Photodynamic methods for inhibiting the growth of microbial pathogens.

Abstract

To provide methods and materials for inhibiting the growth of a microbial pathogen of a plant.SOLUTION: This method comprises the steps of applying to the plant a combination of a dye-bearing compound, which is a reactive oxygen species photosensitizer, and a copper compound, which increases the microbial lethality of the dye-bearing compound for the microbial pathogen; and exposing the plant to light to activate the combination of the dye-bearing compound and the copper compound and inhibiting growth of a microbial pathogen of a plant. The combination of the dye-bearing compound and the copper compound may result in a synergistic ability to reduce the growth of a plant pathogen.SELECTED DRAWING: Figure 12

Description

[Technical field]

[0001] (STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT) This invention was made with U.S. Government funds under U.S. Department of Agriculture Contract No. 2017-33610-27303. The Government has certain rights in this invention.

[0002] FIELD OF THEINVENTION The technical field generally relates to the photodynamic inhibition of microbial pathogens in plants using compounds and compositions that include both a photosensitizer compound and a small but synergistically effective amount of a copper compound. More specifically, the technical field relates to the photodynamic inhibition of microbial pathogens in plants, where inhibition is dramatically increased by small amounts of copper compounds. The technical field further relates to methods that use low concentrations of copper compounds that can be used consistent with organic agricultural practices and are exempt from certain environmental regulations. [Background technology]

[0003] The inhibition of plant pathogens by active oxidizing species has the advantage that resistance is slow to develop in the target organisms due to their mechanism of action. A second advantage is that oxidizing species are generally less hazardous to the environment than other antimicrobials, since they tend to decompose by reaction with common materials and therefore do not persist in the environment. A disadvantage of using oxidizing agents as antimicrobials is related to their reactivity: because oxidizing agents react quickly, their effect is not sustained. This can limit their effectiveness and may require frequent reapplication. As an example, hydrogen peroxide is an effective antimicrobial and pesticide, but its usefulness is limited because common materials can react with hydrogen peroxide, such as catalyzing its decomposition into water and oxygen.

[0004] Oxidative antimicrobial species can also be produced where they are needed by reactions driven by light energy. This process is called photodynamic therapy or photodynamic inhibition. Photodynamic inhibition of microbial pathogens involves exposing a photosensitizer to light to generate reactive oxygen species (ROS), such as singlet oxygen, which can have detrimental effects on microbial pathogens. This photodynamic therapy (PDT) consumes oxygen and produces peroxides, superoxide anions (O2 -1 ), hydroxyl radical (·OH), hydroperoxyl radical (·OOH), and singlet oxygen ( 1 Photodynamic therapy generates at least one of several reactive oxygen species (ROS), including O2. PDT requires a photoactivator or photocatalyst that increases the efficiency of converting light energy into reactive oxygen species. This process is also called antimicrobial photodynamic therapy (aPDT). Existing photodynamic inhibition technologies and applications suffer from a variety of shortcomings. For example, photosensitizers are either less effective when used alone, are made from materials that are not consistent with organic farming practices, or both.

[0005] Another class of antimicrobial materials consists of copper compounds (e.g., copper sulfate (CuSO4) and copper hydroxide (Cu(OH)2)). Copper compounds are used as pesticides; however, there are several drawbacks to using copper compounds as pesticides. First, copper salts are widely used as pesticides, so some pests have developed resistance. Second, copper is also an environmental hazard, and discharge of copper into surface waters can be limited. Finally, copper salts used as pesticides are applied to crops at high concentrations in aqueous solutions (e.g., 1% copper sulfate in water), and evaporation can leave precipitates on the crop. These precipitates are undesirable and reduce the value of the crop. The widespread dissemination of high levels of copper in the environment is a severe limitation to the continued use of high concentrations of copper-based antimicrobial agents in agriculture.

[0006] BRIEF DESCRIPTION OF THE PRESENT EMBODIMENT The present invention solves the limitations of the prior art and provides a method for inhibiting the growth of microbial pathogens in plants, the method comprising: applying to a plant a combination comprising a pigment-containing compound that is a reactive oxygen species photosensitizer; and a copper compound for enhancing the microbial lethality of the pigment-containing compound against the microbial pathogen; and exposing the plant to light to activate the combination of the pigment-containing compound and the copper compound and inhibit the growth of the microbial pathogen in the plant.In certain embodiments, the copper compound is a copper salt that enhances the microbial lethality of the pigment-containing compound against the microbial pathogen.The copper salt can be provided in an amount sufficient to increase the microbial pathogen growth inhibition compared to the microbial pathogen growth inhibition of the reactive oxygen species photosensitizer alone.

[0007] In a particularly beneficial embodiment of the invention, the copper salt and dye-containing compound are provided in synergistically effective amounts to inhibit the growth of a microbial pathogen. A synergistically effective inhibition of the growth of a microbial pathogen may be defined as a combination that increases the kill of the microbial pathogen by at least one order of magnitude compared to the kill expected based on an additive effect model. If the kill of the microbial pathogen is measured in log kill units, then a synergistically effective kill is an increase in kill of 1 log unit. This additive effect model is expressed by the formula: P=X+Y(100- X ) / 100, where P is the expected kill rate of the pathogen using the combination of the reactive oxygen species photosensitizer and the copper salt, X is the kill rate of the pathogen when the reactive oxygen species photosensitizer is used alone, and Y is the kill rate of the pathogen when the copper salt is used alone.

[0008] In preferred embodiments, the pigment-containing compound is selected from the group consisting of riboflavin, derivatized riboflavin, lumichrome, derivatized lumichrome, curcumin, fluorescein, eosin Y, erythrosine, flavin-containing compounds and rose bengal B. Most preferably, the pigment-containing compound is riboflavin.

[0009] In a preferred embodiment, the copper salt is selected from the group consisting of copper(II) sulfate, copper(II) nitrate, copper hydroxide, copper carbonate, copper chloride, copper oxide, and copper bromide.

[0010] In any embodiment, the method further comprises applying ascorbate to a plant to increase the microbial lethality of the combination against microbial pathogens.

[0011] In embodiments, the dye-containing compound may be provided at a concentration of about 1-100 ppm by weight and the copper salt at a concentration of about 0.1-100 ppm by weight of copper. More preferably, the dye-containing compound is provided at a concentration of about 5-50 ppm by weight and the copper salt at a concentration of about 2-10 ppm by weight of copper.

[0012] In an embodiment, exposing the plant to light comprises exposing the plant to natural light, or alternatively, exposing the plant to light comprises exposing the plant to artificial light.

[0013] The combination may be applied to the plant by at least one of soil soaking, pipetting, irrigation, spraying, listing, watering, and pouring.

[0014] The microbial pathogen may include any of bacterial pathogens, cyanobacteria, algae, fungal pathogens, or viral pathogens. The plant may be a non-woody crop plant, a woody plant, a turfgrass, a fruit-bearing tree, a nut-bearing tree, or an ornamental plant. The combination may further include at least one agriculturally acceptable adjuvant, which may be selected from the group consisting of surfactants, penetrating agents, wetting agents, spreading agents, moisturizing agents, and emulsifying agents.

