention. Further examples of potentially useful microorganisms include, but are not limited to, Lactobacillus spp. (e.g., Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus plantarum, etc.), Lactococcus spp. (e.g., Lactococcus lactis), Bacillus spp. (e.g., Bacillus amyloliquefaciens), Aspergillus spp. (e.g., Aspergillus oryzae), Candida spp. (e.g., Candida utilis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa, Pseudomonas fluorescens), Saccharomyces spp. (e.g., Saccharomyces cerevisiae), Streptococcus spp. (e.g., Streptococcus lactis), Rhodopseudomonas spp. (e.g., Rhodopseudomonas palustris) and combinations comprising one or more of the above.

[0032] The bacterial composition of the invention may further comprise a herbicide, an insecticide, a fungicide, a bactericide, a nutrient and a mixture thereof.

[0033] Examples of potentially useful herbicides include, but are not limited to, Roundup™, atrazine, 2,4-D (Weed-B-Gon™), dicamba (Banvel™), imazethapyr (Pursuit™), metolachlor (Dual™), S-metolachlor (Dual II Magnum™), pendimethalin (Prowl™), clethodim (Select™), triclopyr (Garlon™), clopyralid (Stinger™), Fluro Star™, flufosinate (Liberty™), halosulfurone (Permit™), isoxaben (Gallery™), sethoxydim (Poast™), bentazone (Basagran™), pyraflufen-ethyl (Epic™), fomesafen (Reflex™), trifluralin (Treflan™), mesotrione (Callisto™), flumioxazine Petition 870250086123, dated 09 / 23 / 2025, page 21 / 87 8 / 56 (Valor), dicamba (Banvel™, Clarity™), and imazapir (Arsenal™).

[0034] Examples of potentially useful insecticides include, but are not limited to, pyrethroids, pyrethrins, neonicotinoids, organophosphates, carbamates, spinosad, imidacloprid, fipronil, malathion, permethrin, cypermethrin, and chlorpyrifos. Examples of potentially useful bactericides include, but are not limited to, copper sulfate, zinc sulfate, peracetic acid, quaternary ammonium compounds, streptomycin, kasugamycin, tetracycline hydrochloride, phyton 27™, aureobasidium pullulans, Actigard™, and Agri-strep™. Examples of potentially useful nutrients include, but are not limited to, nitrogen, phosphorus, potassium, sulfur, magnesium, calcium, and other plant micronutrients.

[0035] Examples of potentially useful fungicides include, among others, copper-based fungicides, which include copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate, and mixtures thereof. Existing commercial examples include, but are not limited to, Kocide 3000™, Kocide 2000™, Cuprofix Ultra 40 Disperse™, Nordox 75 WG™, Champ WG™, Cueva™, Badge SC™, Basic Copper 53™, BOonide™ copper fungicide, Camelot O™, Nu-Cop 3L™, COCS WDG™, Previsto™, Badge X2™, Badge MAXX™, Cuproxat™ fluid copper fungicide, tribasic copper sulfate, Nordox 30 WG™, CopperCount-N™ fungicide, Cuprablau Z 35 WP™, and Captain Jack's™ liquid copper fungicide.

[0036] In some embodiments, the bacterial composition is formulated for application to one or more of the following: a tomato plant, a tomato root, a tomato leaf, a tomato seed, a tomato stem, and a tomato fruit.

[0037] As described in more detail below, the bacterial compositions according to the present invention can be applied using any suitable method or technique, including, but not limited to Petition 870250086123, dated 09 / 23 / 2025, page 22 / 87 9 / 56 limited to, seed coating, application to seeds before and / or during planting, root inoculation, soil soaking, soil application (e.g., injection), foliar spraying (e.g., high or low pressure spraying), etc. In some embodiments, the application comprises dipping the roots of a tomato plant into the bacterial composition before planting it in the soil.

[0038] In a preferred embodiment, the application comprises soil irrigation. When used in this document, the term soil irrigation refers to the process of adding compounds or compositions according to the present invention directly to the base of a plant, preferably to provide deep and targeted treatment.

[0039] When used in this document, the term “tomato plant” refers to any of the more than 10,000 varieties of tomato available in the world. The present composition may exhibit useful anticancer activity against most, if not all, varieties of tomato affected by Cmm, including varieties grown in greenhouses and in the field. The most common tomato cultivars include, among others, Harris Morgan 1823 (HM 1823), Beefsteak Tomato, Cherokee Purple Tomato, Celebrity Tomato, Brandywine Tomato, San Marzano Tomato, Early Girl Tomato, Black Krim Tomato, Sungold Tomato, Roma Tomato, Better Boy Tomato, Rutgers Tomato, Mortgage Lifter Tomato, Big Beef Tomato, Big Boy Tomato, Black Cherry Tomato, Pineapple Tomato, Amish Paste Tomato, Juliet Tomato, Black Beauty Tomato, Green Zebra Tomato.

[0040] Preferably, the bacterial composition and / or method according to the present invention provides at least one of the following benefits: i. increased tomato productivity (e.g., higher productivity for extra-large tomatoes; Petition 870250086123, dated 09 / 23 / 2025, page 23 / 87 10 / 56 ii. increase in the size of the tomatoes; iii. increase in gross tomato yield per acre; iv. bioprotection against Cmm; v. increased resistance against Cmm; vi. reduction of damage caused by Cmm; vii. reduction in the severity of canker and plant tissue affected by canker lesions; viii. reduction of pathological symptoms or lesions resulting from the action of Cmm; and ix. enhancement of the plant's antimicrobial response against Cmm; and x. increased productivity of extra-large tomatoes.

[0041] In some embodiments, the benefit(s) is / are observed or determined by comparing tomatoes in contact with or not in contact with the bacterial composition of the invention. In some embodiments, the benefit(s) is / are observed by comparing tomatoes in contact with the bacterial composition versus tomatoes in contact with a reference treatment. In some embodiments, the reference treatment consists of Kocide™ applied as a foliar spray.

[0042] The bacterial compositions according to the present invention may also find additional useful preventive and / or curative applications for various agricultural crops (e.g., cereals), fruits (e.g., small fruits, citrus), vegetables, grass, peat, ornamental plants, etc.

[0043] The bacterial compositions of the invention may also be useful, for example, against one or more of the following pathogens: Verticillium dahlia, Sclerotinia sclerotiorum, Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp. fragariae, Fusarium spp., Rhizoctonia solani, Phytophthora infestans, Pythium spp., Botrytis cinerea, and Alternaria solani.

[0044] The bacterial compositions of the invention may also Petition 870250086123, dated 09 / 23 / 2025, page 24 / 87 11 / 56 are useful for treating bacterial, fungal, and oomycete pathogens (late blight, mold, rust, scab). They may also be potentially useful for controlling nematodes.

[0045] It may also be possible to market the bacterial compositions of the invention as a kit for protecting tomato plants against Cmm. For example, a kit according to the present invention may comprise: (i) a first container containing a bacterial composition as defined herein; and (ii) a second container containing at least one of a herbicide, an insecticide, a fungicide, a bactericide, and a nutrient. In this kit, the contents of the first and / or second container may be a ready-to-use solution or a concentrated solution to be mixed and / or diluted before use. Protection methods

[0046] Another aspect of the invention relates to methods for protecting tomato plants against Clavibacter michiganensis subsp. michiganensis (Cmm). In one embodiment, the method comprises applying an effective amount of a bacterial composition, as defined herein, to a tomato plant, a tomato root, a tomato leaf, a tomato seed, and / or a tomato stem. The present invention covers the protection of tomatoes in a variety of plant environments, such as fields, greenhouses, vertical gardens, urban afforestation systems, and hydroponic systems.

[0047] In some modalities, the method provides one or more of the benefits listed above for the bacterial composition.

[0048] The bacterial compositions according to the present invention can be applied using any suitable method, including, but not limited to, seed coating, application to seeds before and / or during planting, root inoculation, etc. Petition 870250086123, dated 09 / 23 / 2025, page 25 / 87 12 / 56 Soil dredging, soil application (e.g., injection), foliar spraying (e.g., high or low pressure spraying), etc.

[0049] In some embodiments, the application involves immersing the roots of a tomato plant in the bacterial composition before planting it in the soil.

[0050] In a preferred embodiment, the application comprises soil irrigation. When used in this document, the term soil irrigation refers to the process of adding compounds or compositions according to the present invention directly to the base of a plant, preferably to provide deep and targeted treatment.

[0051] In some forms, the method also includes the application of at least one of the following: herbicide, insecticide, fungicide, bactericide and nutrient.

[0052] In specific embodiments, the method further includes the application of at least one copper fungicide. In some embodiments, the copper fungicide comprises a compound of copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate, and mixtures thereof. In specific embodiments, the copper fungicide is selected from existing commercial products, including, but not limited to, Kocide 3000™, Kocide 2000™, Kocide LF™, Kocide DF™, Kocide 101™, Cuprofix™ Ultra 40 Disperss™, Nordox™, Nordox 75 WG™, Champ WG™, Cueva™, Badge SC™, Basic Copper 53™, Bonide™, Camelot O™, Nu-Cop 3L™, COCS WDG™, Previsto™, Badge X2™, Badge MAXX™, Cuproxat™, Nordox 30 WG™, Copper-Count-N™ fungicide, Cuprablau Z 35 WP™, and Captain liquid copper fungicide. Jack's™, Bordeaux Blend™, Cuprofix Disperss™, Champion™, Nu-Cop™, Copper Count-N™, Mastercop Instill™, Phyton™, and Petition 870250086123, dated 09 / 23 / 2025, page 26 / 87 13 / 56 CS 2005™.

