Bacterial composition and method for protecting tomatoes from tomato canker fungus (Clavibacter michiganensis subsp. michiganensis: Cmm)

Abstract

This document describes a bacterial composition and its use for controlling, suppressing, and / or preventing infection of tomatoes with the tomato canker fungus (Cmm). The bacterial composition comprises an aqueous mixture of live Bacillus subtilis and live Bacillus pumilus. The bacterial composition can be combined with existing herbicides, insecticides, mycicides, bactericidal agents, and nutrients, including but not limited to copper-containing compounds. Combination with copper mycicides such as Kocide® imparts synergistic efficacy.

Description

[Technical Field]

[0001] Cross-reference of related applications none

[0002] This invention relates to the agricultural field, and more particularly to the prevention and control of tomato canker (tomato canker fungus (Clavibacter michiganensis pv.michiganensis)). [Background technology]

[0003] Tomato canker is the most devastating bacterial disease affecting tomatoes. It is caused by the bacterium Clavibacter michiganensis subsp. michiganensis (Cmm). This microorganism is primarily introduced into farmland through infected seeds or seedlings and, due to its ability to spread rapidly, causes catastrophic losses. Controlling this disease is particularly difficult, not only because there is no cure, but also because Cmm can be extremely difficult to eradicate once introduced into greenhouses, fields, or farms. Therefore, there is a strong need to reduce the economic and environmental costs associated with Cmm infection.

[0004] International Publication No. 2020 / 069438 describes the use of bacterial cultures containing Bacillus pumilus to protect tomatoes from Cmm. However, there is still a need for improved methods and compositions for controlling, suppressing, and / or preventing infection of tomatoes with Cmm.

[0005] In particular, there is a need for a method to impart a measurable effect in the field by applying an effective amount of bacterial composition containing Bacillus subtilis and Bacillus pumilus. There is also a need for a method to bring the effective bacterial composition into contact with the roots of tomato plants before planting them in the soil.

[0006] There is a need for improved yield predictability, as well as a reduction in the economic and environmental costs associated with the overuse of currently available citrus canker control agents. Therefore, it is desirable to provide bacterial compositions and methods for protecting tomatoes from Cmm, which can be combined with existing herbicides, insecticides, fungicides, bactericides, and nutrients, including but not limited to copper-containing compounds.

[0007] As will become apparent from the following discussion of this disclosure and the description of the features of the present invention, the present invention addresses these and other needs. [Overview of the Initiative]

[0008] According to one embodiment, the present invention relates to a bacterial composition comprising an aqueous mixture of live Bacillus subtilis and live Bacillus pumilus, which is effective in controlling, suppressing, and / or preventing infection of tomatoes with Clavibacter michiganensis subsp. michiganensis (Cmm).

[0009] In another aspect, the present invention relates to a bacterial composition comprising an aqueous mixture of live Bacillus subtilis and live Bacillus pumilus for use in controlling, suppressing, and / or preventing infection of tomatoes with Clavibacter michiganensis subsp. michiganensis (Cmm).

[0010] In another embodiment, the present invention is To prepare a bacterial composition comprising a mixture of Bacillus subtilis and Bacillus pumilus, a culture medium pre-inoculated with Bacillus subtilis and Bacillus pumilus, a cell-free extract of Bacillus subtilis and Bacillus pumilus, and / or one or more metabolites produced by Bacillus subtilis and Bacillus pumilus, and Apply an effective amount of the bacterial composition to one or more of the following: tomato plant body, tomato root, tomato leaf, tomato seed, tomato stem, and tomato fruit. The present invention relates to a method for controlling, suppressing, and / or preventing infection of tomatoes with the tomato canker fungus (Cmm), including the following:

[0011] In the embodiment, the method further includes applying one or more of the following: herbicides, insecticides, fungicides, bactericides, and nutrients.

[0012] In another embodiment, the present invention is To prepare a bacterial composition comprising a mixture of Bacillus subtilis and Bacillus pumilus, a culture medium pre-inoculated with Bacillus subtilis and Bacillus pumilus, a cell-free extract of Bacillus subtilis and Bacillus pumilus, and / or one or more metabolites produced by Bacillus subtilis and Bacillus pumilus. To prepare one or more copper fungicides selected from the group consisting of copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate, and mixtures thereof, and Apply an effective amount of the bacterial composition and copper fungicide to one or more of the following: tomato plant body, tomato roots, tomato leaves, tomato seeds, tomato stems, and tomato fruits. The present invention relates to a method for controlling, suppressing, and / or preventing infection of tomatoes with the tomato canker fungus (Cmm), including the following:

[0013] In another aspect, the present invention relates to a kit for protecting tomatoes from tomato canker fungus (Cmm), comprising (i) a first container containing a bacterial composition as defined herein, and (ii) a second container containing one or more of herbicides, insecticides, fungicides, bactericidal agents, and nutrients.

[0014] In another aspect, the present invention relates to the use of a bacterial composition specified herein for controlling, suppressing, and / or preventing infection of tomatoes with the tomato canker fungus (Cmm).

[0015] In another aspect, the present invention relates to the combined use of (i) a bacterial composition comprising a mixture of live Bacillus subtilis and live Bacillus pumilus, and (ii) one or more of herbicides, insecticides, fungicides, bactericidal agents, and nutrients, for controlling, suppressing, and / or preventing infection of tomatoes with the tomato canker fungus (Cmm).

[0016] Further aspects, advantages, and features of the present invention will become clearer by reading the following non-limiting description of exemplary and preferred embodiments, which should not be construed as limiting the scope of the invention. [Modes for carrying out the invention]

[0017] Embodiments of the present invention are shown in the following description. It will be understood that other embodiments can be made without departing from the scope of the disclosed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains.

[0018] overview The present invention aims to address the high demand for the prevention and control of tomato canker.

[0019] As used herein, the terms "Clavibacter michiganensis subsp. michiganensis" and "Cmm" are used synonymously to refer to the Gram-positive aerobic plant pathogenic bacterium that causes wilting and canker in tomatoes.

[0020] As described below, the present invention proposes novel compositions and methods for controlling, suppressing, and / or preventing Cmm infection in tomatoes.

[0021] bacterial composition One aspect of the present invention relates to a bacterial composition effective against Cmm. The present invention particularly relates to a combination of Bacillus subtilis and Bacillus pumilus. The composition according to the present invention preferably comprises a mixture of live Bacillus subtilis and Bacillus pumilus. In some embodiments, the present invention also includes culture media pre-inoculated with Bacillus subtilis and Bacillus pumilus, cell-free extracts of Bacillus subtilis and Bacillus pumilus, and / or any metabolites produced by Bacillus subtilis and Bacillus pumilus.

[0022] As used herein, the reference to “culture media pre-inoculated with Bacillus subtilis and Bacillus pumilus” means media in which the two bacteria are pre-inoculated together or separately. Therefore, this term encompasses mixtures of two or more culture media pre-inoculated with one or both of the bacteria.

[0023] As used herein, "cell-free extracts of Bacillus subtilis and Bacillus pumilus" refers to a culture medium from which bacteria have been removed (e.g., by filtration, centrifugation, etc.) from a previously defined pre-inoculated culture medium. In embodiments, the cell-free extract is the filtered fraction and / or cell-free supernatant obtained from a culture of Bacillus subtilis and / or Bacillus pumilus.

[0024] As used herein, the reference to "metabolites produced by Bacillus subtilis and Bacillus pumilus" refers to a mixture of peptides, proteins, small molecules, lipids, etc., produced separately or in combination by bacteria (e.g., through synthesis, expression, extraction, secretion).