[0015] The pigment-containing compound and the copper salt may be applied to the plant simultaneously or sequentially. The combination may be applied to the plant by applying to the plant a composition containing the components of the combination.

[0016] In a preferred embodiment, the copper salt is used at a concentration of less than 100 ppm by weight to avoid unnecessary damage to the plant or the environment. In another embodiment, the pigment-containing compound and the copper salt are suitable for organic farming.

[0017] In any embodiment, the method further comprises applying to the plant ascorbic acid and sodium bicarbonate to react with the copper salt to generate reactive oxygen species, or alternatively an ascorbate salt to react with the copper salt to generate reactive oxygen species. The ascorbate salt of the method can be an agriculturally acceptable salt.

[0018] In a preferred embodiment, the dye-containing compound that is a reactive oxygen species photosensitizer is selected from the group consisting of riboflavin, derivatized riboflavin, lumichrome, derivatized lumichrome, curcumin, fluorescein, eosin Y, erythrosine, and rose bengal B; and the copper compound is a copper ion-containing organic complex that increases the microbial lethality of the dye-containing compound against microbial pathogens.

[0019] In any embodiment, the copper ion-containing organic complex is provided in an amount sufficient to increase the inhibition of microbial pathogen growth as compared to the inhibition of microbial pathogen growth of the reactive oxygen species photosensitizer alone.

[0020] In a preferred embodiment, the copper ion-containing organic complex and the dye-containing compound are provided in synergistically effective amounts to inhibit the growth of a microbial pathogen, the combination enhancing the inhibition of the growth of the microbial pathogen to an extent greater than the sum of the inhibition of the growth of the microbial pathogen by the reactive oxygen species photosensitizer and the copper ion-containing organic complex when each is used alone. In another embodiment, the copper ion-containing organic complex and the dye-containing compound are provided in synergistically effective amounts to inhibit the growth of a microbial pathogen, the combination increasing the log kill of the microbial pathogen by at least one log, or an order of magnitude, compared to the log kill expected based on an additive effect model, the additive effect model being represented by the formula: P=X+Y(100- X) / 100, where P is the expected kill rate of the pathogen using the combination of the reactive oxygen species photosensitizer and the copper ion-containing organic complex, X is the kill rate of the pathogen when the reactive oxygen species photosensitizer is used alone, and Y is the kill rate of the pathogen when the copper ion-containing organic complex is used alone. In a preferred embodiment, the dye-containing compound is riboflavin. In another preferred embodiment, the copper ion-containing organic complex is selected from the group consisting of copper ethanolamine, copper EDTA, copper ions complexed to crown ethers, and copper ions complexed to polyethylene glycol. The method may further include at least one agriculturally acceptable adjuvant, which may optionally be selected from the group consisting of surfactants, penetrating agents, wetting agents, spreading agents, moisturizing agents, and emulsifiers.

[0021] In any embodiment, the dye-containing compound and the copper ion-containing organic complex may be applied to the plant simultaneously, or, optionally, sequentially. The method includes applying the combination to the plant by applying a composition containing the components of the combination to the plant.

[0022] Preferably, the copper ion-containing organic complex is used at a concentration of less than 100 ppm by weight to avoid unnecessary damage to plants or the environment. Preferably, the dye-containing compound and the copper ion-containing organic complex are suitable for organic farming.

[0023] In any embodiment, the method may include applying to the plant ascorbic acid and sodium bicarbonate for reaction with a copper salt to generate reactive oxygen species, or applying to the plant an ascorbate salt for reaction with a copper salt to generate reactive oxygen species. The ascorbate salt may be an agriculturally acceptable salt. [Brief description of the drawings]

[0024] [Figure 1] The structure of riboflavin.

[0025] [Diagram 2]The structure of lumichrome.

[0026] [Diagram 3] An example of a flavin-containing compound, flavin mononucleotide.

[0027] [Figure 4] Structure of the modified flavin, FLASH-01a.

[0028] [Diagram 5] Structure of the modified flavin, FLASH-07a.

[0029] [Figure 6] Structures of curcumin, the enol and keto forms.

[0030] [Figure 7] Structure of halogenated xanthene rose bengal.

[0031] [Figure 8] The structure of phenothiazinium methylene blue.

[0032] [Figure 9] Representative structure of triscationic fullerene BB6.

[0033] [Figure 10] The structure of fluorescein.

[0034] [Figure 11] HIGROW HG-GL36B output light 6 spectrum

[0035] [Figure 12A] Results of Example 1 with slopes. [Figure 12B] Results of Example 1 without slopes.

[0036] [Figure 13] Data for Example 1.

[0037] [Figure 14] E. coli reduction after 15 minutes exposure

[0038] [Figure 15] Results of Example 2.

[0039] [Figure 16] Results for Xanthomonas hortorum pv. Geranium Pelargonii (see Example 5). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description of the Invention Various compositions and methods for photodynamic inhibition of microbial pathogens on plants are described herein. Photodynamic inhibition of bacterial, fungal, viral, or microbial pathogens can be carried out by applying a photosensitizer compound and a copper compound (optionally a copper salt or a copper ion-containing organic complex) to a plant and exposing the plant to light. The photosensitizer compound can be optionally selected from the group consisting of riboflavin, derivatized riboflavin, lumichrome, derivatized lumichrome, curcumin, fluorescein, eosin Y, erythrosine, flavin-containing compounds, and rose bengal B.

[0041] Surprisingly, it was found that the antimicrobial effect of light-driven generation of reactive oxygen species (ROS) is dramatically enhanced by the presence of low levels of copper compounds, such as copper salts. For example, the combination of a photosensitizer and a copper salt produces a much greater antimicrobial effect than either the light-driven antimicrobial process or the copper salt individually, exceeding the additive effects of each. One example is the combination of a light-driven process in which the photoactivator is riboflavin and copper sulfate is added (see Example 1 below). Riboflavin (vitamin B2) is a natural material that is safe for human consumption and the environment. As a vitamin, it is an essential nutrient for humans. Both riboflavin and copper salts are on the Generally Recognized as Safe (GRAS) list of the U.S. Food and Drug Administration (FDA). Antimicrobial or insecticidal formulations containing riboflavin and copper qualify as organic pesticides and can be used in the cultivation of organic produce.

[0042] Certain embodiments of the invention use a combination of a photoactivator and a copper salt. These two components may be combined as a mixture in a solvent. For example, both the photoactivator and the copper salt may be dissolved in water. The mixture does not have to be completely homogenous. When copper is used, the copper salt is Cu +1 or Cu +2 It could be.

[0043] Photosensitizers A photosensitizer can be any material that absorbs light (UV and / or visible light) and efficiently converts it into energetic intermediates that can interact with copper to produce a synergistic effect. As shown in Example 2, we tested riboflavin, erythrosine, rose bengal, and eosin Y; all were effective, with riboflavin being the most effective under the conditions tested. Lumichrome, which is produced by photolysis of riboflavin, and riboflavin tetraacetate (2',3',4',5'-tetraacetylriboflavin) are effective in photosensitization reactions. Charged, e.g., positively charged, functional groups can be added to flavin derivatives to attach these molecules to the negatively charged surfaces of bacteria or to increase their solubility in water (Figure 2). Compounds containing suitable chromophores with a range of additional functional groups, including partial decomposition products of photosensitizers, can be suitable photosensitizers for use in the present invention.