[0053] The bacterial composition can be applied at a specific time, or one or more times, depending on parameters that include, among others, the Cmm population in a tomato plant or soil planted with a tomato plant, environmental conditions, tomato susceptibility, planting time, etc.

[0054] In some embodiments, the bacterial composition is applied: when a plant rooted in it exhibits a pathological symptom associated with Cmm; when a plant currently rooted in it exhibits a pathological symptom associated with Cmm; and / or when a tomato plant to be planted in it is expected to exhibit a pathological symptom associated with Cmm. In some embodiments, the bacterial composition is applied to the seeds that will be planted in such soil. In some embodiments, the bacterial composition is applied to the seeds of a parent tomato plant that was planted in such soil. In some embodiments, the bacterial composition is applied to the plant that is rooted in such soil. In some embodiments, the bacterial composition is applied to a plant that exhibits a pathological symptom associated with Cmm.

[0055] The bacterial composition can be applied before or after Cmm infection. In some embodiments, the composition is applied at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months before planting a seed or plant. In some embodiments, the bacterial composition is applied at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months after planting a seed or plant. In some embodiments, the bacterial composition is applied 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 5 to 10 weeks before harvesting a tomato.

[0056] When treating seeds, the bacteria can be applied by a Petition 870250086123, dated 09 / 23 / 2025, p. 27 / 87 14 / 56 variety of techniques, including, but not limited to, high or low pressure spraying, coating, immersion and injection. Once treated, the seeds can be planted in natural or artificial soil and grown using conventional procedures to produce plants. After plants propagate from seeds treated with the bacterial composition, they can be further treated with one or more applications of the described bacterial composition.

[0057] The described bacterial compositions can be applied to all or part of the tomato plant. For example, a described composition can be applied to stems, roots, leaves and / or propagules (e.g., cuttings). The plant can be treated at one or more developmental stages. In one embodiment, a described composition is applied to the roots.

[0058] In some embodiments, the bacterial composition is mixed or diluted in an agriculturally acceptable carrier before use.

[0059] In some embodiments, the bacterial composition is applied to a delivery vehicle, wherein the delivery vehicle serves as a means of transporting the bacteria from the bacterial composition to the soil, plant, seed, field, etc. For example, the bacterial compositions described may be applied to a delivery vehicle (e.g., a particle, a polymer, or a substrate) for use in filtration systems for irrigation water treatment. In some embodiments, the bacterial composition is to be applied to a polymer as a wetting agent and / or gel that releases water as needed.

[0060] The bacterial composition of the present invention is preferably applied in an amount effective for the bioprotection of a tomato plant against Cmm (e.g., controlling, suppressing and / or preventing a Cmm infection). In some embodiments, the amount Petition 870250086123, dated 09 / 23 / 2025, page 28 / 87 15 / 56 is sufficient to prevent Cmm infection. In some modalities, the amount is sufficient to treat or reduce one or more symptoms associated with Cmm.

[0061] In some embodiments, the quantity is sufficient to reduce the concentration of Cmm in a tomato plant tissue treated with the bacterial composition. In some embodiments, the concentration of Cmm measured in a tomato plant tissue 10 days after the application step is less than 109 CFU / g. In some embodiments, the concentration of Cmm measured in a tomato plant tissue 21 days after the application step is less than 109 CFU / g. In some embodiments, the concentration of Cmm measured in a tomato plant tissue 3, 5, 7, 14, 21, 28, 35 or 42 days after the application step is less than 109 CFU / g. In some embodiments, the concentration of Cmm measured in a tomato plant tissue 3, 5, 7, 14, 21, 28, 35 or 42 days after the application step is less than 108 CFU / g. In some modalities, the concentration of Cmm measured in a tomato plant tissue 3, 5, 7, 14, 21, 28, 35 or 42 days after the application step is less than 107 CFU / g.In some embodiments, the concentration of Cmm measured in a tomato plant tissue 3, 5, 7, 14, 21, 28, 35 or 42 days after the application step is less than 10⁶ CFU / g. In some embodiments,

[0062] In some embodiments, Cmm infection is measured in a tomato plant tissue by assessing canker severity or incidence on a scale of 0 to 100%, where 0% indicates absence of disease severity and 100% indicates complete disease dominance in the plant. Canker control in the % of the whole plant can be calculated using Abbott's formula for SAUDPC (i.e., standardized area under the disease progress curve). In some embodiments, the bacterial composition of the present invention is applied in Petition 870250086123, dated 09 / 23 / 2025, page 29 / 87 16 / 56 an effective amount to control, suppress and / or prevent canker severity by at least 1%, or at least 2%, or at least 3%, or at least 4%, or at least 5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, or at least 10%, when compared to untreated tomato plants or crops.

[0063] Those skilled in the art will understand that the specific amounts and frequency of application required may vary depending on the types and conditions of the soil, the types and conditions of the tomatoes, the potency and activity of the Cmm, etc. The specific amounts may also vary depending on the environment, for example, whether the plant is in a pot, a greenhouse, a field, etc.

[0064] Specific quantities can be determined using methods known in the art, for example, by testing the dose-dependent response. In some embodiments, the specific quantity is determined by testing the dose-dependent response in a culture plate with Cmm, for example, by measuring a zone of inhibition. In some embodiments, the specific quantity is determined by testing the dose-dependent response in a vessel or in a field. In some embodiments, the specific quantity is determined based on measuring the concentration of Cmm or the quantity of a specific Cmm of a gene in a tissue of a tomato plant treated with the bacterial composition. In some embodiments, the specific quantity is determined based on the concentration of Bacillus pumilus, Bacillus subtilis, or both.

[0065] In some embodiments, the bacterial culture applied to a tomato plant comprises Bacillus pumilus at a concentration between 10⁶ and 10⁹ CFU / mL, or between 10⁷ and 10⁹ CFU / mL, or between 2.5 x 10⁷ and 10⁹ CFU / mL, or between 2.5 x 10⁷ and 8.5 x 10⁸ CFU / mL, or between 5 x 10⁷ and 8.5 x 10⁸ CFU / mL, or between 2 x 10⁸ and 8.5 x 10⁸ CFU / mL, or 10⁸ Petition 870250086123, dated 09 / 23 / 2025, page 30 / 87 17 / 56 CFU / mL. In some embodiments, the bacterial culture applied to a tomato plant comprises Bacillus subtilis at a concentration between 10⁶ and 10⁹ CFU / mL or 10⁷ and 10⁹ CFU / mL, or between 2.5 x 10⁷ and 10⁹ CFU / mL, or between 2.5 x 10⁷ and 8.5 x 10⁸ CFU / mL, or between 5 x 10⁷ and 8.5 x 10⁸ CFU / mL, or between 2 x 10⁸ and 8.5 x 10⁸ CFU / mL, or 10⁸ CFU / mL.

[0066] In some embodiments, the bacterial composition is applied to the tomato plant to produce a final concentration of Bacillus pumilus measured in the root, stem or leaf of the tomato plant to vary between 106 and 109 CFU / mL, or between 107 and 109 CFU / cm3, or between 2.5 x 107 and 109 CFU / cm3, or between 2.5 x 107 and 8.5 x 108 CFU / cm3, or between 5 x 107 and 8.5 x 108 CFU / cm3, or between 2 x 108 and 8.5 x 108 CFU / cm3, or between 3 x 108 and 8 x 108 CFU / cm3, or 108 CFU / cm3.

[0067] In some embodiments, the bacterial composition is applied to the tomato plant to produce a final concentration of Bacillus subtilis measured in the root, stem or leaf of the tomato plant to vary between 106 and 109 CFU / mL, or between 107 and 109 CFU / cm3, or between 2.5 x 107 and 109 CFU / cm3, or between 2.5 x 107 and 8.5 x 108 CFU / cm3, or between 5 x 107 and 8.5 x 108 CFU / cm3, or between 2 x 108 and 8.5 x 108 CFU / cm3, or between 3 x 108 and 8 x 108 CFU / cm3, or 108 CFU / cm3.

[0068] In some embodiments, the bacterial composition is applied to the tomato plant to produce a final concentration of Bacillus pumilus and Bacillus subtilis measured in the root, stem or leaf of the tomato plant to vary between 106 and 109 CFU / mL, or between 107 and 109 CFU / cm3, or between 2.5 x 107 and 109 CFU / cm3, or between 2.5 x 107 and 8.5 x 108 CFU / cm3, or between 5 x 107 and 8.5 x 108 CFU / cm3, or between 2 x 108 and 8.5 x 108 CFU / cm3, or between 3 x 108 and 8 x 108 CFU / cm3, or 108 CFU / cm3.

[0069] The composition can be applied in an amount ranging from 0.2 to 3 gal / A, from 0.5 to 2.5 gal / A, from 0.75 to 2 gal / A, 0.5 gal / A, 1 gal / A, 1.25 gal / A, 1.5 gal / A, or 2 gal / A.

