Bacterial compositions and methods for the protection of tomato from bacterial speck

Abstract

Compositions for the prevention and control of bacterial speck of tomato are described. The compositions comprises comprises an aqueous mixture of living Bacillus subtilis and living Bacillus pumilus. The compositions have been shown to be highly effective in the field. Also described are methods of use and kits comprising the compositions.

Description

BACTERIAL COMPOSITIONS AND METHODS FOR THE PROTECTION OF TOMATO FROM BACTERIAL SPECK CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to United States patent application U.S. 63 / 694,944 filed on September 16, 2024, the content of which is incorporated herein in its entirety. FIELD OF THE INVENTION

[0002] The invention relates to the field of agriculture, and more particularly to prevention and control of bacterial speck of tomato. BACKGROUND OF THE INVENTION

[0003] Bacterial speck of tomato is a common disease caused by the bacterium Pseudomonas syringae pv. tomato. This disease can significantly impact tomato plants, affecting both the leaves and fruit. The symptoms on leaves include small black spots, often with yellow halos. These spots are typically more prominent on the undersides of the leaves. The spots on the fruit are very small, brown to black, and can be raised, flat, or sunken. On unripe fruits, the spots often have darker green halos, while on ripe fruits, the halos can be yellow. Unfortunately, there is no cure once the plants are infected.

[0004] Accordingly, there is a long felt need for compositions and methods effective in preventing infections and / or treating tomato from bacterial speck.

[0005] The present invention addresses these needs and other needs as it will be apparent from the review of the disclosure and description of the features of the invention hereinafter. BRIEF SUMMARY OF THE INVENTION

[0006] According to one aspect, the invention relates to a bacterial composition for use in controlling, suppressing and / or preventing bacterial speck in tomato, wherein said composition comprises an aqueous mixture of living Bacillus subtilis and living Bacillus pumilus.

[0007] According to another aspect, the invention relates to a method for controlling, suppressing and / or preventing bacterial speck in tomato, comprising: ^ 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; wherein said applying provides for control, suppression and / or prevention of bacterial speck infection(s) in tomato.

[0008] According to another aspect, the invention relates to a method for controlling, suppressing and / or preventing bacterial speck in tomato, comprising: ^ 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 of a salicylic acid mimic, an antibiotic, and a copper-based fungicide / bactericide; and ^ applying an effective amount of said bacterial composition and an effective amount of said at least one salicylic acid mimic, antibiotic, and copper-based fungicide / bactericide to at least one of a tomato plant, a tomato root, a tomato leaf, a tomato seed, a tomato stem and a tomato fruit; wherein said applying provides for control, suppression and / or prevention of bacterial speck infection(s) in tomato.

[0009] In embodiments, for the above methods, the bacterial composition comprises a mixture of living Bacillus subtilis and living Bacillus pumilus. In embodiments, the bacterial composition consists essentially of a mixture of living Bacillus subtilis and living Bacillus pumilus.

[0010] According to another aspect, the invention relates to a kit for the protection of tomato from bacterial speck, comprising: (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 salicylic acid mimic, an antibiotic, an herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

[0011] According to another aspect, the invention relates to the combined use of: (i) a bacterial composition comprising a mixture of living Bacillus subtilis and living Bacillus pumilus; and (ii) at least one of a salicylic acid mimic, an antibiotic, an herbicide, an insecticide, a fungicide, a bactericide, and a nutrient, for controlling, suppressing and / or preventing a bacterial speck infection in tomato.

[0012] According to another aspect, the invention relates to the use of a bacterial composition as defined herein, for controlling, suppressing and / or preventing a bacterial speck infection in tomato.

[0013] Additional aspects, advantages and features of the present invention will become more apparent upon reading of 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 EMBODIMENTS

[0014] The following description provides embodiments by which the invention may be practised. It will be understood that other embodiments may be made without departing from the scope of the invention disclosed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. General overview

[0015] The invention aims to address an important need in the prevention and control of bacterial speck in tomato.

[0016] As used herein, the term “bacterial speck” refers to a tomato disease caused by the bacterium Pseudomonas syringae pv. tomato.

[0017] As described hereinafter, the present invention proposes new compositions and methods for controlling, suppressing and / or preventing bacterial speck-related infections in tomato. Bacterial compositions

[0018] One aspect of the invention concerns bacterial compositions that are effective against bacterial speck. The present invention relies particularly on the combined use of Bacillus subtilis and Bacillus pumilus. Compositions in accordance with the present invention preferably comprises a mixture of living 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. In embodiments, the bacterial composition comprises minimally a mixture of living Bacillus subtilis and living Bacillus pumilus. In embodiments, the bacterial composition consists essentially of a mixture of living Bacillus subtilis and living Bacillus pumilus. In embodiments, the bacterial composition simply consists of a mixture of living Bacillus subtilis and living Bacillus pumilus.