[0044] As mentioned above, photosensitizer compounds may be used to enable photodynamic inhibition of microbial pathogens present in plants. Photosensitizer compounds respond to light by generating reactive oxygen species (ROS). Depending on the type of ROS generated, photosensitizers may be divided into two classes, namely type I photosensitizers and type II photosensitizers. On the one hand, type I photosensitizers, when excited at the appropriate wavelength in the presence of oxygen, form short-lived free radicals by electron abstraction or transfer from a substrate. On the other hand, type II photosensitizers form highly reactive oxygen states known as "singlet oxygen," which are also referred to herein as "reactive singlet oxygen species." Singlet oxygen is generally relatively long-lived and may have a large radius of action.

[0045] It should be understood that photosensitizer compounds can generally be metallized or non-metallized. A preferred embodiment of the present invention is non-metallized. It is known that certain metallized photosensitizers are less effective when they are in a metallized state. For example, if a chlorin photosensitizer compound is metallized with copper, the ROS (type I) generated tends to be less available for microbial inhibition, for example because of a very short half-life. In contrast, if the same photosensitizer compound is metallized with another metal, such as magnesium, the ROS generated is more available for microbial inhibition. Thus, copper used in near stoichiometric amounts with the photosensitizer (to metallize the photosensitizer) is not advantageous. It should be understood that copper, at least at high concentrations, is known to adversely affect type II photosensitizers. It should be understood that selecting a metal that does not allow for the formation of type II photosensitizers will usually result in much less inhibition of microbial pathogen growth, at least because no or less reactive singlet oxygen species are generated. Non-limiting examples of metals known not to form Type II photosensitizers when complexed with chlorins are Cu, Co, Fe, Ni, and Mn. Thus, the synergistic effect of combining a photosensitizer with a small amount of a copper-containing compound, particularly a preferred embodiment of the present invention, is surprising and runs counter to existing understanding of how copper can adversely affect the suppression of plant pathogens using photosensitizers.

[0046] The metallated state should not be confused with a mixture of a separate nonmetallated photosensitizer with a metal salt or other metal-containing compound. Preferred photosensitizers of the invention are not used in a form in which the photosensitizer compound itself is bound to or chemically complexed with a metal ion.

[0047] It should be understood that the term "singlet oxygen photosensitizer" as used herein refers to a compound that generates reactive singlet oxygen species when excited by light. In other words, this term refers to a photosensitizer in which the Type II process defined above predominates compared to the Type I process.

[0048] Also relevant to the selection of potential photosensitizers of the present invention is the work of researchers at Procter & Gamble Company, as described, for example, in the following U.S. patent applications: US2015 / 0208896, US2015 / 0208898, US2015 / 0209808, US2015 / 0210960, US2015 / 0210963, US2015 / 0210964, US2015 / 0211165, and US2015 / 0211170, which are incorporated herein by reference.

[0049] The photosensitizer is optionally selected from the group consisting of 1,1'-biphenyl-4,4'-diamine, 1,1'-biphenyl-4-amine, benzophenone, 1,1'-biphenyl-4,4'-diol, 1,1'-biphenyl-4-amine, 1,1'-biphenyl-4-ol, 1,1':2',1''-terphenyl, 1,1':3',1''-terphenyl, 1,1':4',1'':4'',1'''-quaterphenyl, 1,1':4',1''-terphenyl, 1,10-phenanthroline ...-biphenyl-4,4'-diol, 1,1'-biphenyl-4,4'-diol, 1,1'-biphenyl Phenyl, 1,2,3,4-dibenzanthracene, 1,2-benzenedicarbonitrile, 1,3-isobenzofurandione, 1,4-naphthoquinone, 1,5-naphthalenediol, 10H-phenothiazine, 10H-phenoxazine, 10-methylacridone, 1-acetonaphthone, 1-chloroanthraquinone, 1-hydroxyanthraquinone, 1-naphthalenecarbonitrile, 1-naphthalenecarboxaldehyde, 1-naphthalenesulfonic acid, 1-naphthalenol, 2(1H)-chi Nolinone, 2,2'-biquinoline, 2,3-naphthalenediol, 2,6-dichlorobenzaldehyde, 21H,23H-porphine, 2-aminoanthraquinone, 2-benzoylthiophene, 2-chlorobenzaldehyde, 2-chlorothioxanthone, 2-ethylanthraquinone, 2H-1-benzopyran-2-one, 2-methoxythioxanthone, 2-methyl-1,4-naphthoquinone, 2-methyl-9(10-methyl)-acridinone, 2-methylanthraquinone, 2-methylbenzaldehyde 2-Naphthalenamine, 2-Naphthalenecarboxylic acid, 2-Naphthalenol, 2-Nitro-9(10-methyl)-acridinone, 9(10-ethyl)-acridinone, 3,6-Acridinediamine, 3,9-Dibromoperylene, 3,9-Dicyanophenanthrene, 3-Benzoylcoumarin, 3-Methoxy-9-cyanophenanthrene, 3-Methoxythioxanthone, 3'-Methylacetophenone, 4,4'-Dichlorobenzophenone, 4,4'-Dimethoxybenzophenone, 4-Bromobenzophenone, 4-Chlorobenzophenone, 4'-Fluoroacetophenone, 4-Methoxybenzophenone, 4'-Methylacetophenone, 4-Methylbenzaldehyde, 4-Methylbenzophenone, 4-Phenylbenzophenone, 6-Methylchromanone, 7-(Diethylamino)coumarin, 7H-Benz[de]anthracen-7-one, 7H-Benz[c]xanthen-7-one, 7H-Furo[3,2-g][1]benzopyran-7-one, 9 (10H)-acridinone, 9(10H)-anthracenone, 9(10-methyl)-acridinone, 9(10-phenyl)-acridinone, 9,10-anthracenedione, 9-acridinamine, 9-cyanophenanthrene, 9-fluorenone, 9H-carbazole, 9H-fluoren-2-amine, 9H-fluorene, 9H-thioxanthen-9-ol, 9H-thioxanthen-9-one, 9H-thioxanthen-2,9-diol, 9H-xanthen-9-one, acetophenone, acridinone, dene, acridine, acridone, anthracene, anthraquinone, anthrone, α-tetralone, benz[a]anthracene, benzaldehyde, benzamide, benzo[a]coronene, benzo[a]pyrene, benzo[f]quinoline, benzo[ghi]perylene, benzo[rst]pentaphene, benzophenone, benzoquinone, 2,3,5,6-tetramethyl, chrysene, coronene, dibenz[a,h]anthracene, dibenzo[b,def]chrysene, dibenzo[c,g]phenanthrene, di Benzo[def,mno]chrysene, dibenzo[def,p]chrysene, DL-tryptophan, fluoranthene, fluoren-9-one, fluorenone, isoquinoline, methoxycoumarin, methylacridone, Michler's ketone, naphthacene, naphtho[1,2-g]chrysene, N-methylacridone, p-benzoquinone, p-benzoquinone, 2,3,5,6-tetrachloro, pentacene, perylenequinonoid pigments, phenanthrene, phenanthrenequinone, phenanthridine, phenanthro[3,4-c]phenanthrene, phenazine, phenothiazine, p-methoxyacetophenone, pyranthrene, pyrene, quinoline, quinoxaline, riboflavin 5'-(dihydrogen phosphate), thioxanthone, thymidine, xanthen-9-one, xanthone, and mixtures thereof. Other suitable water-soluble photoactivators include fluorescein and its derivatives; preferably, halogen-substituted fluoresceins; more preferably, bromofluoresceins and iodofluoresceins, such as dibromofluorescein, diiodofluorescein, rose bengal, erythrosine, eosin (e.g., eosin Y), curcumin, protoporphyrin IX, resazurin, dichlorodihydrofluorescein diacetate, dihydrorhodamine 123 and related chromophores, methylene blue, and rose bengal.