[0070] Those versed in the technique will recognize, or be able to Petition 870250086123, dated 09 / 23 / 2025, p. 31 / 87 18 / 56 verify, using only routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered within the scope of this invention and covered by the appended claims. The invention is further illustrated by the following example, which should not be construed as further or specifically limiting. EXAMPLE 1 - Evaluation of CXC effectiveness

[0071] Field trials were conducted in Florida over a 3-month period (September 2022 to December 2022) to evaluate the efficacy and activity of a composition according to the present invention (hereinafter referred to as CXC™) against bacterial canker of tomato (Clavibacter michiganensis pv. michiganensis) when applied as a root dip and soil irrigation. The activity and efficacy of CXC were compared to Kocide 3000™, a currently available product against bacterial canker of tomato, as well as to an untreated control. Materials and methods CXC Composition

[0072] The bacterial composition tested, (referred to below as “CXC” (or “CXC biocontrol” in Examples 2 and 3), comprised an aqueous mixture of 1) live Bacillus subtilis (ATCC® deposit number PTA-125303) and 2) live Bacillus pumilus (ATCC® deposit number PTA-125304). The two bacterial strains were present in the mixture in a 1:1 ratio, with each strain at a concentration of approximately 10⁷–10⁹ bacteria / ml. To obtain the CXC composition, the two microbes were cultured separately for at least 72 hours at 28 °C in a shaker incubator (125 rpm) in LB broth containing tryptone (10 g), yeast extract (5 g), and NaCl (5 g) in 1 liter of water. The bacterial count was determined by counting CFU per mL using the serial dilution method. Bacterial cultures Petition 870250086123, dated 09 / 23 / 2025, p. 32 / 87 19 / 56 rianas were serially diluted and aliquots were seeded onto LB agar medium, incubated at 28 °C, and CFUs were counted after at least 48 hours of incubation. After final harvest, the cultures of the two microorganisms were mixed (1:1) and kept refrigerated until use. Field conditions

[0073] The test was conducted on soil with an overall pH of 6.6 and an overall Cation Exchange Capacity level of 3.7. The soil composition consisted of 0.5% organic matter, 90.4% sand, 4.4% silt, and 5.2% clay.

[0074] Fresh tomatoes of the Harris Morgan 1823 variety (HM 1823) were transplanted on September 21, 2022, under simulated commercial conditions (48 cm spacing, in 1.8 m wide beds), for a total planting density of 1,280 plants / acre. The beds were 9.7 m wide in a single bed design, replicated four times in a randomized block design and irrigated by drip irrigation.

[0075] Bacterial canker was inoculated with a backpack sprayer (246 spores / ml) two weeks after planting (October 2, 2022).

[0076] CXC treatments were applied once, twice, or three times, as drops manually. Treatments were initially applied at planting (designated treatment A, week 0, September 21, 2022), again 2 weeks later (designated treatment B, week 2, October 6, 2022), and finally again 3 weeks after treatment B (designated treatment C, week 5, November 3, 2022).

[0077] A reference treatment of Kocide 3000™ (referred to herein as “Kocide”) was applied as a foliar spray at different concentrations (see Table 1 below) using a tractor-mounted sprayer weekly from October 5, 2022 (designated DK, weeks 2 to 9). The tractor-mounted sprayer operated at 200 PSI.

[0078] An untreated control was included as a reference. Petition 870250086123, dated 09 / 23 / 2025, page 33 / 87 20 / 56 cia (control). Reviews

[0079] Canker severity was assessed in ten plants per plot on a scale of 0% to 100%, where 100% is a plant with all tissue affected by lesions. Assessments were performed on whole plants, new shoots, and stems of 10 plants. Whole-plant assessments were performed on September 30 (9 DA-A), October 7 (2 DA-D), October 15 (3 DA-B), October 21 (2 DA-G), November 9 (7 DA-H), November 22 (7 DA-J), December 1 (8 DAK), and December 8 (15 DA-K). New plant growth and stem disease assessment were performed on November 3 (1 DAH). Canker control was calculated using the Abbott formula from the Standardized Area Under Disease Progress Curve (SAUDPC) (average severity over time).

[0080] Tomatoes were harvested manually and organized into five categories: Extra Large, Large, Medium, Sunscald, and Other Discarded. Collections were carried out on November 23 (8 DA-J) and November 30 (7 DA-K). Counts and weights for marketable small, medium, large, and extra-large tomatoes, and isolated counts for non-marketable and cancer-infected tomatoes, were recorded. Results

[0081] The results of whole-plant cancer severity (%) are presented in Table 1. Cancer severity was classified on a scale of 0 to 100%, where 0% showed no disease severity and 100% indicated complete disease dominance in the crop. Whole-plant assessments were performed on September 30 (9 DA-A), October 7 (2 DA-D), October 15 (3 DA-B), October 21 (2 DA-G), November 9 (7 DA-H), November 22 (7 DA-J), December 1 (8 DA-K), and December 8 (15 DA-K). Petition 870250086123, dated 09 / 23 / 2025, p. 34 / 87 21 / 56 CM .ο ο ό -Q ro CM i va 03 / 8 _ _ o xP xP o'- 0s o' lo lo co। σ> m OÓ sf CXÍ 'í CXI LO

[0082] The results for whole-plant cancer incidence (% of plants) are presented in Table 2. Whole-Plant Cancer Incidence (%). Cancer incidence was classified on a scale of 0 to 100%. The Petition 870250086123, dated 09 / 23 / 2025, p. 35 / 87 22 / 56 whole-plant assessments were conducted on September 30 (9 DA-A), October 7 (2 DA-D), October 15 (3 DA-B), October 21 (2 DA-G), November 9 (7 DA-H), November 22 (7 DA-J), December 1 (8 DA-K), and 8 Petition 870250086123, dated 09 / 23 / 2025, p. 36 / 87 23 / 56 Petition 870250086123, dated 09 / 23 / 2025, p. 37 / 87 24 / 56 Table 4: Cancer of New Plant Growth (%) Treatment No. Treatment Name Rate Unit Code Canker of new plant growth CXC 100% v / v ABC 9.10% c 35.00% c 71.01% a Kocide3000 1.5 Ib / a DEFGHIJK 20.80% b 72.50% b 32.61% b Witness without application 31.90% to 87.50% to 0.00% c Petition 870250086123, dated 09 / 23 / 2025, page 38 / 87 25 / 56

[0086] The count of canker-infected tomatoes per plot is presented in Table 6. Canker-infected tomatoes in the field were counted from 50 tomatoes and the average calculated for the plot on 20th of no- ω lo co CO w CO TQ CO co o LO CO CD _Q O .Qco CO CO 00CO ó o C\J T- CO'st ro n > > oo CM CO LO X LU X LU ó l— JD E φ > oc Φ I CO Φ O 1— OE φ > oc Φ I CO Φ i < Q oo O s— _Q E φ > Petition 870250086123, dated 09 / 23 / 2025, p. 39 / 87 26 / 56 Table 7: Total Count of Extra Large Tomatoes Harvested (# / plot) Treatment No. Treatment Name Fee Unit Application Code 11 / 23 / 2022 11 / 30 / 2022

[0088] Tables 9 and 10 show the count and weight (lbs per bed (#), respectively, of the large tomatoes harvested, organized into five categories: Extra Large, Large, Medium, Sun-Scalded and Other. [Selected Petition 870250086123, dated 23 / 09 / 2025, page 40 / 87] 27 / 56 of the samples were collected on November 23 (8 DA-J) and November 30 (7 DA-K). Table 9: Total Count of Large Tomatoes Harvested (# / plot)

[0089] Tables 11 and 12 show the count (#) and total weight (lbs) of average tomatoes per bed of the harvest. Petition 870250086123, dated 09 / 23 / 2025, page 41 / 87 28 / 56 ro co The Crazy Ο O t- COCO sf st St COsf ro cor ro ns ro CO CD O 05 <ώ <ώ σι id σι CXÍ CXÍ CXÍ CXÍ t-' 1- CM CO 'st LO

[0090] Tables 13 and 14 show the counts per plot of sunburnt tomatoes and other selected types, respectively, harvested on November 23 (8 DA-J) and November 30 (7 DA K). Petition 870250086123, dated 09 / 23 / 2025, page 42 / 87 29 / 56 Table 13: Total Count of Sun-Scalded Tomatoes Harvested (# / bed) Treatment No. Treatment Name Fee Unit Application Code 11 / 23 / 2022 11 / 30 / 2022 CO LO CXI φ TJ ro c ZD ro ro o Έ Φ E ro ro o TJ Φ E ozoc (DE ro ro o TJ ó Z * < Q < Q CO ro ro ro ππ CO q oolo 'st LO lo cd~id ro ro ro roro CD O CO CD00 LO CO 'sf <D’sf o LU ro D LO ooo CO The Φ Ç CM WITH LO