[0019] As used herein, reference to a “culture medium previously inoculated with Bacillus subtilis and Bacillus pumilus” means a medium previously inoculated with the two bacteria, either together or separately. Therefore, this term encompasses mixtures of two or more culture pooled media, previously inoculated with one bacterium and / or both bacteria.

[0020] As used herein, 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, by centrifugation, etc.). In embodiments the cell-free extract is a filtered fraction and / or a cell-free supernatant obtained from culturing Bacillus subtilis and / or Bacillus pumilus.

[0021] As used herein, reference to “metabolites produced by Bacillus subtilis and Bacillus pumilus” refers to a mixture comprising peptides, proteins, small molecules, lipids etc. which have been produced by one or both bacteria (synthesis, expression, extraction, secretion, etc.), either separately or in combination. In embodiments, the strain of Bacillus pumilus is a strain that produces a biologically active metabolite known as “Micrococcin P1” (see PCT publication WO 2020 / 069438, incorporated herein by reference). In some embodiments, the composition of the invention comprises Micrococcin P1 at a concentration of at least 5 µg / L, 10 µg / L, 25 µg / L, 50 µg / L or 100 µg / L. In some embodiments, the composition of the invention comprises Micrococcin P1 at a concentration above 100 µg / L, 150 µg / L, 200 µg / L, 300 µg / L, 500 µg / L, 600 µg / L, 1000 µg / L, or 5000 µg / L.

[0022] In embodiments, the Bacillus pumilus consists of “Bacillus pumilus strain deposited with the American Type Culture Collection (ATCC) on September 26, 2018 and bearing deposit number PTA-125304. In embodiments, the Bacillus pumilus consists of strain NES-CAP-1 (GenBank Accession No. MF079281.1).

[0023] In embodiments, the Bacillus subtilis consists of strain “Bacillus subtilis strain deposited with the ATCC® September 26, 2018 and bearing deposit number PTA-125303.

[0024] In embodiments, the Bacillus subtilis consists of strain Bacillus subtilis strain deposited with the ATCC® on September 26, 2018, and bearing deposit number PTA-125302.

[0025] In embodiments, the composition comprises living Bacillus subtilis and living Bacillus pumilus in a 1:1 ratio. In other embodiments, that ratio is 2:1. In other embodiments, that ratio is 1:2.

[0026] In embodiments, the composition comprises between 1 x 109and 1 x 106Bacillus pumilus bacteria / ml. In embodiments, the composition comprises at least 1 x 109Bacillus pumilus bacteria / ml, or at least 1 x 108Bacillus pumilus bacteria / ml, or at least 1 x 107Bacillus pumilus bacteria / ml, or at least 1 x 106Bacillus pumilus bacteria / ml.

[0027] In embodiments, the composition comprises between 1 x 109and 1 x 106Bacillus subtilis bacteria / ml. In embodiments, the composition comprises at least 1 x 109Bacillus subtilis bacteria / ml, or at least 1 x 108Bacillus subtilis bacteria / ml, or at least 1 x 107Bacillus subtilis bacteria / ml, or at least 1 x 106Bacillus subtilis bacteria / ml.

[0028] It is also envisionable to include additional bacteria, culture medium, cell-free extracts and / or metabolites thereof, to the composition of the invention. Additional 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., Apergillus oryzae), Candida spp. (e.g., Candida utilis), Pseudomonas spp. (e.g., Pseudomonas aeuroginosa, 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.

[0029] The bacterial composition of the invention may further comprise an herbicide, an insecticide, a fungicide, a bactericide, a nutrient, and mixtures thereof.

[0030] 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™), pendimenthalin (Prowl™), clethodim (Select™), triclopyr (Garlon™), clopyralid (Stinger™), Fluro Star™, flufosinate (Liberty™), halosulfuron (Permit™), isoxaben (Gallery™), sethoxydim (Poast™), bentazon (Basagran™), pyraflufen-ethyl (Epic™), fomesafen (Reflex™), trifluralin (Treflan™), mesotrione (Callisto™), flumioxazin (Valor), dicamba (Banvel™, Clarity™), and imazapyr (Arsenal™).

[0031] Examples of potentially useful insecticides include, but are not limited to, pyrethoids, pyrethins, neonicotinoids, organophosphates, carbamates, spinosad, imidacloprid, fipronil, malathion, permethrin, cypermethrin, and chlorpyrifos. Examples of potentially useful bactericide 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, sulphur, magnesium, calcium and other plant micronutrients.