[0025] In this embodiment, Bacillus pumilus consists of the Bacillus pumilus strain with deposit number PTA-125304, deposited with the American Type Culture Collection (ATCC) on September 26, 2018. In this embodiment, Bacillus pumilus consists of strain NES-CAP-1 (GenBank accession number MF079281.1).

[0026] In an embodiment, the Bacillus subtilis consists of a Bacillus subtilis strain with the deposit number PTA-125303 deposited with ATCC (registered trademark) on September 26, 2018.

[0027] In an embodiment, the Bacillus subtilis consists of a Bacillus subtilis strain with the deposit number PTA-125302 deposited with ATCC (registered trademark) on September 26, 2018.

[0028] In an embodiment, the composition contains live Bacillus subtilis and live Bacillus pumilus in a ratio of 1:1. In other embodiments, this ratio is 2:1. In other embodiments, this ratio is 1:2.

[0029] In an embodiment, the composition contains 6 ~1x10 9 Bacillus pumilus per ml. In an embodiment, the composition contains 1x10 9 or more Bacillus pumilus per ml, or 1x10 8 or more Bacillus pumilus per ml, or 1x10 7 or more Bacillus pumilus per ml, or 1x10 6 or more Bacillus pumilus per ml.

[0030] In an embodiment, the composition contains 1x10 6 ~1x10 9 Bacillus subtilis per ml. In an embodiment, the composition contains 1x10 9 or more Bacillus subtilis per ml, or 1x10 8 or more Bacillus subtilis per ml, or 1x10 7 or more Bacillus subtilis per ml, or 1x10 6 or more Bacillus subtilis per ml.

[0031] Other bacteria, their culture media, their cell-free extracts, and / or their metabolites may also be included in the compositions of the present invention. Examples of other potentially useful microorganisms include Lactobacillus species (e.g., Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus plantarum, etc.), Lactococcus species (e.g., Lactococcus lactis), Bacillus species (e.g., Bacillus amyloliquefaciens), and Aspergillus species (e.g., Aspergillus nihonkoujika). Examples include, but are not limited to, species of the genus oryzae, Candida (e.g., Candida utilis), Pseudomonas (e.g., Pseudomonas aeruginosa, Pseudomonas fluorescens), Saccharomyces (e.g., Saccharomyces cerevisiae), Streptococcus (e.g., Streptococcus lactis), Rhodopseudomonas (e.g., Rhodopseudomonas palustris), and combinations including one or more of the above.

[0032] The bacterial composition of the present invention may further comprise herbicides, insecticides, fungicides, bactericides, nutrients, and mixtures thereof.

[0033] Examples of potentially useful herbicides include Roundup™, Atrazine, 2,4-D (Weed-B-Gon™), Dicamba (Banvel™), Imazetapir (Pursuit™), Metrachlor (Dual™), S-Metrachlor (Dual II Magnum™), Pendimethalin (Prowl™), Cretodim (Select™), Triclopyr (Garlon™), Clopyralide (Stinger™), and Fluro Examples include, but are not limited to, Star™, glufosinate (Liberty™), halosulfuron (Permit™), isoxaben (Gallery™), cethoxydim (Poast™), bentazone (Basagran™), pyraflufen ethyl (Epic™), fomesaphen (Reflex™), trifluralin (Treflan™), mesotrione (Callisto™), flumioxazine (Valor), dicamba (Banvel™, Clarity™), and imazapir (Arsenal™).

[0034] Examples of potentially useful insecticides include, but are not limited to, pyretoids, pyretins, neonicotinoids, organophosphates, carbamates, spinosad, imidacloprid, fipronil, malathion, permethrin, cypermethrin, and chlorpyrifos. Examples of potentially useful bactericidal agents include, but are not limited to, copper sulfate, zinc sulfate, peracetic acid, quaternary ammonium compounds, streptomycin, kasugamycin, tetracycline hydrochloride, phyton 27 (trademark), Aureobasidium pullulans, Actigard (trademark), and Agri-strep (trademark). 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, but are not limited to, copper fungicides containing copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate, and mixtures thereof. Existing commercially available examples include, but are not limited to, Kocide 3000(trademark), Kocide 2000(trademark), Cuprofix Ultra 40 Disperss(trademark), Nordox 75 WG(trademark), Champ WG(trademark), Cueva(trademark), Badge SC(trademark), Basic Copper 53(trademark), Bondide(trademark) copper fungicide, Camelot O(trademark), Nu-Cop 3L(trademark), COCS WDG(trademark), Previsto(trademark), Badge X2(trademark), Badge MAXX(trademark), Cuproxat(trademark) liquid copper fungicide, tribasic copper sulfate, Nordox 30 WG(trademark), Copper-Count-N(trademark) fungicide, Cuprablau Z 35 WP(trademark), and Captain Jack's(trademark) Liquid Copper Fungicide.

[0036] In the embodiment, the bacterial composition is formulated for application to one or more of the tomato plant, tomato roots, tomato leaves, tomato seeds, tomato stems, and tomato fruits.

[0037] As described in more detail below, the bacterial compositions according to the present invention may be applied by any preferred method or technique, including, but not limited to, seed coating, application to seeds before and / or at planting, root inoculation, soil drenching, application to soil (e.g., injection), and foliar application (e.g., high-pressure or low-pressure spraying). In embodiments, application involves immersing the roots of tomato plants in the bacterial composition before planting the tomato plants in the soil.

[0038] In one preferred embodiment, application includes soil drenching. As used herein, the term “soil drenching” refers to the process of directly applying the compound or composition according to the present invention to the base of a plant, preferably to deep and targeted areas.

[0039] As used herein, the term “tomato” refers to any of the more than 10,000 tomato varieties available worldwide. This composition may exhibit useful anti-cancer activity against most, if not all, tomato varieties that are susceptible to Cmm, including greenhouse and open-field varieties. The most common tomato varieties include Harris Moran 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, and Black Cherry Tomato. This includes, but is not limited to, Tomato, Pineapple Tomato, Amish Paste Tomato, Juliet Tomato, Black Beauty Tomato, and Green Zebra Tomato.

[0040] Preferably, the bacterial composition and / or method according to the present invention has the following effects: i. Increase in tomato yield (e.g., increase in yield of extra-large tomatoes), ii. Increase in tomato size, iii. Increase in total tomato yield per acre, iv. Biological defense against Cmm Improved resistance to v.Cmm Mitigation of losses caused by vi.Cmm vii. Severity of citrus canker and reduction of plant tissue affected by citrus canker lesions. viii. Reduction of pathological symptoms or lesions induced by the action of Cmm, and Enhancement of plant antimicrobial response against ix.Cmm, and x. Increased yield of extra-large tomatoes Assign one or more of the following.

[0041] In the embodiments, the effect is observed or evaluated by comparing tomatoes that have been in contact with the bacterial composition of the present invention with tomatoes that have not been in contact with it. In the embodiments, the effect is observed when tomatoes that have been in contact with the bacterial composition are compared with tomatoes that have been in contact with a reference treatment. In the embodiments, the reference treatment consists of Kocide® applied as a foliar spray.

[0042] Furthermore, the bacterial composition according to the present invention may also be found to have other useful preventive and / or therapeutic uses for various crops (e.g., grains), fruits (e.g., small fruits, citrus fruits), vegetables, pasture grasses, turfgrasses, ornamental plants, and the like.

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

[0044] The bacterial composition of the present invention may also be useful for treating bacterial, fungal, and oomycotic pathogens (leaf blight fungi, powdery mildew fungi, mold fungi, rust fungi, scab fungi). It may also be useful for controlling nematodes.

[0045] The bacterial composition of the present invention may also be commercialized as a kit for protecting tomatoes from Cmm. For example, a kit according to the present invention may include (i) a first container containing the bacterial composition as defined herein, and (ii) a second container containing one or more of herbicides, insecticides, fungicides, bactericidal agents, and nutrients. In such a kit, the contents of the first and / or second containers may be ready-to-use solutions or concentrated solutions that have been mixed and / or diluted before use.