[0091] Table 15 presents the total containment of tomatoes picked by quarry, by category, in both code 870250086123, dated 9 / 23 / 2025, p. 43 / 87 30 / 56 I THE Ό Φ Φ ω ω ο L— Ο ω ο Ό CÜ C LO σΓ 05 with co CXj ο Ο Ό 05 Ε ϕ ο ο ω ο φ ο. 05 05 0505 THE CD WITH NECK with t- τ- οT- with_ CM 0505 The Crazy with the crazy CXI CXJ Ο Ε φ σ: 05 ο −φ 05 05 0505 cd lo lo lolo CX| T- coT- CXI CXI CXI CXICXI Iheitas, on November 23 (8 CE-W) and November 30 (7 CE-K). Table 15: Total Contagem of Pickled Tomatoes (# / plot). with < the co < ΞE o LU Q co Witness without application 122.0 a Petition 870250086123, dated 09 / 23 / 2025, page 44 / 87 31 / 56 ro ro ro ro σ> sr ο σ> CO τ-_ cd CN cd of b- cd ro ro ro ro ^p ^p ^p ^p 0^ 0^ 0^ 0^ CO b~ O> CD CO CD CO CD b- sf Cd' cd ST ro ro ro ro ^p ^p ^p ^p 0^ 0^ 0^ 0^ CXI CD tCN O- b- O_ CO CO sf sf CXI CXI CXI CXI xc CQ CQ LU < < Q CN CO ST LO CO CD OO ro c φ E ro c Φ E ro ro CN CD ST CO sf co' sr sr ro CD CD_ cd' ro ro CO 05 sr o_ cd' co sr sr ro roro LO 0co CO CD-b~cd' cd'cd' CN CNCN -Q 05 σ>' o CQ < CN CO sr ro 00 CDLO' sr ro LO CDcd' ro 05 CO00' CN -Q ro sr CDθ' LO iro ro the. ro E Φ ω Petition 870250086123, dated 09 / 23 / 2025, p. 45 / 87 32 / 56

[0094] Table 18 presents the estimate of marketable return. The estimated marketable weight of harvested tomatoes (PM) was converted to pounds per acre (lbs / acre). The estimated gross rental return was calculated using a Petition 870250086123, dated 09 / 23 / 2025, p. 46 / 87 33 / 56 Discussion

[0095] The percentage of plants with any symptoms reached 100% (all plants) approximately 4 weeks after infection, with a delay in complete infestation in the Kocide-treated plots. Overall, untreated control plots consistently showed significantly higher mean canker severity than treated plots, and a positive dose response was observed for CXC treatments, with the lowest mean severity in the three-times-treated plots.

[0096] Treatment with the CXC composition, according to a three-application regimen, demonstrated a consistent improvement in controlling cancer severity and progression, as shown in Tables 1 to 3. Treatment with CXC, according to all regimens, demonstrated a net improvement in controlling cancer severity compared not only to no treatment, but also to Kocide 3000™, a commercial product currently widely used for this purpose. Consequently, the composition of the present invention may provide better cancer control and greater predictability of production and reduction of economic and environmental costs associated with the more intensive use of currently available cancer control agents.

[0097] Control of canker in new shoots and stems was greater for tomatoes treated with one and two applications of CXC (Tables 4 and 5).

[0098] On average, there were significantly more tomatoes infected with canker in each harvest interval, and the lowest number of infected tomatoes was collected from plots treated with the high CXC rate (Table 6).

[001] As shown in Tables 7 to 14, three applications of CXC produced a larger harvest of extra-large tomatoes (n° per Petition 870250086123, dated 09 / 23 / 2025, p. 47 / 87 34 / 56 plot and total pounds harvested) than Kocide 3000™ and the untreated control. Furthermore, a single application of CXC produced a larger harvest of large tomatoes (number per plot and total pounds harvested) than Kocide™ or the untreated control. Finally, three applications of CXC produced a larger harvest of medium tomatoes (number per plot and total pounds harvested) than Kocide™.

[002] Marketable tomato yields by count did not differ significantly, and most of the harvest consisted of large or extra-large tomatoes (Tables 15 to 16). However, yield weights were significantly higher for extra-large tomatoes grown in plots treated with three CXC applications (Table 17).

[003] Estimated gross returns were higher for the treatment with three (3) CXC applications (Table 18). Key findings:

[004] Bacterial canker control was dose-dependent for CXC, with significantly greater mean control when treatment was applied three times.

[005] Tomato yields infected with canker were higher in untreated control plots or those treated with Kocide and lower for CXC applied three times, thus demonstrating the effectiveness of CXC treatments.

[006] These results also suggest greater productivity predictability with the use of CXC composition.

[007] These results also demonstrate that the CXC composition is a very interesting alternative to currently available cancer control agents. EXAMPLE 2 - Synergistic efficacy of Kocide™ with the compositions of the invention

[008] Field trials were conducted in Thonotosassa, Fló Petition 870250086123, dated 09 / 23 / 2025, page 48 / 87 35 / 56 rida, during a period of 4 months (May 2023 to August 2023) to evaluate the efficacy and activity of the compositions according to the present invention (hereinafter referred to as CXC ABIO™ and CXC Biocontrol™), alone or in combination with Kocide™, against bacterial canker of tomato. Materials and methods Tested CXC Compositions

[009] The following bacterial CXC compositions were tested: 1) CXC Biocontrol™ and 2) CXC ABIC. The CXC Biocontrol™ composition corresponds to the CXC composition of Example 1, as defined above. The “CXC ABIO” composition is a biostimulant containing Bacillin 20.

[0010] Bacillin 20 used in field assays consists of an aqueous solution of Bacillin 20. Bacillin 20 was purified from the MS20 strain of Bacillus thuringiensis. The process used to isolate Bacillin 20 involved the following steps: 1) production of the microbial culture; 2) centrifugation and membrane filtration; 3) absorption of organic metabolites with butanol; 4) evaporation of butanol and concentration of the extract; 5) fractionation of the organic extract with acetonitrile solvent; 6) purification of Bacillin 20 by HPLC; and 7) mass spectrometry analysis of Bacillin 20 to confirm its identity.

[0011] During step 1), an MS20 starter culture of Bacillus thuringiensis was prepared and large-scale production was carried out in a bioreactor. In step 2), the bacterial mass was removed by centrifugation and membrane filtration to obtain a cell-free supernatant. The butanol extraction of step 3 was performed by vigorously mixing and stirring the cell-free supernatant with 1-butanol and separating the 1-butanol phases. In step 4, the butanol was evaporated and an organic extract was obtained by Petition 870250086123, dated 09 / 23 / 2025, page 49 / 87 36 / 56 Vacuum evaporation of 1-butanol using a rotary evaporator. Then, in step 5, the organic extract was fractionated to remove undesirable materials and debris, retaining only the active fractions of Bacillin 20 for further purification. In step 6, Bacillin 20 was purified by high-performance liquid chromatography (HPLC) using an acetonitrile / water gradient until a target purity of 85 to 95% of Bacillin 20 was achieved. In step 7, mass spectrometry analysis was performed on a purified sample to identify various fractions, using a pure Bacillin 20 as a comparative internal standard.

[0012] Purified Bacillin 20 (i.e., with a purity between 85 and 95%) was diluted in phosphate-buffered saline to obtain a stock solution containing a concentration of 10⁻⁹M Bacillin 20. The Bacillin 20 stock solution was diluted 100 times in water, resulting in a final diluted working solution of 10⁻¹¹M, ready for use, which was used for seed treatment.

[0013] Kocide 3000™ (hereinafter referred to as Kocide) was applied as a foliar spray at a rate of 1.5 lb / a using a tractor-driven sprayer. The tractor-driven sprayer operated at 14.06 kg / cm² (200 psi). When used in combination with CXC BioControl and / or CCX ABIO, Kocide™ was added to different treatment tanks at the desired rate (see, for example, the preparation of treatment 4 and treatment 5, as described below). Field conditions

[0014] The experiment was conducted in soil with an overall pH of 6.4 and an overall Cation Exchange Capacity of 4. The soil composition consisted of 2.1% organic matter, 99.5% sand, virtually no silt, and 0.5% clay. The average air temperature ranged between 22 °C (72 °F) and 30 °C (87 °F), and the measured accumulated precipitation was Petition 870250086123, dated 09 / 23 / 2025, page 50 / 87 37 / 56 of 77.6 cm (30.54 inches).

[0015] Tomatoes of variety 2255 were manually transplanted on May 4th, under simulated commercial conditions (48 cm spacing, in 1.8 m wide beds), to a total planting density of 4,585 plants / acre. CXC Biocontrol was applied as a soil moistening agent on May 5th and then as a soil moistening agent on May 27th and June 23rd.

[0016] CXC ABIO, when used, was applied as a foliar spray with Kocide™ weekly, starting on May 5 and continuing until July 18. Treatment 4 was created by diluting 80 mL of the CXC ABIC stock solution with 8 L of water to create a 10⁻⁹M concentration, and then Kocide™ was added to the tank at a rate of 1.5 lb / A. Treatment 5 was created by taking 80 mL from the tank mixed with treatment 4 at the 10⁻⁹M concentration, prior to the addition of Kocide™. The 80 mL of solution taken from the tank from treatment 4 was then mixed with 8 L of water to create a 10⁻¹¹ solution. After the 10⁻¹¹ solution was created, Kocide™ was added to the tank at a rate of 1.5 lb / A. An untreated control was included as a reference. The plots were 8.7 m long and were replicated four times in a randomized block design. An untreated control was included as a reference (control). Reviews

[0017] The severity of bacterial canker was assessed in whole plants at eleven intervals and in plant canopies twice. In mid-June, in whole plants, the incidence of bacterial canker was 100%.

[0018] Fifty tomato samples were collected three times and the counts of fruits infected with canker were reported.