[0032] Examples of potentially useful fungicides include, but are not limited to, copper fungicide that includes 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 3000TM, Kocide 2000™, Cuprofix Ultra 40 Disperss™, Nordox 75 WG™, Champ WG™, Cueva™, Badge SC™, Basic Copper 53™, BOonide™ copper fungicide, Camelot O™, Nu-Cop 3L™, C-O- C-S WDG™, Previsto™, Badge X2™, Badge MAXX™, Cuproxat™ flowable copper fungicide, tribasic copper sulfate, Nordox 30 WG™, Copper-Count-N™ fungicide, Cuprablau Z 35 WP™ and Captain Jack’s™ liquid copper fungicide.

[0033] In embodiments, the composition of the invention comprises at least one of a salicylic acid mimic, an antibiotic, and a copper-based fungicide / bactericide.

[0034] In one particular embodiment, the salicylic acid mimic is acibenzolar S-methyl (CAS No.135158-54-2). In one particular embodiment, the composition of the invention is mixed or used in combination with Actigard™ (Syngenta™) a fungicide which active ingredient is acibenzolar S-methyl.

[0035] In one particular embodiment, the antibiotic comprises streptomycin and / or a salt thereof. In one particular embodiment, the composition of the invention is mixed or used in combination with FireWall™ (AgroSource, Inc.) is a fungicide / bactericide which active ingredient is streptomycin sulfate (CAS No.3810-74-0).

[0036] In one particular embodiment, the copper-based fungicide / bactericide comprises copper hydroxide. In one particular embodiment, the composition of the invention is mixed or used in combination Kocide™ (Dupont™) a copper-based fungicide / bactericide which active ingredient is copper hydroxide (CAS No.20427-59-2). Additional examples of potentially useful copper-based fungicide / bactericide include the copper fungicides mentioned hereinbefore.

[0037] In embodiments, the bacterial composition is formulated for application to one or more of a tomato plant, a tomato root, a tomato leaf, a tomato seed, a tomato stem and a tomato fruit.

[0038] As described with more details hereinafter, the bacterial compositions in accordance with the present invention may be applied using any suitable method or technique including, but not limited to, seed coating, application to seeds prior to and / or during planting, root inoculation, soil drenching, soil application (e.g., injection), foliar spray (e.g., high- or low-pressure spraying), etc. In embodiments, the applying comprises dipping roots of a tomato plant into the bacterial composition prior to planting the tomato plant into soil.

[0039] In one preferred embodiment, the applying comprises soil drenching. As used herein the term “soil drenching” refers to the process of adding compounds or compositions in accordance with the present invention to directly to the base of a plant, preferably to provide deep, targeted treatment.

[0040] As used herein, the term “tomato” refers to any one of the more than 10000 tomato varieties available in the world. The present composition may find useful anti- bacterial speck activity against most if not all tomato varieties affected by bacterial speck, including greenhouse and field-grown varieties. Most common tomato cultivars include but are not limited to, 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.

[0041] Preferably, the bacterial composition and / or method in accordance with the present invention provides at least one of the following benefits: i. bioprotection against bacterial speck; ii. enhancing resistance against bacterial speck; iii. reducing damages caused by bacterial speck;iv. reducing severity and plant tissue affected by bacterial speck lesions; v. reducing pathological symptoms or lesions resulting from actions of bacterial speck; vi. enhancing plant antimicrobial response against bacterial speck; vii. increasing tomato yield; viii. increasing tomato size; and ix. increasing gross return of tomato per acre.

[0042] In embodiments, benefit(s) is(are) observed or determined by comparing tomato contacted or not with the bacterial composition of the invention (i.e., untreated control). In embodiments, benefit(s) is(are) observed when comparing tomato contacted with the bacterial composition versus tomatoes contacted with a commercial reference treatment. In embodiments the commercial reference treatment consists of Kocide™ applied as a foliar spray. In embodiments the commercial reference treatment consists of Actigard™ applied as a foliar spray. In embodiments the commercial reference treatment consists of Firewall™ applied as a foliar spray.

[0043] In some embodiments, bacterial speck infection is measured in a tissue of a tomato plant by assessing bacterial speck severity or bacterial speck incidence rated on a 0-100% scale, where 0% displays no disease severity and 100% indicates complete disease takeover of the plant. In embodiments, the bacterial compositions and / or methods in accordance with the present invention reduces bacterial speck severity on the whole plant by at least 5%, or at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, when compared to an untreated control and / or when compared to a commercial reference treatment. In embodiment, bacterial speck control or reduced infection expressed as % of the whole plant may be calculated using Abbott’s formula for SAUDPC (i.e., standardized area under the disease progress curve). In embodiments, the reduction above is achieved by applying the bacterial composition(s) as a root dip. In embodiments, the reduction above is achieved by a combination treatment comprisingapplying the bacterial composition(s) as a root dip and a commercial reference treatment as a foliar spray.

[0044] Bacterial compositions in accordance with 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, turf, ornamental plants, etc.