[0050] copper-containing compounds The copper compound of the present invention is a copper salt (preferably Cu +2 , and optionally Cu +1 Non-limiting examples include copper(II) sulfate, copper(II) nitrate, copper hydroxide, copper carbonate, copper chloride, copper oxide, copper octoate, copper bromide (or the reduction products of these salts in their copper+1 state), as well as copper ethanolamine complex, copper gluconate, copper orthophosphate, copper pyrophosphate, or agriculturally acceptable salts thereof and hydrated forms thereof.

[0051] When defining a plant protection product using the synergy between photoactivators that generate reactive oxygen species (ROS) and copper, we need to consider which copper compounds are acceptable. Although experiments with E. coli using elemental copper did not show significant performance, successful testing does not necessarily have to be based solely on soluble salts or soluble compounds. Furthermore, the practice of the invention should not be limited to formulations using highly soluble copper water-soluble salts, as barely soluble salts are also effective. Other forms of suspended copper may be effective.

[0052] Cu +2The solubility of salts in water is a function of pH and is also affected by the presence of carbonates and bicarbonates. Copper salts are generally soluble in distilled water at pH 5.5. At higher pH values, the amount of soluble copper is a function of both pH and total copper concentration, with a maximum of 4 mg / l soluble copper at pH 6.5 and 1.3 mg / l soluble copper at pH 7.4. The present invention teaches the important contribution of low levels of copper compounds to the synergistic effect with photosensitizers in reducing the growth of plant pathogens. In a preferred embodiment, 10 mg / l copper sulfate or only 4 mg / l copper is used. It should be clear that the present invention is operable even when using copper compounds with limited solubility in water, including formulations designed for controlled release.

[0053] Combination of photosensitizer and copper-containing compound The combination of the photosensitizer and the copper-containing compound leads to a reduction in the growth of plant pathogens that is much greater than the individual contribution of either component when used separately. Plant pathogens whose growth may be retarded in this manner include gram-negative bacteria, gram-positive bacteria, cyanobacteria, algae, fungi, and viruses.

[0054] Where combinations of a photosensitizer compound, a copper-containing compound and other optional additives or adjuvants are described throughout this specification and claims, it is understood that an agriculturally effective amount of each one of the components of the combination may be used to provide antimicrobial activity while being minimally or non-phytotoxic to the host plant.

[0055] In some embodiments, the methods and compositions described herein include one or more agriculturally suitable adjuvants.In some embodiments, each of the one or more agriculturally suitable adjuvants is independently selected from the group consisting of one or more activator adjuvants (e.g., one or more surfactants; one or more oil adjuvants, e.g., one or more penetrants); one or more wetting or spreading agents; one or more humectants; one or more emulsifiers; one or more drift control agents; one or more thickening agents; one or more deposition agents; one or more water conditioning agents; one or more buffering agents; one or more defoaming agents; one or more UV blocking agents; one or more antioxidants; one or more fertilizers, nutrients, and / or micronutrients; and / or one or more herbicides.Exemplary adjuvants are provided in Hazen, JL Weed Technology 14: 773-784 (2000), which is incorporated by reference in its entirety.

[0056] The photosensitizer compound and the copper-containing compound may be provided as part of an antimicrobial composition. The antimicrobial composition may also include a delivery fluid, such as water. The antimicrobial composition may be provided to have a particular concentration and relative ratio of components. For example, the antimicrobial composition may have between about 100 nM and about 50 mM, between 1 micromolar and about 1000 micromolar, between 5 micromolar and about 200 micromolar of the photosensitizer compound, between about 10 micromolar and about 150 micromolar, between about 20 micromolar and about 100 micromolar of the photosensitizer compound, or between about 25 micromolar and about 75 micromolar of the photosensitizer compound.

[0057] The antimicrobial composition may also have, for example, between about 2 micromolar and about 10,000 micromolar copper-containing compounds, between about 5 micromolar and about 5,000 micromolar copper-containing compounds, between about 10 micromolar and about 1,000 micromolar copper-containing compounds, between about 25 micromolar and about 500 micromolar copper-containing compounds, between about 50 micromolar and about 100 micromolar copper-containing compounds. Note that 1 micromolar / L of copper is 0.0635 mg / L. Also, 10 ppm of CuSO4 is equivalent to 4 ppm of copper, which is 62.7 micromolar.

[0058] The relative proportions by weight of the photosensitizer compound and the copper-containing compound in the antimicrobial composition can be, for example, between about 1000:1 and about 1:1000, between about 500:1 and about 1:500, between about 100:1 and about 1:100, or between about 10:1 and about 1:10.

[0059] Most preferably, the relative molar ratio of copper to photosensitizer is about 1:1.

[0060] Regarding other additives that may be present in the antimicrobial composition, a surfactant may be present as an adjuvant to aid in the coverage of the plant foliage. The surfactant may be an acceptable polysorbate type surfactant (e.g., Tween® 80), a non-ionic surfactant blend (e.g., Altox™ 3273), or other suitable surfactant.

[0061] Application of photosensitizers and copper-containing compounds The photosensitizer compound and enhancer compound of the present invention can be applied to plants for photodynamic inhibition of microbial pathogens. The photosensitizer compound and enhancer compound can be applied to plants simultaneously. For example, an antimicrobial composition can be prepared to include the photosensitizer compound and enhancer compound and a delivery liquid. The antimicrobial composition can be applied to plants by spraying, misting, watering, pouring, or any other suitable method. The antimicrobial composition can be applied to the leaves, roots, and / or stems of plants. Other additives can also be included in the antimicrobial composition, and other application methods can also be implemented.

[0062] The plants to which the antimicrobial composition is applied may be outdoors or indoors (e.g., in a greenhouse) where they are exposed to natural light, or indoors where they are exposed to artificial light. Exposure to incident light is provided such that the photosensitizer compound can generate ROS, which in turn promotes the destruction of microbial growth.

[0063] In operation, the photosensitizer compound and the enhancer compound are contacted with a microbial pathogen that is infecting a plant, and both the photosensitizer and the enhancer compound come into contact with the cell walls and intercellular material of the pathogenic microorganism.