[0019] The fruits were harvested twice and the counts and pe Petition 870250086123, dated 09 / 23 / 2025, page 51 / 87 38 / 56 of the marketable and non-marketable tomatoes were recorded. Productivity was uniform. Results

[0020] The severity of bacterial canker throughout the plant (0 to 100) is shown in Table 19. Bacterial canker severity was calculated from ten plants and rated on a scale of 0 to 100, where 0 indicates a completely healthy plant and 100 indicates a completely diseased plant. Assessments were carried out on May 13 (8 DA-A), May 20 (3 DA-E), May 29 (2 DA-B), June 8 (7 DA-G), June 16 (15 DA-G), June 24 (1 DA-C), July 3 (10 DA-C), July 11 (6 DA-J), July 26 (8 DA-K), August 10 (14 DA-K), and August 9 (22 DA-K). Petition 870250086123, dated 09 / 23 / 2025, p. 52 / 87 39 / 56 Table 19: Severity of bacterial canker throughout the plant (0 to 100) 6 / 24 / 2023 1 DA-C 20.6 b 10.9 c 7.8 d 7.0 d 8.1 d 29.8 to 6 / 16 / 2023 15 DA-G 27.3 to 31.6 to 30.4 to 31.6 to 32.3 to 33.9 to 6 / 8 / 2023 7 DA-G 9.1 b 7.5 b 6.7 b 7.6 b 7.1 b 15.5 a 5 / 29 / 2023 2 DA-B 6.0 b 3.9 b 3.5 b 3.5 b 4.0 b 13.1 a 5 / 20 / 2023 3 DA-E 2.9 b 0.6 b 0.7 b 1.0 b 3.6 b 8.7 to 5 / 13 / 2023 8 DA-A 0.0 a 0.0 a 0.0 a 0.0 a 0.4 a 0.0 a Application Code Immersion / Irrigation Spraying Immersion / Irrigation Spraying Removed Spraying Tank Mixing Spraying Rate Unit % v / v lb / a % v / v lb / a % v / v lb / a % v / v lb / a Rate 100 1.5 s « s « s « Treatment Name CXC Biocontrol Kocide 3000 CXC Biocontrol Kocide 3000 CXC ABIC Kocide 3000 CXC ABIC Kocide 3000 Control without treatment Treatment No. C\| CO LO CO 9 / 8 / 2023 22 DA-k 20.6 bc 15.3 c 13.5 c 23.6 b 23.4 b 47.0 to 1 / 8 / 2023 14 DA-k 17.9 b 11.9c 8.1 c 20.0 b 20.3 b 42.5 to 7 / 26 / 2023 8 DA-K _Q CO CO 8.6 c 5.6 d 15.8 b 16.8 b 26.4 a 7 / 11 / 2023 6 DA-J 24.9 b 15.9 c 14.0 c 9.3 d 9.4 d 35.0 a 7 / 3 / 2023 10 DA-C 21.6 b 15.8 c 13.3 c 12.4c 12.3c 30.9 Application Code Immersion / Irrigation Spraying Immersion / Irrigation Spraying Removed Spraying Tank Mixing Spraying Rate Unit % v / v lb / a % v / v lb / a % v / v lb / a % v / v lb / a Rate 100 1.5 s « s « s « Treatment Name CXC Biocontrol Kocide 3000 CXC Biocontrol Kocide 3000 CXC ABIC Kocide 3000 CXC ABIC Kocide 3000 Control without treatment Treatment No. C\| CO LO CO Petition 870250086123, dated 09 / 23 / 2025, p. 53 / 87 40 / 56

[0021] Table 20 shows the incidence of bacterial canker throughout the plant, in percentage. The severity of bacterial canker was averaged from ten plants and classified on a scale of 0 to 100, where 0 in(Λ CO CO N φ φ φ M—* C φ ο φ φ C φ Ε co Η—» φ ο. Ξ ο Ç0 co ο. co E φ φ Ç0 Φ CO ω φ M—' C φ E co Μ—* Φ ο. Ε ο ω çq c co Cl co Ε φ LU I Q co ο 'co Ε φ φ ο CM co Μ-> C co Q. (0 (0 φ ο 4-< Ε φ ​​ο C Ç0 '1— Φ ο (0 42 ο ίο Q ο 'co Ε φ φ co E φ CM CM φ) co φ φ co ο C <φ φ õ cÒ CM (0 φ (0 6 / 24 / 2023 1 DA-C 100.0% to 100.0% to 92.5% ab 90.0% b 100.0% to 100.0% to 6 / 16 / 2023 15 DA-G 100.0% to 100.0% to 100.0% to 100.0% to 100.0% to 100.0% on 8 / 6 / 2023 7 DA-G 92.5% to 100.0% to 95.0% to 97.5% a5 97.5% to 100.0% on 5 / 29 / 2023 2 DA-B 77.5% to 57.5% to 37.5% to 57.5% to 62.5% at 95.0% 5 / 20 / 2023 3 DA-E 50.0% ab 17.5% b 15.0% b 25.0% b 40.0% b 87.5 a 5 / 13 / 2023 8 DA-A 0.0 a 0.0 a 0.0 a 0.0 a 5.0 a 0.0 a Application Code Immersion / Irrigation Spraying Immersion / Irrigation Spraying Removed Spraying Tank Mixing Spraying Rate Unit % v / v lb / a % v / v lb / a % v / v lb / a % v / v lb / a Rate 100 1.5 s « s « s « Treatment Name CXC Biocontrol Kocide 3000 CXC Biocontrol Kocide 3000 CXC ABIO Kocide 3000 CXC ABIO Kocide 3000 Witness without treatment No. of Treatment C | CO LO CO Petition 870250086123, dated 09 / 23 / 2025, p. 54 / 87 41 / 56 9 / 8 / 2023 22 DA-k 100.0% to 100.0% to 100.0% to 100.0% to 100.0% to 100.0% to 1 / 8 / 2023 14 DA-k 100.0% to 100.0% to 100.0% to 100.0% to 100.0% at 100.0% on 7 / 26 / 2023 8 DA-K 100.0% to 100.0% at 90.0% to 97.5% 100.0% a 100.0% on 7 / 3 / 2023 10 DA-C 100.0% to 100.0% to 100.0% to 100.0% to 100.0% to 100.0% to Application Code Immersion / Irrigation Spraying Immersion / Irrigation Spraying Removed Spraying Tank Mix Spraying Rate Unit % v / v lb / a % v / v lb / a % v / v lb / a % v / v lb / a Rate 100 1.5 s « s « s « Treatment Name CXC Biocontrol Kocide 3000 CXC Biocontrol Kocide 3000 CXC ABIC Kocide 3000 CXC ABIC Kocide 3000 Control without treatment Treatment No. C\| CO LO CO Application Code Immersion / Irrigation Spraying Immersion / Irrigation Spraying Removed Spraying ω χ ω Φ σ σ 'c Ζ) ο Έ φ Ε ω E ο σ ό ζ τ- c\i co CXCABIO 100% v / v Tank mixing 12.29 c 52.55 b Kocide 3000 1.5 lb / a Spray Witness without treatment 25.97 to 0.00% d Petition 870250086123, dated 09 / 23 / 2025, p. 55 / 87 42 / 56

[0023] Table 22 shows the severity of bacterial canker in the upper part of the plant. The severity of bacterial canker was calculated from ten plants and classified on a scale of 0 to 100, where 0 indicates one plant. Completely healthy and 100 indicates a completely diseased plant. Assessments were carried out on June 16 (15 DA-G) and July 19 (75 DA-A). Table 22: Severity of bacterial canker in the upper part of the plant (0 to 100) 7 / 19 / 2023 75 DA-A 3.5 c 3.8 c 4.3 bc o 'd- 6.9 b 13.3 a 6 / 16 / 2023 15 DA-G 11.4 b 7.8 bc 4.5 c 7.1 bc 5.4 c 27.0 a Application Code Immersion / irrigation Spraying Immersion / irrigation Spraying Removed Spraying Tank Mix Spraying Rate Unit % v / v lb / a % v / v lb / a % v / v lb / a % v / v lb / a Rate 100 1.5 s « s « s « Treatment Name CXC Biocontrol Kocide 3000 CXC Biocontrol Kocide 3000 CXC ABIC Kocide 3000 CXC ABIC Kocide 3000 Witness without treatment Treatment No. C | CO LO CO Petition 870250086123, dated 09 / 23 / 2025, pp. 56 / 87 43 / 56

[0024] Table 23 shows the percentage incidence of bacterial canker in the upper part of the plant. The incidence of bacterial canker was averaged from ten plants and classified on a scale of 0 to 100, where 0 j completely healthy and 100 indicates a completely diseased plant. Assessments were carried out on June (15 DA-G) and July 19 (75 DA-A), incidence of bacterial canker in the upper part of the plant (%) 7 / 19 / 2023 75 DA-A 57.5% c 62.5% bc 60.0% c 67.5% bc 85.0% ab 100.0% a