[0046] Control methods Another aspect of the present invention relates to a method for controlling tomatoes from the tomato canker fungus (Cmm). In one embodiment, the method comprises applying an effective amount of the bacterial composition specified herein to tomato plants, tomato roots, tomato leaves, tomato seeds, and / or tomato stems. The present invention encompasses the protection of tomatoes in various plant environments, such as fields, greenhouses, vertical farms, urban greening systems, and hydroponic systems.

[0047] In this embodiment, the method imparts one or more of the effects listed above to the bacterial composition.

[0048] The bacterial compositions according to the present invention may be applied by any preferred method, including, but are not limited to, seed coating, application to seeds before and / or at planting, inoculation to roots, soil drenching, application to soil (e.g., injection), and foliar spraying (e.g., high-pressure or low-pressure spraying).

[0049] In the embodiment, application includes immersing the roots of tomato plants in the bacterial composition before planting the tomato plants in the soil.

[0050] In one preferred embodiment, application includes soil drenching. As used herein, the term “soil drenching” refers to the process of directly applying the compound or composition according to the present invention to the base of a plant, preferably to deep and targeted areas.

[0051] In the embodiment, the method further includes applying one or more of the following: herbicides, insecticides, fungicides, bactericides, and nutrients.

[0052] In certain embodiments, the method further comprises applying one or more copper fungicides. In embodiments, the copper fungicides include compounds derived from copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate, and mixtures thereof. In certain embodiments, copper fungicides include, but are not limited to, Kocide 3000(trademark), Kocide 2000(trademark), Kocide LF(trademark), Kocide DF(trademark), Kocide 101(trademark), Cuprofix(trademark) Ultra 40 Disperss(trademark), Nordox(trademark), Nordox 75 WG(trademark), Champ WG(trademark), Cueva(trademark), Badge SC(trademark), Basic Copper 53(trademark), Bondide(trademark), Camelot O(trademark), Nu-Cop 3L(trademark), COCS WDG(trademark), Previsto(trademark), Badge X2(trademark), Badge MAXX(trademark), Cuproxat(trademark), Nordox 30 WG(trademark), Copper-Count-N(trademark) fungicide, and Cuprablau Z 35. The selection is made from existing commercially available formulations, including WP (trademark), Captain Jack's (trademark) Liquid Copper Fungicide, Bordeaux (trademark) mixture, Cuprofix Disperss (trademark), Champion (trademark), Nu-Cop (trademark), Copper Count-N (trademark), Mastercop Instill (trademark), Phyton (trademark), and CS 2005 (trademark).

[0053] Without limiting these, the bacterial composition may be applied a specific number of times, i.e., one or more times, depending on parameters including the Cmm population in the tomato plant or in the soil in which the tomato plant is planted, environmental conditions, tomato susceptibility to disease, and planting time.

[0054] In some embodiments, the bacterial composition is applied if Cmm-related pathological symptoms have occurred in plants that were planted there, if Cmm-related pathological symptoms are currently occurring in plants that are planted there, and / or if Cmm-related pathological symptoms are expected to occur in tomato plants that will be planted there. In some embodiments, the bacterial composition is applied to seeds that will be planted in such soil. In some embodiments, the bacterial composition is applied to seeds derived from tomato parent plants that have been planted in such soil. In some embodiments, the bacterial composition is applied to plants that are planted in such soil. In some embodiments, the bacterial composition is applied to plants that are exhibiting Cmm-related pathological symptoms.

[0055] The bacterial composition may be applied after or before Cmm infection. In some embodiments, the composition is applied at least one week, two weeks, three weeks, one month, two months, three months, four months, five months, or six months before planting seeds or plants. In some embodiments, the bacterial composition is applied at least one week, two weeks, three weeks, one month, two months, or three months after planting seeds or plants. In some embodiments, the bacterial composition is applied one week, two weeks, three weeks, four weeks, five weeks, or five to ten weeks before harvesting tomatoes.

[0056] When treating seeds, the bacterial composition may be applied by various techniques, including, but not limited to, high-pressure or low-pressure spraying, coating, dipping, and injection. After treating the seeds, they can be planted in natural or artificial soil and cultivated according to conventional procedures to obtain plants. After the plants have grown from seeds treated with the bacterial composition, they may be further treated by applying one or more of the disclosed bacterial compositions.

[0057] The disclosed bacterial composition may be applied to the whole or a portion of a tomato plant. For example, the disclosed composition may be applied to the stem, roots, leaves, and / or the reproductive body (e.g., cuttings). The plant may be treated at one or more growth stages. In one embodiment, the disclosed composition is applied to the roots.

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

[0059] In some embodiments, the bacterial composition is supported on a delivery vehicle, which functions as a means of transporting bacteria from the bacterial composition to soil, plants, seeds, fields, etc. For example, the disclosed bacterial composition may be supported on a delivery vehicle (e.g., particles, polymers, or substrates) used in a filtration system for treating irrigation water. In some embodiments, the bacterial composition is supported on a polymer as a gel that releases a wetting agent and / or water as needed.

[0060] The bacterial composition of the present invention is preferably applied in an amount effective for the biological defense of tomatoes against Cmm (e.g., control, suppression, and / or prevention of Cmm infection). In some embodiments, the amount is sufficient to prevent Cmm infection. In some embodiments, the amount is sufficient to treat or reduce one or more symptoms associated with Cmm.

[0061] In some embodiments, the amount is sufficient to reduce the Cmm concentration in the tissue of tomato plants treated with the bacterial composition. In some embodiments, the Cmm concentration measured in the tissue of tomato plants 10 days after the application process is 10 9 It is less than CFU / g. In some embodiments, the Cmm concentration measured in the tissue of tomato plants 21 days after the application process was 10 9 The Cmm concentration is less than CFU / g. In some embodiments, the Cmm concentration measured in the tissue of tomato plants 3, 5, 7, 14, 21, 28, 35, or 42 days after the application process is 10 9The Cmm concentration is less than CFU / g. In some embodiments, the Cmm concentration measured in the tissue of tomato plants 3, 5, 7, 14, 21, 28, 35, or 42 days after the application process is 10 8 The Cmm concentration is less than CFU / g. In some embodiments, the Cmm concentration measured in the tissue of tomato plants 3, 5, 7, 14, 21, 28, 35, or 42 days after the application process is 10 7 The Cmm concentration is less than CFU / g. In some embodiments, the Cmm concentration measured in the tissue of tomato plants 3, 5, 7, 14, 21, 28, 35, or 42 days after the application process is 10 6 In some embodiments,

[0062] In some embodiments, Cmm infection is measured by evaluating the canker severity or incidence rate in the tissues of tomato plants on a scale of 0 to 100%, where 0% indicates no disease severity and 100% indicates the plant is completely infected. The canker control rate (%) for the entire plant may be calculated from SAUDPC (i.e., area under the standardized disease progression curve) using Abbott's formula. In some embodiments, the bacterial composition of the present invention is applied in an amount effective to control, suppress, and / or prevent canker severity, compared to untreated control tomato plants or crops, at a rate of 1% or more, or 2% or more, or 3% or more, or 4% or more, or 5% or more, or 6% or more, or 7% or more, or 8% or more, or 9% or more, or 10% or more.

[0063] Those skilled in the art will understand that the prescribed application rate and required application frequency may vary depending on the type and condition of the soil, the type and condition of the tomato, the pathogenicity and activity of Cmm, etc. Furthermore, the prescribed amount may also vary depending on the environment, for example, whether the plants are in pots, greenhouses, fields, etc.