[0064] Ascorbic acid or ascorbate additives Ascorbic acid (also known as vitamin C) is an optional additive that may further increase the lethality of the antimicrobial composition of the present invention. Ascorbic acid is a solid with a pKa of 4.17. The plant protection formulation of the present invention is buffered to a solution pH of 7, so that it exists primarily in the form of ascorbate. Small amounts of copper have been shown to catalyze the oxidation of ascorbate, producing ROS including hydrogen peroxide (Zhu et al, RSC Adv., 2016, 6, 38541). These are useful for controlling plant pathogens even in the dark (e.g., at night).

[0065] When disease pressure is high or light intensity is expected to be low (e.g., overcast days, evening or nighttime applications), ascorbic acid may be added to supplement the photosensitized antimicrobial performance. The effect of ascorbate oxidation in the presence of copper can be controlled by controlling the concentration of added ascorbate; higher concentrations will have a longer effect, while lower concentrations will have a shorter effect. In plant protection applications, including high concentrations of ascorbic acid can be problematic because the ionic strength of the resulting solution that must be included to maintain a neutral pH and convert ascorbic acid to ascorbate is excessively high and therefore salty.

[0066] In photodynamic therapy for plant protection applications as described in the present invention, the inclusion of ascorbic acid in the formulation has been found to be beneficial at weight percent levels of between 10 and 0.0001%, between 5 and 0.005%, and between 1 and 0.01%.

[0067] Microbial pathogens and plants The microbial pathogens to which the antimicrobial composition can be applied include fungal pathogens and bacterial pathogens.The fungal pathogens to which the antimicrobial composition can be applied include Alternaria solani, which can infect plants such as tomato and potato; Botrytis cinerea, which can infect grapes, as well as soft fruit and bulb crops; or Sclerotinia homoeocarpa, which can generally infect turfgrass.Other fungal pathogens of the Alternaria, Botrytis, or Sclerotinia genus can also be applied with the antimicrobial composition. The antimicrobial compositions may be applied to plants that are infected with or susceptible to pathogens that cause various plant diseases, such as, for example, Colletotrichum, Fusarium, Puccinia, Erysiphaceae, Cercospora, Rhizoctonia, Bipolaris, Microdochium, Venturia inaequalis, Monilinia fructicola, Gymnosporangium juniperi-virginianae, Plasmodiophora brassicae, Ustilago zeae, Phytophthora, Pythium, Fusarium oxysporum, Phytophthora infestans, Taphrina deformans, Powdery Mildew, Phragmidium spp., and other fungal pathogens.

[0068] Bacterial pathogens to which the antimicrobial composition can be applied include gram-negative bacteria such as Erwinia amylovara, or other bacterial pathogens of the genus Erwinia that can infect woody plants. E. amylovara causes fire blight in a variety of plants, including pear, apple, and other rose crops. The antimicrobial composition can be applied to plants that are diseased or susceptible to pathogens that cause various plant diseases, such as Pseudomonas, Xanthomonas, Agrobacterium, Curtobacterium, Streptomyces, Escherichia coli, Xylella fastidiosa (which causes Olive Quick Decay Syndrome (OQDS) disease), or other bacterial pathogens.

[0069] The antimicrobial composition can be used on various kinds of plants affected by microbial pathogens. Crop plants, turf plants, trees, and other plants infected with microbial pathogens can be treated. It should also be noted that the antimicrobial composition described herein can have various inhibitory effects on microbial pathogens, depending on the type of plant and pathogen, and the state of microbial infection. Although it is described herein that the antimicrobial composition can inhibit the growth of microbial pathogens on plants, such expression should not be limited and should be understood to generally include inhibition of microbial pathogens, prevention against microbial pathogens, destruction of microbial pathogens, or increase toxicity against microbial pathogens.

[0070] Plant types Compound or composition can be used for various kinds of plants that can be affected by microbial pathogens.Plants can be non-woody crop plants, woody plants or turfgrass.Plants can be selected from the group consisting of food crop plants, non-food crop plants, fruit plants, vegetable plants, legume plants, cereal plants, forage plants, oilseed plants, field crops, garden crops, greenhouse plants, house plants, flowers, turfgrass, trees such as fruit trees, and other plants that can be affected by microbial pathogens.

[0071] In some embodiments, the plant is a turfgrass. As used herein, the term "turfgrass" refers to cultivated grasses that provide ground cover, such as turf or lawn grasses that are periodically cut or mowed to maintain a constant height. Grasses belong to the family Poaceae, which are subdivided into six subfamilies, three of which include common turfgrasses: the subfamily Festucoideae of cool-season turfgrasses; the subfamily Panicum and the subfamily Eragrostoideae of warm-season turfgrasses. A limited number of species are widely used as turfgrasses, and generally meet the criteria of forming a uniform soil cover and tolerating mowing and traffic. Turfgrasses generally have a compressed crown that facilitates mowing without cutting the growing point. In this context, the term "turfgrass" includes areas where one or more types of grass are cultivated to form a relatively uniform soil, including blends that combine different varieties of the same species, or mixtures that combine different species and / or varieties.

[0072] In some scenarios, the combination may show a synergistic reaction to suppress the growth of microbial pathogens in plants. It should be understood that the term "synergistic" or "synergistic" as used herein refers to the interaction of two or more components of a combination (or composition) such that their combined effect is greater than the sum of their individual effects. This may include, in the context of this specification, the action of two or more photosensitizers, copper-containing compounds.

[0073] In some scenarios, the approach set out in SR Colby, "Calculating synergistic and antagonistic responses of herbicide combinations", Weeds 15, 20-22 (1967) may be used to assess synergistic effects. The expected effect, E, is expressed as follows: E=X+Y-[(X*Y / 100], where X is the effect of the first component of the combination, expressed as a % of the untreated control, and Y is the effect of the second component of the combination, expressed as a % of the untreated control. Two components are said to be present in synergistically effective amounts when the observed effect is higher than the expected effect. More preferably, in the present invention, two components are said to be present in synergistically effective amounts when the observed effect is at least one order of magnitude higher than the expected effect, more preferably when the observed effect is at least two orders of magnitude higher than the expected efficacy, and most preferably when the observed effect is three or more orders of magnitude higher than the expected effect.

[0074] Application of the formulations of the invention can be made to the plant either before or after the plant has become infected with a plant pathogen, and thus the method can be used as a preventative measure or as a treatment for infected plants.

[0075] Examples and Experiments Example 1: Photodynamic treatment of E. coli with 10 ppm CuSO4 and 18 ppm riboflavin using an LED blue light source.