[0025] Table 24 shows the incidence of bacterial canker in the upper part of the plant SAUDPC and in percentage in relation to the control group. 6 / 16 / 2023 15 DA-G 95.0% to 87.5% to 80.0% to 90.0% to 90.0% to 100.0% to Application Code Immersion / Irrigation Spraying Immersion / Irrigation Spraying Removed Spraying Tank Mix Spraying Rate Unit % v / v lb / a % v / v lb / a % v / v lb / a % v / v lb / a Rate 100 1.5 s « s « s « Treatment Name CXC Biocontrol Kocide 3000 CXC Biocontrol Kocide 3000 CXC ABIC Kocide 3000 CXC ABIC Kocide 3000 Control without treatment indicates a plantlet located on 16 of Table 23: Incidence No. of Treatment C\| CO LO CO Petition 870250086123, dated 09 / 23 / 2025, p. 57 / 87 44 / 56 Table 24: Incidence of bacterial canker in the upper part of the plant (SAUDPC and % of control) Φ Ό CO O 'c Z) O Ό Φ And ozone Έ ϕ And 05 And o T5 Ó z X Φ ϕ They ro CQ The OX EEE 05 Φ 05 Φ 05 Φ 05 CQ The OX CQ ooo CO Φ Ό T- CM CO LO CO ω or _£Z — to CD -g Φ THE Φ WO Total 23.8 c 15.8 d 13.0 e 33.3 b 34.5 b 47.0 a 8 / 3 / 2023 16 DA-K 12.0 c 9.3 d 6.5 e 16.8 b 17.8 b 24.0 a 7 / 25 / 2023 7 DA-K 8.0 c 5.5 d 3.0 e 12.5 b 13.3 b 18.0 a 7 / 17 / 2023 12 DA-J 3.8 ab 1.0 c 3.5 b 4.0 ab 3.5 b 5.0 a Application Code Immersion / Irrigation Spraying Immersion / Irrigation Spraying Removed Spraying Tank Mix Spraying Rate Unit % v / v lb / a % v / v lb / a % v / v Ib / a % v / v Ib / a Rate 100 1.5 s « s « s « Treatment Name CXC Biocontrol Kocide 3000 CXC Biocontrol Kocide 3000 CXC ABIO Kocide 3000 CXC ABIO Kocide 3000 Control without treatment No. Treatment C\| CO LO CO Petition 870250086123, dated 09 / 23 / 2025, p. 58 / 87 45 / 56

[0027] Table 26 shows the percentage of fruit infected with canker. Fruit infected with bacterial canker was counted before harvest. Counts were performed on July 17 (12 DA-J), July 25 (7 DAI < Q to o ω oo cc Φ I CO Φ Table 26: Fruits infected with canker (%) Total 0s· 00 Lí) T- 10.5% d 8.7% eq 23.0% b 31.3% a 8 / 3 / 2023 16 DA-K 24.0% c 18.5% d 13.0% e 33.5% b 35.5% b 48.0% a 7 / 25 / 2023 7 DA-K 16.0% c 11.0% d 6.0% e 25.0% b 26.5% b 36.0% a 7 / 17 / 2023 12 DA-J 7.5% ab 2.0% c 7.0% b 8.0% ab 7.0% b 10.0% a Application Code Immersion / irrigation Spraying Immersion / irrigation Spraying Removed Spraying Tank Mixing Spraying Rate Unit % v / v lb / a % v / v lb / a % v / v lb / a % v / v lb / a Rate 100 1.5 s « s « s « Treatment Name CXC Biocontrol Kocide 3000 CXC Biocontrol Kocide 3000 CXC ABIC Kocide 3000 CXC ABIC Kocide 3000 Control without treatment No. Treatment C\| CO Lí) CO Petition 870250086123, dated 09 / 23 / 2025, page 59 / 87 46 / 56 Discussion

[0028] Control plants without treatment showed significantly higher average pest severity (Table 19). Untreated plots showed significantly more canker-infected fruits, and plants treated with CXC Biocontrol™ and Kocide™ showed the lowest number (Tables 25 and 26).

[0029] The combination of Kocide™ and CXC Biocontrol™ resulted in significantly better cancer suppression compared to either treatment alone (Tables 25 and 26).

[0030] These results show that combined treatment with CXC Biocontrol™ and Kocide achieves better results than treatment with CXC Biocontrol™ or Kocide alone™, indicating a synergistic effect of the treatments.

[0031] In the plant canopies, the incidence of canker was lower with the CXC biocontrol (Treatment 1). Treatment suspension (Treatment 4) was the second most effective in reducing canker incidence, followed by Treatment 5 (tank mixture alone) (Table 23).

[0032] Overall, these results clearly demonstrate that the compositions according to the present invention are effective and can successfully improve and / or complement current means of cancer control. Therefore, the compositions and methods according to the present invention can also be useful in reducing the economic and environmental costs associated with the intensive use of commercially popular cancer control agents. EXAMPLE 3 - Beneficial effects of CXC Biocontrol™ applied as a dip bath and foliar spray, when used alone or in combination with Kocide™

[0033] Field trials were conducted in Thonotosassa, Florida, over a period of 4 months (October 2023 to February 2024). Petition 870250086123, dated 09 / 23 / 2025, pp. 60 / 87 47 / 56 2024) to evaluate the efficacy and activity of the compositions according to the present invention (hereinafter referred to as CXC Biocontrol™), when applied as a dip bath and / or foliar spray, alone or in combination with Kocide™, against bacterial canker of tomato. Materials and methods Treatments

[0034] The following treatments were tested in this study: 1. Control without Treatment 2. CXC Biocontrol 1% v / v (A, Root Dip), CXC Biocontrol 0.01% (BN, AA-AC, Drench) 1 3. Kocide™ 1.5 Ib / a (OZ, AD-AG, Foliar) 4. CXC Biocontrol 1% v / v (A, Root Dip), CXC Biocontrol 0.01% (CEGIKM, Drench), Kocide™ 1.5 Ib / a (PRTVXZ) 5. CXC Biocontrol 1% v / v (OZ AD-AG, Foliar), Kocide™ 1.5 Ib / a (OZ, AD-AG, Foliar) 6. CXC Biocontrol 1% v / v (A, Root Dip), CXC Biocontrol 0.1% v / v (DHL, Drench), Actigard™ 0.75 oz / a (QUY, Foliar), Firewall™ 200 ppm pr (RVZ, Foliar), Kocide™ 1.5 Ib / a (PTX, Foliar)

[0035] "CXC Biocontrol™" corresponds to the "CXC" composition of Example 1, as defined above. Experimental Unit

[0036] Tomatoes of the 1823 variety were manually transplanted into rows 1.8 m wide, spaced 48 cm apart, on October 17, 2023. The roots were immersed in CXC Biocontrol™ 1% v / v before transplanting, as the first application. Applications B to N and AA to AC were applied as soil moistening directly at the base of the plant. The remaining applications (O to Z, AD to AG) were applied using a tractor-mounted Nifty Fifty sprayer, with an operating pressure of 200 PSI. Additional applications of Petition 870250086123, dated 09 / 23 / 2025, pp. 61 / 87 48 / 56 AA-AG were added to the original spraying program to extend applications until harvest. Treatments included CXC Biocontrol, Kocide, CXC Biocontrol applied foliarly with Kocide or as a wetting with Kocide, and CXC Biocontrol in rotation with Actigard, Firewall, and Kocide. A control without treatment was also included for reference. The plots were 7.6 m long and were replicated four times in a randomized block design. Application Equipment

[0037] Application A was a root bath prior to transplanting. Applications BN and AA-AC were applied as soil moistening directly at the base of the plant. Applications OZ and AD-AG were foliar sprays, using a Nifty-Fifty tractor-mounted operating at 14.06 kg / cm2 (200 psi). Reviews

[0038] Bacterial Canker severity was averaged from ten plants and rated on a scale of 0 to 100, where 0 indicates a completely healthy plant and 100 indicates a completely diseased plant. Assessments were carried out on October 28 (5 DA-P), November 3 (4 DA-Q), November 9 (3 DA-R), November 18 (5 DA-S), November 23 (3 DA-T), November 30 (3 DA-U), December 10 (6 DA-V), December 21 (3 DA-X), December 28 (2 DA-Y), January 5 (3 DA-Z), January 15 (7 DA-AD) and January 22 (7 DA-AE).

[0039] The severity of bacterial spot was assessed on December 29 (3 DA-A) on a scale of 0 to 100, with an average of ten plants per plot.

[0040] Tomatoes were harvested three times throughout the growing season for extra-large, large, medium, worm-infested, sunscalded, and bacterial canker-infected tomatoes. The harvests Petition 870250086123, dated 09 / 23 / 2025, p. 62 / 87 49 / 56 were carried out on January 19 (4 DA-AA), February 1 (3 DAAG) and February 12 (14 DA-AG). Results