[0064] The predetermined amount can be determined by using methods known in the art, for example, by testing a dose-dependent response. In some embodiments, the predetermined amount is determined by testing a dose-dependent response in a culture plate containing Cmm, for example, by measuring the inhibition zone. In some embodiments, the predetermined amount is determined by testing a dose-dependent response in a pot or field. In some embodiments, the predetermined amount is determined based on the measurement of the concentration of Cmm or the amount of Cmm-specific genes in the tissue of tomato plants treated with the bacterial composition. In some embodiments, the predetermined amount is determined based on the concentration of Bacillus pumilus, Bacillus subtilis, or both.

[0065] In some embodiments, the bacterial culture applied to the tomato plant is 10 6 ~10 9 CFU / mL, or 10 7 ~10 9 CFU / mL, or 2.5 x 10 7 ~10 9 CFU / mL, or 2.5 x 10 7 ~8.5x10 8 CFU / mL, or 5x10 7 ~8.5x10 8 CFU / mL, or 2x10 8 ~8.5x10 8 CFU / mL, or 10 8 It contains Bacillus pumila at a concentration of CFU / mL. In some embodiments, the bacterial culture applied to tomato plants is 10 6 ~10 9 CFU / mL, or 10 7 ~10 9 CFU / mL, or 2.5 x 10 7 ~10 9 CFU / mL, or 2.5 x 10 7 ~8.5x10 8 CFU / mL, or 5x10 7 ~8.5x10 8 CFU / mL, or 2x10 8 ~8.5x10 8 CFU / mL, or 10 8Contains Bacillus subtilis at a concentration of CFU / mL.

[0066] In some embodiments, the bacterial composition is applied to tomato plants to increase the final concentration of Bacillus pumilus measured in the roots, stems, or leaves of the tomato plants to 10 6 ~10 9 CFU / mL, or 10 7 ~10 9 CFU / cm 3 , or 2.5x10 7 ~10 9 CFU / cm 3 , or 2.5x10 7 ~8.5x10 8 CFU / cm 3 , or 5x10 7 ~8.5x10 8 CFU / cm 3 , or 2x10 8 ~8.5x10 8 CFU / cm 3 , or 3x10 8 ~8x10 8 CFU / cm 3 , or 10 8 CFU / cm 3 Keep it within the range.

[0067] In some embodiments, the bacterial composition is applied to tomato plants to increase the final concentration of Bacillus subtilis measured in the roots, stems, or leaves of the tomato plants to 10 6 ~10 9 CFU / mL, or 10 7 ~10 9 CFU / cm 3 , or 2.5x10 7 ~10 9 CFU / cm 3 , or 2.5x10 7 ~8.5x10 8 CFU / cm 3 , or 5x10 7 ~8.5x10 8 CFU / cm 3 , or 2x10 8 ~8.5x10 8 CFU / cm 3 , or 3x10 8~8 x 10 8 CFU / cm 3 、 or 10 8 CFU / cm​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0071] Example 1 - Evaluation of CXC efficacy To evaluate the efficacy and activity of the composition according to the present invention (hereinafter referred to as "CXC®") against tomato canker (tomato canker fungus) when applied by root immersion and soil drenching, a field trial was conducted in Florida over a period of three months (September 2022 to December 2022). The activity and efficacy of CXC were compared with Kocide 3000®, a currently available formulation for tomato canker, and an untreated control group.

[0072] Materials and methods

[0073] CXC composition

[0074] The bacterial composition tested (hereinafter referred to as "CXC" (or "CXC biocontrol" in Examples 2 and 3)) contained 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 present at approximately 10 7 ~10 9 The concentration was 1 / ml. To obtain the CXC composition, two microorganisms were grown separately in LB culture medium containing tryptone (10g), yeast extract (5g), and NaCl (5g) in 1 liter of water at 28°C for at least 72 hours using a shaking incubator (125 rpm). The bacterial count was measured by counting the CFU per 1 mL using the serial dilution method. The bacterial cultures were serially diluted, and a portion of each was seeded onto LB agar medium and incubated at 28°C for at least 48 hours before counting the CFU. After final collection, the cultures of the two microorganisms were mixed together (1:1) and stored in a refrigerator until use.

[0075] Field conditions

[0076] The experiment was conducted in soil with an overall pH of 6.6 and an overall cation exchange capacity level of 3.7. The soil composition included 0.5% organic matter, 90.4% sand, 4.4% silt, and 5.2% clay.

[0077] On September 21, 2022, Harris Moran 1823 (HM 1823) variety of fresh market tomato was transplanted under simulated commercial conditions (row spacing 6 feet, plant spacing 19 inches) at a total planting density of 4,585 plants / acre. Each experimental plot consisted of four replicates on a single row, with a row length of 32 feet, using the randomized block method, and was drip-irrigated.

[0078] Two weeks after planting (October 2, 2022), the plants were inoculated with canker fungus using a backpack sprayer (246 cells / ml).

[0079] CXC treatment was applied one, two, or three times by manual pouring. The first application was at planting time (referred to as Treatment A, Week 0, September 21, 2022), followed by a second application two weeks later (referred to as Treatment B, Week 2, October 6, 2022), and finally a third application three weeks after Treatment B (referred to as Treatment C, Week 5, November 3, 2022).

[0080] As a reference treatment, Kocide 3000 (trademark) (hereinafter referred to as "Kocide") was applied weekly from October 5, 2022, as a foliar spray at different concentrations (see Table 1 below) using a tractor-towed sprayer (referred to as D to K, weeks 2 to 9). The sprayer attached to the tractor was operated at 200 PSI.

[0081] An untreated group was included as a reference (control).

[0082] evaluation

[0083] The severity of canker disease in 10 plants per test plot was evaluated on a scale from 0% to 100%. 100% represents plants with lesions in all tissues. Scoring was performed on the entire plant, newly growing parts, and stems of the 10 plants. Evaluation of the entire plant was 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). Disease evaluation of newly growing parts and stems was performed on November 3 (1 DA-H). Canker disease control was calculated from the area under the standardized disease progression curve (SAUDPC) using Abbott's correction formula (average severity over time).

[0084] Tomatoes were harvested by hand and sorted into five categories: extra-large, large, medium, sunburnt, and other discarded. Harvesting took place on November 23rd (8 DA-J) and November 30th (7 DA-K). The number and weight of small, medium, large, and extra-large tomatoes that were shippable were recorded, while only the number of unshippable tomatoes and tomatoes infected with canker were recorded.

[0085] result Table 1 shows the results regarding the severity (%) of canker disease in the entire plant. The severity of canker disease was scored on a scale from 0 to 100%. 0% indicates no disease severity, and 100% indicates that the crop was completely infected with the disease. Evaluations of the entire plant 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). Table 1 Severity of canker disease in the entire plant (%) [Table 1]

[0086] Table 2 shows the results regarding the incidence rate of canker disease in the entire plant population (%). The incidence rate of canker disease in the entire plant population (%). The incidence rate of canker disease was scored on a scale from 0 to 100%. Evaluations of the entire plant population 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). Table 2 : Percentage of canker disease incidence in the entire plant (%) [Table 2]

[0087] Table 3 shows the overall canker control rate (%) of the plant, calculated from SAUDPC using Abbott's correction formula. Table 3 : SAUDPC (Sea Canker) disease rate and control rate (%) across the entire plant [Table 3]

[0088] Table 4 shows the results regarding canker disease in newly grown parts. Canker disease severity was rated on a scale of 0-100%. 0% indicates no disease severity, and 100% indicates the crop is completely infected with the disease. Disease evaluation of the new growing parts and stems was performed on November 3 (1 DA-H). Table 4 : Canker disease in newly growing parts (%) [Table 4]