[0076] Tests were conducted using Escherichia coli (E. coli) bacteria (ATCC 25922) exposing the organisms to various test conditions. The light source for photoactivation was a blue LED light (HIGROW 2019) placed 7 cm away from the container containing the E. coli. The photoactivator was riboflavin (Aldrich R7649, Lot WXBB4048V, 98.6% purity) at a concentration of 18 ppm. Copper sulfate was used at a concentration of 10 ppm (aqueous solution). Tests included the wetting agent Silwet® L-77 at a concentration of 0.0125%. Silwet® L-77 is a product of Helena Agri-Enterprises, LLC of Collierville, TN, and is a nonionic organosilicone surfactant copolymer that provides improved wetting and spreading properties when used in aqueous sprays. Silwet® L-77 has a CAS Registry Number of 27306-78-1. Each test was performed for a defined period of time, which varied between 5 and 30 minutes. After the exposure period, the bacteria were diluted and plated, colonies were counted, and the corresponding reduction was determined by comparison with an untreated control.

[0077] The results are shown in Figure 12. A and Figure 12B The results are shown in Figure 1 and summarized below. Riboflavin at 18 ppm in the dark produced no measurable kill. Copper sulfate at 10 ppm in the dark gave only a 0.226-log kill in 30 minutes. Blue light alone (no copper or photosensitizer) produced a 0.2775-log kill after 30 minutes. 18 ppm riboflavin alone exposed to blue light produced a 0.83-log kill after 30 minutes, providing a small kill. 10 ppm copper sulfate alone exposed to blue light produced a 0.954-log kill after 30 minutes, providing a small kill. Surprisingly, 10 ppm copper sulfate and 18 ppm riboflavin exposed to blue light produced a 3.67-log kill at 5 minutes, a 6.89-log kill at 15 minutes, and a complete kill (7.73-log kill) at 30 minutes.

[0078] The expected efficacy, E, based on 10 ppm copper sulfate alone and 18 ppm riboflavin alone can be calculated as follows: E=X+Y(100-X) / 100, where X is the efficacy of the first component of the combination, expressed as a % of the untreated control, and Y is the efficacy of the second component of the combination, expressed as a % of the untreated control. First, the log kill data above must be converted to percent E. coli presence (compared to the control inoculum). The formula for converting Log reduction to percent reduction is % reduction=(1-10 -L )X100, where L is the Log reduction. Thus, 18 ppm riboflavin alone exposed to blue light resulted in a 0.83-log kill after 30 minutes, which is a reduction of 85.208%. 10 ppm copper sulfate alone exposed to blue light resulted in a 0.954-log kill after 30 minutes, which is a reduction of 88.88%. Thus, the expected efficacy would be predicted to be 85.208+88.88-(85.208*88.88) / 100=98.355. A reduction of 98.355% is only a 1.785-log kill. The actual efficacy is 7.73-log kill, which is 5.945-log greater than would be predicted by classical antimicrobial additivity theory. The synergistic effect accounts for nearly six orders of magnitude more bacteria killed.

[0079] The combination of 18 ppm riboflavin and 10 ppm copper sulfate has a dramatic synergistic effect of pathogen reduction. It is a million times better than additive. This is clearly not expected. Further combinations of antimicrobials and antibiotics can lead to additive, synergistic, or in some cases antagonistic interactions (Ocampo 2014, Bollenbach 2015). Thus, it is not clear that the addition of copper to riboflavin in a light-treatment antimicrobial system would result in significantly improved results, let alone results that are over a million times better. The raw data are listed in the table seen in Figure 13.

[0080] We also performed tests to evaluate the effect of light intensity. These tests used 10 ppm copper sulfate and 18 ppm riboflavin (in water) with 15 min exposure under blue light and reduced light intensity using neutral density filters. The total light intensity in the range of 450 nm to 460 nm was 353 W / m 2 The results are shown in Table 14. We used 16 W / m 2 (4.5% of total light intensity), we observed a 0.71 log kill at 56 W / m 2 (15.9% of total light intensity), we observed a 1.8 log kill at 195 W / m 2 (55% of the total light intensity), a 3.12 log kill was observed at full intensity (353 W / m 2 , 100%) produced a 6.9 log kill. These results confirm that this is a photoactivated process, as increasing light intensity increases the desired effect.

[0081] It is important to recognize the difference between the concentration of copper sulfate used in Example 1 and the concentrations used in the prior art for general-purpose pesticides. The Bordeaux mixture (CuSO4 and Ca(OH)2) and Burgundy mixture (CuSO4 and Na2CO3) use copper sulfate at 1% or higher (Burgundy mixture up to 20%). In a preferred embodiment, the present invention teaches the use of copper sulfate at a concentration of 10 ppm, which is 1,000 times lower. The widely used copper-based pesticide CuPRO® 5000 (CuPRO® 2019) contains copper hydroxide. According to the label instructions, the recommended mixing rate for the lowest copper concentration is 4 lbs of CuPRO® (2.45 lbs of copper hydroxide) per 100 gallons (834 lbs) of water, which corresponds to a copper concentration of 0.030 M or 0.294.%. That concentration is 294 times higher than the 10 ppm value used in the preferred formulation of the present invention. Thus, when comparing just the levels of copper used in this invention to the minimum concentrations recommended by commonly used products, we end up using nearly 300 times lower concentrations. Several studies cited herein use 10 ppm copper sulfate; at higher concentrations (e.g., 100 ppm), the antimicrobial properties of copper itself may result in some antimicrobial activity in the absence of riboflavin.

[0082] Example 2: Synergistic killing of E. coli using a copper salt-based photosensitizer and a broad spectrum light source.

[0083] In this example, we compare different photosensitizers (50 μM in water) in combination with and without copper sulfate. The agricultural adjuvant, sticker spreader, Silwet® L-77, was also included at a concentration of 0.0125%. The experimental conditions were the same as in Example 1. E. coli was the test organism, copper sulfate was 10 ppm, and the light exposure time was 30 minutes. However, in this test series, we used a light source that reproduced the spectrum of sunlight, including UV. It is used in terrariums to house reptiles, some of which require a UV component in their light. Herein, it is referred to as the "Reptile Light" (Zoo Med 2019). We tested riboflavin (R), erythrosine (ER), rose bengal (RB), eosin Y (Y), and riboflavin monophosphate (RMP); with and without copper. The results (average of 3 tests) are shown in Figure 15. All dyes showed synergistic killing effects, with riboflavin in combination with copper unexpectedly producing the greatest synergistic log kill.

[0084] Using the median values ​​for each test (see Figure 15), it is clear that the combination of photosensitizer and copper results in a synergistic effect. First, 30 minutes of exposure to 10 ppm copper sulfate under a reptile lamp results in only a 0.131-Log kill (26.04% kill), and photosensitizers used alone are not very effective either: ER has a 0.13-Log kill (25.88% kill); RB has a 0.16-Log kill (30.81% kill); R has a 0.115-Log kill (23.26% kill); EY has an average of 0.87-Log kill (6.46% kill); and RMP has an average of 0.042-Log kill (3.17% kill).

[0085] Using classical additivity theory, the expected kill for the combination of 18 ppm ER and 10 ppm copper sulfate is 45.18% (0.261-Log kill). The actual median kill was 1.22-Log kill, 0.959-Log higher than expected, which indicates a synergistic effect.

[0086] Using classical additivity theory, the expected kill for the combination of 18 ppm EY and 10 ppm copper sulfate is 30.82% (0.160-Log kill). The actual median kill was 1.86-Log kill, 1.70-Log higher than expected, which indicates a synergistic effect.