[0041] The Severity of Whole Plant Bacterial Canker (0 to 100) is shown in Table 27. Bacterial canker severity was calculated by averaging ten plants and classified on a scale of 0 to 100, where 0 indicates a completely healthy plant and 100 indicates a completely diseased plant. Assessments were carried out on October 28 (5 DAAP), November 3 (4 DA-Q), November 9 (3 DA-R), November 18 (5 DA-S), November 23 (3 DA-T), November 30 (3 DA-U), December 10 (6 DA-V), December 21 (3 DAX), December 28 (2 DA-Y), January 5 (3 DA-Z), January 15 (7 DA-AD), and January 22 (7 DA-AE). Petition 870250086123, dated 09 / 23 / 2025, p. 63 / 87 50 / 56 Table 27: Severity of bacterial canker throughout the plant (0 to 100) 11 / 30 / 2023 3 DA-U 14.1 a 6.4 bc 4.9 c 8.7 b 5.8 c 6.9 bc 11 / 23 / 2023 3 DA-T 10.5 a 2.8 b 1.9 b 4.7 b 5.0 b 4.9 b 11 / 18 / 2023 5 DA-S 8.4 a 2.5 a 1.5 a 4.5 a B0'9 5.6 a 11 / 9 / 2023 3 DA-R 3.8 a LO o' CO o' 0.9 b 1.9 b 2.1 ab 11 / 3 / 2023 4DA-Q 2.8 a 0.4 b CO o' 0.3 b 1.8 ab 1.3 ab 10 / 28 / 2023 5 DA-P 2.4 a 0.3 c 0.4 bc 0.4 bc 0.9 b 0.7 bc Treatment Description Root Immersion Foliar Irrigation Root Immersion Foliar Irrigation Foliar Foliar Root Immersion Foliar Irrigation Foliar Foliar Application Code A BN OZ A CEGIKM PRTVXZ NN Ó Ó < □ σ κ l Rate Unit % v / v % v / v lb / a % v / v % v / v lb / a % v / v lb / a EE ra E φ φ 75 g. ™ O CL £ Rate 1 0.01 LO 1 0.01 1.5 LO LO o Q ο 'Λ OO OJ Treatment Name Untreated Control CXC Bioconrol CXC Bioconrol Kocide CXC Bioconrol CXC Bioconrol Kocide CXC Bioconrol Kocide CXC Bioconrol CXC Bioconrol Actigard Firewall Kocide Treatment No. CO LO <0 1 / 22 / 2024 7 DA-AE 29.5 a P 9'8 15.4 b 8.3 d 14.1 bc O 00 o' 1 / 15 / 2024 7 DA-AD 20.5 a 6.5 c CO o' 6.6 c 10.9 b 9.1 bc 1 / 5 / 2024 3 DA-Z 21.3a P 0'9 16.3 b 7.9 cd 15.1 b 11.0c 12 / 28 / 2023 3 DA-Y 17.9 a 00 CO 12.5 b 5.3 cd 11.5 b 8.6 bc 12 / 21 / 2023 3DA-X 14.4 a 3.4 c 9.0 b 4.3c 8.5b 6.5bc 12 / 10 / 2023 6 DA-V 13.6 a 2.8 c 00 oo 44.5 c 9.5 b 5.1 c Treatment Description Root Immersion Foliar Irrigation Root Immersion Foliar Irrigation Foliar Foliar Root Immersion Foliar Irrigation Foliar Foliar Application Code A BN OZ A CEGIKM PRTVXZ NN Ó Ó x È 2 £ < QOK tL Rate Unit % v / v % v / v lb / a % v / v % v / v lb / a % v / v lb / a EEB | φ φ 75 g. 5 O CL £ Rate 1 0.01 LO 1 0.01 1.5 LO LO o Q ο 'Λ OO CN Treatment Name Untreated Control CXC Bioconrol CXC Bioconrol Kocide CXC Bioconrol CXC Bioconrol Kocide CXC Bioconrol Kocide CXC Bioconrol CXC Bioconrol Actigard Firewall Kocide Treatment No. Petition 870250086123, dated 09 / 23 / 2025, p. 64 / 87 51 / 56

[0042] As demonstrated in Table 27, pest pressure was relatively low throughout the trial, with the highest observed in the untreated control.

[0043] Plots treated only with CXC Biocontrol showed significantly better control of bacterial canker, followed by plots treated with CXC Biocontrol applied as a Kocide bath (Table 28). Table 28: Bacterial Canker (SAUDPC) and % Whole Plant Control. Bacterial Canker SAUDPC % Control 0.00% and 72.15% a 45.65% cd 63.53% ab 41.19% d 53.33% bc 13.59 a 3.75 d 7.31 b 4.93 cd 7.93 b 6.32 bc Treatment Description Root Immersion Foliar Irrigation Root Immersion Foliar Irrigation Foliar Foliar Root Immersion Foliar Irrigation Foliar Foliar Application Code A BN N Ó A CEGIKM PRTVXZ NN Ó Ó < q σ tr o.Rate Unit % in v / v % in v / v Ib / a EE Φ Φ 03 25 O'· O'· _ % in v / v Ib / a EE „ £ â Π Rate 1 0.01 1.5 1 0.01 1.5 1 1.5 V- LO — Q « T- OO CM T- Name of Treatment Control without Treatment CXC Bioconrol CXC Bioconrol Kocide CXC Bioconrol CXC Bioconrol Kocide CXC Bioconrol Kocide CXC Bioconrol CXC Bioconrol Actigard Firewall Kocide Treatment No. V CM CO Tt LO CO.

[0044] A bacterial stain was observed on December 29 (3 days). CXC Biocontrol proved to be the Petition 870250086123, dated 09 / 23 / 2025, pp. 65 / 87 52 / 56 best control against bacteria for one day observation (Table 29). Table 29: % Control of Bacterial Spot in the Whole Plant (%) 12 / 29 / 2023 3 DA-Y 0.00% d 74.02% a O <o 0s· o 00 cd t- 33,98% b 5,22% cd q %^ό^ descrição do tratamento imersão da raiz irrigação foliar código aplicação a b-n n ó cegikm prtvxz í 5 s g < σ cr o. unidade de taxa % em v ib a e φ 03 d _ „ £ § π 1 0,01 1,5 ίθ — q ά cm nome controle sem cxc bioconrol kocide actigard firewall no. v co tt lo coo ω o 0) Q. LOs infested with worms and infected with bacterial canker. Kocide and CXC Biocontrol, in rotation, showed the lowest number of fruits infected with bacterial canker (Table 30). Petition 870250086123, dated 09 / 23 / 2025, pp. 66 / 87 53 / 56 Table 30: Total Count of Tomatoes Harvested (# / plot) Bacterial 44.8 a 20.5 b 8.3 d 17.5 bc 15.3 c 9.3 d Sunburned 8.0 a 8.0 a 8.3 a 8.5 a 9.0 a 7.8 a Earthworm 10.8 a 17.5 a 9.8 a 9.5 a 9.0 a 9.5 a Total Count Medium 147.5 a 161.3 a 137.5 a 165.5 a 153.8 a 157.5 a Large 213.8 a 228.5 a 180.0 a 199.0 a 213.8 a 189.0 a Extra Large 100.3 a 119.0 a 96.0 a 131.3 a 123.8 a 99.5 a Treatment Description Root Immersion Foliar Irrigation Root Immersion Foliar Irrigation Foliar Foliar Root Immersion Foliar Irrigation Foliar Foliar Application Code A BN N Ó A CEGIKM PRTVXZ NN Ó Ó í □ 3 ? < q σ tr o. Rate Unit % in v / v % in v / v Ib / a Ε E Φ Φ 03 O'· O'· _ % in v / v Ib / a Ε E „ S' â i â Rate 1 0.01 1.5 1 0.01 1.5 1 1.5 T- ίθ — Q « T- OO CM T- Name of Treatment Control without Treatment CXC Bioconrol CXC Bioconrol Kocide CXC Bioconrol CXC Bioconrol Kocide CXC Bioconrol Kocide CXC Bioconrol CXC Bioconrol Actigard Firewall Kocide No.

[0046] No significant differences were reported in the overall estimate of marketable productivity. However, numerically, the highest productivity estimates were reported in plots treated with CXC Biocontrol soil or CXC Biocontrol with Kocide, either by irrigation or spraying (Table 31). Petition 870250086123, dated 09 / 23 / 2025, pp. 67 / 87 54 / 56 Table 31: Estimated Marketable Weight (lbs / acre) Total 56421.10 to 62541.28 to 53771.20 to 62929.69 to 64047.73 to 54635.14 to Medium 11223.96 to 12236.73 to 10770.21 to 12694.11 to 12273.03 to 12149.61 to EST MW / A Large 34869.78 to 37810.08 to 32662.74 to 36060.42 to Π5 CO O) o' 00 00 00 CO 32125.50 to Extra Large 10327.35 to 12494.46 to 10338.24 to 14175.15 to 12893.76 to 10360.02 to Treatment Description Root Immersion Foliar Irrigation Root Immersion Foliar Irrigation Foliar Foliar Root Immersion Foliar Irrigation Foliar Foliar Application Code A BN N Ó A CEGIKM PRTVXZ NN Ó Ó í □ 3 í < q σ tr o. Rate Unit % in v / v % in v / v Ib / a EE Φ Φ 03 O'· O'· _ % in v / v Ib / a EE „ S' â I s Rate 1 0.01 1.5 1 0.01 1.5 1 1.5 V- LO — Q « T- OO CM T- Name of Treatment Control without Treatment CXC Bioconrol CXC Bioconrol Kocide CXC Bioconrol CXC Bioconrol Kocide CXC Bioconrol Kocide CXC Bioconrol CXC Bioconrol Actigard Firewall Kocide Treatment No. V CM CO Tt LO CO Petition 870250086123, dated 09 / 23 / 2025, pp. 68 / 87 55 / 56 Discussion

[0047] Overall, these results clearly demonstrate that the compositions according to the present invention are effective when applied as an irrigation to control, suppress, and / or prevent infections by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato plants. The study further demonstrates that the compositions according to the present invention are compatible with existing commercial fungicides and can be tank-mixed without concern that the fungicide will denature, affect, or damage the composition of the invention. The compositions according to the present invention have proven useful when used alone and also in combination with existing canker control methods, such as Kocide™.

[0048] The headings are included herein for reference and to assist in locating certain sections. These headings are not intended to limit the scope of the concepts described herein, and these concepts may have applicability in other sections throughout the descriptive report. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but should have the broadest scope consistent with the innovative principles and features described herein.

[0049] The singular forms a, an, and the include corresponding plural references unless the context clearly indicates otherwise. Thus, for example, a reference to a tomato includes one or more tomato plants or tomato fruits (i.e., tomatoes), and a reference to the method includes a reference to equivalent steps and methods known to those with common knowledge in the art that could be modified or replaced by the methods described herein.