[0089] Table 5 shows the results regarding the control of stem canker disease. Canker disease severity was scored on a scale of 0 to 100%. 0% indicates no disease severity, and 100% indicates that the crop was completely infected with the disease. Disease evaluation of new growing parts and stems was performed on November 3 (1 DA-H). Table 5Stem canker (%) [Table 5]

[0090] Table 6 shows the number of tomatoes infected with canker disease per test plot. On November 20th (5 DA-J), November 25th (2 DA-K), and November 30th (7 DA-K), 50 tomatoes in each test plot were counted and the average number of infected tomatoes in the field was calculated. Table 6 : Number of tomatoes infected with citrus canker (tomatoes / test plot, average of 50 tomatoes) [Table 6]

[0091] Tables 7 and 8 show the number (units) and weight (lb) of extra-large tomatoes harvested per test plot on November 23rd and November 30th, respectively. The harvested tomatoes were classified into five categories: extra-large, large, medium, sunburnt, and other discarded tomatoes. Harvesting took place on November 23rd (8 DA-J) and November 30th (7 DA-K). Table 7 : Total number of extra-large tomatoes harvested (tomatoes / test plot) [Table 7] Table 8 Total weight (lb) of harvested extra-large tomatoes [Table 8]

[0092] Tables 9 and 10 show the number (pieces) and weight (lb) of large tomatoes per experimental plot, respectively, that were harvested and classified into five categories (extra-large, large, medium, sunburned, and other discarded fruits). Harvesting took place on November 23 (8 DA-J) and November 30 (7 DA-K). Table 9 : Total number of large tomatoes harvested (tomatoes / test plot) [Table 9] Table 10 Total number of large tomatoes harvested (lb) [Table 10]

[0093] Tables 11 and 12 show the number of medium-sized tomatoes (pieces) and total weight (lb) per test plot for harvests taken on November 23rd and November 30th. Harvesting took place on November 23rd (8 DA-J) and November 30th (7 DA-K). Table 11 : Total number of medium-sized tomatoes harvested (tomatoes / test plot) [Table 11] Table 12 Total number of medium-sized tomatoes harvested (lb) [Table 12]

[0094] Tables 13 and 14 show the number of sunburnt tomatoes and other discarded tomatoes per experimental plot, harvested on November 23 (8 DA-J) and November 30 (7 DA-K), respectively. Table 13 : Total number of sunburnt tomatoes harvested (tomatoes / test plot) [Table 13] Table 14 : Total number of other discarded tomatoes harvested (tomatoes / test plot) [Table 14]

[0095] Table 15 shows the total number of tomatoes harvested per test plot, categorized, for both November 23 (8 DA-J) and November 30 (7 DA-K). Table 15 : Total number of tomatoes harvested (tomatoes / test plot) [Table 15]

[0096] Table 16 shows the composition of the total harvest conducted on November 23rd and November 30th, broken down by category. Table 16 : Composition of all harvested tomatoes (%) [Table 16]

[0097] Table 17 shows the total weight (lb) of harvested, marketable tomatoes per test plot, broken down by category. Table 17 Total weight (lb) of harvested, marketable tomatoes [Table 17]

[0098] Table 18 shows estimated sales revenue. The estimated weight (MW) of harvested, marketable tomatoes was converted to pounds per acre (lb / acre). Estimated total revenue was calculated based on a tomato price of $18.80 per 25 lb (dollars / acre). Table 18: Estimated Sales Revenue [Table 18]

[0099] Consideration

[0100] The proportion of plants exhibiting some symptoms reached 100% (all plants) approximately four weeks after infection, and the spread of the disease was delayed in the Kocide-treated plots. Overall, the untreated plots consistently had significantly higher mean canker severity than the treated plots, a positive dose-response was observed with CXC treatment, and the plots treated three times had the lowest mean severity.

[0101] As shown in Tables 1 to 3, consistent improvements in the control of canker disease severity and progression were observed when treated with the CXC composition according to a three-application system. Treatment with CXC according to all systems showed substantial improvements in the control of canker disease severity, not only compared to untreated cases but also compared to Kocide 3000™, a commercially available formulation currently widely used for this purpose. Therefore, the composition of the present invention can lead to improved canker disease control, enhanced yield predictability, and reductions in economic and environmental costs associated with the heavy use of currently available canker disease control agents.

[0102] In new growth areas and stems, tomato plants treated with CXC once and twice showed the best control of citrus canker (Tables 4 and 5).

[0103] On average across all harvest seasons, a considerable number of tomatoes were infected with citrus canker, but tomatoes collected from the high-concentration CXC treatment plots showed the lowest infection rates (Table 6).

[0104] As shown in Tables 7 to 14, when CXC was applied three times, it was possible to harvest more extra-large tomatoes (number of tomatoes per plot and total harvest in lbs) than with Kocide 3000 (trademark) or the untreated plot. Furthermore, when CXC was applied once, it was possible to harvest more large tomatoes (number of tomatoes per plot and total harvest in lbs) than with Kocide (trademark) or the untreated plot. In addition, when CXC was applied three times, it was possible to harvest more medium-sized tomatoes (number of tomatoes per plot and total harvest in lbs) than with Kocide (trademark).

[0105] There was no significant difference in the yield of marketable tomatoes based on the number of fruits, and the harvested produce consisted mostly of large or extra-large tomatoes (Tables 15-16). However, the yield weight was significantly higher in the extra-large tomatoes grown in the experimental plots treated with CXC three times (Table 17).

[0106] The estimated total revenue was highest with the treatment that included three applications of CXC (Table 18).

[0107] Important insights: Canker control was dose-dependent in CXC, and the mean control was significantly higher after three treatments.

[0108] The yield of tomatoes infected with citrus canker was highest in the untreated or Kocide-treated plots and lowest in the plot treated with CXC three times, demonstrating the effectiveness of CXC treatment.

[0109] Furthermore, these results suggest that the use of CXC compositions improves yield predictability.

[0110] Furthermore, these results suggest that the CXC composition is a very interesting alternative to currently available canker control agents.

[0111] Example 2 - Synergistic effect with the composition of the present invention Kocide(trademark) To evaluate the efficacy and activity of the compositions according to the present invention (hereinafter referred to as "CXC ABIO®" and "CXC Biocontrol®"), either alone or in combination with Kocide®, against tomato canker, a field trial was conducted in Thonotosassa, Florida, over a period of four months (May 2023 to August 2023).

[0112] Materials and methods

[0113] Test CXC composition

[0114] The following CXC bacterial compositions, 1) "CXC Biocontrol (trademark)" and 2) "CXC ABIO," were tested. As previously defined, composition "CXC Biocontrol (trademark)" corresponds to composition "CXC" in Example 1. Composition "CXC ABIO" is a biostimulant containing bacillin 20.

[0115] The bacillin 20 used in this field trial consisted of a bacillin 20 aqueous stock solution. Bacillin 20 was purified from Bacillus thuringiensis MS20 strain. The process used to isolate bacillin 20 included the following steps: 1) culturing of microbial culture, 2) centrifugation and membrane filtration, 3) butanol extraction of organic metabolites, 4) evaporation of butanol and concentration of the extract, 5) fractionation of the organic extract using acetonitrile solvent, 6) purification of bacillin 20 by HPLC, and 7) mass spectrometry of bacillin 20 to confirm its identity.