[0045] The bacterial compositions of the invention may also possibly be useful, for instance, against one or more of the following pathogens: Clavibacter michiganensis subsp. michiganensis (Cmm), 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.

[0046] The bacterial compositions of the invention may also possibly be useful, for the treatment of bacterial, fungal and oomycetes pathogens (blight, mildew, mold, rust, scabs). It could also potentially be useful to control nematodes.

[0047] It may also be envisionable to commercialize the bacterial compositions of the invention as a kit for the protection of tomato from bacterial speck. For instance, a kit in accordance with the present invention may comprise: (i) a first container comprising a bacterial composition as defined herein; and (ii) a second container comprising at least one of a salicylic acid mimic, an antibiotic, herbicide, an insecticide, a fungicide, a bactericide, and a nutrient. In such kit the content of the first and / or second container may be a ready to use solution or it may be a concentrated solution to be mixed and / or diluted prior use. Methods of protection

[0048] Another aspect of the invention concerns methods for protecting tomato from bacterial speck. 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, a tomato stem and / or a tomato fruit. The present inventionencompasses protection of tomato in a variety of plant environments such as fields, greenhouse facilities, vertical farms, urban greening systems, and hydroponic systems.

[0049] In embodiments, the method provides for one or more of the benefits listed hereinbefore for the bacterial composition.

[0050] The bacterial compositions in accordance with the present invention may be applied using any suitable method including, but not limited to, seed coating, application to seeds prior to and / or during planting, root inoculation, soil drenching, soil application (e.g., injection), foliar spray (e.g., high- or low-pressure spraying), etc.

[0051] In embodiments, the applying comprises dipping roots of a tomato plant into the bacterial composition prior to planting the tomato plant into soil.

[0052] In one preferred embodiment, the applying comprises soil drenching. As used herein the term “soil drenching” refers to the process of adding compounds or composition(s) in accordance with the present invention directly to the base of a plant, preferably to provide deep, targeted treatment.

[0053] In embodiments, the method further comprises applying at least one of an herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

[0054] In particular embodiments, the method further comprises applying at least one of a salicylic acid mimic, an antibiotic, and a copper-based fungicide / bactericide as defined hereinbefore.

[0055] The bacterial composition can be applied at a particular time, or one or more times, depending on parameters including, but not limited to, bacterial speck population in a tomato plant or soil planted with a tomato plant, environmental conditions, tomato susceptibility, time of planting, etc.

[0056] In some embodiments, the bacterial composition is applied: where a plant rooted therein showed a pathological symptom associated with bacterial speck; where a plant currently rooted therein shows a pathological symptom associated with bacterial speck; and / or where a tomato plant which will be planted therein is expected to show apathological symptom associated with bacterial speck. In some embodiments, the bacterial composition is applied to the seeds that will be planted to such a soil. In some embodiments, the bacterial composition is applied to the seeds from a parent tomato plant that has been planted to such a soil. In some embodiments, bacterial composition is applied to the plant that is rooted in such a soil. In some embodiments, the bacterial composition is applied to a plant that shows one or more pathological symptoms associated with bacterial speck.

[0057] The bacterial composition can be applied after or prior to infection by bacterial speck. In some embodiments, the composition is applied at least 1 week, 2 weeks, 3 weeks, 1 months, 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 months, 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 5-10 weeks before harvesting a tomato.

[0058] When treating seeds, the bacterial composition of the invention can be applied by a variety of techniques including, but not limited to, high- or low-pressure spraying, coating, immersion, and injection into the soil. Once treated, seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants. After plants have propagated from seeds treated with the bacterial composition, the plants may further be treated with one or more applications of the disclosed bacterial composition.

[0059] Disclosed bacterial compositions can be applied to all or part of the tomato plant. For example, a disclosed composition can be applied to the stems, roots, leaves, and / or propagules (e.g., cuttings). The plant may be treated at one or more developmental stages. The bacterial composition of the invention can be applied by a variety of techniques including, but not limited to, high- or low-pressure spraying, coating, root drip, soil drench, etc. In one embodiment, the disclosed composition of the invention is applied to roots.

[0060] In some embodiments, the bacterial composition is mixed with or diluted in an agriculturally acceptable carrier before used.

[0061] In some embodiments, the bacterial composition is applied to a delivery vehicle, wherein the delivery vehicle serves as a means of transporting the bacteria that are present in the bacterial composition to the soil, plant, seed, field, etc. For example, disclosed bacterial compositions can be applied to a delivery vehicle (e.g., a particle, a polymer, or a substrate) to be used in filtration systems for the treatment of irrigation water. 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.

[0062] The bacterial composition of the present invention is preferably applied in an amount effective for bioprotection of a tomato from bacterial speck (e.g., controlling, suppressing and / or preventing a bacterial speck infection). In some embodiments, the amount is sufficient to prevent bacterial speck infection. In some embodiments, the amount is sufficient to treat or reduce one or more symptoms associated with bacterial speck.