[0050] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, concentration Petition 870250086123, dated 09 / 23 / 2025, p. 69 / 87 56 / 56 tractions, properties, and so forth, used in the descriptive report and claims, should be understood as modified in all cases by the term approximately. At a minimum, each numerical parameter should be interpreted in light of the reported number of significant digits and applying common rounding techniques. Consequently, unless otherwise indicated, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending on the properties sought. Although the ranges and numerical parameters that define the broad scope of embodiments are approximations, the numerical values ​​presented in the specific examples are reported to the greatest possible accuracy. Any numerical value, however, inherently contains certain errors resulting from variations in experiments, test measurements, statistical analyses, and others.

[0051] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or alterations in light thereof will be suggested to persons skilled in the art and should be included in the present invention and within the scope of the appended claims. Petition 870250086123, dated 09 / 23 / 2025, pp. 70 / 87< / o>

Claims

1 / 7 CLAIMS 1. Bacterial composition, comprising an aqueous mixture of live Bacillus subtilis and live Bacillus pumilus, said composition characterized in that it is effective in controlling, suppressing and / or preventing infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato plants.

2. Bacterial composition for use in the control, suppression and / or prevention of infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato, said composition characterized by the fact that it comprises an aqueous mixture of live Bacillus subtilis and live Bacillus pumilus.

3. Bacterial composition, according to claim 1 or 2, characterized in that Bacillus subtilis and Bacillus pumilus are present in a ratio of about 1:

1.

4. Bacterial composition, according to any one of claims 1 to 3, characterized in that each of Bacillus subtilis and Bacillus pumilus is present in the composition at a concentration of about 107 to about 109 bacteria / ml.

5. Bacterial composition, according to any one of claims 1 to 4, characterized in that Bacillus pumilus consists of the ATCC® strain with deposit number PTA-125304 and Bacillus subtilis consists of the ATCC® strain with deposit number PTA 125303.

6. Bacterial composition, according to any one of claims 1 to 5, characterized in that it is formulated for soil irrigation.

7. Bacterial composition, according to any one of claims 1 to 5, characterized in that it is formulated for foliar spraying.

8. Bacterial composition, according to any one of claims 1 to 7, characterized in that the application of said composition to at least one of a tomato plant, a tomato root, a tomato leaf, a tomato seed, a tomato stem and a tomato fruit provides at least one of the following benefits: (i) increased tomato productivity; (ii) increased tomato size; (iii) increased gross tomato yield per acre; (iv) bioprotection against Cmm; (v) enhanced resistance against Cmm; (vi) reduced damage caused by Cmm; (vii) reduced severity of canker and plant tissue affected by canker lesions; (viii) reduced pathological symptoms or lesions resulting from the action of Cmm; (ix) enhanced antimicrobial response of the plant against Cmm; and (x) increased productivity of extra-large tomatoes.

9. Method for controlling, suppressing and / or preventing an infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato plants, characterized in that it comprises: - providing a bacterial composition comprising at least one of a mixture of Bacillus subtilis and Bacillus pumilus, a culture medium previously inoculated with Bacillus subtilis and Bacillus pumilus, a cell-free extract of Bacillus subtilis and Bacillus pumilus and / or metabolites produced by Bacillus subtilis and Bacillus pumilus; and - applying an effective amount of said bacterial composition to at least one of a tomato plant, a tomato root, a tomato leaf, a tomato seed, a tomato stem and a tomato fruit.

10. Method according to claim 9, characterized in that it further comprises the application of at least one of a herbicide, an insecticide, a fungicide, a bactericide and a nutrient.

11. Method according to claim 10, characterized in that at least one copper fungicide is selected from the group consisting of copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate and mixtures thereof.

12. Method for controlling, suppressing and / or preventing an infection caused by Clavibacter michiganensis subsp.A bacterial fungicide is applied to tomato plants, characterized in that it comprises: - providing a bacterial composition comprising at least one of a mixture of Bacillus subtilis and Bacillus pumilus, a culture medium previously inoculated with Bacillus subtilis and Bacillus pumilus, a cell-free extract of Bacillus subtilis and Bacillus pumilus and / or metabolites produced by Bacillus subtilis and Bacillus pumilus; - providing at least one copper fungicide selected from the group consisting of copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate and mixtures thereof; and - applying an effective amount of said bacterial composition and said copper fungicide to at least one of a tomato plant, a tomato root, a tomato leaf, a tomato seed, a tomato stem and a tomato fruit.

13. Method, according to claim 11 or 12, characterized in that the copper fungicide is selected from the group Petition 870250086123, dated 09 / 23 / 2025, page. 73 / 87 4 / 7 consisting of Kocide 3000™, Kocide 2000™, Kocide LF™, Kocide DF™, Kocide 101™, Cuprofix™ Ultra 40 Disperss™, Nordox™, Nordox 75 WG™, Champ WG™, Cueva™, Badge SC™, Basic Copper 53™, Bonide™, Camelot O™, Nu-Cop 3L™, COCS WDG™, Previsto™, Badge X2™, Badge MAXX™, Cuproxat™, Nordox 30 WG™, Copper-Count-N™ fungicide, Cuprablau Z 35 WP™ and Captain Jack's™ liquid copper fungicide, Bordeaux mixture™, Cuprofix Disperss™, Champion™, Nu-Cop™, Copper Count-N™, Mastercop Instill™, Phyton™, and CS 2005™.

14. Method, according to any one of claims 9 to 13, characterized in that the application comprises immersing the roots of a tomato plant in said bacterial composition before planting the tomato plant in the soil.

15. A method, according to any one of claims 9 to 14, characterized in that the application comprises irrigating the soil.

16. Method according to claim 11 or 12, characterized in that the copper fungicide is applied as a foliar spray.

17. A method according to any one of claims 9 to 11, characterized in that it further comprises the application of at least one of a herbicide, an insecticide, a second fungicide, a bactericide, and a nutrient.

18. A method according to any one of claims 8 to 16, characterized in that the bacterial composition comprises a mixture of live Bacillus subtilis and live Bacillus pumilus.

19. Method, according to claim 18, characterized in that the bacterial composition comprises live Bacillus subtilis and live Bacillus pumilus in a 1:1 ratio, or in a 2:1 ratio or in a 1:2 ratio.

20. Method, according to any one of claims 8 to 19, characterized in that Bacillus pumilus consists of the ATCC® strain with deposit number PTA-125304.

21. Method, according to any one of claims 19 to 20, characterized in that Bacillus subtilis consists of the ATCC® strain with PTA deposit number 125303 or ATCC® with PTA deposit number 125302.

22. Method, according to any one of claims 9 to 21, characterized in that the bacterial composition comprises Bacillus pumilus at a concentration of about 109 CFU / ml to about 106 CFU / ml and Bacillus subtilis at a concentration of about 109 CFU / ml to about 106 CFU / ml.

23. Method, according to any one of claims 9 to 22, characterized in that the effective quantity comprises the application of between 107 and 109 CFU / cm3 of each of Bacillus pumilus and Bacillus subtilis.

24. A method, according to any one of claims 9 to 23, characterized in that it provides at least one of the following benefits: (i) increased tomato productivity; (ii) increased tomato size; (iii) increased gross tomato yield per acre; (iv) bioprotection against Cmm; (v) increased resistance against Cmm; (vi) reduced damage caused by Cmm; (vii) reduced severity of canker and plant tissue affected by canker lesions; (viii) reduced pathological symptoms or lesions resulting from the action of Cmm; Petition 870250086123, dated 23 / 09 / 2025, p. 75 / 87 6 / 7 (ix) increased antimicrobial response of the plant against Cmm; and (x) increased productivity of extra-large tomatoes.

25. Method, according to claim 24, characterized in that the said benefit is determined by comparing the tomato plant in contact with or without said bacterial composition and / or by comparing the tomato plant in contact with said bacterial composition and the tomato plant in contact with a reference treatment.

26. Kit for protecting tomato plants against Clavibacter michiganensis subsp. michiganensis (Cmm), characterized in that it comprises: (i) a composition comprising at least one of a mixture of Bacillus subtilis and Bacillus pumilus, a culture medium previously inoculated with Bacillus subtilis and Bacillus pumilus, a cell-free extract of Bacillus subtilis and Bacillus pumilus and / or metabolites produced by Bacillus subtilis and Bacillus pumilus; and (ii) a second container comprising at least one of a herbicide, an insecticide, a fungicide, a bactericide and a nutrient.

27. Kit according to claim 26, characterized in that the second container comprises at least one copper fungicide.

28. Kit, according to claim 26 or 27, characterized in that the copper fungicide comprises a compound selected from the group consisting of copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate and mixtures thereof.

29. Kit, according to any one of claims 26 to 28, characterized in that the said fungicide comprises Kocide™.

30. Combined use, characterized by the fact that it is contained in Petition 870250086123, dated 09 / 23 / 2025, page 76 / 87 7 / 7: (i) a bacterial composition comprising a mixture of live Bacillus subtilis and live Bacillus pumilus; and (ii) at least one of a herbicide, an insecticide, a fungicide, a bactericide and a nutrient, to control, suppress and / or prevent infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato plants.

31. Combined use, according to claim 30, characterized in that it provides synergistic efficacy in controlling, suppressing and / or preventing infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato plants.

32. Use of a bacterial composition, as defined in any one of claims 1 to 8, characterized in that it is for controlling, suppressing and / or preventing infection by Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato plants. Petition 870250086123, dated 09 / 23 / 2025, p. 77 / 87