[0116] In step 1), the inoculum Bacillus thuringiensis MS20 was prepared and cultured on a large scale in a bioreactor. In step 2), bacterial clumps were removed by centrifugation and membrane filtration to obtain a cell-free supernatant. In step 3), butanol extraction was performed by mixing the microbial cell-free supernatant with 1-butanol and shaking vigorously to separate the 1-butanol into phases. In step 4), butanol was evaporated by vacuum evaporation of 1-butanol using a rotary vaporizer to obtain an organic extract. Next, in step 5), the organic extract was fractionated to remove undesirable "junk" components, and only the active fraction of bacillin 20 was retained for further purification. In step 6), bacillin 20 was purified by high-performance liquid chromatography (HPLC) using an acetonitrile / water gradient to obtain bacillin 20 with a purity of 85-95%. In step 7), the purified sample was subjected to mass spectrometry to identify various fractions using pure bacillin 20 as a comparative internal standard. Dilute purified bacillin 20 (i.e., purity approximately 85-95%) with phosphate-buffered saline to a concentration of 10 -9 A stock solution containing M basilin 20 was obtained. The stock solution of basilin 20 was diluted 100 times with water to obtain the final usable solution. -11 A diluted working solution was obtained and used for seed treatment.

[0117] Kocide 3000® (hereinafter referred to as "Kocide") was applied as a foliar spray at a rate of 1.5 lb / a using a tractor-towed sprayer. The tractor-mounted sprayer was operated at 200 PSI. When used in conjunction with CXC BioControl and / or CXC ABIO, Kocide® was added to each treatment tank at the desired rate (see, for example, the preparation of treatments 4 and 5 described below).

[0118] Field conditions

[0119] The experiment was conducted in soil with an overall pH of 6.4 and an overall cation exchange capacity level of 4. The soil composition included 2.1% organic matter, 99.5% sand, and 0.5% clay, with virtually no silt. The average temperature ranged from approximately 22°C (72°F) to 30°C (87°F), and the cumulative measured rainfall was 77.6 cm (30.54 inches).

[0120] On May 4, 2,255 varieties of tomatoes were manually transplanted under simulated commercial conditions (row spacing of 6 feet, plant spacing of 19 inches) at an overall planting density of 4,585 plants per acre. CXC Biocontrol was applied as a soil drenching on May 5, followed by further soil drenching on May 27 and June 23.

[0121] When using CXC ABIO, it was applied in conjunction with foliar application of Kocide (trademark) during each week from May 5th to July 18th. 80 ml of undiluted CXC ABIO was diluted with 8 L of water to a concentration of 10. -9 Prepared treatment 4 by setting it to M, and then added Kocide (trademark) to the tank at a rate of 1.5 LB / A. Before adding Kocide (trademark), the concentration was 10-9 80 mL was taken from the tank containing the mixture of treatment 4 (M) to prepare treatment 5. Next, 80 mL of the solution taken from treatment 4 was mixed with 8 L of water, and 10 -11 Solution M was prepared. 10 -11 After preparing the M solution, Kocide (trademark) was added to the tank at a rate of 1.5 LB / A. An untreated control group was included as a reference. Each test group consisted of 4 replicates using a randomized block method with a row length of 29 feet. An untreated control group was included as a reference (control).

[0122] evaluation

[0123] The severity of canker disease was assessed for the entire plant at 11 harvest times and for new shoots at 2 times. The incidence of canker disease in the entire plant was 100% in mid-June.

[0124] Fifty tomato samples were collected three times, and the number of fruits infected with citrus canker was reported.

[0125] The tomatoes were harvested twice, and the number and weight of both marketable and unmarketable tomatoes were recorded. The yields were similar.

[0126] result

[0127] Table 19 shows the severity of canker disease in the entire plant (0-100). The severity of canker disease in 10 plants was averaged and scored on a scale of 0-100. 0 indicates a completely healthy plant, and 100 indicates a plant completely infected with the disease. The evaluation was conducted on May 13 (8 DA-A), May 20 (3 DA-E), May 29 (2 DA-B), June 8 (7 DA-G), June 16 (15 AD-G), June 24 (1 DA-C), July 3 (10 DA-C), July 11 (6 DA-J), July 26 (8 DA-K), August 1 (14 DA-K), and August 9 (22 DA-K). Table 19 Severity of canker disease in the entire plant (0-100) [Table 19]

[0128] Table 20 shows the percentage of canker disease incidence across the entire plant population. The severity of canker disease was averaged across 10 plants and scored on a scale from 0 to 100. 0 indicates a completely healthy plant, and 100 indicates a plant completely infected with the disease. The evaluation was conducted on May 13 (8 DA-A), May 20 (3 DA-E), May 29 (2 DA-B), June 8 (7 DA-G), June 16 (15 AD-G), June 24 (1 DA-C), July 3 (10 DA-C), July 11 (6 DA-J), July 26 (8 DA-K), August 1 (14 DA-K), and August 9 (22 DA-K). Table 20 : Percentage of canker disease incidence in the entire plant (%) [Table 20]

[0129] Table 21 shows the percentages of SAUDPC (Severe Artificial Canker) in the entire plant population and the control group. Table 21 : Canker disease affecting the entire plant (SAUDPC and control rate (%)) [Table 21]

[0130] Table 22 shows the severity of canker disease on new shoots of plants. The severity of canker disease was averaged from 10 plants and scored on a scale from 0 to 100. 0 indicates a completely healthy plant, and 100 indicates a plant completely infected with the disease. The evaluation was conducted on June 16 (15 DA-G) and July 19 (75 DA-A). Table 22 Severity of canker disease on new plant shoots (0-100) [Table 22]

[0131] Table 23 shows the incidence rate (%) of canker disease on new plant shoots. The incidence rate of canker disease was averaged from 10 plants and scored on a scale from 0 to 100. 0 indicates a completely healthy plant, and 100 indicates a plant completely infected with the disease. The evaluation was conducted on June 16 (15 DA-G) and July 19 (75 DA-A). Table 23 : Incidence rate of canker disease on new plant shoots (%) [Table 23]

[0132] Table 24 shows the incidence of canker disease in plant shoots (SAUDPC) as a percentage of the control group. Table 24 : Incidence rate of canker disease on plant shoots (SAUDPC and control rate (%)) [Table 24]

[0133] Table 25 shows the number of fruits infected with citrus canker (number of fruits per test plot). Fruits infected with citrus canker were counted before harvest. Counting was performed on July 17 (12 DA-J), July 25 (7 DA-K), and August 3 (16 DA-K). Table 25 : Number of fruits infected with citrus canker (fruits / test plot) [Table 25]

[0134] Table 26 shows the percentage of fruits infected with citrus canker. Fruits infected with citrus canker were counted before harvest. Counting was performed on July 17 (12 DA-J), July 25 (7 DA-K), and August 3 (16 DA-K). Table 26 Fruit infected with citrus canker (%) [Table 26]

[0135] Consideration

[0136] The average disease severity was significantly higher in the untreated plants (Table 19). The number of fruits infected with canker was significantly higher in the untreated groups and lowest in plants treated with both CXC Biocontrol® and Kocide® (Tables 25 and 26).

[0137] When Kocide™ and CXC Biocontrol™ were used in combination, canker disease was suppressed significantly better compared to when either was used alone (Tables 25 and 26).

[0138] These results clearly demonstrate that combined treatment with CXC Biocontrol® and Kocide yields better results than treatment with either CXC Biocontrol® or Kocide® alone, showing a synergistic effect in combined treatment.

[0139] The incidence of canker disease in new plant shoots was lowest when CXC biocontrol was used (Treatment 1). The isolated treatment (Treatment 4) was the second most effective in reducing the incidence of canker disease, followed by Treatment 5 (tank mixing alone) (Table 23).

[0140] Overall, these results clearly demonstrate that the compositions according to the present invention are effective and can effectively improve and / or complement existing canker control methods. Therefore, the compositions and methods according to the present invention may also be useful in reducing the economic and environmental costs associated with the heavy use of existing commercially available canker control agents.