[0087] Using classical additivity theory, the expected kill for the combination of 18 ppm R and 10 ppm copper sulfate is 43.24% (0.246-Log kill). The actual median kill is 3.473-Log kill, 3.23-Log higher than expected, which is a synergistic effect. It is also on average over 1,000 times more effective than expected from additivity theory.

[0088] Using classical additivity theory, the expected kill for the combination of 18 ppm RB and 10 ppm copper sulfate is 48.82% (0.159-Log kill). The actual median kill was 1.38-Log kill, 1.22-Log higher than expected, which indicates a synergistic effect.

[0089] Using classical additivity theory, the expected kill for the combination of 18 ppm RMP and 10 ppm copper sulfate is 28.38% (0.144-Log kill). The actual median kill was 4.13-Log kill, 3.98-Log higher than expected, so this is a synergistic effect. This is almost 10,000 times more effective than expected.

[0090] Example 3: Further studies with higher photosensitizer concentrations.

[0091] In this experiment, we also used a reptile light. In this test, the light intensity was 8,520 Lux, or 26 W / m 2(For comparison, this could be full daylight on a cloudy day, or the typical light level in a greenhouse). We observed the following results: Riboflavin (10 ppm) + Silwet (0.0125%) for 30 minutes showed a log reduction of 0.15. Riboflavin at 50 ppm for 1 hour showed a log reduction of 1.6. Silwet (see Helena 2019) is an agricultural non-ionic organosilicon surfactant wetting adjuvant and spreading agent commonly used with pesticides, also commonly known as a sticker spreader). Silwet® L-77 has little to no antimicrobial activity.

[0092] Copper sulfate (10 ppm) + Silwet (0.0125%) for 30 minutes showed a log reduction of 0.13. Copper sulfate at 40 ppm for 30 minutes also showed a log reduction of 0.13. Riboflavin (18 ppm) + copper sulfate (10 ppm) + Silwet (0.0125%) for 30 minutes showed a log reduction of 3.8. Again, we see a more than 1,000-fold improvement in results with the combination of photoactivator and copper. This example shows that the combination of photoactivator and copper salt is highly effective under the most relevant conditions: general light levels, with the lowest effective concentrations of both photoactivator and copper, which is also desirable for economic reasons. In this example, the light source includes UV light. However, as shown in the example with blue LEDs, UV is not necessary.

[0093] Example 4: Copper ethanolamine as the copper compound

[0094] In this example, we tested Cutrine®, a commercial algaecide product containing copper ethanolamine (from the product label: Copper Ethanolamine Complex, Mixed (Mono CAS#14215-52-2 and Tri CAS#82027-59-6) 27.9%; Other ingredients 72.1% Total 100.0% Metallic copper equivalent, 9%; Contains 0.909 lbs of elemental copper per gallon) (EPA Registration No. 8959-10). Copper ethanolamine is effective in keeping the copper salts in solution at a neutral pH. Testing was performed under a reptile light for 30 minutes as described above. Test data to date has shown that riboflavin alone provides well below a 1-log kill under these conditions.

[0095] The above test with CuSO4 at 10 ppm corresponds to 3.98 ppm copper. We initially tested Cutrine® at low and high concentrations, but now the low concentration is adjusted back to about 3.98 ppm copper as before and the high concentration is adjusted to 39.8 ppm copper (same copper concentration as 100 ppm CuSO4). We tested them with and without riboflavin at 18 ppm. Tests were also done in triplicate. Also, riboflavin alone at 18 ppm and 30 minutes under a reptile lamp has a 0.115-Log kill (E. coli). Cutrine® at 3.98 ppm copper has a 0.30-Log kill and Cutrine® at 39.8 ppm has a 3.1-Log kill.

[0096] A 2.5-Log kill was observed with a combination of 3.98 ppm copper Cutrine® and 18 ppm riboflavin. The expected kill based on additivity theory is only 0.314-Log kill. Thus, the actual kill is 2.18-Log higher than expected, which is a synergistic effect.

[0097] A 7.4-Log kill was observed with a combination of 39.8 ppm copper Cutrine® and 18 ppm riboflavin (essentially complete kill). The expected kill based on additivity theory is only a 3.11-Log kill. Thus, the actual kill is 4.29-Log higher than expected, which is clearly a synergistic effect.

[0098] Example 5: Reduction of Xanthomonas hortorum pv. Pelargonii in geranium plants.

[0099] In these trials, geranium cuttings, "American Bright Red," were obtained from Syngenta Flowers (Gilroy, CA). Cuttings were rooted and planted in 3.5-inch pots with Joly Ranch Potting Mix #2. Plants were initially fertilized with 1.5 g / pot (Osmocote Plus, 15-9-12 with micronutrients) and hand watered three times a week. Plants were allowed to establish and grow to approximately 15 cm in height. The experiment was conducted in a greenhouse with temperatures maintained at 65-90°F and light levels of 1000-2000 footcandles. The experiment was set up in a randomized block design with 3 blocks per treatment (10 plants per treatment, 60 total). Single product applications were made on 02 / 27 / 19. The fungicide CuProT / N / O 2005 (copper hydroxide, containing 35% metallic copper equivalent) was used at 2 lbs / 100 gallons (0.7 lbs metallic copper equivalent per 100 gallons) and was applied on the same day as a control. All products were sprayed onto plant surfaces until runoff using a hand sprayer.

[0100] For production of bacterial inoculum, cultures of Xanthomonas hortorum pv. pelargonii (formerly X. campestris pv. Pelargoni, X575) were grown for 48 hours at 28 ± 1 °C on Difco Nutrient Agar (Difco Laboratories, Detroit, MI) amended with 5% sucrose. Bacteria were harvested from NA plates, suspended in saline (NaCl, 8.5 g / l) and adjusted spectrophotometrically to an A600 of 1X105 colony forming units / ml. Leaves and stems were sprayed with bacterial suspension until runoff, and they were sealed in clear polyethylene bags for 24 hours. Treatments were inoculated 24 hours after fungicide application (02 / 28 / 19). For treatment 1, non-inoculated plants (sprayed with saline) served as controls. The number of spots on the leaves of each plant was counted two weeks after inoculation. Each treatment was compared using ANOVA and LSD. A visual comparison was also made to determine whether there were any signs of phytotoxicity.

[0101] The data shows that Treatment E, containing only riboflavin at 20 ppm, is only partially effective. However, Treatment C, containing riboflavin at 20 ppm and copper sulfate at 10 ppm, is effective. Treatment D, containing riboflavin and copper sulfate with sodium ascorbate, is as effective as commercial CuPRO. The advantage of the present invention is that it significantly reduces the amount of copper required. Comparing Treatment F, a commercial CuPRO product containing 0.7 lbs metallic copper equivalent per 100 gallons, or 0.839 g copper per liter, to Treatment D, containing 10 ppm copper sulfate, or 0.00398 g copper per liter, it can be seen that the solution of the present invention provides comparable results while reducing the copper concentration by more than 200 times. (See Figure 16).

[0102] Although the present invention has been described in some detail with reference to certain preferred versions thereof, other versions are possible, and therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained herein, except as required by law.