[0063] In some embodiments, the applied amount is sufficient to reduce bacterial speck concentration in a tissue of a tomato plant treated with the bacterial composition. In some embodiments, bacterial speck concentration measured in a tissue of a tomato plant 10 days after the step of applying is lower than 109CFU / g. In some embodiments, bacterial speck concentration measured in a tissue of a tomato plant 21 days after the step of applying is lower than 109CFU / g. In some embodiments, bacterial speck concentration measured in a tissue of a tomato plant 3, 5, 7, 14, 21, 28, 35, or 42 days after the step of applying is lower than 109CFU / g. In some embodiments, bacterial speck concentration measured in a tissue of a tomato plant 3, 5, 7, 14, 21, 28, 35, or 42 days after the step of applying is lower than 108CFU / g. In some embodiments, bacterial speck concentration measured in a tissue of a tomato plant 3, 5, 7, 14, 21, 28, 35, or 42 days after the step of applying is lower than 107CFU / g. In some embodiments, bacterial speck concentration measured in a tissue of a tomato plant 3, 5, 7, 14, 21, 28, 35, or 42 days after the step of applying is lower than 106CFU / g. In some embodiments,

[0064] Those skilled in the art will appreciate the fact that specific amounts and required frequency of applications may vary depending on the types and condition of soils, the types and conditions of tomatoes, potency and activity of bacterial speck, etc. Thespecific amounts can also vary depending on the environment, for example, whether the plant is in a pot, a greenhouse, a field, etc.

[0065] The specific amounts can be determined by using methods known in the art, for example, by testing dose dependent response. In some embodiments, the specific amount is determined by testing dose dependent response on a culture plate with bacterial speck, for example, by measuring a zone of inhibition. In some embodiments, the specific amount is determined by testing dose dependent response in a pot or in a field. In some embodiments, the specific amount is determined based on the measurement of bacterial speck concentration or amount of a gene specific bacterial speck in a tissue of a tomato plant treated with the bacterial composition. In some embodiments, the specific amount is determined based on the concentration of Bacillus pumilus, Bacillus subtilis or both, or an active metabolite thereof (e.g., Micrococcin P1).

[0066] In some embodiments, the bacterial culture applied to a tomato plant comprises Bacillus pumilus at a concentration between 106and 109CFU / mL, or between 107and 109CFU / mL, or between 2.5x107and 109CFU / mL, or between 2.5x107and 8.5x108CFU / mL, or between 5x107and 8.5x108CFU / mL, or between 2x108and 8.5x108CFU / mL, or 108CFU / mL. In some embodiments, the bacterial culture applied to a tomato plant comprises Bacillus subtilis at a concentration between 106and 109CFU / mL or 107and 109CFU / mL, or between 2.5x107and 109CFU / mL, or between 2.5x107and 8.5x108CFU / mL, or between 5x107and 8.5x108CFU / mL, or between 2x108and 8.5x108CFU / mL, or 108CFU / mL.

[0067] In some embodiments, the bacterial composition is applied to the tomato plant to make a final concentration of Bacillus pumilus measured in root, stem or leaf of the tomato plant to range between 106and 109CFU / mL, or between 107and 109CFU / mL , or between 2.5x107and 109CFU / mL, or between 2.5x107and 8.5x108CFU / mL, or between 5x107and 8.5x108CFU / mL, or between 2x108and 8.5x108CFU / mL, or between 3x108and 8x108CFU / mL, or 108CFU / mL.

[0068] In some embodiments, the bacterial composition is applied to the tomato plant to make a final concentration of Bacillus subtilis measured in root, stem or leaf of the tomato plant to range between 106and 109CFU / mL, or between 107and 109CFU / mL, orbetween 2.5x107and 109CFU / mL, or between 2.5x107and 8.5x108CFU / mL, or between 5x107and 8.5x108CFU / mL, or between 2x108and 8.5x108CFU / mL, or between 3x108and 8x108CFU / mL, or 108CFU / mL.

[0069] In some embodiments, the bacterial composition is applied to the tomato plant to make a final concentration of Bacillus pumilus and Bacillus subtilis measured in root, stem or leaf of the tomato plant to range between 106and 109CFU / mL, or between 107and 109CFU / mL, or between 2.5x107and 109CFU / mL, or between 2.5x107and 8.5x108CFU / mL, or between 5x107and 8.5x108CFU / mL, or between 2x108and 8.5x108CFU / mL, or between 3x108and 8x108CFU / mL, or 108CFU / mL.

[0070] The composition can be applied in an amount that ranges between 0.2 and 3 gal / A, between 0.5 and 2.5 gal / A, between 0.75 and 2 gal / A, 0.5 gal / A, 1 gal / A, 1.25 gal / A, 1.5 gal / A, or 2 gal / A.