[0141] Example 3 - Beneficial effects of CXC Biocontrol (trademark) applied as a drenching and foliar spray, either alone or in combination with Kocide (trademark). To evaluate the efficacy and activity of the compositions according to the present invention (hereinafter referred to as "CXC Biocontrol" or "CXC Biocontrol" in combination with Kocide®) against tomato canker when applied by drenching and / or foliar spray, a field trial was conducted in Thonotosassa, Florida, over a period of four months (October 2023 to February 2024).

[0142] Materials and methods

[0143] process

[0144] The following processes were tested for this study. 1. Untreated control 2.CXC Biocontrol 1% v / v (A, root soaking), CXC Biocontrol 0.01% (B~N, AA~AC, canning) 3.Kocide (trademark) 1.5lb / a (O~Z, AD~AG, foliar) 4.CXC Biocontrol 1% v / v (A, root soak), CXC Biocontrol 0.01% (CEGIKM, can injection), Kocide (trademark) 1.5lb / a (PRTVXZ) 5.CXC Biocontrol 1% v / v (O~Z AD~AG, foliar), Kocide(TM) 1.5lb / a (O~Z, AD~AG, foliar) 6.CXC Biocontrol 1% v / v (A, root soak), CXC Biocontrol 0.1% v / v (DHL, can injection), Actigard (trademark) 0.75oz / a (QUY, foliar), Firewall (trademark) 200ppm pr (RVZ, foliar), Kocide (trademark) 1.5lb / a (PTX, foliar)

[0145] As defined earlier, "CXC Biocontrol (trademark)" corresponds to the composition "CXC" in Example 1.

[0146] Experimental System

[0147] On October 17, 2023, 1823 varieties of tomatoes were manually transplanted in rows 6 feet wide and 19 inches apart. As the initial application, the roots were immersed in 1% v / v CXC Biocontrol™ before transplanting. Applications B-N and AA-AC were applied directly to the base of the plants as soil drenching. The remaining applications (O-Z, AD-AG) were applied using a tractor fitted with a Nifty Fifty sprayer at an operating pressure of 200 PSI. The remaining applications AA-AG were added to the original spraying system, and application was extended until harvest. Treatments included CXC Biocontrol, Kocide, CXC Biocontrol applied as foliar in combination with Kocide, or CXC Biocontrol applied as drenching in combination with Kocide, as well as CXC Biocontrol applied sequentially in combination with Actigard, Firewall, and Kocide. An untreated control was also included for reference. Each test plot consisted of four replicates using a randomized block method, with a row length of 25 feet.

[0148] Application equipment

[0149] Application A was root immersion before transplanting. Applications B-N and AA-AC were applied directly to the base of the plant as soil drenching. Applications O-Z and AD-AG were foliar sprays applied using a tractor equipped with a nifty-fifty system operating at 200 psi.

[0150] evaluation

[0151] The severity of canker disease in 10 plants was averaged and scored on a scale from 0 to 100. 0 indicates a completely healthy plant, and 100 indicates a plant completely infected with the disease. The evaluations were conducted 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).

[0152] On December 29 (3 DA-Y), the severity of bacterial spot disease was evaluated on a scale of 0 to 100 (averaging the severity of 10 strains per test plot).

[0153] Tomatoes were harvested three times throughout the season, targeting extra-large, large, medium, pest-damaged, sunburnt, and canker-infected varieties. Harvesting took place on January 19th (4 DA-AA), February 1st (3 DA-AG), and February 12th (14 DA-AG).

[0154] result

[0155] Table 27 shows the severity of canker disease in the entire plant (0-100). The severity of canker disease in 10 plants was averaged and scored on a scale of 0-100. 0 indicates a completely healthy plant, and 100 indicates a plant completely infected with the disease. The evaluation was conducted 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). Table 27 Severity of canker disease in the entire plant (0-100) [Table 27]

[0156] As shown in Table 27, the disease outbreak pressure remained relatively low throughout the test period, and was highest in the untreated area.

[0157] In the treatment group treated with CXC Biocontrol alone, control of canker disease was significantly better, followed by the treatment group treated with CXC Biocontrol administered as an injection in combination with Kocide (Table 28). Table 28: SAUDPC and control rate (%) of canker disease throughout the entire plant. [Table 28]

[0158] Bacterial spot disease was observed on December 29 (3 DA-Y). Observations over 1 day showed that CXC Biocontrol alone provided the best control of the bacteria (Table 29). Table 29: Control rate of bacterial leaf spot disease throughout the entire plant (%) [Table 29]

[0159] In each experimental plot, extra-large fruits, large fruits, medium-sized fruits, sunburnt fruits, fruits damaged by insects, and tomatoes infected with citrus canker were harvested three times. The number of fruits infected with citrus canker was lowest when Kocide and CXC Biocontrol were applied sequentially (Table 30). Table 30: Total number of tomatoes harvested (tomatoes / test plot) [Table 30]

[0160] No significant differences were reported in the overall estimated marketable yield. However, numerically, the highest estimated yields were reported in the experimental plots treated with CXC Biocontrol alone, or as a drenching or spraying agent in combination with Kocide (Table 31). Table 31 Estimated shippable weight (lb / acre) [Table 31] Consideration Overall, these results clearly demonstrate that when the compositions according to the present invention are applied as irrigation, they are effective in controlling, suppressing, and / or preventing infection by the tomato canker fungus (Cmm) in tomatoes. Furthermore, this study shows that the compositions according to the present invention can be used in combination with existing commercially available fungicides and can be mixed together in tanks without concern that the fungicides will denature, affect, or damage our compositions of the present invention. It has been confirmed that the compositions according to the present invention are useful when used alone or in combination with existing canker control methods such as Kocide®.

[0161] For reference and to help locate certain sections, this specification includes headings. These headings are not intended to limit the scope of the concepts set forth therein, and these concepts may be applicable to other sections throughout this specification. Accordingly, the present invention is not intended to be limited to the embodiments shown herein, but rather to encompass the broadest scope consistent with the principles and novel features disclosed herein.

[0162] The singular forms “a,” “an,” and “the” include references to the corresponding plural unless otherwise explicitly indicated by the context. For example, “a tomato” includes one or more tomato plants or tomato fruits (i.e., tomatoes), and “the method” includes references to equivalent steps and methods known to those skilled in the art that can be used to carry out modifications or substitutions of the methods described herein.

[0163] Unless otherwise specified, all figures representing quantities, reaction conditions, concentrations, properties, etc., of components used herein and in the claims should be understood to be modified in all cases by the term "approximately." At a minimum, each numerical parameter should be interpreted by applying the usual rounding in light of the reported number of significant figures. Thus, unless otherwise stated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the properties to be obtained. While the numerical ranges and parameters representing a wide range of embodiments are approximations, the figures shown in specific examples are reported as accurately as possible. However, all figures inherently contain certain errors arising from variations in experiments, test measurements, statistical analyses, etc.

[0164] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in light of them will be understood to those skilled in the art as being suggested and included within the scope of the present invention and the appended claims.

Claims

1. A bacterial composition comprising an aqueous mixture of live Bacillus subtilis and live Bacillus pumilus, effective for controlling, suppressing, and / or preventing infection of tomatoes with Clavibacter michiganensis subsp. michiganensis (Cmm).

2. A bacterial composition comprising an aqueous mixture of live Bacillus subtilis and live Bacillus pumilus, for use in controlling, suppressing, and / or preventing infection of tomatoes with Clavibacter michiganensis subsp. michiganensis (Cmm).

3. The bacterial composition according to claim 1 or claim 2, wherein the Bacillus subtilis and Bacillus pumilus are present in a ratio of approximately 1:

1.

4. Each of the aforementioned Bacillus subtilis and Bacillus pumila is present in the composition at a rate of about 10 ７ ~about 10 ９ A bacterial composition according to any one of claims 1 to 3, which is present at a concentration of cells / ml.