[0103] The reader's attention is directed to all references filed contemporaneously herewith and incorporated herein by reference.

[0104] All features in this specification (including the accompanying claims, abstract and drawings), unless otherwise stated, may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless otherwise stated, each disclosed feature is described only as a generic example of a series of equivalent similar features.

Claims

1. 1. A method for synergistically inhibiting the growth of a microbial pathogen in a plant, comprising: applying to said plant a combination comprising: a dye-containing compound that is a reactive oxygen species photosensitizer, the dye-containing compound being provided at a concentration of 1 to 100 ppm by weight; and a copper compound for enhancing microbial lethality of said dye-containing compound against said microbial pathogen, said copper compound being provided at a concentration of 0.1 to 100 ppm copper by weight; exposing the plant to light to activate the combination of the pigment-containing compound and the copper compound to inhibit the growth of microbial pathogens of the plant; The method, wherein the copper compound and the dye-containing compound are provided in synergistically effective amounts to inhibit the growth of the microbial pathogen.

2. The method of claim 1, wherein the copper compound is a copper salt, and the copper salt and the dye-containing compound are provided in amounts synergistically effective to inhibit growth of the microbial pathogen, and the combination increases the log kill of the microbial pathogen by at least one order of magnitude compared to the log kill expected based on an additive effect model, the additive effect model determining an expected kill rate as calculated by the formula: P=X+Y(100-X) / 100, where P is the expected kill rate of a pathogen with the combination of the reactive oxygen species photosensitizer and the copper salt, X is the kill rate of the pathogen when the reactive oxygen species photosensitizer is used alone, and Y is the kill rate of the pathogen when the copper salt is used alone.

3. 3. The method of claim 2, wherein the pigment-containing compound is selected from the group consisting of riboflavin, derivatized riboflavin, lumichrome, derivatized lumichrome, curcumin, fluorescein, eosin Y, erythrosine, flavin-containing compounds, and rose bengal B.

4. The method of claim 3 , wherein the pigment-containing compound is riboflavin.

5. 2. The method of claim 1, wherein the copper compound is a copper salt selected from the group consisting of copper (II) sulfate, copper (II) nitrate, copper hydroxide, copper carbonate, copper chloride, copper oxide, and copper bromide.

6. 10. The method of claim 1, wherein the dye-containing compound is provided in a concentration of 5 to 50 ppm by weight and the copper compound is provided in a concentration of 2 to 10 ppm by weight of copper.

7. The method of claim 1 , wherein exposing the plant to light comprises exposing the plant to natural light.

8. The method of claim 1 , wherein exposing the plant to light comprises exposing the plant to artificial light.

9. 10. The method of claim 1, wherein the combination is applied to the plant by at least one of soil soaking, pipetting, irrigating, spraying, listing, watering, and pouring.

10. 10. The method of claim 1, wherein the microbial pathogen comprises any of a bacterial pathogen, a cyanobacterium, an alga, a fungal pathogen, or a viral pathogen.

11. 10. The method of claim 1, wherein the plant is a non-woody crop plant, a woody plant, a turfgrass, a fruit-bearing tree, a nut-bearing tree, or an ornamental plant.

12. The method of claim 1 , wherein the combination further comprises at least one agriculturally acceptable adjuvant.

13. 13. The method of claim 12, wherein the agriculturally acceptable adjuvant is selected from the group consisting of surfactants, penetrating agents, wetting agents, spreading agents, humectants and emulsifiers.

14. The method of claim 1, wherein the copper compound is a copper salt, and the pigment-containing compound and the copper salt are applied to the plant simultaneously.

15. The method of claim 1, wherein the copper compound is a copper salt, and the pigment-containing compound and the copper salt are applied to the plant in sequence.

16. The method of claim 1 , wherein applying the combination to the plant comprises applying a composition comprising the components of the combination to the plant.

17. The method described in claim 1, wherein the copper compound is a copper salt, and the copper salt is used at a concentration of less than 100 ppm by mass to avoid unnecessary damage to the plant or the environment.

18. The method of claim 1, wherein the copper compound is a copper salt, and the pigment-containing compound and the copper salt are suitable for organic farming.

19. 2. The method of claim 1, wherein the dye-containing compound that is a reactive oxygen species photosensitizer is selected from the group consisting of riboflavin, derivatized riboflavin, lumichrome, derivatized lumichrome, curcumin, fluorescein, eosin Y, erythrosine, and rose bengal B; and the copper compound is a copper ion-containing organic complex that increases the microbial lethality of the dye-containing compound against the microbial pathogen.

20. 20. The method of claim 19, wherein the copper ion-containing organic complex is provided in an amount sufficient to increase the microbial pathogen growth inhibition compared to the microbial pathogen growth inhibition of the reactive oxygen species photosensitizer alone.

21. 21. The method of claim 20, wherein the copper ion-containing organic complex and the dye-containing compound are provided in synergistically effective amounts for inhibiting the growth of the microbial pathogen, and the combination enhances the inhibition of the microbial pathogen growth to an extent greater than the sum of the inhibition of the microbial pathogen growth of the reactive oxygen species photosensitizer and the copper ion-containing organic complex when each is used alone.

22. 21. The method of claim 20, wherein the copper ion containing organic complex is provided in a synergistically effective amount to inhibit the growth of the microbial pathogen, and the combination increases the log kill of the microbial pathogen by at least one log or at least one order of magnitude compared to the expected log kill based on an additive effect model, the additive effect model determining an expected kill rate as calculated by the formula: P=X+Y(100-X) / 100, where P is the expected kill rate of a pathogen with the combination of the reactive oxygen species photosensitizer and the copper ion containing organic complex, X is the kill rate of the pathogen when the reactive oxygen species photosensitizer is used alone, and Y is the kill rate of the pathogen when the copper ion containing organic complex is used alone.

23. 23. The method of claim 22, wherein the pigment-containing compound is riboflavin.

24. 23. The method of claim 22, wherein the copper ion-containing organic complex is selected from the group consisting of copper ethanolamine, copper EDTA, copper ions complexed with crown ethers, and copper ions complexed with polyethylene glycol.

25. 23. The method of claim 22, wherein the combination further comprises at least one agriculturally acceptable adjuvant.

26. 26. The method of claim 25, wherein the agriculturally acceptable adjuvant is selected from the group consisting of surfactants, penetrating agents, wetting agents, spreading agents, humectants and emulsifiers.

27. 23. The method of claim 22, wherein the pigment-containing compound and the copper ion-containing organic complex are applied to the plant simultaneously.

28. 23. The method of claim 22, wherein the pigment-containing compound and the copper ion-containing organic complex are applied sequentially to the plant.

29. 23. The method of claim 22, wherein applying the combination to the plant comprises applying a composition comprising the components of the combination to the plant.

30. 23. The method of claim 22, wherein the copper ion-containing organic complex is used at a concentration of less than 100 ppm by weight to avoid unnecessary damage to the plant or the environment.

31. 23. The method of claim 22, wherein said dye-containing compound and said copper ion-containing organic complex are suitable for organic farming.

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