[0071] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and covered by the claims appended hereto. The invention is further illustrated by the following example which should not be construed as further or specifically limiting. EXAMPLE 1 – Field study in Florida

[0072] Objective of the Study: The primary objective of this study was to evaluate the efficacy of a particular embodiment of a composition according to the present invention, referred hereinafter as “CXC Biocontrol™”, in managing bacterial speck in tomato. The study aimed to identify the most effective treatment methods, including root dips, soil drenches, and foliar sprays, both as standalone applications and in combination with other fungicides / bactericides like Kocide™, Actigard™, and Firewall™. By rigorously testing these treatments under controlled conditions, the study sought to provide actionable insights for improving tomato crop health and reducing the severity and incidence of bacterial speck.

[0073] Composition of CXC Biocontrol™. The particular bacterial composition tested, referred hereinafter as “CXC biocontrol™” comprised an aqueous mixture of 1) living Bacillus subtilis (ATCC® deposit number PTA-125303) and 2) living Bacillus pumilus (ATCC® deposit number PTA-125304). The two strains of bacteria were present in the mixture at a 1:1 ratio, each strain at a concentration of about 107- 109bacteria / ml. For obtaining the CXC biocontrol™ composition, the two microbes were grown separately for at least 72 hours at 28^C in a shaking incubator (125 rpm) in LB broth comprising tryptone (10g), yeast extract (5g), and NaCl (5g) in 1 liter water. Bacterial number was determined by counting CFU per mL using serial dilution method. Bacterial cultures were serially diluted and aliquots were plated on LB agar medium, incubated at 28^C and CFUs counted after at least 48 hours of incubation. After final harvest, the cultures of the two microbes were mixed together (1:1) and kept in the fridge until use.

[0074] Methodology: The study focused on evaluating the efficacy of various treatments against bacterial speck in 1823 variety tomatoes. The experimental design was a randomized block with four replications. Treatments included an untreated control, CXC Biocontrol™ at 1% v / v applied as a root dip (Application A) and as a soil drench (Applications B-N, AA-AC), and Kocide™ at 1.5 lb / a applied as a foliar spray (Applications O-Z, AD-AG) using a tractor-mounted sprayer operating at 200 psi. Combination treatments involved various mixes where CXC Biocontrol™ was mixed with Kocide™, Actigard™ at 0.75 oz / a, and Firewall™ at 200 ppm in different application sequences to evaluate their synergistic effects against bacterial speck in tomatoes

[0075] The severity of bacterial speck was assessed approximately 2.5 to 3 months after planting, using a 0-100 scale averaged from ten plants per plot. The incidence of bacterial speck, defined as the percentage of plants affected, was also recorded on the same date. Control efficiency was calculated by comparing the treatments to the untreated control.

[0076] Agricultural practices: The experiment adhered to standard agricultural practices, with tomatoes being manually transplanted into rows spaced 6 feet apart and 19 inches between plants. The irrigation was managed through a drip system, which maintained consistent soil moisture levels throughout the study. These practices werecritical in maintaining plant health and ensuring that the effects of the treatments were not confounded by other environmental factors.

[0077] Weather Conditions: The study was conducted in Thonotosassa, Florida, USA under specific environmental conditions, with the weather being closely monitored throughout the experimental period.

[0078] Evaluation Timeline: The severity and incidence of bacterial speck were meticulously evaluated approximately two and a half months after the initial planting. This assessment was conducted using a 0-100 scale for severity, averaged across ten plants per plot, ensuring a comprehensive evaluation of each treatment's efficacy.

[0079] Results: The results indicated that CXC Biocontrol™, especially when used as a root dip, significantly reduced the severity of bacterial speck compared to the untreated control (Table 1). While the untreated control had the highest severity score of 32.1, the CXC Biocontrol™ root dip treatment achieved the best treatment with a severity score of only 8.3. In contrast, Kocide™ as a foliar spray had a severity score of 26.8. Combination treatments showed varying degrees of effectiveness; for instance, CXC Biocontrol™ combined with Kocide™ (root dip and drench) had a severity score of 21.0, while the same combination as a foliar application scored 31.1. The combination of CXC Biocontrol™ with Actigard™ and Firewall™ plus Kocide™ resulted in a severity score of 19.0.Table 1: Bacterial Speck Severity of the Whole Plant (expressed as 0-100)

[0080] of that all plants were affected to some extent (data not shown). Nevertheless, as shown in Table 2, CXC Biocontrol™ applied as a root dip provided the highest control efficiency at 74.02%, followed by the combination of CXC Biocontrol™ with Actigard™, Firewall™, and Kocide™ at 40.44%. Therefore, the present study underscores the superior performance of CXC Biocontrol™, particularly as a root dip, in managing bacterial speck severity in tomatoes.Table 2: Bacterial Speck Severity of the Whole Plant (expressed as % of control)

[0081] performance of CXC Biocontrol™, particularly when applied as a root dip, in reducing the severity of bacterial speck in tomatoes. The CXC Biocontrol™ root dip treatment achieved a severity score of 8.3, which was significantly lower than the score of 32.1 for the untreated control, reflecting a control efficiency of 74.02%. In contrast, Kocide™ as a foliar spray showed limited efficacy, with a severity score of 26.8 and a control efficiency of 16.80%. The most effective combination was CXC Biocontrol™ with Actigard™, Firewall™, and Kocide™ (treatment #6), achieving a control efficiency of 40.44%.