5. The bacterial composition according to any one of claims 1 to 4, wherein the Bacillus pumilus is ATCC (registered trademark) strain deposit number PTA-125304, and the Bacillus subtilis is ATCC (registered trademark) strain deposit number PTA-125303.

6. A bacterial composition according to any one of claims 1 to 5, formulated for soil drenching.

7. A bacterial composition according to any one of claims 1 to 5, formulated for foliar application.

8. By applying the above composition to one or more of the tomato plant, tomato roots, tomato leaves, tomato seeds, tomato stems, and tomato fruits, the following effects are obtained: (i) Increase in tomato yield, (ii) Increase in tomato size, (iii) Increase in total tomato yield per acre, (iv) Biological defense against Cmm (v) Improved resistance to Cmm, (vi) Reduction of losses caused by Cmm, (vii) Reduction of the severity of canker disease and the plant tissue affected by canker lesions, (viiii) Reduction of pathological symptoms or lesions induced by the action of Cmm, Enhancement of the plant antimicrobial response to (ix)Cmm, and (x) Increased yield of extra-large tomatoes A bacterial composition according to any one of claims 1 to 7, wherein one or more of the following are granted.

9. To prepare a bacterial composition comprising a mixture of Bacillus subtilis and Bacillus pumilus, a culture medium pre-inoculated with Bacillus subtilis and Bacillus pumilus, a cell-free extract of Bacillus subtilis and Bacillus pumilus, and / or one or more metabolites produced by Bacillus subtilis and Bacillus pumilus, and Apply an effective amount of the bacterial composition to one or more of the following: tomato plant body, tomato root, tomato leaf, tomato seed, tomato stem, and tomato fruit. A method for controlling, suppressing, and / or preventing infection of tomatoes with the tomato canker fungus (Cmm), including the following.

10. The method according to claim 9, further comprising applying one or more of the following: herbicides, insecticides, fungicides, bactericides, and nutrients.

11. The method according to claim 10, wherein the one or more copper fungicides are selected from the group consisting of copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate, and mixtures thereof.

12. To prepare a bacterial composition comprising a mixture of Bacillus subtilis and Bacillus pumilus, a culture medium pre-inoculated with Bacillus subtilis and Bacillus pumilus, a cell-free extract of Bacillus subtilis and Bacillus pumilus, and / or one or more metabolites produced by Bacillus subtilis and Bacillus pumilus. To prepare one or more copper fungicides selected from the group consisting of copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate, and mixtures thereof, and Apply an effective amount of the bacterial composition and the copper fungicide to one or more of the tomato plant, tomato roots, tomato leaves, tomato seeds, tomato stems, and tomato fruits. A method for controlling, suppressing, and / or preventing infection of tomatoes with the tomato canker fungus (Cmm), including the following.

13. The copper fungicide may be Kocide 3000(TM), Kocide 2000(TM), Kocide LF(TM), Kocide DF(TM), Kocide 101(TM), Cuprofix(TM) Ultra 40 Disperss(TM), Nordox(TM), Nordox 75 WG(TM), Champ WG(TM), Cueva(TM), Badge SC(TM), Basic Copper 53(TM), Bonide(TM), Camelot O (trademark), Nu-Cop 3L (trademark), C-O-C-S WDG (trademark), Previsto (trademark), Badge X2 (trademark), Badge The method according to claim 11 or claim 12, selected from the group consisting of MAXX (trademark), Cuproxat (trademark), Nordox 30 WG (trademark), Copper-Count-N (trademark) fungicide, Cuprablau Z 35 WP (trademark), and Captain Jack's (trademark) Liquid Copper Fungicide, Bordeaux mixture (Bordeaux (trademark) mixure), Cuprofix Disperss (trademark), Champion (trademark), Nu-Cop (trademark), Copper-Count-N (trademark), Mastercop Instill (trademark), Phyton (trademark), and CS 2005 (trademark).

14. The method according to any one of claims 9 to 13, wherein the application includes immersing the roots of the tomato plants in the bacterial composition before planting the tomato plants in the soil.

15. The method according to any one of claims 9 to 14, wherein the application includes soil drenching.

16. The method according to claim 11 or claim 12, wherein the copper fungicide is applied as a foliar spray.

17. The method according to any one of claims 9 to 11, further comprising applying one or more of the following: a herbicide, an insecticide, a second fungicide, a bactericide, and a nutrient.

18. The method according to any one of claims 8 to 16, wherein the bacterial composition comprises a mixture of live Bacillus subtilis and live Bacillus pumila.

19. The method according to claim 18, wherein the bacterial composition comprises live Bacillus subtilis and live Bacillus pumilus in a ratio of 1:1, 2:1, or 1:

2.

20. The method according to any one of claims 8 to 19, wherein the Bacillus pumilus is ATCC (registered trademark) stock deposit number PTA-125304.

21. The method according to any one of claims 19 to 20, wherein the Bacillus subtilis strain consists of ATCC® strain deposit number PTA-125303 or ATCC® strain deposit number PTA-125302.

22. The bacterial composition is about 10 ９ CFU / ml ~ approx. 10 ６ Bacillus pumilus at a concentration of CFU / ml and approximately 10 ９ CFU / ml ~ approx. 10 ６ The method according to any one of claims 9 to 21, comprising Bacillus subtilis at a concentration of CFU / ml.

23. The effective amount is 10 for each of Bacillus pumilus and Bacillus subtilis ７ to 10 ９ CFU / cm ３ and applying at, a method according to any one of claims 9 to 22.

24. The following effects, (i) Increase in tomato yield, (ii) Increase in tomato size, (iii) Increase in total tomato yield per acre, (iv) Biological defense against Cmm (v) Improved resistance to Cmm, (vi) Reduction of losses caused by Cmm, (vii) Reduction of the severity of canker disease and the plant tissue affected by canker lesions, (viiii) Reduction of pathological symptoms or lesions induced by the action of Cmm, Enhancement of the plant antimicrobial response to (ix)Cmm, and (x) Increased yield of extra-large tomatoes The method according to any one of claims 9 to 23, wherein one or more of the following are added.

25. The method of claim 24, wherein the effect is evaluated by comparing tomatoes that have been in contact with the bacterial composition with tomatoes that have not been in contact with it, and / or by comparing tomatoes that have been in contact with the bacterial composition with tomatoes that have been in contact with the reference treatment.

26. A kit for protecting tomatoes from tomato canker fungus (Cmm), comprising (i) a composition comprising a mixture of Bacillus subtilis and Bacillus pumilus, a culture medium pre-inoculated with Bacillus subtilis and Bacillus pumilus, a cell-free extract of Bacillus subtilis and Bacillus pumilus, and / or one or more metabolites produced by Bacillus subtilis and Bacillus pumilus, and (ii) a second container comprising one or more of herbicides, insecticides, fungicides, bactericidal agents, and nutrients.

27. The kit according to claim 26, wherein the second container contains one or more copper-based fungicides.

28. The kit according to claim 26 or claim 27, wherein 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. The kit according to any one of claims 26 to 28, wherein the fungicide comprises Kocide (trademark).

30. A combination of (i) a bacterial composition comprising a mixture of live Bacillus subtilis and live Bacillus pumilus, and (ii) one or more of the following: herbicides, insecticides, fungicides, bactericidal agents, and nutrients, for controlling, suppressing, and / or preventing infection of tomatoes with the tomato canker fungus (Cmm).

31. The combination according to claim 30, which provides synergistic effectiveness in controlling, suppressing, and / or preventing infection of tomatoes with the tomato canker fungus (Cmm).

32. Use of the bacterial composition according to any one of claims 1 to 8 for controlling, suppressing, and / or preventing infection of tomatoes with the tomato canker fungus (Cmm).

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