[0082] Yield Data: While the primary focus of the study was on managing bacterial speck, yield data were also collected. The study recorded the total number and weight of harvested tomatoes, including extra-large, large, and medium-sized fruits, as well as those affected by bacterial speck. Plots treated with CXC Biocontrol™, particularly in combination with Kocide™ demonstrated a trend toward higher marketable yields (data not shown).

[0083] Discussion: The results of the study underscore the potential of a composition in accordance with the present invention as a critical component in managing bacterial speck in tomatoes. Particularly, the study's findings suggest that CXC Biocontrol™,especially when used as a root dip, is highly effective in reducing the severity of bacterial speck in tomatoes. The root dip application likely enhances the plant's systemic resistance, providing a more consistent and thorough defense against the pathogen. The lower efficacy of Kocide™ as a foliar spray could be attributed to factors such as inadequate coverage or the limited residual activity on the leaf surface. * * *

[0084] Headings are included herein for reference and to aid in locating certain sections. These headings are not intended to limit the scope of the concepts described therein, and these concepts may have applicability in other sections throughout the entire specification. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0085] The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a tomato" includes one or more tomato plants or tomato fruit (i.e., tomatoes) and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0086] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, concentrations, properties, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that may vary depending upon the properties sought to be obtained. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors resulting from variations in experiments, testing measurements, statistical analyses, and such.

[0087] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.

Claims

AMENDED CLAIMS received by the International Bureau on 19 February 2026 (19.02.2026)1. A bacterial composition for use in controlling, suppressing and / or preventing bacterial speck in tomato, wherein said composition comprises an aqueous mixture of living Bacillus subtilis and living Bacillus pumilus, and wherein the Bacillus pumilus consists of strain ATCC® deposit number PTA-125304 and wherein the Bacillus subtilis consists of strain ATCC® deposit number PTA-125303.

2. The bacterial composition as defined in claim 1 , wherein said Bacillus subtilis and Bacillus pumilus are present at a ratio of about 1 :1 ratio.

3. The bacterial composition as defined in claim 1 or 2, wherein each of said Bacillus subtilis and Bacillus pumilus are present in the composition at a concentration of about 107to about 109CFU / ml.

4. The bacterial composition as defined in any one of claims 1 to 3, further comprising at least one of an herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

5. The bacterial composition as defined in any one of claims 1 to 3, further comprising at least one of a salicylic acid mimic, an antibiotic, and a copper-based fungicide / bactericide.

6. The bacterial composition of claim 5, wherein the salicylic acid mimic is acibenzolar S- methyl, wherein the antibiotic is streptomycin or a salt thereof, and wherein the copper-based fungicide / bactericide comprises copper hydroxide.

7. The bacterial composition as defined in any one of claims 1 to 6, wherein said bacterial composition is formulated for at least one of root dripping, soil drenching and foliar spray.

8. The bacterial composition as defined in any one of claims 1 to 7, wherein 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. bioprotection against bacterial speck; ii. enhancing resistance against bacterial speck; iii. reducing damages caused by bacterial speck; iv. reducing severity and plant tissue affected by bacterial speck lesions;V. reducing pathological symptoms or lesions resulting from actions of bacterial speck; vi. enhancing plant antimicrobial response against bacterial speck; vii. increasing tomato yield; viii. increasing tomato size; and ix. increasing gross return of tomato per acre.

9. A method for controlling, suppressing and / or preventing bacterial speck in tomato, comprising:- 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, wherein the Bacillus pumilus consists of strain ATCC® deposit number PTA-125304 and wherein the Bacillus subtilis consists of strain ATCC® deposit number PTA-125303; 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; wherein said applying provides for control, suppression and / or prevention of bacterial speck infection(s) in tomato.

10. The method of claim 9, further comprising applying at least one of an herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

11. The method of claim 9, further comprising applying at least one of a salicylic acid mimic, an antibiotic, and a copper-based fungicide / bactericide.

12. The method of claim 11 , wherein the salicylic acid mimic is acibenzolar S-methyl, wherein the antibiotic is streptomycin or a salt thereof, and wherein the copper-based fungicide / bactericide comprises copper hydroxide.

13. A method for controlling, suppressing and / or preventing bacterial speck in tomato, comprising:- 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, wherein the Bacillus pumilus consists of strain ATCC® deposit number PTA-125304 and wherein the Bacillus subtilis consists of strain ATCC® deposit number PTA-125303;- providing at least one of a salicylic acid mimic, an antibiotic, and a copper-based fungicide / bactericide; and- applying an effective amount of said bacterial composition and an effective amount of said at least one salicylic acid mimic, antibiotic, and copper-based fungicide / bactericide to at least one of a tomato plant, a tomato root, a tomato leaf, a tomato seed, a tomato stem and a tomato fruit; wherein said applying provides for control, suppression and / or prevention of bacterial speck infection(s) in tomato.

14. The method of claim 13, wherein the salicylic acid mimic is acibenzolar S-methyl, wherein the antibiotic is streptomycin or a salt thereof, and wherein the copper-based fungicide / bactericide comprises copper hydroxide.

15. The method of any one of claims 9 to 14, wherein said applying comprises dipping roots of a tomato plant into said bacterial composition prior to planting said tomato plant into soil.

16. The method of any one of claims 9 to 15, wherein said applying comprises soil dripping.

17. The method of claim 14, wherein the copper-based fungicide / bactericide is applied as a foliar spray.

18. The method of any one of claims 9 to 17, wherein said method further comprises applying at least one of an herbicide, an insecticide, a second fungicide, a second bactericide, and a nutrient.

19. The method of any one of claims 9 to 18, wherein said bacterial composition comprises a mixture of living Bacillus subtilis and living Bacillus pumilus.

20. The method of any one of claims 9 to 18, wherein said bacterial composition consists essentially of a mixture of living Bacillus subtilis and living Bacillus pumilus.

21. The method of claim 19 or 20, wherein said bacterial composition comprises living Bacillus subtilis and living Bacillus pumilus in a 1 :1 ratio, or in a 2:1 ratio or in a 1 :2 ratio.

22. The method of any one of claims 9 to 21 , wherein the bacterial composition comprises Bacillus pumilus at a concentration of about 109CFU / ml to about 106CFU / ml and Bacillus subtilis at a concentration of about 109CFU / ml to about 106CFU / ml.

23. The method of any one of claims 9 to 22, wherein the effective amount comprises applying between 107and 109CFU / ml of each of Bacillus pumilus and Bacillus subtilis.

24. The method of any one of claims 9 to 23, wherein said method provides for at least one of the following benefits: i. bioprotection against bacterial speck; ii. enhancing resistance against bacterial speck; iii. reducing damages caused by bacterial speck; iv. reducing severity and plant tissue affected by bacterial speck lesions; v. reducing pathological symptoms or lesions resulting from actions of bacterial speck; vi. enhancing plant antimicrobial response against bacterial speck; vii. increasing tomato yield; viii. increasing tomato size; and ix. increasing gross return of tomato per acre.

25. The method of claim 24, wherein said reduces bacterial speck severity on the whole plant by at least 5%, or at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, when compared to an untreated control and / or when compared to a commercial reference treatment.

26. The method of claim 24 or 25, wherein said benefit is determined by comparing tomato contacted or not with said bacterial composition, and / or when comparing tomato contacted with said bacterial composition versus tomato contacted with a reference treatment.

27. A kit for the protection of tomato from bacterial speck, comprising: (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, wherein the Bacillus pumilus consists of strain ATCC® deposit number PTA-125304 and wherein the Bacillus subtilis consists of strain ATCC® deposit number PTA-125303; and (ii) asecond container comprising at least one of a salicylic acid mimic, an antibiotic, an herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

28. The kit of claim 27, wherein the salicylic acid mimic is acibenzolar S-methyl, wherein the antibiotic streptomycin or a salt thereof.

29. The kit of claim 27 or 28, wherein and wherein the fungicide and / or bactericide comprises copper hydroxide.

30. Combined use of: (i) a bacterial composition comprising a mixture of living Bacillus subtilis and living Bacillus pumilus, wherein the Bacillus pumilus consists of strain ATCC® deposit number PTA- 125304 and wherein the Bacillus subtilis consists of strain ATCC® deposit number PT A-125303; and (ii) at least one of a salicylic acid mimic, an antibiotic, an herbicide, an insecticide, a fungicide, a bactericide, and a nutrient, for controlling, suppressing and / or preventing a bacterial speck infection in tomato.

31. The combined use of claim 30, wherein said combined use provide for a synergistic efficacy in the controlling, suppressing and / or preventing an infection from bacterial speck in tomato.

32. Use of a bacterial composition as defined in any one of claims 1 to 8, for controlling, suppressing and / or preventing a bacterial speck infection in tomato.

Images